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 intraperitoneal 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 immunotherapy with an antitoxic IgG.

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro.

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 micrograms/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the 'sleep peptide.'

Degradation and aggregation of delta sleep-inducing peptide (DSIP) and two analogs in plasma and serum.

The biostability of DSIP (delta sleep-inducing peptide) and two analogs in blood was investigated in order to determine if rates of inactivation contribute to variable effects in vivo. Incubation of DSIP in human or rat blood led to release of products having retention times on a gel filtration column equivalent to Trp. Formation of products was dependent on temperature, time, and species. Incubation of 125I-N-Tyr-DSIP and 125I-N-Tyr-P-DSIP, a phosphorylated analog, revealed slower degradation and, in contrast to DSIP, produced complex formation. An excess of unlabeled material did not displace the radioactivity supporting the assumption of non-specific binding/aggregation. It was concluded that the rapid disappearance of injected DSIP in blood was due to degradation, whereas complex formation together with slower degradation resulted in longer persistence of apparently intact analogs. Whether this could explain the sometimes stronger and more consistent effects of DSIP-analogs remains to be examined.

DSIP/DSIP-P and circadian motor activity of rats under continuous light.

Daily intravenous injection of 30 nmol/kg DSIP (delta sleep-inducing peptide) in rats under constant illumination produced marked changes of their motor activity as compared to saline controls. Similar marked but distinctly different effects on the circadian pattern of locomotor behavior partially abolished by constant illumination were also obtained after repeated administration of 0.1 nmol/kg DSIP-P (the phosphorylated analogue of DSIP) which enhanced overall motor activity. In both instances the results additionally differed from those reported for a normal 12 hr light:dark cycle. The present results support the hypothesis that DSIP might primarily act by influencing circadian rhythmicity.

Amphetamine-induced locomotor behavior of mice is influenced by DSIP.

The delta sleep-inducing peptide (DSIP) has been shown to induce effects other than only delta sleep. One of these effects was the paradoxical thermoregulatory and locomotor response of rats to amphetamine after DSIP administration. In the present investigation we found similar effects of DSIP on the locomotor activity in mice. However, two different doses of DSIP (30 and 120 nmol/kg) and 3 doses of amphetamine (4, 10, and 15 mg/kg) produced a complex pattern of effects in mice tested at 22 degrees C. In general, DSIP-treated mice showed lower locomotor activity after amphetamine than controls, but under two conditions, both using 15 mg/kg amphetamine, DSIP produced higher scores; this occurred in the first two hours after amphetamine for the 30 nmol/kg DSIP group and in the third hour for mice given 120 nmol/kg DSIP. The results indicate that the effects of DSIP on locomotor behavior were dependent on the dosage of the peptide and the time of measurement as well as the level of amphetamine stimulation.

Isoenzyme specific inhibition of the reactivation of in vitro dissociated lactic dehydrogenase isoenzymes by two different peptides isolated from human liver.

The catalytic activity of the LDH-isoenzymes depends on their tetrameric structure. Low pH or other denaturants leads to dissociation into monomers and to the loss of the specific activity. After removal of the denaturing conditions reassociation and reactivation occur spontaneously. Neither NADH nor NAD+ shows a significant effect on the reactivation. We have isolated two different peptides which isoenzyme specifically inhibit the reactivation of dissociated LDH. Inhibition was abolished by treating with proteases. Additionally, NAD+ and NADH were found to be antagonists of the inhibitors. The heart-type enzyme-inhibitor system is especially susceptible for NADH whereas NAD+ affects the inhibition only slightly. The muscle-type system shows the opposite behavior, e.g., the completely inhibited system can be fully reactivated by NAD+ but not by NADH. These findings together with first kinetic studies suggest a possible specific regulatory function of these peptides.

Inhibition of the reactivation of acid-dissociated lactate dehydrogenase isoenzymes by their aminoterminal CNBr fragments.

The catalytic activity of lactate dehydrogenase isoenzymes (LDH) depends on their tetrameric structure. Stabilization of this quaternary structure is achieved by interaction of the N-terminal part of one subunit with the C-terminal region of the other subunit. The N-terminal peptides from pig M-LDH and H-LDH which are responsible for this stabilization were obtained by CNBr-fragmentation and purification on reversed-phase HPLC. The effect of these peptides on the formation of the quaternary structure of LDH-isoenzymes was investigated by monitoring the reconstitution of the catalytic activity after acid-dissociation. Low concentrations of the N-terminal peptides led to an increased, and high concentrations to a decreased yield of reconstituted LDH activity. The effects of these two peptides were isoenzyme specific. The 32 residue peptide derived from M-LDH showed the highest effect when tested with M-LDH as target enzyme but only a poor effect with H-LDH. On the other side the 33 residue peptide generated from H-LDH showed a moderate effect with both isoenzymes. The effects of the N-terminal LDH peptides are antagonized by the coenzymes NAD+ and NADH. The most significant influence was observed with NAD+ in the M-LDH peptide-M-LDH enzyme system. Comparison of the properties of the reactivation antagonists isolated from human origin with the N-terminal CNBr-peptides of LDH revealed identity in all essential properties, suggesting that the former peptides are generated by degradation of LDH.

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and -H4) isozymes.

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375--1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1.1.27) isoenzymes (LDH-M4 = muscle type; LDH-H4 = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M4 or LDH-H4. From the amino acid analyses, gel filtrations and PAGE + SDS, molecular weights of 1800 for the M4 and approximately 2700 for the H4 inhibitor were calculated. An apparent Ki of approximately 3 X 10(-5) mM for the H4 and approximately 7 X 10(-5) mM for the H4 inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M4-specific) and 2.4 (LDH-H4-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides react with monomers, dimers or a transition state during the tetramerisation process. kappa 1 for the dimerisation step of M4 = 2.0 X 10(5) M-1 . S-1 and of H4 = 8.2 X 10(4) M-1 . S-1; kappa 2 for the tetramerisation step of M4 = 2.8 X 10(5) M-1 . S-1 and of H4 = 1.2 X 10(5) . M-1 S-1, were calculated, the second step still being the faster one (Rudolf, R. and Jaenicke, R. (1976) Eur. J Biochem. 63, 409--417).

Autoradiographic localization of binding sites for the delta sleep-inducing peptide ( [3H]DSIP) on neurons of cultured rat brainstem.

Binding of the delta sleep-inducing peptide (DSIP) was studied in cultures of rat brainstem by means of autoradiography. Binding sites for [3H]DSIP were observed on small, medium-sized and large brainstem neurons but not on glial cells. Addition of unlabeled DSIP inhibited or markedly reduced binding of [3H]DSIP. It is suggested that brainstem neurons might possess receptors for this sleep-inducing peptide.

Comparison of DSIP- (delta sleep-inducing peptide) and P-DSIP-like (phosphorylated) immunoreactivity in cerebrospinal fluid of patients with senile dementia of Alzheimer type, multi-infarct syndrome, communicating hydrocephalus and Parkinson's disease.

The concentrations of delta sleep-inducing peptide (DSIP)-like (DSIP-LI) and P-DSIP-like (phosphorylated, Ser7) immunoreactivity (P-DSIP-LI) were measured by specific radioimmunoassay in the cerebrospinal fluid (CSF) of patients with senile dementia of the Alzheimer type [SDAT, subdivided into early (S1), middle (S2) and late dementia (S3)], multi-infarct dementia (MD), Parkinson's disease (PD), vascular disease (VD) and communicating hydrocephalus (H), as well as in control patients (C1, C2). Mean DSIP-LI and P-DSIP-LI concentrations were found to be significantly higher in the elderly control group (C1, mean age 83 +/- 5 years) than in the middle-aged control group (C2, mean age 40 +/- 16 years). DSIP-LI and P-DSIP-LI were positively correlated with age in both control groups. Significant decreases of DSIP-LI compared with age-matched controls (C1) were observed for S2, S3, MD, PD, VD and H. In contrast, no significant differences corresponding to pathology were found for P-DSIP-LI.

Delta sleep-inducing peptide in spontaneously hypertensive rats.

Delta sleep-inducing peptide has been shown to exert extra-sleep effects as well as effects on sleep. In this study, the concentrations of DSIP-like immunoreactivity were measured by radioimmunoassay in the plasma of spontaneously hypertensive rats (SHR). They were found to be about 25% higher in SHR plasma than in the plasma of the normotensive Wistar-Kyoto (WK) controls. DSIP was then infused for 10 days by osmotic minipump (200 micrograms/kg/day) into SHR. This resulted in in maintenance of BP at a level of about 200 mm Hg as compared with the significant increase to about 220 mm Hg after 10 days in the SHR controls infused with 0.9% NaCl. After daily SC injection of a single dose of 200 micrograms/kg DSIP for each of 5 days in SHR, findings were similar. The results raise the possibility of an involvement of DSIP in the regulation of BP in SHR.

DSIP-induced changes of the daily concentrations of brain neurotransmitters and plasma proteins in rats.

The influence of delta sleep-inducing peptide (DSIP) on the brain neurotransmitters 5-HT, dopamine and norepinephrine and plasma proteins/corticosterone concentrations for four time points within the 24 hr following IV injection of 30 nmol/kg was investigated in rats. DSIP administered in the morning or in the evening respectively induced changes in nearly all measured parameters. Different effects were observed for different times of administration. The most marked changes were found in the level of serotonin during daytime. In view of the multivariate results obtained by measuring several parameters at multiple time points, a method was developed to describe the time-dependent changes. By means of "circadian rhythm statistics" based on a statistical likelihood analysis we found that multiple and different changes within the factor's daily variation are induced by one injection of DSIP. A multidimensional scaling of the results provides further insights into the correlations of the DSIP-induced effects on plasma and brain factors which are therefore tentatively termed "programming functions." These apparently involve not just sleep induction but also act on multiple parameters within the 24 hr rest-activity period.

Effects of repeated DSIP and DSIP-P administration on the circadian locomotor activity of rats.

Daily intravenous evening injections of 30 nmol/kg DSIP (Delta Sleep-Inducing Peptide) in rats adapted to a constant 24 hr light:dark cycle produced changes in the circadian locomotor behavior. After 3 days the normally high locomotor activity during the dark phase was reduced while during the light (sleeping) phase the animals became relatively more active. Similar, but more rapid and more marked changes were observed (with the same schedule of injections) after 0.1 nmol/kg DSIP-P (the analogue of DSIP phosphorylated at the serine in position 7). In fact the peptide and its analogue induced a relative reversal or shift of the circadian locomotor activity phases opposite to the persisting light:dark conditions (=Zeitgeber). This suggests that DSIP exerts rather complex "programming" effects on the circadian activities and has more than just a sleep-inducing activity.

Influence of burn-induced lipid-protein complex on IL1 secretion by PBMC in vitro.

The lipid-protein complex (LPC) formed by thermal injury to skin, which has been shown to have a toxic effect on mice, and which suppresses the immune response, was tested for its specific influence on monocytes. Growth of bacterial endotoxin-stimulated peripheral blood mononuclear cells (PBMC) was inhibited in the presence of LPC, however, the inhibition was less at the time of the optimal rate of cell proliferation. Inhibition was proportional to LPC concentration. ConA-stimulated PBMC were also inhibited by LPC in a dose-related manner. PBMC, in the presence of LPC, secreted interleukin 1 (IL1) at an increasing rate as LPC concentration rose from 5 to 40 micrograms/ml, and the levels of IL1 which could be induced by endotoxin were increasingly amplified in the presence of LPC. In comparison to LPC, the native tissue proteins which were isolated from unburned skin by the same techniques which produced LPC from burned skin, were tested for their effect on PBMC. Native proteins had no effect on IL1 secretion, whether on background or endotoxin-stimulated levels. Thus, the thermally induced change in skin proteins has a specific effect on monocyte IL1 secretion which is not matched by the native proteins, indicating that burn injury to skin specifically affects the lymphokine cascade and consequent immune function.

Plasma levels of cutaneous burn toxin and lipid peroxides in thermal injury.

Lipid peroxides, formed as a consequence of oxygen free radical formation, are responsible for tissue damage in a great variety of pathological conditions including thermal injury. 'Cutaneous burn toxin', formed by application of heat to skin, is thought to be specific to the burn injury. It causes dose-dependent damage to mitochondrial and red cell membranes, and dose-dependent inhibition of interleukin-2-dependent growth of lymphocytes. The possibility that the toxicity of 'cutaneous burn toxin', a lipid-protein, is exerted through lipid peroxides, was examined by measuring the levels of both agents in plasmas of eight burn patients during the first week after their injury. It was observed that plasma lipid peroxides did not appear in parallel with absorption into the circulation of 'cutaneous burn toxin'. Lipid peroxide levels equally common to very low and very high burn toxin levels, were recorded. The pair of agents correlated negatively (r = -0.26) at a significance of only 0.1. In addition, isolated purified 'cutaneous burn toxin' contained no measurable lipid peroxide. No relationship was therefore demonstrated between plasma levels of 'cutaneous burn toxin' and lipid peroxides.

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.

Burn sepsis and burn toxin.

The salient steps of a 20-year programme of research into the nature of burn disease are described. By burn disease we mean the late mortality and morbidity following burns. We have isolated a burn toxin which is derived from a thermal polymerization of cell membrane lipoproteins within the dermis and have studied its influence on the effects of sepsis. We have also used it in the development of active and passive immunization therapy of severe burns.