Subtherapeutic Dose Of Cyclosporine Research Articles

Spiegelmer Inhibition of MCP-1/CCR2--Potential as an Adjunct Immunosuppressive Therapy in Transplantation.

The rejection process remains the key unsolved issue after transplantation of disparate tissue. The CC chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) has been reported to be involved in the process of alloimmune interaction. Spiegelmers are l-oligonucleotides that can be designed to bind to pharmacologically relevant target molecules. Here, we tested a high-affinity Spiegelmer-based MCP-1 inhibitor (mNOX-E36) in an allogeneic heart transplant model. Fully vascularized allogeneic heterotopic heart transplantations from BALB/c to C57BL/6 mice were performed. Mice were either treated with the anti-MCP-1-Spiegelmer (mNOX-E36) in monotherapy or in combination with subtherapeutic doses of cyclosporine A (CsA) (10 mg/kgBW/day) for 10 days. Controls received equivalent doses of a non-functional Spiegelmer (revmNOX-E36). Graft survival of allogeneic heart transplants was slightly but significantly prolonged under mNOX-E36 monotherapy (median graft survival 10 day ± 0.7) compared to revmNOX-E36 (median graft survival 7 day ± 0.3; P = 0.001). A synergistic beneficial effect could be seen when mNOX-E36 was administered in combination with subtherapeutic doses of CsA (18 day ± 2.8 versus 7 day ± 0.3; P < 0.0001). Levels of inflammatory cytokines and 'alarmins' were significantly reduced, and the number of F4/80(+) cells was lower under combination therapy (1.8% ± 1.3%; versus 14.6% ± 4.4%; P = 0.0002). This novel inhibitor of the MCP-1/CCR2 axis (mNOX-E36), which has already proven efficacy and tolerability in early clinical trials, alleviates acute rejection processes in allogeneic transplantation especially when combined with subtherapeutic doses of CsA. Thus, mNOX-E36 may have potential as an adjunct immunomodulatory agent.

Interleukin (IL)-10 Induced by CD11b+ Cells and IL-10-Activated Regulatory T Cells Play a Role in Immune Modulation of Mesenchymal Stem Cells in Rat Islet Allografts

Mesenchymal stem cells (MSCs) are suggested to be immune modulators because of their therapeutic potential in transplantation. In the present study, we evaluated the therapeutic potential of autologous MSCs for preventing graft rejection after allogeneic rat islet transplantation. We assessed the ability of MSCs to elicit an antiproliferative response in alloreactive lymphocytes and tested the immunosuppressive effect of MSCs in allogeneic islet transplantation. In islet allotransplantation, injection of autologous MSCs or a subtherapeutic dose of cyclosporine A (CsA; 5 mg/kg) alone did not prolong allograft survival. However, graft survival was attained for >100 d in 33% of autologous MSC-plus-CsA-treated recipients, indicating that graft acceptance was achieved in a subgroup of allograft recipients. Splenocytes from autologous MSC-plus-CsA-treated rats exhibited a reduced mixed lymphocyte reaction (MLR)-proliferative response to donor stimulators and increased interleukin (IL)-10 release. Interestingly, after excluding host CD11b(+) cells, splenic T cells from autologous MSC-plus-CsA-treated rats did not produce IL-10 or did not inhibit proliferative responses under the same conditions. The use of autologous MSC-plus-CsA downregulated immune responses, inducing donor-specific T-cell hyporesponsiveness by reducing the production of proinflammatory cytokines and inducing antiinflammatory cytokine production, especially that of IL-10, during the early posttransplantation period. T-regulatory cells made a contribution at a later phase. In conclusion, the combined use of autologous MSCs and low-dose CsA exerted a synergistic immunosuppressive effect in an islet allograft model, suggesting a role for autologous MSCs as an immune modulator.

Molecular and cellular pathways involved in the therapeutic functions of MHC molecules; a novel approach for mitigation of chronic rejection

The mutated major histocompatibility complex (MHC) class I that contains donor-type epitopes displayed on recipient-type molecule was show- n to inhibit acute and chronic rejection and in-duce indefinite survival of heterotopic cardiac allografts when administered in combination with a sub-therapeutic dose of cyclosporine (CsA) in a rat transplantation model. To eluci-date the molecular pathways involved in the immunosuppressive effects of the mutated MHC molecule, we analyzedgene and protein expres-sion profile during early and late phase follow-ing post-transplantation. Cytoskeletal structure analysis and expression status of Rho GTPase proteins, vacuolar transport and cytoskeleton regulatory pathways involved in immune re-sponse in T and dendritic cells demonstrated the novel mechanism for the abrogation of chronic rejection. Our studies confirm a new role of Rho GTPase pathway in the modification of T cell motility and infiltration of the graft. We discuss these results within the framework of the most recent literature on MHC and molecu-lar machinery controlling T cell functions and dendritic cell antigen presentation.

Induction of Lymphocyte Apoptosis in Rat Liver Allograft by Adenoviral Gene Transfection of Human Interleukin-10

Background/Aims: Gene therapy can provide a possible avenue in organ transplantation to treat acute allograft rejection. This study was designed to investigate the effect of adenovirus-mediated human IL-10 (hIL-10) gene transfer on the apoptosis of infiltrating lymphocytes and examine the efficacy of hIL-10 gene transfer in combination with subtherapeutic doses of cyclosporine A (CsA) in a rat liver transplantation model. Methods: Inbred male DA and LEW rats were used for liver donors and recipients, respectively. The rats were divided into saline, Ad-lacZ, CsA, Ad-hIL-10 and Ad-hIL-10 + CsA groups. Graft survival, histopathological, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction and flow cytometry were performed in liver specimens obtained from different time points after transplantation in the 5 groups. Results: Ad-hIL-10 pretreatment inhibited allograft rejection, prolonged the survival of hepatic allografts, and downregulated the expression of IFN-γ and IL-2 mRNA, with simultaneous upregulation of IL-4 mRNA. In addition, Ad-hIL-10 pretreatment upregulated the expression of Fas mRNA in the isolated graft-infiltrating lymphocytes and induced graft-infiltrating lymphocyte apoptosis. A single subtherapeutic dose of CsA acted synergistically with it. Conclusion: hIL-10 gene therapy induced alloreactive lymphocyte apoptosis via Fas/FasL pathway. hIL-10 gene transfection in combination with subtherapeutic doses of CsA facilitates the long-term survival of liver grafts.

Down Regulation of Genes Involved in T Cell Polarity and Motility during the Induction of Heart Allograft Tolerance by Allochimeric MHC I

BackgroundThe allochimeric MHC class I molecule [α1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) epitopes displayed on recipient-type (ACI, RT1a) administered in conjunction with sub-therapeutic dose of cyclosporine (CsA) induces indefinite survival of heterotopic cardiac allografts in rat model. In vascularized transplantation models, the spleen contributes to graft rejection by generating alloantigen reactive T cells. The immune response in allograft rejection involves a cascade of molecular events leading to the formation of immunological synapses between T cells and the antigen-presenting cells.Methodology/Principal FindingsTo elucidate the molecular pathways involved in the immunosuppressive function of allochimeric molecule we performed microarray and quantitative RTPCR analyses of gene expression profile of splenic T cells from untreated, CsA treated, and allochimeric molecule + subtherapeutic dose of CsA treated animals at day 1, 3 and 7 of post transplantation. Allochimeric molecule treatment caused down regulation of genes involved in actin filament polymerization (RhoA and Rac1), cell adhesion (Catna1, Vcam and CD9), vacuolar transport (RhoB, Cln8 and ATP6v1b2), and MAPK pathway (Spred1 and Dusp6) involved in tubulin cytoskeleton reorganization and interaction between actin and microtubule cytoskeleton. All these genes are involved in T cell polarity and motility, i.e., their ability to move, scan and to form functional immunological synapse with antigen presenting cells (APCs).ConclusionsThese results indicate that the immunosuppressive function of allochimeric molecule may depend on the impairment of T cells' movement and scanning ability, and possibly also the formation of immunological synapse. We believe that these novel findings may have important clinical implications for organ transplantation.

The Chemokine Receptor Cxcr3 Is Not Essential for Acute Cardiac Allograft Rejection in Mice and Rats

Chemokine receptors have gained attention as potential targets for novel therapeutic strategies. We investigated the mechanisms of allograft rejection in chemokine receptor Cxcr3-deficient mice using a model of acute heart allograft rejection in the strain combination BALB/c to C57BL/6. Allograft survival was minimally prolonged in Cxcr3-deficient mice compared to wild-type (wt) animals (8 vs. 7 days) and treatment with a subtherapeutic dose of cyclosporine A (CsA) led to similar survival in Cxcr3-deficient and wt recipients (13 vs. 12 days). At rejection grafts were histologically indistinguishable. Microarray analysis revealed that besides Cxcr3 only few genes were differentially expressed in grafts or in spleens from transplanted or untransplanted animals. Transcript analysis by quantitative RT-PCR of selected cytokines, chemokines, or chemokine receptors or serum levels of selected cytokines and chemokines showed similar levels between the two groups. Furthermore, in a rat heart allograft transplantation model treatment with a small molecule CXCR3 antagonist did not prolong survival despite full blockade of Cxcr3 in vivo. In summary, Cxcr3 deficiency or pharmacologic blockade does not diminish graft infiltration, tempo and severity of rejection. Thus, Cxcr3 does not appear to play a pivotal role in the allograft rejection models described here.

Efficacy of sirolimus (rapamycin) administered concomitantly with a subtherapeutic dose of cyclosporin in the treatment of severe psoriasis: a randomized controlled trial

Background The identification of a highly potent immunosuppressive/antiproliferative agent with an acceptable toxicity profile has long been a goal for the management of severe plaque psoriasis. Objectives To investigate the efficacy and safety of sirolimus (Rapamune®) for severe psoriasis when given alone or in association with cyclosporin. Methods In a randomized, double-blind, eight parallel group, pilot study in 24 out-patient centres in seven European countries, 150 patients, 18 years and older, with severe chronic plaque psoriasis were given sirolimus 0·5, 1·5 and 3·0 mg m−2 daily for 8 weeks, either alone or in association with a subtherapeutic dose of cyclosporin (1·25 mg kg−1 daily). Cyclosporin 5 mg kg−1 daily was the positive control and cyclosporin 1·25 mg kg−1 daily the negative control. The primary efficacy variable was the mean percentage reduction in Psoriasis Area and Severity Index (PASI). Safety assessments included monitoring of adverse events, clinical laboratory parameters and sirolimus/cyclosporin blood concentrations. Results The greatest mean percentage decreases in PASI were seen with cyclosporin 5·0 mg kg−1 daily (70·5%) and with sirolimus 3·0 mg m−2 daily + cyclosporin 1·25 mg kg−1 daily (63·7%). Both groups demonstrated significantly better results than cyclosporin 1·25 mg kg−1 daily (mean decrease 33·4%). Serum creatinine levels were significantly lower for groups with sirolimus alone and sirolimus plus reduced-dose cyclosporin when compared with cyclosporin 5·0 mg kg−1 daily. Adverse events associated with sirolimus included thrombocytopenia (5%), hyperlipidaemia (9%), aphthous stomatitis (9%) and acne (13%), whereas adverse events associated with cyclosporin included hot flushes (12%), hyperlipidaemia (9%) and increased serum creatinine (9%). Conclusions The concomitant administration of sirolimus with a subtherapeutic dose of cyclosporin in severe psoriasis may permit a reduction in their respective toxicities, notably cyclosporin-induced nephrotoxicity.

C. sinensis ablates allograft vasculopathy when used as an adjuvant therapy with cyclosporin A

Immunosuppressive treatments are available to suppress acute cardiac rejection; however, no viable treatment exists for long-term cardiac graft failure. Moreover, extended use of calcineurin inhibitor immunosuppressants, the mainstay of the current therapeutic for cardiac transplantation, leads to significant associated pathologies such as nephrotoxicity and increased risk of cardiac disease. For the last ten years alternatives to calcineurin inhibitors, or adjuvant therapies designed to complement their activities, have been explored. In tandem with this development, there has been considerable interest in Traditional Chinese Medicines (TCM) as sources for novel therapeutics. Our study examines the ability of the TCM Cordyceps sinensis to reduce acute and chronic rejection associated with cardiac transplantation. The objectives of this study were to first determine if oral delivery of the extract could reduce acute rejection in a rat heterotopic heart model of transplantation. The second objective was to determine, in vitro, if a sterile, aqueous extract of C. sinensis could decrease CD8+ T cell activity. The third objective was to determine if oral delivery of the extract could ablate allograft vasculopathy in a mouse abdominal aortic transplant model. We found that oral delivery of the extract demonstrated a reduction in acute rejection when used in conjunction with a sub-therapeutic dose of Cyclosporine. Further, we found, using a mixed lymphocyte reaction, that the extract was able to significantly reduce CD8+ T cell activity. Finally, we demonstrate that oral delivery of the extract, used with a therapeutic dose of Cyclosporine to suppress acute rejection, ablates allograft vasculopathy.

Use of Sphingosine-1-Phosphate 1 Receptor Agonist, KRP-203, in Combination with a Subtherapeutic Dose of Cyclosporine A for Rat Renal Transplantation

We demonstrate the long-term effectiveness of KRP-203 treatment in combination with a subtherapeutic dose of cyclosporine A (CsA) on rat renal allografts. We tested the effect of KRP-203 in combination with CsA using a rat skin allograft model. The Pharmacokinetic interaction between CsA and KRP-203 was evaluated. The selectivity of KRP-203 for sphingosine-1-phosphate (S1P)1 and S1P3 receptors were investigated in vitro. Heart rate alteration following bolus injection of phosphorylated KRP-203 (KRP-203-P) or FTY720 (FTY720-P) was also monitored in rats. Finally, the long-term effectiveness of KRP-203 in conjunction with a low dose of CsA was investigated in a rat renal transplantation model. Administration of KRP-203 with CsA prolonged skin allograft survival. KRP-203 and CsA had no effect on the pharmacokinetics of the other. While FTY720-P activated both S1P1 and S1P3 receptors, KRP-203-P selectively activated S1P1, but not the S1P3 receptor (EC50:>1000 nM). Compared to FTY720-P, a tenfold higher dose of KRP-203-P was necessary to induce transient bradycardia. With a low dose of CsA (1 mg/kg/day), KRP-203 (0.3 mg/kg/day) significantly prolonged renal allograft survival (P<0.05, survival time: 9.8 days (CsA) vs. >27.4 days (CsA+KRP)). Although a higher dose of CsA (3 mg/kg/day) alone kept recipients alive, this caused severe renal graft dysfunction. Use of KRP-203 (3 mg/kg/day) in conjunction with CsA markedly improved graft function (P<0.05, creatinine clearance: 0.41+/-0.25 ml/min [CsA] vs. 1.15+/-0.16 ml/min [CsA+KRP]). The selectivity of KRP-203 for S1P1 reduces the risk of bradycardia, and the combination therapy of KRP-203 with CsA represents a safe and effective strategy for use in renal transplantation.

KRP-203, a Novel Synthetic Immunosuppressant, Prolongs Graft Survival and Attenuates Chronic Rejection in Rat Skin and Heart Allografts

A novel immunomodulator, KRP-203, the molecular structure of which has some similarity to FTY720, has been developed for use in organ transplantation. The present study was designed to investigate the potency and safety of KRP-203 on allograft survival against both acute and chronic rejection in rat skin and heart transplantation. KRP-203 significantly prolonged skin or heart allograft survival of a minor histocompatibility complex (mHC)-disparate (LEW to F344) rat combination. Histopathological and immunohistochemical analysis at 100 days after mHC-disparate rat heart transplantation revealed that KRP-203 treatment significantly inhibited infiltration of inflammatory cells, including macrophages and T cells; expression of endothelin-1 and transforming growth factor-beta1; and IgG deposition and eventually attenuated neointimal formation and myocardial fibrosis. KRP-203 also prolonged heart allograft survival in a major histocompatibility complex (MHC)-incompatible (DA to LEW) rat combination, but the efficacy was not as significant. However, KRP-203 combined with a subtherapeutic dose of cyclosporin A synergistically prolonged the heart allograft survival. Flow cytometric analysis demonstrated that KRP-203 reduced the number of peripheral blood mononuclear cells (lymphocytes and monocytes) but not granulocytes and enhanced lymphocyte homing into peripheral lymph nodes. The influence of KRP-203 on heart rate changes in Hartley guinea pigs was examined. KRP-203 had less of a tendency to cause bradycardia than FTY720. These findings demonstrated that KRP-203 prolonged skin and heart allograft survival and significantly attenuated chronic rejection and bradycardia as an adverse effect. Therefore, KRP-203 offers considerable potential as a novel therapeutic immunosuppressant in patients with organ transplantation.

A novel immunomodulator KRP-203 combined with cyclosporine prolonged graft survival and abrogated transplant vasculopathy in rat heart allografts

To find more effective and less toxic immunosuppressive strategies in long-term treatment for organ transplantation patients, we examined the effects on rat heart allograft survival of a novel sphigosine-1-phosphate receptor agonist, KRP-203, combined with a subtherapeutic dose of cyclosporine (CsA). Rat heart transplantation was performed across a major histocompatibility complex-incompatible (DA to LEW) rat combination. KRP-203 alone showed little or no effect on heart allograft survival. In contrast, KRP-203 combined with a subtherapeutic dose of CsA led to prolonged allograft survival. Histologic analyses showed that the combination completely suppressed acute rejection, as characterized by allograft vasculopathy, mononuclear cell infiltration, and myocardial necrosis in the heart allografts. RT-PCR analysis showed that the allografts treated with CsA or KRP-203 alone showed no suppression of IL-10, IFN-γ, and TNF-α mRNA expression, but when combined with a subtherapeutic dose of CsA it completely suppressed their mRNA expressions. Furthermore, the combination treatment reduced donor-specific antibody production. KRP-203 combined with a subtherapeutic dose of CsA synergistically prolonged rat heart allograft survival. The combination of CsA with KRP-203 may provide an option to prevent allograft rejection and reduce adverse effects.

Prevention of lung allograft rejection by combined treatment with adhesion molecule antibodies and cyclosporine

Infiltration of leukocytes into the lung allograft is regulated by adhesion molecules during acute rejection. The purpose of this study was to assess the effect of monoclonal antibodies against ICAM-1 (1A29) to prevent rejection after lung transplantation. Left lateral orthotopic lung transplantation was performed using Dark Agouti rats as donors and Lewis rats as recipients. Recipients received 1A29 alone (group A), cyclosporine A alone (group B), a combination of both drugs (group C) or no therapy (group D). Animals were killed on day 5 and 10, respectively. Rejection was graded by histology. Myeloperoxidase activity (MPO) was measured in the graft. In allografts treated with cyclosporine and 1A29 histologically a lower grade of rejection was seen and less MPO were detected compared to groups A, B and D. Anti-ICAM-1 monoclonal antibodies alone as well as a subtherapeutic dose of cyclosporine are not effective to prevent acute allograft rejection after lung transplantation. However, the combination of both strategies significantly reduces rejection in this model.

A novel nitric oxide scavenger in combination with cyclosporine A ameliorates experimental autoimmune encephalomyelitis progression in mice

Immunotherapy improves experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), while excessive production of nitric oxide (NO) has been implicated in the pathogenesis of this disease. Here, we show that disease progression in SJL/J mice with EAE is improved after treatment with either a subtherapeutic dose of cyclosporine A (CsA) or NOX-100, a nitric oxide scavenger. Importantly, the impact of subtherapeutic doses of CsA in combination with NOX-100 on disease progression in EAE was greater than that attained with either agent alone and led to near total protection. CNS inflammation and gene expression of proinflammatory cytokines and iNOS were also significantly reduced after treatment. These observations point to the potential therapeutic utility of NOX-100 as a dose-reducing agent for CsA in the treatment of MS.

Chemokines and allograft rejection: narrowing the list of suspects.

Chemokines and allograft rejection: narrowing the list of suspects.

Efficacy of sirolimus (rapamycin) administered concomitantly with a subtherapeutic dose of cyclosporin in the treatment of severe psoriasis: a randomized controlled trial

The identification of a highly potent immunosuppressive/antiproliferative agent with an acceptable toxicity profile has long been a goal for the management of severe plaque psoriasis. To investigate the efficacy and safety of sirolimus (Rapamune) for severe psoriasis when given alone or in association with cyclosporin. In a randomized, double-blind, eight parallel group, pilot study in 24 out-patient centres in seven European countries, 150 patients, 18 years and older, with severe chronic plaque psoriasis were given sirolimus 0.5, 1.5 and 3.0 mg m(-2) daily for 8 weeks, either alone or in association with a subtherapeutic dose of cyclosporin (1.25 mg kg(-1) daily). Cyclosporin 5 mg kg(-1) daily was the positive control and cyclosporin 1.25 mg kg(-1) daily the negative control. The primary efficacy variable was the mean percentage reduction in Psoriasis Area and Severity Index (PASI). Safety assessments included monitoring of adverse events, clinical laboratory parameters and sirolimus/cyclosporin blood concentrations. The greatest mean percentage decreases in PASI were seen with cyclosporin 5.0 mg kg(-1) daily (70.5%) and with sirolimus 3.0 mg m(-2) daily + cyclosporin 1.25 mg kg(-1) daily (63.7%). Both groups demonstrated significantly better results than cyclosporin 1.25 mg kg(-1) daily (mean decrease 33.4%). Serum creatinine levels were significantly lower for groups with sirolimus alone and sirolimus plus reduced-dose cyclosporin when compared with cyclosporin 5.0 mg kg(-1) daily. Adverse events associated with sirolimus included thrombocytopenia (5%), hyperlipidaemia (9%), aphthous stomatitis (9%) and acne (13%), whereas adverse events associated with cyclosporin included hot flushes (12%), hyperlipidaemia (9%) and increased serum creatinine (9%). The concomitant administration of sirolimus with a subtherapeutic dose of cyclosporin in severe psoriasis may permit a reduction in their respective toxicities, notably cyclosporin-induced nephrotoxicity.

Prolongation of allograft survival with viral IL-10 transfection in a highly histoincompatible model of rat heart allograft rejection.

The ability to express genes with potential immunoregulatory capacity could reduce the immunogenicity of allografts and result in long-term graft survival. In this study, we examine the feasibility of transferring viral interleukin-10 (vIL-10) gene into rat hearts using adenovirus by intracoronary administration. The subsequent effects of delivered vIL-10 alone or with subtherapeutic doses of cyclosporine A (CsA) on parameters of allograft rejection (AR) were also examined. Recombinant adenovirus vectors containing vIL-10 (Ad-vIL-10) or beta-galactosidase (Ad-beta-gal) were derived from adenovirus type 5. vIL-10 expression in supernatants of transfected COS7 cell cultures and in transfected heart allografts were examined by enzyme immunoassay (EIA) and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Rat heart transplants (LEWS->ACI) were performed in five groups [group 1: no treatment, group 2: Ad-beta-gal, group 3: AdvIL-10, group 4: CsA (10 mg/kg), and group 5: Ad-vIL10+CsA (10 mg/kg)]. Allograft survival was determined by palpating heartbeats. Allograft tissues were also submitted for histological study. vIL-10 expression was shown in both transfected COS7 cells and heart isografts. Animals transfected with vIL-10 showed prolongation of graft survival (19.6 vs. 12 days, P<0.001) when compared to beta-gal transfected controls. Animals treated with a single low dose injection of CsA showed no significant prolongation of graft survival compared to controls (11.7 vs. 10.5 days). Animals treated with both vIL-10 and CsA demonstrated a synergistic prolongation of allograft survival compared with controls and with animals treated with CsA or vIL-10 treatment alone (36.7 days vs. 11.7, P<0.001 or 36.7 vs.19.6, P<0.001, respectively). Histological study showed that allografts from untreated controls exhibited extensive AR with loss of graft architecture by day 7 posttransplant while those from the vIL-10 group showed less AR. The best pathological scores were seen in vIL-10 + CsA-treated animals. 1) Delivering Ad-vIL-10 into donor hearts by intracoronary perfusion results in overexpression of vIL-10 and significantly prolongs cardiac allograft survival in a highly histoincompatible rat model. 2) Subtherapeutic doses of CsA do not prolong allograft survival, but act synergistically with vIL-10 to significantly prolong graft survival beyond that achieved with either agent alone.

A Non-peptide Functional Antagonist of the CCR1 Chemokine Receptor Is Effective in Rat Heart Transplant Rejection

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

Prolongation of canine liver allograft survival by a novel immunosuppressant, FTY720: effect of monotherapy and combined treatment with conventional drugs.

The immunosuppressive effect and other properties of a novel immunosuppressant, FTY720, have been studied mostly in the experimental transplantation of various extrahepatic organs. In this experiment, we evaluated the antirejection potency and adverse effects of this agent on liver grafts using a canine liver transplantation model. Forty-eight orthotopic liver transplantations were performed by the standard technique under a veno-venous bypass. Liver recipients were divided into two studies: a single-dose study with FTY720 at various doses and a combined dose study with conventional immunosuppressants (cyclosporine or tacrolimus) alone and combined with FTY720. Survival, biochemical and hematological tests, blood levels of immunosuppressants, and postmortem histology were determined. The median survival of untreated control animals was 9 days, whereas treatment with FTY720 at a dose of 0.1 mg/kg/day prolonged graft survival to 49.5 days. FTY720 at 1 mg/kg/day showed a slight but insignificant prolongation to 16 days, but when the dose was increased to 5 mg/kg/day, the graft was rejected at 10 days. The combination of FTY720, 0.1 mg/kg/day, with a subtherapeutic dose of cyclosporine, 5 mg/kg/ day, prolonged median animal survival from 40 days with cyclosporine alone to 74 days. A combination of FTY720 (0.1 mg/kg/day) with tacrolimus (0.5 mg/kg/ day) compromised animal survival, reducing survival from 83.5 days with tacrolimus alone to 30.5 days due to infectious complication and emaciation by overimmunosuppression. No evident drug-induced side effects were observed. FTY720 has a potent immunosuppressive effect when used alone at 0.1 mg/kg/day in canine liver transplantation. FTY720 is a promising candidate for future clinical application in orthotopic liver transplantation.

In vitro and in vivo immunomodulatory effects of RDP1258, a novel synthetic peptide.

Peptides derived from certain regions of human class I MHC molecules are known to have immunomodulatory effects. In particular, amino acid residues 75-84 of the HLA-B7 and HLA-B2702 molecules have demonstrated allele nonspecific immunosuppression in several animal transplant models. There is evidence that these effects are mediated by binding to intracellular heat shock proteins, including heme oxygenase-1. A new derivative of these peptides, RDP1258, was developed using a novel computer-assisted rational design technique. In vitro, RDP1258 peptide inhibited rat heme oxygenase activity in a dose-dependent manner. Similar to observations made with other in vitro heme oxygenase inhibitors, in vivo administration of RDP1258 peptide to naive rats resulted in upregulation of splenic heme oxygenase activity. The effects of the peptide on alloimmune responses were then tested. Addition of RDP1258 to rat and human mixed leukocyte reactions inhibited proliferation in a dose-dependent manner. In a rat renal transplantation model, peptide therapy combined with a sub-therapeutic dose of cyclosporin A significantly prolonged allograft survival. These data provide further evidence that modulation of the heat shock protein heme oxygenase by rationally designed peptides affects immune effector functions and may allow the development of novel immunomodulatory strategies in organ transplantation.

PROPHYLAXIS OF RENAL ALLOGRAFTS FROM REJECTION BY FTY720 IN CYNOMOLGUS MONKEYS TREATED WITH SUBTHERAPEUTIC DOSES OF CYCLOSPORINE

378 Background: The new immunosuppressive drug FTY720 is a modification of a natural compound extracted from the fungus Isaria sinclairii. FTY720 therapy significantly reduces the number of circulating peripheral blood lymphocytes (PBL). The present study examined the in vivo effect of FTY720 in combination with subtherapeutic doses of cyclosporine (CsA) on the survival of kidney allografts in cynomolgus monkeys. Methods: Monkeys transplanted with highly incompatible kidney allografts were treated with daily intramuscular (i.m.) injections of CsA that were adjusted to maintain the blood levels between 40-200 ng/ml. In three experimental groups, recipients received in addition to CsA either 0.1, 0.3, or 1.0 mg/kg/day FTY720 delivered daily by intravenous (i.v.) bolus injections. Results: Recipients treated with CsA alone rejected kidney allografts at a median survival time of 8.5 days (n=4). Recipients that received in addition to subtherapeutic CsA therapy either 0.1 or 0.3 mg/kg/day FTY720 showed significant prolongation of kidney allograft survivals to 71 days (range 36-109 days; n=3; p<0.04) and 63 days (range 10-125 days; n=5; p<0.05), respectively. Transplant biopsy on day 7 in animals treated with 0.1 mg/kg/day FTY720 documented a mild to moderate grade I rejection with focal areas of tubular destruction. Two out of three recipients displayed increased creatinine levels at day 20, and the third recipient at day 41, postgrafting. In contrast, biopsy on day 7 in all five transplants from recipients treated with 0.3 mg/kg/day FTY720 had only a minimal interstitial inflammatory infiltrates without evidence of tubular or arterial damage. Consequently, three out of five recipients in the latter group maintained low creatinine levels up to 45 days postgrafting; in addition, one of these recipients displayed stable kidney function for 125 days. Although recipients treated with CsA and 1.0 mg/kg/day FTY720 had a median of 48 days (range 9-73 days; n=5), two out of five recipients succumbed within 9 and 17 days postgrafting, suggesting possible complications caused by over-immunosuppression. Blood FTY720 through concentrations correlated with significantly decreased levels of PBLs.Conclusions: Addition of FTY720 therapy to a subtherapeutic CsA immunosuppressive regimen effectively blocks the rejection of renal allografts in monkeys.