Search

The rat model has been used to present evidence of the effect of surgical damage on the immune system. Syngeneic small bowel transplantation (SBT) has been used to show an increased incidence of graft-versus-host disease (GVHD) as well as thymic atrophy and altered host T cell proliferative response. Syngeneic auxiliary SBT was carried out between (LEW x BN)F1 hybrids. Varying amounts of LEW mesenteric lymphocytes were injected into the last animals to induce GVHD. Results showed that in the SBT recipients the incidence of lethal GVHD was increased when compared with untreated or sham-laparotomy controls. Marked thymic atrophy was also observed, while the number of hepatic lymphocytes increased transiently. Lymphocyte proliferation in response to concanavalin A or interleukin-2 was impaired for up to 21 days postoperatively, whereas the mixed lymphocyte reaction reactivity was not affected. These results show that the number and proliferative activity of thymic T cells were impaired after major small bowel transplantation surgery and that extrathymic lymphocytes were developed in the liver.

Immunological aspects after orthotopic rat liver retransplantation (re-OLT) were examined in association with cell migration and mixed chimerism. At day 2 after the first orthotopic liver transplantation (day 0) in the combination of DA (MHC haplotype, RT1a) donor into PVG (RT1c) recipient, the grafted DA liver was removed and a new PVG liver was implanted into the same PVG recipient (re-OLT). In the PVG recipient at various times after the re-OLT, DA-derived antigen and cells were detected using a DA-specific anti-class I mAb R3/13 in conjunction with ELISA, immunoblotting, and immunohistochemistry. The level of soluble class I antigen, which had risen to 270 ng/ml after the first OLT, substantially decreased within 24 hr after re-OLT. Using immunoblotting, DA class I antigen was detected in the PVG recipient's lymphoid organs at day 3 after DA liver grafting and persisted for up to 21 days after the DA liver was replaced by a new PVG liver. Immunohistochemistry on sections of spleen from re-OLT rats showed that the level of migratory cells expressing DA class I correlated with the findings obtained by immunoblotting. While the DA-derived antigen and cells were detected in the re-OLT recipient, the DA-specific inhibition of mixed lymphocyte reaction was observed in re-OLT serum. Our results suggest that the implanted DA liver graft was the source of DA soluble class I antigen, but DA-derived antigen and cells detected in the re-OLT recipient organs could persist for a relatively long time under immunosuppression after the implanted DA liver was removed by re-OLT.

Small bowel transplantation (SBT) is thought to be the only radical treatment for short bowel syndrome in childhood. It is very important that the length of the graft and the type of intestine be chosen carefully because this will determine the outcome of transplantation. This model of short bowel syndrome in the rat confirms that total intestinal resection results in malnutrition and failure to gain weight. After 10 cm of ileum was transplanted orthotopically in a syngeneic combination in rats with total intestinal resection, the animals gained weight. The authors determined that 10 cm of terminal ileum is the minimum length required for survival. Second, the immunologic basis of lethal graft-versus-host reaction (GVHR) as it relates to the intestinal graft length was also evaluated. Ileal grafts of 10 cm from LEW (RT1l) rats were implanted heterotopically into (LEW x BN)F1 rats. Ileal grafts of 10 or 40 cm were implanted from BN(RT1n) rats into (LEW x BN)F1 animals. A lethal GVHR always occurred after grafting a 10 cm ileum in the LEW/F1 combination, whereas only 17% of the BN/F1 recipients died of typical GVHR. However, in the latter combination, 67% lethal GVHR was induced when 40 cm of the intestinal graft was implanted. These results indicate that mesenteric lymph nodes are a major source of lethal GVHR, but gut-associated lymphoid tissue can also induce this.

Two models of pancreas transplantation were examined in the rat with a view to choosing one for regular use in functional experiments. A cuff technique applied to the renal vessels of the recipient was used. The histology and endocrine functioning of whole pancreas-duodenal transplant (Tx) plus bladder drainage (drainage model), and the pancreatic duct ligated segmental pancreas Tx (ligated model) were examined. Syngeneic operations were performed using either Lewis or Wistar rats. Streptozotocin (STZ) 60 mg/kg intravenously (i.v.) was used to render the recipient rats diabetic. A cuff technique was used with both models to anastomose the grafts to the renal vessels instead of the conventional technique of hand suturing to the abdominal vessels. This allows a shorter warm ischaemia time for the donor pancreas and leaves the systemic circulation intact leading to a high success rate for both techniques. Operation survival rates were 93% (n = 30) and 90% (n = 10) in the ligated and drainage models, respectively. The recipients in both groups were normoglycaemic for > 100 days. Histological examination revealed atrophic exocrine tissue early in the ligated group but only two from the drainage group showed exocrine atrophy at > 100 days. There was no statistical difference in i.v. glucose tolerance tests with both models showing a normal pattern. Thus, endocrine function remained independent of exocrine function. Both models are quicker than the conventional techniques. The duct ligation model was a simpler transplant to perform, suggesting that this should be the technique of choice in future experiments.

50 open thymectomies were performed in adult rodents using intubation combined with a fibrin glue able to prevent hemorrhage and pulmonary air leakage. This method had a 100% success rate and lower mortality than the ordinal suction procedure. Although the conventional suction thymectomy has been widely used, the open thymectomy method would permit more complete thymectomies for immunological studies.

Despite several technical improvements, orthotopic liver transplantation (OLT) in the rat remains the most difficult experimental microsurgical transplant to perform. The short anhepatic phase tolerated by the rat makes it difficult for beginners to perform the suprahepatic and portal vein anastomoses in the limited time available. To overcome this obstacle, we examined the ability of a portosystemic shunt, induced previously by subcutaneous transposition of the spleen (STS), to decompress the portal system during hepatic pedicle clamping and thus prolong the anhepatic phase of OLT in the rat. Effective drainage was shown by the ability of rats subjected to STS 3 weeks previously to survive portal pedicle clamping of 120 min, while normal rats all died after 90 min of clamping. We demonstrated that, in STS rats, OLT performed with an anhepatic phase of 1 hr had 100% survival at 1 week, while this procedure caused 100% death in normal rats. The ablation in STS rats of the drastic pH decrease and appearance of endotoxins seen in the portal blood of normal rats subjected to "OLT-like" clamping corroborated these results. This application of the STS model to the field of OLT has already proved in our laboratory to be extremely useful during the surgical learning phase of liver transplantation in the rat.

In the rat combination of DA (MHC haplotype RT1av1) donor into PVG(RT1c) recipient, liver grafts are not rejected and allow the acceptance of other organ grafts from the same donor strain. Here we show that an existing DA liver graft allows the acceptance of a DA small bowel graft in the PVG; the liver graft also prevented the graft-versus-host reaction normally associated with small bowel grafting. In addition, a liver graft could suppress the GVHR in F1 hybrid recipients of parental lymphoid cells, a classic GVHR model. GVHR suppression was immunologically specific and required that donor lymphocytes and liver be of the same strain. Besides their clinical implications, these results demonstrate the capacity of the liver for tolerance induction and suggest that it may play a physiological role in negative selection of T cells.

Although small bowel transplantation (SBT), or pancreas-spleen transplantation (PST) often lead to lethal graft-versus-host reaction (GVHR) in experimental animals, fatal GVHR is rare after clinical liver transplantation. This study describes a modified model of SBT and PST in the rat using cuff techniques applied to the renal artery and vein of the recipient. The ability of LEW (RT1(1)) or BN (RT1n) lymphocytes accompanying intestinal, splenic, or hepatic grafts to induce lethal GVHR in (LEW x BN) F1 hybrid recipients was compared. SBT and PST experiments showed that lethal GVHR always occurred in LEW-into-F1 combination, but was much less frequent in BN-into-F1 SBT. In mixed lymphocyte reaction (MLR), LEW mesenteric or splenic T cells showed significantly higher proliferative responses against BN stimulators than did BN mesenteric or splenic T cells against LEW. Adoptive cell transfer experiments using mesenteric or splenic cells also showed that LEW cells were higher responders than BN. In contrast with SBT and PST results, a lethal GVHR was not induced after liver or pancreas grafting alone in either parent-to-F1 combination. In MLR, hepatic T cells from either parent failed to elicit a proliferative response against allostimulators. These results indicate that the occurrence of lethal GVHR is dependent upon the reactivity of parental lymphocytes against allo-antigenicity of F1 hybrids and also upon the lymphoid tissue transplanted. The lack of alloreactivity of hepatic T cells accounts for the absence of lethal GVHR after liver grafting.

The prior transfusion of heat-treated (60 degrees C for 1 hr) allogeneic spleen cells is known to bring about specific prolongation of the survival of subsequent donor strain heart allografts. In this communication we show that some polymorphic and monomorphic class 1 determinants on spleen cells are heat denatured so that they no longer provoke antibody formation in naive allogeneic hosts. By contrast, the heated cells remain able to provoke the formation of anti-class 2 antibodies. When measured in a binding assay, the levels of anti-class 2 antibodies are similar irrespective of whether the immunizing inoculum consists of normal or heated cells. In a cytotoxic assay, the antibodies produced following exposure to normal cells are cytotoxic; this activity is substantially reduced when the cellular immunizing inoculum is heated. Cells heated to 60 degrees C for 1 hr are unable to stimulate in a mixed lymphocyte reaction, but reactivity can be partially restored by the addition of exogenous IL-2 to the culture. From previous evidence it seems unlikely that suppressor cells play a major role in the immunosuppression effected by cells heated to 60 degrees C. The results presented in this communication suggest a possible role for anticlass 2 antibodies and also imply the defective production of a costimulatory signal that normally follows the presentation of allogeneic MHC antigens.

Adult male recipients could accept allogeneic skin graft with CyA treatment alone for considerably long periods, whereas young male and female, and adult females were not susceptible to the immunosuppression. When testosterone or prednisolone was administered with CyA as a combination therapy to young male and adult female recipients, the survival time of the graft was significantly prolonged. On the contrary, estradiol totally inhibited the immunosuppressive activity of CyA in any recipients. These present data strongly suggested that immunoreactivity against transplantation antigens can be influenced by sex steroid hormones, and that the usage of steroid combination therapy may overcome the lack of immunosuppression by CyA induced by the female sex steroid estradiol.

Three-week-old DA (RT1a) male and female rats were pretreated with either orchiectomy or ovariectomy and administration of the opposite-sex steroid hormones, estradiol or testosterone. Skin from same-sex AO (RT1a) rats was then grafted when these pretreated rats reached 14 weeks of age, with a short course of immunosuppressive treatment of cyclosporine. The survival times of the grafts were reversed in that the male recipients pretreated to be like females acutely rejected the graft within 10 days as do normal adult female recipients. On the other hand, the female recipients pretreated to be like males accepted the graft as do normal adult male recipients. In addition, the synergistic effects of either testosterone or estradiol with CsA on the survival time of the graft were examined. Testosterone successfully prolonged graft survival on normal adult female and young male recipients, but negligible prolongation was observed on young females. In contrast, estradiol abrogated the immunosuppressive activity of CsA and accelerated graft rejection in both male and female recipients.

As reported previously, only male rats were susceptible to immunosuppression by CsA in allogeneic skin grafts. We have further investigated the sex differences observed in the prolongation of skin allograft survival time, focusing on the role of sex-related organs. Adult male DA (RT1a) rats (14 weeks old), which had been preorchiectomized at 5 weeks old or just 2 days prior to the operation, had fully allogeneic skin grafts from male AO (RT1u) rats, followed by CsA treatment. A small but significant reduction of graft survival time was observed in the recipients orchiectomized when fully mature. On the other hand, adult rats preorchiectomized while immature (five weeks old), showed a graft survival time similar to that of the comparable sham-operated group. DA female rats with syngeneic or allogeneic pregnancies always rejected AO skin, even in the course of CsA treatment, without showing any differences in graft survival time compared with nonpregnant rats.

A single intraperitoneal dose(1 ml/kg body weight) of carbon tetrachloride (CCl4) caused a rapid and drastic induction of choline kinase activity in rat liver cytosol. The administration of either cycloheximide or actinomycin D completely blocked the CCl4-mediated induction of choline kinase activity, indicating that the elevated activity could be due to the change in the enzyme level. The pretreatment of rats with phenobarbital did not cause any significant effect on hepatic choline kinase induction by CCl4, suggesting that the induction may not be directly related to the metabolic rate of CCl4. A considerable part of induced form(s) of choline kinase appeared not to be a form present in the liver of untreated rats. The contribution of adrenals to the CCl4-mediated hepatic choline kinase induction could be ruled out.

Focusing on sex-difference in prolongation of allograft survival time, we have performed skin grafts between fully allogeneic rat strains, AO (RT1u) and DA (RT1a) with an immunosuppressant, cyclosporine. Isografted skins survived indefinitely, whereas allografts were severely rejected at days 7-9 without immunosuppressive treatment. When adult male DA rats received CsA (15 mg/kg/day, i.m., for 14 days postoperatively), allogeneic skin was accepted from either male or female AO rats for 38.8 +/- 20.5 days (mean survival time [MST] +/- SD) and for 44.7 +/- 43.3 days (MST +/- SD), respectively, with normal hair growth at around day 17. Additional CsA administration every 5 days after the initial short course treatment was also effective in preventing chronic rejection. Male AO rat skin grafted onto adult male DA rats survived for over 50 days as long as the treatment was carried out. In contrast, when adult female DA rats were used as recipients, only a few days' prolongation was observed in comparison with a non-treated group. The rejection always occurred, even during the initial course of treatment (MST +/- SD): 10.9 +/- 1.6 days). Younger male DA recipients, 5 and 10 weeks old, rejected AO skin within a shorter time, depending on the age. The maximal graft survival was observed when male adult rats more than 14 weeks old were used as recipients. On the other hand, the CsA serum level of female recipients at day 14 was considerably lower than that of males. However, even when the level of females was adjusted to that of males by the administration of a double dosage (30 mg/kg/day), the female recipients consistently rejected the skins (MST +/- SD: 14.5 +/- 1.9 days). Therefore, these results clearly indicate that this male-associated immunosuppressive effect depends upon the sex and age of the recipient animals.

Fractionation of yeast mitochondria by controlled hypotonic treatment revealed that the enzyme for heme attachment to apocytochrome c was localized in mitochondrial inner membrane. Trypsin digestion of mitoplasts resulted in a considerable loss of enzymatic activity, whereas the enzyme in intact mitochondria resisted the digestion. Triton X-100 solubilized the enzyme from the membrane but high concentration of salt did not. These results reveal that the enzyme for heme attachment is localized in mitochondrial inner membrane facing the cytoplasmic surface.

A method for assaying the covalent attachment of heme to apoprotein of cytochrome c was developed. 125I-labeled apoprotein was chemically prepared from 125I-labeled yeast cytochrome c (iso-1-cytochrome c). After incubation of 125I-apocytochrome c with yeast mitochondria, the product was extracted with Triton X-100, digested with trypsin in the presence or absence of a reducing agent, and then precipitated in trichloroacetic acid. The resulting precipitates were collected on nitrocellulose membranes and counted for radioactivity. The radioactivity correlated well with the appearance of a heme-containing peptide in the trypsin digested peptide fragments of cytochrome c. This procedure is simpler and faster than the previously reported methods.

The effect of CHCl3 on the composition of hepatic microsomal cytochrome P450 species was compared with that of CCl4 in rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3MC). The administration of CHCl3 hardly affected cytochrome P450 content in non-treated rat liver, but caused a similar degree of depletion in the content as observed after CCl4 administration in PB-pretreated rats. In the pretreatment with 3MC, the administration of CHCl3 brought about a marked decrease in the content to 24% of control after 12 hr, while CCl4 reduced the content only to one-half of control. It was demonstrated by SDS-polyacrylamide gel electrophoresis and Whatman DE-52 anion-exchange chromatography that 3MC-induced P450 species decreased with CHCl3, while it was affected little by CCl4 treatment. The activity of benzo[a]pyrene hydroxylase was altered together with the change in the content of cytochrome P450 species. The administration of CHCl3 to PB-pretreated rats caused the depletion in PB-induced P450. These findings indicate that cytochrome P450 species induced with 3MC as well as PB are highly susceptible to CHCl3 intoxication, whereas the administration of CCl4 depletes the PB-induced species without affecting the 3MC-induced species.