Dual cell seeding and the use of zymogen tissue plasminogen activator to improve cell retention on polytetrafluoroethylene grafts.

OBJECTIVE: The purpose of this study was to enhance the retention of seeded endothelial cells (EC) on prosthetic vascular grafts. Dual-layer EC and smooth muscle cell (SMC) seeding and gene transfer of a zymogen tissue plasminogen activator gene (tPA) into seeded EC were studied. METHODS: Polytetrafluoroethylene (PTFE) grafts were precoated with fibronectin, seeded with SMC followed by EC a day later, and then, 24 hours later, exposed to an in vitro flow system for 1 hour. Cell retention rates were determined for grafts seeded with EC only, a dual layer of EC on top of SMC, EC transduced with wild-type tPA, and EC transduced with zymogen tPA. RESULTS: Seeding efficiency of PTFE pretreated with fibronectin was 260 +/- 8 cell/mm(2). After exposure to flow, only 39% +/- 14% of the EC were retained when EC were seeded alone, whereas 73% +/- 22% of EC remained on grafts when EC were seeded on top of SMC (P <.001, n = 10). The enzyme activity of a mutant zymogen tPA in absence of fibrin was 14 +/- 1 IU/mL, which is 3.6-fold lower than that in the presence of fibrin (50 +/- 19 IU/mL), whereas fibrin has no effect on the wild-type tPA activity. EC expressing a high level of wild-type tPA had a lower retention rate (37%) when compared with normal EC (45%). EC expressing the mutant zymogen tPA had an improved retention rate (54%, P =.001, n = 10) in absence of fibrin, whereas its retention rate was reduced to 43% when the cells were exposed to fibrin. CONCLUSION: SMC seeded between EC and PTFE improves EC retention in vitro. Transduction of zymogen tPA increases thrombolytic ability of seeded cells with less adverse impact on cell retention than wild-type tPA.

Functional metabolic characteristics of intact pig livers during prolonged extracorporeal perfusion: potential for a unique biological liver-assist device.

BACKGROUND: The clinical development of liver-support devices based on perfusion of either pig hepatocytes cartridges or whole pig livers has been hampered by the ability to use sufficient liver cell mass to provide adequate metabolic support, limited perfusion times, and the potential for patient exposure to pig zoonotic diseases. METHODS: We designed an original system in which an isolated intact pig liver was perfused extracorporeally under physiological conditions in a closed loop circuit with allogeneic pig blood and constant monitoring of major physiological and functional parameters. The perfusion circuit further included an interface membrane to provide for separation of patient and liver perfusion circulation. RESULTS: Prolonged (6-21 hr) liver perfusion did not produce significant liver damage as reflected by modest rises in the levels of the serum transaminases, stability of main biochemical parameters (including potassium), and the maintenance of normal cellular morphology. Optimal liver function was documented as measured by lactate consumption, control of glycemia, and the results of clotting studies and functional assays. The perfused liver cleared 82% and 79% of peak bilirubin and ammonia concentrations with clearing kinetics identical throughout perfusion. Indocyanine green clearance was identical to that observed in the living donor before explant surgery. CONCLUSIONS: In conclusion, the extracorporeal pig liver perfusion apparatus described here allows optimal pig liver function for prolonged periods of time. The microporous membrane to provide separation of donor organ and recipient and the high level of functional activity suggest that this form of liver metabolic support may have important clinical applications.

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. I. Correlation of graft survival with antidonor IgG antibody subclasses.

We have recently demonstrated that cardiac allograft rejection in the PVG.R8-to-PVG.1U rat strain combination involves the recognition of a isolated class I (RT1.Aa) molecules as peptides in the context of the recipient MHC molecules. Three synthetic peptides (P1, P2, and P3) corresponding to the alpha-helices of the RT1.Aa molecule served as T-cell epitopes for graft rejection. In this study, we demonstrate that two of these peptides (P2 and P3) are sufficient to induce immune nonresponsiveness (median survival time >237 days) to cardiac allografts when presented to the recipient immune system in the thymus 7 days before transplantation. This effect was time dependent, as intrathymic inoculation 60 days before transplantation did not prolong graft survival (median survival time=12 days). Previous studies have demonstrated a critical role for alloantibody responses in mediating graft rejection in this rat strain combination. We, therefore, studied the role alloantibody responses may play in the observed immune nonresponsiveness. The titers of alloantibody in serum samples harvested from graft recipients at different times after transplantation were measured. We used recipient primary aortic endothelial cells genetically manipulated to express the donor RT1.Aa molecule as targets in an enzyme-linked immunosorbent assay. High titers of anti-RT1.Aa IgM antibody were detected in unmanipulated controls at the time of graft rejection. The IgM antibody switched to high IgG titers in intrathymically inoculated rats with accelerated or delayed rejection. Graft rejection in intrathymically manipulated recipients that had achieved a transient state of immunological nonresponsiveness correlated with higher titers of the IgG2b alloantibody. In marked contrast, the long-term graft survivors expressed undetectable or low levels of the IgG2b antibody and moderate to high levels of the IgG1 and IgG2a subclasses. These data suggest that the IgG2b alloantibody may contribute to the rejection reaction, whereas IgG1 and IgG2a may be involved in active enhancement of graft survival.

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. II. Evidence for persistent chronic rejection despite high levels of donor microchimerism.

We have recently demonstrated that three synthetic peptides corresponding to the donor class I RT1.Aa molecule induce long-term survival of cardiac allografts in the PVG.R8-to-PVG.1U rat strain combination (disparate for one isolated class I, RT1.A, molecule) when presented to the recipient immune system in the thymus. Long-term graft survivors had measurable levels of donor-reactive alloantibodies in their serum. In this study, we examined long-term allografts for the presence of chronic rejection and donor microchimerism to assess whether this regimen of immune modulation establishes true tolerance and whether this tolerance is dependent upon the presence of donor-recipient microchimerism. Histological examination of long-term heart grafts (>100 days) demonstrated chronic rejection, including a mild degree of myocardial infiltration by mononuclear cells, mild to moderate myocardial fibrosis, and various vascular changes ranging from focal intimal thickening to total vascular lumen blockade due to smooth muscle cell proliferation. In contrast, long-term syngeneic hearts transplanted under similar experimental conditions lacked these pathological manifestations. Donor microchimerism was analyzed using the polymerase chain reaction with a pair of oligonucleotides specific for the donor class I RT1.Aa gene and genomic DNA harvested from various tissues from graft recipients. We detected high levels of donor microchimerism in the heart, kidney, liver, skin, bone marrow, thymus, and lymph nodes of long-term graft recipients. Donor microchimerism was also detected in unmanipulated control graft recipients at rejection (7 days) and in intrathymically manipulated recipients that rejected allografts in a delayed fashion (12-82 days). These data clearly demonstrate that intrathymic inoculation of donor class I allopeptides induces long-term graft survival but does not prevent chronic rejection. Allograft rejection occurred despite high levels of donor microchimerism, providing direct evidence that donor-recipient microchimerism is not sufficient for the prevention of acute or chronic rejection in this model.

Genetic control of the humoral immune response to xenografts. I. Functional characterization of rat monoclonal antibodies to hamster heart xenografts.

The rejection of cardiac xenografts in the hamster-to-rat combination is characterized by the production of IgM antibodies that result in the rapid loss of the graft. We have recently produced rat monoclonal antibodies (mAb) to hamster heart xenografts in an attempt to develop reagents for use in identifying the target antigens for this reaction and to study the nature of the genetic control of the humoral response. The monoclonals were created by the fusion of myeloma cells with splenic lymphocytes from LEW rat recipients of hamster cardiac xenografts. The hybridomas were screened for antibody production, reactivity to hamster cell surface antigens, and the ability to mediate hyperacute rejection of hamster heart xenografts. A panel of monoclonal antibodies has been identified that are capable of inducing hyperacute rejection. All of these mAbs are IgM and bind strongly to hamster vascular endothelium. None of the mAbs were lymphocytotoxic or bound to hamster lymphocytes or erythrocytes. Immunopathologic studies demonstrated that these mAbs react specifically with hamster vascular endothelium and mediate a complement-dependent humoral reaction leading to the destruction of the cardiac xenografts. One of the mAbs (designated as HAR-1) has been characterized in detail. HAR-1 detects antigens distributed in the vascular endothelium, epithelium of bronchi in the lung, small intestine, tubules of kidney, and selective components of lymphoid organs--e.g., the stromal cells of the spleen and thymic medullary epithelium. Western blot analysis of hamster heart proteins with HAR-1 showed multiple bands with two major bands migrating at 80 kDa and 48 kDa. Absorption of the HAR-1 antibody with 48 individual carbohydrate molecules demonstrated that the strongest reactivity of the antibody is with a sialyl-Lea carbohydrate antigen.

Differential patterns of reaction of human natural antibodies to pig hepatocytes and vascular endothelium.

We have recently conducted a series of experiments to characterize the pattern of reaction of human natural antibodies (NA) with individual pig liver cells. Pooled normal human serum (PHS) was incubated with cultured pig hepatocytes (HEP), aortic endothelial cells (AEC), and portal endothelial cells (PEC), and the reaction of NA to different cell types was measured by antibody-mediated cytotoxic (MTT assay), antibody binding (ELISA), and flow cytometric analysis. The human NA displayed a differential pattern of binding with hepatocytes exhibiting a more limited expression of xenoantigen expression than either aortic or portal endothelial cells. These differences in reaction patterns were also noted for Western blot analysis of individual cell membrane extracts. Preincubation of the pig cells with anti-pig MHC antibodies did not inhibit the binding of human IgM natural antibodies to the pig cells. Comparison of the pattern of NA absorption following the use of bioartificial liver support in patients with acute hepatic failure demonstrated limited ability of pig hepatocytes to absorb substantial amounts of NA. These studies indicate that pig hepatocytes are less vulnerable to NA cytotoxicity than pig vascular endothelial cells and that pig vascular endothelial cells express xenoantigens that are unique and not found on hepatocytes.

Peptides derived from alpha-helices of allogeneic class I major histocompatibility complex antigens are potent inducers of CD4+ and CD8+ T cell and B cell responses after cardiac allograft rejection.

We studied the rejection of cardiac allografts in a rat strain combination (PVG.R8 to PVG.1U) disparate for a single class I MHC antigen (RT1.Aa) to test the extent by which this molecule is recognized as peptides in association with recipient MHC molecules during graft rejection and the contribution of this recognition process to the rejection reaction. Three synthetic peptides that correspond to the portions of alpha-helices of the alpha 1 (P1, P2) and alpha 2 (P3) domains of the donor RT1.Aa molecule were used in this study. Splenocytes from heart allograft recipients at rejection responded in a proliferation assay to all 3 peptides and in a cytotoxic assay to peptides P1 and P2. The peptide-mediated proliferation and cytolytic reactions were blocked by antibodies against CD4/class II MHC and CD8 molecules. Serum from graft recipients at rejection contained significant titers of antibodies to peptides. Presensitization of graft recipients with the peptides resulted in a marked increase in peptide-mediated T cell and antibody responses. Although all 3 peptides were effective in eliciting active immune responses, the P3-mediated response was minimal when compared with those mediated by P1 and P2. Recipients presensitized with the peptides rejected their grafts in 5 days compared with 6 days for unsensitized animals. Recipients presensitized with donor-irradiated splenocytes and aortic endothelial cells, on the other hand, rejected their grafts in 1 and 3 days, respectively, which suggests that immunization with the whole RT1.Aa molecule is required to stimulate accelerated rejection of the graft. This rejection was associated with high titers of donor cell-specific antibodies that exhibited moderate cross-reactivity with the peptides. Our results clearly demonstrate that (1) the donor RT1.Aa molecule is recognized as peptides in the context of recipient class I and class II MHC molecules during the rejection of heart allografts, and (2) peptides derived from this molecule are highly immunogenic in that they contain epitopes recognized by CD4+ and CD8+ T cells and alloantibodies. Immune responses elicited by these peptides, however, did not significantly affect the rate of rejection. These results suggest that acute rejection of allografts may be mediated primarily by the direct recognition of intact MHC molecules.

Intragraft cytokine gene expression in human liver allografts.

Cytokines are thought to play an important role in the inflammatory and immune responses of allograft rejection. We evaluated the pattern of cytokine gene expression in 36 liver biopsy specimens obtained from 20 recipients of primary orthotopic liver allografts. Specific mRNA expression was identified by a polymerase chain reaction (PCR) using oligonucleotide primers specific for human interleukin (IL)-1 beta, IL-2, IL-4, IL-6, IL-10 Interferon (IFN) gamma, tumor necrosis factor (TNF)-alpha and beta-actin. We detected IL-1 beta, IL-6 and IFN-gamma cytokine message most consistently in patients with rejecting liver allografts. TNF-alpha and IL-2 were also observed in rejecting livers, but only during the early phases of the reaction. IL-4 was expressed in the majority of liver allograft biopsy specimens, regardless of the presence or absence of clinical or pathological evidence of rejection. Sequential biopsy specimens in rejecting allografts showed decreased cytokine expression after the induction of a positive response to immunosuppressive therapy. The analysis of biopsy specimens from stable liver grafts showed a predominance in the expression of IL-10. These results may reflect a differential production of inflammatory and regulatory cytokines in response to liver allograft rejection in transplant recipients. They suggest that three cytokines, IL-1 beta, IL-6 and IFN-gamma, may play an important role as markers for liver allograft rejection. Conversely, IL-10 expression was noted in patients with stable graft function. This pattern of expression may correlate with host immune responses that allow for prolonged, rejection-free survival of the graft.

Combination therapy with Brequinar sodium and cyclosporine synergistically prolongs hamster-to-rat cardia xenograft survival.

The accelerated rejection of hamster cardiac xenografts by Lewis rat recipients is due primarily to a humoral immune response. Traditional immunosuppressive agents, including cyclosporine, FK 506, and rapamycin, have not been effective in prolonging the rejection of xenografts in this model. We have recently examined the ability of the combination of cyclosporine and a new immunosuppressive agent, Brequinar sodium, to prevent the rejection of hamster xenografts. Prolongation of xenograft survival by treatment with Brequinar sodium alone (3 mg/kg/day) was minimally effective, with a median survival of 5.5 days. Conversely, a combination of Brequinar sodium (3 mg/kg/day) and cyclosporine (10 mg/kg/day), was associated with a significant prolongation of median graft survival (> 100 days). A quantitative analysis of the dose-effect relationship of Brequinar sodium and cyclosporine demonstrated a potent synergistic interaction for this combination therapy (combination index < 1). A sharp increase occurred in the binding of immunoglobulin M antibodies at day 4 after transplantation in the serum of untreated and cyclosporine-treated Lewis recipients of cardiac xenografts, whereas a clear decrease occurred in anti-hamster immunoglobulin M antibody production during this period of time in the animals treated by combined immunosuppression. Inhibition of anti-donor immunoglobulin M antibody production, as evaluated by the direct enzyme-linked immunosorbent assay and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide measurements, extended for more than 100 days after transplantation in the combination therapy group.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of orthotopic liver allograft rejection in rats with a short-term brequinar sodium therapy. Analysis of intragraft cytokine gene expression.

Brequinar sodium (BQR) is a new immunosuppressive drug that is highly effective in preventing graft rejection in several different experimental settings, including primary allografts and xenografts. A short course of BQR treatment during the onset of allograft rejection can induce the permanent survival of liver and kidney allografts in rats. To study the molecular basis of BQR-induced prolongation of allograft survival, we analyzed the intragraft pattern of IL-1 alpha, IL-2, IL-2R, IL-4, IL-6, IL-10, and TNF gene expression in the ACI-to-LEW liver allograft model. A semiquantitative polymerase chain reaction was developed to measure cytokine gene expression in control and BQR-treated liver graft recipients at various days after transplantation. Untreated control liver allografts expressed all of the cytokines analyzed. There was a marked increase in the steady state level of transcripts for each cytokine as graft rejection proceeded. The treatment of liver graft recipients with 12 mg/kg/day of BQR on days 6, 7, and 8 after transplantation suppressed the expression of all these cytokines within 24 hr of administration. The early suppression of cytokine expression was associated with a modest but distinct reduction in the infiltration of inflammatory cells into the liver grafts. The reduction in the level of transcripts for IL-4, IL-6, and IL-10 persisted in long-term survivors (30 days after transplantation). In contrast, there was a significant increase in the level of transcripts for IL-1 alpha, IL-2, and IL-2R in these long-term survivors. Our results demonstrated clearly that the pattern of cytokine gene expression during allograft rejection is significantly altered by a 3-day course of therapy with BQR. The temporary down-regulation of cytokine gene expression may be responsible for an altered immunological state that results in the prolonged survival of liver allografts.

The prevention of accelerated cardiac allograft rejection in sensitized recipients after treatment with brequinar sodium.

Brequinar sodium (BQR) is a novel immunosuppressive agent that is highly effective in preventing B lymphocyte-mediated antibody production. We have examined the effects of BQR treatment in sensitized recipients on graft survival, donor-specific antibody responses (IgM and IgG), and the appearance of immunopathological lesions present in the grafts. LEW rat recipients were sensitized with single ACI skin graft on day 7 and received heterotopic ACI cardiac grafts on day 0. The recipients rejected the cardiac grafts in an accelerated fashion at day 2.5 post-transplantation, compared to day 7.0 in unsensitized recipients. The animals were treated with low (3 mg/kg/day) or high (12 mg/kg/3x weekly) doses of BQR during skin graft sensitization and/or after challenge with ACI heart allografts. All groups treated with BQR showed significant prolongation of graft survival in the sensitized recipients. The best survival was observed following high-dose BQR therapy during both sensitization and effector phases (median survival time = 40.0 days, P << 0.001). Daily treatment with BQR (3 mg/kg/day) prevented IgM (but not IgG) antibody responses. Treatment with higher doses of BQR (12 mg/kg/3x weekly) before and after skin graft sensitization was effective in preventing both IgM and IgG production. In general, BQR treatment resulted in effective suppression of anti-donor antibody responses, stable graft function, and a reduction in the severity of the acute vascular lesions in the graft. The effectiveness of BQR in preventing accelerated graft rejection when used at 12 mg/kg/3x weekly was comparable to that seen with treatment of sensitized animals with CsA at 15 mg/kg/day for 30 days. Daily treatment with cyclophosphamide at 5 or 15 mg/kg/day was ineffective for preventing graft rejection in sensitized recipients. These results indicated that BQR may provide an important addition to treatment protocols designed to prevent transplantation rejection in presensitized patients. BQR has the ability to significantly inhibit host cellular and humoral immune responses to the donor graft and this facet of the immunosuppressive activity of the drug may be responsible for preventing this aggressive form of rejection.

Lymphocytic subsets and histopathologic changes associated with the development of heart transplant arteriosclerosis.

Transplantation of vascularized organ allografts is associated with the development of arteriosclerosis in the vessels of the donor graft. We have recently examined the distribution of lymphocytic/mononuclear inflammatory cell subsets and histopathologic changes seen in the sequential development of transplant arteriosclerosis in cardiac allografts exchanged between inbred rat strain combinations (LEW-to-F344) that differ for weak histocompatibility loci. The donor grafts were harvested at 7, 14, 21, 28, 50 to 65, and 90 days after transplantation, and the vascular lesions were characterized for the immunopathologic changes associated with the chronic rejection of the grafts. The inflammatory cell infiltration of the graft myocardium was present as early as 7 days after transplantation (53.37 +/- 9.06 inflammatory cells/high-power field), and the accumulation of cells increased at 3 and 4 weeks after transplantation (112.12 +/- 16.58 and 130.40 +/- 21.24 cells/high-power field, respectively). The infiltrating inflammatory cells in the grafts at 4 weeks after transplantation were predominantly ED-1+ macrophages (63.39%), with a substantial number of OX19+ T lymphocytes (28%), OX8+ Tc/s cells (25.4%), 3.2.3+ natural killer cells (16.89%), W3/25+ Th cells (20.2%), and a small number of OX33+ B lymphocytes (1.13%) and interleukin-2 receptor+ T lymphocytes (2.6%). Among the T-cell population, most (87.6%) were positive (R73+) for T-cell receptors. The arteriosclerotic lesions present in the cardiac grafts exhibited a gradual increase in severity of the vascular intimal proliferation and a similar pattern of increased intensity of cellular infiltration. The lesions were infiltrated with inflammatory cells beginning in the first week and increasing for the first month after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

FK506 suppression of heart and liver allograft rejection. II: The induction of graft acceptance in rats.

Lewis recipients of orthotopic ACI livers had permanent graft acceptance induced with 3 doses of i.m. FK506 in the early postoperative period. They were studied 100 and 300 days posttransplantation. The recipients rejected ACI as well as Brown Norway (BN) (third-party) skin grafts, and had lymphocytes with substantial reactivity by mixed lymphocyte culture testing against ACI and third-party (BN) alloantigens. Lymphocyte subset redistribution had not occurred in the peripheral blood or spleens of these animals, and there was no evidence of suppressor cell activation by in vitro and in vivo tests. Graft-versus-host reactivity in splenic lymphoid tissues of these recipients was demonstrated with the popliteal lymph node assay. Attempts at adaptive transfer with recipient lymphocytes were unsuccessful. Heart graft acceptance was far more difficult to accomplish than liver graft acceptance, and probably was never permanent. ACI heart graft prolongation in LEW recipients after a brief induction with FK506 lasted for no more than 3 months in most animals. The temporary heart graft acceptance was specific for hearts of the original ACI donor strain but not for ACI skin. Results of studies of lymphocyte subsets and suppressor cell activity were similar to those in the liver recipients. These studies illustrate how poorly graft acceptance is understood and how badly further work is needed to clarify its mechanism.