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Experimental observations on immune burn serum
EXPERIMENTAL
OBSERVATIONS ON I M M U N E
B U R N SERUM
By B. N. BAILEY, F.R.C.S. 1 Senior Registrar, Plastic Surgery and Jaw Injuries Unit, Stoke Mandeville
DEFINITE toxmmia occurs in the burned patient as a result of specific and non-specific bacterial invasion. Many clinicians have thought that some part of the burned patient's early illness, before the onset of bacterial invasion, is due to absorption into the circulation of products from the burn site. The number of substances variously incriminated raises the usual doubts as to whether any of them is causative of, or merely co-existent with, the burn "toxmmia." Avdakoff (z876) first claimed that toxicity was demonstrable by injecting blood from burned to unburned animals. Since then experiments have been reported, repeated, refuted, and rejected time and time again, and with the growth of knowledge about burn pathology the need for a toxin has diminished. At the present time most authorities would reject the existence of a burn toxin on the principle of Occam's razor. Rosenthal (x937 a) described the presence of a toxin in the blood of burned experimental animals and human beings, and in the same year (z937 b) described the presence of a corresponding antitoxin. He further demonstrated that a diffusate of burned skin was lethal to mice and rats and that precipitins were produced against this toxin after injection into the rabbit (z955, I956, I957, I959, z96o). Fedorov (z956) demonstrated a high level of antigen in the circulation after burning, followed by the development of an antibody which could be demonstrated by the cold agglutination method. Pavkova and Dolezalova (I96O) and Dobrkovski et al. (I96o) describe a rising antibody level against burned skin antigen, and a close relation between the antibody level and the patient's clinical condition. Rosenthal et al. (I96O a) reported that serum from patients up to sixteen days after burning inhibited HeLa cells in fourteen out of fifteen cases, while, following transfusion with healed burn donor blood the toxicity to HeLa cells disappeared. They claim that aqueous extract of burned skin inhibits HeLa cells and gives precipitation reactions with convalescent burn serum. Rosenthal et al. (I96O b) used convalescent burn serum in the I958 Chicago school fire and claim dramatic clinical improvement. Pushkar (I958) also reports successful results from immuno-ha:motherapy. There appears to be a rift between the demonstration of the toxin-antitoxin phenomenon in vitro and the demonstration of clinical improvement in humans. This should logically be bridged by carefully controlled animal work, but other than that of Malme and Slawikowski (I96o), who report increased survival time in the rat following the use of convalescent burn serum, and that of Fedorov (I96O), there is little or none. Fedorov gives no comparative figures on which to base deductions, and one must be prepared to accept his conclusions uncritically. 1 Research Associate, University of Texas Medical Branch, z959-6o.
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The work reported here was undertaken at the University of Texas in order to :-L Confirm or refute the value of convalescent serum in burned animals. 2. Determine the specificity of the response. 3. Find under what circumstances immunity could be produced. E x p e r i m e n t I.--This was performed in order to find the degree of protection produced by previous scalding. Fifty 200 g. (plus or minus I g.) female Holtzman albino rats were divided into two sets of twenty-five, and epilated with electric clippers and calcium
FIG. I
Apparatus used to mass-produce standard scalds. Exact position of the rat is obtained and depth of immersion, temperature and time are automatically controlled.
thyoglycollate paste. The first twenty-five were subjected to a 15 per cent. weight immersion scald in water at 90 ° C. for fifteen seconds while anazsthetised with intraperitoneal Nembutal (3 mg. per IOO g.). The rats in this and subsequent series were scalded using the apparatus in Fig. I. Previous experiments had shown that we were able to produce the desired surface area burn with automatic control of temperature and time to a very high degree of accuracy. The 15 per cent. weight immersion scald represents a scald of I5 per cent. body surface. The rats were untreated, placed in individual cages in a room air-conditioned to 780 F., and allowed Purina Chow and water ad lib. All recovered and the eschar started to separate after two weeks. The raw surface left by the eventual separation was not completely healed by the time of the second scald fifty-six days later. The other twenty-five rats had I5 per cent. of their back skin excised down to deep fascia under intraperitoneal Nembutal. The skin edges were sutured to the fascia to prevent too early healing (Fig. 2).
EXPERIMENTAL
OBSERVATIONS
ON
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BURN
SERUM
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The purpose of the control set was to eliminate difficulties with the depth of the scald, for clearly, scalding would produce deeper necrosis over an already raw surface than over normal skin and panniculus. At the end of fifty-six days both sets had a residual raw surface (Fig. 3) of approximately equal area and were subjected to a 65 per cent. weight immersion scald (about 40 per cent. surface area) at 9°0 C. for fifteen seconds. Post-operative treatment given to both sets was io ml. normal saline intraperitoneally immediately after scalding, and a further Io ml. intraperitoneally
FIG. 2
FIG. 3
Fig. 2 . - - S c a l d e d rat on the left and excisec] control for Experiment I. Fig. 3.--Scalded rat on the left and excised control after epilation prior to scalding fifty-six days after first challenge.
after six hours. They were allowed Chow and water ad lib, and observed every hour on the hour. The average survival time of the previously scalded rats was fifty-one hours compared with that of the excised rats which was twenty-four and a half hours (Fig. 4). A 65 per cent. scald was chosen because in this type of rat it is uniformly fatal, and it was considered easier to assess survival times when these were short than when they were long, and complicated by the late effects of burning. The doubling of survival time suggests that pre-treatment by scalding, though not by excision, effects some useful modification of the body defence, whether glandular, metabolic, nervous, or humoral. E x p e r i m e n t 2.--In this experiment a wide variety of pre-treatments was used to see if any had an influence on survival after scalding. Two hundred 14o g. (plus or minus ! g.) female Holtzman albino rats were 3 EI
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divided into four sets of fifty, each set being divided into couples which were caged together and, apart from the specific trauma, identically treated. Set Ia was given 5 rag. intraperitoneal Nembutal every third day for two weeks, and three weeks later scalded. Set 2a was bled of 4 to 6 ml. blood by severing the jugular vein with a scissor cut through the skin on two occasions at weekly intervals, and three weeks after the second bleeding scalded. Set 3a was subjected to tourniquet trauma by applying a very tight rubber band to one hind limb for six hours. This was repeated after a week on the opposite hind limb and followed by scalding after a further three weeks. Experiment 1 CUMULATIVE MORTALITY A f t e r 40°/o s u r f o c e area sceld at 90~C f o r 15secs. PREVIOUSLY EXCISED
25
/J SET
/
PREVIOUSLY SCALDED
SET
o
5 f----
O
J 20
10
J 30
4to
510
TIME OF DEATH IN HOURS AFTER
J 60
'(3
7
SCALDING
FIG. 4
Set 4a.--The right femur was fractured under Nembutal anaesthesia without breaking the skin, and four weeks later the animals were scalded. Sets ib, 2b, 3b, and 4b were the paired untreated controls. All animals had a 65 per cent. body weight scald at 9 °° C. for fifteen seconds, and were treated as in Experiment I. Average survival times are shown below :-Pre-treated.
Set Set Set Set
Ia, 2a, 3a, 4a,
26 hours. 23 hours. z9½ hours. 22 hours.
Control.
Set Set Set Set
Ib, 2b, 3b, 4 b,
27½ hours. 21½ hours. 24 hours. 2I hours.
There is not a significant difference in survival times between pre-treated and control rats, but it is heartening to find that the controls show such a close survival time. It may be suggested that it is unfair to compare rats which have been subjected to major trauma with healthy controls. This point is clarified in Experiment 4, but it should be pointed out that the rats protected by a previous scald and their controls in Experiment I were also severely treated. As it is possible that there is some development of protection against scalding
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BURN
SERUM
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by previous trauma, which is, however, overwhelmed by the severe test scald in conjunction with debility of the rat, a further series of experiments was performed after the question of transferred immunity had been elucidated. E x p e r i m e n t 3 . - - T o demonstrate whether passive immunity can be achieved a large number of rats was severely scalded. The supposition was made that if serological immunity occurred at all it would surely be manifested by the small number of tough survivors from an almost uniformly fatal scald. One thousand adult male and female Holtzman albino rats weighing about 20o g. each were epilated, ana:sthetised with intraperitoneal Nembutal, and scalded at 55 per cent. weight immersion (about 35 per cent. surface area) at 90 ° C. for fifteen seconds. They were untreated in order that only the hardiest (and most competent immunologically ?) would survive. Most of the rats died within the first three days after scalding, but any lasting longer than a week usually recovered. There were approximately one hundred survivors who went through the usual phases of escharification, very slow sloughing, cicatrisation, and epithelialisation. At the time of slough separation the rats were thin and obviously ill. During the ensuing slow period of healing they gradually increased in weight, and (like the human patient) at about the tenth week showed a sudden recovery, healed rapidly, and became heavier and more active. Healing was complete in most of the rats at the twelfth week, and the few remaining unhealed ones were discarded. The rats which at eighty-four days after scalding weighed 35o to 4oo g. were exsanguinated in the following way. Each received an intravenous injection of 20 ml. sterile normal saline containing 5,ooo units penicillin and 5 mg. streptomycin. This put the rats into congestive failure and they were then sacrificed by cutting their throats through an epilated area cleaned with phisohex, and exsanguinated into a sterile beaker. Each rat yielded 15 to 2o ml. mixture of diluted blood which was pooled, and centrifuged after dotting. Ha~matocrit indicated that the serum was roughly a 5o-5o mixture of serum and saline. This was stored in. sterile flasks at - IO° C. At the same time IOO rats were exsanguinated and their diluted and antibiotic impregnated serum stored similarly. One hundred 200 g. (plus or minus I g.) female Holtzman albino rats were split into two sets of fifty. They were epilated, an~esthetised, and scalded to 65 per cent. weight immersion at 9o ° C. for fifteen seconds and treated as follows :-I. Immediately after scalding, 2 ml. serum-saline mixture intravenously and Io ml. intraperJtoneal normal saline. 2. Four hours after scalding, I ml. serum-saline mixture subcutaneously and IO ml. intraperitoneal saline. 3. Eight hours after scalding, I ml. serum-saline mixture subcutaneously. 4- Twenty-four, forty-eight, seventy-two, etc., hours after scalding, i ml. serum-saline mixture subcutaneously. Half the animals received convalescent serum, while the other half received normal serum. The cumulative mortality chart (Fig. 5) shows that with the exception of a few rats that never recovered consciousness (probably due to the anzesthetic) the time of death of the control set lies between twenty and forty hours, while in the convalescent serum set it is between forty and eighty hours, with the exception 3 E2
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of a few stragglers in the ends of the main part of both curves. The average survival time of the untreated rats is twenty-seven hours and the convalescent serum treated set sixty hours. The behaviour of the rats after scalding is of interest. Those dying within the first ten hours failed to recover consciousness and were probably anmsthetic deaths. All the other rats recovered consciousness to the extent that they moved their limbs and responded to stimuli within an hour, but thereafter showed little Experiment
3
CUMULATIVE MORTALITY After 40°/o surface area s c a l d at 9 0 ° C f o r 15 sees
CONTROL SET
50
i
]O
!
20
30
i
40
,
50
__.r-...~
i
60
i
70
T I M E O F D E A T H IN H O U R S A F T E R
f
80
90
'
~
'
100
1 0
130
IMMUNE SERUM
SET
1~0
SCALDING
FIG. 5
activity for up to four hours. At this time they would be on their feet and wandering about the cage, eating occasionally and drinking frequently. The behaviour of the two sets is sharply contrasted. In the racks of cages the rats were arranged in alternate rows of treated and control animals. Though the rats given convalescent serum were known only to the writer, laboratory assistants and visitors were able without hesitation to select the treated rats. They were more alert and active and usually eating and particularly drinking eagerly. On the other hand, the untreated rats wandered around listlessly and rather aimlessly without eating or drinking much. Most of the rats in both sets had hmmoglobinuria after about twelve hours and up to twenty-four hours after scalding. Their mode of death was of two types. The rat, after appearing well and active, would slow down, have convulsions, and die within minutes. On the other hand, animals which survived more than thirty-six hours lapsed slowly into coma which might last up to twenty-four hours before death. There appears to be little doubt after this experiment that convalescent serum collected three months after scalding contains some factor capable of prolonging life in a severely scalded rat. As far as it is valid to base the deduction on animal work, it would appear that the rats felt better in the early post-scald period, and this was reflected by their greater activity. It is obvious that this work will have to be repeated on larger laboratory animals and humans in order to assess the actual changes (other than possible serological ones) by which the beneficial effects of convalescent serum are effected.
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BURN
S ER U M
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E x p e r i m e n t 4.--It was apparent by this stage that not only does previous scalding confer some active immunity against subsequent scalding, but that passive immunity can be conferred by using convalescent serum as a transfusion substance in the scalded rat. This made a better planned follow-up of Experiment 2 possible. In this experiment sets of animals traumatised as in Experiment 2 were sacrificed three months after trauma to provide convalescent serum for testing. Twenty 20o g. (plus or minus I g.) female Holtzman rats were used in each of six different sets and were all subjected to a scald of 65 per cent. weight immersion at 9°o C. for fifteen seconds, and given the same post-operative treatment as in Experiment 3, using the different types of convalescent serum. I. Received normal serum-saline mixture. 2. Received serum-saline mixture from multiple an~esthetised rats. 3. Received serum-saline mixture from twice-bled rats. 4. Received serum-saline mixture from rats with fractured femora. 5. Received serum-saline mixture from rats after two tourniquets. 6. Received serum-saline mixture from convalescent scalded rats--stored for two months from Experiment 3. Cumulative mortality charts are not given as they are largely superimposed, but the average survival times of the rats are as follows : (I) 29 hours, (2) 32 hours, (3) 3° hours, (4) 281 hours, (5) 31 hours, (6) 58~- hours. It is apparent that the original batch of convalescent serum had not lost its potency after two months' storage at - i o ° C. and that serum from other types of trauma does not have a protective action against scalding. The serum donors were left three months on this occasion after the initial challenge, as this time had proved effective in Experiment 3. E x p e r i m e n t 5.--BY this time it appeared that a specific response of the antigenantibody type was occurring, and further tests were made with sera. of different vintage. It was found that sera extracted two weeks, four weeks, and eight weeks after a 55 per cent. standard scald were ineffective in giving protection. It may well be that the serum does need to be convalescent. Certainly the rats at two, four, and eight weeks are not convalescent. On the other hand, the clear demonstration of actively acquired immunity in Experiment I probably occurred because the scald was very much smaller and the animals were truly convalescent at the time of the second scalding. It was decided to proceed from this point on the assumption that skin and the subjacent tissues are altered by scalding and some diffusable product reaches the blood stream capable of promoting an antibody response. Whether any toxic product could be demonstrated in the skin or serum remained to be seen. A set of 2o0 g. female Holtzman rats was given the standard scald, and sacrificed two hours later. The skin, panniculus, and as much of the (edematous areolar layer above the deep fascia as possible were removed aseptically. The scalded skins were cut into tiny pieces and ground in sterile sand in a pestle and mortar until only the irreducible skin was left. This was extracted by multiple washings in sterile distilled water, centrifuged, filtered, and lyophilised. Intravenous injections of this crude extract into healthy 2o0 g. female rats were immediately fatal, but so were equal injections of normal skin similarly extracted.
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Subcutaneous injections of scalded skin extract produced variable results. M o r e than 3 ° per cent. surface area made the rats ill ; normal skin did not do this. After half an hour or so, rats became listless, hypotonic muscularly, and their hair bristled (Fig. 6). After about forty-eight hours they recovered. Occasional deaths followed subcutaneous injection o f scalded and normal skin, but these were not related to the dose. T h e intention to establish the minimum lethal dose of scalded skin extract was abandoned owing to the failure to produce death. In general, doses of more than IO per cent. scalded skin extract made the animals i11, but did not kill them. In case there was some change in the products from the skin following absorption into the blood stream, a series o f rats was exsanguinated (without dilution) twenty
FIG. 6 A p p e a r a n c e of rat two hours after s u b c u t a n e o u s i n j e c t i o n of 20 p e r cent. surface area scalded skin extract.
hours after a 65 per cent. standard scald. Intravenous injection of 5 ml. undiluted normal serum (if spread over about ten minutes to avoid heart failure) was found to be without effect. Injection of 5 ml. scalded rat serum produced the same effects as subcutaneous injection o f scalded skin extract, but even this quantity, which represented the serum from two scalded rats, did not prove fatal. It was concluded that if there is a toxin in scalded skin extract or serum from scalded rats, it is not sufficiently potent to cause death without the other concomitants o f scalding. E x p e r i m e n t 6 . 1 S i n c e rats had been made ill by toxic extracts, a number of adult female Holtzman rats was actively immunised against scalded skin extract, normal skin extract, post-scald serum, and normal serum. In view of the possible objections to Experiment 2 and the demonstration of the occurrence of passive immunity in Experiment 3, direct testing o f these immunised animals was not performed. Instead they were used as donors of i m m u n e serum. Preparation ofSera--Set i a . m T w e n t y - f i v e 200 g. female Holtzman rats received a subcutaneous injection of IO per cent. surface area of scalded skin extract, 5,000 units penicillin, 5 mg. streptomycin, and 0"5 ml. complete Freund's adjuvant. One
EXPERIMENTAL OBSERVATIONS ON IMMUNE BURN SERUM
25I
week later a repeat injection of 2 to 3 per cent. surface area scalded skin extract with penicillin and streptomycin but without adjuvant was given. Set ib.wTwenty-five similar rats received similar treatment except that normal skin was substituted. Set 2a.wTwenty-five similar rats were treated with a subcutaneous injection of 5 ml. serum from twenty-hour post-scald rats, with penicillin, streptomycin, and Freund's adjuvant. After one week they had a repeat injection of I ml. with penicillin and streptomycin but no adjuvant. Set 2b.--Twenty-five similar rats were treated in the same way as 2a except that normal serum was substituted. In all cases the injected skin extracts and injected post-scald serum were pooled. Four weeks after the second injection the rats were exsanguinated by the usual technique. Their number had shrunk by this time. About a quarter of the rats receiving normal or scalded skin injections had developed abscesses at the site of the first injection and were sacrificed. Of the serum sets, only two in the scalded serum and three in the normal serum group developed infection and were discarded. Sets of fifteen 200 g. (plus or minus I g.) female Holtzman rats were subjected to the 65 per cent. standard scald and treated as in Experiment 3 except for the substitution of the appropriate serum. Set IA received serum-saline mixture from the immunised set Ia. Set I~ received serum-saline mixture from the immunised set lb. Set 2A received serum-saline mixture from the immunised set 2a. Set 2B received serum-saline mixture from the immunised set 2b. Set 3 received normal serum-saline mixture from healthy rats. No control with the original three-month post-scald serum was possible as it had all been used. The number of rats in each set (owing to shortage of serum) is not large enough to enable detailed comparison between sets to be made. For this.reason, and because they are largely superimposed, cumulative mortality charts are not given. The average survival times of the different sets are as follows : (Ii) 65 hours, (IB) 20 hours, (2A) 5 O1 hours, (2B) 24½ hours, (3) 25 hours. It seems clear that normal skin extract and serum, potentiated by Freund's adjuvant, do not provoke any immunity. Even (or perhaps particularly) crude scalded skin extract and early post-scald serum give rise to immunity against subsequent scalding which is of the same order as that produced by previous scalding. Again it was easy for uninformed observers to pick out the treated animals as opposed to the controls within the first twenty-four hours of scalding. Groups (IA) and (2A) were selected by five independent observers. The possibility of one person making this selection by chance is one in ten. The chance of five independent observers doing it is I in IOO,OOO.
SUMMARY AND CONCLUSION
Under the experimental conditions used it appears that the rat is capable of developing active immunity to scalding. Convalescent serum from previously scalded rats exerts a marked protective influence on other scalded rats.
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After a severe scald this immunity reaches a useful level after three months. This is a specific response to scalding and is not produced by some other forms of trauma. Toxic but not lethal effects have been produced by injection of scalded skin extract and serum from severely scalded rats. After a month these injections produce a degree of immunity which enables their serum to protect other scalded rats. It appears that an effective period after a major scald is three months. A less severe scald produced active immunity after two months. In both sets the rats were truly convalescent at the time of their high immunity. It is tempting to speculate that only when the rat shows obvious clinical signs of recovery, analogous to those seen in the burned human, does it have a high enough antibody titre to be of value. One might guess that up to that time any antibody produced was immediately neutralised by still present antigen. This fits well with the findings, and is additionally confirmed in Experiment 6 by the immunisation against scalded skin extract. The rats--not suffering from the major depressing effect of a burn, and being given a comparatively small quantity of antigen--appear to achieve a useful immune level earlier. Dobrkovski's claims at the Washington meeting seem to be based on an impartial serological and clinical survey. Although the number of cases is small he has carefully related the patient's clinical progress with the serum antibody level. There appears to be a maximum antibody level in the early post-burn period which falls off during recovery, rising finally after three months during the patient's convalescence. He has been impressed (personal communication) by the marked subjective improvement in severely burned cases treated with convalescent serum, although the extent to which this influences the eventual outcome is difficult to assess at this stage. It is clear that in this series of rat experiments, although "subjective" wellbeing and prolongation of life were obtained, immune serum did not prevent eventual mortality. Although attempts were made to give adequate fluid therapy to control and test rats, it may be that supportive therapy was less than Ioo per cent. If this is the case, it could well be that if supportive treatment were improved, the difference apparently obtained with the use of convalescent serum might disappear. Fluid therapy in the burned human is painstakingly and accurately controlled, and it may be that some minor benefits from convalescent serum would not be apparent under these circumstances. In most burn units early death is not a problem, and the incidence of death after about ten days is due to a multiplicity of pathological processes. It is difficult to see how these deaths could be influenced by the use of convalescent serum, unless its early use were to prevent some organ damage which later rendered the patient more susceptible to infection, exhaustion, and the other late consequences of burning. This is, however, only conjecture, and the possibility that a borderline case may be influenced by the early use of convalescent serum is sufficient incentive to continue the study of this scientifically exciting field. M y thanks are due to Dr Blocker who provided the facilities, Sally Abston who helped to prepare and scald the rats, Harvey Williams who ground down the skin, and Taylor Wharton who lyophilised the watery extract.
EXPERIMENTAL
OBSERVATIONS
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IMMUNE
BURN
SERUM
REFERENCES AVDAKOFF (1876). St Petersburg reed. Wschr. Quoted by S. Sevitt (1957) in " Burns : Pathology and Therapeutic Applications." London : Butterworth. DOBRKOVSKI, M., DOLEZALOVA, J., and PAVKOVA, L. (196o). " Report to the ISt International Congress of Research in Burns." Washington. FEDOROV, N. A. (1956). Proceedings of the 6th Congress of the International Society of Blood Transfusion, p. 54. --(196o). " Report to the ISt International Congress on Research in Burns." Washington. MALME, O. J., and SLAWIKOWSKI, J. M. (196o). " Report to the ISt International Congress on Research in Burns." Washington. PAVKOVA, L., and DOLEZALOVA, J. (I96o). " Report to Symposium on Plastic Surgery and Burns." Marianske Lazne. PUSHKAR, L. I. (1958). Ter. Ark., Moskva. ROSENTHAL, S. R. (I937 a). Ann. Surg., xo6, III. - - - - (I937 b). Ann. Surg., xo6, 257. - - - - (I959). Surgery, 46, 932. ROSENTHAL, S. R., FINAMORE, F. J., HUNTER, F. R., and WILLIAMS, A. D. (I956). Fed. Proc., xS, I56. ROSENTHAL, S. R., HARTNEY, J. B., and SI'URRIER, W. A. (I96O a). J. Amer. reed. Ass., I74, 957. ROSENTHAL, S. R., HUNTER, F. R., and FINAMORE, F. J. (196o b). Arch. int. Pharmacodyn., x26, 43. ROSENTHAL, S. R., HUNTER, F. R., HUNTER~ A, S., FINAMORE, F. J., WILLIAMS, f . , and ROMAN, I.-N. (1955). Fed. Proc., I4, I24. ROSENTr{AL, S. R., and TRAHAN, H. (I957). Fed. Proc., I6, 370.
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OBSERVATIONS ON I M M U N E
B U R N SERUM
By B. N. BAILEY, F.R.C.S. 1 Senior Registrar, Plastic Surgery and Jaw Injuries Unit, Stoke Mandeville
DEFINITE toxmmia occurs in the burned patient as a result of specific and non-specific bacterial invasion. Many clinicians have thought that some part of the burned patient's early illness, before the onset of bacterial invasion, is due to absorption into the circulation of products from the burn site. The number of substances variously incriminated raises the usual doubts as to whether any of them is causative of, or merely co-existent with, the burn "toxmmia." Avdakoff (z876) first claimed that toxicity was demonstrable by injecting blood from burned to unburned animals. Since then experiments have been reported, repeated, refuted, and rejected time and time again, and with the growth of knowledge about burn pathology the need for a toxin has diminished. At the present time most authorities would reject the existence of a burn toxin on the principle of Occam's razor. Rosenthal (x937 a) described the presence of a toxin in the blood of burned experimental animals and human beings, and in the same year (z937 b) described the presence of a corresponding antitoxin. He further demonstrated that a diffusate of burned skin was lethal to mice and rats and that precipitins were produced against this toxin after injection into the rabbit (z955, I956, I957, I959, z96o). Fedorov (z956) demonstrated a high level of antigen in the circulation after burning, followed by the development of an antibody which could be demonstrated by the cold agglutination method. Pavkova and Dolezalova (I96O) and Dobrkovski et al. (I96o) describe a rising antibody level against burned skin antigen, and a close relation between the antibody level and the patient's clinical condition. Rosenthal et al. (I96O a) reported that serum from patients up to sixteen days after burning inhibited HeLa cells in fourteen out of fifteen cases, while, following transfusion with healed burn donor blood the toxicity to HeLa cells disappeared. They claim that aqueous extract of burned skin inhibits HeLa cells and gives precipitation reactions with convalescent burn serum. Rosenthal et al. (I96O b) used convalescent burn serum in the I958 Chicago school fire and claim dramatic clinical improvement. Pushkar (I958) also reports successful results from immuno-ha:motherapy. There appears to be a rift between the demonstration of the toxin-antitoxin phenomenon in vitro and the demonstration of clinical improvement in humans. This should logically be bridged by carefully controlled animal work, but other than that of Malme and Slawikowski (I96o), who report increased survival time in the rat following the use of convalescent burn serum, and that of Fedorov (I96O), there is little or none. Fedorov gives no comparative figures on which to base deductions, and one must be prepared to accept his conclusions uncritically. 1 Research Associate, University of Texas Medical Branch, z959-6o.
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The work reported here was undertaken at the University of Texas in order to :-L Confirm or refute the value of convalescent serum in burned animals. 2. Determine the specificity of the response. 3. Find under what circumstances immunity could be produced. E x p e r i m e n t I.--This was performed in order to find the degree of protection produced by previous scalding. Fifty 200 g. (plus or minus I g.) female Holtzman albino rats were divided into two sets of twenty-five, and epilated with electric clippers and calcium
FIG. I
Apparatus used to mass-produce standard scalds. Exact position of the rat is obtained and depth of immersion, temperature and time are automatically controlled.
thyoglycollate paste. The first twenty-five were subjected to a 15 per cent. weight immersion scald in water at 90 ° C. for fifteen seconds while anazsthetised with intraperitoneal Nembutal (3 mg. per IOO g.). The rats in this and subsequent series were scalded using the apparatus in Fig. I. Previous experiments had shown that we were able to produce the desired surface area burn with automatic control of temperature and time to a very high degree of accuracy. The 15 per cent. weight immersion scald represents a scald of I5 per cent. body surface. The rats were untreated, placed in individual cages in a room air-conditioned to 780 F., and allowed Purina Chow and water ad lib. All recovered and the eschar started to separate after two weeks. The raw surface left by the eventual separation was not completely healed by the time of the second scald fifty-six days later. The other twenty-five rats had I5 per cent. of their back skin excised down to deep fascia under intraperitoneal Nembutal. The skin edges were sutured to the fascia to prevent too early healing (Fig. 2).
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BURN
SERUM
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The purpose of the control set was to eliminate difficulties with the depth of the scald, for clearly, scalding would produce deeper necrosis over an already raw surface than over normal skin and panniculus. At the end of fifty-six days both sets had a residual raw surface (Fig. 3) of approximately equal area and were subjected to a 65 per cent. weight immersion scald (about 40 per cent. surface area) at 9°0 C. for fifteen seconds. Post-operative treatment given to both sets was io ml. normal saline intraperitoneally immediately after scalding, and a further Io ml. intraperitoneally
FIG. 2
FIG. 3
Fig. 2 . - - S c a l d e d rat on the left and excisec] control for Experiment I. Fig. 3.--Scalded rat on the left and excised control after epilation prior to scalding fifty-six days after first challenge.
after six hours. They were allowed Chow and water ad lib, and observed every hour on the hour. The average survival time of the previously scalded rats was fifty-one hours compared with that of the excised rats which was twenty-four and a half hours (Fig. 4). A 65 per cent. scald was chosen because in this type of rat it is uniformly fatal, and it was considered easier to assess survival times when these were short than when they were long, and complicated by the late effects of burning. The doubling of survival time suggests that pre-treatment by scalding, though not by excision, effects some useful modification of the body defence, whether glandular, metabolic, nervous, or humoral. E x p e r i m e n t 2.--In this experiment a wide variety of pre-treatments was used to see if any had an influence on survival after scalding. Two hundred 14o g. (plus or minus ! g.) female Holtzman albino rats were 3 EI
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divided into four sets of fifty, each set being divided into couples which were caged together and, apart from the specific trauma, identically treated. Set Ia was given 5 rag. intraperitoneal Nembutal every third day for two weeks, and three weeks later scalded. Set 2a was bled of 4 to 6 ml. blood by severing the jugular vein with a scissor cut through the skin on two occasions at weekly intervals, and three weeks after the second bleeding scalded. Set 3a was subjected to tourniquet trauma by applying a very tight rubber band to one hind limb for six hours. This was repeated after a week on the opposite hind limb and followed by scalding after a further three weeks. Experiment 1 CUMULATIVE MORTALITY A f t e r 40°/o s u r f o c e area sceld at 90~C f o r 15secs. PREVIOUSLY EXCISED
25
/J SET
/
PREVIOUSLY SCALDED
SET
o
5 f----
O
J 20
10
J 30
4to
510
TIME OF DEATH IN HOURS AFTER
J 60
'(3
7
SCALDING
FIG. 4
Set 4a.--The right femur was fractured under Nembutal anaesthesia without breaking the skin, and four weeks later the animals were scalded. Sets ib, 2b, 3b, and 4b were the paired untreated controls. All animals had a 65 per cent. body weight scald at 9 °° C. for fifteen seconds, and were treated as in Experiment I. Average survival times are shown below :-Pre-treated.
Set Set Set Set
Ia, 2a, 3a, 4a,
26 hours. 23 hours. z9½ hours. 22 hours.
Control.
Set Set Set Set
Ib, 2b, 3b, 4 b,
27½ hours. 21½ hours. 24 hours. 2I hours.
There is not a significant difference in survival times between pre-treated and control rats, but it is heartening to find that the controls show such a close survival time. It may be suggested that it is unfair to compare rats which have been subjected to major trauma with healthy controls. This point is clarified in Experiment 4, but it should be pointed out that the rats protected by a previous scald and their controls in Experiment I were also severely treated. As it is possible that there is some development of protection against scalding
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by previous trauma, which is, however, overwhelmed by the severe test scald in conjunction with debility of the rat, a further series of experiments was performed after the question of transferred immunity had been elucidated. E x p e r i m e n t 3 . - - T o demonstrate whether passive immunity can be achieved a large number of rats was severely scalded. The supposition was made that if serological immunity occurred at all it would surely be manifested by the small number of tough survivors from an almost uniformly fatal scald. One thousand adult male and female Holtzman albino rats weighing about 20o g. each were epilated, ana:sthetised with intraperitoneal Nembutal, and scalded at 55 per cent. weight immersion (about 35 per cent. surface area) at 90 ° C. for fifteen seconds. They were untreated in order that only the hardiest (and most competent immunologically ?) would survive. Most of the rats died within the first three days after scalding, but any lasting longer than a week usually recovered. There were approximately one hundred survivors who went through the usual phases of escharification, very slow sloughing, cicatrisation, and epithelialisation. At the time of slough separation the rats were thin and obviously ill. During the ensuing slow period of healing they gradually increased in weight, and (like the human patient) at about the tenth week showed a sudden recovery, healed rapidly, and became heavier and more active. Healing was complete in most of the rats at the twelfth week, and the few remaining unhealed ones were discarded. The rats which at eighty-four days after scalding weighed 35o to 4oo g. were exsanguinated in the following way. Each received an intravenous injection of 20 ml. sterile normal saline containing 5,ooo units penicillin and 5 mg. streptomycin. This put the rats into congestive failure and they were then sacrificed by cutting their throats through an epilated area cleaned with phisohex, and exsanguinated into a sterile beaker. Each rat yielded 15 to 2o ml. mixture of diluted blood which was pooled, and centrifuged after dotting. Ha~matocrit indicated that the serum was roughly a 5o-5o mixture of serum and saline. This was stored in. sterile flasks at - IO° C. At the same time IOO rats were exsanguinated and their diluted and antibiotic impregnated serum stored similarly. One hundred 200 g. (plus or minus I g.) female Holtzman albino rats were split into two sets of fifty. They were epilated, an~esthetised, and scalded to 65 per cent. weight immersion at 9o ° C. for fifteen seconds and treated as follows :-I. Immediately after scalding, 2 ml. serum-saline mixture intravenously and Io ml. intraperJtoneal normal saline. 2. Four hours after scalding, I ml. serum-saline mixture subcutaneously and IO ml. intraperitoneal saline. 3. Eight hours after scalding, I ml. serum-saline mixture subcutaneously. 4- Twenty-four, forty-eight, seventy-two, etc., hours after scalding, i ml. serum-saline mixture subcutaneously. Half the animals received convalescent serum, while the other half received normal serum. The cumulative mortality chart (Fig. 5) shows that with the exception of a few rats that never recovered consciousness (probably due to the anzesthetic) the time of death of the control set lies between twenty and forty hours, while in the convalescent serum set it is between forty and eighty hours, with the exception 3 E2
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of a few stragglers in the ends of the main part of both curves. The average survival time of the untreated rats is twenty-seven hours and the convalescent serum treated set sixty hours. The behaviour of the rats after scalding is of interest. Those dying within the first ten hours failed to recover consciousness and were probably anmsthetic deaths. All the other rats recovered consciousness to the extent that they moved their limbs and responded to stimuli within an hour, but thereafter showed little Experiment
3
CUMULATIVE MORTALITY After 40°/o surface area s c a l d at 9 0 ° C f o r 15 sees
CONTROL SET
50
i
]O
!
20
30
i
40
,
50
__.r-...~
i
60
i
70
T I M E O F D E A T H IN H O U R S A F T E R
f
80
90
'
~
'
100
1 0
130
IMMUNE SERUM
SET
1~0
SCALDING
FIG. 5
activity for up to four hours. At this time they would be on their feet and wandering about the cage, eating occasionally and drinking frequently. The behaviour of the two sets is sharply contrasted. In the racks of cages the rats were arranged in alternate rows of treated and control animals. Though the rats given convalescent serum were known only to the writer, laboratory assistants and visitors were able without hesitation to select the treated rats. They were more alert and active and usually eating and particularly drinking eagerly. On the other hand, the untreated rats wandered around listlessly and rather aimlessly without eating or drinking much. Most of the rats in both sets had hmmoglobinuria after about twelve hours and up to twenty-four hours after scalding. Their mode of death was of two types. The rat, after appearing well and active, would slow down, have convulsions, and die within minutes. On the other hand, animals which survived more than thirty-six hours lapsed slowly into coma which might last up to twenty-four hours before death. There appears to be little doubt after this experiment that convalescent serum collected three months after scalding contains some factor capable of prolonging life in a severely scalded rat. As far as it is valid to base the deduction on animal work, it would appear that the rats felt better in the early post-scald period, and this was reflected by their greater activity. It is obvious that this work will have to be repeated on larger laboratory animals and humans in order to assess the actual changes (other than possible serological ones) by which the beneficial effects of convalescent serum are effected.
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E x p e r i m e n t 4.--It was apparent by this stage that not only does previous scalding confer some active immunity against subsequent scalding, but that passive immunity can be conferred by using convalescent serum as a transfusion substance in the scalded rat. This made a better planned follow-up of Experiment 2 possible. In this experiment sets of animals traumatised as in Experiment 2 were sacrificed three months after trauma to provide convalescent serum for testing. Twenty 20o g. (plus or minus I g.) female Holtzman rats were used in each of six different sets and were all subjected to a scald of 65 per cent. weight immersion at 9°o C. for fifteen seconds, and given the same post-operative treatment as in Experiment 3, using the different types of convalescent serum. I. Received normal serum-saline mixture. 2. Received serum-saline mixture from multiple an~esthetised rats. 3. Received serum-saline mixture from twice-bled rats. 4. Received serum-saline mixture from rats with fractured femora. 5. Received serum-saline mixture from rats after two tourniquets. 6. Received serum-saline mixture from convalescent scalded rats--stored for two months from Experiment 3. Cumulative mortality charts are not given as they are largely superimposed, but the average survival times of the rats are as follows : (I) 29 hours, (2) 32 hours, (3) 3° hours, (4) 281 hours, (5) 31 hours, (6) 58~- hours. It is apparent that the original batch of convalescent serum had not lost its potency after two months' storage at - i o ° C. and that serum from other types of trauma does not have a protective action against scalding. The serum donors were left three months on this occasion after the initial challenge, as this time had proved effective in Experiment 3. E x p e r i m e n t 5.--BY this time it appeared that a specific response of the antigenantibody type was occurring, and further tests were made with sera. of different vintage. It was found that sera extracted two weeks, four weeks, and eight weeks after a 55 per cent. standard scald were ineffective in giving protection. It may well be that the serum does need to be convalescent. Certainly the rats at two, four, and eight weeks are not convalescent. On the other hand, the clear demonstration of actively acquired immunity in Experiment I probably occurred because the scald was very much smaller and the animals were truly convalescent at the time of the second scalding. It was decided to proceed from this point on the assumption that skin and the subjacent tissues are altered by scalding and some diffusable product reaches the blood stream capable of promoting an antibody response. Whether any toxic product could be demonstrated in the skin or serum remained to be seen. A set of 2o0 g. female Holtzman rats was given the standard scald, and sacrificed two hours later. The skin, panniculus, and as much of the (edematous areolar layer above the deep fascia as possible were removed aseptically. The scalded skins were cut into tiny pieces and ground in sterile sand in a pestle and mortar until only the irreducible skin was left. This was extracted by multiple washings in sterile distilled water, centrifuged, filtered, and lyophilised. Intravenous injections of this crude extract into healthy 2o0 g. female rats were immediately fatal, but so were equal injections of normal skin similarly extracted.
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Subcutaneous injections of scalded skin extract produced variable results. M o r e than 3 ° per cent. surface area made the rats ill ; normal skin did not do this. After half an hour or so, rats became listless, hypotonic muscularly, and their hair bristled (Fig. 6). After about forty-eight hours they recovered. Occasional deaths followed subcutaneous injection o f scalded and normal skin, but these were not related to the dose. T h e intention to establish the minimum lethal dose of scalded skin extract was abandoned owing to the failure to produce death. In general, doses of more than IO per cent. scalded skin extract made the animals i11, but did not kill them. In case there was some change in the products from the skin following absorption into the blood stream, a series o f rats was exsanguinated (without dilution) twenty
FIG. 6 A p p e a r a n c e of rat two hours after s u b c u t a n e o u s i n j e c t i o n of 20 p e r cent. surface area scalded skin extract.
hours after a 65 per cent. standard scald. Intravenous injection of 5 ml. undiluted normal serum (if spread over about ten minutes to avoid heart failure) was found to be without effect. Injection of 5 ml. scalded rat serum produced the same effects as subcutaneous injection o f scalded skin extract, but even this quantity, which represented the serum from two scalded rats, did not prove fatal. It was concluded that if there is a toxin in scalded skin extract or serum from scalded rats, it is not sufficiently potent to cause death without the other concomitants o f scalding. E x p e r i m e n t 6 . 1 S i n c e rats had been made ill by toxic extracts, a number of adult female Holtzman rats was actively immunised against scalded skin extract, normal skin extract, post-scald serum, and normal serum. In view of the possible objections to Experiment 2 and the demonstration of the occurrence of passive immunity in Experiment 3, direct testing o f these immunised animals was not performed. Instead they were used as donors of i m m u n e serum. Preparation ofSera--Set i a . m T w e n t y - f i v e 200 g. female Holtzman rats received a subcutaneous injection of IO per cent. surface area of scalded skin extract, 5,000 units penicillin, 5 mg. streptomycin, and 0"5 ml. complete Freund's adjuvant. One
EXPERIMENTAL OBSERVATIONS ON IMMUNE BURN SERUM
25I
week later a repeat injection of 2 to 3 per cent. surface area scalded skin extract with penicillin and streptomycin but without adjuvant was given. Set ib.wTwenty-five similar rats received similar treatment except that normal skin was substituted. Set 2a.wTwenty-five similar rats were treated with a subcutaneous injection of 5 ml. serum from twenty-hour post-scald rats, with penicillin, streptomycin, and Freund's adjuvant. After one week they had a repeat injection of I ml. with penicillin and streptomycin but no adjuvant. Set 2b.--Twenty-five similar rats were treated in the same way as 2a except that normal serum was substituted. In all cases the injected skin extracts and injected post-scald serum were pooled. Four weeks after the second injection the rats were exsanguinated by the usual technique. Their number had shrunk by this time. About a quarter of the rats receiving normal or scalded skin injections had developed abscesses at the site of the first injection and were sacrificed. Of the serum sets, only two in the scalded serum and three in the normal serum group developed infection and were discarded. Sets of fifteen 200 g. (plus or minus I g.) female Holtzman rats were subjected to the 65 per cent. standard scald and treated as in Experiment 3 except for the substitution of the appropriate serum. Set IA received serum-saline mixture from the immunised set Ia. Set I~ received serum-saline mixture from the immunised set lb. Set 2A received serum-saline mixture from the immunised set 2a. Set 2B received serum-saline mixture from the immunised set 2b. Set 3 received normal serum-saline mixture from healthy rats. No control with the original three-month post-scald serum was possible as it had all been used. The number of rats in each set (owing to shortage of serum) is not large enough to enable detailed comparison between sets to be made. For this.reason, and because they are largely superimposed, cumulative mortality charts are not given. The average survival times of the different sets are as follows : (Ii) 65 hours, (IB) 20 hours, (2A) 5 O1 hours, (2B) 24½ hours, (3) 25 hours. It seems clear that normal skin extract and serum, potentiated by Freund's adjuvant, do not provoke any immunity. Even (or perhaps particularly) crude scalded skin extract and early post-scald serum give rise to immunity against subsequent scalding which is of the same order as that produced by previous scalding. Again it was easy for uninformed observers to pick out the treated animals as opposed to the controls within the first twenty-four hours of scalding. Groups (IA) and (2A) were selected by five independent observers. The possibility of one person making this selection by chance is one in ten. The chance of five independent observers doing it is I in IOO,OOO.
SUMMARY AND CONCLUSION
Under the experimental conditions used it appears that the rat is capable of developing active immunity to scalding. Convalescent serum from previously scalded rats exerts a marked protective influence on other scalded rats.
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After a severe scald this immunity reaches a useful level after three months. This is a specific response to scalding and is not produced by some other forms of trauma. Toxic but not lethal effects have been produced by injection of scalded skin extract and serum from severely scalded rats. After a month these injections produce a degree of immunity which enables their serum to protect other scalded rats. It appears that an effective period after a major scald is three months. A less severe scald produced active immunity after two months. In both sets the rats were truly convalescent at the time of their high immunity. It is tempting to speculate that only when the rat shows obvious clinical signs of recovery, analogous to those seen in the burned human, does it have a high enough antibody titre to be of value. One might guess that up to that time any antibody produced was immediately neutralised by still present antigen. This fits well with the findings, and is additionally confirmed in Experiment 6 by the immunisation against scalded skin extract. The rats--not suffering from the major depressing effect of a burn, and being given a comparatively small quantity of antigen--appear to achieve a useful immune level earlier. Dobrkovski's claims at the Washington meeting seem to be based on an impartial serological and clinical survey. Although the number of cases is small he has carefully related the patient's clinical progress with the serum antibody level. There appears to be a maximum antibody level in the early post-burn period which falls off during recovery, rising finally after three months during the patient's convalescence. He has been impressed (personal communication) by the marked subjective improvement in severely burned cases treated with convalescent serum, although the extent to which this influences the eventual outcome is difficult to assess at this stage. It is clear that in this series of rat experiments, although "subjective" wellbeing and prolongation of life were obtained, immune serum did not prevent eventual mortality. Although attempts were made to give adequate fluid therapy to control and test rats, it may be that supportive therapy was less than Ioo per cent. If this is the case, it could well be that if supportive treatment were improved, the difference apparently obtained with the use of convalescent serum might disappear. Fluid therapy in the burned human is painstakingly and accurately controlled, and it may be that some minor benefits from convalescent serum would not be apparent under these circumstances. In most burn units early death is not a problem, and the incidence of death after about ten days is due to a multiplicity of pathological processes. It is difficult to see how these deaths could be influenced by the use of convalescent serum, unless its early use were to prevent some organ damage which later rendered the patient more susceptible to infection, exhaustion, and the other late consequences of burning. This is, however, only conjecture, and the possibility that a borderline case may be influenced by the early use of convalescent serum is sufficient incentive to continue the study of this scientifically exciting field. M y thanks are due to Dr Blocker who provided the facilities, Sally Abston who helped to prepare and scald the rats, Harvey Williams who ground down the skin, and Taylor Wharton who lyophilised the watery extract.
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REFERENCES AVDAKOFF (1876). St Petersburg reed. Wschr. Quoted by S. Sevitt (1957) in " Burns : Pathology and Therapeutic Applications." London : Butterworth. DOBRKOVSKI, M., DOLEZALOVA, J., and PAVKOVA, L. (196o). " Report to the ISt International Congress of Research in Burns." Washington. FEDOROV, N. A. (1956). Proceedings of the 6th Congress of the International Society of Blood Transfusion, p. 54. --(196o). " Report to the ISt International Congress on Research in Burns." Washington. MALME, O. J., and SLAWIKOWSKI, J. M. (196o). " Report to the ISt International Congress on Research in Burns." Washington. PAVKOVA, L., and DOLEZALOVA, J. (I96o). " Report to Symposium on Plastic Surgery and Burns." Marianske Lazne. PUSHKAR, L. I. (1958). Ter. Ark., Moskva. ROSENTHAL, S. R. (I937 a). Ann. Surg., xo6, III. - - - - (I937 b). Ann. Surg., xo6, 257. - - - - (I959). Surgery, 46, 932. ROSENTHAL, S. R., FINAMORE, F. J., HUNTER, F. R., and WILLIAMS, A. D. (I956). Fed. Proc., xS, I56. ROSENTHAL, S. R., HARTNEY, J. B., and SI'URRIER, W. A. (I96O a). J. Amer. reed. Ass., I74, 957. ROSENTHAL, S. R., HUNTER, F. R., and FINAMORE, F. J. (196o b). Arch. int. Pharmacodyn., x26, 43. ROSENTHAL, S. R., HUNTER, F. R., HUNTER~ A, S., FINAMORE, F. J., WILLIAMS, f . , and ROMAN, I.-N. (1955). Fed. Proc., I4, I24. ROSENTr{AL, S. R., and TRAHAN, H. (I957). Fed. Proc., I6, 370.
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