prolonged survival of glutaraldehyde-treated skin homografts

5
Prolonged Survival of Glutaraldehyde-Treated Skin Homografts Author(s): Israel Schechter Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 68, No. 7 (Jul., 1971), pp. 1590-1593 Published by: National Academy of Sciences Stable URL: http://www.jstor.org/stable/60748 . Accessed: 07/05/2014 14:39 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . National Academy of Sciences is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of the National Academy of Sciences of the United States of America. http://www.jstor.org This content downloaded from 169.229.32.136 on Wed, 7 May 2014 14:39:09 PM All use subject to JSTOR Terms and Conditions

Upload: israel-schechter

Post on 05-Jan-2017

214 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Prolonged Survival of Glutaraldehyde-Treated Skin Homografts

Prolonged Survival of Glutaraldehyde-Treated Skin HomograftsAuthor(s): Israel SchechterSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 68, No. 7 (Jul., 1971), pp. 1590-1593Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/60748 .

Accessed: 07/05/2014 14:39

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

National Academy of Sciences is collaborating with JSTOR to digitize, preserve and extend access toProceedings of the National Academy of Sciences of the United States of America.

http://www.jstor.org

This content downloaded from 169.229.32.136 on Wed, 7 May 2014 14:39:09 PMAll use subject to JSTOR Terms and Conditions

Page 2: Prolonged Survival of Glutaraldehyde-Treated Skin Homografts

Proc. Nat. Acad. Sci. USA Vol. 68, No. 7, pp. 1590-1593, July 1971

Prolonged Survival of Glutaraldehyde-Treated Skin Homografts (mice/poly(L-lysine)/polypeptides)

ISRAEL SCHECHTER*

Department of Chemical Immunology, The Weizmann Institute of Scienice, Rehovot, Israel

Communicated by Albert B. Sabin, May 13, 1971

ABSTRACT Treatment of mouse skin homografts in vitro with glutaraldehyde prolonged their average sur- vival time from 12.4 to 39.2 days, presumably because the reagent became covalently bound to the histocompati- bility antigen sites (or in their close vicinity) and shielded them from the immune apparatus of the recipient. The attachment to skin of the inert polymer poly(L-lysine) via this bifunctional reagent increased the average survival time to 52.9 days. The simplicity and versatility of this approach might make it possible to screen a large number of reagents that can bind covalently to tissue constituents under physiological conditions. It seems possible that a particular treatment leading to a new contact surface in the transplant might favor the acceptance of homografts as well as of heterografts.

The main obstacle encountered in organ transplantation is the immune rejection of the transplant. The rejection phenomenon originates from antigenic differences between the cells of the recipient and the transplant, as well as from the natural im- mune response of the organism toward "non-self" antigens. Attempts to prolong the survival of homografts, both in ex- perimental models and in medical practice, have been mainly aimed at the suppression of the immune apparatus of the recipient. This has been achieved by means of cytotoxic drugs, antimetabolites, corticosteroids, and antilymphocytic serum. The generalized immunosuppression, however, is accompanied by undesirable toxic effects, decreased resistance to infectionl, and reduction in the level of the hemopoietic stem cells. Another possibility is to attenuate, or to abolish completely, the antigenicity of the homograft with preservation of its biological funietions. The advantage of this approach is that the immune capacity of the recipient is not affected. In- vestigations along this line have for the most part been unsuc- cessful, and have not been subjected to clinical evaluation. Treatment of homografts in vitro with cortisone (1), thalido- mide (2), or urethane (3) prolonged their survival by a factor of about two. The amount of drug locally applied to the skiin was smaller than the amount required to achieve a similar effect by injecting the drug systemically. In attempts to mod- ify the antigenic properties of homografts, the donor skin has been treated in vitro with streptokinase/streptodornase (4), or with RNA and DNA preparations of the recipient (5). Homo- graft survival was not prolonged by exposure of donor skin to transplantation antigens of the recipient (6). Minimal immune

Abbreviations: PBS, 0.01 MI phosphate buffer (pH 7.3)-0.15 l/I sodium chloride; AST, average survival time; C57, C57BL/6 mice. * Present address: National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014.

reactioii was observed toward homografts in which cellular viability was destroyed by in vitro treatment with formalin (7) or cyanide (8) or by freeze-drying (9). The majority of the dead grafts were retained by the host for a limited period of time.

The present study was based on the hypothesis that, if homografts were treated in vitro with certain chemical rea- gents, the reacting groups might be directly and covalently bound to the histocompatibility antigen molecules, or in their close vicinity. In consequence the histocompatibility antigens might be masked and become inaccessible to the immune apparatus of the recipient. The purpose in this approach is to achieve functional elimination of the antigens responsible for tissue rejection. A large number of chemical reagents can react under physiological conditions with functional groups present in tissue constituents. The extent and nature of the masking effect might conceivably be controlled by choosing the appro- priate reagent. Moreover, chemically inert molecules can also be bound to tissues by means of bifunctional reagenits. Another feature inherent in such an approach might be the capacity to generate new properties (say, positive or negative charges, hydrophobic groups, etc.) on the surface of the transplant. The simplicity and versatility of such a procedure could make it possible to screen a large number of parameters affecting the surface properties of the transplant. It seemed possible that a particular treatment leading to a new contact surface in the homograft (or heterograft) might favor its acceptance by the host.

Preliminary findings of experiments designied to test this hypothesis are described in this communication. It was found that in vitro treatment of skin homografts by glutaraldehyde (a bifunctional reagent) significantly prolonged their average survival time (from 12.4 to 39.2 days). The effect of other compounds coupled to the skin graft via glutaraldehyde was also examined.

MATERIALS AND METHODS

Glutaraldehyde (25% w/v, aqueous solution) was purchased from Fluka AG, Switzerland. Poly(L-lysine) hydrobromide (average number of lysine residues per mole, 360) was a gift from Mr. I. Jacobson, and the polymer Try35Lys84Glu530, mol wt 97,000) was donated by Dr. Bila Schechter of the Weiz- mann Institute of Science. Sterile solutions of 0.01 M phos- phate buffer (pH 7.3)-0.15 M sodium chloride (PBS) were used.

Female mice (20-25 g) of the strains C57BL/6 and SWR, which share an identical H-2 locus (H-2b) but differ in a number of other histocompatibilit;y antigens (10), were used. Full- thickness skin grafts were performed according to Billingham

159(0

This content downloaded from 169.229.32.136 on Wed, 7 May 2014 14:39:09 PMAll use subject to JSTOR Terms and Conditions

Page 3: Prolonged Survival of Glutaraldehyde-Treated Skin Homografts

Proc. Nat. Acad. Sci. USA 68 (1971) Glutaraldehyde-Treated Skin Homografts 1591

80 -

F-- z

0 -

10 20 30 40 50 60 70 80 103 DAYS

FIG. 1. Effect of in vitro treatment of C57 skin homografts on their survival time in SWR recipients. A, PBS alone (group 1); 0, PBS + 2.8 mg/ml glutaraldehyde (group 4); *, PBS + 2.8 mg/ml glutaraldehyde and then PBS + 5.2% poly(L-lysine)- HBr (group 11).

and Medawar (11). Grafts were protected by vaseline gauze and plaster jackets, which were removed 10 days after trans- plantation. All grafts were inspected daily until they were rejected. The time of rejection was taken as the day when about 80% of the graft had become nonviable. In the control and experimental groups the course of graft rejection was different. Nonviability was therefore considered when the graft exhibited either necrosis and maceration, or dryness and separation from the recipient bed. Results for the groups are expressed as average survival time (AST) i SD.

Skin grafts were excised from the donor animal, treated in vitro, and then applied to the recipient. Twelve skin patches were placed in a glass vessel containing 20 ml of PBS and glutaraldehyde, and kept at room temperature for 20 min with gentle manual shaking to prevent clumping. The grafts were freed from the glutaraldehyde in two ways: by washing with PBS alone (50 ml X 5), or by washing with a solution of amino acids (or polyamino acids, see Table 2) dissolved in PBS (50 ml X 2) and then with PBS (50 ml X 4). In every experiment fresh glutaraldehyde solution was prepared by adding the appropriate volume of glutaraldehyde (25% w/v) to PBS. In the control groups the grafts were exposed only to PBS.

RESULTS

Untreated homografts

In the course of this study, three control groups in which C57 skin was grafted to SWR mice were examined. In these experi- ments (9, 10, and 11 mice per group) the average survival times obtained were quite similar (11.5, 12.8, and 13.1 days); therefore, all the data were pooled. The result was that C57 homografts had an AST of 12.4 days on SWR recipients (Group 1 in Table 1, and Fig. 1). When the casts were removed at day 10 after transplantation, most of the grafts appeared normal. Within 1-3 days, however, the normal course of acute rejection was observed. It involved shrinkage of the graft, maceration, and serum exudation with slough formation. Eventually, remnants of the dead graft fell from the recipient animal, leaving a small scar (Fig. 2D).

Homografts treated with glutaraldehyde

Results given in Table 1 and Fig. 1 show that the AST of 057 donor grafts on SWR recipients could be considerably pro-

FIG. 2. Homografts (C57 -~SWR). The same C57 skin graft, treated in vitro with 10.6 mg/ml glutaraldehyde in PBS at (A) 14 days, (B) 24 days, (C) 42 days (it was rejected at 66 days). D, untreated C57 skin graft at 18 days (it was rejected at 13 days).

longed (up to 3.2-fold) by in vitro inlcubation of the homografts in glutaraldehyde solutions. The AST increased with increas- ing concentration of glutaraldehyde up to a certain limit. The maximal effect (prolongation to 39.2 days) was obtained by soaking the grafts in PBS containing 2.8 mg/ml glutaralde- hyde. Further increase of glutaraldehyde concentration to 10.6 mg/ml did not improve the results (Group 5 in Table 1). Removal of the casts (day 10) was carried out carefully, since some of the grafts were not yet tightly bound to the recipient bed. At this stage all grafts were soft and pliable. However, 1-3 days later they began to harden, but small soft areas persisted for a long period of time. In addition, the grafts became firmly adherent, and even when separation from the bed started (see below), the portion of the graft that was attached to the host was tightly bound. In contradistinction to untreated homo- grafts, in this case shrinkage of the graft was either delayed, or it did not occur at all. In most cases, the grafts retained their original size and only toward the end showed some diminu- tion in size (Fig. 2A,B,C). The rejection process was char- acterized by progressive desiccation of the graft and separa- tion from the host bed. Separation started at peripheral por- tions of the graft and proceeded in a continuous fashion; inl

TABLE 1. Prolongation of skin homograft survival as a function of glutaraldehyde concentration

Glutaraldehyde Average survival No. of (mg/mi) time i SD

Group mice in medium (days)

1 30 -12.4+ 2.1 2 11 0.3 18.1?i 1.8 3 10 1.0 23.9?z 2.6 4 22 2.8 39.2+i18.8 5 10 10.6 39.5+ 16.7

This content downloaded from 169.229.32.136 on Wed, 7 May 2014 14:39:09 PMAll use subject to JSTOR Terms and Conditions

Page 4: Prolonged Survival of Glutaraldehyde-Treated Skin Homografts

1592 Medical Sciences: Scheclhter Proc. Nat. Acad. Sci USA 68 (1971)

- ,~~~~~~~~~~~~~~~~~~~~~~~~~~~~....:.;.....,.,j:

FIG. 3. Isografts (SWR -~SWR). The same SWR skin treated in vitro with 10.6 mg/ml glutaraldehyde in PBS at (A) 16 days, (B) 34 days (it was rejected at 30 days). C, untreated SWR skin graft (it was retained permanently).

other words, there were no scattered foci of graft separation. Occasionally, by the end of this process a dry graft having its original dimension was detached in one piece from the host. Concurrently with the graft separation, the recipient bed was healed and did not expose a denuded surface. At all stages neither infection nor inflammation were observed.

It was now of interest to explore the effect of further modifi- cations of the surface properties of glutaraldehyde-treated homografts on susceptibility to rejection. Glutaraldehyde is a bifunctional reagent (i.e., it has two reactive functions per molecule) capable of cross-linking molecules possessing a (or e)-amino groups. The glutaraldehyde molecules that reacted with the skin can be divided into two categories: molecules that cannot react any further because both reactive functions were coupled to skin constituents, and those that can still combine with other molecules because only one reactive func- tion reacted with the skin. Therefore, it might be possible to couple amino acids, polymers of lysine, and proteins to glu- taraldehyde-treated skin. Reaction with alanine, for example, would result in the attachment of an alanine residue bearing a free a-carboxyl group. In consequence, the surface area of the glutaraldehyde-treated graft would be negatively charged. Reaction with phenylalanine would also generate negative charges, but in addition it would endow the surface with hydrophobic properties by virtue of the benzyl side chain. Reaction with macromolecules should be much more efficient than reaction with amino acids since, for each covalent bond formed, many charges and considerable mass would be added. Accordingly, the glutaraldehyde-treated grafts were soaked in PBS solutions containing each of the following compounds: alanine (which upon coupling to skin would generate negative charge), phenylalanine (negative charge and hydrophobic properties), cysteine (negative charge and a thiol group), lysine (both a negative and a positive charge), the acidic copolymer

TyLsGu (ne neatv chrg an hyrohoi prop

eris,* n h basi plye poyI-yie .B (oitv

TABLE 2. Effect of different compounds on the survival of skin homografts treated with glutaraldehyde

Average survival No. of time 4SD

Group mice Compound (days)

4 22 - 39.2+ 18.8 6 10 0.5 M L-alanine 40.4+ 12.4 7 9 0.2ML-phenylalanine 37. 0 + 17. 3 8 12 0.5 ML-cysteine 41.0 i 16.5 9 10 0.5ML-lysine * HCl 34.9+i 13.5

10 11 2. 0% Try35LysmGluwo 34.9 i 12.6 11 10 5.2%poly(L-lysine) * HBr 52.9 + 18.0

The skin grafts were first treated with PBS containing 2.8 mg/ml glutaraldehyde and then with PBS containing each of the above compounds.

charges). The results (Table 2) show that the AST was not altered by treatment with alanine, phenylalanine, or cysteine, was somewhat shortened by treatment with cysteine or Tyr3s- Lysg4Glu53o (from 39.2 to 34.9 days), and was extended from 39.2 to152.9 days by exposure of the glutaraldehyde-treated graft to poly(ilysine) * HBr (see also Fig. 1). The last-men- tioned finding indicates that it might be possible to improve this system, that is, to prolong graft survival by further chemi- cal modifications of the skin.

Isografts

In order to gain some information on the mechanism of rejec- tion of glutaraldehyde-treated homografts, the effect of glutar- aldehyde on isografts was studied. All untreated isografts (10 mice) were accepted and survived permanently, as expected (Fig. 3C). On the other hand, SWR skin grafts treated with PBS containing 10.6 mg/ml glutaraldehyde were rejected by the SWR recipients (10 mice). The pattern of rejection (Fig. 3A,B) and the AST of the glutaraldehyde-treated isografts (35.5 + 22.2) were indistinguishable from similarly treated homografts (Group 5 in Table 1). Thus, a similar response was elicited by the host towards glutaraldehyde-treated homo- grafts and isografts. This response is different from the normal homograft rejection as judged by macroscopic inspection and AST values. Apparently, the rejection of glutaraldehyde- treated grafts does not involve the usual immune reaction directed against histocompatibility antigens.

DISCUSSION

The experiments described here show that a skin homograft can be retained by the host for 39.2 + 18.8 days when it is treated in vitro with the optimal dose of glutaraldehyde prior to transplantation, as compared with 12.4 + 2.1 days for the PBS-treated control. This effect can be attributed, in part at least, to the "coating" of the histocompatibility antigens by glutaraldehyde molecules which can bind directly to them, or in their close vicinity. Thus, these antigens are shielded from the immune apparatus of the host. This is analogous to the finding that the coupling of polyalanine chains to protein car- riers in sufficient amounts have led to a considerable diminu- tion in the immune response towards the protein moiety (12). Previous investigations have shown that homografts and iso- grafts rendered nonviable by cyanide (8) or by freeze-drying (9) were rejected in identical fashion and that they possessed diminished antigenicity. On the basis of these reports and our

This content downloaded from 169.229.32.136 on Wed, 7 May 2014 14:39:09 PMAll use subject to JSTOR Terms and Conditions

Page 5: Prolonged Survival of Glutaraldehyde-Treated Skin Homografts

Proc. Nat. Acad. Sci. USA 68 (1971) Glutaraldehyde-Treated Skin Homografts 1593

finding that glutaraldehyde-treated isografts were eventually rejected similarly to glutaraldehyde-treated homografts, one might assume that the glutaraldehyde effect sterms from the fact that it killed the cells. In the absence of histologic exami- nations it is impossible at the present time to evaluate the ex- tent of viability of the glutaraldehyde-treated grafts. How- ever, the AST of freeze-dried grafts was extended from 10 to 13 days, and no extension was observed in cyanide-treated grafts; on the contrary, they shrank even more rapidly than fresh, viable homografts. In our system the AST of glutaraldehyde- treated grafts was increased more than three-folcl and graft contraction was minimal. The observed dose-response effect of glutaraldehyde treatment (Table 1) and the capacity to im- prove retention of the graft by further treatmenit with poly- (L-lysine).HBr (Table 2) suggest that it may be possible to (levelop a mild procedure by which most of the physiological functions of the skin would be preserved for a long period of time.

Homografts, both fresh and preserved, have beeni used extensively and successfully in the treatment of seriously burned patients (13, 14). For this purpose large quantities of skin are required, more than is available, because the grafts have to be replaced within 4 days. After this time the graft becomes adherent to the recipient bed, and open septic wounds develop because of rejection of the graft. Under spec- ified conditions, the graft can remain in place until rejection (14). The approach described here permits the introduction of many modifications in the surface properties of the skin, some of which may lead to grafts that may not become adherent, as well as to grafts that will be retained for long periods of time. The coupling to tissue of compounds either directly or via bifunctional reagents is a nonspecific manipulation which might be effective in prolonging survival not only of homo- grafts but perhaps also of heterografts. If this should prove to be so, it might become possible to overcome the difficulties encountered in the limited skin supplies from human sources.

In skin homografts, permanent survival is not required (14), and the preliminary results obtained justify further studies along this line. It might also be desirable to apply this ap- proach to internal organs, since it is known that the immune

rejection of these is much less vigorous than that elicited toward skin transplants (15). For example, kidneys might be perfused in vitro with reagent solution in such a way that the resulting modification of the surface endothelium would be compatible with kidney function. Even a small and temporary diminution in antigenicity would be beneficial since it might attenuate or decrease the incidence of rejection crises (16, 17). The applicability of this approach to experimentation with internal organs is warranted because of the availability of a large number of reagents which can react with tissue con- stituents under physiological conditions.

The author thanks Mr. I. Serusi for excellent technical assist- ance, Dr, Bila Schechter for help and discussion, and Dr. C. B. Anfinsen for comments.

1. Billingham, R. E., P. L. Krohn, and P. B. Medawar, Brit. Med. J., 2, 1049 (1951).

2. Hellmann, K., D. I. Duke, and D. F. Tucker, Brit. Med. J., 2, 687 (1965).

3. Bonmassar, E., G. Francesconi, S. C. Manzoni, and M. Prelli-Ercolini, Nature, 209, 1141 (1966).

4. Dukes, C. D., and T. G. Blocker, Ann. Surg., 136, 999 (1962).

5. Lemperle, G., J. Surg. Res., 8, 511 (1968). 6. Hellmann, K., and D. I. Duke, Transplantation, 5, 184

(1967). 7. Parrish, R. A., J. K. Tippens, M. M. Pulliam, and W. H.

Moretz, Amer. Surg., 30, 793 (1964). 8. Grillo, H. C., and C. F. McKhann, Transplantation, 2, 48

(1964). 9. Abbott, W. M., and A. M. Pappas, Ann. Surg., 172, 781

(1970). l. Staats, J., Cancer Res., 28, 391 (1968). 11. Billingham, R. E., and P. B. Medawar, J. Exp. Biol., 28, 385

(1951). 12. Bauminger, S., I. Schechter, and M. Sela, Immunochemistry,

4, 169 (1967). 13. Pruitt, B. A., and J. A. Moncrief, J. Surg. Res., 7, 332

(1967). 14. Zaroff, L. I. W. Mills, Jr., J. W. Duckett, Jr., W. E.

Switzer, and J. A. Moncrief, Surgery, 59, 368 (1966). 15. White, E., and W. H. Hildemann, Science, 162, 1293 (1968). 16. Williams, G. M., D. M. Hume, R. P. Hudson, P. J. Morris,

K. Kano, and F. Milgrom, N. Engl. J. Med., 279, 611 (1968). 17. Williams, G. M., B. DePlanque, W. H. Graham, and R. R.

Lower, N. Engl. J. Med., 281, 1145 (1969).

This content downloaded from 169.229.32.136 on Wed, 7 May 2014 14:39:09 PMAll use subject to JSTOR Terms and Conditions