Dongyuan Xia

Evidence for a genetic predisposition towards acute rejection after kidney and simultaneous kidney-pancreas transplantation.

Background. In vitro production of tumor necrosis factor-&agr; (TNF-&agr;), interferon-&ggr; (IFN-&ggr;), interleukin 10 (IL-10), and transforming growth factor-&bgr; (TGF-&bgr;) correlate with their respective genetic polymorphisms. We analyzed the relationship between these genetic polymorphisms and posttransplant outcome. Methods. Using DNA polymerase chain reaction (PCR) technology, polymorphisms for TNF- &agr;, IFN-&ggr;, IL-10, and TGF-&bgr; were determined for 82 kidney (K) and 19 simultaneous kidney-pancreas (SKP) recipients. These results were analyzed with regard to the incidence of acute rejection (AR), and the timing and severity of the first AR episode. Results. A high TNF-&agr; production phenotype correlated with recurrent acute rejection (AR) episodes (P <0.026). Compared with the low TNF-&agr; production phenotype, more patients with the high production phenotype had a post-AR serum creatinine >2.0 mg/dl, but this was not statistically significant (64 vs. 35%, P =0.12). There was no relationship between TNF-&agr; genotype and the time to first AR episode or incidence of graft loss. IFN-&ggr; production phenotype showed no correlation with any of these clinical outcome parameters. There was an increase in AR incidence as the IL-10 production phenotype increased (low, intermediate, high), but only in low TNF-&agr; producer phenotypes (P =0.023). Conclusions. Patients with a polymorphic cytokine genotype putatively encoding for high in vivo TNF-&agr; production, and to a lesser extent IL-10 cytokine genotypes putatively encoding for higher levels of in vivo IL-10 production, had a worse clinical outcome regarding AR episodes. These data support the hypothesis that the strength of alloimmune responsiveness after transplantation in part is genetically determined.

Real-time polymerase chain reaction analysis reveals an evolution of cytokine mrna production in allograft acceptor mice

BACKGROUND The relative contribution of pro-inflammatory and anti-inflammatory cytokines in promoting the rejection or acceptance of experimental cardiac allografts remains controversial. We hypothesized that the posttransplantation induction of a new immune response to graft alloantigens at a distant delayed-type hypersensitivity (DTH) site would force the immune system to reveal its current disposition toward graft alloantigen as it initiates the new immune response. Thus, we should be able to monitor the evolution of the immunologic relationship between allograft recipients and their grafts at any time posttransplantation by challenging the recipient for DTH responses to donor alloantigen and evaluating the cytokine profiles displayed at the DTH site. METHODS We have used the sensitive and quantitative technique of real-time polymerase chain reaction to evaluate the patterns of donor alloantigen-induced cytokine mRNA production for interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-10, and transforming growth factor (TGF)-beta. We evaluated cytokine mRNA expression in cardiac allografts and in donor alloantigen-challenged DTH sites in mice that have either accepted or rejected cardiac allografts. RESULTS We observed the following. (1) Normal hearts and pinnae exhibited detectable baseline production of cytokine mRNAs: TGF-beta>IFN-gamma=IL-10>IL2->IL-4. (2) Both the accepted and rejecting cardiac allografts produced increased amounts of all cytokine mRNAs tested and displayed few quantitative differences in cytokine mRNA production. Notably, accepted allografts displayed enhanced IL-10 mRNA production on day 7 posttransplantation, but not on day 60 posttransplantation and reduced IFN-gamma mRNA production on day 60, but not day 7. (3) There was a high degree of variability in production levels among the various cytokine mRNAs, both for background levels and for allograft-stimulated levels. (4) Donor-reactive DTH sites of allograft rejector mice displayed a broad array of cytokine mRNAs, whereas the DTH sites of allograft acceptor mice displayed only IL-4 mRNA production. (5) Provision of exogenous TGF-beta or IL-10 at a DTH challenge site of allograft rejector mice caused a shift in the cytokine mRNA profile that minimized IFN-gamma and IL-2 mRNA production but spared IL-4, IL-10, and TGF-beta mRNA production. CONCLUSIONS A broad array of cytokine mRNAs may be stockpiled for future use in cardiac allografts, regardless of whether the grafts will be accepted or rejected. This stockpile is continuously replenished for as long as the graft survives, thereby obscuring any changes in immune disposition of the graft recipient toward graft alloantigens. However, such changes can be revealed by challenge with donor alloantigens at a distant site (DTH challenge). In allograft acceptor mice, this disposition evolves from pro-inflammatory to anti-inflammatory.

Mechanisms of Graft Acceptance: Evidence That Plasminogen Activator Controls Donor-Reactive Delayed-Type Hypersensitivity Responses in Cardiac Allograft Acceptor Mice

We have used delayed-type hypersensitivity (DTH) responses to probe the mechanisms of drug-induced cardiac allograft acceptance in mice. DBA/2→C57BL/6 cardiac allograft recipients treated transiently with gallium nitrate accept their grafts for >90 days and fail to display DBA/2-reactive DTH responses. These DTH responses are restored when anti-TGF-β Abs are included at the challenge site, and cell depletion studies showed that this DTH inhibition is mediated by CD4+ cells. Real-time PCR analysis revealed that allograft acceptor mice produce no more than background levels of TGF-β mRNA at DTH challenge sites. This suggests that DTH regulation in allograft acceptor mice may involve TGF-β activation, rather than TGF-β production. The protease, plasmin, can activate TGF-β, and activated T cells can express a receptor for the plasmin-producing enzyme urokinase-type plasminogen activator (uPA), and can also produce both uPA and tissue-type plasminogen activator (tPA). We observed that Abs to tPA or uPA can replace anti-TGF-β mAb for the restoration of donor-reactive DTH responses in allograft acceptor mice. Histologic analysis revealed that accepted cardiac allografts express uPA, tPA, and active TGF-β, whereas accepted cardiac isografts express only tPA, but not uPA or activated TGF-β. These data demonstrate that local tPA and uPA contribute to DTH regulation in allograft acceptor mice and suggest that these elements of the fibrinolytic pathway are used to control donor-reactive cell-mediated immunity in allograft acceptor mice.

Acute Versus Chronic Graft Rejection: Related Manifestations of Allosensitization in Graft Recipients

I n the earlier years of clinical transplantation, there was a tendency among clinicians and investigators to focus on the immediate problem of acute graft rejection, which occurred soon arter transplantation in a large number or transplant recipients and was a major cause ofgraft loss. In the current clinical era, acute rejection can be pharmacologically controlled in most patients, and clinical concerns have begun to focus on the less immediate problem of chronic graft rejection. This phenomenon occurs in about half of all transplant patients, and is a majot cause of late graft loss. Chronic rejection is generally unresponsive to therapies that are effective against acute rejection, and to date, there is no accepted clinical therapy for chronic graft rejection. As a result, chronic rejection now claims more grafts than acute rejection. Chronic rejection is not simply delayed acute rejection. Indeed, acute and chronic rejection are diKerent in many ways. Acute rejection is histologically characterized by a rapid, intense and destructive leukocytic inliltration of graft tissues, and generally represents a pathologic inflammatory response.‘” In contrast, chronic rejection is characterized by a slower development within the graft or interstitial librosis and neointimal formation within the large1 vessels, and seems to represent a pathologic tissue remodeling response .+a For some time, these disparate characteristics led clinicians and investigators to consider acute and chronic rejection as separate and unrelated entities. However, there is developing cvidence that the two pathologies are closely related manifestations of post-transplant cellular and humoral sensitization to graft alloantigens. provided by several investigators,‘* who indcpendently showed that long-term (5-10 year) graft survival decreases signilicantly in direct relation to the number of acute rejection episodes experienced by the patients. For esample, Matas et al” have calculated that the half-life of kidney allografts in patients with no clinical acute rejection episodes is 733 years, compared with 5 1 years in patients with one rejection episode, and 6 years in patients with more than one acute rejection episode. Conversely, these data indicate that late graft loss due to chronic rejection is relatively rare among patients who do not esperience an early acute rejection episode. This suggests that the two types of graft rejection are somehow related. It also suggests that control of chronic rejection in transplant patients might be achieved by developing immunosuppressive strategies that efTectively avoid, rather than reverse, acute rejection episodes. Indeed, the current clinical tendency to accept acute rejection, because it is treatable, may indirectly promote chronic rejection development. The purpose of this discussion is to review some current concepts regarding the related mechanisms of acute and chronic grart rejection in the light of some key observations in experimental transplant models. This is not meant to be a comprehensive review of the subject, and many interesting studies will not be cited for the sake of clarity and brevity. What should emerge from this discussion is a broadened view of transplant biology, and a working model of the continuum of pathologic events associated with acute and chronic graft rejection.

Allograft acceptance despite differential strain-specific induction of TGF-beta/IL-10-mediated immunoregulation.

We examined the immune approaches that C57BI/6 and BALB/c mice take when treated to accept cardiac allografts. C57Bl/6 mice accept DBA/2 cardiac allografts when treated with gallium nitrate (GN) or anti‐CD40L mAb (MR1). These allograft acceptor mice fail to mount donor‐reactive delayed type hypersensitivity (DTH) responses, and develop a donor‐induced immunoregulatory mechanism that inhibits DTH responses. In contrast, BALB/c mice accept C57BI/6 cardiac allografts when treated with MR1 but not with GN. These allograft acceptor mice display modest donor‐reactive DTH responses, and do not develop donor‐induced immune regulation of DTH responses. Real‐time PCR analysis of rejecting graft tissues demonstrated no strain‐related skewing in the production of cytokines mRNAs. In related studies, C57Bl/6 recipients of cytokine and alloantigen educated syngeneic peritoneal exudate cells (PECs) failed to mount DTH responses to the alloantigens unless neutralizing antibodies to transforming growth factor‐beta (TGF‐β were present at the DTH site demonstrating regulation of cell‐mediated alloimmune responses. In contrast, BALB/c recipients of cytokine‐and alloantigen‐educated PECs expressed strong DTH responses to alloantigens demonstrating a lack of regulated alloimmunity. In conclusion, C57BI/6 mice respond to immunosuppression by accepting cardiac allografts and generating TGF‐β‐related regulation of donor‐reactive T cell responses, unlike BALB/c mice that do not generate these regulatory responses yet still can accept cardiac allografts.

Gene therapy in transplantation: pathological consequences of unavoidable plasmid contamination with lipopolysaccharide

Experimental studies evaluated the responses of murine cardiac graft recipients to high and low levels of lipopolysaccharide (LPS) contaminating plasmid DNA preparations. Immediately prior to transplantation, graft recipients were transfected by injecting the quadriceps muscles with plasmids that encoded the murine interleukin (IL)-4 gene and beta-galactosidase (beta-gal) gene. Graft recipients transfected with plasmids encoding only the beta-gal gene served as negative plasmid controls. Three groups of mice were transfected with plasmids containing high levels of contaminating LPS: (a) nontransplanted C57B1/6 mice, (b) C57B1/6 cardiac isograft recipients, (c) DBA/2 (H-2d)-->C57BL/6 (H-2b) cardiac allograft recipients. Unexpectedly, graft failure within 24 h was observed in IL-4 transfected isograft and allograft recipients, but not in mice transfected with the beta-gal gene alone. However, histopathological findings, for example, vascular cell adhesion moelcule-1 (VCAM-1) expression in cardiac grafts and mononuclear lung infiltration, were remarkably similar for both treatment groups and consistent with LPS-induced pathology. LPS assays were used to evaluate four different methods of plasmid purification for degree of LPS contamination. A successful strategy for reducing levels of LPS contamination was identified and transfection experiments repeated in cardiac allograft recipients receiving LPS inoculum that were minimized and standardized (6.4 EU/mouse) for all treatment groups. Despite receiving substantially lower levels of LPS, in all treatment groups there was persistent cardiac graft endothelial cell activation manifested by VCAM-1 expression and persistent, albeit less severe, lung pathology. We found that plasmid contamination with LPS was unavoidable and that even very low levels can alter immune responses in transplant recipients confounding data interpretation. Thus, it is imperative to account for LPS contamination in experiments utilizing plasmid DNA for gene transfer, especially in experimental models of immunity and inflammation.

In vivo immune response to allogeneic hepatocytes

T HE PURPOSE of the following study was to determine the in vivo fate of allogeneic hepatocytes (Hc) in a functional model of hepatocyte transplantation (Hc TX) in mice. The Hc TX model used in this study involved the creation of mice which expressed the transgene human alpha-1-antitrypsin (hAlAT) (under control of the hAlAT promoter) in the liver. Hc isolated from the transgenic donors produce and secrete hAlAT such that detection of the genetic “marker” protein (hAlAT) in recipient serum after Hc TX reflects the survival and intact metabolic function of the transplanted Hc. The advantages of this model include (1) the use of a direct method for monitoring graft function and survival (secretion of hAlAT), (2) histologic confirmation of hepatocellular graft survival can be determined easily by immunohistochemical analysis for Hc bearing the transgene hAlAT, (3) transgenic donor Hc may be transplanted into a variety of murine hosts which allows us to assess the influence of particular immunodeficient states as well as differences in host/donor MHC disparities on hepatocellular graft survival, and (4) permits the distinction between immunologic and nonimmunologic factors (including primary nonfunction, poor engraftment, and senescence of transplanted Hc) which may affect graft function and survival after Hc TX. One potential disadvantage of the model is that the genetic marker protein (hAlAT) may elicit an immune response which adversely affects graft survival. In the current study, this functional model of Hc TX was used to determine the fate of the transplanted Hc in syngeneic, allogeneic, and immunoincompetent hosts.