Prevention of autoimmune recurrence and rejection by adenovirus-mediated CTLA4Ig gene transfer to the pancreatic graft in BB rat.

Type 1 diabetes is the result of a selective destruction of pancreatic islets by autoreactive T-cells. Therefore, in the context of islet or pancreas transplantation, newly transplanted beta-cells are threatened by both recurrent autoimmune and alloimmune responses in recipients with type 1 diabetes. In the present study, using spontaneously diabetic BB rats, we demonstrate that whereas isolated islets are susceptible to autoimmune recurrence and rejection, pancreaticoduodenal grafts are resistant to these biological processes. This resistance is mediated by lymphohematopoietic cells transplanted with the graft, since inactivation of these passenger cells by irradiation uniformly rendered the pancreaticoduodenal grafts susceptible to recurrent autoimmunity. We further studied the impact of local immunomodulation on autoimmune recurrence and rejection by ex vivo adenovirus-mediated CTLA4Ig gene transfer to pancreaticoduodenal grafts. Syngeneic DR-BB pancreaticoduodenal grafts transduced with AdmCTLA4Ig were rescued from recurrent autoimmunity. In fully histoincompatible LEW-->BB transplants, in which rejection and recurrence should be able to act synergistically, AdmCTLA4Ig transduced LEW-pancreaticoduodenal allografts enjoyed markedly prolonged survival in diabetic BB recipients. In situ reverse transcription-polymerase chain reaction revealed that transferred CTLA4Ig gene was strongly expressed in both endocrine and exocrine tissues on day 3. These results indicate the potential utility of local CD28-B7 costimulatory blockade for prevention of alloimmune and autoimmune destruction of pancreatic grafts in type 1 diabetic hosts.

A simple technique for circular-stapled Billroth I reconstruction in laparoscopic gastrectomy.

A simple surgical technique that facilitates circular-stapled Billroth I gastroduodenostomy in laparoscopic distal gastrectomy is described. After standard laparoscopic mobilization of the distal stomach, a small duodenotomy is made just distal to the pyloric ring. The anvil of a circular-stapling device, secured with a Vicryl suture, is introduced via the duodenotomy. The Vicryl suture is advanced anteriorly so that a center rod penetrates the anterior duodenal wall. The duodenum is staple-transected at this point, and the center rod is wrapped with the stapled duodenal stump by approximation of both edges using a suturing device. The circular-stapled gastroduodenostomy then is completed in a standard fashion. The authors have used this technique for three patients, and their early outcomes are promising.

Beneficial effects of pancreas transplantation: regeneration of pancreatic islets in the spontaneously diabetic Torii rat.

AIMS: Type 2 diabetes is characterized by a combination of insulin resistance and pancreatic beta-cell dysfunction. Although pancreas transplantation (PTx) is mainly performed in patients with type 1 disease, both clinical and experimental data have demonstrated that PTx improves insulin sensitivity in type 2 diabetic recipients. However, it remains unclear whether PTx has the potential to induce islet neogenesis in a recipient's native pancreas. METHODS: Nondiabetic 10-week-old and diabetic (defined as blood glucose level >250 mg/dL) 25-week-old (average onset age of diabetes) male spontaneously diabetic Torii (SDT; RT1(a)) rats served as donors and recipients, respectively. RESULTS: In nontreated control SDT rats, beta-cell mass gradually decreased and blood glucose levels progressively increased (>600 mg/dL after 40 weeks of age). In PTx rats, however, the onset of diabetes was significantly delayed (>47.5 +/- 18.2 [graft age] versus 25.2 +/- 3.9 weeks in control rats). On immunohistochemical staining, insulin-secreting islets were observed in the naive pancreata of 40-week-old recipients with PTx (PTx40w), whereas no islets were found in 40-week-old control SDT rats. Moreover, the islets in the native pancreata of PTx40w recipients were located close to ductal structures, and PDX-1 (pancreatic duodenal homeobox-1)-positive cells were more clearly visible. These results indicate the possibility of beta-cell regeneration in the recipient native pancreas by avoiding glucose toxicity under normoglycemic condition achieved by PTx. CONCLUSIONS: Pancreas transplantation has beneficial effects on impaired islet, inducing regeneration in the spontaneously diabetic Torii rat.

Preventing human CD8+ cytotoxic T lymphocyte-mediated cytotoxicity against swine endothelial cells by overexpression of human decoy Fas antigen.

Although the birth of homozygous alpha1, 3 galactosyltransferase gene-knockout pigs raised hopes for an imminent breakthrough in the prevention in the antibody-mediated rejection of pig to human discordant xenotransplants, human CD8(+) cytotoxic T lymphocyte (CTL)-mediated killing may represent a new immunological barrier to long-term survival in xenograft recipients. In this study, we demonstrated that the cytotoxicity of human CD8(+) CTL against swine endothelial cells (SEC) is highly detrimental and mediated at least in part by the Fas/FasL pathway. To prevent this CTL-mediated xenocytotoxicity, we overexpressed the human decoy Fas antigen, which does not contain a death domain in its cytoplasmic region, by means of binding competition with endogenous pig Fas antigen on SEC for the common ligand, human FasL. Furthermore, we generated a membrane-bound form of human FasL that cannot be cleaved by a putative metalloproteinase to produce a soluble form, which was assessed as an inhibitor of CTL cytotoxicity. Both human decoy Fas and membrane-bound FasL were effective to prevent CTL-mediated killing, suggesting that these novel molecules may represent a step forward toward preventing CD8(+) CTL-mediated xenograft rejection. The combined expression of both molecules may be more beneficial to protect xenograft cells.

Synergistic effects on the inhibition of human CD8+ cytotoxic T lymphocytes-mediated killing against xenograft cells by coexpression of membrane-bound Human FasL and decoy Fas antigen.

The principal barrier to the use of pigs as donors to humans is hyperacute rejection mediated by the interaction of alpha-gal abundantly expressed on pig cells and the natural anti-Gal antibody, abundantly produced in humans. This antibody-mediated hyperacute rejection may be overcome by an alpha1, 3 galactosyltransferase gene-knockout pig. However, xenograft cells could be rejected by T cells, especially CD8+ cytotoxic T lymphocytes (CTL)-mediated response, because these elements show great cytotoxicity against xenografted cells. We previously demonstrated that the Fas/FasL pathway is a major contributor to CD8+ CTL function. Furthermore, we sought to prevent this cytotoxicity by overexpression of membrane-bound FasL carrying the deletion at the metalloproteinase cleavage site or by decoy Fas antigen that does not contain the death domain in its cytoplasmic region. To investigate the effects of coexpression of these molecules, we cotransfected both genes into swine endothelial cells (SEC). The double-overexpression effectively prevented CD8+ CTL-mediated killing. Although cotransfectants and single transfectants of either membrane-bound FasL or decoy Fas gene showed similar inhibition of cytotoxicity, the expression levels of decoy Fas in SEC cotransfectants were much lower than those of decoy Fas single transfectants. These data suggest that beneficial effects for prevention of CTL-mediated xenocytotoxicity may be produced by the double expression of these molecules. The overexpression of both molecules on xenografted cells may decrease the innate cellular response to xenografts creating a window of opportunity to facilitate xenograft survival.

Restoration of immune abnormalities in diabetic BB rats after pancreas transplantation. I. Macrochimerism of donor-graft-derived RT6+ T cells responsible for restoration of immune responsiveness and suppression of autoimmune reaction.

Diabetes-prone (DP) BB rats (RT1(u), RT6.1) spontaneously develop insulin-dependent diabetes mellitus (IDDM) and the disease manifestation resembles that in human IDDM. DP rats are immunodeficient with severe T lymphocytopenia due to the absence of T cells expressing the RT6 differential alloantigen, which have immunoregulatory functions. MHC- and non-MHC-compatible Wistar Furth (WF; RT1(u), RT6.2) pancreases were transplanted into DP rats. WF pancreas grafts were destroyed by IDDM recurrence (insulitis), but not by rejection, with a mean survival time of 65.3 +/- 21.7 days. To prevent the recurrence of IDDM in the grafts, monoclonal antibodies to intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 were administered. WF pancreas grafts were indefinitely accepted (>108.0 +/- 26.8 days) in monoclonal antibody-treated DP recipients. The number of T cells was increased and cellular immune responses restored only in the DP rats that had accepted grafts. The increased number of T cells was due to the peripheral appearance of donor-type RT6.2+ T cells, which represented 34.3 +/- 7.0% of total splenic T cells. The cytotoxicity of splenic T cells to WF islet cells was suppressed in the presence of RT6+ T cells in vitro. These findings demonstrated that stable macrochimerism of donor-derived RT6+ T cells could restore the immune responses and prevent the recurrence of IDDM in the DP recipients.

The rejection mechanism of rat pancreaticoduodenal allografts with a class I MHC disparity.

The present study has demonstrated for the first time that PVG.R1 (RT1.AaBcDcCc) pancreatic grafts are rejected by so-called "low"-responder PVG (RT1.AcBcDcCc) recipients with an isolated class I MHC disparity (mean survival time; MST: 21.4 +/- 1.8 days, n = 5), whereas PVG.R1 heart grafts are able to survive indefinitely (MST: > 100 days, n = 5). Splenic CD4+ T cells but not CD8+ T cells from the PVG recipients of PVG.R1 pancreatic grafts show a remarkable proliferative response against donor class I RT1.Aa alloantigens, while only a minimal proliferation is observed in the PVG recipients of PVG.R1 heart grafts or naive PVG rats. Naive PVG rats display an extremely low frequency of IL-2-producing helper T cell precursors (fThp) of 1/40,609 +/- 15,441 against class I RT1.Aa alloantigen. The PVG recipients of PVG.R1 heart grafts have a slightly greater fThp of 1/17,326 +/- 6822. On the other hand, the PVG recipients that rejected PVG.R1 pancreatic grafts show a significantly increased fThp of 1/5030 +/- 3396 compared with those of PVG.R1 heart grafts (P < 0.05) or naive PVG rats (P < 0.01). The frequency of cytotoxic T cell precursors (fTcp) increases slightly in the PVG recipients of PVG.R1 pancreatic grafts (1/1848 +/- 330) compared with those of PVG.R1 heart grafts (1/2215 +/- 2131) or naive PVG rats (1/2476 +/- 585). The size of cytotoxic T cell clones alone does not adequately account for a proliferation sufficient to complete the rejection of pancreatic grafts. The PVG recipients of PVG.R1 pancreatic grafts, but not heart grafts, demonstrate a strong cytotoxic alloantibody response to donor class I RT1.Aa alloantigens. In the study of alloantibodies, IgM is detected mainly in the early phase and IgG in the late phase during the course of pancreatic rejection. It is determined that in blocking studies by FACS analysis these antibodies target class I MHC antigens. These results suggest that cytotoxic T cells do not appear to be responsible for the rejection of PVG.R1 pancreatic grafts in PVG recipients. Rather, the rejection is mediated by CD4+ T cells and complement-fixing antibodies directed at class I MHC antigens.

Generation of nitric oxide as a rejection marker in rat pancreas transplantation.

In clinical pancreas transplantation, no reliable marker for the early diagnosis of acute rejection has been reported. This is one reason why the graft survival rate of pancreas transplantation alone is much lower than that of other organs, such as hearts, livers, and kidneys. We designed an experiment to investigate acute rejection of pancreas allografts in hyperglycemic rats by measurement of blood glucose levels and nitric oxide (NO) products (nitrite plus nitrate, and nitrosyl hemoglobin). As recipients, Lewis rats were rendered hyperglycemic by intravenous injection of streptozotocin before transplantation. F344 rats were used as donors of pancreas allografts. Lewis rats were also used as donors of syngeneic pancreas grafts. After transplantation, the blood glucose level returned to a normal level and rejection was defined as the recurrence of hyperglycemia. The mean survival time of pancreas allografts was 14 +/- 0.7 days. The plasma level of nitrite plus nitrate in allografted rats peaked on postoperative day 7. Electron spin resonance spectra of NO bound to hemoglobin were detected in the blood from allografted rats with a peak on postoperative day 7, whereas NO bound to hemoglobin was not detected in the blood from recipients of syngeneic grafts at any sampling time. The results show that NO was synthesized in the earlier period than the elevation of the blood glucose level during rejection after pancreas transplantation in rats.

Donor-specific tolerance by perioperative intrathymic injection of bone marrow cells in the rat cardiac allograft model: use of FK506 can shorten the necessary duration of pretransplant intrathymic conditioning.

BACKGROUND: Many strategies of tolerance induction by intrathymic (IT) injection of donor alloantigens have been reported to date; however, the timing of IT injection is usually 1-3 weeks before transplantation. METHODS: To apply IT injection to cadaveric organ transplantation, 1 x 10(8) fully allogeneic bone marrow cells (BMC) of Buffalo (BUF; RT1b) rats were intrathymically injected into Wistar Furth (WF; RT1u) rats at the time of BUF cardiac allografting with short-course therapy of antilymphocyte serum (ALS) and FK506 in our experimental model. RESULTS: Allogeneic IT injection of BUF BMC with ALS and FK506 indefinitely prolonged graft survival (mean survival time > 210 days) in all WF rats. On day 130 after grafting, tolerant WF rats accepted donor BUF skin grafts (> 120 days) but not third-party Lewis skin grafts. In control groups, syngeneic IT injection of WF BMC or intravenous injection of donor BUF BMC in combination with ALS/FK506 therapy failed to induce tolerance. In vivo testing was performed during induction (1 month) or during maintenance (6 months of tolerance. In the mixed lymphocyte reaction (MLR), spleen T cells of tolerant rats at 1 month after grafting displayed hyporesponsiveness after stimulation with donor cells. The addition of interleukin (IL)-2 to MLR culture did not restore T-cell responsiveness. Tolerant rats had a significantly decreased frequency of T cytotoxic cell precursors (fTcp) of 1:4,926, and frequency of IL-2-producing T helper cell precursors (fThp) of 1:23,925, compared with naive rats (1: 2,158 and 1:4,266, respectively). By 6 months after grafting, however, the anti-donor MLR proliferative responses of tolerant rats had been restored to the levels of naive splenic T cells. These tolerant rats displayed restoration of the (fTcp) of 1:2,842 and of the (fThp) of 1:5,630, which were comparable frequencies of naive rats. Suppressor T cells did not contribute in this model. In cardiac grafts of tolerant rats induced by IT injection, expression of both Th1 (interferon-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokines was detected in the early phase; thereafter, expression was completely inhibited, except for interferon-gamma in the chronic phase. CONCLUSIONS: Perfect donor-specific tolerance was obtained by IT injection of donor BMC at the time of transplantation, while alloimmune responses were maintained at levels similar to those of naive rats.

Advantages of laparoscope-assisted surgery for recurrent Crohn's disease.

BACKGROUND: Laparoscopic surgery has been applied to patients with primary Crohn's disease, and its beneficial outcomes have been already investigated. However, there is no systematic study of laparoscopic surgery for patients with recurrent diseases. METHODS: We performed reoperation for 43 patients with recurrent Crohn's disease, including 23 patients who underwent laparoscope-assisted surgery. RESULTS: For all the patients, laparoscope-assisted surgery could be performed safely, even if the patients had been treated previously by open surgery or had undergone multiple abdominal procedures. Conversion to open or hand-assisted laparoscopic surgery was necessary for 16 patients (69.6%) because of dense adhesions (11 cases) or bulky tumor (5 cases). Importantly, even if the procedure was converted, the skin incision was significantly shorter than with open surgery, and postoperative recovery was faster, especially for the patients who underwent conversion to hand-assisted laparoscopic surgery. CONCLUSIONS: Laparoscope-assisted surgery is feasible and advantageous in reoperation for patients with recurrent Crohn's disease.

A new method of laparoscopic cholecystectomy using three trocars combined with suture retraction of gallbladder.

BACKGROUND AND PURPOSE: Since the establishment of laparoscopic cholecystectomy (LC) for benign gallbladder lesions, the four-trocar method has been the standard procedure. However, the fourth trocar generally is used just for fundic retraction of the gallbladder. We have developed a three-trocar method for LC and performed it in 132 patients. PATIENTS AND METHODS: After the creation of the pneumoperitoneum, the first 10-mm trocar sheath was inserted in the subumbilicus for the endoscope, the second 5-mm trocar in the epigastric paramedian point for the working port, and the third 5-mm trocar in the subcostal area for grasping forceps. Monofilament nylon with a straight needle was inserted through the right 7th intercostal space in the anterior axillary line, and the seromuscular layer of the gallbladder fundus was punctured and retracted toward the anterior abdominal wall. After that, usual cholecystectomy was performed. RESULTS: Among the 132 patients who underwent the three-trocar method, 10 cases (8%) needed a fourth trocar. No patient was converted to open cholecystectomy. There were no significant differences in the operating time, the length of hospital stay after the operation, or the use of analgesics between the three-trocar and the four-trocar methods. No major complication was recognized. CONCLUSION: This method also has cosmetic advantages. Therefore, we believe this method might be recommended for LC.