Long-term survival of DLA-matched segmental small-bowel allografts in dogs.

The aim of this study was to investigate the combined effect of DLA matching and immunosuppressive therapy on the survival of segmental small-bowel allografts in dogs. Orthotopic segmental small-bowel transplantations (25 to 30% of total small bowel length) were performed in two stages: first a heterotopic segmental small bowel transplantation, followed after 5 to 8 weeks by a second-stage operation during which the heterotopic graft was placed in an orthotopic position and the native small bowel was resected. All dogs received cyclosporine immunosuppression. Control dogs (n = 4), subjected to total enterectomy, survived 37.3 +/- 7.1 days (mean +/- SEM). Recipients of DLA-mismatched small bowel grafts (n = 6) survived 113.2 +/- 37.0 days, which was a significantly shorter time than dogs with a DLA-matched graft (n = 6, 211.5 +/- 38.8 days, P < 0.05). None of the matched allografts was rejected during CsA treatment, whereas four of six mismatched grafts were (P < 0.05). The control dogs uniformly showed progressive weight loss, steatorrhea, and hypoalbuminemia. The dogs with DLA-mismatched grafts did not regain initial body weight, whereas animals with DLA-matched grafts recovered preoperative weight after 20 weeks. Both transplanted groups showed near-normal fecal fat excretions and constant serum albumin, cholesterol, and triglyceride levels, whereas serum total protein levels increased during follow-up. We conclude that segmental small bowel transplantation between DLA-matched donor-recipient pairs results in long-term survivors with an adequate nutritional status. This may have important implications for future living-related small-bowel transplantation.

Porcine islet cells of Langerhans are destroyed by human complement and not by antibody-dependent cell-mediated mechanisms.

In the near future, xenotransplantation of porcine islets of Langerhans might be an alternative in the treatment of patients with diabetes mellitus. However, xenotransplantation of islets of Langerhans in large animals has been shown to result in an exceedingly short graft survival, which suggests that a humoral immune response might play a major role in islet demise. This study was performed to assess binding human preformed antibodies to isolated porcine islet cells (PIC) and to determine the lysis of PIC using human sera in complement-mediated cytotoxicity (CMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Ten Dutch Landrace pigs were used for the isolation of PIC. Sera from 30 healthy blood donors (1/10 diluted) were used in a 51Cr release assay to assess CMC. Heat-inactivated normal human sera and fresh sera from patients with agammaglobulinemia were used as controls. Binding of human IgM IgG, and IgA antibodies to PIC was tested in an ELISA using isotype-specific secondary monoclonal antibodies ADCC was tested in a 51Cr release assay using normal human sera and sera from newly diagnosed type I diabetics with peripheral blood mononuclear cells as effector cells and PIC as targets. It was found that PIC were recognized by human IgM and IgG preformed antibodies and that fresh human sera had strong CMC activity inducing a percentage-specific PIC lysis of 61 +/- 10% (mean +/- SD) within 60 min. Agammaglobulinemic sera killed 42 +/- 12% of PIC. No significant cytotoxic activity was found in ADCC assays using normal sera or sera from diabetic patients. These results show that all tested human sera lyse PIC via CMC, even in the absence of human antibodies, as concluded from the use of agammaglobulinemic sera. In pig-to-human transplantation, islets may be hyperacutely rejected by antibody-dependent and antibody-independent activation of complement and not by antibody-dependent cell-mediated mechanisms.

Interleukin-6: historical background, genetics and biological significance.

Interleukin-6 (IL-6) is a pleiotropic cytokine previously known as B cell stimulatory factor (BSF-2), interferon-beta 2 (IFN-beta 2), 26-kDa protein, and hepatocyte stimulating factor (HSF). The name IL-6 was proposed when the nucleotide sequences of the cDNAs for these proteins had been determined and the molecules were found to be identical. IL-6 production can be induced by a wide variety of agents in a wide range of cells, although IL-6 gene expression seems to be regulated in a tissue and stimulus specific manner. At least 3 different signal pathways regulate IL-6 gene expression, emphasizing its multiply inducible nature. The currently known activities of IL-6 include regulatory functions in hematopoiesis, immune reactions and acute phase responses. IL-6 appears to be a key member of the IL family; however, it is still poorly understood how IL-6 interacts with other lymphokines within the network. The anti-viral activity of IL-6 seems to be negligible. Elevated IL-6 levels have been found in diseases like rheumatoid arthritis, multiple myeloma and systemic lupus erythematosus. The abnormal expression and dysregulation of IL-6 in certain disorders may be a typical feature of this cytokine, making it the first cytokine that may be directly related to pathogenesis.

Cautionary note on the use of end-labelling DNA fragments for detection of apoptosis.

The gastrointestinal epithelum undergoes a continual process of cell renewal which is partly regulated by apoptosis. The process of growth and differentiation is greatly enhanced in the terminal ileum after massive small bowel resection, a well-established model of intestinal adaptation. We have applied the terminal deoxynucleotidyl transferase (TdT)-mediated UTP nick end-labelling (TUNEL) method to establish the role of apoptosis in intestinal adaption in terminal ileum of control animals after small bowel resection. Healing skin wound and lymph node were used as positive control tissue for apoptotic cells. We report that considerable inter-animal variation was observed in the intestinal tissue. We believe that caution is required in the interpretation of TUNEL staining in intestinal tissue and in its use as a specific marker for apoptosis in this setting.

Segmental intestinal transplantation can be an adequate therapy for short bowel syndrome in growing dogs.

This study was undertaken to investigate whether two-stage segmental small bowel allotransplantation can maintain growth and development of young dogs (16 weeks, 5 to 6 kg) with surgically created short bowel syndrome (SBS). After near-total small bowel resection (group 1; n = 3), irreversible weight loss was noted. After a sham operation (group 2; n = 3), no growth disturbances were found. Major histocompatibility matched small bowel transplantation (SBT) with cyclosporine A as immunosuppressant, was performed in two stages (group 3; n = 7). During the first stage, one meter of jejunoileum from an adult donor was placed as a Roux loop. Four weeks later, the native small bowel was removed and replaced by the graft. Only one dog survived long-term; the dogs died from infectious complications. The addition of selective decontamination of the digestive tract and early gastrostomy feeding (group 4; n = 10) resulted in long-term survival in 60%. Follow-up at 4 months showed that their growth was about 20% compromised compared with that of the sham-operated animals. Functional analysis showed that electrolytes, urea, and D-xylose were normal, but there was an increase in the lactulose:mannitol ratio, fecal fat excretion, and postheparin diamine oxidase release. These results show that under the conditions described, segmental SBT functions sufficiently to treat SBS but does not maintain normal growth.

Follicular development in cryopreserved Common Wombat ovarian tissue xenografted to Nude rats.

The Northern Hairy-nosed Wombat (Lasiorhinus krefftii) is a highly endangered marsupial species and every possible option for sustaining the species needs to be explored. One important approach may be the development of assisted reproductive technologies in the non-endangered Common Wombat (Vombatus ursinus) and Southern Hairy-nosed Wombat (Lasiorhinus latifrons) for application in breeding the Northern Hairy-nosed Wombat. In this study, it was examined whether cryopreserved Wombat ovarian tissue would develop following xenografting to immunologically deficient rats. Ovarian tissue was collected from Common Wombats (n = 3) and cryopreserved as small cortical pieces. After thawing the cortical pieces were grafted underneath the kidney capsule of Nude rats (n = 16). The grafts were recovered at 2, 4, and 10 weeks after transplantation and their gross and histological appearance investigated. Two weeks after grafting (n = 2), the tissue was revascularized and healthy primordial follicles were present. At week 4 (n = 2), some follicular development was present. At week 10, six rats received human chorionic gonadotrophin (hCG) to trigger follicle and oocyte maturation while another six rats were not given any treatment. The administration of hCG did not induce preovulatory follicles and oocyte maturation although type 5 follicles were present in ovarian tissue collected 10 weeks posttransplantation in both treated and untreated groups. This study demonstrates for the first time that Wombat ovarian tissue can survive and function when grafted into immunocompromized rats and that Wombat ovarian follicles can be recruited to growth and development in an ovarian xenograft. This model system has the potential to produce mature oocytes from endangered species for use in assisted reproductive technologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and mature oocytes from non-endangered species for nuclear transfer which may be necessary for the preservation of critically endangered species.

Diamine oxidase: an overview of historical, biochemical and functional aspects.

This article is a review of the historical, biochemical, and functional aspects of the enzyme diamine oxidase (DAO). The amine oxidase DAO, formerly called histaminase, is found in various tissues, but is especially active in the intestinal mucosa. Its function is the oxidative deaminating of several polyamines, essential substances for cell proliferation. DAO is thus a regulating enzyme in rapidly proliferating tissues such a bone marrow and intestinal mucosa. Results from several studies have demonstrated that both ornithine decarboxylase (ODC) and DAO activity rise during adaptive hyperplasia seen after small bowel resection. The ODC-dependent increase in polyamine content and subsequent increase in cell proliferative activity is probably downregulated locally in the villus tip by the increased DAO activity. DAO is normally present in very small amounts in the circulation and its basal plasma levels are positively correlated with the maturity and integrity of the intestinal mucosa. After intravenous administration of heparin, DAO is released from its capillary binding sites in the lamina propria into the peripheral circulation. Measurement of postheparin DAO release enhances its sensitivity and is now extensively studied to assess its value as follow-up or screening test for several enteropathies. Measuring basal as well as postheparin DAO levels has potential relevance following small bowel transplantation. Rejection of the small bowel graft leads to mucosal damage, which could conceivably lead to changes in DAO activity.

Towards understanding the process of intestinal adaptation.

When bowel is lost due to disease or surgery, the residual bowel increases its functional capacity in order to compensate for the loss of absorptive capacity. The success and extent of this adaptive process is critical to recovery. This article reviews the current understanding of the intestinal adaptation. Adaptation is a complex process which as yet is poorly understood and thus it is difficult to develop strategies to enhance the out-come of the adaptation phase. Extensive loss of bowel brings about functional as well as morphologic changes in the remaining intestine. The exact relationship between histologic changes and function is still unknown and some studies show that histologic changes may precede functional changes. Both nonnutritive and nutritive factors have been identified as major stimuli to the adaptive process. The importance of oral intake as a positive stimulus has been demonstrated and the role of diet composition rather than diet complexity being recognized. Recent studies attempt to understand the molecular basis of intestinal adaptation by seeking to characterize the nature of humoral factors involved and to establish alterations in the patterns of gene expression. These new approaches may facilitate the identification of both nutritional and pharmacological methods to manipulate the intestinal adaptation process.

Primary nonfunction of islet xenografts: the role of macrophages.

Primary nonfunction (PNF) of hamster islets occurs when they are transplanted under the kidney capsule of diabetic Lewis rat recipients. PNF is absent if the islets are grafted into the liver. Previous results have indicated that macrophages might be involved in the phenomenon of PNF. To test this hypothesis, two procedures affecting macrophages were employed. First, recipients were pretreated with liposomes containing dichloromethylene disphosphonate (L-MDP), killing macrophages upon phagocytosis. Second, recipients were pretreated with the arginine analogue, L-NA, in order to inhibit the synthesis of nitric oxide (NO), a molecule believed to mediate beta cell dysfunction. Injection of L-MDP into the peritoneal cavity had no effect on PNF. However, co-transplantation of L-MDP with hamster islets under the kidney capsule reduced PNF significantly. Eradication of Kupffer cells with L-MDP did not prolong the survival of islets transplanted into the liver, indicating that acute xenogeneic rejection in this model is not mediated by macrophages. A single injection of L-NA, 3 h before transplantation resulted in complete annihilation of PNF, all recipients became normoglycemic within 1 day and remained so for 1.4+/-0.5 days. These results confirmed the finding that macrophages and NO play a crucial role in PNF of islet grafts.

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.