Felix T. Rapaport

Pemphigus-Like Antibodies in Patients with Skin Burns

Previous stu&es in our laboratories have demonstrated that severe thermal injury may be associated in man and in experimental animals with an inhibition of delayed hypersensitivity to tuberculin [4, lo], and a prolongation of skin allograft survival [ll], as well as with the development of autohemagglutinins [8], heterophile antibodies and anti-gamma globulin factors [6]. More recently an antigenic component was demonstrated in burned skin, which is absent from normal skin [7]. The present study was undertaken on the assumption tha t skinspecific antigens, partially altered by the thermal injury, may stimulate formation of tissue-specific antibodies in the host. Similar mechanisms have been implicated in the production of many tissuespecific autoantibodies [9]. The demonstration that sera obtained from patients suffering from pemphigus contain autoantibodies which combine with the intercellular areas of stratified squamous epithelium [l, 21 has provided a convenient model for these studies. Serum samples from 15 patients of the New York University Medical Center Surgical Services, who had suffered skin burns involving 20% t o 80% of their body surface area, were studied. Indirect immunofluorescence tests with sections of rhesus monkey

Homograft Sensitivity Induction by Group A Streptococci

Streptococcal cells can induce in the guinea pig a state of altered reactivity to skin homografts similar to that resulting from sensitization with homologous tissue.

TRANSPLANTATION ANTIGEN ACTIVITY OF BACTERIAL CELLS IN DIFFERENT ANIMAL SPECIES AND INTRACELLULAR LOCALIZATION

The observation that the homograft response obtained following sensitization of guinea pigs with heat-killed group A Streptococci or Staphylococci is indistinguishable from that resulting from pretreatment with homologous tissues has raised the possibility that the antigens concerned with the induction of homograft sensitivity may exist in heterologous form throughout nature. In this respect, mammalian transplantation antigens may fit into the category of surface structure principles shared by widely separated phylogenetic groups, as described in the studies of F o r ~ s m a n n , ~ of Springer et al . , and of other investigators. The present report describes results obtained with this experimental system in the guinea pig and, subsequently, in other mammalian hosts. In addition, an attempt is made to analyze the intracellular location of the streptococcal antigen(

An Approach To Organ Salvage From Non-heartbeating Cadaver Donors Under Existing Legal And Ethical Requirements For Transplantation

concerned with the induction of homograft sensitivity in the guinea pig. 1 3

Heterophile antibodies in human transplantation

Effective utilization of nonheartbeating cadaver donor organs is limited by the time required to obtain the necessary family consent prior to organ retrieval (a delay of at least 4-6 hr); this exceeds by far the maximum tolerance of kidneys to warm ischemia. Measures that could theoretically permit use of such organs include: (1) rapid in situ flush cooling; (2) continued in situ kidney cooling until permission for donation is secured; and (3) cell-membrane stabilization of vital organs, with only minimal invasion of the donor body. These measures were tested experimentally in dogs. Hemorrhagic shock was produced in mongrel dogs. One hour after cessation of heartbeat, a rapid perfusion tube was placed into the femoral artery; it was advanced, and its balloon was inflated in the aorta above the renal vessels. The kidneys were then flushed in situ with 1000 cc of cold preservation solution containing a calmodulin inhibitor, trifluoperazine. Two other catheters were inserted percutaneously into the peritoneal cavity for continuous intraperitoneal cold perfusion. Core temperatures of 4 degrees C were maintained in situ in the kidneys for 5 hr. Six hours after cardiac arrest, the kidneys were removed and preserved ex vivo at 4 degrees C for 24 hr, and were then transplanted into their respective hosts (n = 11), where they sustained life uneventfully. This method requires a 2-inch incision in the groin of the prospective donor, and two small stab wounds of the abdomen; i.e., semi-invasive procedures which are commonly performed in emergency rooms. The perfused body could then be released to the family if donation is denied. The recently documented increased willingness of the public to donate organs when the termination of life support is not an issue, and court decisions that have authorized the performance of nondeforming diagnostic procedures in cadavers without consent, suggest that the salvage of transplantable semi-invasive procedures described in this study may be useful in helping to alleviate the current shortage of transplantable organs. This technique can provide the time needed for families to consider the option of organ donation from nonheartbeating cadaver donors in an unhurried and unpressured manner, while preserving the viability of vital organs during the decision-making process.

OBSERVATIONS ON IMMUNOLOGICAL MANIFESTATIONS OF THE HOMOGRAFT REJECTION PHENOMENON IN MAN: THE RECALL FLARE

Sensitization of human recipients with transplantation antigens (leucocytes, skin, or kidney allografts) has resulted in the appearance of serum hemagglutinins directed against sheep, guinea pig, and rat erythrocytes. Such hemagglutinins have been identified as IgG and IgM antibodies. Their appearance was not related to AB0 erythrocyte group incompatibility between donors and recipients, and the antibodies were not of the Forssman or Paul-Bunnel type. The antibody responses appeared to be primarily directed against antigen(s) present on rat erythrocytes, but shared to varying extents by other species. The peak antibody titers occurred in association with allograft rejection. In this regard, they may be of interest as a possible early warning system for the diagnosis and prompt management of rejection crises in clinical organ transplantation.

Mechanisms of antibody formation. I. Early in vivo proliferation of mouse spleen t cells as an initial step in antibody formation.

This investigation of homografts in man was undertaken to study the reactions to repeated skin homografts from the same donor and to evaluate the duration of the resulting sensitivity. Immunological aspects of the rejection of skin homografts have been reported by Medawar (1954)’ and Billingham, Brent, and Medawar (1954).2 These investigators described the active sensitization pattern expressed by the host’s reaction to skin transplants, and also the host’s behavior when again placed in contact with an antigenic challenge from the same source. Secondset skin homografts have exhibited an accelerated form of rejection in the species studied (Medawar, 1944; 1945: 1946;6 Dempster, Lennox, and Boag, 1950;6 Lehrfeld, Taylor, and Converse, 1955;7 Dempster, 1952 ;” Sparrow, 1953;e Gibson and Medawar, 1942;’O Longmire and Smith, 1951;” and Baxter and Entin, 1951’*). Reactions of the host to additional skin grafts from the same source have been described in rabbits, in rats, and in man (Billingham and Boswell, 1953 ;13 Lehrfeld, Taylor, and Converse, 195S7). Data obtained in our laboratories previously (Rogers, Converse, Taylor, and Campbell1*), support the results described in this report. The experimental method outlined below is similar to that used in previous studies. The results obtained in each series were also similar.

Immunohistologic analysis of human renal allograft dysfunction.

Abstract Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10 6 −2 × 10 8 sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement. Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr. Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function. In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10 8 sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro , even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.

Kinetics of humoral responsiveness in severe thermal injury.

The value of percutaneous core needle biopsy in the immunohistological evaluation of renal allograft dysfunction was studied in 72 consecutive biopsies performed in 42 patients. The phenotypes of infiltrating cells mediating graft destruction were identified with monoclonal antibodies and immunoperoxidase staining techniques. Light microscopy, electron microscopy, and immunofluorescence staining were performed in all biopsies. Biopsies were divided into groups depending on their classification on the basis of standard histologic criteria, i.e., acute tubular necrosis (ATN), acute interstitial rejection, acute vascular rejection, chronic rejection and renal disease in native kidneys (RDNK) of nontransplant patients. Immunohistologic analysis of graft biopsies showed a significant increase in Leu 1 (pan-T cells), (P<0.001), Leu 2 (cytotoxic/suppressor cells) (P<0.001), and Leu 3 cells (P<.05) in acute interstitial rejection. The expression of BR antigen was significantly increased in both acute (P<.0.25) and chronic (P<.05) rejection, when compared with the findings in ATN biopsies. Leu M1 (monocytes/activated T cells) and Leu 10 (B cells/macrophages) were significantly increased (P<0.05 and P<.005, respectively) in acute interstitial rejection only. The helper/suppressor ratio of infiltrating cells showed no significant change in any clinopathologic category. There was no correlation between the cell populations infiltrating the graft and those monitored in the peripheral blood. Allograft mononuclear ceil infiltrates in cyclosporine (CsA) vs. azathioprine-treated patients revealed significantly fewer Leu 2 (P<.05) and Leu M1 (P<.05) cell populations in CsA patients during acute rejection. In 32 of these 72 biopsies (44.4%), the biopsy results provided a direct contraindication to the use of steroids, by allowing differentiation between allograft rejection and other causes of graft dysfunction. A total of 38% of the biopsies yielded a histological diagnosis that contradicted the clinical pre-biopsy diagnosis. All allografts showing evidence of severe small vessel disease and/or antibody-mediated rejection eventually were lost. These data highlight the usefulness of needle biopsy material as a guide to the study of intragraft immune events and to clinical management of recipients.

Ranks of Donor-Recipient Histocompatibility for Human Transplantation

Severe thermal injury has the capacity to increase the rate of generation of antibody-forming cells in mice. The intensity of stimulation appears to be proportional to the extent of injury. The effect has been observed in animals burned within 1 hr before or after sensitization with test antigen(s), and persists up to 14 days after injury. Thereafter, the stimulatory effect wanes, and disappears by the 21st day after burning. Responses to T-cell (thymus derived lymphocytes) dependent antigens (sheep erythrocytes; sheep erythrocytes coupled to TNP) and to antigens not requiring T and B-cell (bone marrow derived lymphocytes) cooperation (DNP-Ficoll) appear to be equally affected by thermal injury. The mechanisms underlying this form of enhanced antibody response are not clear. The data, however, support the possibility that the burn wound may release factor(s) capable of enhancing humoral responsiveness in the injured animal. Such factor(s) do not appear to be endotoxins.