Meike Schaub

Inhibition of CD26/dipeptidyl peptidase IV activity in vivo prolongs cardiac allograft survival in rat recipients

The CD26 antigen, one of the major costimulatory molecules in T cell activation, was shown to possess dipeptidyl peptidase IV (DPP IV) activity. Previously, we demonstrated that immunosuppressed kidney transplant patients exhibit lower DPP IV serum activity as compared with healthy individuals. In the present study, we analyzed the role of CD26/DPP IV in the immune cascade triggered by organ transplantation and leading to acute rejection of cardiac allografts in rat recipients. Transplantation of hearts from (Lewis x Brown Norway)F1 donors into Lewis hosts resulted in an early (24 hr) increase in cellular CD26 expression, followed by a rise in DPP IV serum activity, which peaked at day 6, i.e., before the time of actual graft loss. Specific targeting of DPP IV activity with a novel, low-molecular-weight inhibitor of the diphenyl-phosphonate group (prodipine) abrogated acute rejection and prolonged cardiac allograft survival to 14.0+/-0.9 days (P<0.0001). Prodipine treatment prevented the early peak of cellular CD26 expression and thoroughly suppressed systemic DPP IV activity. The inhibition of DPP IV was associated with severely impaired host cytotoxic T lymphocyte responses in vitro. These results demonstrate the role of CD26/DPP IV in alloantigen-mediated immune regulation in vivo and provide the first direct evidence that CD26/DPP IV plays an important role in the mechanism of allograft rejection. The model of targeting CD26/DPP IV may reveal essential interactions on the level of costimulatory alternate T cell activation pathways, allowing a more subtle approach for more selective immunosuppression in transplant recipients.

Prevention of Cold‐Preservation Injury of Cultured Endothelial Cells by Catecholamines and Related Compounds

The present study was conducted to dissect the underlying mechanisms by which catecholamines protect cells against preservation injury.

Costimulatory signal blockade in murine relapsing experimental autoimmune encephalomyelitis

Blockade of the CD28-B7 or CD40L-CD40 T cell costimulatory signals prevents induction of experimental autoimmune encephalomyelitis (EAE). However, the effect of simultaneous blockade of these signals in EAE is unknown. We show that administration of either MR1 (to block CD40L) or CTLA4Ig (to block B7) after immunization or after the first attack protects from EAE. Treatment with a combination of CTLA4Ig and MR1 provides additive protection, and is associated with complete absence of mononuclear cell infiltrates in the central nervous system, and marked suppression of proliferation of primed T cells in the periphery. Selective B7-1 blockade did not protect from EAE. These observations have implications for therapy of autoimmune diseases.

Immune parameters affecting adenoviral vector gene therapy in the brain

Gene therapy utilizing replication deficient adenoviral vectors represents a potentially promising approach to the treatment of brain tumors. Limited duration of systemic transgene expression and inefficient transduction following repeat systemic vector administration secondary to an effective anti-vector immune response limits the potential application of first generation adenoviral vectors. Whether host immune responses will significantly limit the use of these vectors within the immunopriviledged environment of the central nervous system remains to be elucidated. Following a single intravenous injection of a beta-galactosidase expressing adenoviral vector (Ad.CMV-betagal), we found maximal betagal transgene expression in systemic sites (i.e. liver) at day 4, with almost complete disappearance by day 7. In contrast, significant beta-galactosidase activity was seen for greater than 28 days following a single intracerebral inoculum of virus. Rechallenge experiments demonstrated complete protection against repeat systemic vector administration, whereas intracerebral transgene expression was not affected by prior systemic or intracerebral exposure to adenoviruses. These data suggest that systemic anti-adenoviral vector immune responses are attenuated within the central nervous system and may not pose as significant a problem for the treatment of brain tumors as for other systemic indications.

Treatment of chronic renal allograft rejection in rats with a low-molecular-weight heparin (reviparin).

BACKGROUND Low-molecular-weight heparin (LMWH) has been shown to prolong survival of rat cardiac allografts independently from immunosuppressive treatment. Furthermore, long-term treatment reduces the development of chronic graft vascular disease after experimental heart transplantation. The aim of the present study was to determine whether treatment with the LMWH reviparin has a beneficial effect on chronic rejection in a rat renal allograft model. METHODS Kidneys of Fisher (F344) rats were transplanted into unilaterally nephrectomized Lewis (LEW) recipients. LEW-->LEW isografts served as controls. Animals were treated with cyclosporine (5 mg/kg/d) for the first 10 days. Nephrectomy of the remaining kidney was performed after 10 days. Allografted animals were treated either with reviparin (2 mg/kg/d subcutaneously) for 24 weeks (Allo-24), from week 12 to 24 (Allo-12), or with vehicle for 24 weeks. Proteinuria was determined at regular intervals. Kidneys were harvested after 24 weeks for histomorphological and immunohistochemical evaluation. RESULTS No major bleeding complications were observed in reviparin-treated animals. Proteinuria was significantly reduced in allografted animals both by early as well as by late-onset treatment with reviparin. Transplant glomerulopathy was diminished in Allo-24 and in Allo-12 groups compared to vehicle-treated animals, whereas tubulointerstitial inflammation was influenced only in animals immediately treated with reviparin. Immunohistochemical studies demonstrated a marked reduction of renal monocyte and T-cell infiltration as well as expression of MHC II by treatment with reviparin. CONCLUSIONS Treatment with the LMWH reviparin significantly improved chronic renal allograft rejection in the F344-to-LEW rat model, both after early and late start of therapy. Although the exact mechanisms of this beneficial effect remain unclear, our data offer a potential new therapeutical approach for prevention of chronic allograft nephropathy.

Intrathymic immunomodulation in sensitized rat recipients of cardiac allografts: requirements for allorecognition pathways

Abstract Background: Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models. We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days. The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection. Methods Lewis RT1 1 (LEW) rats sensitized by transplantation with Wistar-Furth RT1 u (WF) skin grafts received WF cardiac allografts 7 days later—a classic model of accelerated rejection. At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides. Results Control animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours. Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6 ± 0.6 days ( p n cells was ineffective in this regard. Intrathymic injection of γ-irradiated donor cells marginally extended cardiac allograft survival to 3.0 ± 0.9 days ( p Conclusion The superiority of non-modified donor spleen cells over γ-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients. Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.

The influence of antigen-dependent and antigen-independent factors on the development of graft vasculopathy in a fully allogeneic cardiac allograft model in the rat.

Graft vasculopathy (GVP) is one of the major obstacles to long-term graft and patient survival after cardiac transplantation and a major reason for morbidity and mortality. Antigen-dependent and antigen-independent factors play causal roles in the development of GVP. The aim of this study was to evaluate antigen-dependent and -independent factors in the development of GVR in a clinically relevant fully allogeneic rat cardiac model under immunosuppression with cyclosporine (CyA). Lewis rats were challenged with Wistar-Furth cardiac allografts. Acute rejection occurred within 10 days after engraftment (n = 6). Daily SC administration of CyA (2.5 mg/kg body weight, n = 12) led to long-term graft survival (>100 days) but did not prevent GVP (Adams Score: 1.7 +/- 1.9, n = 4). Isografts did not develop GVP. In allografts, the dose modification of CyA to 5 mg or 1.25 mg/kg body weight as well as the prolongation of ischemia from 45 minutes to 4 hours did not increase the development of GVP. In isografts, the prolongation of ischemic time from 45 minutes to 4 hours significantly increased the development of GVP (Adams score, 0.3 +/- 0.8 [n = 7] vs 1.2 +/- 1.9 [n = 6]; P < .05). In this fully allogeneic cardiac allograft model with clinically relevant immunosuppressive therapy, GVP was induced independent of the applied CyA dose. In addition, the prolongation of ischemic time did not increase the development of GVP. Isografts only developed significant GVP with long ischemia times. Therefore, an initial injury, either prolonged ischemia time or an allogeneic immune response, predispressed to the development of GVP.

FR167653 Ameliorates Expression of Proinflammatory Mediators in Human Umbilical Venous Endothelial Cells and Human Monocytes

p38MAP kinase plays a crucial role in intracellular signal transduction of inflammation. The inhibitor of p38 MAP kinase, FR167653, has been proven to be effective to suppress proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in various animal models. The aim of our study was to investigate p38MAP kinase inhibition by FR167653 on the inflammatory profile of cells involved in vascular injury. HUVEC incubated with FR167653 in concentrations of 0.1 to 20 mumol for 24 hours were stimulated with TNF-alpha (20 ng/mL). Human monocytes were incubated with equal concentrations of FR167653 and stimulated with lipopolysaccharides (LPS; 10 microg/mL). In monocytes, p38 MAP kinase could be inhibited by FR167653 (Western blot). The cytokines IL-6 and IL-8 were dose dependently downregulated by FR167653 (enzyme-linked immunosorbent assay) [ELISA]. These results were confirmed at a transcriptional level by real-time polymerase chain reaction (PCR). Gene expression of IL-6 and IL-8 was dose dependently downregulated. The expression pattern of ICAM-1 and VCAM-1 was not altered by FR167653 (ELISA). In HUVEC, the cytokines IL-6 and IL-8 were dose dependently downregulated by FR167653 (ELISA). These results were confirmed on a transcriptional level by real-time PCR. Gene expression of IL-6 and IL-8 were also dose dependently suppressed by FR167653. In addition FR167653 downregulated the expression of the adhesion molecules ICAM-1 and VCAM-1 (ELISA). FR167653 suppressed the development of a proinflamatory profile of HUVEC and human monocytes after stimulation with TNF-alpha or LPS, respectively. These results indicated anti-inflammatory properties of FR167653 on endothelial and inflammatory cells, which may be therapeutically useful to ameliorate vascular injury.

Intrathymic immunomodulation of sensitized rat recipients of cardiac allografts: Requirements for allorecognition pathways☆

BACKGROUND Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models. We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days. The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection. METHODS Lewis RT1(1) (LEW) rats sensitized by transplantation with Wistar-Furth RT1(u) (WF) skin grafts received WF cardiac allografts 7 days later-a classic model of accelerated rejection. At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides. RESULTS Control animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours. Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6+/-0.6 days (p<0.001), whereas intrathymic administration of syngeneic (LEW) or allogeneic third party Brown Norway RT1(n) cells was ineffective in this regard. Intrathymic injection of gamma-irradiated donor cells marginally extended cardiac allograft survival to 3.0+/-0.9 days (p< 0.001), but the grafts were still rejected in an accelerated fashion. Intrathymic injection of donor-derived class I and/or class II MHC allopeptides at the same time period also failed to prolong cardiac allograft survival beyond 3 days. In the group receiving unmodified donor cells, elevated immunoglobulin M (IgM) and immunoglobulin G (IgG) allo-antibodies were found at the time of cardiac transplantation; this pattern was not observed with any other treatment. CONCLUSION The superiority of non-modified donor spleen cells over gamma-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients. Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.

Apoptotic Cell Death During Accelerated Rejection in Sensitized Rat Recipients of Cardiac Allografts

Accelerated rejection due to host sensitization to major histocompatibility complex antigens is a critical problem in clinical organ transplantation in patients who have previously received an organ transplant, experienced acute rejection episodes, received blood transfusions, or been pregnant. The precise pathologic mechanisms underlying accelerated rejection have not been characterized. Herein, we describe apoptosis during T- and B-cell-driven accelerated rejection of cardiac allografts in presensitized recipients. In an established accelerated rejection model, Lewis rats were sensitized to skin grafts from Wistar-Furth rats; after 7 days, they received Wistar-Furth hearts. These grafts were rejected within 24 hours posttransplantation compared with 10 days in nonsensitized recipients (acute rejection, n = 5). Apoptosis was observed during accelerated rejection of cardiac allografts but not in naïve recipients of hearts, as demonstrated at DNA laddering and TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling) assay. Apoptosis was discovered as a thus far unknown effector mechanism in accelerated cardiac transplant rejection that accompanies combined cellular and humoral immune alloreactivity. Apoptotic cell death in accelerated rejection and the cascade of upstream and downstream events leading to or resulting from this process should be considered critical steps in the pathogenesis of accelerated rejection.