Markers of operational immune tolerance after pediatric liver transplantation in patients under immunosuppression.

A prospective identification of the estimated 20-50% of pediatric LTX recipients developing operational tolerance would be of great clinical advantage. So far markers of immune tolerance - T-cell subpopulations or gene expression profiles - have been investigated only retrospectively in successfully weaned patients. Fifty children aged 8-265 months (median 89) were investigated 1-180 months (median 44) after LTX under ongoing immunosuppression. T-cell subpopulations were measured during regular post-transplant visits using FACS (Vδ1- vs. Vδ2-γδ-T cells and Tregs). A Vδ1/Vδ2-γδ-T-cell ratio ≥1.42 previously reported in operational tolerance was found in 12 of 50 (24%) patients. In analogy, a Treg count ≥44 per µL was found in 35 of 50 (70%) patients and a Treg proportion ≥2.23% of CD3(+) -T cells in 39 of 50 (78%) patients. Only 9 of 50 patients (18%) fulfilled both criteria. The parameters Vδ1/Vδ2-γδ-T-cell ratio and Tregs were not significantly correlated to each other or with donor type or immunosuppression. Vδ1/Vδ2-γδ-T-cell ratio was more stable in serial examinations compared with Treg analyses. The observed proportion of 18% pediatric LTX patients with potential operational tolerance is in accordance with previous reports. However, clinical experience shows that rejections may happen even after long-time weaning of immunosuppression. This suggests that operational tolerance is a dynamic process, with uncertain prediction by Vδ1/Vδ2-γδ-T-cell ratio and/or Tregs under immunosuppression.

Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function.

In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in pancreatic tumor cells.

Audit of a series of 40 gastrointestinal stromal tumour cases.

AIMS: To analyze prognostic factors influencing survival and tumour recurrence after resection of gastrointestinal stromal tumours. METHODS: Forty patients who underwent surgery for a GIST at our institution were reviewed. Patients were classified on the basis of tumour size, mitotic rate and CD117 positivity. The overall survival and disease free survival were calculated using Kaplan-Meier method considering the extent of surgery comparing local tumour excisions with segmental organ resections. RESULTS: Tumours were localized in the oesophagus, stomach, duodenum, small bowel and large bowel and rectum. Sixty-five percent of the patients had an intermediate or high risk GIST according to tumour size and mitotic count. In 26/40 patients tumour resection was performed using segmental organ resection, in all other patients local tumour excision was carried out. The mean overall survival was 73 months. Disease free survival was significantly better after local tumour excision compared to segmental organ resection (73 months versus 53 months; p=0.05). Large tumour size (p=0.07) and high mitotic count (p=0.14) were negative prognostic factors for disease free survival, although statistical significance was not reached yet. CONCLUSION: Primary surgery remains the cornerstone in the treatment of primary and recurrent GIST. Risk adapted surgery is the most important factor to avoid early tumour recurrence. In case of small tumour size segmental organ resections can be avoided favouring local tumour excisions with a low risk of tumour recurrence.

Different in vivo tolerogenicity of MHC class I peptides.

The efficacy of MHC class I-derived peptides to induce tolerance was tested in a cardiac transplantation model. Two 25-mer peptides from the polymorphic region of the DA class I molecule (RT1.Aa) were synthesized by F-moc chemistry and injected intrathymically or intraperitoneally into LEW (RT1.1) responder animals. Intrathymic treatment of the recipient animals with peptide 1 (residues 56-80) accompanied by intraperitoneal treatment with peptide 4 (residues 96-120) led to indefinite survival of allogeneic DA cardiac allografts (n = 7; > 100 days). The tolerogenicity of both peptides differed according to the site of inoculation, as donor-specific tolerance was only observed after administration of peptide 1 into the thymus and injection of peptide 2 into the abdominal cavity of LEW recipients, but not vice versa. Donor-specific tolerance was confirmed in vivo by grafting of full-thickness skin and in vitro by appropriate proliferation and cytotoxicity assays using donor and third-party rats. Donor-specific tolerance was associated with up-regulation of interleukin-4, transforming growth factor beta, and interleukin-10 gene expression within cardiac allografts, thus suggesting intrathymic clonal deletion and external suppression with expansion of T-helper 2-type lymphocytes as the underlying mechanisms of tolerance induction.

Oral feeding of an immunodominant MHC donor-derived synthetic class I peptide prolongs graft survival of heterotopic cardiac allografts in a high-responder rat strain combination.

The efficacy of two synthetic major histocompatibility complex (MHC)-derived DA (RT1.Aa) 25-mer peptides (residues 56-80 and 96-120) to modulate alloreactivity was tested in Lewis (RT1.A1) responder animals. The DA peptide 56-80, but not peptide 96-120, induced delayed-type hypersensitivity (DTH). DTH was significantly reduced by oral feeding of peptide 56-80, P = 0.004. In addition, oral feeding of this peptide in combination with a short course of cyclosporin A (CsA) prolonged graft survival of 60% of heterotope transplanted DA cardiac allografts in Lewis recipient rats. Long-term survivors developed low levels of allo-antibodies against donor tissue as compared to rejecting animals and increased levels of interleukin-4 (IL-4) within the allograft. Similarly, IL-4-secreting splenocytes were identified by flow cytometry in these animals, indicating a Th2-type cytokine pattern. However, graft survival was particularly limited to cardiac allografts because donor-type skin grafts were acutely rejected in tolerant animals. It is interesting that residue alignment of peptide 56-80 to the motif of the RT1.A1 molecule showed a preferred class I motif within this sequence, suggesting indirect presentation of this peptide to recipient T cells. Thus, peptide 56-80 appears to represent a dominant epitope that can be exploited for establishing tolerance in this transplantation strain combination.

IL-2 antagonists: the European perspective.

Acute rejection remains the main risk factor following intestinal transplantation. New immunosuppressive agents have substantially reduced the incidence of severe acute rejection. The question arises, which is the most powerful immunosuppressive combination with the lowest incidence of side effects? According to International Intestinal Transplant Registry data, anti-IL-2 antibodies are slightly advantageous compared with antilymphocyte preparations with respect to long-term patient survival. However, different antilymphocyte preparations are used in different doses and at different time points. The anti-IL-2 antibodies daclizumab and basiliximab were also used in different protocols. Therefore, final results on efficacy are awaited. The most important difference between IL-2 antibodies and antilymphocyte preparations is the suppression of CD4+ CD25+ T lymphocytes by anti-IL-2 antibodies. Antilymphocyte preparations do not affect CD4+ CD25+ T cells. Because regulatory CD4+ CD25+ T cells are essential for tolerance induction, protocols attempting tolerance may omit anti-IL-2 antibodies in the future.

Migration and activation pattern of specialized dendritic cells after heterotopic small bowel transplantation in a graft-versus-host model of the rat.

Besides specific cellular-mediated T cell responses, B cell related humoral responses have been demonstrated during the course of graft-versus-host disease after semiallogeneic transplantation of cellular antigen. Following semiallogeneic small bowel transplantation, there are, besides others, two specific forms of antigen-presenting cells, namely sinus lining cells (SLCs) and follicular dendritic cells (FDCs) which mediate primary and secondary humoral immune responses, respectively. This study was aimed to clarify the role of these dendritic cell entities after transplantation of small bowel grafts in a one-sided graft-versus-host (GvH) model for untreated and immunosuppressed (15-deoxyspergualin) recipient animals. As graft-versus-host disease progressed, SLCs and FDCs were eliminated in donor and recipient graft-versus-host associated target tissues (spleen and mesenteric lymph nodes) of untreated animals, whereas these dendritic cells prevailed in immunosuppressed recipients. 15-deoxyspergualin successfully prevented GvHD and significantly prolonged the mean survival time of untreated rats (16.0 +/- 4.5 d) for at least 21 d. Based on the immunosuppressive efficacy of 15-deoxyspergualin on the survival and function of SLCs and FDCs, an unaltered development of germinal centers and B cell proliferation within mesenteric lymph nodes and spleen was maintained

Donor-derived soluble MHC antigens plus low-dose cyclosporine induce transplantation unresponsiveness independent of the thymus by down-regulating T cell-mediated alloresponses in a rat transplantation model.

BACKGROUND: In vitro, soluble MHC (sMHC) antigens modulate and induce apoptosis in alloreactive and antigen-specific T cells, demonstrating their potency to regulate T cell-mediated immune responses. However, their efficacy to regulate immunological responses in vivo remains unclear. Here, we report that repetitive intraperitoneal injection of recombinant Lewis rat-derived MHC class I antigens in Dark Agouti (DA) rats modulates alloreactivity. METHODS: RT1.A1 (Lewis derived) genes were cloned into mammalian expression vectors, and RT1.Aa (DA derived) genes were used to transfect a rat myeloma cell line. RT1.A1 molecules were injected intraperitoneally in DA recipients that subsequently underwent transplantation with Lewis-derived cardiac allografts. RESULTS: Soluble class I antigens were secreted by the transfected cells and were shown to be heterodimeric, peptide-loaded, and conformationally folded. Injection of donor-derived soluble MHC significantly reduced the ability of recipient animals to mount a cytotoxic T-cell response to donor-derived tissue. More interestingly, this treatment significantly prolonged donor-graft survival and allowed 60% of treated animals to develop graft tolerance (>120 days), when donor sMHC were combined with a single subtherapeutic dosage of cyclosporine. Thymectomy of recipient animals before transplantation did not interfere with induction of peripheral tolerance. CONCLUSIONS: Donor-derived sMHC are potential tolerogens for down-regulating the cytotoxic T-cell response of animals that undergo transplantation. Thus, these data provide for the first time a rationale for the application of directly injected sMHC in vivo to down-regulate immunological responses and aid the induction of graft tolerance.

Co-transplantation of donor-derived hepatocytes induces long-term tolerance to cardiac allografts in a rat model.

BACKGROUND: Liver allografts transplanted between MHC-disparate mice, rats, and swine are spontaneously accepted in most strain combinations without requirement for immunosuppression. The underlying mechanism has, however, remained elusive. Here, we demonstrate that co-transplantation of donor-derived hepatocytes protect Lewis (RT1.A1) cardiac allografts from acute and chronic rejection in DA (RT1.Aa) recipients indefinitely. METHODS: Livers of donor Lewis rats were harvested and the hepatocytes separated from hepatic leukocytes by collagenase digestion and gradient separation. DA recipient animals were transplanted Lewis cardiac allografts and simultaneously intraportally infused either Lewis-derived hepatocytes or hepatic leukocytes. Recipient animals were either not further treated or received a single dose of 15 mg/kg cyclosporine. RESULTS: Donor hepatocytes alone significantly protected syngeneic cardiac allografts from rejection, whereas hepatic leukocytes failed to influence graft survival. In combination with cyclosporine, recipient cardiac allografts were indefinitely protected from rejection. Graft-infiltrating cells in tolerant animals presented as clusters of CD4+ T cells and stained mostly positive for interleukin-4, whereas graft-infiltrating cells in rejected allografts were predominantly positive for interferon-gamma. Adoptive transfer of splenocytes derived from tolerant animals protected Lewis cardiac allografts from rejection in DA recipients without immunosuppression. In contrast, hepatic leukocytes protected only 50% of the allografts from rejection. CONCLUSION: We propose that donor hepatocytes induce permanent engraftment of syngeneic allografts by establishing a Th2 type alloresponse that is transferable to new graft recipients. The results of this study demonstrate that liver parenchymal cells significantly mediate spontaneously liver-induced tolerance.

Glutamine dipeptides-supplemented parenteral nutrition reverses gut mucosal structure and interleukin-6 release of rat intestinal mononuclear cells after hemorrhagic shock.

Glutamine is an essential substrate for gut mucosal structure, but the role for gut immune function is not fully known. To determine the effect on gut cytokine release in relation to bacterial translocation and gut morphology, a nonlethal hemorrhagic shock (30 min, 30 mmHg) was performed in male Wistar rats followed by 4 days of different way of feeding. A conventional total parenteral nutrition (TPN) solution was compared with an isocaloric and isonitrogenous TPN solution supplemented with alanin-L-glutamine and glycyl-L-glutamine. An enteral chow-fed control group was included. Gut mononuclear cells and splenic macrophages were obtained and endotoxin-induced supernatant tumor necrosis factor-alpha (TNF) and interleukin-6 (IL-6) bioactivity was measured. Histological specimen of the small bowel were taken and mesenteric lymph nodes (MLN) were separated. Enteral feeding following hemorrhagic shock was accompanied by a normal mucosal structure and no bacterial translocation could be detected. TPN was characterized by suppression of cytokine release in gut mononuclear cells and splenic macrophages compared with the enteral-fed control (p < .05). Decreased TNF and IL-6 release was associated with a significantly increased mucosal injury score (p < .05) and a high incidence of bacterial translocation to MLN (66%, p < .05 vs. control). Supplementation of glutamine-dipeptides did not prevent TPN-induced bacterial translocation to MLN (p < .05 vs. control) but significantly improved mucosal injury (p < .05 vs. TPN). Down-regulation of TNF release in TPN-fed rats could not be reversed by glutamine dipeptides while IL-6 release was significantly increased compared with TPN-fed animals (p < .05), and no difference to enteral-fed controls could be found. Enteral nutrition following hemorrhagic shock is superior to parenteral nutrition with regard to mucosal structure, cytokine release, and bacterial translocation. Supplementation of TPN with glutamine dipeptides could reverse TPN-induced suppression of IL-6 release and improved mucosal structure, which may be beneficial in various disease conditions in which TPN is an integrated part of patients management.

WOFIE augments the immunosuppressive potency of FK-506. Window of opportunity for immunological engagement.

UNLABELLED: Bidirectional recognition of donor- and recipient-derived immunocompetent cells has been proven to play a pivotal role for the induction of long-term unresponsiveness to allogeneic grafts. This study investigated the fate of heterotopic heart grafts with respect to the timing of subtherapeutic doses of FK-506 and with respect to the time point and type of donor antigen application, leaving space for mutual adaptation of alloreactive lymphocytes, designated as the 'WOFIE-concept' (window of opportunity for immunological engagement), originally described by R Calne. METHODS: Heterotropic heart transplantation was performed using male DA (RT1.a) donor and LEW (RT1.1) recipient rats in the following groups (n = 6). FK-506 was applied intramuscularly (i.m.) using doses of 2 mg/kg x body weight per day. Donor antigen application was performed either by DA blood transfusion, 2 ml intravenously (i.v.), or by i.v. transfusion of 5 x 10(7) DA splenocytes. (i) LEW --> LEW, untreated; (ii) DA --> LEW, untreated; (iii) DA --> LEW, FK-506 days 0, 4-7; (iv) DA --> LEW, FK-506 as group (iii) plus 2 ml of DA blood 6 h post-Tx; (v) same as group (iv) but DA blood transfusion 24 h post-Tx; (vi) DA --> LEW, FK-506 as group (iii) plus DA splenocytes 6 h post-Tx; (vii) same as group (vi) but DA splenocyte transfusion 24 h post-Tx; (viii) DA --> LEW, FK-506 days 0-4 and (ix) DA --> LEW, FK-506 as group (viii) plus DA blood 6 h post-Tx. Immunohistochemical stainings (APAAP-method) of the allografts and flow cytometric analysis of recipient spleens were performed electively 3, 7 and 14 days after organ reperfusion. RESULTS: The mean graft survival differed significantly between groups and comprised (mean +/- SD days): (i) >100, (ii) 6.5 +/- 1.0, (iii) 31.6 +/- 12.1, (iv) 44.8 +/- 10.1, (v) 29.8 +/- 14.2, (vi) 27.2 +/- 4.7, (vii) 14.6 +/- 4.2, 17.5 +/- 4.2, (viii) 17.5 +/- 4.2 and (ix) 18.8 +/- 2.8 days. Prolongation of graft survival and long-term unresponsiveness (group iv) revealed a substantially different pattern of graft infiltration. CONCLUSIONS: Effective treatment with unspecific immunosuppressants like FK-506 can be substantially improved if (i) mutual antigen recognition between donor and recipient immunocompetent cells is warranted, (ii) donor-derived blood-borne antigens are given immediately after graft reperfusion, and (iii) the type of inoculated donor antigen has a strong impact on graft survival as splenocytes which contain a large population of professional antigen-presenting cells failed to prolong graft survival after interrupted FK-506 treatment.

The role of natural killer cell mediated caspases activation in a graft-versus-host disease model of semiallogeneic small bowel transplantation.

In the clinical setting of solid organ transplantation the event of graft-versus-host disease (GvHD) is rare and not easily predictable. Even intestinal and multivisceral transplants harbour a huge amount of immunocompetent cells and they do not exert a significantly higher risk to trigger serious GvH reactions. A series of our own experimental studies has been conducted to delineate the role of the host's innate immune system in the context of GvHD following parental to F1 hybrid semiallogeneic small bowel transplantation (SBTx). These results clearly demonstrated the immunological significance of the recipient's status of natural killer (NK) cell activity to counteract donor-derived lymphocytes and related cytotoxicity. NK cells and macrophages are both endowed with Ca2+-dependent receptors of the C-type lectin family which interact with a diversity of high-affinity oligosaccharide ligands expressed on potential target cells. One of these proteins of the C-type lectin family, termed NKR-P1, has been cloned and sequenced. Activation of NKR-P1 stimulates activation-induced cell death (AICD) of bound target cells. As intracellular mediators of apoptotic cell death a new family of cysteine proteases, the caspases, have been defined. These proteases appear to be involved in the initiation of apoptosis in response to a number of stimuli. This study was conducted to investigate the impact on the activity level of host NK cells and on target cell lysis of donor-derived lymphocytes after heterotopic semiallogeneic (parental [DA;RT1.aaav1] to F1 [DA x LEW;RT1.(1)]) small bowel transplantation using a rat model. The host's NK activity was either specifically activated (by use of polyinosinic:polycytodilic acid [poly-I:C]) or suppressed (by depletion of host NK cells after intraperitoneal administration of the NKR-P1 monoclonal antibody 3.2.3). The impact of NK-activity on the incidence of GvHD and the recipients' survival was correlated with the frequency of apoptotic cell death and related expression of caspases 1 (ICE) and 3 (CPP-32) from donor and recipient small bowel tissues. Our results confirm that depletion of NK cells in F1 host rats prior to parental small bowel transplantation significantly decreased the mean survival to 11.4 days versus 16.2 days of nondepleted F1 rats (p < 0.01). Conversely, activation of host NK activity with poly-I:C abrogated GvHD in all 12 recipient rats and led to long-term survival in seven of 12 animals. Long-term survival was associated with a substantially higher frequency of apoptotic cell death in donor and recipient small bowel and mesenteric lymph nodes. On day 10 after transplantation, Northern blot analysis of these tissues revealed profound upregulation of mRNA-specific gene expression for caspase 1 and 3 as potential mediators of programmed cell death of activated lymphocytes. Our findings emphasize the importance of NK cell associated innate immunity in the context of GvHD after semiallogeneic small bowel transplantation. Killing of alloreactive donor-derived lymphocytes was mediated by the NKR-P1 protein on NK cells and could be suppressed after pretreatment of F1 hosts with anti-NKR-P1 mAb 3.2.3. Moreover, NK cell-mediated apoptosis induced upregulation of caspases 1 and 3, thus elucidating the involvement of this protein in the context of caspase-mediated target cell killing.

Long-term survival after curative resection for carcinoma of the rectum.

The clinical outcome of 453 patients with histologically confirmed adenocarcinoma surveyed from 1980 to 1992 was evaluated. Special consideration was given to the prognostic significance of local recurrence and distant metastases as the significant contributors to postoperative morbidity and mortality. Of 453 patients, 371 were treated by a curative surgical approach. The remaining 82 patients, with extended disease, received palliative treatment. Among those undergoing surgical treatment, the local recurrence rate was 11.3 percent; the incidence of distant metastases was 16.2 percent, and 2.7 percent of the patients exhibited combined lesions. The five-year survival rate depended on the extent of the primary lesion and lymph node status--it was highest for patients with stage I adenocarcinoma (80 percent), a rate significantly better (p < 0.01) than patients with stage II disease (40 percent), who fared better (p < 0.02) than patients with stage III disease (30 percent) (Union Internatinale Contre le Cancer [International Union Against Cancer] classification). Sixty percent of the patients with local recurrence and almost 70 percent with distant spread showed proof of failure within two years. Of 42 patients with local failure, 12 underwent reoperation without leaving residual tumor (RO-treatment) but exhibited no improvement in five-year survival compared with those with no second surgical approach. The operative techniques were abdominoperineal resection (36.9 percent), low anterior resection (58.2 percent) and transanal resection (4.9 percent). They were without significant influence on long term results. Critical analysis of the data emphasizes the urgency of adjuvant treatment for patients with poor long term prognosis, as given for stages IIB and III.

Glutamine dipeptide-supplemented parenteral nutrition reverses gut atrophy, disaccharidase enzyme activity, and absorption in rats.

BACKGROUND: Total parenteral nutrition (TPN) is associated with intestinal atrophy and dysfunction possibly attributed to the absence of the nonessential amino acid glutamine from commercially available TPN solutions because of the instability of the monoamino acid during heat sterilization and storage. The use of stable dipeptides may overcome this problem. In this study we tested the hypothesis that glutamine dipeptide supplementation with alanyl-L-glutamine during TPN for 10 days would reverse small bowel atrophy and TPN-induced dysfunction in rats. METHODS: A conventional TPN solution (250 kcal/kg bw) was compared with an isocaloric and isonitrogenous TPN supplemented with alanyl-L-glutamine dipeptide. A food-fed control group was included (n = 6 each group). Jejunum mucosal architecture, absorption of water and glucose, and disaccharidase activity of maltase and alkaline phosphatase were evaluated. RESULTS: TPN-induced villous atrophy, significantly reduced absorption rate, and decreased activity of villous enzymes, compared with the TPN group, could be reversed by supplementation of glutamine dipeptide alanyl-L-glutamine to parenteral nutrition solutions with no difference to the control group. CONCLUSIONS: Glutamine dipeptide-enriched parenteral nutrition preserves mucosal structure and reversed atrophy-associated dysfunction.

Xenobiotic metabolizing enzyme activities in isolated and cryopreserved human liver parenchymal cells.

Liver parenchymal cells (hepatocytes) of human organ donors were isolated using a two-step collagenase perfusion technique. The average viability of the freshly isolated liver parenchymal cells, as judged by trypan blue exclusion, was 82% (SD = 7%; n = 6). The inter-individual differences in the determined enzyme activities were less than a factor of 7.5, despite the different sexes and ages of the donors. Freshly isolated parenchymal cells (PC) were cryopreserved using a computer-controlled freezing protocol. After thawing, cryopreserved cells had a mean viability of 57% (SD = 18%; n = 6). The activities of xenobiotic metabolizing enzymes in freshly isolated and cryopreserved cells were compared using PC from two donors. The enzyme activities of phenol sulfotransferase, 1-naphthol UDP-glucuronosyltransferase and microsomal epoxide hydrolase were well maintained after thawing (87-117% of activities in freshly isolated cells), whereas the activities of glutathione S-transferase, monitored with the broad spectrum substrate 1-chloro-2,4-dinitrobenzene, and the major broad spectrum cytosolic epoxide hydrolase were moderately but markedly reduced after cryopreservation (34-64% and 45-89% of levels in fresh cells, respectively). The decrease of both activities was dependent on the viability after thawing. When cryopreserved cells were purified by a Percoll centrifugation after thawing, the viability was increased from 62 to 92% for cells from one of the donors and from 88 to 98% for PC for the other donor. Subsequently the activity of glutathione S-transferase in Percoll-purified PC from the two donors was increased to 71 and 96% of levels in freshly isolated cells. It is concluded that the use of cryopreserved liver parenchymal cells of humans and other species represents a valuable tool in predicting which animal species best represents humans in hepatic metabolism and therefore should be the preferred species for investigations of metabolism and metabolism-dependent toxicities.

A novel technique for long-term vascular access in the unrestrained rat.

A new system for long-term vascular access in the unrestrained rat (n = 380) is described that combines low cost with high reliability and free movement. It consists of a polyurethane catheter that is placed in the right atrium via the external jugular vein. A leather harness is applied to the animal and the catheter passed subcutaneously to the neck into the harness. The harness is connected to the outside of a swivel joint via a silicone tube in which the catheter runs. The catheter is connected to the inside of the swivel. The swivel is a modified conventional 5-mL glass syringe and is positioned in ball bearings and a Johnson joint. Swivel, ball bearings, and the Johnson joint are counterbalanced and can move up and down. With this system all catheters functioned well until they were electively removed after 28 days. Four catheter dislocations resulted from harness failure and three from a twisted catheter. Seven cases of septicemia and eight of thrombosis occurred. All animals gained weight (3.53 +- 0.37 [SEM] g/d) during the time the system was in place. The entire apparatus is simple and inexpensive to construct. The rotary portion of the swivel and the Johnson joint avoid twisting of the catheter, and the counterbalance enables the animal to move up and down. The low thrombogenicity of polyurethane further reduced complications. This system reduces stress for the animal during long-term investigations.

Evaluation of amniotic membrane as adhesion prophylaxis in a novel surgical gastroschisis model.

Adhesions are a leading cause of bowel obstruction and infertility. The coverage of peritoneal defects, as in gastroschisis, is still a crucial problem. Despite biodegradable substitutes and synthetic implants such as PTFE membrane, a satisfactory replacement for gastroschisis has not been identified. The amniotic membrane, which is available at birth with a low antigenicity, was evaluated as a peritoneal substitute. Viable, partially viable, and inversed-used amniotic membranes were compared in a rat model. A full-thickness abdominal wall defect was made and the amniotic membrane sutured into this defect. The skin was closed over the amniotic membrane. Reoperation was performed 3 weeks after initial surgery, and the adhesion formation was measured by computerized area calculation. Viable amniotic membrane showed 0 to 3% area adhesion formation, while partially viable (50%) amniotic membrane demonstrated 33% area adhesion formation. Inversed-used amniotic membrane, with the stromal side directed toward the abdominal cavity, showed 70% of the amniotic membrane area to be covered with adhesions. The same amount was found in the control group, in which no substitute was sutured into the defect. This animal model is suitable for the straightforward evaluation of peritoneal substitutes with regard to adhesion formation. It is easy to perform and mimics surgical needs. Viable amniotic membrane proved to be an excellent antiadhesive tissue.

Isolated adult intestinal transplantation using portal venous drainage.

There is some evidence that portal venous drainage may offer immunologic and metabolic advantages in small bowel transplantation. Isolated small bowel transplantation was performed in 14 adult patients. In all cases, the donor pancreas was transplanted into another patient. During the donor procedure, the superior mesenteric artery and vein were separated below the division of the inferior pancreaticoduodenal artery and below the veins of the pancreatic head. An arterial interposition graft was used in all cases. One donor mesenteric artery was reconstructed in 6 patients; two arteries in 5 patients; and three arteries in 3 patients. Proximal arteries of the graft were ligated and the upper part of the jejunum resected. In 10 patients, a direct anastomosis was performed in an end-to side fashion between donor superior mesenteric vein (SMV) and recipient inferior mesenteric vein (IMV). In 2 patients, a branch of the superior mesenteric vein was used and 2 patients required a venous interposition graft to confluence using the donor iliac vein. Patency of the venous anastomosis was documented by magnetic resonance imaging (MRI) angiography after 6 months. No vascular complications have been observed to date. Portal venous drainage is technically feasible in most cases. An anastomosis to the recipient IMV offers the advantage of being direct despite the short donor vein segment. Furthermore, donor and recipient vessels are well matched for size. Using microsurgical techniques, vascular complications may be avoided.

Expression of E-selectin and its transcripts during intestinal ischemia-reperfusion injury in pigs.

BACKGROUND: Ischemia-reperfusion injury (IRI) can result in severe organ dys- or nonfunction. Interaction of leukocytes and endothelial cells mediated by E-selectin appears to be a key step for disturbed microcirculation. Therefore we studied gene and protein expression as well as localization of E-selectin during intestinal IRI. METHODS: Intestinal tissue samples were obtained from extracorporeal perfused intestines (cold ischemia time [CIT] 2 or 20 hours, each n = 5) and additionally in intestinal transplanted pigs (CIT 2 or 20 hours, each n = 1). Mucosal damage was graded according to the Chiu classification. E-selectin mRNA was determined by PCR and quantitative RT-PCR. Localization of E-selectin mRNA was performed by in situ hybridization and of the protein by immunohistochemistry. RESULTS: Histologically, mucosal damage occurred during reperfusion and was earlier and more severe after 20 hours of CIT. E-selectin mRNA expression was detected by PCR already after laparotomy and was elevated after reperfusion. Interestingly, mRNA expression was already increased after 20 hours of CIT. E-selectin mRNA was localized to the luminal surface of muscular, submucosal, and mucosal endothelial cells and the protein was detected on submucosal arterial endothelium as early as 2 hours after reperfusion. CONCLUSION: Prolongation of CIT results in more severe mucosal damage during reperfusion, which is associated with protein expression of E-selection that might be used as a marker for activated endothelial cells. Increased E-selectin mRNA at end of 20 hours of CIT might indicate a preactivated state of endothelial cells potentially triggered by bacterial translocation or products.

Kinetics and localization of interleukin-2, interleukin-6, heat shock protein 70, and interferon gamma during intestinal-rerfusion injury.

BACKGROUND: Intestinal ischemia-reperfusion injury (IRI) represents an exaggerated inflammatory cascade with a complex pathophysiology. IL-2, IL-6, HSP70, and INF-gamma are mediators of the inflammatory process. Therefore, we investigated their kinetics and localization during intestinal IRI. METHODS: Pig intestinal specimens were obtained during cold preservation (cold ischemia time 2 hours) and extracorporeal perfusion. Mucosal damage was graded according to the Chiu classification. MRNA expression was determined by Northern blot (IL-2, IL-6, IFN-gamma) or by quantitative RT-PCR (IL-6, HSP70) and localized by in situ hybridization. RESULTS: Histologically, mucosal damage occurred during reperfusion. Expression of IL-2 mRNA was up-regulated after HTK perfusion and was highest at the start and 7 hours after reperfusion. Expression of IL-6 mRNA increased at 2 hours after reperfusion and HSP70 at 3 hours after reperfusion. IFN-gamma mRNA was expressed after HTK perfusion, with expression of this cytokine increasing to 1 hour after the start of reperfusion, and decreasing thereafter. IL-2 mRNA was localized to endothelial cells (EC) and leukocytes and in close relation to ganglion cells (GC): IL-6 mRNA in EC, smooth muscle cells (SMC), and GC: HSP70 mRNA in EC and SMC; and IFN-gamma mRNA in leukocytes. CONCLUSION: IL-2, IL-6, HSP70, and INF-gamma are parameters of early mRNA expression during intestinal IRI. EC, SMC, leukocytes, and GC have been identified as sources of transcripts that might afford potential targets for intervention strategies to attenuate IRI.