Larry R. Pennington

Surgical management of Crohn's disease. Influence of disease at margin of resection

To determine the influence of microscopic disease at an anastomosis following intestinal resection for Crohns disease, 97 patients undergoing 103 resections were reviewed. Most resections (85/103) involved both small and large bowel and were followed by an ileocolic anastomosis. All resection margins were available and were reviewed. In 52 instances there was no evidence of Crohns disease at the margins. In 51 instances histologie evidence of Crohns disease varying from chronic inflammation to tissue destruction was present in one or both margins. The incidence of immediate postoperative anastomotic complications (leak with fistula or abscess, or obstruction) was identical in patients with microscopically normal margins (3/52; 6%) and in patients with microscopic Crohns disease at the margins (3/51; 6%). The patients were followed for a mean of 5.4 ± 4.2 years. A clinical recurrence developed during the follow-up period in 50% (26/52) of those patients with normal margins, and in 61% (31/51) of those patients with involved margins. A suture line recurrence developed in 35% (18/52) and required reoperation in 17% (9/52) of those patients with microscopically normal margins. A suture line recurrence developed in 41% of the patients (21/51) and required reoperation in 24% (12/51) of those with microscopically involved margins. None of these differences are statistically significant. The presence or absence of microscopic disease at the anastomosis did not appear to influence immediate anastomotic wound healing or long-term recurrence rates. We therefore recommend conservative resections for Crohns disease to achieve grossly uninvolved margins rather than the sacrifice of normal bowel to achieve histologically normal margins.

Transplantation In Miniature Swine: Viii. Recombination Within The Major Histocompatibility Complex Of Miniature Swine

Offspring of heterozygous parents derived from three herds of miniature swine, each of which is homozygous at the major histocompatibility complex (MHC), were screened for recombination within the MHC. The swine were typed serologically at weaning and later typed by mixed lymphocyte reaction (MLR). Two intra-MHC recombinants were discovered, both of which involved the exchange of D region specificities without apparent dissociation of ABC region specificities, confirming the localization of the SLA-D region outside of the SLA-ABC regions. The first recombinant was the offspring of an SLAc/d (cd) by dd mating and typed serologically as cd but typed by MLR as dd. The second recombinant was the offspring of a cd by cd mating. It typed serologically as cc but stimulated cc in one-way MLR and retained its reactivity to dd, thus suggesting a possible recombination within the D region. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 3H-leucine-labeled lymphocyte surface antigens demonstrated that corresponding la antigens were also exchanged during these recombinant events supporting the hypothesis that genes coding for Ia antigens are identical or closely linked to D region genes encoding the MLR specificities.

Bone Marrow Transplatation In Miniature Swine: I. Development of the Model

Procedures for successful autologous and MHC-matched allogeneic bone marrow transplantation in partially inbred, MHC-defined miniature swine have been established. All marrow recipients were conditioned with single-dose total-body irradiation at the upper level of tolerance, and supported with antibiotics and irradiated blood products during aplasia. Surgical harvest of autologous and allogeneic marrow yielded sufficient numbers of cells to successfully reocnstitute recipients. Radiation control animals that reveived no marrow failed to show recovery of marrow function. Pigs transplanted with autologous marrow at dosed greater than108 cells/kg/ routinely engrafted and recovered normal marrow function. The major clinical complications were acute and chronic infections and hemorrhage. T cell-depleted autologous marrow also engrafted, and there was no observed increase in clinical complications. In bone marrow transplantation across non-MHC allogeneic differences, engraftment and survival were similar

Immunologic characterization of MHC recombinant swine. Production of SLA class specific antisera and detection of Ia antigens on both B and non-B PBL.

Two intra-MHC recombinant haplotypes have been examined to document the nature of the recombination and to generate MHC-specific alloantisera. Cells from progeny of recombinant pigs have been compared by mixed lymphocyte reaction, by complement-dependent cytotoxicity with standard alloantisera, and by sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of immune precipitates of radio-labeled extracts. The results demonstrate that both recombinant haplotypes, f and g, have inherited the SLA-A, B loci of the c haplotype and the SLA-D loci of the d haplotype, and that no differences between the two recombinant chromosomes are detectable. Class specific anti-SLA-A, B and anti-SLA-DR sera were produced in or against the g haplotype. In terms of cytotoxicity and SDS-PAGE these sera exhibited the expected reactivities, except that the high-titered anti-SLA-DR sera gave an unexpectedly high percentage of lysis of swine peripheral blood lymphocytes. That cells other than pig B cells were being lysed by the anti-SLA-DR sera was confirmed by analyses of subpopulations of peripheral blood lymphocytes. Approximately 50% of nylon nonadherent T cells were specifically lysed by allo-anti-Ia sera. Similar lysis of T cells was found with crossreactive mouse anti-Ia sera. Thus, unlike other species in which Ia antigens are expressed on T cells at low levels and are difficult to detect, the SLA-D region products are readily detectable on swine peripheral blood T lymphocytes.

Transplantation in miniature swine. X. Evidence for non-SLA-linked immune response gene(s) controlling rejection of SLA-matched kidney allografts.

The survival of renal allografts between SLA-matched swine has been found to be subject to non-SLA-linked Ir gene control. Using three herds of miniature swine, each homozygous for a different SLA haplotype (designated aa, cc, and dd, respectively), we initially observed that all animals of the c haplotype rejected SLA-matched renal allografts. In contrast, the subset of dd animals which were the product of continuous homozygous matings since establishment of the dd herd (ddR) accepted SLA-matched grafts indefinitely without any immunosuppression. A formal backcross study was therefore performed in which offspring of (cd x ddR) matings (designated cdbc and ddbc) were bilaterally nephrectomized and transplanted with SLA-matched renal allografts. Acceptors and rejectors were found among both backcross types, with a total of 6 of 17 (35%) of the animals dying of renal failure secondary to rejection. When ddbc animals were used as donors for ddR recipients, all grafts were accepted, ruling out the possibility that rejection was attributable to strong non-SLA antigens segregating within the cc herd. These results are consistent with a model in which rejection of SLA-matched renal allografts is controlled by either one or two non-SLA-linked immune response genes. These findings raise the possibility that the observed 5 to 15% frequency of rejection of HLA-identical living related donor renal allografts in man could involve similar immune response gene control.

Bone marrow transplantation in miniature swine. II: Effect of selective genetic differences on marrow engraftment and recipient survival

In order to stusy the effct of defined genetic differences of one bone marrow transplantation in miniature swain, five different conninations of major histocompatibility complex (MHC)-matched adn mismatched bone marrow transplants were perfomed. Eight of nine fully MHC-mismatched allogeneic bone a marrow transplants faile to reconstitute, and one animal reconstituted briefly but then died quickly therafter. Five of six class I-matched/class II-mismatched (gc) bone marrow transplants engrafted, showed a skin rash trypical of graft-versus-host (GVH) reaction, and died 3 weeks after the marrow transplants into F1 hosts engrafted and caused GVH skin rash, with survivals from 1 to 9 montsh (n=5). Serologica typint of the F1 recipients of parental marrow showed only donor-type peripheral blood lymphocytes (PBL), suggesting complete marrow replacemetnt. Conversely, F1 into parental marrow transplants showed no engraftment (n=6).These results indicate that resistance to MHC-mismatched allogeneic bone marrow engraftment donor class I MHC differences. This response appears to interfere with engraftment of donor bone marrow cells despionte host preparation with 900–1100 rads total-body irradiation. In the absebce of donor MHC class IO differences, engraftment was seen despite the existence of multiple non-MHC differences, and even in the presence of class II differeces. Such engraftment also led to GVH, varying in intensity according to the strength of genetic disparioty (i.e., worst in parentF1combination). Thses results suggest that miniature swaine should proviode adn effective model for study of both GVH elimination (in the parentF1 combination) and problems of engraftment (in the F1parent combination), the two most important obstacles to clinical allogeneic transplantation.

Bone marrow transplantation in miniature swine. III: Graft-versus-host disease and the effect of T cell depletion of marrow

Graft-versus-host disease (GVHD) has been evaluated in partially inbred miniature swine in order to study this complication of allogeneic bone marrow transplantation (BMT) in a major histocompatibility complex (MHC) genetically defined large animal model. Bone marrow from MHC homozygous (“parental”) swine was injected into irradiated (900 rads total-body irradiation) MHC heterozygous (“F1”) swine that shared one haplo-type with the donor. All 18 animals successfully engrafted with donor bone marrow, and 17 of these devel-