Sally Chen

Prolongation Of Renal Allograft Survival In A Large Animal Model By Oral Rapamycin Monotherapy

We assessed the efficacy of 5 dose levels of oral rapamycin for prolonging renal allograft survival in pigs. Untreated and triple therapy groups (cyclosporine, azathioprine, and prednisone) served as controls. Immunosuppression was administered for 28 days posttransplant and then stopped. Rapamycin whole-blood concentrations were followed weekly. Chemistry, hematology, and lipid values were monitored post-transplant. For rapamycin-treated pigs, median survival time (MST) correlated with both dose and trough levels (ng/ml). All kidneys had some degree of rejection seen on necropsy. After rejection, pneumonia was the most common cause of death. No specific end-organ toxicity was noted on histopathologic examination. Triglyceride and cholesterol levels increased in all treated pigs (both rapamycin and triple therapy) vs. untreated controls--however, all values were within normal limits. Mean ALT levels increased in weeks 2 to 4 in the higher-dose rapamycin groups but returned to baseline in pigs surviving after the drug was stopped. ALT levels did not increase above twice normal in any group. Creatinine levels correlated with the degree of rejection seen on biopsy. We noted no other toxicities. We conclude that rapamycin, given as oral monotherapy, is an effective and safe immunosuppressant in our large animal renal allograft model. Outcome correlated with dose and whole-blood levels.

Beyond hyperacute rejection : accelerated rejection in a discordant xenograft model by adoptive transfer of specific cell subsets

If hyperacute rejection is prevented in the guineapig (GP)-to-Lewis rat (Lew) cardiac xenograft (CXg) model, an accelerated rejection involving cellular infiltration occurs in 3 to 4 days. In previous work using an adoptive transfer model, we found that this accelerated rejection was facilitated by either sensitized splenocytes or sensitized serum. In the current study, in an attempt to determine which splenocyte subset(s) facilitated this process, sensitized splenocytes, with or without subset depletion were injected, into complement- and natural antibody-depleted Lew recipients of GP CXgs. Graft survival was 4.18 +/- 0.75 days with no injection (n = 11), 4.13 +/- 0.99 days with naive splenocytes (n = 8), 1.80 +/- 0.45 days with sensitized splenocytes (n = 5), 2.67 +/- 1.03 days with CD4(W3/25+) depletion of the sensitized splenocytes (n = 6), 3.13 +/- 0.84 days with CD8(OX8+) cell depletion (n = 8), 4.70 +/- 0.68 days with macrophage depletion (n = 10), and 4.22 +/- 0.41 days with B cell depletion (n = 9). Cellular infiltrates, hemorrhage, myocyte necrosis, and endothelial deposition of IgG, IgM, and fibrin were seen in rejected grafts. In most groups, infiltrating cells consisted of CD4 (W3/25+), CD8 (OX8+), IL2R+ cells, macrophages, and natural killer (NK) cells. However, in the macrophages-depleted group, activated (ED2+) macrophages and NK cells were significantly reduced. Total IgM, anti-GP IgM, and anti-GP IgG rebounded in all groups over several days but were not consistent at the time of rejection. Lewis rats rejecting GP CXgs early had lower final titers than those rejecting later. Total IgG titers rebounded to baseline by posttransplant day 1 and were therefore similar in all groups at the time of rejection. These findings suggest that this accelerated rejection requires interaction between macrophages and B cells, since depletion of either significantly alters the rejection tempo. A possible explanation is that xenoreactive IgG antibodies, synthesized by sensitized B cells, bind their target antigens--but also bind sensitized macrophages through their Fc region, thus causing rejection by antibody-dependent cell-mediated cytotoxicity.

Cellular rejection in discordant xenografts when hyperacute rejection is prevented: analysis using adoptive and passive transfer

Hyperacute rejection of discordant xenografts occurs rapidly, precluding cellular infiltration. Thus the role of cellular rejection in discordant xenografts is debated. Using adoptive transfer of sensitized splenocytes and passive transfer of sensitized serum, we evaluated the influence of cellular and humoral elements on cellular infiltration and rejection in the guinea-pig-to-rat discordant xenograft model. Guinea-pig hearts were transplanted into Lewis rats. Pretransplant, rats underwent splenectomy and plasma exchange and were started on daily cobra venom factor injections. Xenografts rejected faster after adoptive (1, 2, 2 and 2 days) or passive (1, 1, 2 and 2 days) transfer than controls (4, 4, 4 and 4 days; p < 0.05). Macrophages and neutrophils were predominant in early prerejection specimens. Over time, cellular infiltrates were dominated by mononuclear cells. Natural killer cells were present in all groups, as were interleukin 2 receptor positive cells. Our data suggest that either sensitized serum or sensitized cells are capable of initiating an accelerated rejection characterized by cellular infiltration. Despite subtle differences, the population of infiltrating cells was similar in each group. Thus, although rejection may be initiated by either cellular or humoral influences, the ultimate result is the same. We have, therefore, established a small animal model to study cellular rejection in discordant xenografts. This model will help evaluate the role of cell subsets and xenoantibodies in xenograft rejection and will help determine the precise relationship between the two when hyperacute rejection is prevented.

Evidence for direct and indirect pathways in the generation of the alloimmune response against pancreatic islets

The role of the direct and indirect pathways of alloantigen presentation in the generation of the alloimmune response was dissected using the murine mixed lymphocyte-islet coculture system (MLIC). Stimulator DBA/2J (H-2d) pancreatic islet populations consisted of whole islets (MHC class I+, II+) or FACS-purified beta cells (MHC class I+, II-). Responding C57B1/6 (H-2b) splenocyte populations were either: (1) untreated; (2) depleted of helper T cells with anti-L3T4 monoclonal antibody plus complement; (3) depleted of cytotoxic T lymphocytes with anti-Lyt2 mAb plus complement; or (4) depleted of antigen-presenting cells by passage through a Sephadex G-10 column. Whole islets were capable of stimulating a significant C57B1/6 anti-DBA cytotoxic T cell response if the responding population was untreated or treated with complement alone. Depletion of responding splenocytes with either anti-Lyt2 or anti-L3T4 mAb plus complement abrogated the generation of allospe-cific CTL. If the responding splenocyte population was depleted of APCs, the allo-CTL response against whole islets was decreased, but still significant. If, however, the stimulator population consisted of FACS-purified DBA 2J beta cells, APC-depleted C57B1 6 splenocytes were incapable of generating any CTL response. Adding responder type (C57B1/6) APCs back to the microwells restored the capacity for both whole islets and purified beta cells to stimulate a strong allo-CTL response. These data demonstrate that both indirect and direct pathways of alloantigen presentation function in the MLIC.

Afferent and efferent pathways in T cell responses to MHC class I+, II-hepatocytes.

We have previously reported that purified hepatocytes stimulate significant in vitro allospecific cytotoxicity when cocultured with naive responder splenocytes in the mixed lymphocyte hepatocyte culture (MLHC). In this report we examined the expression of MHC antigens on the surface of hepatocytes, the phenotypic lymphocyte subset(s) that respond(s) to allogeneic hepatocytes, and the phenotype of allospecific cytolytic effectors generated in MLHC. Hepatocytes expressed MHC class I but not MHC class II antigens by immunofluorescent microscopy and fluorescence activated cell sorting. The lack of MHC class II on the surface of hepatocytes was also indirectly supported by the inability of hepatocytes to stimulate proliferation of a class II-directed allospecific helper T cell clone. The generation of allospecific cytotoxicity in MLHC required the participation of L3T4+, Ly2- T cells and L3T4-, Ly2+ T cells in the naive responder splenocyte population since depletion of these subsets with mAb and complement abrogated the development of allo-CTLs. Furthermore, adherent accessory cells in the naive responder splenocyte population appeared to play a role in the generation of allospecific cytotoxicity in MLHC since depletion of this population by plastic adherence and passage through a Sephadex G10 column resulted in significantly reduced allospecific cytotoxicity. Depletion of day 5 allosensitized cells of Ly2+ but not L3T4+ T cells by mAb and complement eliminated allospecific cytotoxicity--indicating that cytolytic effectors generated in MLHC appear to be L3T4-, Ly2+ T cells.

Rapamycin: immunosuppression, hyporesponsiveness, and side effects in a porcine renal allograft model.

Rapamycin prolongs allograft survival and induces donor-specific tolerance in some small animal transplant models. Large animal studies, however, are limited. We studied rapamycin in a porcine renal allograft model. Donor-recipient combinations were chosen based on high response in pretransplant MLCs. Allografts were anastomosed to the aorta and vena cava and the native kidneys removed. There were 5 treatment groups: (a) no immunosuppression; (b) triple therapy (CsA, 1 mg/kg/day; AZA, 2–3 mg/kg/day; and PRED, 3–4 mg/kg/day); (c) rapamycin (0.75 mg/kg/day i.m.) in carboxymethylcellulose (CMC); (d) rapamycin (0.25 mg/kg/day i.m. in CMC); and (e) a vehicle (CMC) control. Serum creatinine levels were determined every other day. Most allografts were biopsied once a week. Immunosuppression was stopped after 30 days. Mean graft survival in nonimmunosuppressed recipients was 6.8±3.6 days. Mean graft survival in triple therapy recipients (n=10) was 45.7±36 days vs. 59.6±11.4 days in rapamycin (0.25 mg/kg/day) recipients (n=7) (P=0.51). Both triple therapy and rapamycin improved renal allograft survival versus nonimmunosuppressed controls (P=0.0025 and 0.001, respectively). Serum creatinine levels were significantly lower (P<0.05) in rapamycin versus triple therapy recipients. We conclude that rapamycin is a potent immunosuppressant in a porcine renal allograft model and may avoid the elevated serum creatinine levels associated with CsA.

The role of monocytes and macrophages in delayed xenograft rejection

Abstract: Despite the development of successful strategies for averting hyperacute rejection (HAR) in both small and large animal xenograft models, a delayed xenograft rejection (DXR) ultimately occurs. This process is characterized by endothelial cell activation and graft infiltration with activated monocytes and natural killer (NK) cells. We evaluated the role of monocytes and macrophages in a guinea pig‐to‐rat model of DXR. Our results suggest that specific interactions between these cells and the xenograft occur that result in their activation, since adoptive transfer of xenoactivated splenocytes significantly accelerated both DXR and allograft rejection, while adoptive transfer of alloactivated splenocytes did not. Furthermore, while normal splenocytes caused antibody‐dependent cell‐mediated cytotoxicity (ADCC) of xenogeneic endothelial cells, xenoactivated splenocytes caused significantly greater endothelial cytotoxicity by antibody‐independent mechanisms. Both normal and xenoactivated splenocytes were significantly less cytotoxic if adherent cells, consisting predominantly of monocytes and macrophages, were first removed. In vivo recipient macrophage depletion, using liposome‐encapsulated dichloromethylene diphosphonate, did not influence DXR and this may indicate that nonphagocytic circulating monocytes may be more important in DXR. However, adoptive transfer of splenocytes from a macrophage depleted, xenoactivated donor did not accelerate xenograft rejection, while splenocytes from a nondepleted xenoactivated donor did, thereby supporting the importance of monocytes and macrophages in this I phase of xenograft rejection.

Modulation of MHC class I antigen decreases pancreatic islet immunogenicity

Pretreatment regimens directed at reducing the immunogenicity of pancreatic islets have emphasized the elimination or alteration of the major histocompatibility complex (MHC) class II-positive dendritic cells within the islet. Unfortunately, the efficacy of such pretreatment regimens has been extremely variable and the relative contribution of the dendritic cells to the overall immunogenicity of pancreatic islets has remained ambiguous. Recent evidence has suggested that the MHC class I antigen present on the endocrine cells within the islets may play an important role in the alloimmune response. This study utilized the in vitro mixed lymphocyte-islet co-culture system to determine if pretreatment of whole islets with an anti-MHC class I monoclonal antibody specific to the donor strain would block the generation of cytotoxic T lymphocytes (CTL) in the in vitro mixed lymphocyte-islet coculture. Pretreatment of B10.BR (H-2k) and DBA/2J (H-2d) islets with an allospecific anti-MHC class I monoclonal antibody blocked the generation of allospecific CTL when the pretreated islets were placed into coculture with C57Bl/6 (H-2b) splenocytes. If such a pretreatment regimen is similarly effective in vivo, it could potentially be used as an antirejection strategy in pancreatic islet allotransplantation.

Pharmacokinetics of 6-thiouric acid and 6-mercaptopurine in renal allograft recipients after oral administration of azathioprine

SummaryThe immunosuppressive activity of azathioprine (AZA) is unpredictable and depends on the formation of intracellular thiopurine ribonucleotides. However, the quantification of these active thiopurines presents difficult analytical problems. It has recently been postulated that plasma concentrations of 6-thiouric acid (6-TU) and 6-mercaptopurine (6-MP), metabolites of AZA, may provide more readily measurable indices of the pharmacologic activity of AZA. In order to evaluate the utility of 6-TU and 6-MP plasma concentrations in monitoring AZA therapy, we studied their pharmacokinetics in 6 renal transplant patients, and their in vitro immunosuppressive potency in a mixed lymphocyte proliferation assay.A peak plasma 6-TU concentration of 710.7 ng/ml was observed at 3.8 h after oral dosing. Good correlation was observed between the elimination t1/2 of 6-TU and serum creatinine, and between AUC over 24 h and serum creatinine. However, we did not observe a second peak in plasma 6-TU concentration that could be attributed to the degradation of active AZA metabolites. 6-MP plasma concentrations in the patients were low (mean peak concentration 36.0 ng/ml) and rapidly disappeared within 8 h. In vitro immunosuppressive activity could not be demonstrated for 6-TU over a concentration range of 1.25 ng/ml to 0.25 mg/ml.We conclude that 6-TU is pharmacologically inert and is primarily eliminated by the kidneys. Our findings currently do not support the use of plasma concentrations of 6-TU or 6-MP to monitor AZA therapy. In order to optimize AZA therapy, analytical techniques that are technically feasible and that can directly quantify the active intracellular thiopurines are being explored.

Generation of Allospecific Cytolytic T-Lymphocytes Stimulated by Pure Pancreatic β-Cells in Absence of la+ Dendritic Cells

A murine mixed islet-lymphocyte coculture system (MILC) was used to quantitate the immunogenicity of a pure population of pancreatic β-cells to more clearly define whether stimulator major histocompatibility complex (MHC) class ll-positive dendritic cells are a major component leading to islet immunogenicity. Pancreatic β-cells express MHC class I antigen but not class II antigen. These experiments compared the in vitro immunogenicity of fluorescence-activated cell sorted (FACS-IV) pure β-cells (MHC class I-positive cells only) relative to unpurified dispersed islet cells (MHC class I-positive cells and class II-positive cells). The results demonstrated the surprising finding that pure DBA/2J (H-2b) pancreatic β-cells stimulated a strong cytotoxic T-lymphocyte (CTL) response when exposed to C57BL/6 (H-2b) allosplenocytes in the MILC, similar to DBA/2J nonpurified dispersed islet cells. Furthermore, the stimulation of CTL by both purified β-cells and nonpurified dispersed islet cells was blocked by addition of MHC-specific anti-class I monoclonal antibody directed against stimulator MHC antigen. The data imply that the highly immunogenic MHC class II-positive passenger leukocytes present in the islets were not necessary for the generation of the immune response in the presence of MHC class l-positive β-cells. Although most of the pretreatment regimens attempting to decrease islet immunogenicity have been directed at eliminating the MHC class II-positive passenger leukocytes from the islets, this work suggests that modulation of MHC class I antigen may be an important approach.