Safety and Efficacy of Immunoadsorption as an Add-On to Medical Treatment in Patients with Severe Idiopathic Pulmonary Arterial Hypertension.

BACKGROUND: Despite optimized medical therapy, severe idiopathic pulmonary arterial hypertension (IPAH) is a devastating disease with a poor outcome. Autoantibodies have been detected in IPAH that can contribute to worsening of the disease. OBJECTIVES: The objective of this prospective, open-label, single-arm, multicenter trial was to evaluate the safety and efficacy of immunoadsorption (IA) as an add-on to optimized medical treatment for patients with IPAH. METHODS: A total of 10 IPAH patients received IA over 5 days. Their clinical parameters, including hemodynamics measured by right heart catheter, were assessed at baseline and after 3 and 6 months. The primary endpoint was the change in pulmonary vascular resistance (PVR). Secondary endpoints included the change in 6-min walking distance, quality of life, safety, and plasma levels of IgG and autoantibodies. RESULTS: The evaluation of the 10 IPAH patients (75% female; 51 ± 12 years; 166 ± 10 cm; WHO functional class III; 53% on combination therapy) revealed that IA was a safe procedure that efficiently removed IgG and autoantibodies from the circulation. After 3 months, the mean PVR improved significantly by 13.2% (p = 0.03) and the cardiac index improved by 13.1%, but no significant changes were found in 6-min walking distance. The quality of life physical functioning subscale score significantly improved after 6 months. The serious adverse events in 3 patients were possibly related to IA and included pneumonia, temporary disturbance in attention, and thrombocytopenia. CONCLUSIONS: IA as an add-on to targeted medical treatment for IPAH is a safe procedure with beneficial effects on hemodynamics, especially in patients with high levels of autoantibodies. Larger-scale controlled studies are needed to assess its efficacy in IPAH and to identify responders.

Impact of the reduction of calcineurin inhibitors on renal function in heart transplant patients: a systematic review and meta-analysis.

AIMS: Calcineurin inhibitors (CNIs) taken after heart transplantation lead to excellent short-term outcomes, but long-term use may cause chronic nephrotoxicity. Our aim was to identify, appraise, select and analyse all high-quality research evidence relevant to the question of the clinical impact of CNI-sparing strategies in heart transplant patients. METHODS: We carried out a systematic review and meta-analysis of randomized controlled trials on CNI reduction in heart transplant recipients. Primary outcomes were kidney function and acute rejection after 1 year. Secondary outcomes included graft loss, all-cause mortality and adverse events. RESULTS: Eight open-label studies were included, with 723 patients (four tested de novo CNI reduction and four maintenance CNI reduction). Calcineurin inhibitor reduction did not improve creatinine clearance at 12 months 5.46 [-1.17, 12.03] P = 0.32 I(2) = 65.4%. Acute rejection at 12 months (55/360 vs. 52/332), mortality (18/301 vs. 15/270) and adverse event rates (55/294 vs. 52/281) did not differ between the low-CNI and standard-CNI groups. There was significant benefit on creatinine clearance in patients with impaired renal function at 6 months [+12.23 (+5.26, +18.82) ml min(-1) , P = 0.0003] and at 12 months 4.63 [-4.55, 13.82] P = 0.32 I(2) = 75%. CONCLUSIONS: This meta-analysis did not demonstrate a favourable effect of CNI reduction on kidney function, but there was no increase in acute rejection. To provide a better analysis of the influence of CNI reduction patterns and associated treatments, a meta-analysis of individual patient data should be performed.

Pre-explant stability of unloading-promoted cardiac improvement predicts outcome after weaning from ventricular assist devices.

BACKGROUND: Detection of cardiac recovery that allows long-term cardiac stability after ventricular assist device (VAD) explantation is a major goal. After normalization of ventricular diameters during unloading, the pre-explant left ventricular ejection fraction (LVEF) allows the detection of patients with the potential to remain stable after VAD explantation. However, some patients with LVEF >45 before VAD explantation show early recurrence of heart failure (HF). We aimed to find out if unstable improvement can be recognized before VAD explantation. METHODS AND RESULTS: Among 96 patients weaned from VADs since 1995, a relatively homogenous group of 53 patients with nonischemic chronic cardiomyopathy (CCM) was selected for the study. The pre-explant stability of major parameters of LV function, size, and geometry that were measured by echocardiography during serial "off-pump" trials was tested for relationship with cardiac stability after VAD explantation. LVEF, systolic peak wall motion velocity (Sm), end-diastolic diameter (LVEDD), end-diastolic relative wall thickness (RWT(ED)) and end-diastolic short/long-axis ratio (S/L(ED)) were selected for evaluation. In postweaning unstable patients, the selected parameters showed relevant instability already before VAD explantation during the time period between best cardiac improvement and VAD explantation and also during the final off-pump trial just before VAD explantation. For all parameters, there were significant differences (P<0.05) in pre-explant changes between patients with and without postweaning cardiac stability. Using the optimal cutoff values obtained from receiver-operating characteristic analysis, we found for our selected parameters predictive values for postexplant cardiac stability of ≥1 year, ≥3 years, and ≥5 years, ranging between 94 and 100, 92, and 100, and 78 and 100, respectively. Using for all parameter changes the cutoff value of 10, we found similar predictive values for cardiac stability of ≥1 year, ≥3 years, and ≥5 years, ranging between 93 and 97, 90 and 96, and 83 and 92, respectively. CONCLUSIONS: Our results strongly suggest the possibility to improve the prediction of postexplant transplant/VAD-free outcome in CCM patients with cardiac improvement during VAD support by analyzing the pre-explant stability of several LV off-pump echocardiographic parameters during serial off-pump trials.

Sequential treatment with rituximab followed by CHOP chemotherapy in adult B-cell post-transplant lymphoproliferative disorder (PTLD): the prospective international multicentre phase 2 PTLD-1 trial.

BACKGROUND: Post-transplantation lymphoproliferative disorder (PTLD) develops in 1-10% of transplant recipients and can be Epstein-Barr virus (EBV) associated. To improve long-term efficacy after rituximab monotherapy and to avoid the toxic effects of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy seen in first-line treatment, we initiated a phase 2 trial to test whether the subsequent use of rituximab and CHOP would improve the outcome of patients with PTLD. METHODS: In this international multicentre open-label phase 2 trial, treatment-naive adult solid-organ transplant recipients diagnosed with CD20-positive PTLD who had failed to respond to upfront immunosuppression reduction received four courses of rituximab (375 mg/m(2) intravenously) once a week followed by 4 weeks without treatment and four cycles of CHOP every 3 weeks. In case of disease progression during rituximab monotherapy, CHOP was started immediately. Supportive therapy with granulocyte-colony stimulating factor after chemotherapy was mandatory and antibiotic prophylaxis was recommended. The primary endpoint was treatment efficacy measured as response rates in all patients who completed treatment with rituximab and CHOP, per protocol, and response duration, in all patients who completed all planned therapy and responded. Secondary endpoints were frequency of infections, treatment-related mortality, and overall survival. This study is registered at ClinicalTrials.gov, number NCT01458548. FINDINGS: 74 patients were enrolled between Dec 12, 2002 and May 5, 2008, of whom 70 patients were eligible to receive treatment. PTLD was of late type in 53 (76%) of 70 patients, monomorphic in 67 (96%) of 70, and histologically EBV associated in 29 (44%) of 66 cases. Four of 70 patients did not receive CHOP. 53 of 59 patients had a complete or partial response (90%, 95% CI 79-96), of which 40 (68%, 55-78) were complete responses. At data cutoff (June 1, 2011) median response duration in the 53 patients who had responded to treatment had not yet been reached (>79·1 months). The main adverse events were grade 3-4 leucopenia in 42 of 62 patients (68%, 55-78) and infections of grade 3-4 in 26 of 64 patients (41%, 29-53). Seven of 66 patients (11%, 5-21) had CHOP-associated treatment-related mortality. Median overall survival was 6·6 years (95% CI 2·8-10·4; n=70). INTERPRETATION: Our results support the use of sequential immunochemotherapy with rituximab and CHOP in PTLD. FUNDING: F Hoffmann-La Roche, Amgen Germany, Chugaï France.

Cyclosporin A and tacrolimus reduce T-cell polyfunctionality but not interferon-γ responses directed at cytomegalovirus.

Cytomegalovirus (CMV) -specific immunity is often estimated by the number of in vitro CMV antigen-inducible interferon-γ-positive (IFN-γ(+) ) T cells. However, recent work indicates that simultaneous production of IFN-γ, tumour necrosis factor-α (TNF-α) and interleukin-2 (IL-2) (referred to as 'polyfunctionality') is more relevant for anti-viral protection. Here, we compared polyfunctionality of CMV-specific T cells (pp65 and IE-1 proteins) in 23 solid-organ transplant patients and seven healthy controls by flow cytometry. The proportions of TNF-α(+) /IFN-γ(+) /IL-2 cells among the activated cells were significantly reduced in transplant patients but not the frequencies of IFN-γ(+) CD8(+) T cells. Immunosuppression reduces polyfunctionality, which reflects the increased infection risk in this patient group.

Burkitt post-transplantation lymphoma in adult solid organ transplant recipients: sequential immunochemotherapy with rituximab (R) followed by cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or R-CHOP is safe and effective in an analysis of 8 patients.

BACKGROUND: Burkitt lymphoma post-transplantation lymphoproliferative disorder (Burkitt-PTLD) is a rare form of monomorphic B-cell PTLD for which no standard treatment has been established. Currently, the treatment of Burkitt lymphoma outside the post-transplantation setting involves high doses of alkylating agents, frequent dosing, and intrathecal and/or systemic central nervous system prophylaxis. In PTLD, however, such protocols are associated with considerable toxicity and mortality. METHODS: The authors present a retrospective series of 8 adult patients with Burkitt-PTLD. Six patients were reported to the prospective German PTLD registry or were enrolled in the PTLD-1 trial, and 2 patients had received treatment before 2000, thus allowing for comparison with the pre-rituximab era. RESULTS: Seven of the 8 patients were men. The median age at presentation was 38 years, and the median time since transplantation was 5.7 years. Five of 8 patients had histologically established, Epstein-Barr virus-associated disease, and 7 of 7 patients were positive for a MYC translocation. Five of 8 patients received sequential immunochemotherapy (4 courses of rituximab [R] followed by 4 cycles of cyclophosphamide, doxorubicin, vincristine, and prednisolone [CHOP] or R plus CHOP [R-CHOP]). In this group, 5 of 5 patients reached complete remission (CR), and their overall survival (OS) was significantly longer (P = .008) compared with the OS for 2 of 8 patients who received first-line CHOP and did not respond. One of 8 patients (who had stage IV disease with meningiosis) received combination therapy (cyclophosphamide pretreatment, rituximab, intrathecal chemotherapy, whole-brain irradiation, and radioimmunotherapy) and reached CR. Overall, 6 of 8 patients reached CR; and, after a median follow-up of 4.7 years (range, 1.7-4.8 years), the median OS was 36.7 months. There was no treatment-related mortality under first-line therapy. CONCLUSIONS: In the largest adult case series in Burkitt-PTLD to date, sequential immunochemotherapy with rituximab followed by standard CHOP or R-CHOP was a both safe and effective treatment.

Heart failure reversal by ventricular unloading in patients with chronic cardiomyopathy: criteria for weaning from ventricular assist devices.

AIMS: Unloading-promoted reversal of heart failure (HF) allows long-term transplant-free outcome after ventricular assist device (VAD) removal. However, because few patients with chronic cardiomyopathy (CCM) were weaned from VADs (the majority only recently), the reliability of criteria used for weaning decisions to predict long-term post-weaning success is barely known. After 15 years of weaning experience, we assessed this issue. METHODS AND RESULTS: In 47 patients with CCM as the underlying cause for HF, who were part of a total of 90 patients weaned from bridge-to-transplant-designed VADs since 1995, we analysed data on cardiac morphology and function collected before VAD implantation, echocardiographic parameters recorded during 'off-pump' trials, duration of HF before implantation, and stability of recovery before and early after VAD removal. Post-weaning 5 year freedom from HF recurrence reached 66%. Only five patients (10.6%) died due to HF recurrence or weaning-related complications. Pre-explantation off-pump left ventricular ejection fraction (LVEF) of ≥50 and ≥45% revealed predictive values for cardiac stability lasting ≥5 years after VAD removal of 91.7 and 79.1%, respectively. With each unit of LVEF reduction, the risk of HF recurrence became 1.5 times higher. The predictive value of LVEF ≥45% also became >90% if additional parameters like pre-explantation LV size and geometry, stability of unloading-induced cardiac improvement before VAD removal, and HF duration before VAD implantation were also considered. Definite cut-off values for certain parameters (including tissue-Doppler-derived LV wall motion velocity) allowed formulation of weaning criteria with high predictability for post-weaning stability, also in patients with incomplete cardiac recovery. CONCLUSIONS: Ventricular assist device removal in CCM patients is feasible and can be successful even after incomplete cardiac recovery. Parameters of pre-explantation cardiac function, LV size and geometry, their stability during final off-pump trials, and HF duration allow detection of patients with the potential to remain stable for >5 post-weaning years.

Protection from cytomegalovirus after transplantation is correlated with immediate early 1-specific CD8 T cells.

T cells are crucial for the control of cytomegalovirus (CMV) in infected individuals. Although CMV-specific T cells can be quantified by various methods, clear correlates of protection from CMV disease have not been defined. However, responses to the pp65 protein are believed to play an important role. Here, the proportions of interferon gamma-producing T cells following ex vivo activation with pools of overlapping peptides representing the pp65 and immediate early (IE)-1 proteins were determined at multiple time points and related to the development of CMV disease in 27 heart and lung transplant recipients. Frequencies of IE-1-specific CD8 T cells above 0.2 and 0.4% at day 0 and 2 wk, respectively, or 0.4% at any time during the first months discriminated patients who did not develop CMV disease from patients at risk, 50-60% of whom developed CMV disease. No similar distinction between risk groups was possible based on pp65-specific CD8 or CD4 T cell responses. Remarkably, CMV disease developed exclusively in patients with a dominant pp65-specific CD8 T cell response. In conclusion, high frequencies of IE-1 but not pp65-specific CD8 T cells correlate with protection from CMV disease. These results have important implications for monitoring T cell responses, adoptive cell therapy, and vaccine design.

Clinical experience with daptomycin in Europe: the first 2.5 years.

OBJECTIVES: To describe the patient populations and infections being treated with daptomycin, as well as the efficacy and safety outcomes. PATIENTS AND METHODS: Data from the European Cubicin Outcomes Registry and Experience (EU-CORESM), retrospectively collected at 118 institutions between January 2006 and August 2008, were analysed. RESULTS: Daptomycin treatment was documented in 1127 patients with diverse infections, including complicated skin and soft tissue infections (33%), bacteraemia (22%), endocarditis (12%) and osteomyelitis (6%). It was used empirically, before microbiological results became available, in 53% of patients. Staphylococcus aureus was the most common pathogen (34%), with 52% of isolates resistant to methicillin; coagulase-negative staphylococci and enterococci were also frequent, with 22% of Enterococcus faecium isolates resistant to vancomycin. Daptomycin was used as first-line therapy in 302 (27%) patients. When used second line, the most common reasons for discontinuation of previous antibiotic were treatment failure and toxicity or intolerance. The use of concomitant antibiotics was reported in 65% of patients. Most frequent doses were 6 mg/kg (47%) and 4 mg/kg (32%). The median duration of daptomycin therapy was 10 days (range 1-246 days) in the inpatient setting and 13 days (range 2-189 days) in the outpatient setting. The overall clinical success rate was 79%, with a clinical failure rate of <10% for all infection types. Low failure rates were observed in first- and second-line therapy (6% and 8%, respectively). Daptomycin demonstrated a favourable safety and tolerability profile regardless of treatment duration. CONCLUSIONS: Daptomycin has a relevant role in the treatment of Gram-positive infections.

Plasmacytoma-like post-transplant lymphoproliferative disorder, a rare subtype of monomorphic B-cell post-transplant lymphoproliferation, is associated with a favorable outcome in localized as well as in advanced disease: a prospective analysis of 8 cases.

Post-transplantation lymphoproliferative disorder (PTLD) with plasmacellular differentiation has been reported as a rare subtype of monomorphic B-cell post-transplant lympho-proliferation with histological and immunophenotypical features of plasmacytoma in the non-transplant population. Here we present clinical, laboratory and histopathological features, treatment and outcome of 8 patients from the German prospective PTLD registry. Clinically, extranodal manifestations were common while osteolytic lesions were rare and none of the patients had bone marrow involvement. Immunohistochemistry showed light chain restriction and expression of CD138 without CD20 expression in all samples. An association with Epstein-Barr virus was found in 3 out of 8 cases. We suggest that the Ann Arbor classification is most useful for this disease entity and report a generally good response to treatment including reduction of immuno-suppression, surgery and irradiation in localized disease and systemic chemotherapy analogous to plasmacell myeloma in advanced disease.

The effects of bilateral lung transplantation on ventilatory efficiency, oxygen uptake and the right heart: a two-yr follow-up.

BACKGROUND: This study was to examine the course of ventilation/perfusion mismatch (VE/VCO(2)-slope) before and during two-yr follow-up after bilateral lung transplantation (BLTx) and to relate exercise parameters with the reverse right ventricular remodeling. METHODS: We prospectively examined 20 patients (nine women; age 46.0 ± 13.0 yr) by cardiopulmonary exercise testing (before and at 3, 6, 12, and 24 months after BLTx), and by echocardiography and blood gas analysis. Etiology of pulmonary failure was chronic obstructive pulmonary disease as well (n = 8), pulmonary hypertension (n = 7), idiopathic fibrosis (n = 3), others (n = 2). RESULTS: The VE/VCO(2)-slope before BLTx was 47.5 (interquartile range 24.5) and declined at 3 months -25.9%, 6 months -30.9%, 12 months -33.9%, and 24 months -35.1% (all p ≤ 0.003) and was then not different from normal. The right ventricular end diastolic diameter RVEDd narrowed from 35.0 (22.5) before to 31.0 (9.0) mm at 3 months after LTx. Similarly, right ventricular systolic pressure (RV(sys)) decreased from 53.6 ± 28.3 to 26.2 ± 5.2 mmHg (all p < 0.01). RVEDd correlated with VE/VCO(2)-slope before (p < 0.0001) but not after BLTx. PeakVO(2) increased from 10.0 ± 2.3 mL/min per kg before BLTx by 86.5% at 24 months (p < 0.01). CONCLUSIONS: The functional status (VE/VCO(2)-slope, peakVO(2)) improves quickly after lung transplantation and is accompanied by reverse remodeling of the right heart. A correlation between exercise parameters and right heart function was found before BLTx only.

Prediction of cardiac stability after weaning from left ventricular assist devices in patients with idiopathic dilated cardiomyopathy.

BACKGROUND: During ventricular assist device (VAD) unloading, cardiac recovery is possible even in patients with chronic heart failure (HF). We sought parameters predictive of cardiac stability after VAD removal. METHODS AND RESULTS: Among 81 patients weaned since March 1995, a homogenous group of 35 with idiopathic dilated cardiomyopathy weaned from left VADs was selected. We evaluated echo data obtained before left VAD implantation and during "off-pump" trials before explantation, histological changes, and serum anti-beta1-adrenoceptor-autoantibody disappearance during unloading, duration of unloading, and HF duration. Postweaning 10-year survival with native hearts reached 70.7+/-9.2%. During the first 5 years, HF recurred in 13 patients (37.1%). Only 6 (17.1%) died after HF recurrence or noncardiac complications related to left VAD explantation. Comparison of patients with and without long-term cardiac stability showed that stable patients were younger, HF history and recovery time during unloading shorter, and preweaning left ventricular assessment revealed higher left ventricular ejection fraction, lower short/long axis ratios, and higher end diastolic relative wall thicknesses. For left ventricular ejection fraction >/=45% at end diastolic diameter of /=5-year cardiac stability was 87.5%. Left ventricular ejection fraction time course during the first 6 postweaning months appeared predictive for long-term stability. HF history >5 years and preweaning instability of cardiac improvement appeared predictive for HF recurrence. CONCLUSIONS: In idiopathic dilated cardiomyopathy, left VAD removal can be successful for >12 years even with incomplete cardiac recovery. Pre-explantation left ventricular ejection fraction, left ventricular end diastolic diameter and relative wall thicknesses, stability of unloading-induced cardiac recovery, duration of left VAD support, and HF duration before left VAD insertion allow identification of patients able to remain stable for >5 years. Time course of left ventricular ejection fraction during the first 6 postweaning months allows prognostic assessment.

Striking observations during emergency catecholamine treatment of cardiac syncope in a patient with initially unrecognized takotsubo cardiomyopathy.

A case is presented of initially unrecognized takotsubo cardiomyopathy with a dramatic clinical course after emergency catecholamine treatment for circulatory support during stress-induced cardiac syncope followed by complete recovery of cardiac function after catecholamine withdrawal and starting beta-blocker therapy. Echocardiography including 2D-strain imaging suggested that the left ventricle (LV) wall motion abnormality was mainly the consequence of geometry-induced regional differences in wall stress (progressively amplified by catecholamines), which might be another possible pathophysiological mechanism involved in the development of LV dysfunction in takotsubo cardiomyopathy. This case also suggests that in emergency, before coronary angiography is possible, echocardiography can be helpful for initial therapeutic decisions, especially to avoid emergency inotropic therapy in such patients.

Pediatric heart transplant candidates with failed donor heart allocation after Eurotransplant urgency listing profit from pretransplant mechanical circulatory support bridging.

Due to the Eurotransplant organ allocation policy, urgency listing for heart transplantation (HTx) remains in force until ventricular assist device (VAD) implantation in Germany. We studied the prognosis of HTx candidates after failed donor heart allocation in urgent status. We studied all adult and pediatric (<18 years) HTx candidates who underwent primary HTx after Eurotransplant urgency listing between January 2001 and December 2006 (Group A-uHTx [A-"u"rgent status "HTx"], n = 99; Group P-uHTx [P-"u"rgent status "HTx"], n = 24) and those to whom donor heart was not urgently allocated before VAD implantation or death in the same period (Group A-fHA [A-"f"ailed "H"eart "A"llocation], n = 21, Group P-fHA [P-"f"ailed "H"eart "A"llocation], n = 10). Mortality rate after urgency listing or primary VAD implantation was studied in each group. In adult patients, 1-year mortality rate after urgency listing in Group A-fHA was 56.8% and significantly higher than in Group A-uHTx (30.6%, P < 0.001, log-rank test). After failed urgent heart allocation, 15 out of 21 patients in Group A-fHA had VAD implantation and two patients (9.5%) underwent HTx after VAD implantation. In pediatric patients, 1-year mortality rate in Group P-fHA was 40.0% and significantly higher than in Group P-uHTx (8.5%, P < 0.05). In Group P-fHA, all 10 patients underwent VAD implantation after failed urgent heart allocation and six patients (60.0%, P < 0.01 vs. Group A-fHA, Fisher's exact test) underwent HTx after VAD implantation. After failed urgent donor heart allocation, pediatric HTx candidates seem to profit more from mechanical circulatory support than adults.

Prognostic impact of microvasculopathy on survival after heart transplantation: evidence from 9713 endomyocardial biopsies.

BACKGROUND: Epicardial vasculopathy has been shown to be associated with poor outcome after heart transplantation. We demonstrate that histologically proven stenotic microvasculopathy is a novel prognostic factor for long-term survival. METHODS AND RESULTS: In 9713 biopsies harvested within the first posttransplantation year from 873 patients (83% male; mean age, 49.1+/-0.6 years), light microscopic evaluations (x200) were performed for microvasculopathy, defined as stenotic endothelial and/or medial disease. Prevalence of severe epicardial vasculopathy was defined by presence of > or = 75% luminal stenosis in coronary angiography (available in 611 of 873 patients), which was present in 118 of 611 patients (19%). For Kaplan-Meier analysis, we defined fatal cardiac events as lethal acute myocardial infarction, sudden cardiac death, and graft failure. Stenotic microvasculopathy was present in 379 of 873 patients (43%) and was due to medial (345/379; 91%) rather than endothelial disease (2/379; 1%) or a combination of both (31/379; 8%; P<0.001). Endothelial disease (median [95% CI], 12.07 [10.69 to 13.44] versus 12.73 years [10.16 to 15.30]; P=0.3329) and nonstenotic medial disease (12.44 [11.14 to 13.74] versus 12.43 years [10.51 to 14.35]; P=0.4047) did not decrease overall survival or time to fatal cardiac event. Stenotic microvasculopathy was associated with poor overall survival (10.90 [9.16 to 12.60] versus 13.40 years [11.79 to 15.07]; P=0.0374) and decreased freedom from fatal cardiac events (1, 5, 10 years, 95.6+/-1.4%, 86.9+/-2.3%, 75.5+/-3.1% versus 99.1+/-0.5%, 96.8+/-1.0%, 89.8+/-1.9%; P<0.0001). This finding was independent of epicardial transplant vasculopathy (P=0.0031). CONCLUSIONS: Stenotic microvasculopathy is frequent in routinely processed biopsies and a new prognostic factor for long-term survival after heart transplantation.

Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies.

BACKGROUND: Due to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan(R) PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp). RESULTS: T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 +/- 0.33 Ct values. The coefficients for inter- (1.89 +/- 0.48%) and intra-assay variation (0.85 +/- 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 +/- 0.33, without significant differences between self-designed and ABI inventoried Taqman(R) gene assays. Only two of the tested Taqman(R) ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 +/- 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 +/- 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 +/- 0.74), albeit higher inter-assay CVs (5.38 +/- 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts. CONCLUSION: In comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Beneficial effect of female gender on long-term survival after heart transplantation.

BACKGROUND: We aimed to test whether stenotic microvasculopathy affects the more beneficial course in female cardiac transplant recipients. METHODS: We studied 873 patients (35/151 premenopausal women aged < or =40 years) who underwent primary heart transplantation. In 7750 biopsies harvested within the first posttransplant year endothelial disease and stenotic microvasculopathy were evaluated by light microscopy (Hematoxylin and Eosin). Kaplan-Meier and Cox regression analyses were performed for major cardiac events (MACE; lethal myocardial infarction, sudden cardiac death, graft failure, and cardiac retransplantation). RESULTS: Stenotic microvasculopathy was found equally in men (38%) and women (39%). Allografts from premenopausal female-to-male transplants more frequently developed endothelial disease (78% vs. 65%; P=0.021) and stenotic microvasculopathy (46% vs. 28%, P=0.024). Beyond the first 5 posttransplant years women presented MACE less often than men, independently of donor gender and stenotic microvasculopathy (P=0.0001). Multivariate regression analysis found women to be at lower risk for MACE (Relative Risk [RR] 0.38; 95% Confidence Interval [CI] 0.17-0.81), whereas stenotic microvasculopathy (RR 2.15; 95% CI 1.42-3.26) and treated diabetes (RR 1.65; 95% CI 1.08-2.52) indicated a higher risk for MACE. CONCLUSIONS: Stenotic microvasculopathy has prognostic impact on survival of male and female cardiac recipients; however, it does not affect the more beneficial course of women in the long-term follow-up.

Dissection of the CMV specific T-cell response is required for optimized cardiac transplant monitoring.

Despite the success of antivirals in preventing clinically overt CMV disease in cardiac allograft recipients, sub-clinical active CMV infection remains a major concern because of its association with allograft rejection and vasculopathy. The measurement of CMV specific T-cell responses is a promising approach to assessing this situation. For simplicity, class-I MHC/peptide-multimers staining CD8 T-cells directly are often used but this ignores a much wider range of responses including the whole CD4 T-cell compartment. CD4 T-cells, however, were recently shown to be critical to reducing CMV load early after transplantation. To determine how extensive T-cell responses to CMV are, the responses to two dominant CMV proteins, IE-1 and pp65, were dissected in detail accounting for T-cell lineage, frequencies, epitope recognition and changes over time in more than 25 heart transplant recipients. Cross-sectional results from over 30 healthy CMV-carriers were analyzed for comparison. Responses were unexpectedly complex, with considerable inter-individual variation in terms of dominance, breadth, and recognized epitopes. Whereas the use of MHC/peptide-multimers for clinical CD8 T-cell response monitoring alone can be justified in some situations, short term T-cell activation combined with intracellular cytokine staining was clearly found to be of more general usefulness. The performance of IFN-gamma, TNF-alpha, or IL-2 as single read-outs in identifying activated T-cells was examined and confirmed that the frequently used IFN-gamma was best suited. These results should be used to inform the design of clinically applicable and diagnostically useful approaches to monitoring CMV specific responses in heart transplant recipients.

Quilty in biopsy is associated with poor prognosis after heart transplantation.

We tested if Quilty (endocardial infiltration of lymphocytes) in routinely processed endomyocardial biopsy is associated with poor outcome after heart transplantation (HTx). Biopsies (n=9829) harvested within the first post-transplant year from 938 patients (778 men, mean age 49 years) were evaluated for Quilty and acute cellular rejection (according to the International Society for Heart and Lung Transplantation, ISHLT, classification). Transplant vasculopathy was evaluated by coronary angiography, and severe stenosis was found in 19% of patients. Survival was tested by Kaplan-Meier and Cox regression analyses for all-cause mortality and major cardiac events (lethal acute cellular rejection, graft loss or myocardial infarction). We found 1840 (19%) Quilty-positive biopsies in 487 Quilty-positive patients (52%). Quilty was more prevalent in women (p=0.038) and younger men (p=0.001), and was correlated with ISHLT grade 1R (OR 1.45, 95% CI 1.36-1.55; p<0.001) and ISHLT grade 2R (OR 2.48, 95% CI 2.21-3.41; p<0.001). Quilty in any biopsy was associated with a higher all-cause mortality (log rank p=0.045) due to a higher risk for major cardiac event (p=0.0001). Multivariate regression analysis showed Quilty (RR 1.69, 95%CI 1.05-2.73) and transplant vasculopathy (RR 2.78, 95%CI 1.68-4.61) as risk factors for major cardiac events and treated hyperlipidemia as lowering the risk for major cardiac events (RR 0.47, 95%CI 0.28-0.77). Quilty is associated with graft loss and poor outcome post HTx. Index biopsy during the first post-transplant year is a useful tool to identify patients at risk and is recommended during routine post-transplant management.

Oral everolimus inhibits neointimal proliferation in prosthetic pulmonary valved stents in pigs.

BACKGROUND AND AIM OF THE STUDY: Growth factor-dependent cell proliferation can cause in-stent neointimal hyperplasia. The study aim was to evaluate whether oral everolimus inhibits the intimal proliferation associated with the implantation of prosthetic pulmonary valved stents. METHODS: Prosthetic pulmonary valves were implanted in 12 pigs (mean bodyweight 25 kg) using a transcatheter technique. Tricuspid valves were prepared from a titanium-coated polymer and sewn into a self-expanding nitinol stent (diameter 20 mm). Valved stents were implanted in the pulmonary position, where they remained for three months. In six animals, treatment with 2 mg/kg everolimus (Certican; Novartis) per day was started three days before implantation and continued throughout the course of the experiment. The other six pigs acted as controls. Adjuvant anticoagulation treatment consisted of acetylsalicylic acid and oral clopidogrel. After three months, hemodynamic valve function was investigated at catheterization and with MRI. At postmortem investigation the valved stents were explanted and subjected to macroscopic, histological and electron microscopic examination. RESULTS: There were no adverse side effects due to everolimus treatment. The overall mean everolimus plasma level during the study was 4.2 +/- 2.4 ng/ml. MRI revealed intact valve function with a regurgitation fraction of 7.3 +/- 4.2% in controls and 4.3 +/- 3.1% in the everolimus group (p <0.01). On macroscopic inspection and histological examination, the everolimus group showed only a thin tissue coverage of the stent struts. The valve cusps were free from intimal thickening, and electron microscopy showed a thin continuous cellular coating. In contrast, substantial neointimal formation was noted in controls. Tissue neogenesis was pronounced at the base of the valve, extended to the valve cusps, and caused valve thickening and foreshortening. CONCLUSION: The oral administration of everolimus effectively inhibits tissue neogenesis in pulmonary valved stents in pigs.