Junhong Sun

Tolerance to rat liver allografts. IV. Acceptance depends on the quantity of donor tissue and on donor leukocytes

Liver allografts in some rat strains are often spontaneously accepted across a complete major histocompatibility barrier without the requirement for immunosuppression while other nonliver allografts are rejected. In previous studies, we have shown that spontaneous acceptance is dependent on liver passenger leukocytes. Depletion of passenger leukocytes by donor irradiation allows rejection, with DA recipients of irradiated PVG livers having a median survival time (MST) of 16 days. Here we show that, in this model, spontaneous acceptance is reconstituted by intravenous injection of donor leukocytes. Intravenous injection of 3-5x10(7) PVG liver leukocytes significantly prolonged DA survival time (MST=96 days, P=0.026), as did 5x10(7) spleen leukocytes (MST>100 days, P=0.002). Deletion of T cells from the reconstituting inoculum reduced survival time (MST=78 days, P=0.039), whereas deletion of B cells or monocytes/macrophages had no effect on survival time. In contrast, PVG hearts are regularly rejected by DA recipients, and PVG liver or spleen leukocytes, even at doses of greater than 3x10(8) cells/recipient, were unable to induce heart acceptance. To investigate the possibility that acceptance of the irradiated liver but not the heart might be due to the large mass of the liver, two kidneys and two hearts of PVG origin were transplanted to each DA recipient together with 1.5x10(8) PVG leukocytes. These organs survived for greater than 200 days, thereby showing that a large mass of donor tissue, in association with donor leukocytes, leads to acceptance of organs that are rejected if transplanted singly. It appears likely that spontaneous liver transplant tolerance is a high-dose or activation-associated immune phenomenon.

Comparison of porcine hepatocytes with human hepatoma (C3A) cells for use in a bioartificial liver support system.

Cells from primary porcine hepatocytes (PPH) and the immortalized human hepatoma cell line C3A are both used in bioartificial liver support systems (BALSS). In this work the viability and metabolic capacity of PPH and C3A cells cultured in different media were compared. Also, because the cells come into direct or indirect contact with human blood components in BALSS, the effects of human complement on survival and functions of the cells was evaluated. For short-term culture, maintenance of PPH viability was essential for retention of P450IA1 activity (r = 0.882, p < 0.01) and effective ammonia clearance (r = -0.791, p < 0.01). When cell viability was below 60% P450IA1 activity could not be recorded and nitrogen elimination activity significantly diminished. In contrast to PPH, ammonia levels were markedly increased for C3A cells in all culture media tested (p < 0.01). Ammonia increase correlated with C3A viability (r = 0.896, p < 0.05). PPH metabolic function was superior to that of the C3A cell line when evaluated by P450IA1 activity, ammonia removal, and amino acid metabolism. When PPH were incubated in human plasma (HP) or human serum (HS) there was rapid and irreversible deterioration of viability occurring within 9 h. This toxic effect could be prevented by the inactivation of complement. When sodium citrate dissolved in dextrose was added to medium, there was considerable damage to both PPH and the C3A cell line. However, there was no demonstrable toxic effect when hepatic cells of either type were exposed to heparin. We conclude that PPH cultivated in complement-inactivated HP or HS are to be preferred to C3A for clinical application of BALSS, and that heparin should be preferred for anticoagulation in BALSS.

Conversion of pancreas allograft rejection to acceptance by liver transplantation

BACKGROUND Liver allografts of PVG(RT1c)-->DA(RT1a) are spontaneously accepted, whereas pancreas, heart, and kidney allografts are rejected. Our previous studies have shown that simultaneous liver and pancreas transplantation prevents pancreas allograft rejection. The aim of this study was to examine the effect of liver transplantation on subsequent pancreas allografts and on ongoing pancreas rejection. METHODS Heterotopic, duct-ligated segmental pancreas grafts were transplanted into streptozotocin-induced diabetic recipients (60 mg/kg body weight i.p.) with or without orthotopic liver grafting at different times. Experimental design was as follows. Group 1 received PVG-->PVG pancreas syngrafts (n=6); group 2, PVG-->DA pancreas allografts (n=7); groups 3-5, PVG-->DA pancreas allografts followed by liver transplantation on day 2 (n=6), on day 4 (n=5), and on day 6 (n=5), respectively, group 6, PVG-->DA pancreas allografts after liver transplantation at 4 weeks (n=6). RESULTS The results showed that pancreas allografts in group 2 were rejected from postoperative day 7 to 13. Liver transplantation prevented subsequent pancreas allograft rejection in group 6. Ongoing pancreas rejection was reversed by liver transplantation with subsequent graft acceptance in groups 3-5. Significant graft-infiltrating lymphocyte apoptosis was demonstrated at 2 weeks in pancreas transplants associated with liver grafting. Graft-versus-host disease was not detected in the pancreas recipients. CONCLUSIONS We conclude that pancreas allografts in the PVG-->DA combination are rejected rapidly with median survival time of 9 days. Liver transplantation can protect subsequent pancreas grafts from rejection and reverse ongoing pancreas graft rejection with subsequent pancreatic acceptance. Graft-infiltrating lymphocyte apoptosis may be associated with the process of graft acceptance.

Factors Affecting Hepatocyte Viability and Cypial Activity During Encapsulation

Hepatocytes encapsulated in alginate-poly-1-lysine-alginate (APA) are used in transplantation studies and in bioartificial liver support systems. Loss of cell viability in the process of APA encapsulation is usually 20–30% while the effect on cytochrome CYP450 activity is rarely reported. This work investigates the negative influences on hepatocyte viability and CYPIA1 activity during APA encapsulation, and reports methods to alleviate these influences by incorporating certain reagents into the encapsulation solution. The results show that loss of hepatocyte viability and CYPIA1 activity was caused almost entirely by extracellular calcium toxicity rather than by mechanical damage (p < 0.05). Use of 10 mM instead of 100 mM calcium chloride (CaCl2) in the encapsulation process improved CYPIA1 activity (p <0.05), but did not improve hepatocyte viability (p > 0.05) or result in satisfactory microcapsules. Hepatocyte viability was 25% higher (p < 0.05) in CaCl2 than in calcium lactate (CaLa) when the cells were gelled by contact with these calcium solutions at room temperature (RT). Hepatocyte viability showed little improvement by processing at 4°C than at RT in CaCl2 (p > 0.05) but was 23% higher at 4°C than at RT in CaLa (p < 0.05). Calcium used in the process of encapsulation caused cell necrosis rather than apoptosis. Addition of Dulbeccos modified Eagles medium (containing 10% foetal bovine serum) or 20 mM fructose to the calcium solution did not improve cell survival. However, nifedipine at a final concentration of 25 mM modestly improved hepatocyte survival in solution containing 100 mM CaCl2 (p = 0.003). Glutathione and taurine in certain concentrations showed protective effects against loss of CYPIA1 activity (p < 0.05 and <0.01 respectively). In conclusion, to optimise the use of calcium during the process of encapsulation, CaCl2 is preferred to CaLa and inclusion of nifedipine, glutathione or taurine in 100 mM CaCl2 solution is recommended.

Evaluation of MTS, XTT, MTT and3HTdR incorporation for assessing hepatocyte density, viability and proliferation

The new 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric assays for assessing hepatocyte density, viability and proliferation were evaluated and compared with 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and tritiated thymidine (3HTdR) incorporation. OD values of MTS or XTT, which are metabolically reduced in viable cells to a watersoluble formazan product and can be read directly, had a good correlation coefficient with hepatocyte densities in a range of 2.5–40×104 cells/ml (MTSr=0.952; XTTr=0.902) and with hepatocyte viability (MTSr=0.974; XTTr=0.975). At 0, 20, 40 and 60 hr cultures, the correlation coefficients with3HTdR for assessing hepatocyte viability and proliferation (MTSr=0.942–0.981; XTTr=0.953–0.992) were excellent. In contrast with MTS and XTT, MTT OD values had a poor correlation coefficient with hepatocyte densities (r=0.672), viability (r=0.622) and3HTdR incorporation (r=0.701–0.818 at 0, 20 and 40 hour cultures). This study shows that the MTS or XTT colorimetric assays for assessing hepatocyte density, viability and proliferation are more accurate, reliable and simpler than the widely used MTT assay. They avoid use of radioactive material as required for3HTdR incorporation. Of the two, the MTS colorimetric assay is more sensitive than the XTT.

Effect of liver transplantation on islet allografts: up-regulation of Fas ligand and apoptosis of T lymphocytes are associated with islet graft tolerance.

Background. Liver allografts in spontaneously tolerant strain combinations can protect other organs of the same donor origin from rejection and reverse ongoing rejection in previously placed grafts. The aims of this study were to examine whether liver allografts have the same protective effect on islet allografts and to investigate the underlying mechanisms. Methods. PVG islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic DA rats with or without liver allografting. The cellular infiltrate, and the extent of apoptosis and of Fas ligand (FasL) expression in the islet grafts were evaluated on days 2, 4, and 7 after transplantation by means of immunostaining and the in situ terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay. Donor and recipient mixed lymphocyte reactions (MLR) were determined at 7 days or 100 days after islet transplantation. Results. Islet allografts transplanted alone were rapidly rejected within 5-8 days. Rejection was delayed, but not prevented, when islets were transplanted simultaneously with the liver. Liver transplantation 1 month before islet transplantation resulted in long-term survival (>100 days) of islet grafts in three of seven animals, whereas the other four died of liver rejection with functional islet grafts. Liver transplantation on day 4 after islet grafting reversed ongoing islet rejection and led to indefinite islet graft survival in three of seven cases. There was a progressive increase of cellular infiltration in all of the islet allografts, but the intensity of the infiltrate did not correlate with the outcome of the islet allografts. Islet rejection was characterized by an early dominance of monocytes/macrophages and CD25+ T cells in the infiltrates, a high incidence of apoptotic &bgr; cells in grafts, and a sensitized status in the MLR. Tolerance of islet allografts was associated with increased numbers of dendritic cells in the graft infiltrates, up-regulation of FasL, and prominent apoptosis of alloreactive leukocytes in the islet grafts, as well as donor-specific MLR suppression in long-term survivors. Conclusions. These results demonstrate that the extent of the protective effect of liver transplantation on islet allografts varies with the time of liver grafting, ranging from delay in islet rejection to complete islet acceptance. Islet graft tolerance induced by liver transplantation is the result of an immune process that involves up-regulation of Fas ligand expres-sion on, and apoptosis of, islet graft infiltrating lymphocytes.

Outcome of different models of multiorgan transplantation in rats.

In the PVG (RT1c) to DA(RT1a) rat combination, liver allografts are spontaneously accepted across a complete MHC barrier while cardiac and renal allografts are rejected. We postulated that this spontaneous liver acceptance was associated with the large quantity of antigen in the transplanted liver. In order to test this hypothesis, we have developed three different models of multiorgan transplantation: model I, triple heart transplantation; model II, triple kidney transplantation; and model III, double heart and double kidney transplantation. The results showed that prolongation of heart and kidney allografts was achieved with the increasing number of organs transplanted. The models proved reliable and useful in transplant immunological studies.

A new vascularized skin transplant model in rats

The aim of this work was to develop a vascularized skin transplantation model in the rat to allow comparison of the immunological events with those of classic free skin grafts and vascularized organ grafts. Seventy‐nine syngeneic transplants were performed using inbred Wistar and PVG rats to develop the model. Epigastric skin flaps were taken with a vascular pedicle of common femoral artery and vein and implanted on the back of the neck. The graft vessels were anastomosed end‐to‐end and end‐to‐side to the recipient common carotid artery and external jugular vein with interrupted 10‐0 nylon sutures under an operating microscope. A high success rate (11 of 11) was achieved in the final method developed, with light microscopy confirming normal skin structure in the flaps following transplantation. The advantages of the model include comparable skin size to that commonly used for nonvascularized skin grafts, while the physiology in terms of blood supply is similar to that of vascularized organ grafts; the grafts are visible with free access for biopsy; and the size of the graft can easily be varied to allow studies concerning antigen load.

Analysis of multivariables during porcine liver digestion to improve hepatocyte yield and viability for use in bioartificial liver support systems.

In order to achieve optimal BALSS function, preparation of porcine hepatocytes with high yield, viability, and P450 activity is known to be important. To date hepatocyte yields have varied from 0.58 × 1010 to 3.45 × 10 and viabilities from 75% to 95% within and between laboratories, even when using the same digestion methods and procedures, indicating that hepatocyte isolation during porcine liver digestion is not fully optimized. The aim of this work was to identify the critical parameters affecting cell recovery during porcine liver harvesting by investigating 21 variables involved in the process, including pig body and liver weight, different digestion times of perfusates, pH, a range of concentrations of sodium and chloride in EDTA, and collagenase perfusates. Univariate and multivariate analysis of a retrospective study (n = 23) revealed that low perfusate pH during the process of digestion had a positive effect on hepatocyte yield (p < 0.05), while high (relative) concentrations of sodium and chloride in the perfusates had significant negative effects on hepatocyte viability (both p < 0.05). Sodium and chloride had narrow optimal ranges for achieving a >90% viability. These findings were then tested in a prospective study (n = 10) and further verified. High hepatocyte viabilities (91.8 ± 1.6%, p = 0.036) and yields (2.56 ± 0.48 × 1010) were achieved consistently, and P450IA1 activity was increased after sodium and chloride concentrations and pH in the perfusates were controlled. The physiological mechanism by which sodium and chloride affects hepatocyte viability during porcine liver digestion is discussed.