Purpose: HMG-CoA reductase inhibitors, statins, are used to treat patients for elevated serum cholesterol and triglycerides. Statins have been reported to improve heart and renal allograft survivals. Statins may also be immunomodulatory, in that they may reduce the presence of lymphocyte reactive antibodies, positive recipient-donor crossmatches and alter the expression of HLA antigens.

STATs are believed to play key roles in normal and abnormal cell function. In the present work, we investigated the role of STATs in an IL-2-responsive human lymphoblastic lymphoma-derived cell line, YT. Only STAT3 was found constitutively tyrosine phosphorylated, but not other STATs. Hyperactive STAT3 was not attributable to a pre-existing intermediate affinity IL-2R complex and/or hyperactive Jak activity. Depletion of STAT3 protein expression reduced tumor cell viability with protracted kinetics (72–96 h), while TUNEL assays demonstrated cell death occurred via apoptosis. Interestingly, depletion of STAT5 in this same tumor induced more pronounced cell death compared with STAT3 depletion (24 h). Although IL-2 was able to rescue STAT3-depleted cells from death, it could not compensate for the loss of STAT5. To determine the prosurvival function of STAT3 vs STAT5 within the same tumor model, genes were profiled in STAT3- or STAT5-depleted YT cells by apoptosis-specific microarrays. Several differentially expressed genes were identified. Interestingly, those genes involved in NF-κB regulation, such as TNFR-associated factors 2 and 5 and B cell leukemia/lymphoma 10, were readily decreased upon STAT5, but not STAT3, depletion as validated by quantitative RT-PCR. These results suggest that STAT5 and, to a lesser extent, hyperactive STAT3 provide preferential and critical cell survival signals for certain human lymphoid tumors, indicating that nonhyperactive STATs should be considered as therapeutic targets for abrogating tumorigenesis.

We have previously shown that the common feature of both pressure overload-induced hypertrophy and atrophy is a reactivation of the fetal gene program. Although gene expression profiles and signal transduction pathways in pressure overload hypertrophy have been well studied, little is known about the mechanisms underlying atrophic remodeling of the unloaded heart. Here, we induced atrophic remodeling by heterotopic transplantation of the rat heart. The activity parameters of three signal transduction pathways important in hypertrophy, i.e. mitogen-activated protein (MAP) kinase, mammalian target of rapamycin (mTOR), and Janus kinase/signal transducers and activators of transcription (JAK/STAT), were interrogated. Gene expression of upstream stimuli – insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) – and metabolic correlates, i.e. peroxisome proliferator-activated receptor-α (PPARα) and PPARα-regulated genes, of these pathways were also measured. In addition, we measured transcript levels of genes known to regulate skeletal muscle atrophy, all of which are negatively regulated by IGF-1 (Mafbx/Atrogin-1, MuRF-1). Atrophic remodeling of the heart was associated with increased expression of IGF-1 and FGF-2. Transcript levels of the nuclear receptor PPARα were decreased, as were the levels of PPARα-regulated genes. Furthermore, there was phosphorylation of ERK1, STAT3, and p70S6K with unloading. Consistent with the increase in IGF-1, we found a decrease in Mafbx/Atrogin-1 and MuRF-1 transcript levels. Rapamycin administration at 0.8 mg/kg/day for 7 days resulted in enhanced atrophy and attenuated the phosphorylation of ERK1, STAT3, and p70S6K without altering gene expression. We conclude that there is significant crosstalk between the mTOR, MAP kinase, and JAK/STAT signaling cascades. Furthermore, ubiquitin ligases, known to be essential for skeletal muscle atrophy, decrease in unloading-induced cardiac atrophy.

Sirolimus (SRL) has been shown to exacerbate cyclosporine (CsA)-induced nephrotoxicity. The expression of the kidney injury molecule-1 (KIM-1) is markedly upregulated in the postischemic rat kidney. We sought to correlate drug-induced nephrotoxicity and the expression of KIM-1 and aquaporin-2 (AQP-2) in male PVG rats with 2 kidneys (2K), 1 kidney (1K), and half a kidney (1/2K) treated with SRL alone, CsA alone, or a combination of both (SRL-CsA). After 7 days of treatment, the 2K group treated with SRL-CsA showed a significant decrease in creatinine clearance compared with the 2K SRL alone and 2K CsA alone groups (1.2 vs 2.47 vs 2.46 mL/min; P < .001). There was a trend toward deterioration of creatinine clearance in the 1K and 1/2K groups treated with SRL-CsA. The KIM-1 expression in the 2K SRL-CsA group was significantly upregulated compared with that in the 2K SRL alone and 2K CsA alone groups (P = .02). The AQP-2 expression was comparable in the 3 groups. After 1 week of treatment washout, the 2K, 1K, and 1/2K groups treated with SRL alone demonstrated a significantly higher creatinine clearance rate than did the groups treated with SRL-CsA (P = .04, P = .02, and P = .004). The expression of KIM-1 and AQP-2 was similar among the treatment groups. SRL-CsA-induced nephrotoxicity resulted in overexpression of KIM-1, suggesting injury to the proximal tubule. Treatment with SRL alone may enable earlier reversal of tubular injury.

JAK3 is a cytoplasmic tyrosine kinase with limited tissue expression but is readily found in activated T cells. Patients lacking JAK3 are immune compromised, suggesting that JAK3 represents a therapeutic target for immunosuppression. Herein, we show that a Mannich base, NC1153, blocked IL-2-induced activation of JAK3 and its downstream substrates STAT5a/b more effectively than activation of the closely related prolactin-induced JAK2 or TNF-α-driven NF-κB. In addition, NC1153 failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, Src family members, and serine/threonine protein kinases. Although NC1153 inhibited proliferation of normal human T cells challenged with IL-2, IL-4, or IL-7, it did not block T cells void of JAK3. In vivo, a 14-day oral therapy with NC1153 significantly extended survival of MHC/non-MHC mismatched rat kidney allografts, whereas a 90-day therapy induced transplantation tolerance (>200 days). Although NC1153 acted synergistically with cyclosporin A (CsA) to prolong allograft survival, it was not nephrotoxic, myelotoxic, or lipotoxic and did not increase CsA-induced nephrotoxicity. In contrast to CsA, NC1153 was not metabolized by cytochrome P450 3A4. Thus, NC1153 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs.

P987 Aims: The T-cell-growth factors signaling through Janus kinases (Jak1 and 3) and signal transducer and activator of transcription (Stat1, 3, 5a/b and 6) induce full T cell response. Using the Stat5a/b double knockout, we examined the role of both proteins in acute allograft rejection. Methods/Results: Although normal Stat5a/b+/+ (H-2b) recipients acutely rejected C3H (H-2k) heart allografts (7.3 ± 0.5 days; n=4), Stat5a/b−/− mice rejected heart allografts in a delayed fashion (19.3 ± 7.6 days; n=6; p<0.01). In vitro, Stat5a/b−/− T cells failed to proliferate in response to conconavalin A (ConA) or alloantigens and at 24 and 48 hours displayed 30% and 90% apoptosis, respectively, as shown by TUNEL assay; T cells from Stat5a/b+/+ mice proliferated in response to ConA or alloantigen with only 5% apoptosis. Microarray analysis showed that pure Stat5a/b−/− T cells (but not Stat5a/b+/+ T cells) expressed multiple pro-apoptotic mRNAs (at 24 hrs after activation). Western blot confirmed that after activation Stat5a/b−/− T cells expressed elevated level of pro-apototic Bax and no anti-apoptotic Bcl-2; in contrast activated Stat5a/b+/+ T cells expressed Bcl-2 and no Bax. PCR method showed that (at 24 hrs) ConA-stimulated Stat5a/b+/+ or Stat5a/b−/− T cells produced similar amounts of IL2, IL4, IL10 and INF-γ mRNAs. Purified Stat5a/b+/+ and Stat5a/b−/− B cells proliferated in similar fashion after stimulation with LPS and showed no apoptosis. As shown by FACS, sera from both Stat5a/b+/+ and Stat5a/b−/− rejectors had identical concentrations of donor-specific IgM, IgG1, IgG2a and IgG2b alloantibodies. Conclusions: Although Stat5a/b are not required for cytokine production they are critical for regulation of apoptotic activity in T cells. In contrast, Stat5a/b deficiency does not affect B-cell function, including proliferation, differentiation and immunoglobulin class switching. Thus, following T-B cell collaboration the Stat5a/b-deficient recipients mediate allograft rejection by donor-specific IgM and IgG alloantibodies.

P684 Aims: Diabetes mellitus (Type 1) remains a major cause of morbidity and mortality despite significant advances in medical management. Clinical pancreatic islet allotransplantation is a low-risk alternative to whole pancreas transplantation, offering great promise in treating patients. Although an Edmonton therapeutic protocol has dramatically improved the survival of islet allografts, the majority of patients eventually develop impaired graft function and chronic rejection. Therefore, the ultimate goal—to radically improve islet graft survival—is to induce transplantation tolerance. Methods/Results: We have developed a unique mouse model of tolerance to pancreatic islet allografts [C57BL10 (H2b) to C3H (H2k)] by culturing islets in microgravity conditions using rotating bioreactors. Islets cultured in bioreactors for 7 days displayed decreased immunogenicity (lack of dendritic cells), resulting in long-term allograft survival (>100 days; n = 18) with superior islet morphology and function. Indeed, transmission electron microscopy showed gradual loss of dendritic cells and scanning electron microscopy revealed development of multiple nutritional channels in islets cultured in the bioreactor. Similar results were obtained in C57BL10 to Balb/c (H2d) donor/recipient combination. However, although a 7-day culture produced long-term survival (>100 days; n = 4; p = 0.001), a limited 3-day culture shortened the survival to a mean survival time (MST) of 21.6 ± 5.8 days (n = 5; p = 0.026); fresh allogeneic islets survived 13.8 ± 2.7 days (n = 5). Interestingly, the results were different in Stat6 or Stat4 knockout (KO) mice deficient for Th2 and Th1 cells, respectively. Diabetic Stat6 KO mice rejected fresh islet allografts at 12.1 ± 1.6 days (n = 8) and Stat4 KO mice at 13.3 ± 2.4 days. Islets cultured for 3 days in the bioreactor achieved long-term survivals in all Stat4 KO recipients (>100 days; n = 7; p = 0.001). In contrast, Stat6 KO recipients transplanted with 3-day bioreactor islets produced a mixed response with some long-term survivals (>100 days; n = 4; p=0.001) and some rejectors (22.0 ± 1.7 days; n = 3; p = 0.01. Conclusions: Microgravity condition decreases immunogenicity of allogeneic islets. Furthermore, bioreactor-cultured islets induce stable long-term allograft survival by induction of IL-4/Stat6-dependent Th2reg cells.

P924 Aims: Since Janus kinase (Jak)3 activated by common γ-chain cytokines (IL2, IL4, IL7, IL9, IL13, IL15 and IL21) is expressed exclusively in lymphoid tissues it provides a unique target for immunosuppression. Our previous results showed that novel Jak3 inhibitor, NC1153, inhibits allograft rejection and is synergistic with cyclosporine. Present study examined whether extended therapy with NC1153 induces transplantation tolerance Methods/Resuls: Addition of NC1153 to PHA-activated human T cells induced apoptosis (60%), as measured by TUNUL assay. Untreated ACI (RT1Aa) recipients acutely rejected Lewis (LEW; RT1l) kidney allografts at a mean survival time (MST) of 8.8±0.5 days. A 14-day oral therapy with 40-240 mg/kg NC1153 produced dose-dependent effects with 240 mg/kg dose producing a mean survival time of 50.6 ± 14.3 days. However, when a 14-day course of 160 mg/kg was combined with subsequent thrice-weekly continuous drug administration up to 90 days, 75% of recipiects displayed graft survivals beyond 200 days. Induction of tolerance was confirmed by acceptance of LEW donor- (>100 days; n=3) but not third-party BUF (7.0±1.0 days; n=3) heart allografts by long-term surviving recipients. In order to examine the mechanism, irradiated (400 rads) ACI recipients of LEW heart allografts were adoptively transferred with 30x106 purified T cells or 1 ml serum from tolerant recipients. Recipients transferred with tolerant T cells displayed significantly delayed rejection of LEW heart allografts (40.0±15.0 days; n=6 vs 15±1.0 days; n=5 in irradiated controls) with 2 hearts beating well for >100 days, but rejected third-party heart allografts (12±1.0 days; n=2). These results document that therapy with NC1153 alone induces transplantation tolerance to kidney allografts. Structure your abstract in aims, methods, results and conclusions. The text should be in justified alignment. Please make sure that the body of your abstract does not exceed 3100 characters. Abstracts will be reproduced exactly as submitted and not be edited in any way. You may include any symbols or tables necessary in the body of your abstract, however, you do so at your own risk. Every effort will be made to maintain the formatting of the submitted abstract. Abstracts must be written and presented in English. Linguistic accuracy is the responsibility of the authors. Conclusions: A new and selective Jak3 inhibitor, NC1153, induces transplantation tolerance to organ allografts.

P923 Aims: Little is known about molecules controlling generation of CD4+ IL4/IL10-producing T helper regulatory (Th2reg) cells that maintain transplantation tolerance. We examined involvement of suppressor of cytokine signaling (SOCS)1-3, GATA-3 and c-Maf in regulation of IL4-dependent signaling in Th2reg cells. Methods/Results: Balb/c recipients were treated with anti-CD40L (MR-1) MAb (0.5 mg; days 0, 2, 4, and 6) alone or with CTLA4-Ig (day 0) to induce tolerance to B6 heart allografts (>200 days; n=10); untreated controls produced mean survival time of 7.8±0.8 days. The same protocol failed to induce tolerance in Th2reg-deficient Stat6−/− recipients (20.2±7.0 days; n=5). The expression of SOCS1-3 mRNA was measured by the real-time PCR and GATA-3 and c-maf by RT-PCR. Purified T cells from untreated rejectors (day 7; n=3) stimulated with Th1-promoting IL12 showed increased levels of SOCS2 at 6 hours (0.06±0.001; SOCS2/β-actin ratio x 10000; p=0.001) in comparison to tolerant recipients (none; day 200). In contrast, T cells from tolerant host had 5-fold increased levels of IL4-inducible SOCS3 (0.3±0.02; p=0.02) when compared to rejectors (0.07±0.002). In vitro, purified Stat4−/− T cells were cultured (ConA/IL4/anti-IL12 Ab) to generate Th2, and Stat6−/− T cells (ConA/IL12/anti-IL4 Ab) to obtain Th1. Regression coefficient analysis at 7, 14 and 35 days (n=3) showed that Th2 had increasing and Th1 had decreasing SOCS3 mRNA levels. In particular, normal T cells showed low levels of SOCS3 mRNA (0.3±0.015); Th2 culture conditions elevated SOCS3 levels at 7 (1.5±0.08), 14 (1.4±0.08), and 35 (3.3±0.6) days; and Th1 commitment showed that SOCS3 levels observed at day 7 (1.1±0.2) decreased at days 14 (0.3±0.1; p<0.01) and 35 (0.05±0.001; p<0.01; n=3). Th2 cells (but not Th1) and tolerant recipients expressed significantly more GATA-3 and c-Maf mRNAs. Conclusions: SOCS3, GATA-3 and c-Maf maintain the function of Th2reg cells in transplantation tolerance.

Background-Mechanical unloading of the heart results in atrophic remodeling. In skeletal muscle, atrophy is associated with inactivation of the mammalian target of rapamycin (mTOR) pathway and upregulation of critical components of the ubiquitin proteosome proteolytic (UPP) pathway. The hypothesis is that mechanical unloading of the mammalian heart has differential effects on pathways of protein synthesis and degradation.Methods and Results-In a model of atrophic remodeling induced by heterotopic transplantation of the rat heart, we measured gene transcription, protein expression, polyubiquitin content, and regulators of the mTOR pathway at 2, 4, 7, and 28 days. In atrophic hearts, there was an increase in polyubiquitin content that peaked at 7 days and decreased by 28 days. Furthermore, gene and protein expression of UbcH2, a ubiquitin conjugating enzyme, was also increased early in the course of unloading. Transcript levels of TNF-alpha, a known regulator of UbcH2-dependent ubiquitin conjugating activity, were upregulated early and transiently in the atrophying rat heart. Unexpectedly, p70S6K and 4EBP1, downstream components of mTOR, were activated in atrophic rat heart. This activation was independent of Akt, a known upstream regulator of mTOR. Rapamycin treatment of the unloaded rat hearts inhibited the activation of p70S6K and 4EBP1 and subsequently augmented atrophy in these hearts compared with vehicle-treated, unloaded hearts.Conclusions-Atrophy of the heart, secondary to mechanical unloading, is associated with early activation of the UPP. The simultaneous activation of the mTOR pathway suggests active remodeling, involving both protein synthesis and degradation.

It has been observed that opposite changes in cardiac workload result in similar changes in cardiac gene expression. In the current study, the hypothesis that altered gene expression in vivo results in altered substrate fluxes in vitro was tested. Hearts were perfused for 60 minutes with Krebs-Henseleit buffer containing glucose (5 mmol/L) and oleate (0.4 mmol/L). At 30 minutes, either insulin (1 mU/mL) or epinephrine (1 [mu ]mol/L) was added. Hearts weighed 35% less after unloading and 25% more after aortic banding. Contractile function in vitro was decreased in transplanted and unchanged in banded hearts. Epinephrine, but not insulin, increased cardiac power. Basal glucose oxidation was initially decreased and then increased by aortic banding. The stimulatory effects of insulin or epinephrine on glucose oxidation were reduced or abolished by unloading, and transiently reduced by banding. Oleate oxidation correlated with cardiac power both before and after stimulation with epinephrine, whereas glucose oxidation correlated only after stimulation. Malonyl-coenzyme A levels did not correlate with rates of fatty acid oxidation. Pyruvate dehydrogenase was not affected by banding or unloading. It was concluded (1) that atrophy and hypertrophy both decrease insulin responsiveness and shift myocardial substrate preference to glucose, consistent with a shift to a fetal pattern of energy consumption; and (2) that the isoform-specific changes that develop in vivo do not change the regulation of key metabolic enzymes when assayed in vitro.

118 Identification of immunogenic epitopes that induce allograft rejection is essential to understanding the mechanisms of the rejection cascade, and it may facilitate the development of novel tolerance strategies. This study was aimed at investigating the immunogenic epitopes in RT1.Aa, RT1.A1, and RT1.Au rat class I molecules. Gene splicing with overlap extension was used to construct allochimeric rat [α1h1/∊]-RT1.Aa. The α1-helical region (a.a. 51-90) of wild type (WT)-RT1.Aa cDNA was altered to Lewis (LEW, RT1.A1) sequences by changing nucleotides encoding 10 a.a: (Glu58⇒Asp, Gln62⇒Arg, Gln63⇒Glu, Arg65⇒Gln, Ile66⇒Lys, Glu69⇒Gly, Trp70⇒Asn, Ile73⇒Asn, Asp77⇒Ser, Thr80⇒Asn). Wistar Furth rats (WF, RT1u) share the four underlined a.a. in this region with LEW. Thus, [α1h1/∊]-RT1.Aa bears both RT1.A1 and RT1.A∊ α1-helical epitopes. Chimeric and WT cDNAs were transfected and expressed on the non-professional Buffalo (BUF; RT1b) hepatoma antigen presenting cells (APCs). In BUF (RT1b) hosts, subcutaneous (s.c.) pretransplant injection of 2×107 WT-RT1.Aa bearing transfectants caused accelerated rejection of ACI (RT1a) heart allografts (mean survival time, MST±SD=3.3±0.5 days), compared to non-transfected cells (5.7±0.5 days, P<0.001). On the one hand, transfectants bearing [α1h1/∊]-RT1.Aa, which lacks α1ha determinants, failed to immunize recipients against ACI (RT1a, 5.5±0.5 vs 5.7±0.5 days in controls; NS) hearts, indicating the presence of immunogenic epitopes in the α1ha region. On the other hand [α1h1/∊]-RT1.Aa, which exhibits α1h1 and α1h∊ determinants, immunized hosts toward LEW (RT11, 4.25±0.5 vs 5.75±0.5 days; P<0.01) and WF (RT1∊, 4.3±0.5 vs 6.0±0.0 days; P<0.001) hearts. Further, [α1h1/∊]-RT1.Aa transfectants significantly increased the cytotoxic T cell frequency (fTc) toward WF (1:9751±39) and LEW (1:9039±55) in BUF hosts compared with untreated contols (1:13, 726±386, P<0.02 and 1:12,433±613, P<0.04). Analysis of the Vβ usage of the T-cell receptor repertoire from splenocytes of BUF rats that were primarily in vivo immunized with the [α1h1/∊]-RT1.Aa allochimeric antigen and in vitro restimulated with alloantigen for 7 days, did not demonstrate selective expression of Vβ receptors, but exhibited a repertoire that was similar to rejecting controls or polyclonaly stimulated T cells. In ACI hosts, s.c. administration of [α1h1/∊]-RT1.Aa class I, that bears WF immunogenic epitopes and recipient-type RT1.Aa backbone sequences, expressed on the non-professional BUF APCs; induced prolongation, rather than shortening, of WF heart allograft survival (MST=14±10.3 vs 5.4±0.6 days, P <0.01). Thus, immunogenic epitopes that cause accelerated allograft rejection are localized in the α1-helical region of rat class I MHC molecule. Modification of the immunogenic donor epitopes may deviate the host immune response from rejection toward acceptance of allografts.