Historical Matching Strategies in Kidney Paired Donation: The 7-Year Evolution of a Web-Based Virtual Matching System.

Failure to convert computer-identified possible kidney paired donation (KPD) exchanges into transplants has prohibited KPD from reaching its full potential. This study analyzes the progress of exchanges in moving from "offers" to completed transplants. Offers were divided into individual segments called 1-way transplants in order to calculate success rates. From 2007 to 2014, the Alliance for Paired Donation performed 243 transplants, 31 in collaboration with other KPD registries and 194 independently. Sixty-one of 194 independent transplants (31.4%) occurred via cycles, while the remaining 133 (68.6%) resulted from nonsimultaneous extended altruistic donor (NEAD) chains. Thirteen of 35 (37.1%) NEAD chains with at least three NEAD segments accounted for 68% of chain transplants (8.6 tx/chain). The "offer" and 1-way success rates were 21.9 and 15.5%, respectively. Three reasons for failure were found that could be prospectively prevented by changes in protocol or software: positive laboratory crossmatch (28%), transplant center declined donor (17%) and pair transplanted outside APD (14%). Performing a root cause analysis on failures in moving from offer to transplant has allowed the APD to improve protocols and software. These changes have improved the success rate and the number of transplants performed per year.

Anti-TCRß mAb induces long-term allograft survival by reducing antigen-reactive T cells and sparing regulatory T cells.

TCR specific antibodies may modulate the TCR engagement with antigen-MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRß (H57-597), TCRα or CD3 promptly reduced the number of CD4(+) and CD8(+) T cells in normal mice, but H57-597 mAb most potently increased the frequency of CD4(+) Foxp3(+) Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57-597 mAb, the expansion of SEB-reactive Vß8(+) T cells was completely abrogated while SEB-nonreactive Vß2(+) T cells remained unaffected. More importantly, transient H57-597 mAb treatment exerted long-lasting effect in preventing T cell responses to alloantigens, and produced long-term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor-specific long-term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57-597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin-sensitized recipients. Thus, transient modulation of the TCRß chain by H57-597 mAb exhibits potent, long-lasting therapeutic effects to control alloimmune responses.

Correlation between cyclosporine-induced nephrotoxicity in reduced nephron mass and expression of kidney injury molecule-1 and aquaporin-2 gene.

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.

New approaches to transplant immunosuppression.

Although considerable progress has been achieved using immunosuppressive drugs that inhibit lymphocyte activation and T-cell cytokine signal transduction pathways, the widespread tissue distribution of the molecular targets exploited to date, calcineurin, mammalian target of rapamycin, and inosine monophosphate dehydrogenase, engenders a constellation of collateral toxicities. One strategy to develop new immunosuppressants seeks to identify targets that are critical for and specific to the adaptive immune response. Three approaches have been used to guide this enterprise; molecular design based on steric resemblance of the antagonist to the natural ligand; construction of complementary DNA oligonucleotides that hybridize with the leader sequence of messenger RNA encoding the synthesis of the specific target, thereby preventing production of that protein; and functional comparisons based on similar inhibitory profiles of candidate compounds and a probe that blocks the target nonselectively. Use of these 3 technologies has led to identification of antagonists blocking selectins, intercellular adhesion molecule-1, or Janus kinase 3, respectively. These lead compounds have been tested for their effects on the alloimmune response and/or the ischemia-reperfusion injuries.

Preclinical results of sirolimus treatment in transplant models.

Sirolimus (SRL; rapamycin) is a macrolide antibiotic, which modest anticandidal and tumoricidal activities were superseded by its immunosuppressive potential to block allograft rejection. The most intriguing biological characteristic of SRL emerged after demonstration of its potent synergism with cyclosporine (CsA). Naïve T cells, residing in the G(0) phase of the cell cycle, become activated by three signals. Signal 1 (T cell antigen receptor/alloantigen) and Signal 2 (CD28/B7) progress T cell to the early G(1) phase inducing production of interleukin-2 (IL-2) and other T cell growth factors (TGFs). Signal 3 (cytokine/cytokine receptor) initiate cell division and differentiation in the late G(1)/S phase. Whereas CsA binding to calcineurin blocks Signal 1/2, SRL binding to mammalian target of rapamycin (mTOR) blocks Signal 3. Our preclinical studies have established the in vivo principles of the effects exhibited by SRL alone on allograft survival, synergism between SRL and CsA as well as two drugs pharmacokinetic and pharmacodynamic interactions. In our experimental model, a 14-day i.v. continuous infusion of SRL by osmotic pump into rat recipients extended the survivals of heart allografts in a dose-dependent fashion. In comparison to untreated controls (MST of 6.3 +/- 0.5 days), 0.08 mg/kg SRL extended MST to 34.4 +/- 12.1 days, and 0.8 mg/kg to 74.1 +/- 20.2 days, with 6/18 allografts surviving for more than 100 days. Since almost identical results were produced by 10-fold higher SRL doses delivered by oral gavage, we estimated its bioavailability at 10%. Similarly, SRL prolonged the survivals of kidney, pancreas, and small bowel allografts in rats. At the same time large animal models cautioned about potential toxicities, namely intestinal vasculitis. The synergistic interactions of CsA and SRL may be explained by sequential effects in the early G(0)/G(1) versus late G1/S phases of cell cycle progression, respectively. The in vivo interaction of SRL with other immunosuppressive drugs was evaluated by the median effect analysis and the combination index (CI) values (CI = 1 shows additive, CI < 1, synergistic, and CI > 1, antagonistic, interactions). Oral SRL proved to be synergistic in both CsA-resistant mouse (CI = 0.4-1.5) and CsA-sensitive rat (CI = 0.3-0.6) models. The pharmacokinetic interactions of SRL and/or CsA were evaluated in rats for i.v. and oral formulations. Although low CsA and SRL i.v. doses did not affect each other levels, potent interaction was observed after oral gavage: CsA increased SRL levels by 2-11 folds; and, SRL increased CsA levels by 2-3-folds. Our results suggested that both pharmacodynamic and pharmacokinetic interactions contribute to the synergism between SRL and CsA. We also estimated the impact of CsA/SRL interaction on renal dysfunction, myelosuppression, and hyperlipidemia. Salt-depleted rats treated with SRL (0.4-6.4 mg/kg) and/or CsA (2.5-20 mg/kg) were examined for glomerular filtration rates (GFR), lipid levels, and bone marrow cellularity. CsA-induced kidney function deficiency was exacerbated by SRL. This exacerbation of renal dysfunction correlated with increased CsA levels in kidneys when combined with SRL. Furthermore, CsA potentiated SRL-mediated toxicities, namely myelosuppression and increased cholesterol. In conclusion, SRL therapy is synergistic with CsA but both drug levels should be carefully monitored to avoid toxic effects.

Selective inhibition of IL-2 gene expression by IL-2 antisense oligonucleotides blocks heart allograft rejection.

PURPOSE: We tested the effects of selective inhibition of interleukin (IL)-2 gene expression by IL-2 antisense oligonucleotide (oligo) with phosphorothioate (PS)/phosphodiester (PD)/2'-methoxyethyl (ME) modifications (17359) on T-cell function and the survival of heart allografts in mice. METHODS: The PS- (17328) or PS/PD/ME- (17359) IL-2 oligo was electroporated to mouse T cell lymphoma cells (TIB 155) stimulated with concanavalin A (Con A). Expression of IL-2 was analyzed by an ELISA spot assay and a reverse transcript polymerase chain reaction method. C3H (H-2k) mice transplanted with BALB/c (H-2d) heart grafts were treated i.v. with a 7-day osmotic pump with 20 mg/kg 17359 alone or in combination with sirolimus (SRL). RESULTS: In comparison with untreated controls, 500 to 2000 nM 17328 inhibited IL-2 protein production by 21.8% to 47.2%, whereas 500 to 2000 nM 17359 did so by 35.5% to 83.5% (both P<0.001). In vivo, 20 mg/kg 17359 prolonged survivals to a mean survival time (MST) of 18.3+/-2.6 days (P<0.001) in comparison with only 8.2+/-0.8 days in untreated controls. Although 0.2 mg/kg SRL alone produced a MST of 18.8+/-6.0 days (P<0.01), addition of 20 mg/kg 17539 synergistically extended survivals to 54.3+/-12.1 days (P<0.001). As expected, IL-2 mRNA, but not IL-7, IL-9, or IL-15 mRNA, was reduced in allografts from recipients treated with 17359 compared with untreated controls. Lymph node cells from the same recipients displayed reduction in proliferative response to donor alloantigen and in generation of alloantigen-specific cytotoxic T cells. CONCLUSION: Selective inhibition of IL-2 mRNA in vivo inhibits T-cell function and extends allograft survival.

Load-induced changes in vivo alter substrate fluxes and insulin responsiveness of rat heart in vitro.

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 micromol/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 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 that the isoform-specific changes that develop in vivo do not change the regulation of key metabolic enzymes when assayed in vitro.

Effect of low dose cyclosporine and sirolimus on hepatic drug metabolism in the rat1.

BACKGROUND: We examined the effect cyclosporine (CsA) and sirolimus (SRL) alone and in combination on hepatic cytochrome P450-mediated metabolism in rats. METHODS: Rats were given 1 mg/kg of CsA or 0.4 mg/kg of SRL alone or in combination via constant intravenous infusion. Renal function was evaluated at the end of treatment. Blood samples were obtained to estimate CsA and SRL concentrations. Hepatic microsomes were prepared for immunoblotting and catalytic assays. RESULTS: CsA alone did not alter serum creatinine levels. SRL given alone or in combination with CsA produced a significant increase in urine output without changes in fluid balance. Although CsA and SRL administered alone caused damage to renal proximal tubules, the two-drug combination dramatically increased the renal structural damage. CsA alone suppressed cytochrome P450 (CYP) 3A2 protein levels by 39% (P=0.012) and catalytic activity by 30% (P=0.042). SRL alone reduced catalytic activity by 38% (P=0.012). Combination therapy reduced both CYP3A2 levels by 55% (P<0.001) and catalytic activity by 55% (P=0.001). CYP2C11 protein expression or catalytic activity were not changed in any group. CYP2A1 protein expression and catalytic activity were both significantly reduced in rats given CsA or/and SRL. Steady-state CsA levels were increased during concurrent SRL dosing, however, SRL concentrations were not changed by CsA coadministration. CONCLUSIONS: Concurrent SRL dosing increases CsA concentrations due to inhibition of hepatic CYP3A2 protein expression. Nephrotoxicity caused by combination therapy is due to CsA elevating levels of SRL or by SRL itself. Concurrent administration of CsA and SRL in transplant patients should be performed with caution.