Malignancies after heart transplantation: incidence, risk factors, and effects of calcineurin inhibitor withdrawal.

The objectives of the present study were to evaluate the incidence of malignancies and to describe the effects of immunosuppression on survival and recurrence of malignancies after heart transplantation (HTX). Data were analyzed in 211 cardiac allograft recipients, in whom HTX was performed between 1989 and 2005. All of these patients survived for more than 2 years after HTX and received induction therapy with antithymocyte globulin (RATG) guided by T-cell monitoring since 1994. An immunosuppressive regimen consisting of cyclosporine A (CsA) combined with azathioprine was followed by CsA and mycophenolate mofetil (MMF) in 2001; mammalian target of rapamycin (mTOR) inhibitors (everolimus/sirolimus) were used since 2003. Mean patient age at HTX was 51.4 ± 10.5 years; mean follow-up time after HTX 9.2 ± 4.7 years. Overall incidence of neoplasias was 30.8%. Individual risk factors associated with a higher risk of malignancy after HTX were higher age at transplantation (P = .003), male gender (P = .005) and ischemic cardiomyopathy before HTX (P = .04). Administration of azathioprine (P < .0001) or a calcineurin inhibitor (CNI) (P = .02) for more than 1 year was associated with development of malignancy, whereas significantly fewer malignancies were noticed in patients receiving an mTOR-inhibitor (P < .0001). Kaplan-Meier analysis demonstrated a strong statistical trend toward an improved survival in patients with a noncutaneous neoplasia switched to a CNI-free protocol (P = .05). This study demonstrated the impact of a variety of individual risk factors and immunosuppressive drugs on development of malignancy after HTX. Markedly fewer patients with noncutaneous malignancies died after switch to a CNI-free regimen, not quite reaching statistical significance by Kaplan-Meier analysis, however.

Proinflammatory and prothrombotic effects on human vascular endothelial cells of immune-cell-derived LIGHT.

OBJECTIVE: LIGHT (TNFSF 14) belongs to the tumor necrosis factor superfamily and is expressed by activated T cells as well as various types of antigen presenting cells. LIGHT binds to its cellular receptors TR2 and LTbetaR and has a co-stimulatory role in T cell activation. Here, we compared the relative expression of LIGHT in different immune cells and the biological activity of immune cell-derived LIGHT on endothelial cells. METHODS AND RESULTS: Surface expression of LIGHT and mRNA production by PBMC and isolated T cells (CD4+ or CD8+) significantly increased after stimulation with PMA (Phorbolester-12- Myristat-13-Acetat)+ionomycin. No LIGHT expression on PMA stimulated monocytes or monocytic-like THP-1 cells could be detected; differentiation of monocytes and THP-1 cells into macrophages, however, resulted in up-regulation of LIGHT. Supernatants of stimulated T cells contained higher concentrations of soluble LIGHT than macrophage supernatants normalized to cell numbers; release of soluble LIGHT was found to be dependent on metalloproteinase activity. Size determination of released soluble LIGHT by size exclusion chromatography revealed a molecular mass of approximately 60 kDa, suggesting a trimeric form. Released soluble LIGHT induced expression of proinflammatory antigens ICAM-1, tissue factor and IL-8 in human endothelial cells and caused apoptosis of IFN-g pretreated endothelial cells. Soluble LIGHT was detected at low levels in sera of healthy controls and was significantly enhanced in sera of patients with chronic hepatitis C and rheumatoid arthritis (24.93+/-9.41 vs. 129.53+/-49.14 and 172.13+/-77.64; p<0.0005). CONCLUSION: These findings suggest that among immune cells activated T lymphocytes are the main source of soluble LIGHT with released amounts of soluble LIGHT markedly higher compared to platelets. Immune cell-derived membrane-bound and soluble trimeric LIGHT is biologically active, inducing proinflammatory changes in endothelial cells. Enhanced plasma levels of soluble LIGHT in patients with chronic infections suggest a role of LIGHT in systemic inflammatory activation.

[Risk stratification and treatment of cardiac amyloidoses]. / Risikostratifizierung und Therapie kardialer Amyloidosen.

Cardiac amyloidoses are a heterogeneous group of cardiomyopathies that are resistant to treatment and are associated with a poor outcome. Standard heart failure treatment is usually not well tolerated and the underlying disease remains unaffected. The clinical picture is uncharacteristic. Cardiac amyloidosis is often associated with dysfunction of additional organs. Early cardiac amyloid involvement usually reveals left ventricular hypertrophy, impairment of longitudinal shortening and diastolic ventricular function. Without adequate therapy (bi-)ventricular hypertrophy will progress to severe systolic ventricular function decrease. The combination of low voltage pattern, left ventricular hypertrophy and granular sparkling is characteristic for advanced cardiac amyloid involvement. Cardiac magnetic resonance imaging and scintigraphy yield further information on the pattern and severity of cardiac involvement. In unclear cases (left ventricular) endomyocardial biopsy is necessary. Detection of early cardiac involvement and proper identification of patients at high risk for sudden cardiac death due to rapid progressive amyloidosis is still incompletely defined. Referral to specialized centers is strongly recommended.

[MR imaging in cardiac amyloidosis--morphology, function and late enhancement]. / Magnetresonanztomografie bei kardialer Amyloidose--Morphologie, Funktion und late enhancement.

PURPOSE: Since limited data is available using MR imaging in cardiac amyloidosis, the purpose of our study was to evaluate morphological and functional differences of the heart using cardiac MRI. MATERIALS AND METHODS: 19 consecutive patients (14 males, 5 females, mean age 59 +/- 6 years) with histologically proven cardiac amyloidosis were evaluated with MRI at 1.5 T. Results were compared with data of 10 healthy, age-matched control subjects (5 males, 5 females, mean age 60 +/- 6 years). Functional and morphological data including late enhancement (LE) was acquired. RESULTS: Compared to the control group, patients with cardiac amyloidosis had thickened atrial walls and dilated atriums. Both ventricles and the interventricular septum were thickened. The LV hypertrophy was focal in 11 / 19 (58 %) and global in 4 / 19 (21 %) of patients. A myocardial edema occurred in 2 / 19 patients with cardiac amyloidosis (11 %). An edema of the myocardium was visible in 2 / 19 (11 %) of patients. The LV ejection fraction was statistically significantly decreased. The prevalence of LE was 74 % (14 / 19 of patients). LE was detected predominantly in the LV anterior wall and in the interventricular septum. Within the segments LE was located predominantly in a subendocardial location. Between patients with and without LE no statistically significant differences of functional and morphological results were able to be established. CONCLUSION: There are three major outcomes of our assessment: 1. The LV hypertrophy is focal in the majority of patients with cardiac amyloidosis. 2. No statistically significant differences can be established in regard to the functional and morphological features between patients with and without LE. 3. Myocardial edema is a possible feature in cardiac amyloidosis.

Interstitial leukocytes in right ventricular endomyocardial biopsies after heart transplantation in patients with complicated versus uneventful postoperative course.

BACKGROUND: Infections and rejections play key roles in morbidity and mortality in the early postoperative period after orthotopic heart transplantation (HTX). The aim of this study was to evaluate whether qualitative and quantitative analyses of various interstitial leukocytes in endomyocardial biopsies during the first 2 weeks after HTX provided early information on these complications. PATIENTS AND METHODS: During and after HTX, endomyocardial biopsies were obtained in 51 patients. By immunohistochemistry we determined the CD3-, CD4-, CD8-, CD15-, CD20-, CD57-, and CD68-positive cell numbers projected to planimetrically measured areas. To compare morbidity in the postoperative course, the patients were subdivided into complicated versus uncomplicated after 3 months. RESULTS: In the uncomplicated group, the cell counts of CD3-, CD8-, CD57-, and CD68-positive cells were significantly lower than in the complicated group. CD3-, CD4-, and CD8-positive cell numbers showed a significant decrease in the first week among the uncomplicated group. In the complicated group, the cell counts increased significantly in the second week. The numbers of CD57-positive cells were significantly lower during the first and second weeks among the uncomplicated group. CONCLUSIONS: Increased T lymphocytes, natural killer cells, and macrophages observed in the second week after HTX indicated increased morbidity. A reduction in CD3-positive cells in the first week indicated a low morbidity risk; an increase indicated a higher risk.

Detection of cardiac allograft rejection by real-time PCR analysis of circulating mononuclear cells.

BACKGROUND: Detection of cardiac allograft rejection is based on the histological examination of endomyocardial biopsies (EMB). We have explored the possibility of whether graft rejection could be detected by characteristic gene expression patterns in peripheral blood mononuclear cells (PBMC) of heart-transplant recipients. METHODS: The study included 58 blood samples of 44 patients. On the day of EMB, mononuclear cells were isolated from peripheral blood, and gene expression was measured by quantitative real-time PCR. Thirty-nine parameters, including cytokine and chemokine genes were analyzed. Gene expression results were correlated with histological assessment of concomitant evaluated EMB according to International Society for Heart and Lung Transplantation (ISHLT) nomenclature. RESULTS: Gene expression of perforin, CD95 ligand, granzyme B, RANTES, CXCR3, COX2, ENA 78 and TGF-beta1 was significantly different in PBMC of patients with mild to moderate degrees of allograft rejection (> or =grade 2) compared with patients exhibiting no or minor forms of rejection ( or =grade 2 vs.

Do cytokines enable risk stratification to be improved in NYHA functional class III patients? Comparison with other potential predictors of prognosis.

AIMS: Elevated plasma levels of proinflammatory cytokines have been reported in patients with congestive heart failure. The purpose of this study was to assess whether cytokines improve risk stratification in a homogeneous group of NYHA class III patients with a left ventricular ejection fraction <40%. METHODS AND RESULTS: Plasma concentrations of big endothelin, tumour necrosis factor alpha, interleukins -1, -6, -10 and -12, sCD14 and GM-CSF were measured by ELISA in 91 NYHA III patients [mean (SD) age: 55 (10) years, 69% male, 34% coronary artery disease, 66% dilated cardiomyopathy] with a left ventricular ejection fraction and a peak oxygen uptake (peak VO2) of 19 (9)% and 12.1 (3.6) ml x min(-1) x kg(-1), respectively. During follow-up [22 (13) months], 31 patients (34%) died due to cardiovascular causes. In non-survivors, interleukin-6 was twice as high as in survivors [12.8 (16.9) pg x ml(-1)vs 5.6(5.3) pg x ml(-1), P<0.003], whereas plasma concentrations of the other cytokines showed no significant differences. Concerning long-term survival (> or =1 year), multivariate Cox regression analysis revealed an independent prognostic power for interleukin-6, which was further improved by combining with left ventricular ejection fraction and peak VO2, while for short-term survival (up to 6 months) interleukin-6 did not allow risk stratification. CONCLUSION: In NYHA class III patients, plasma concentrations of interleukin-6 are predictive of long-term survival. However, its value may be limited for clinical decision-making for cardiac transplantation (short-term survival).

Ex vivo evaluation of PBMNCs collected with a new cell separator.

BACKGROUND: This study reports on an evaluation of the ability of a cell separator (Amicus, Baxter Healthcare) and the integral MNC computer software program to collect a variety of MNC subsets. The collection efficiency (CE) of the Amicus for these MNC subsets was compared to that of another cell separator (CS-3000 Plus, Baxter). The collected MNCs were also assayed ex vivo to determine if these cells remained functional. STUDY DESIGN AND METHODS: Healthy volunteer blood donors were recruited to provide PBMNCs for the isolation of CD3+, CD4+, CD8+, CD19+, NK, and gammadelta+ cells and monocytes. Cells were collected with an Amicus (test arm; n = 16) or a CS-3000 Plus (control arm; n = 11) cell separator. Cells were counted on a flow cytometer and CEs were calculated. For functional studies, the Amicus-collected MNC data were compared to CS-3000 Plus historical data. Functional studies performed included surface antigen expression assays (CD8+), proliferation assays (CD4+ and CD8+ cells), NK cytotoxicity assays for K562 and HUVE cells, and E-selectin induction on endothelial cells through NK+ contact dependency. Dendritic cells (DCs) were generated from CD34+ cells collected on the Amicus, positively selected by the use of antibody-bound, magnetic bead technology, and then cultured ex vivo with a combination of growth factors to generate the DCs. RESULTS: CEs were higher on the Amicus than on the CS-3000 Plus for CD3+ (68 vs. 54%), CD4+ (70 vs. 56%), CD8+ (68 vs. 52%), and CD19+ (60 vs. 48%) cells (p<0.05). For the two separators, CEs were equivalent for monocytes, NK+, and gammadelta+ cells. The Amicus separator collected significantly fewer platelets than did the CS-3000 Plus (p<0.00001). CD4+, CD8+, and NK cells proliferated normally. NK cells appropriately stimulated E-selectin expression on endothelial cells. Culture-generated DCs obtained by using Amicus-collected CD34+ cells expressed appropriate cell surface markers. CONCLUSION: The Amicus separator is acceptable for the collection of PBMNC subsets. The device collects CD3+, CD4+, CD8+, and CD19+ T- and B-cell subsets with greater efficiency and collects MNCs with significantly fewer contaminating platelets than does the CS-3000 Plus. Cells collected on the Amicus are suitable for use in a variety of research and clinical immunobiologic studies.

Systolic and diastolic properties and myocardial blood flow in the heterotopically transplanted rat heart during acute cardiac rejection.

The aim of the study was to characterize the course of systolic and diastolic function, myocardial blood flow, and histologic changes during acute rejection in a model of heterotopic transplantation in rats. For this purpose isogenic Lewis-to-Lewis and allogenic DA (Dark Agouti)-to-Lewis rat cardiac transplants were studied 1 hour and 1, 3, and 5 days, respectively, after heterotopic intraabdominal transplantation. Myocardial tissue blood flow (MBF) was assessed by the hydrogen-clearance method. An implanted balloon was used to measure pressure-volume relations in the transplanted heart. Myocardial water content was determined at the end of the experiments, and histologic examinations were performed. The MBF recovered during the first day postoperatively in both groups and decreased again in the allogenic group after 3 and 5 days (p < 0.05); it remained stable in the isogenic group. Myocardial relaxation was already prolonged in the allogenic group after 3 days and deteriorated further. Left ventricular end-diastolic pressure progressively increased in the allogenic group, whereas it remained unchanged in the isogenic group up to 5 days. After recovery from ischemia, the left ventricular peak systolic pressure was stable in the isogenic group for the entire further observation period, but it significantly decreased in the allogenic group after 5 days (p < 0.05). Myocardial water content showed a significant increase in the allogenic group compared to that in the isogenic group after 5 days. In the allogenic group histologic examination confirmed mild to moderate rejection after 3 days and severe acute rejection after 5 days. Thus, after recovery from ischemia, mild to moderate cardiac rejection was associated with reduced MBF and impaired relaxation. In a typical sequence, generation of edema and impaired diastolic compliance were terminally followed by systolic dysfunction during severe rejection.

Human vascular endothelial cells stimulate a lower frequency of alloreactive CD8+ pre-CTL and induce less clonal expansion than matching B lymphoblastoid cells: development of a novel limiting dilution analysis method based on CFSE labeling of lymphocytes.

We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8(+) T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8(+) T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8(+) T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8(+) T cells by either BLC or HUVEC derived from the same donor. CD8(+) T cells expanded by allostimulation were identified as CD8(+), CFSE(low) cells and were categorized as CTL by the expression of intracellular perforin and IFN-gamma. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p < 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8(+) T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8(+) T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.

Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses.

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-kappaB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.

Cytomegalovirus infection of vascular cells induces expression of pro-inflammatory adhesion molecules by paracrine action of secreted interleukin-1beta.

BACKGROUND: Infection with human cytomegalovirus (HCMV) has been associated with vascular disease processes such as vascular allograft rejection, transplantation vasculopathy, restenosis after angioplasty, and native atherosclerosis. To elucidate underlying pathomechanisms, the effect of acute HCMV infection on the expression of pro-inflammatory adhesion molecules on human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (hvSMC) was examined. METHODS AND RESULTS: Cells were infected in vitro with clinical strains of HCMV and the resulting changes in adhesion molecule expression were quantified by histology and flow cytometric analysis. On HUVEC, surface expression of vascular cell adhesion molecule-1 and E-selectin was induced de novo on HCMV infection and intercellular adhesion molecule-1 expression was increased by >200%. On hvSMC, intercellular adhesion molecule-1 surface expression induced de novo, although vascular cell adhesion molecule-1 and E-selectin were not changed. Expression of major histocompatibility complex (MHC) class II, lymphocyte-function associated antigen 3 (LFA-3; CD58), and CD40 was not altered by HCMV infection in either cell type. In partially infected cultures, up-regulation of surface molecules also occurred on noninfected cells, suggesting a paracrine mechanism via a soluble factor. Expression of surface molecules could be enhanced in noninfected HUVEC and hvSMC by incubation with virus-free conditioned supernatant from HCMV-infected cells or by coincubation in transwells with infected cells. The responsible agent could be identified as IL- interleukin- (IL) 1beta by detection of de novo secretion of IL-1beta by HCMV-infected cells and by prevention of adhesion molecule up-regulation after addition of an IL-1-converting enzyme inhibitor or IL-1 receptor antagonist. Surface molecule up-regulation could be suppressed by UV inactivation of virus, but not by treatment of cell cultures with inhibitors of viral replication (ganciclovir). CONCLUSION: We propose that HCMV infection induces IL-1beta release and subsequent up-regulation of pro-inflammatory adhesion molecules on noninfected neighboring cells through a paracrine mechanism. This may lead to local potentiation of the inflammatory effects of HCMV infection, not amenable to current therapeutic antiviral strategies.

[Long-term management after heart transplantation--an assessment of current status]. / Langzeitbetreuung nach Herztransplantation--eine Bestandsaufnahme.

Due to an improvement of results after heart transplantation, there is a continuously growing number of long-term surviving patients. Aimed at a characterization of established diagnostic and therapeutic protocols, the Working Group of Thoracic Organ Transplantation within the German Society of Cardiology performed a survey among all German heart transplantation centers. Based on the experience of 1,500 patients, the clinical relevance as well as approaches for prevention and treatment of rejection, infection, cardiac allograft vasculopathy, malignancy, hypertension, renal insufficiency, and quality of life were assessed by a questionnaire. As a result, a time dependency of expected complications could clearly be shown. While early after HTX acute rejection and infection episodes were judged as clinically important, later on cardiac allograft vasculopathy, malignancy, and renal insufficiency predominate as relevant complications. This spectrum was reflected by a differentiated diagnostic protocol (early after HTX more frequent diagnostic procedures for rejection and infection, later intensified examinations to identify cardiac allograft vascular disease and malignancy) as well as by different intensities of immunosuppression and concomittant medication. Regarding further improvement of survival rates and quality of life, future clinical and scientific activities should be focused on the prevention of late complications after heart transplantation.

Prolonged allograft survival but no tolerance induction by modulating CD28 antibody JJ319 after high-responder rat heart transplantation.

BACKGROUND: Allograft rejection depends on T cell immune responses requiring antigen recognition and costimulatory signals through accessory T cell receptors, including CD28. Inhibition of CD28 signaling with a CTLA-4-immunoglobulin (Ig) fusion protein has resulted in immunosuppression and occasional T cell anergy in mouse transplant models, but not in rats. Because this approach also inhibits a potentially tolerizing signal through CTLA-4, selective blockade of CD28 ligation might induce more profound immunosuppression and transplant tolerance. METHODS: The effects of escalating doses of the rat CD28 monoclonal antibody JJ319 on allograft survival were studied after vascularized heterotopic heart transplantation in a high responder strain combination (DA to Lewis). CD28 antigen modulation and circulating antibody levels were monitored by flow cytometry. RESULTS: CD28 antibody JJ319 markedly prolonged cardiac graft survival compared with untreated controls (7 days, range: 6-8). A strictly dose-dependent increase in median graft survival time was demonstrated with a maximum of 36 days (range: 30-40; p <0.001) after the administration of 8 x 1 mg JJ319 i.p. (days -1 to +6 before/after transplantation). However, indefinite graft survival and tolerance could not be induced by JJ319 treatment. At the maximal dose, flow cytometry showed complete down modulation of the CD28 receptor for 10-14 days without T cell depletion in close temporal relation to antibody presence in serum. In vitro, CD28-modulated T cells showed significantly reduced responses to activation. CONCLUSIONS: CD28 antibody JJ319 induces profound immunosuppression after rat heart transplantation, however without development of transplant tolerance. The underlying mechanism seems to be receptor modulation during primary alloantigen recognition. While still potentially applicable clinically, there are no qualitative or quantitative differences to the treatment with CTLA-4/lg or the blockade of CD2 or LFA-1, as reported elsewhere. Thus, a CD28-modulating approach seems not to allow therapeutic exploitation of a tolerizing signal delivered by CTLA-4 but may still be clinically applicable, especially in combined immune interventions.

Elevated serum concentrations of cardiac troponin T in acute allograft rejection after human heart transplantation.

OBJECTIVES: This study evaluates the concept and diagnostic efficacy of using serum troponin T for the detection of cardiac graft rejection. BACKGROUND: Cardiac troponin T is a cardiospecific myofibrillar protein, which is only detectable in the circulation after cardiac myocyte damage. It might be expected to be released during acute heart allograft rejection, allowing noninvasive rejection diagnosis. METHODS: In 35 control subjects and in 422 samples from 95 clinically unremarkable heart allograft recipients more than 3 months postoperatively, troponin T serum concentrations were compared to the histological grade of acute graft rejection in concurrent endomyocardial biopsies. RESULTS: Mean troponin T serum concentrations were identical in control subjects (23.2 +/- 1.4 ng/liter) and in heart transplant recipients without graft rejection (International Society for Heart and Lung Transplantation [ISHLT] grade 0; 22.4 +/- 1.7 ng/liter). Mean troponin T concentrations increased in parallel with the severity of graft rejection (ISHLT grade 1: 27.8 +/- 1.8 ng/liter; grade 2: 33.2 +/- 2.7 ng/liter; grade 3A: 54.6 +/- 6.5 ng/liter; grade 3B and 4: 105.4 +/- 53.7 ng/liter; p < 0.001 for grades 3 and 4 vs. grades 0 and 1). The proportion of positive samples also increased in parallel with rejection severity, reaching 100% in rejections of grade 3B and 4. Sensitivity and specificity for the detection of significant graft rejection (ISHLT grade 3/4) were 80.4% and 61.8%, respectively. The negative predictive value was most remarkable with 96.2%. Intraindividual longitudinal analysis of troponin T levels and biopsy results in 15 patients during long-term follow-up confirmed these findings. CONCLUSIONS: The present data demonstrate that acute allograft rejection after human heart transplantation is often associated with increased serum concentrations of troponin T. All cases of serious forms of graft rejection would have been detected before the development of clinical symptoms. Measurement of troponin T levels may become a useful ancillary parameter for noninvasive rejection diagnosis, being most valuable in the exclusion of severe cardiac graft rejection.

L-arginine: effect on reperfusion injury after heart transplantation.

Global myocardial ischemia and reperfusion injury play a major role in early postoperative myocardial graft dysfunction. The aim of the present study was to investigate the effects of the nitric oxide (NO) precursor L-arginine on myocardial and endothelial function after hypothermic ischemia and reperfusion in a heterotopic rat heart transplantation model. After 1 hour ischemic preservation, reperfusion was started after application of placebo (control, n = 12) or L-arginine (L-Arg 40 mg/kg, n = 12), a substrate of NO synthesis. Myocardial blood flow (MBF) was assessed by the hydrogen clearance method. An implanted balloon was used to obtain pressure-volume relations of the transplanted heart. Left ventricular developed pressure (LVDP), rate of pressure development (dP/dt), end-diastolic pressure (LVEDP), isovolumic relaxation constant (TE), and MBF were measured after 60 minutes and 24 hours of reperfusion. endothelium-dependent vasodilatation in response to acetylcholine (ACh) and endothelium-independent vasodilatation in response to sodium nitroprusside (SNP) were also determined. After 1 hour the MBF was significantly higher in the L-Arg group (3.6 +/- 0.6 vs. 1.9 +/- 0.2 ml/min/g, p < 0.05). The L-Arg group showed better recovery of systolic function and myocardial relaxation (LVDP 106 +/- 6 VS. 70 +/- 7 mmHg, p < 0.05; maximal dP/dt 5145 +/- 498 vs. 3410 +/- 257 mmHg/s, P < 0.05; TE 12.1 +/- 0.9 vs. 16.1 +/- 1.5 ms, p < 0.05, at an intraventricular volume of 80 microliters). LVEDP was similar in the two groups. After 24 hours no difference was found between the groups for basal MBF, LVP dP/dt, TE, LVEDP, or the response of MBF to SNP. However, ACh led to a significantly higher increase in MBF in the L-Arg group (52 +/- 8% vs. 29 +/- 7%, p < 0.05). These results indicate that (1) NO donation improves myocardial and endothelial functional recovery during early reperfusion after heart transplantation; and (2) initial treatment with L-Arg has a persisting beneficial effect against reperfusion-induced graft coronary endothelial dysfunction during late reperfusion.

Endothelin plasma levels in acute graft rejection after heart transplantation.

BACKGROUND: Endothelin is an oligopeptide of endothelial origin with potent vasoconstrictive and mitogenic properties, implicated in the pathogenesis of cyclosporine-induced hypertension, graft vasculopathy, and renal failure. Experimental animal data suggest a role for endothelin in allograft rejection also. METHODS: To determine the role of endothelin in acute graft rejection after heart transplantation, we determined endothelin plasma levels in 165 blood samples from 79 cardiac allograft recipients (2 to 81 months after the operation) with normal graft function and correlated our findings with the histologic severity of acute graft rejection according to International Society for Heart and Lung Transplantation grading. For comparison endothelin levels were determined in 30 healthy controls and in 22 early postoperative transplant recipients (< 2 months after the operation). RESULTS: Endothelin plasma levels were significantly higher in transplant recipients than in controls (early postoperative: 7.97 = 7.53 pg/ml; late postoperative: 3.68 +/- 1.72 pg/ml; controls: 1.55 +/- 0.89 pg/ml). Endothelin plasma levels were not significantly different between groups of rejection grades 0 to 4. In the comparison of two groups of no rejection or lower (International Society for Heart and Lung Transplantation grade 0 and 1, n = 134) and higher (International Society for Heart and Lung Transplantation grade > or = 2, n = 31) rejection severity or comparing patients requiring rejection therapy (n = 20) with those not requiring therapy (n = 145), endothelin levels did not differ significantly between the groups. In 22 patients with three to six available consecutive biopsy scores and endothelin levels, intraindividual longitudinal analysis did also not show any significant correlation. The only positive correlation of endothelin levels with other laboratory parameters was found with serum creatinine concentrations (p < 0.001). In the early postoperative recipients, no correlation of endothelin plasma levels with rejection severity was seen; furthermore the only significant association was found with time after operation. CONCLUSIONS: In this study endothelin plasma levels were not influenced by acute allograft rejection after heart transplantation. Therefore endothelin levels do not appear to be a useful marker for noninvasive rejection diagnosis. Furthermore, a relevant pathogenetic role of endothelin in the rejection process cannot be derived from these data.

A soluble form of the adhesion receptor CD58 (LFA-3) is present in human body fluids.

The human adhesion receptor CD58 (LFA-3) is expressed on most human cell types. Here we report on a soluble form of CD58 (sCD58) in human serum, human urine, and culture supernatants of several cell lines. sCD58 partially purified from human serum, from supernatant of the Hodgkin cell line L428, and purified sCD58 from human urine were found to have a molecular mass of 40-70 kDa under denaturating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting). However, gel filtration of sCD58 purified from human urine gave a molecular mass of 118-166 kDa, suggesting a noncovalent homotrimer conformation or its association with other molecules. Using an enzyme-linked immunosorbent assay specific for CD58 we found that sera from patients suffering from different forms of hepatitis contained elevated sCD58 levels (n = 108). Accordingly, there was a fivefold increase of supernatant sCD58 when the hepatocellular carcinoma cell line Hep G2 was incubated with 25 ng/ml recombinant tumor necrosis factor-alpha in vitro. In contrast, sCD58 serum levels of 337 additional patients suffering from various other immunological disorders were not found to be raised. At high concentrations sCD58 binds to CD2-positive cells and inhibits rosette formation of human T cells to human erythrocytes. Thus, local release of large quantities of naturally occurring sCD58 may interfere with intercellular adhesion in vivo.

Biological response modifiers render tumor cells susceptible to autologous effector mechanisms by influencing adhesion receptors.

Adhesion molecules such as CD2 and its ligand CD58 (LFA-3), as well as CD11a/18 (LFA-1) and CD54 (ICAM-1) regulate not only cell to cell attachment but also participate in lymphocyte activation, recirculation, and effector function including cytolytic activity towards tumor cells. We have investigated the role of CD2/CD58 and CD11a/18/CD54 interactions in cellular immune responses directed towards freshly recovered human T cell leukemias. Downregulation of CD54 and CD58 were observed to correlate with enhanced numbers of blasts in circulation and lack of susceptibility to killing by autologous cytotoxic lymphocytes. Furthermore, culturing tumor cells with recombinant TNF-alpha conditioned medium resulted in reinduction of CD54 and CD58 expression and susceptibility to lymphocyte mediated resulted in reinduction of CD54 and CD58 expression and susceptibility to lymphocyte mediated lysis in vitro. Our findings support the view that adhesion molecules play a pivotal role for tumor cell biology in vivo and stress the point that successful immunotherapy of malignant disease may be facilitated by influencing not only the immune response itself but also adhesion molecules on the malignant tumor targets.