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Experimental skin homografts
Experimental
Skin Homografts
EFFECT OF HOMOHEMOTHERAPY
ON THEIR SURVIVAL TIME
HECTOR MARINO, M.D. ANDFORTUNATOBENAIM, M.D., Buenos
Aires, Argentina
tissues beIonging to another rat not geneticaIIy reIated to the donee but aIso susceptibIe to the same tumor. Billingham and Sparrow [@ have recentIy reported on the increase of surviva1 time of skin homografts by inducing a state of specific immunoparaIysis with injection of a suspension of skin ceIIs from the donor. Having knowIedge of former work on the subject, the senior author of this paper one year ago had the idea of trying to influence the immunoIogica1 mechanisms of a prospective host by injecting it with whoIe blood from the skin donor. He concIuded that theoreticaIIy such an effect couId be obtained by massive, repeated injections, so as to alter the organic defenses of the host toward the graft. The reaction would be suppressed onIy for that given combination of genetic and immunoIogicaI eIements and wouId be preserved for any other kind of antigen. Thus clinica uses couId aIso be found for the procedure as the host would be abIe to respond to other (and pathoIogica1) antigens, which wouId not be the case with tota radiation, agammagIobuIinemia or hormonotherapy. ResuIts of experiments conducted according to these ideas are reported in the following section.
From tbe Laboratory jar Tissue Transplantation, tbe Williams Foundation. tbe Buenos Aires Naval Hosoital and tbe National Institute for Burns, Buenos Aires, Argentina.
UNDAMENTALresearch work conducted by and co-workers [25-291 has shown that rejection of skin homografts is due to an immunoIogica1 reaction of the active acquired type. So far this theory has been demonstrated onIy by indirect means, as specific antibodies circmating in the host have not been isolated [ 1,6,7, r4,3 I]. Therefore, there are two possibIe ways to prevent the rejection reaction: frrst, to act directIy upon the antibodies which wiI1 not be feasibIe unti1 they are known and isolated; second, to act upon the mechanisms of production and destruction of these antibodies. In this heId success has been attained by inff uencing the immunoIogica1 mechanisms of the host with hormones such as cortisone [2] and by subIetha1 tota radiation of the host (Trentin [33] and others). Both procedures couId not be accepted on clinica grounds. So far the best prospects are offered by the creation of a state of acquired specific immunoparalysis as shown by the aIready cIassic works of SneII [30], FeIton [12] and others. These authors have found that the injection of the host with massive, repeated doses of an antigen from the donor (suspension of Iiving ceIIs, tumor Iysate, specific proteic poIysaccharides, etc.) provokes a specific receptivity for tissues from the donor, for tissues of individuals geneticaIIy reIated to the donor, for tumors to which the donor is susceptibIe, and a lack of response to such specific proteic groups. A fitting exampIe are the experiments of Kaliss [zr-221 in which a rat not susceptibIe to a given maIignant tumor becomes susceptible after receiving a series of injections of a suspension of Iymphatic ceIIs or of other
F Medawar
EXPERIMENTS Two different technics were adopted in order to compare the evoIution in animaIs treated with blood injections and in contro1 groups (autografts-pIain homografts) : (I) the “ transparent chamber” technic in mice, and (2) the “three-graft” technic in rats. The first technic aIIowed us to control the vascuIar penetration into the grafts and to observe by means of the microscope the aIterations produced since the graft was applied, up to its vitaIization or rejection. 267
American Journal of Surgery, Volume
93, February,
1958
Marino
and Benaim
FIG. I. Plan of experiments. Group A, treated homograft. Homohemotherapy from the same donor. Group B, homograft pIus homohemotherapy different donors. Group C, contro1 homograft (without injection of bIood). Group D, contro1 autograft.
The second technic permitted macroscopic of the comparative surviva1 time between grafts of different kinds. We used the Transparent Cbamber Technic. transparent chamber technic, as described by JosIin [19] and Conway et a1. [~-II], with some personal modifications devised to increase the duration of the experiment. The most important modification was suturing the periphery of the skin foId to the frame with a running suture of stainIess stee1 wire. This suture aIso
incIuded two layers of poIyethyIene fiIm. Thus the skin foId couId be heId by the bronze wire spIint for periods of up to forty-five days with IittIe addition of new holding sutures. Microscopic observation of the chamber showed the evolution of the graft as compared with contro1 grafts. In the case of autografts a characteristic pIasmatic circuIation was seen during the first week. Starting on the seventh day new growth of bIood vesseIs was observed. These grew toward the graft and entered it.
controI
268
Experimental
Skin Homografts TABLE
GENERAL
COMPARISON
OF Microscopic
EVOLUTION
TECHNIC
No. of AnimaIs Operated Upon
Group
I OBSERVED USING THE TRANSPARENT
IN NINETY-TWO
-
I No. of Surviving AnimaIs
No. of Deaths
Takes
FaiIures
CHAMBER
MICE Visible CapiIlaries within Graft
_~___. First Week
CapiIIaries with Circulation within Graft _____-.-.
Second Week
First Week
1 Second Week
-__ A ........
57
B .........
c.
....
D.. Totals...../
1
12
15 gz
Once the microscopic control showed visible circulation in the newgrown vesseIs within the graft, one couId admit that the graft had taken. A series of more than IOO mice have been operated upon with this technic. Because of standardization requirements onIy the last ninety-two were considered acceptable for this study. These ninety-two mice were divided into four groups, named A, B, C and D. Their weight was 20 gm. each. (Fig. I.) Group A mice had one homograft each and were treated with injections of 0.01 cc. of bIood every other day, totaling ten injections (skin homograft pIus homohemotherapy with bIood of the skin donor). Group B mice (controls) were aIso prepared with homografts, but injections of bIood (using the same technic and dosage as in group A) came from a different donor (homograft pIus homohemotherapy from different donors). Group C mice (controIs) received onIy homografts, without injections of bIood. Group D mice (test group) had autografts onIy, without injections of blood. From the comparative study (TabIe I) in which the microscopic evoIution of the grafts in these groups is anaIyzed, it can be seen that the grafts were successfu1 in thirty-six of the fifty-seven mice beIonging to group A (homograft pIus homohemotherapy) as against twenty-one faiIures; this is more than 50 per cent positive. In group C (contro1 homograft) we had five takes and eIeven faiIures of sixteen mice operated upon, which is more than a 50 per cent faiIure.
In group D, to which the test autografts belong, we observed that Iess than 50 per cent of the grafts took, although no mice died. We wish to state that we do not assign an). fina value to these figures from a statistica point of view. Due to the limited number of observations an estimate of the standard deviation wiI1 show their reIative significance. Nevertheless, it is evident that there is a difference between the evolution of group A and that of group C (control). Three-graft Technic. This technic was developed in our laboratory in order to make simultaneous and comparative observations on the behavior of homografts appIied to rats receiving homohemotherapy, of control homografts and of test autografts. The animal selected was the rat, because of its larger size (200 gm.). Each experiment required three animals: one as the donor and two others as the hosts. Special care was taken in seIecting the strains of animaIs to avoid genetical similarities. Our first experiments were made with white rats, because of the difficulties in getting grey rats. This has been overcome in Iater work and we now use grey donors and white hosts. As shown in Figures 2 and 3, each rat has: (I) an autograft (with skin of the same rat), (2) a contro1 homograft (with skin from the other host) and (3) a homograft (with skin from the bIood donor). The bIood from the donor rat is injected intramuscuIarIy into the two hosts in a dosage of 0.1 cc. every other day, totaling ten injections. Up to the writing of this report twenty-seven 269
Marino and Benaim
FIG. 2. The three-graft technic. D, donor. Provides skin and bIood to be applied to hosts (H.I and H.2) as grafts and homohemotherapy. H.I, host. Its skin wiII be reappEed as an autograft and appIied to H.2 as a controI homograft. H.2, host. Will receive a homograft and bIood from the donor, a controI homograft from H.I and its own skin as control autograft.
/
FIG. 3. The three-graft technic. H.I, host I. H.2, host 2. Dots = ControI homograft (without blood). Crosshatching = Homograft with blood. 270
autograft.
DiagonaI
Iines = Control
Experimental
Skin Homografts
FIG. 4. Rat No. I I twenty days postoperatively. First sectionshows autograft; second section, contro1 homograft; third section, homograft plus homohemotherapy.
activity of intramuscuIar injections of donor’s bIood as a specific agent for stopping, for a Iength of time, the immunoIogica1 reactions Iinked with rejection phenomena. Our experiments with the transparent chamber technic coincide with those of Taylor and Lehrfeld [32], Converse and Rappaport [8] and others, in that during the period of acceptance of the homograft (ten to twelve days) vascuIar penetration is simiIar to that observed in contro1 autografts. However, a fundamenta1 difference is found from the time of the vascuIar thrombosis and the onset of the rejection reaction in the contro1 homograft; a number of the treated homografts preserve a vascuIar pattern simiIar to that of test autografts. Even without definite proof we believe that these observations can be attributed to manifestations of specific immunoparaIysis eIicited by repeated injections of bIood antigens. Our experiments do not differ from simiIar experiments in the technica procedures. The difference Iies in the use of whoIe blood as a specific antigen, demonstrating its activity on skin homografts by the intramuscurar route. This route had the advantage of eIiminating the hazards involved when immunoIogicaIIy active ceIIuIar suspensions are injected by the intravenous route. Moreover, these hosts were not in the fetal or neutra1 neonata1 states, but were adult subjects. The choice of blood was aIso dictated by the idea that it was a practica1 conditioning antigen, being a Iiquid living tissue, and therefore easiIy injectabIe. It was aIso chosen because it couId not possibIy differ geneticaIIy and in its
rats have been grafted, of which five have died due to different complications. Eight are &II under observation and fourteen are analyzed in TabIe II. ResuIts show a noticeable difference in the evoIution of homografts coming from the donor whose bIood was periodicaIIy injected TABLE COMPARISON
OF
EVOLUTION
II OF
GRAFTS
IN
FOURTEEN
RATS
Takes
Type of Graft
I __---
.___
___-
Autograft..................... ControI homograft. Homograft homohemotherapy
~~~~ Totals
I
I ~.._~_ __
IO .
.
I4
2
I”:
7
7
13 I4
into the hosts (homograft pIus homohemotherapy, Figure 4), as compared to the control homografts. In the first group (homograft plus homohemotherapy) of fourteen animals we had seven takes and seven faiIures which compares favorabIy with two takes and eleven faiIures observed in the contro1 homografts. As expected, in the autografts we had ten takes and four failures. The same considerations of Iack of statistical value made for the transparent chamber technic appIy aIso for these results. COMMENTS
First, we wish to state it is evident that we do not pretend to soIve the problem of homograft acceptance by the experiments described herein. We onIy expect to show the 271
Marino immunological effects from other conditioning agents such as the skin suspensions of BiIIingham and Brent [5], the spIenic ceIIuIar suspensions of Jordan or the successive grafts of Hardin et a1. [17]. Regarding the viabiIity of treated homografts it can onIy be said that observation with the transparent chamber technic was Iimited to a maximum of forty-five days due to its impIicit limitations. HistoIogicaI controIs confirmed the in vivo findings. We have been abIe to control the three-graft technic onIy for a period of three months. It is probabIe that viabiIity wiI1 be preserved as long as the immunoparaIytic state lasts, but if it disappears the homograft wiI1 be rejected. On the other hand, the injected specific antibody (with the genetic combination of the prospective skin donor) must be seIected with extreme precision. This has been demonstrated by persistent faiIures with injection of bIood not beIonging to the skin donor or of blood altered by heat or other causes. In this way perhaps the genetic and the immunoIogica1 theories of homograft rejection can be Iinked together. CeIIuIar histoindividuaIity (chromosomic dissimilarity) would thus preserve its ruIing significance, and the immunologic mechanism wouId be the means by which the host shows its capacity to differentiate the grafted tissue and gets rid of it. With a given dosage of antigen (in this particuIar case a skin homograft) the dissimilarity wiI1 start an inevitabIe chain of events ending with the rejection of the homograft. If the dose of skin antigen is repeated two or three times, the rejection reaction wiI1 be more prompt and vioIent each time [3,25]. However, if, as in other bioIogica1 phenomena, we continue to insist that with further doses of antigen the reactivity of that specific section of the mechanism wiI1 be used up (immunoparaIysis), we wiII get a transitory or permanent acceptance. A11 we can say about the possibIe use of blood injections as a conditioning agent in human beings is that we are starting to try them with due caution. ResuIts so far are dificuIt to assess and wiI1 be reported in a later paper. In any case the procedure couId not be appIied in its present form. We must remember that 0.01 cc. of bIood injected into a 20 gm. mouse or 0.1 cc. of bIood injected into a 200 gm. rat are equal to 35 cc. of bIood injected intramuscuIarIy every other day into a 70 kg. adult.
and Benaim Moreover, the dangers of induced chimerism shown by recent research [5] caI1 attention to the indiscriminate use of biood antigens in humans especiaIIy in the first days of Iife. FinaIIy (as a comment which does not exactIy beIong to this subject) we would Iike to point out that going a step further, this demonstration of the activity of intramuscuIar injections of bIood couId give a hypothetical expIanation of the effects of autohemotherapy. In this case it couId be supposed that bIood components suffer some sort of aIteration due to their heterotopic dispIacement into the body (intramuscular injection) and act as some sort of conditioning agent foIIowing their repeated administration. SUMMARY
Significant survivaIs of skin homografts in aduIt mice not genetically reIated to the donors have been obtained by repeated intramuscuIar injections of donor’s bIood. Experiences were conducted empIoying the transparent chamber technic (JosIin) and the three-graft technic. (origina1). REFERENCES I.
2.
3. 4. 5. 6.
9. IO. II. 12. ‘3. 14. 15. 16. 17. 18. ‘9.
20.
ALLGOWER,M., BLOCKER,T. G.. JK. and ENC,LEY, B. W. D. Plast ti Reconstruct. Surg., g: I, 1952. BILLINGHAM,R. E., KROHN, P. L. and MEDAWAR, P. B. Brit. M. J., I : I I 57, Ig5 I. BILLINGHAM,R. E. Transplant. Bull., 3: 68, 1956. BILLINGHAM,R. E. and BRENT, L. Transplant. Bull., 3: 122, 1956. BILLINGHAM,R. E. and BRENT, L. Transplant. Bull., 5: 67, 1957. BILLINGHAM.R. E. and SPARROW.E. M. J. Exaer. Biol., 1953. BOLLAG,W. S. Transplant. Bull., 3: 43, 1956. CONVERSE,J. M. and RAPPAPORT, F. T. Ann. Surg., 143: 306, 1956. CONWAY, H., JOSLIN, D. and STARK, R. B. Plast. d Reconstruct. Surg., 8: 194, 1951. CONWAY, H., JOSLIN,D., REES, T. D. and STARK, R. B. P&t. ti Reconstruct. Surg., g: 557, 1952. CONWAY, H., SEDAR, J. and STARK, R. B. Plast. c!? Reconstruct. Surg., 14: 417, 1954. FELTON,L. D. J. Immunol., 61: 107, Ig4g. FELTON, L. D., KAUFMAN, G., PRESCOTT,B. and &-~INGER, B. J. Immunol., 74: r7, ,955. _ GORER, P. A. Brit. J. Cancer, 4: 372, 1950. HARDIN.C. A. and WERDER. A. A. Plast. @ Reconstruct. Surg., 15: 107, 1955. HARDIN,C. A. and WERDER,A. A. Ann. New York Acad. SC., 59: 381, 1955. HARDIN, C. A., WERDER, A. A., LIGGETT, M. S. and HOOFER,W. D. Surgery, 38: 566, 1955. HARRIS. R. J. C. Transvlant. Bull.. 3: 122. 1026. JOSLIN,‘D. Science, I 15; 601, Ig5z.. . __ KALISS, N. Transplant. Bull., 2: 8, 1955.
Experimenta 21. KALISS, N. Cancer Res., 12: 379, 1952. 22. KALISS, N. and SNELL, G. D. Cancer Res., ‘95’. 23. KALISS, N. Transplant. Bull., 2: 52, 1955. 24. LEHRFELD, J. W. and TAYLOR, A. C. Pk. CO?lstrUCt..%rg., 12: 432, 1953. 25. MEDAWAR, P. B. J. Anat., 78: 176, 1944.
I I : I 22 IY Re-
26. MEDAWAR, P. B. J. Anat., 79: 157, 1945. 27. MEDAWAR, P. B. Brit. J. Exper. Path., 27: 9, 1946.
Skin Homografts 28. MEDAWAK, P. B. Brit. J. Exper. Path., 27: 15, 1946. 29. MEDAWAR, P. B. Quart. J. Micr. SC., 89: 23g, 194.8. 30. SNELL, G. D. Cancer Res., 12: 543, 1952. 31. SPARROW, E. M. J. Endocrinol., 9: IOI, 1953. 32. TAYLOR, A. C. and LEHRFELD, J. W. Ann. New York Acad. SC., 59: 351, 1955. 33. TRENTIN, J. J. Transplant. Bull., 5: 67, 1957. 34. WERDER, A. A. and HARDIN, C. A. Surgery, 36: 371, 1954.
273
Skin Homografts
EFFECT OF HOMOHEMOTHERAPY
ON THEIR SURVIVAL TIME
HECTOR MARINO, M.D. ANDFORTUNATOBENAIM, M.D., Buenos
Aires, Argentina
tissues beIonging to another rat not geneticaIIy reIated to the donee but aIso susceptibIe to the same tumor. Billingham and Sparrow [@ have recentIy reported on the increase of surviva1 time of skin homografts by inducing a state of specific immunoparaIysis with injection of a suspension of skin ceIIs from the donor. Having knowIedge of former work on the subject, the senior author of this paper one year ago had the idea of trying to influence the immunoIogica1 mechanisms of a prospective host by injecting it with whoIe blood from the skin donor. He concIuded that theoreticaIIy such an effect couId be obtained by massive, repeated injections, so as to alter the organic defenses of the host toward the graft. The reaction would be suppressed onIy for that given combination of genetic and immunoIogicaI eIements and wouId be preserved for any other kind of antigen. Thus clinica uses couId aIso be found for the procedure as the host would be abIe to respond to other (and pathoIogica1) antigens, which wouId not be the case with tota radiation, agammagIobuIinemia or hormonotherapy. ResuIts of experiments conducted according to these ideas are reported in the following section.
From tbe Laboratory jar Tissue Transplantation, tbe Williams Foundation. tbe Buenos Aires Naval Hosoital and tbe National Institute for Burns, Buenos Aires, Argentina.
UNDAMENTALresearch work conducted by and co-workers [25-291 has shown that rejection of skin homografts is due to an immunoIogica1 reaction of the active acquired type. So far this theory has been demonstrated onIy by indirect means, as specific antibodies circmating in the host have not been isolated [ 1,6,7, r4,3 I]. Therefore, there are two possibIe ways to prevent the rejection reaction: frrst, to act directIy upon the antibodies which wiI1 not be feasibIe unti1 they are known and isolated; second, to act upon the mechanisms of production and destruction of these antibodies. In this heId success has been attained by inff uencing the immunoIogica1 mechanisms of the host with hormones such as cortisone [2] and by subIetha1 tota radiation of the host (Trentin [33] and others). Both procedures couId not be accepted on clinica grounds. So far the best prospects are offered by the creation of a state of acquired specific immunoparalysis as shown by the aIready cIassic works of SneII [30], FeIton [12] and others. These authors have found that the injection of the host with massive, repeated doses of an antigen from the donor (suspension of Iiving ceIIs, tumor Iysate, specific proteic poIysaccharides, etc.) provokes a specific receptivity for tissues from the donor, for tissues of individuals geneticaIIy reIated to the donor, for tumors to which the donor is susceptibIe, and a lack of response to such specific proteic groups. A fitting exampIe are the experiments of Kaliss [zr-221 in which a rat not susceptibIe to a given maIignant tumor becomes susceptible after receiving a series of injections of a suspension of Iymphatic ceIIs or of other
F Medawar
EXPERIMENTS Two different technics were adopted in order to compare the evoIution in animaIs treated with blood injections and in contro1 groups (autografts-pIain homografts) : (I) the “ transparent chamber” technic in mice, and (2) the “three-graft” technic in rats. The first technic aIIowed us to control the vascuIar penetration into the grafts and to observe by means of the microscope the aIterations produced since the graft was applied, up to its vitaIization or rejection. 267
American Journal of Surgery, Volume
93, February,
1958
Marino
and Benaim
FIG. I. Plan of experiments. Group A, treated homograft. Homohemotherapy from the same donor. Group B, homograft pIus homohemotherapy different donors. Group C, contro1 homograft (without injection of bIood). Group D, contro1 autograft.
The second technic permitted macroscopic of the comparative surviva1 time between grafts of different kinds. We used the Transparent Cbamber Technic. transparent chamber technic, as described by JosIin [19] and Conway et a1. [~-II], with some personal modifications devised to increase the duration of the experiment. The most important modification was suturing the periphery of the skin foId to the frame with a running suture of stainIess stee1 wire. This suture aIso
incIuded two layers of poIyethyIene fiIm. Thus the skin foId couId be heId by the bronze wire spIint for periods of up to forty-five days with IittIe addition of new holding sutures. Microscopic observation of the chamber showed the evolution of the graft as compared with contro1 grafts. In the case of autografts a characteristic pIasmatic circuIation was seen during the first week. Starting on the seventh day new growth of bIood vesseIs was observed. These grew toward the graft and entered it.
controI
268
Experimental
Skin Homografts TABLE
GENERAL
COMPARISON
OF Microscopic
EVOLUTION
TECHNIC
No. of AnimaIs Operated Upon
Group
I OBSERVED USING THE TRANSPARENT
IN NINETY-TWO
-
I No. of Surviving AnimaIs
No. of Deaths
Takes
FaiIures
CHAMBER
MICE Visible CapiIlaries within Graft
_~___. First Week
CapiIIaries with Circulation within Graft _____-.-.
Second Week
First Week
1 Second Week
-__ A ........
57
B .........
c.
....
D.. Totals...../
1
12
15 gz
Once the microscopic control showed visible circulation in the newgrown vesseIs within the graft, one couId admit that the graft had taken. A series of more than IOO mice have been operated upon with this technic. Because of standardization requirements onIy the last ninety-two were considered acceptable for this study. These ninety-two mice were divided into four groups, named A, B, C and D. Their weight was 20 gm. each. (Fig. I.) Group A mice had one homograft each and were treated with injections of 0.01 cc. of bIood every other day, totaling ten injections (skin homograft pIus homohemotherapy with bIood of the skin donor). Group B mice (controls) were aIso prepared with homografts, but injections of bIood (using the same technic and dosage as in group A) came from a different donor (homograft pIus homohemotherapy from different donors). Group C mice (controIs) received onIy homografts, without injections of bIood. Group D mice (test group) had autografts onIy, without injections of blood. From the comparative study (TabIe I) in which the microscopic evoIution of the grafts in these groups is anaIyzed, it can be seen that the grafts were successfu1 in thirty-six of the fifty-seven mice beIonging to group A (homograft pIus homohemotherapy) as against twenty-one faiIures; this is more than 50 per cent positive. In group C (contro1 homograft) we had five takes and eIeven faiIures of sixteen mice operated upon, which is more than a 50 per cent faiIure.
In group D, to which the test autografts belong, we observed that Iess than 50 per cent of the grafts took, although no mice died. We wish to state that we do not assign an). fina value to these figures from a statistica point of view. Due to the limited number of observations an estimate of the standard deviation wiI1 show their reIative significance. Nevertheless, it is evident that there is a difference between the evolution of group A and that of group C (control). Three-graft Technic. This technic was developed in our laboratory in order to make simultaneous and comparative observations on the behavior of homografts appIied to rats receiving homohemotherapy, of control homografts and of test autografts. The animal selected was the rat, because of its larger size (200 gm.). Each experiment required three animals: one as the donor and two others as the hosts. Special care was taken in seIecting the strains of animaIs to avoid genetical similarities. Our first experiments were made with white rats, because of the difficulties in getting grey rats. This has been overcome in Iater work and we now use grey donors and white hosts. As shown in Figures 2 and 3, each rat has: (I) an autograft (with skin of the same rat), (2) a contro1 homograft (with skin from the other host) and (3) a homograft (with skin from the bIood donor). The bIood from the donor rat is injected intramuscuIarIy into the two hosts in a dosage of 0.1 cc. every other day, totaling ten injections. Up to the writing of this report twenty-seven 269
Marino and Benaim
FIG. 2. The three-graft technic. D, donor. Provides skin and bIood to be applied to hosts (H.I and H.2) as grafts and homohemotherapy. H.I, host. Its skin wiII be reappEed as an autograft and appIied to H.2 as a controI homograft. H.2, host. Will receive a homograft and bIood from the donor, a controI homograft from H.I and its own skin as control autograft.
/
FIG. 3. The three-graft technic. H.I, host I. H.2, host 2. Dots = ControI homograft (without blood). Crosshatching = Homograft with blood. 270
autograft.
DiagonaI
Iines = Control
Experimental
Skin Homografts
FIG. 4. Rat No. I I twenty days postoperatively. First sectionshows autograft; second section, contro1 homograft; third section, homograft plus homohemotherapy.
activity of intramuscuIar injections of donor’s bIood as a specific agent for stopping, for a Iength of time, the immunoIogica1 reactions Iinked with rejection phenomena. Our experiments with the transparent chamber technic coincide with those of Taylor and Lehrfeld [32], Converse and Rappaport [8] and others, in that during the period of acceptance of the homograft (ten to twelve days) vascuIar penetration is simiIar to that observed in contro1 autografts. However, a fundamenta1 difference is found from the time of the vascuIar thrombosis and the onset of the rejection reaction in the contro1 homograft; a number of the treated homografts preserve a vascuIar pattern simiIar to that of test autografts. Even without definite proof we believe that these observations can be attributed to manifestations of specific immunoparaIysis eIicited by repeated injections of bIood antigens. Our experiments do not differ from simiIar experiments in the technica procedures. The difference Iies in the use of whoIe blood as a specific antigen, demonstrating its activity on skin homografts by the intramuscurar route. This route had the advantage of eIiminating the hazards involved when immunoIogicaIIy active ceIIuIar suspensions are injected by the intravenous route. Moreover, these hosts were not in the fetal or neutra1 neonata1 states, but were adult subjects. The choice of blood was aIso dictated by the idea that it was a practica1 conditioning antigen, being a Iiquid living tissue, and therefore easiIy injectabIe. It was aIso chosen because it couId not possibIy differ geneticaIIy and in its
rats have been grafted, of which five have died due to different complications. Eight are &II under observation and fourteen are analyzed in TabIe II. ResuIts show a noticeable difference in the evoIution of homografts coming from the donor whose bIood was periodicaIIy injected TABLE COMPARISON
OF
EVOLUTION
II OF
GRAFTS
IN
FOURTEEN
RATS
Takes
Type of Graft
I __---
.___
___-
Autograft..................... ControI homograft. Homograft homohemotherapy
~~~~ Totals
I
I ~.._~_ __
IO .
.
I4
2
I”:
7
7
13 I4
into the hosts (homograft pIus homohemotherapy, Figure 4), as compared to the control homografts. In the first group (homograft plus homohemotherapy) of fourteen animals we had seven takes and seven faiIures which compares favorabIy with two takes and eleven faiIures observed in the contro1 homografts. As expected, in the autografts we had ten takes and four failures. The same considerations of Iack of statistical value made for the transparent chamber technic appIy aIso for these results. COMMENTS
First, we wish to state it is evident that we do not pretend to soIve the problem of homograft acceptance by the experiments described herein. We onIy expect to show the 271
Marino immunological effects from other conditioning agents such as the skin suspensions of BiIIingham and Brent [5], the spIenic ceIIuIar suspensions of Jordan or the successive grafts of Hardin et a1. [17]. Regarding the viabiIity of treated homografts it can onIy be said that observation with the transparent chamber technic was Iimited to a maximum of forty-five days due to its impIicit limitations. HistoIogicaI controIs confirmed the in vivo findings. We have been abIe to control the three-graft technic onIy for a period of three months. It is probabIe that viabiIity wiI1 be preserved as long as the immunoparaIytic state lasts, but if it disappears the homograft wiI1 be rejected. On the other hand, the injected specific antibody (with the genetic combination of the prospective skin donor) must be seIected with extreme precision. This has been demonstrated by persistent faiIures with injection of bIood not beIonging to the skin donor or of blood altered by heat or other causes. In this way perhaps the genetic and the immunoIogica1 theories of homograft rejection can be Iinked together. CeIIuIar histoindividuaIity (chromosomic dissimilarity) would thus preserve its ruIing significance, and the immunologic mechanism wouId be the means by which the host shows its capacity to differentiate the grafted tissue and gets rid of it. With a given dosage of antigen (in this particuIar case a skin homograft) the dissimilarity wiI1 start an inevitabIe chain of events ending with the rejection of the homograft. If the dose of skin antigen is repeated two or three times, the rejection reaction wiI1 be more prompt and vioIent each time [3,25]. However, if, as in other bioIogica1 phenomena, we continue to insist that with further doses of antigen the reactivity of that specific section of the mechanism wiI1 be used up (immunoparaIysis), we wiII get a transitory or permanent acceptance. A11 we can say about the possibIe use of blood injections as a conditioning agent in human beings is that we are starting to try them with due caution. ResuIts so far are dificuIt to assess and wiI1 be reported in a later paper. In any case the procedure couId not be appIied in its present form. We must remember that 0.01 cc. of bIood injected into a 20 gm. mouse or 0.1 cc. of bIood injected into a 200 gm. rat are equal to 35 cc. of bIood injected intramuscuIarIy every other day into a 70 kg. adult.
and Benaim Moreover, the dangers of induced chimerism shown by recent research [5] caI1 attention to the indiscriminate use of biood antigens in humans especiaIIy in the first days of Iife. FinaIIy (as a comment which does not exactIy beIong to this subject) we would Iike to point out that going a step further, this demonstration of the activity of intramuscuIar injections of bIood couId give a hypothetical expIanation of the effects of autohemotherapy. In this case it couId be supposed that bIood components suffer some sort of aIteration due to their heterotopic dispIacement into the body (intramuscular injection) and act as some sort of conditioning agent foIIowing their repeated administration. SUMMARY
Significant survivaIs of skin homografts in aduIt mice not genetically reIated to the donors have been obtained by repeated intramuscuIar injections of donor’s bIood. Experiences were conducted empIoying the transparent chamber technic (JosIin) and the three-graft technic. (origina1). REFERENCES I.
2.
3. 4. 5. 6.
9. IO. II. 12. ‘3. 14. 15. 16. 17. 18. ‘9.
20.
ALLGOWER,M., BLOCKER,T. G.. JK. and ENC,LEY, B. W. D. Plast ti Reconstruct. Surg., g: I, 1952. BILLINGHAM,R. E., KROHN, P. L. and MEDAWAR, P. B. Brit. M. J., I : I I 57, Ig5 I. BILLINGHAM,R. E. Transplant. Bull., 3: 68, 1956. BILLINGHAM,R. E. and BRENT, L. Transplant. Bull., 3: 122, 1956. BILLINGHAM,R. E. and BRENT, L. Transplant. Bull., 5: 67, 1957. BILLINGHAM.R. E. and SPARROW.E. M. J. Exaer. Biol., 1953. BOLLAG,W. S. Transplant. Bull., 3: 43, 1956. CONVERSE,J. M. and RAPPAPORT, F. T. Ann. Surg., 143: 306, 1956. CONWAY, H., JOSLIN, D. and STARK, R. B. Plast. d Reconstruct. Surg., 8: 194, 1951. CONWAY, H., JOSLIN,D., REES, T. D. and STARK, R. B. P&t. ti Reconstruct. Surg., g: 557, 1952. CONWAY, H., SEDAR, J. and STARK, R. B. Plast. c!? Reconstruct. Surg., 14: 417, 1954. FELTON,L. D. J. Immunol., 61: 107, Ig4g. FELTON, L. D., KAUFMAN, G., PRESCOTT,B. and &-~INGER, B. J. Immunol., 74: r7, ,955. _ GORER, P. A. Brit. J. Cancer, 4: 372, 1950. HARDIN.C. A. and WERDER. A. A. Plast. @ Reconstruct. Surg., 15: 107, 1955. HARDIN,C. A. and WERDER,A. A. Ann. New York Acad. SC., 59: 381, 1955. HARDIN, C. A., WERDER, A. A., LIGGETT, M. S. and HOOFER,W. D. Surgery, 38: 566, 1955. HARRIS. R. J. C. Transvlant. Bull.. 3: 122. 1026. JOSLIN,‘D. Science, I 15; 601, Ig5z.. . __ KALISS, N. Transplant. Bull., 2: 8, 1955.
Experimenta 21. KALISS, N. Cancer Res., 12: 379, 1952. 22. KALISS, N. and SNELL, G. D. Cancer Res., ‘95’. 23. KALISS, N. Transplant. Bull., 2: 52, 1955. 24. LEHRFELD, J. W. and TAYLOR, A. C. Pk. CO?lstrUCt..%rg., 12: 432, 1953. 25. MEDAWAR, P. B. J. Anat., 78: 176, 1944.
I I : I 22 IY Re-
26. MEDAWAR, P. B. J. Anat., 79: 157, 1945. 27. MEDAWAR, P. B. Brit. J. Exper. Path., 27: 9, 1946.
Skin Homografts 28. MEDAWAK, P. B. Brit. J. Exper. Path., 27: 15, 1946. 29. MEDAWAR, P. B. Quart. J. Micr. SC., 89: 23g, 194.8. 30. SNELL, G. D. Cancer Res., 12: 543, 1952. 31. SPARROW, E. M. J. Endocrinol., 9: IOI, 1953. 32. TAYLOR, A. C. and LEHRFELD, J. W. Ann. New York Acad. SC., 59: 351, 1955. 33. TRENTIN, J. J. Transplant. Bull., 5: 67, 1957. 34. WERDER, A. A. and HARDIN, C. A. Surgery, 36: 371, 1954.
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