Lex P.W.M. Maat

Susceptibility of human mesenchymal stem cells to tacrolimus, mycophenolic acid, and rapamycin.

Background. Mesenchymal stem cells (MSC) have multilineage differentiation and immunomodulatory capacities and are potentially useful for therapeutic applications, such as tissue regeneration and control of alloreactivity. MSC are present in most tissues including the transplantable organs. It is therefore unavoidable that MSC will be exposed to immunosuppressive drugs in a clinical transplantation setting. The molecular targets of these drugs are expressed in MSC, but the effect of their inhibition on MSC functioning is unknown. Methods. MSC were isolated and expanded from heart tissue and the effects of the calcineurin inhibitor tacrolimus, the cell cycle inhibitor mycophenolic acid (MPA), and the mammalian target of rapamycin inhibitor on MSC survival, proliferation, differentiation, and immunosuppressive capacity were examined. Results. Short-term exposure to the immunosuppressants did not induce toxicity or apoptosis in MSC, but high-dose tacrolimus induced toxicity after 7 days. MPA and rapamycin inhibited MSC proliferation at therapeutic doses. The immunosuppressants had differential effects on the differentiation capacity of MSC. Tacrolimus reduced the expression of troponin T type 2 and desmin during cardiomyogenic differentiation of MSC, whereas MPA decreased the deposition of calcified minerals during osteogenic differentiation. Rapamycin stimulated lipid production during adipogenic differentiation. Unexpectedly, MSC had adverse effects on the immunosuppressive efficacy of tacrolimus and rapamycin. There was no such effect of MSC on the function of MPA. Preincubation of MSC with tacrolimus increased the immunosuppressive capacity of MSC. Discussion. This study demonstrates that therapeutic concentrations of immunosuppressive drugs affect MSC function. MSC affect the efficacy of immunosuppressive medication. These findings are important for potential clinical use of MSC in combination with immunosuppressants.

Renal failure after clinical heart transplantation is associated with the TGF-β1 codon 10 gene polymorphism

BACKGROUND To determine whether genetic factors are involved in the development of renal dysfunction due to cyclosporine nephrotoxicity, we analyzed 2 polymorphisms in the signal sequence of the transforming growth factor (TGF)-beta 1 gene; codon 10 (Leu(10) --> Pro) and codon 25 (Arg(25) --> Pro). METHOD Using sequence specific oligonucleotide probing, we analyzed both TGF-beta1 gene polymorphisms in cardiac allograft recipients (n = 168) who survived at least 1 year with minimal follow-up of 7 years. Patients received cyclosporine and steroids as maintenance immunosuppressive therapy. Renal dysfunction was defined as a serum creatinine > or = 250 micromol/liter. RESULTS Renal dysfunction was observed in 2% (3/168) of the patients at 1 year, in 7% (11/160) at 3 years, in 12% (18/152) at 5 years, and in 20% (26/131) at 7 years post-transplantation. The genotypic distributions for TGF-beta1 codon 10 were 7% Pro/Pro, 61% Pro/Leu, and 32% Leu/Leu, and for codon 25 these percentages were 1% Pro/Pro, 12% Pro/Arg, and 87% Arg/Arg. We found an association between the TGF-beta 1 genotype encoding proline at codon 10 and renal dysfunction. At 7 years post-transplantation, 26% (23/89) of the patients with the heterozygous Pro/Leu or homozygous Pro/Pro genotype had renal dysfunction vs only 7% (3/42) of the patients with the homozygous Leu/Leu genotype (p = 0.017). For the TGF-beta1 codon 25 genotypes, we found no association between TGF-beta 1 genotypes and renal dysfunction. CONCLUSION Our data support the hypothesis that TGF-beta 1 is involved in the process leading to renal insufficiency in cyclosporine-treated cardiac allograft recipients. In these patients the presence of TGF-beta 1 Pro(10) might be a risk factor.

Anti-CD25 therapy reveals the redundancy of the intragraft cytokine network after clinical heart transplantation

BACKGROUND Despite blockade of the interleukin-2/interleukin 2 receptor (IL-2/IL-2R) pathway by the murine anti-CD25 (i.e., IL-2R alpha chain) monoclonal antibody BT563, cardiac rejection can still occur. In these cases, growth factors other than IL-2 may contribute to allograft rejection. We studied the expression of IL-15, a macrophage-derived cytokine associated with T-cell activation, which interacts with the beta and gamma chains of the IL-2R during rejection episodes under anti-CD25 therapy. METHODS We measured intragraft IL-15 mRNA expression and the number of IL-15- and CD68-positive cells in posttransplantation endomyocardial biopsies (EMBs; n=45) and in nontransplanted, donor-heart specimens (n=11) by competitive template reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS IL-15 mRNA expression was present in the majority of posttransplantation EMB specimens (91%, 41/45) and in nontransplanted donor-heart specimens (91%, 10/11). Relative IL-15 mRNA levels were neither associated with transplantation nor with rejection status. After transplantation, the number of IL-15- and CD68-positive cells significantly increased (P<0.001), but IL-15-positive cell counts did not reflect the histological rejection grade. Anti-CD25 treatment, in contrast to its effects on the IL-2/IL-2R complex, had no influence on intragraft IL-15 mRNA and protein production. In rejection EMB specimens, during (n=5) and after (n=8) anti-CD25 therapy, no differences in relative IL-15 mRNA levels, or in IL-15- and CD68-positive cell counts, were measured. CONCLUSIONS After heart transplantation, high numbers of IL-15- and CD68-positive cells infiltrate the graft. This phenomenon is independent of the rejection status. IL-15 remains present during blockade of the IL-2/IL-2R pathway by anti-CD25 monoclonal antibodies, and it may participate in T cell-dependent donor-directed immune responses, thereby explaining the occurrence of rejection in the absence of IL-2.

FUNCTIONAL RESPONSES OF T CELLS BLOCKED BY ANTI-CD25 ANTIBODY THERAPY DURING CARDIAC REJECTION

BACKGROUND Despite anti-CD25 (interleukin [IL]-2 receptor alpha chain) monoclonal antibody (mAb) therapy, rejection can still occur. T-cell activation through the IL-2 receptor beta and gamma chains by IL-2 or other growth factors may contribute to this rejection. Recently, we have demonstrated that the T-cell growth factor IL-15 was abundantly present in rejecting cardiac grafts during anti-CD25 mAb treatment. METHODS To test whether IL-2- and IL-15-responsive T cells play an active role in rejection during anti-CD25 mAb therapy, we measured the frequency of IL-2- and IL-15-proliferative T cells in peripheral blood from treated patients during rejection (n=12). Measurements were made by limiting dilution analysis in the absence and presence of extra in vitro-added mouse anti-human CD25 mAb. RESULTS In the absence of anti-CD25 mAb, the frequencies of peripheral T cells responding to recombinant human (rh)IL-2 and rhIL-15 from patients were lower than those measured in samples of healthy controls (n=7): median of IL-2-responding T cells 78 per 10(6) (range 31-210 per 10(6)) vs. 154 per 10(6) (122-484 per 10(6), P=0.008) and median of IL-15-responding T cells 62 per 10(6) (range 19-207 per 10(6)) vs. 129 per 10(6) (range 79-192 per 10(6), P=0.02), respectively. In the presence of extra in vitro-added anti-CD25 mAb, frequencies of IL-2-responding T cells from patients significantly decreased, although a considerable number of T cells still proliferated on rhIL-2 (median 85%, range 46-100%). In contrast, the frequencies of IL-15 T cells still responding remained stable (median 2%, range 0-50%, P<0.001). CONCLUSIONS Treatment with anti-CD25 mAbs cannot provide complete suppression of T-cell function because significant numbers of IL-2- and IL-15-responsive T cells remain present in the peripheral blood of allograft recipients during anti-CD25 mAb treatment.

Conversion from cyclosporin A to tacrolimus is safe and decreases blood pressure, cholesterol levels and TGF-β1 type I receptor expression

To determine whether conversion from cyclosporin A (CsA) to tacrolimus (TAC)‐based immunosuppressive therapy is safe and might lead to improvement in the clinical side effect profile we studied 55 cardiac allograft recipients. Ten stable patients were electively converted (0.2–1.5 yr after transplantation; group I) and 45 patients were converted on indication (0.5–14 yr after transplantation; group II). 
We studied blood pressure, cholesterol level and renal function in all patients. To unravel the mechanisms by which CsA may exert its toxic effects and to evaluate whether conversion is associated with immune activation, we analyzed the transforming growth factor (TGF)‐β1 system and intragraft interleukin (IL)‐2 and IL‐15 mRNA expression by real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) and quantitative flow cytometry in the selectively converted patients (group I). 
Conversion did not result in immune activation as no clinical, histological or molecular signs of immune activation (increased intragraft IL‐2 and IL‐15 messenger RNA (mRNA) expression) leading to rejection were found. It did not improve renal function neither in patient group I nor in patient group II. However, after conversion the blood pressure decreased (group I: systolic 154±16 vs 143±21 mmHg, p=0.03, diastolic: 99±11 vs 90±11, p=0.02 and group II: systolic 155±17 vs 142±14, p<0.001, diastolic: 99±11 vs 91±8 mmHg, p<0.001). Likewise, the cholesterol levels improved (group I: 6.6±0.5 vs 5.7±0.3 mmol/L, p=0.001 and group II: 7.1±1.7 vs 6.1±1.7 mmol/L, p=0.001). When patients were treated with TAC the ongoing rejections (n=4) resolved and gum hyperplasia disappeared (n=5). Conversion was associated with a two‐fold lower TGF‐β1 type I receptor expression on peripheral lymphocytes and monocytes (p=0.02 and p=0.002, respectively). 
Conversion from CsA to TAC results in improvement of blood pressure and cholesterol levels and does not induce immune activation. These beneficial effects were accompanied with lower TGF‐β1 type I receptor expression.

Interleukin-17-producing CD4 + cells home to the graft early after human heart transplantation

BACKGROUND Interleukin-17 (IL-17) is regarded as a major effector cytokine with pro-inflammatory actions. It has pleiotropic and environment-specific functions by promoting adaptive cytotoxic T-lymphocyte responses during inflammation. Therefore, it is tempting to speculate that IL-17 plays a major role in inflammatory responses in transplant recipients. We questioned whether IL-17 is expressed in the transplanted heart during acute rejection (AR), or during immunologic quiescence, and which graft-infiltrating lymphocytes produce IL-17. In addition, we analyzed donor-specific IL-17-producing cells in peripheral blood cells in comparable periods after transplantation. METHODS Endomyocardial biopsies from heart transplant recipients with early or late AR or in an immunologic quiescence period were analyzed for the presence of IL-17 mRNA. In addition, the capacity of graft-infiltrating lymphocytes (GILs) to produce IL-17 was analyzed. Moreover, we determined the frequency of donor-reactive IL-17-producing peripheral blood mononuclear cells (PBMCs) using an Elispot assay. RESULTS Twenty-one percent (14 of 67) of the biopsies assessed were positive for IL-17 mRNA. Thirteen of 41 biopsies were observed in the early period (≤3 months) after transplantation. One (of 26) of the late biopsies expressed IL-17 (p = 0.006). Specifically, IL-17 was expressed during early AR (57%, or 8 of 14), whereas biopsies from late AR (0 of 5) did not express IL-17 mRNA (p = 0.02). During AR, IL-17 is derived from IL-17-producing CD4(+)CD161(+), and not CD8(+), GILs. In contrast to the graft findings, we detected circulating donor-reactive IL-17-producing cells mostly during immunologic quiescence. CONCLUSIONS Particularly early after heart transplantation, IL-17-producing CD4(+) T cells home to the graft, which contributes to the AR process.

Spark erosion myectomy in hypertrophic obstructive cardiomyopathy

The design features of the cutting electrode and the electrical characteristics of a monopolar electrosurgical device were specially adapted for performing a septal myectomy in patients with hypertrophic obstructive cardiomyopathy. Both the cutting behavior and electrode design were found to facilitate myectomy.

An unusual problem during surgical removal of a broken guidewire.

At operation, it proved impossible using traction alone to remove an angioplasty guidewire that was lodged in an intermediate artery. The problem was solved by passing a Teflon catheter over the wire, thus protecting the left main coronary artery while freeing the entrapped distal end of the guidewire.

Impact of heart transplantation on the safety and feasibility of the dobutamine stress test

BACKGROUND Dobutamine myocardial perfusion imaging is a useful method for evaluation of coronary artery disease. However, this technique does not allow for ischemia monitoring, which may have an impact on the safety of the test in heart transplant recipients due to cardiac sensory denervation. The aim of this study was to assess the impact of heart transplantation on the feasibility and complications of the dobutamine stress test. METHODS We studied 225 heart transplant recipients (mean age 57 +/- 7 years) and a control group of 225 patients without previous transplant matched for age and gender by dobutamine (up to 40 microg/kg per minute) stress myocardial perfusion imaging. RESULTS During the test, transplant recipients had a lower prevalence of premature ventricular contractions (23% vs. 37%, p < 0.001) and ventricular tachycardia (0.04% vs 7.5%, p < 0.0001) compared with control patients. By multivariate analysis, heart transplantation was a powerful independent variable associated with a reduced risk of ventricular arrhythmias (chi(2) = 20.8, p < 0.0001) and minor side effects (nausea, dizziness, anxiety, flushing, chills) (chi(2) = 20, p < 0.0001) during dobutamine stress. The target heart rate was reached in 82% of transplant recipients and in 77% of the control group. Overall feasibility (achievement of the target heart rate and/or an ischemic end-point) was 87% in the transplant and 86% in the control group. CONCLUSIONS Dobutamine stress myocardial perfusion imaging is a safe and feasible method for evaluation of coronary artery disease in heart transplant recipients. The prevalence of arrhythmias and minor complications using the dobutamine stress test is lower in heart transplant recipients compared with control patients. The independent association between heart transplantation and reduced risk of arrhythmias and minor side effects of the dobutamine stress test indicates that cardiac sensory and autonomic nerve function plays a major role in the induction of these complications during the test.