R. L. Marquet

Total orthotopic allogeneic small bowel transplantation in rats. Attempts to ameliorate the graft-versus-host disease by irradiation and transfusions of the donor.

The effect of pretransplant specific blood transfusions and irradiation of the donor were studied in total orthotopic small bowel transplantation in rats, with the aim of ameliorating the graft-versus-host reaction. Syngeneic transplantation with or without irradiation of the donor with 10 Gy showed that small bowel transplantation is compatible with a normal nutritional status, when no rejection is involved. Transplantation in the histoincompatible WAG-to-BN combination without pretreatment resulted in rejection of the small bowel grafts in 16.6 +/- 2.7 days. Pretreatment with 3 BN blood transfusions to the WAG donor did not prolong the survival time; surprisingly, the transfusions induced a more-severe GVH reaction. Irradiation of the donor with 10 Gy was very effective in ameliorating the GVH disease, but the absence of the immunosuppressive effect of the GVH disease did lead to an accelerated graft rejection (7.5 +/- 0.9 days).

Detection of canine intestinal allograft rejection by in vivo electrophysiologic monitoring.

The aim of this study was to evaluate the significance of in vivo measurements of electrophysiologic parameters for the detection of canine small bowel (SB) allograft rejection. In dogs of group I (n=17) a heterotopic SB autotransplantation was performed. Dogs of group II (n=8) received a heterotopic SB allograft in a fully mismatched donor-recipient combination. No immuno-suppression was given. All grafts were monitored regularly by in vivo measurements of transepithelial potential differences (PDs) and by biopsies of the grafts. The overall technical failure rate was 36% caused by thrombosis at the vascular anastomosis in most cases. All successful autografts survived the experimental period and showed physiologic PD responses after stimulation by both a theophylline solution and a glucose solution. The successful allografts survived 5.5±0.2 days (mean ± SEM); the transepithelial PDs showed normal responses at postoperative day 3, but showed decreased responses at day 5 (P<0.05) and reversed responses at day 6 (P<0.05). The diminished PD responses correlated well with the onset of histologic alterations characteristic of rejection. This study demonstrates that serial monitoring of transepithelial PD responses is a non-invasive method to detect acute SB allosraft rejection.

Value of in vivo electrophysiological measurements to evaluate canine small bowel autotransplants.

This study aimed to develop a non-invasive method for in vivo measurement of the transepithelial potential difference in the canine small bowel and to evaluate this parameter in small bowel autotransplants. In group 0 (control group, n = 4), two intestinal loops were created without disturbing their vascular, neural, and lymphatic supplies. In group I (successful autotransplants, n = 11), two heterotopic small bowel loops were constructed. Long term functional sequelae of vascular, neural, and lymphatic division were studied. Group II (n = 6) consisted of dogs with unsuccessful autotransplants suffering thrombosis of the vascular anastomosis, which resulted in ischaemic small bowel autografts. In group I, values of spontaneous transepithelial potential difference, an index of base line active electrolyte transport, were significantly lower compared with group 0 (p less than 0.05), probably as a result of denervation of the autotransplants. Both theophylline and glucose stimulated potential difference responses, measuring cyclic adenosine monophosphate mediated chloride secretion and sodium coupled glucose absorption respectively, showed negative luminal values in group I at all time points after transplantation. These transepithelial potential difference responses diminished progressively with time. From day 21 onwards both theophylline and glucose stimulated potential difference responses were significantly less than the corresponding responses at day seven (p less than 0.05). Morphometric analysis showed that the reduction of transepithelial potential difference responses preceded degenerative mucosal changes in the heterotopic small bowel autografts. In group II, potential difference responses to theophylline and glucose showed positive luminal values (p<0.01 v group I), probably as a result of passive potassium effusion from necrotic enterocytes.

The value of in vivo electrophysiological measurements for monitoring functional adaptation after massive small bowel resection in the rat.

The process of functional adaptation after extensive small bowel resection is complex and imprecisely understood. In vivo electrophysiological measurements for monitoring the functional adaptive process after massive small bowel resection in Brown-Norway rats were evaluated. Rats underwent either a sham operation (SH) or a 90% small bowel resection (SB). Standard rat chow was fed in unlimited quantities. At three or 10 weeks after operation, jejunal and ileal transepithelial potential differences (PD, mV) were determined. Electrogenic ion transport in the villus was measured after glucose (sodium coupled active glucose absorption; PD-glu) and in the crypt, after theophylline infusion (theophylline stimulated chloride secretion; PD-theo). Biopsies were taken simultaneously. Each experimental group consisted of three to five animals. At three weeks the PD-theo and PD-glu in SB rats were significantly lower than in SH rats in both jejunal and ileal segments. At 10 weeks PD-theo and PD-glu were significantly diminished in the jejunal segment of the SB rats compared with the SH rats. The values of PD-theo and PD-glu in the ileal segments were, however, no longer different between the two groups. Three and 10 weeks after operation the length of the villi in the SB group was increased significantly compared with the SH controls. These results indicate that in the early phase of adaptation in vivo electrophysiological variables do not correlate with histological changes in the SB rats. This might be due to cell immaturity resulting from an increased rate of cell turnover or lack of intercellular tight junctions. This hypothesis is supported by a recovery of PD responses in the ileum 10 weeks after resection.

Small bowel transplantation in rats. The effect of pretransplant donor-specific blood transfusions on various segments of small bowel grafts.

The effects of pretransplant donor-specific blood transfusion on the survival of orthotopic small bowel transplants in rats were investigated in the fully allogeneic BN (Rt1n) to WAG (Rt1u) donor-host combination. Previous studies show that in this combination DSTs lead to permanent survival of heterotopically transplanted hearts, marked prolongation of kidney grafts, and moderate prolongation of pancreas grafts but have no effect on skin grafts. Without pretreatment, total small bowel grafts (+/- 45 cm) were rejected in 12.2 +/- 1.8 days (mean +/- SD), and 10-cm segments of jejunum or ileum in 11.2 +/- 4.0 and 11.6 +/- 0.5 days, respectively. Three DSTs given on days -21, -14, and -7 before transplantation had no effect on graft survival in any of the groups tested. Total small bowel grafts were rejected in 12.8 +/- 2.5 days, and 10-cm-long segments or jejunum or ileum in 17.0 +/- 7.2 days and 11.5 +/- 2.7 days, respectively. Graft-versus-host disease, which was mild and transient, occurred in 50% of the nontreated rats engrafted with a total small bowel, in 40% of the animals transplanted with an ileum segment, and in none of the rats that received a jejunal transplant. In the DST-pretreated groups, none of the animals transplanted with a total small bowel or ileum segment and 16.6% of the animals transplanted with a jejunum segment showed clinical signs of GVHD. When DST pretreatment was combined with cyclosporine, grafts did not survive any longer than with cyclosporine treatment alone. It is concluded that DSTs ameliorate GVHD but do not prolong the survival of small bowel allografts nor act additively with cyclosporine treatment.

Contrast in the efficacy of hDAF mouse hearts between ex vivo perfusion and transplantation into primates

Abstract: In recent experiments, in which we compared hDAF transgenic rat hearts perfused with 15% human serum in the Langendorff device and hDAF rat hearts transplanted into cynomolgus monkeys, we demonstrated that in the ex vivo heart perfusion model both homozygous and heterozygous hDAF hearts survived longer as nontransgenic controls. Surprisingly, we found that only homozygous hDAF hearts were protected against hyperacute rejection in vivo. The first aim of this study was to determine whether perfusion of mouse hearts with higher human serum concentrations or human blood might explain some of the differences found in survival time of the recently performed experiments with rat heart xenografts. Secondly, we investigated whether the observed differences in survival times of rat xenografts between in vivo and ex vivo transplantation would also hold for mouse hearts transgenic for hDAF. An ex vivo model was used to perfuse hDAF mouse hearts and controls with human serum or blood, and hDAF transgenic hearts and controls were transplanted into cynomolgus monkeys. hDAF transgenic mouse hearts survived significantly longer than their controls when perfused with 15% human serum, but no difference was found when 30% human serum was used, or when these hearts were transplanted into cynomolgus monkeys. However, in both the in vivo and ex vivo models the amount of PMNs adhering to the vascular endothelium was significantly lower in hDAF transgenes as compared with their controls. In conclusion, in the ex vivo situation, the efficacy of hDAF transgenesis in preventing HAR is limited by serum complement concentration.

Total orthotopic allogeneic small bowel transplantation in rats: effect of allograft irradiation combined with cyclosporine-A therapy.

Rejection and graft versus host disease are prominent features in small bowel allotransplantation in rats. Cyclosporine treatment of the recipient and irradiation of the donor were used to circumvent these phenomena in the WAG to brown Norway rat model. Irradiation of the donor with five or 10 Gy did prevent graft versus host disease but resulted in a more vigorous rejection of small bowel allografts in untreated recipients (mean (SEM) survival times of 11.5 (0.4) (n = 8) and 7.5 (0.9) (n = 11) days respectively, versus 16.6 (2.6) days (n = 17), p less than 0.01). Cyclosporine treatment of the recipient (25 mg/kg on days 0, 1, 2, 4, and 6 after transplantation) led to a mean (SEM) survival time of 38.3 (8.5) days (n = 10); 20% of the animals developed graft versus host disease. Combined with 5 Gy donor pretreatment, a similar survival was obtained without occurrence of graft versus host disease. However, cyclosporine treatment combined with 10 Gy led to a significant shortening of graft survival (23.1 (6.8) days, n = 9). These results suggest that although irradiation is very effective in preventing graft versus host disease, high dosages may accelerate rejection either by making the graft more vulnerable to rejection or by completely removing the immuno-suppressive effect of graft versus host disease.

Site-specific antitumour effects of 2 pyrimidinone compounds in rats

Recently a series of 5-halo-6-phenyl pyrimidinones has been found to induce interferon production in several animal species (Stringfellow et al., 1980) and in man (Earhart et al., 1985). These compounds modulate a variety of immune responses. Most prominently they enhance the activity of natural killer (NK) cells (Taggert et al., 1980) and macrophages (Li et al., 1985). In the experiments reported here we evaluated the effects of two pyrimidinones that differ greatly in their ability to induce interferon (IFN) production. ABPP (2amino-5-bromo-6-phenyl-4-pyrimidinone) induces high serum levels of IFN whereas AIPP (2-amino-5iodo-6-phenyl-4-pyrimidinone) does not. Yet, both agents are equally active in enhancing NK cell activity (Lotzova et al., 1983). In a previous communition (Marquet et al., 1983) we have shown that subcutaneous growth of liposarcoma LS175 in BN rats is inhibited by IFN. This nonimmunogenic liposarcoma is of spontaneous origin and has been found to be NK resistant. In order to investigate whether the induction of IFN plays a part in the antitumour effects of ABPP and AIPP we compared the two agents in the subcutaneous and in the artificial lung metastasis model, using tumour LS175. Ten to 12-week old male BN rats were used in all experiments. The NK cell activity in peripheral blood lymphocytes (PBL) after the intraperitoneal administration of 250mg kg-1 of ABPP or AIPP was assessed by a standard 3 h IICr release assay as described by Ortaldo et al. (1977). Control animals received an equal volume of PBS i.p. The results are shown in Figure 1. A rapidly established and longlasting +4-fold increase in NK cell cytotoxicity is seen after the administration of either agent. The influence on macrophage activity was assessed by measuring the ingestion of latexFigure 1 Enhancement of NK cell activity inperipheral blood lymphocytes (PBL) 1, 3 and 9 daysafter a single i.p. injection of250mgkg-1 ofAIPP orABPP. Control animals received PBS i.p. Thepercentage ofspecific lysis is shownas determined in a3h5Cr release assay at an effector-to-target (Yac-1)ration of40:1.

Experimental Xenotransplantation in Rodents — II: Skin Versus Heart Grafts

When studying xenotransplantation in a small animal model, one has a choice between two different types of models. In one, rejection of the graft takes place within minutes after transplantation — so-cahed hyperacute rejection. In the other type, rejection takes more time — usually 3-5 days — and is generally referred to as accelerated acute rejection as it occurs some days earlier than the acute graft rejection seen in allogeneic transplantation.

Moderate effect of preoperative blood transfusions on pancreas allograft survival in rats and dogs.

Treatment of type I diabetes by early pancreas transplantation requires the availability of a safe and effective transplantation technique. With the currently available immunosuppressive drugs it is difficult to obtain long-term pancreatic allograft survival. In this respect pancreas grafts compare unfavorably with heart or kidney grafts. Using a relatively simple and safe subcutaneous transplantation technique we investigated the effect of blood transfusions combined with low-dose immunosuppressive drugs in rats and dogs in order to attain an immunosuppressive schedule of low toxicity. Subcutaneous pancreas transplantation appeared to be a feasible technique, with long-term graft survival in syngeneically transplanted rats and autotransplanted dogs. Only a moderate prolongation of pancreatic allograft survival by blood transfusions was demonstrated in both models. In rats one or three preoperative donor-specific blood transfusions significantly prolonged pancreas graft survival to 23±15 and 29±15 days, respectively, compared with 12±2 days in untreated controls. Low-dose cyclosporine (15 mg/kg on the day of operation) led to improved graft survival in nontransfused recipients (17±4 days), however, this treatment could not further prolong graft survival in transfused animals (34±20 days). In dogs, treated postoperatively with azathioprine and prednisolone, three preoperative third-party blood transfusions led to 29±19 days of pancreas graft survival, which was not significantly different from nontransfused controls (17±5 days). These results indicate that, in rats as well in dogs, pancreatic allografts are less sensitive to the immunomodulating effect of blood transfusions than heart and kidney grafts.