Elevated serum interleukin-6 (IL-6) in solid-organ transplant recipients is positively associated with tissue destruction and IL-6 gene expression in the periodontium.

BACKGROUND: The number of transplanted solid organs and life expectancy after transplantation are steadily rising worldwide. Inflammation is widely recognized as playing a pivotal role in transplant rejection, and several studies have shown that serum interleukin-6 (IL-6) levels can identify individuals who are at greater risk for rejection. Given the known association between IL-6 and chronic periodontitis, the aim of our study was to assess the periodontal status of solid-organ transplant subjects compared to systemically healthy controls, to quantify the IL-6 levels in the serum and periodontal tissues, and to explore their association. METHODS: Forty-seven heart and kidney transplant and 18 systemically healthy age-matched individuals were recruited. Subjects received a complete periodontal examination, and blood and periodontal tissue samples were collected for quantification of IL-6 protein and mRNA levels, respectively. RESULTS: Transplant subjects had significantly higher serum IL-6 levels and slightly but statistically significantly increased mean probing depths than healthy controls. Multivariable linear regression analysis adjusting for gender, diabetes, smoking, and immunosuppressant dose showed that the mean probing depth, number of missing teeth, and mean percentage of sites with > or =4 mm attachment loss were independent predictors for elevated serum IL-6 levels. Transplant subjects with chronic periodontitis had higher mean serum IL-6 levels than those without chronic periodontitis, and there was a positive correlation between periodontal IL-6 gene expression levels and serum IL-6 protein levels. CONCLUSIONS: Periodontal tissue destruction and local IL-6 synthesis are associated with elevated serum IL-6 levels in transplant recipients. This may have serious implications in solid-organ transplant deterioration and chronic rejection.

DNA-liposome versus adenoviral mediated gene transfer of transforming growth factor beta1 in vascularized cardiac allografts: differential sensitivity of CD4+ and CD8+ T cells to transforming growth factor beta1.

We have developed a model of transforming growth factor (TGF)beta1 gene transfer into mouse vascularized cardiac allografts to study the use of gene transfer as an immunosuppressive therapy in transplantation. Donor hearts were perfused with either DNA-liposome complexes or adenoviral vectors that encode the active form of human TGFbeta1. DNA-liposome mediated transfection prolonged allograft survival in approximately two-thirds of transplant recipients, while adenoviral delivery of TGFbeta1 was not protective. Protective TGFbeta1 gene transfer was associated with reduced Th1 responses and an inhibition of the alloantibody isotype switch. The protective effects of TGFbeta1 gene transfer were overridden by exogenous interleukin-12 administration. Interestingly, alloreactive CD4+ and CD8+ cells exhibited distinct sensitivities to TGFbeta1 gene transfer: CD4+ Th1 function was abrogated by this modality, although CD8+ Th1 function was not. Transient depletion of recipient CD8+ cells markedly prolonged the survival of grafts transfected with either DNA-liposome complexes or adenoviral vectors. Transgene expression persisted for at least 60 days, and Th1 responses were not detectable until CD8+ T cells repopulated the periphery. However, long-term transfected allografts appeared to exhibit exacerbated fibrosis and neointimal development. These manifestations of chronic rejection were absent in long-term transfected isografts, suggesting that long-term expression of active TGFbeta1 alone is not sufficient to induce fibrosis of the grafts. Collectively, these data illustrate the utility of immunosuppressive gene therapy as a treatment for transplantation when combined with additional conditioning regimens. Further, they illustrate that alloreactive CD4+ and CD8+ cells may be differentially influenced by cytokine manipulation strategies.

Efficient gene transfer method into the whole heart through the coronary artery with hemagglutinating virus of Japan liposome.

OBJECTIVE: To confirm gene transfer techniques especially into the whole heart, we tried out a gene transfer method involving liposome with the viral envelope hemagglutinating virus of Japan liposome as an alternative to existing techniques such as cationic lipofection or other viral vectors. METHOD: For this study, hemagglutinating virus of Japan liposome (H group) or cationic liposome (L group) was used to compare the efficacy of gene transfection of oligonucleotide labeled with fluorescein isothiocyanate and cDNA of beta-galactosidase and human manganese-superoxide dismutase. Fluorescein-labeled oligonucleotide, cDNA of beta-galactosidase, or manganese-superoxide dismutase was complexed with liposomes, DNA-binding nuclear protein, and the viral protein coat of hemagglutinating virus of Japan. After donor rat hearts arrested by cardioplegia had been harvested, the coronary artery during cardioplegic arrest was infused via an aortic cannula with the liposome-gene complex. Next, the hearts were transplanted into the abdomen of recipient rats of the same strain, and all recipients were put to death after 3 days of transfection. RESULTS: Fluorescein isothiocyanate was detected in the nuclei of more than 70% of the myocytes (75% +/- 14%, n = 5) in the H group compared with fewer than 10% in the L group (7% +/- 5%, n = 5). The intensity of fluorescein isothiocyanate was significantly higher in the H group (979 +/- 112 FI) than in the L group (116 +/- 68 FI). beta-Galactosidase was expressed in the cytosol of more than 50% of the myocytes in the H group (61% +/- 7%, n = 5) compared with none in the L group (0%, n = 5). After 3 days of gene transfection, and when exposed to ischemia (30 minutes, 37 degrees C) and reperfusion (30 minutes, 37 degrees C) with Langendorff apparatus, the hearts transfected with manganese-superoxide dismutase (S group, n = 5) showed a significantly higher percentage of recovery of left ventricular end-diastolic pressure (S vs C, 86% +/- 3% vs 54% +/- 12%) and coronary flow (98% +/- 2% vs 66% +/- 12%) than did the control hearts (C group, n = 5). Western blotting analysis showed an apparent increased expression of manganese-superoxide dismutase in the hearts transfected with manganese-superoxide dismutase compared with the control hearts. These results clearly demonstrated that the donor hearts were transfected with fluorescein-labeled oligonucleotide and the beta-galactosidase gene as a result of coronary infusion of the hemagglutinating virus of Japan liposome during cardioplegic arrest at the time of harvest. Furthermore, the hearts transfected with manganese-superoxide dismutase showed significant improvement in tolerance against ischemia reperfusion injury. CONCLUSION: We believe that this method represents a novel in vivo gene transfer technique for the heart and thus may provide a new tool for research and therapy of heart transplantation.

Alteration in diastolic function following cardiac cryopreservation at subzero temperatures.

BACKGROUND: We have studied the alterations produced in the diastolic function of the left ventricle after applying a protocol of cryopreservation at subzero temperatures. METHODS: Isolated rabbit hearts and 5% polyethylene glycol (PM 4000) as the cryoprotective agent were used for the study. RESULTS-CONCLUSIONS: Following cryopreservation we found a statistically significant increase in systolic function. However, the diastolic function shows worsening, with a statistically significant increase (p < 0.05) in mean stiffness, decrease in differential stiffness, (p < 0.05) and upward and leftward displacement of the diastolic pressure-volume curve.

Twenty-four-hour ice storage of rabbit heart.

Although cardioplegia is limited to 4 hours of ice storage, University of Wisconsin solution has successfully extended this period to approximately 12 hours. In this study we have substituted polyethylene glycol for hydroxyethyl starch in a simplified University of Wisconsin solution (Cardiosol). Rabbit hearts were ice stored for 24 hours at 0 degrees C in either University of Wisconsin solution or Cardiosol (containing either 5% or 10% polyethylene glycol). Fresh control hearts were tested immediately after cardiectomy. Function was evaluated in an in vitro working heart model for 1 hour with aortic afterload at 100 cm H2O. Total cardiac output or the proportion of hearts reaching 100 cm H2O were compared. Hearts stored in University of Wisconsin solution for 24 hours functioned at 6% of control levels at 15 minutes of observation. None reached 100 cm H2O or deteriorated further with time (p < 0.05). By contrast, hearts stored in 5% Cardiosol showed progressive recovery during the 1-hour observation. Of the 13 hearts, 11 reached 100 cm H2O with a mean cardiac output of 51% of the control value. Increasing the concentration of polyethylene glycol to 10% improved cardiac output at all observation times, reaching 80% of control heart performance at 1 hour (control > 10% > 5% > University of Wisconsin solution [p < 0.05]). We concluded that 10% polyethylene glycol significantly improved 24-hour ice storage and, hence, viability to a functional level that matched our previously reported microperfusion results.

Cholesterol-lowering therapy after heart transplantation: a 12-month randomized trial.

BACKGROUND: Hypercholesterolemia, a common problem after heart transplantation, may be important in the genesis and progression of allograft coronary artery disease. The current study was performed to compare the efficacy of gemfibrozil, simvastatin, and cholestyramine for cholesterol lowering in heart transplant recipients. METHODS: In this prospective 1-year study, 48 heart transplant recipients with moderate hypercholesterolemia were randomized to therapy with gemfibrozil 600 mg twice daily (n = 17), simvastatin 10 mg daily (n = 13), and cholestyramine 4 gm twice daily (n = 18). Detailed lipoprotein analysis was performed at baseline and after 3, 6, and 12 months of treatment. RESULTS: Total cholesterol and low-density lipoprotein cholesterol were reduced 19% and 29%, respectively, after 3 months of simvastatin therapy (p < 0.0001) with a sustained reduction in total cholesterol (25%) and low-density lipoprotein cholesterol (39%) at 1 year. Gemfibrozil and cholestyramine treatment did not result in a reduction in cholesterol levels. Apolipoprotein B levels were reduced by 29% at the end of 1 year with simvastatin but not with the other treatments. Serum triglyceride levels were reduced significantly by treatment with gemfibrozil (up to 36%, p < 0.01) but not by the other treatments. High-density lipoprotein cholesterol initially rose in patients treated with simvastatin and gemfibrozil; however, this effect did not persist to 12 months. However, the ratio of low-density lipoprotein/high-density lipoprotein was favorably affected by simvastatin, with a 38% reduction by 12 months (p < 0.0001) but not by the other treatments. Over the course of 1 year, 14 patients dropped out of the study: four from the gemfibrozil arm and ten from the cholestyramine arm. Gastrointestinal intolerance was the most common reason for study termination (8 of 14). All patients in the simvastatin treatment arm completed 12 months of therapy. No biochemical abnormalities resulted from any therapy, and no therapy caused significant alteration in cyclosporine blood levels. CONCLUSIONS: Of the three therapies studied, simvastatin was found to be the most efficacious and well tolerated for cholesterol lowering in patients after heart transplantation.

Circulatory dynamics during dental extractions in normal, cardiac and transplant patients.

BACKGROUND: The authors conducted a study to evaluate the relationship between the circulatory dynamics of patients with heart disease and these patients' cardiac status when undergoing dental extractions. METHODS: Subjects with minimal heart disease, or MHD; with severe heart disease, or SHD; or who received a heart transplant, or TRAN; as well as control, or NOR, subjects were enrolled in the study. The authors recorded subjects' systolic blood pressure, or SBP; diastolic blood pressure, or DBP; and heart rate, or HR, while they were under basal conditions and during postanesthesia and dental extraction periods. The authors estimated a general linear model and performed analysis of variance. RESULTS: Under basal conditions, MHD subjects did not show significantly different mean blood pressure values compared with NOR subjects but did show slightly significantly higher mean HR values. Mean DBP and HR values were significantly higher in SHD and TRAN subjects than in MHD and NOR subjects, while SBP values in SHD subjects were significantly lower than those in MHD, TRAN and NOR subjects. During dental extraction sessions, SBP, DBP and HR mean values increased significantly compared with basal conditions and post-anesthesia periods in MHD and NOR subjects. SHD and TRAN subjects showed no significant time-dependent variation during dental extraction sessions in any circulatory parameter. CONCLUSIONS: MHD subjects had cardiovascular responses to stress similar to those of NOR subjects, while SHD and TRAN subjects had similar slight and dulled cardiac responses. CLINICAL IMPLICATIONS: Patients with SHD may not be able to adapt their cardiac performance to an emotional stress such as a dental appointment, while it seems to be easier for MHD and TRAN patients. Managing TRAN patients is relatively easier than managing SHD patients.

Significant prolongation of guinea pig heart contraction transplanted in rat after removal of anti-xeno-antibodies by whole body rinse-out (WBRO) with hemoglobin solution.

PURPOSE: In order to investigate effectiveness of removal of the anti-xeno- antibodies in xenotransplantation (xeno Tx), WBRO using pyridoxalated-human hemoglobin-polyethyleneglycol conjugate (PHP solution) was performed prior to transplantation (Tx) of a guinea pig heart in a rat. MATERIALS & METHOD: Experiment I. Removal of the immunoglobulins and the anti-guinea pig lymphocytotoxic antibody (ALA) by WBRO. Exchange transfusion with the PHP solution was done in the Tx-expected rats until a hematocrit lowered below 5% (n = 11). Experiment II. Xeno heart Txs. Guinea pig hearts were transplanted into rats without immunosuppressants 1) without (n = 8) or 2) with the WBRO (n = 8). RESULTS: Experiment I. Levels lowered to 14% in IgG, 17% in IgA and 6% in IgM, respectively, to initial values after the WBRO. An ALA titer lowered from 4 X (+) to 1 X (-) after the WBRO. Experiment II. An average heart contraction period was 10.4 +/- 1.8 minutes 1) without the WBRO in contrast to 472.5 +/- 4.8 minutes with the WBRO (p < 0.01). CONCLUSION: WBRO using PHP solution is effective in removal of the anti-xeno-antibodies and consequent prolongation of survival of the xenograft.

Efficiency, efficacy, and adverse effects of adenovirus vs. liposome-mediated gene therapy in cardiac allografts.

Virus- and nonvirus-mediated immunosuppressive cytokine gene therapy prolongs cardiac allograft survival in various nonfunctional heart transplant animal models, but its cardiac adverse effects have not been addressed. Recently, we developed a functional heterotopic heart transplant model in rabbits. For the first time, we were able to systematically compare the efficiency, efficacy, and adverse effects of optimized adenovirus- and liposome-mediated ex vivo interleukin (IL)-10 gene transfer in functional donor hearts. The efficiency of liposome-mediated gene transfer was greatly improved in physiologically functioning donor hearts and was only three- to fourfold lower than adenovirus-mediated gene transfer. The efficacy of liposome-mediated IL-10 gene transfer was much higher than that mediated by adenovirus. Significant negative inotropic and arrhythmogenic adverse effects on transplanted hearts were observed due to viral cytotoxicity and immunogenesis, which greatly abated the therapeutic efficacy of this first generation adenovirus-mediated gene therapy.

Tacrolimus: the good, the bad, and the ugly.

The aim of this study was to evaluate the efficacy and side-effects of tacrolimus in pediatric transplant patients previously receiving cyclosporin A (CsA). This study was a retrospective chart review strengthened by a concomitant patient interview. Eleven pediatric cardiac or renal transplant patients, who had been converted from CsA to tacrolimus from October 1995 to January 1999 at The Cleveland Clinic Foundation, were included; there were six renal and five cardiac transplant patients. Each chart was reviewed to assess transplanted organ function pre- and post-conversion. For the six renal transplant patients, creatinine levels and biopsy findings were evaluated. For the five cardiac transplant patients, cardiac catheterization and routine biopsy data were analyzed likewise. Epstein Barr virus (EBV) status was also evaluated in each patient. In addition, each parent or patient was interviewed to ascertain dates of transplant, current medications, and side-effects. The patients' ages ranged from 6 to 20 yr (mean age 14.6 yr). All patients had been converted to tacrolimus. Eight patients were converted for treatment of refractory rejection, two were converted because of CsA-associated side-effects, and one patient was converted empirically for a history of multiple previous transplant rejections. Seven out of eight patients who received tacrolimus for rejection therapy improved. One patient had complete resolution of gingival hyperplasia. Another patient who previously developed hemolytic uremic syndrome on CsA had no further evidence of hemolysis. Four patients were weaned off steroid therapy. Despite conversion, two renal transplant patients progressed to chronic rejection. Five patients exhibited no side-effects. Side-effects experienced included transient hyperglycemia in conjunction with steroid use, headaches, and tremors that subsided rapidly. Four of 11 patients developed post-transplant lymphoproliferative disease (PTLD). Fortunately, reducing the dose of tacrolimus and/or surgical resection of the mass (if present), eradicated the disease. In conclusion, conversion therapy successfully provides an alternate treatment for acute rejection. It also enabled some patients to discontinue steroid therapy, maximizing growth potential. PTLD is a severe, potentially life-threatening complication that needs to be recognized and monitored closely. In conclusion, tacrolimus has been shown to be a very effective agent for the treatment of refractory organ rejection, but must be used cautiously.