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Introduction: Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are susceptible to haemostatic disturbances. Monitoring the haemostatic capacity by conventional clotting tests is challenging., Materials and Methods: Thrombin generation (TG) by Calibrated Automated Thrombography, clotting tests and tissue factor pathway inhibitor (TFPI) measurements were performed to describe the relationship between haemostatic changes and alterations in these tests. Blood samples were collected before, during and after CPB. Furthermore, it was investigated whether TG measured intraoperatively, is associated with increased risk of bleeding postoperatively., Results: TG diminished significantly (p<0.01) after heparinization in the presence and absence of platelets (37% and 50%) compared to baseline. After the start of CPB, TG elevated and persisted till the end of surgery but remained lower than preoperatively. Activated clotting time increased after heparinization and after the start of bypass compared to baseline (400% and 500%). Anti-FXa activity reduced on the start of CPB compared to the level after heparinization, to almost the baseline value following protamine reversal of heparin. The plasma levels of total and free TFPI elevated 9 and 14 fold during bypass and remained after protamine administration higher than preoperatively. Plasma D-dimer levels reduced (p<0.01) when bypass started. However, a marked elevation was observed in the following time points. TG in platelet-rich plasma measured after heparinization and after the start of CPB associated (p<0.05) with postoperative blood loss., Conclusions: TG can be determined during CPB despite the high heparinization level, it reflects the haemostatic capacity better than clotting-based assays and might better predict bleeding when performed intraoperatively., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: In this study the value of thrombin generation parameters measured by the Calibrated Automated Thrombography for prediction of blood loss after cardiac surgery with cardiopulmonary bypass was investigated., Methods: Thirty male patients undergoing first-time coronary artery bypass grafting were enrolled. Blood samples were taken pre-bypass before heparinisation (T1) and 5 min after protamine administration (T2). Thrombin generation was measured both in platelet-rich plasma and in platelet-poor plasma. Besides thrombin generation measurements, activated clotting time, haematocrit, haemoglobin, platelet number, fibrinogen, antithrombin, D-dimers, prothrombin time and activated partial thromboplastin time were determined. Blood loss was measured and the amount of transfusion products was recorded postoperatively until 20 hours after surgery. Patients were divided into two groups based on the median volume of postoperative blood loss (group 1: patients with median blood loss <930 ml; group 2: patients with median blood loss ≥930 ml)., Results: On T1, patients of group 2 had a significantly lower endogenous thrombin potential and peak thrombin (p<0.001 and p=0.004 respectively) in platelet-rich plasma, a significantly lower endogenous thrombin potential (p=0.004) and peak thrombin (p=0.014) in platelet-poor plasma, and a lower platelet count (p=0.002). On T2 both endogenous thrombin potential and peak thrombin remain significantly lower (p=0.011 and p=0.010) in group 2, measured in platelet-rich plasma but not in platelet-poor plasma. In addition, platelet number remains lower in group 2 after protamine administration (p=0.002)., Conclusions: The key finding is that the Calibrated Automated Thrombography assay, performed preoperatively, provides information predictive for blood loss after cardiac surgery.

Objective: Although failure to achieve an adequate activated coagulation time (ACT) after full heparinization before cardiopulmonary bypass often is attributed to antithrombin (AT) deficiency, it remains unclear if this is a causative mechanism of decreased heparin responsiveness. Therefore, the authors determined the relationship between AT and other coagulation-related factors that affect the ACT measurement and heparin sensitivity index before the establishment of cardiopulmonary bypass., Design: Observational study., Setting: University medical center., Participants: Adult elective cardiac surgical patients., Interventions: Preoperative data collection included demographics, type of preoperative medical therapy, hemoglobin, platelet count, and AT. Intraoperative measurements included ACT and anti-Xa activity., Results: Of the 203 patients enrolled in this study, 10% (n = 21) did not achieve an adequate ACT (≥400 seconds) after full heparinization. Subnormal AT activity (55%-79%) was not related to a low ACT and a low heparin sensitivity index. Preoperative low-molecular-weight heparin therapy did not cause a decreased ACT response. However, preoperative low hemoglobin levels and high platelet counts were associated with a decreased ACT., Conclusions: All these observations suggest that failure to achieve an adequate ACT is, in general, not an indicator of AT deficiency but could be affected by high platelet counts and low hemoglobin levels., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: In thoracic stent graft repair, the importance of segmental artery (SA) occlusion and the role of blood pressure management during the intraoperative and directly postoperative period are not clear. To study these aspects in relation to spinal cord ischemia, our protocol in the endovascular treatment of descending thoracic aneurysms covering segmental arteries T8 and lower includes preoperative assessment of the spinal cord circulation using magnetic resonance angiography, intraoperative cerebrospinal fluid drainage, and spinal cord function monitoring using motor evoked potentials (MEPs)., Methods: Thirteen patients with thoracic aortic aneurysms and dissections needing stent graft coverage of T8 and lower were included. In 9 patients, spinal cord circulation was evaluated preoperatively by magnetic resonance angiography. In 12 patients, MEPs were recorded during the endovascular procedure. A combination of both techniques was used in 8 patients., Results: The distal stent graft landing zone covered the intercostal arteries up to T10 in 4 patients, up to T11 in 7 patients, up to T12 in 1 patient, and all SAs to the aortic bifurcation in 1 patient. In 6 patients, the SA feeding the Adamkiewicz artery was covered by the stent graft. In three patients, intersegmental collaterals were present to the SA feeding the Adamkiewicz artery. The MEPs decreased to 50% and 30% in 2 patients, recovering to levels above 50% by elevation of the mean arterial pressure. Postoperatively, no signs of paraplegia were present., Conclusions: We believe that the presence of intersegmental collaterals decreases the risk of spinal cord ischemia during endovascular thoracic aortic aneurysm repair. Monitoring of MEPs during endovascular thoracic procedures shows no decrease in most cases. However, if a decrease of MEPs occurs, this can be reversed by elevation of the mean arterial pressure.

Objective: Paraplegia after thoracoabdominal aortic aneurysm (TAAA) repair mainly occurs in patients with Crawford extent I and II. We assessed the impact of monitoring spinal cord integrity and the subsequent adjusted surgical maneuvers on neurologic outcome in repairs of type I and II TAAAs., Methods: Surgical repair of TAAAs was performed in 112 consecutive patients with extent type I (n = 42) and type II (n = 70) aneurysms. The surgical protocol included cerebrospinal fluid drainage, moderate hypothermia, and left heart bypass with selective organ perfusion. Spinal cord function was assessed by means of monitoring motor evoked potentials (MEPs). Significant decreased MEPs always generated adjustments, including raising distal aortic and mean arterial pressure, reattachment of visible intercostal arteries, or endarterectomy of the excluded aortic segment with revascularization of back bleeding intercostal arteries., Results: Motor evoked potential monitoring could be achieved in all patients. By maintaining a mean distal aortic pressure of 60 mm Hg, MEPs were adequate in 82% of patients. Increasing distal aortic pressure restored MEPs in all patients. In 19 patients (17%), MEPs decreased significantly during aortic cross-clamping because of critical spinal cord ischemia. MEPs returned in all patients after spinal cord blood flow was re-established except in three patients with type II TAAA in whom MEPs could not be restored, and absent MEPs at the end of the procedure corresponded with neurologic deficit. Delayed paraplegia developed in two patients owing to hemodynamic instability with insufficient mean arterial blood pressure to maintain adequate spinal cord perfusion., Conclusion: Monitoring MEPs is a highly reliable technique to assess spinal cord ischemia during TAAA repair. A surgical protocol including cerebrospinal fluid drainage, left heart bypass, and monitoring of MEPs can reduce the paraplegia rate significantly. Adjusted hemodynamic and surgical strategies induced by changes in MEPs could restore spinal cord ischemia in most patients, preventing early and late paraplegia in all type I patients. In type II patients, early paraplegia occurred in 4.2% and delayed neurologic deficit in 2.9%. Despite all available measures, complete prevention of paraplegia in type II aneurysms seems to be unrealistic.

Objective: Renal failure is a potential complication after thoracoabdominal aortic aneurysm (TAAA) repair and is a significant risk factor for postoperative mortality. We assessed the results of distal aortic perfusion and continuous volume-controlled and pressure-controlled blood perfusion of the kidneys during TAAA repair in patients with preoperative normal and impaired renal function., Methods: Surgical repair of TAAA was performed in 279 consecutive patients (type I, n = 90; type II, 117; type III, 42; type IV, 30). In 195 patients preoperative renal function was normal; however, in 84 patients renal insufficiency was diagnosed (serum creatinine concentration [SCr], 1.4-2.0 mg/dL, n = 46; SCr, 2.0-2.5 mg/dL, n = 20; and SCr, >2.5 mg/dL, n = 18). Renal perfusion was established with catheters connected to the left-sided heart bypass. Volume flow was assessed with ultrasound, and pressure channels in the catheters enabled pressure- controlled perfusion of the kidneys., Results: Selective renal artery perfusion was achieved in all patients without technical problems or complications. In each catheter, mean arterial pressure was 69 mm Hg and volume flow was 275 mL/min. During aortic cross-clamping, urine output was uninterrupted, irrespective of clamp time. Most patients demonstrated limited or moderate increase in SCr concentration. In 17 patients (6%) SCr doubled, and peaked above 3 mg/dL, but returned to baseline levels within several days. Three patients (1%) required temporary dialysis but were discharged without further need for dialysis. In general, preoperative renal impairment did not worsen., Conclusion: Distal aortic and selective renal blood perfusion is an effective measure to protect renal function during TAAA repair, but only if perfusion is provided with adequate volume and pressure. This technique also averts dialysis in most patients with preoperative renal failure.

Background and Aim of the Study: Preoperative left ventricular (LV) function is the strongest predictor of outcome after valve replacement for aortic stenosis (AS). Although pressure-volume analysis with the conductance catheter technique can provide detailed information on LV systolic and diastolic function, this technique has not yet been used in AS patients. The present study examined the potential use of LV function measurements using pressure-volume analysis with a conductance catheter during surgery for AS., Methods: In six patients with severe symptomatic AS, a conductance catheter was placed under transesophageal echocardiographic guidance in the left ventricle via the right superior pulmonary vein., Results: The procedure was successful in all patients and lengthened the duration of surgery by <30 min, but with no increase in bypass or aortic cross-clamp times. Pressure-volume analysis showed that systolic function was normal in all patients (ejection fraction 42-59%, end-systolic elastance 1.6-4.5 mmHg/ml), while diastolic dysfunction was found in all patients (Tau 32-96 ms, LV end-diastolic pressure 7-42 mmHg, atrial kick 25-60%). After valve replacement, systolic function improved, but diastolic function did not., Conclusion: The conductance catheter placed via the pulmonary vein can determine LV systolic and diastolic dysfunction in detail in an individual patient with AS before and after valve replacement. The technique may be used to extend diagnostic data from less-invasive modalities and to determine prognosis in the individual patient.

Background: Monitoring motor-evoked potentials (MEPs) is an accurate technique to assess spinal cord integrity during thoracoabdominal aortic aneurysm (TAAA) repair, guiding surgical strategies to prevent paraplegia., Methods: In 210 consecutive patients with type I (n = 75), type II (n = 103), and type III (n = 32) TAAA surgical repair was performed using left heart bypass, cerebrospinal fluid drainage, and MEPs monitoring., Results: Reliable MEPs were registered in all patients. The median total number of patent intercostal and lumbar arteries was five. After proximal aortic crossclamping, MEP decreased below 25% of base line in 72 patients (34%) indicating critical spinal cord ischemia, which could be corrected by increasing distal aortic pressure. By using sequential clamping it appeared that in 43% of type I and II cases spinal cord circulation was supplied between T5 and L1, and 57% between L1 and L5. In type II and III cases cord perfusion was dependent upon lower lumbar arteries in 16% and pelvic circulation in 8%, necessitating reattachment of these segmental arteries. In 9% of patients critical ischemic MEP changes occurred without visible arteries, requiring aortic endarterectomy and selective grafting. One patient suffered early paraplegia and 2 delayed, and 2 patients had temporary neurologic deficit (5 of 210; 2.4%)., Conclusions: In patients with TAAA, blood supply to the spinal cord depends upon a highly variable collateral system. Monitoring MEPs is an accurate technique for detecting cord ischemia, guiding surgical tactics to reduce neurologic deficit (2.4%).

Background: In this feasibility study, early results are presented of our first series of patients with microwave ablation for atrial fibrillation (AF) on the beating heart., Methods: From June 2001 until December 2001, a total of 24 patients underwent beating-heart epicardial ablation for AF. With a microwave antenna, the left and right pulmonary veins were isolated and connected to each other followed by amputation of the left atrial appendage. Subsequently, patients underwent either off-pump coronary artery bypass graft or valve surgery on pump. The mean age of the patients was 67.4 +/- 6 years. Three patients experienced paroxysmal atrial fibrillation and all others chronic AF. Mean left atrial diameter was 5.4 +/- 0.6 cm, and mean ablation time was 13 min., Results: All procedures but one were completed successfully on the beating heart. All patients were in sinus rhythm after the procedure. A total of 15 patients experienced periods with postoperative AF during hospital stay; 9 of these patients were discharged with AF. All patients received either sotalol or amiodarone. At latest follow-up (3 to 9 months), 20 of 23 patients were in sinus rhythm., Conclusions: With microwave ablation, electrical isolation of the pulmonary veins can be achieved epicardially without cardiopulmonary bypass support.

Objectives: Aprotinin is frequently administered systemically to patients undergoing cardiopulmonary bypass to inhibit activation of platelets and plasma protein systems and thus reduce postoperative blood loss. Two reports on local aprotinin administration, that is, into the pericardial cavity, also indicated improvement in postoperative blood loss, but the underlying mechanism was not investigated. We previously reported the disappearance of glycoprotein Ib from the platelet surface and the appearance of platelet-derived microparticles in the pericardial cavity of patients undergoing cardiopulmonary bypass as signs of platelet activation. Here, we investigated whether such local aprotinin administration reduced platelet activation., Methods: In a double-blind study, 6 patients received aprotinin (500,000 KIU) into the pericardial cavity during the operation and 7 patients received a placebo. Platelet surface glycoprotein Ib expression, concentration of microparticles, and concentration of complexes of platelets with leukocytes, erythrocytes, or each other, were measured by flow cytometry., Results: We confirmed the reduced glycoprotein Ib expression and the increased concentration of microparticles in the pericardial cavity, as previously reported, and found no increased concentration of platelet complexes. However, no differences between aprotinin and placebo treatments were observed in these platelet activation parameters in the pericardial cavity or the systemic circulation., Conclusion: We conclude that administration of aprotinin into the pericardial cavity during cardiopulmonary bypass and at concentrations similar to the systemic application does not reduce platelet activation in that compartment or the systemic circulation.

Studies on graft infiltrating cells demonstrated that accumulation of cytotoxic T lymphocytes (CTL) with high avidity for donor antigens (Ag) coincided with acute cardiac rejection. In the present study, we analyse whether such high-avidity CTL are present within the peripheral blood of cardiac transplant recipients and whether their kinetics correspond with the rejection status of the allograft. Using limiting dilution analysis (LDA), donor-specific CTL were enumerated in serial blood samples of seven patients. From each patient, 7-11 samples were obtained during the first year after transplantation and up to three samples were obtained at a later date. Enumerated donor-specific CTL were divided into CTL with high or low avidity for donor Ag, depending on their sensitivity to CD8-blocking. In contrast to the situation in the graft, the donor-specific CTL present within the peripheral blood were CTL precursors (pCTL) and not fully mature CTL (cCTL). The number of donor-specific pCTL among peripheral blood cells fluctuated irrespective of the rejection grade of the allograft, indicating that the frequency of circulating donor-specific CTL does not reflect the immunological status of the allograft. During acute cardiac rejection, 66% (median) of the circulating donor-specific pCTL had a high avidity for donor Ag. This percentage significantly exceeded pre- and postrejection values obtained during the first year post-transplantation (median, 39% and 37%, respectively). The disparity in avidity increased even further more than 1 year after transplantation, when stable engraftment was achieved. Among donor-specific pCTL in peripheral blood, those with a high avidity were absent (median, 0%). Hence the avidity of circulating donor-specific CTL might inform us about the immune status of the cardiac allograft.

Background: It is assumed that not all donor-specific cytotoxic T lymphocytes (CTLs), but only those with a high avidity for donor antigens, can function as terminal effector cells in transplant rejection., Methods: In the present study, we searched for markers that would exclusively designate these high-avidity CTL., Results: FACS analysis of donor-specific CTL clones obtained from heart transplant patients revealed that high- and low-avidity CTL varied in their expression of p38, a surface molecule involved in signal transduction, which is stained by the antibody C1.7. High- and low-avidity CD8+ CTL and high-avidity CD4+ CTL expressed p38, whereas low-avidity CD4+ CTL did not. Noncytotoxic and naive CD4+ lymphocytes also lacked p38 surface expression., Conclusion: Therefore, we conclude that p38 is a marker for CD4+ lymphocytes with the potency to damage the transplanted heart. Accordingly, p38 might be used to analyze the contribution of CD4+ CTL in immune responses, such as transplant rejection.

Background: To determine mechanisms that trigger graft vascular disease (GVD) after heart transplantation, we studied parameters that reflect both early and late intragraft allogeneic reactions., Method: With reverse transcriptase-polymerase chain reaction analysis, mRNA expression of interleukin-2 (IL-2), interleukin-4, interleukin-6, interleukin-10, interferon-gamma, platelet-derived growth factor-alpha, and transforming growth factor-beta was measured in endomyocardial biopsy (EMB) specimens obtained from 34 recipients during the first acute rejection episode (n = 29) or at a comparable time after transplantation for patients without rejection (n = 5) and at time of assessment of GVD by coronary angiography at 1 year (n = 34)., Results: At the time of assessment of GVD, mRNA expression of IL-2, interleukin-4, and interleukin-6 were barely detectable, whereas messenger coding for interferon-gamma, interleukin-10, transforming growth factor-beta, and platelet-derived growth factor-alpha genes were constitutively expressed. Moreover, intragraft mRNA patterns of cytokines and growth factors between patients with GVD (n = 17) or without GVD (n = 17) were comparable. In contrast, during the first acute rejection episode a completely different pattern was found. Development of GVD was associated with IL-2 mRNA expression and not with the other cytokines analyzed. IL-2 mRNA was present in 77% of rejection EMB specimens obtained from patients with GVD versus 33% of the EMB specimens obtained from patients without GVD (p = 0.03) and not detectable in EMB specimens obtained from patients with no rejection. Also nonimmunologic risk factors such as longer ischemia time (median 193 vs 141 minutes; p = 0.002) and higher donor age (median 32 vs 23 years; p = 0.02) were associated with GVD. But no relation was found between these nonimmunologic risk factors and IL-2-positive acute rejections., Conclusions: Nonspecific risk factors and IL-2-positive rejections may independently trigger GVD after clinical heart transplantation.

Background: Treatment policy of acute rejection after heart transplantation has been changed after adopting the ISHLT endomyocardial biopsy grading system in 1991., Objective: To determine the effect of this policy change on clinical outcome after transplantation., Methods: The outcome of 147 patients who had a transplant before (early group, median follow up 96 months) and 114 patients who had a transplant after (late group, median follow up 41 months) the introduction of the ISHLT biopsy grading system was studied retrospectively. Initially "moderate rejection" according to Billingham's conventional criteria was treated. From January 1991 grade 3A and higher was considered to require intensification of immunosuppression., Results: There were some differences between the two groups: recipients (50 v 44 years) as well as donors (28 v 24 years) were older in the "late group" and more patients of this group received early anti-T cell prophylaxis (92% v 56%). Despite more extensive use of early prophylaxis more rejection episodes were diagnosed (2.4 v 1.4) and considerably more courses of rejection treatment were instituted in the late compared with the early group (3.2 v 1.5). There were no deaths because of rejection in the late group, however, more infections occurred within the first year (mean 1.8 v 1.4) and more non-skin malignancies within the first 41 months were diagnosed (8 of 57 v 6 of 147, 95% CIs of difference includes 0). The incidence of graft vascular disease in the late group has been comparable to the early group until now., Conclusion: The interpretation of the ISHLT grading system resulted in lowering of the threshold for the diagnosis of rejection thereby increasing the number of rejections and subsequently the immunosuppressive load and its complications.

During cardiac rejection we studied the kinetics of IL-2 and IL-4 mRNA and subsequent protein production by in vivo primed graft-infiltrating lymphocytes (GIL), using semiquantitative RT-PCR and ELISA. Following in vitro stimulation with either donor or third-party antigens, mRNA expression of IL-2 and IL-4 were already detectable 1-2 h after stimulation, while their protein production could be measured from 4 h onwards at least until 48 h. At both the mRNA and protein level, we measured a donor-specific signal for IL-2 and for IL-4 production (p = 0.02), while the relative donor-specific IL-2 mRNA level was significantly higher than the relative IL-4 mRNA level (p = 0.002). These observations suggest that after in vitro challenge with donor antigens, GIL obtained from rejecting cardiac allografts predominantly produce IL-2 mRNA and protein.

Background: The development of graft vascular disease (GVD) in the allograft is a major impediment for long-term survival of heart transplant recipients. GVD may be mediated by cellular processes, in response to the transplanted heart, and regulated by cytokines., Methods: We studied donor-specific cytokine production patterns in graft-infiltrating lymphocyte cultures propagated from endomyocardial biopsies. The biopsies were derived from patients with and without signs of GVD, as diagnosed by angiography at 1 year after heart transplantation., Results: In the first year after transplantation, significantly more T-helper (Th) 1 cytokines (interleukin [IL]-2: P=0.04; interferon-gamma: P=0.01), but not Th2 (IL-4 and IL-6) cytokines, were produced by cultures of patients with GVD compared with patients without GVD. Thereafter, the Th1 cytokine levels in patients with GVD normalized to the levels of patients without GVD. Detectable levels of IL-6 were produced significantly more often (P=0.009) by cultures obtained more than 1 year after transplantation from patients with GVD., Conclusions: The results suggest that high levels of Th1 cytokines produced by graft-infiltrating lymphocytes early after transplantation may be responsible for activation of vascular endothelium, leading to the migration and proliferation of smooth muscle cells that is characteristic of GVD. IL-6, produced later after transplantation, continues this process by promoting smooth muscle cell proliferation.

Background: To analyze the relevance of CD4-positive cytotoxic T-lymphocytes (CTL) in clinical heart rejection, we studied the frequency and avidity of donor human leukocyte antigen class II-specific CTL present within the graft during a rejection episode and during a period without rejection., Methods: For this analysis endomyocardial biopsies of heart transplant recipients were cultured to obtain graft-infiltrating lymphocytes (GIL). GIL cultures exhibiting donor class II-directed cytotoxicity were considered for this study. With limiting dilution analysis, the frequency of donor class II-specific CTL that had been primed by donor antigens in vivo (designated cCTL) was determined in GIL cultures established from endomyocardial biopsies taken during a rejection episode (n = 10) or during a period without rejection (n = 11). Addition of anti-CD4 to the limiting dilution analysis revealed the fraction of donor class II-specific cCTL having a high avidity for donor antigen., Results: During a rejection episode, 196 (median) donor class II-specific cCTL/106 GIL were present. In a period without rejection, the frequency of donor class II-specific cCTL was not significantly different (median = 330/10(6); p = 0.1). Addition of anti-CD4, however, revealed that donor class II-specific cCTL with a high avidity for donor antigen are predominant during a rejection episode (median = 100%) but are in minority during a period without rejection (median = 35%; p < 0.0001)., Conclusions: These results suggest that graft-infiltrating CD4+ CTL can mediate heart rejection provided they have a high avidity for donor antigen.

Background: Previously, we showed that lymphocytes cultured from cardiac allografts can lyse lymphoid cell lines from donor origin. Because endothelial cell form the first allogeneic barrier to be encountered in vivo, the reactivity of graft-infiltrating cells against donor heart-derived endothelial cells (DoHEC) may be more relevant for understanding the clinical course after heart transplantation., Methods: Endomyocardial biopsies (EMB) were taken at different times after transplantation, both during acute rejection and during quiescence. Lymphocytes cultured from these EMB were assayed for cell-mediated cytotoxicity in a 4-hour chromium 51 release test by use of target panels of different cell types derived from donor or third party. Cell specificity was investigated by addition of a tenfold excess of unlabelled target cells to 51Cr-labeled DoHEC., Results: These experiments show that DoHEC can be lysed by lymphocytes derived from EMB. Antigens that were recognized on DoHEC were often donor human leukocyte antigen class I antigens. Some cultures in addition recognized alloantigens present only on DoHEC and not on donor-derived B cells as suggested by lack of or partial inhibition of DoHEC lysis by addition of excess competitor B cells. Endothelial cell-specific reactivity was unequivocally identified after limiting dilution of EMB-derived cultures., Conclusion: We conclude that not only DoHEC-reactive but also DoHEC-specific cells are present among the cardiac graft infiltrating cells.

Background: We studied the effect of antirejection therapy on intragraft cytokine mRNA expression., Methods: Therapy consisted of three doses of 1 gm of intravenous methylprednisolone. We determined its effect on intragraft mRNA expression of immunoregulatory (interleukin-2, interleukin-4) and inflammatory cytokines (interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha), and the high-affinity interleukin-2 receptor (p55 chain) in endomyocardial biopsy specimens from cardiac allograft recipients., Results: By reverse-transcriptase polymerase chain reaction methods, we detected mRNA transcription for interleukin-2 in 56% of the pretreatment endomyocardial biopsy specimens (n = 16), for interleukin-4 in 31%, and for interleukin-6 in 56% of the specimens, and interleukin-2 receptor, interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha were constitutively expressed. Individual cytokine mRNA profiles were not helpful in differentiating between rejections that proved to be methylprednisolone resistant (n = 9) or methylprednisolone sensitive (n = 7). After successful antirejection therapy, the overall intragraft mRNA expression was downregulated. None of the posttreatment endomyocardial biopsy specimens taken from six patients with methylprednisolone-sensitive rejections expressed the interleukin-2 gene, in contrast to 88% of the endomyocardial biopsy specimens obtained from eight patients with methylprednisolone-resistant rejections (p = 0.005). Moreover, intragraft interleukin-4 and interleukin-6 mRNA transcripts were hardly detectable (both 17%) in methylprednisolone-reversible rejections, but in ongoing rejections interleukin-4 mRNA expression was found in 62% (p = 0.14), and interleukin-6 was found in 88% of the endomyocardial biopsy specimens (p = 0.03). Semiquantitative analysis showed that the intragraft interleukin-2 receptor, interleukin-1 beta, and tumor necrosis factor-alpha mRNA levels were lower in posttreatment endomyocardial biopsy specimens from methylprednisolone-reversible rejections than in endomyocardial biopsy specimens from methylprednisolone-irreversible rejections (p = 0.03)., Conclusions: Our data suggest that the efficacy of antirejection therapy with methylprednisolone is reflected in intragraft cytokine mRNA profiles.

We studied the reactivity of cardiac graft-infiltrating cells, cultured from endomyocardial biopsy specimens, toward heart endothelial cells (Hec). In two cases, Hec derived from the specific donor heart or Hec compatible with the donor were lysed, but not the syngeneic B cell line. A vessel-derived endothelial cell line was not lysed. Panel studies suggest that the epitopes recognized are, in one case, a Hec-specific peptide presented in the context of HLA-Bw41 and, in the other case, a Hec-specific peptide presented by a subtype of HLA-B44. In conclusion, we showed that cardiac graft-infiltrating cells cultured from endomyocardial biopsy specimens can exhibit cytotoxic reactivity specifically directed against HLA-peptide complexes on Hec.

In a prospective randomized trial, BT563, a murine IgG, anti-interleukin-2 receptor antibody, was compared with OKT3 for use as an early rejection prophylaxis after heart transplantation. Patients received either BT563 (n=31) or OKT3 (n=29) during the first 7 days after transplantation; cyclosporine was started on day 3. Median follow-up was 34 months. A cytokine release syndrome occurred in the majority of patients of the OKT3-treated group but in none of the BT563 recipients. The mean duration of electrical stimulation of the heart in the BT563 group was longer than in the OKT3 group (5.1 vs. 2.1 days). In both groups, one patient required insertion of a permanent pacemaker. Freedom from acute rejection at 3 months was not significantly different between the two groups (BT563: 5/29, 17%; OKT3: 6/29, 21%). In the BT563 group, however, rejection tended to occur earlier after transplantation. There was no difference in the overall incidence of rejection. The incidence of infectious complications was evenly distributed in both groups. Malignancies occurred in two patients, both in the OKT3 group. In conclusion, the use of this anti-interleukin-2 receptor monoclonal antibody in heart transplant recipients is safe and devoid of the side effects that accompany the use of OKT3. OKT3 and BT563 result in a similar freedom from rejection at 3 and 12 months after heart transplantation.

We used RT-PCR and ELISA to study the kinetics of IL-2 (Th1) and IL-4 (Th2) both on mRNA and protein level from "naive" PBMC and "primed" graft-infiltrating lymphocytes (GIL) obtained from a heart transplant recipient. For this purpose, these cells were stimulated for 1-48 h with donor and control third-party antigens. Only stimulation of GIL with donor-specific antigen resulted in early detectable IL-2 and IL-4 mRNA and protein levels. Maximal relative IL-2 mRNA levels were significantly higher than maximal relative IL-4 mRNA levels (100-fold) in both GIL and PBMC after donor-specific stimulation. This was accompanied by a maximum protein production of 908 pg/ml IL-2 and 19 pg/ml IL-4 by GIL, and of 82 pg/ml IL-2 and undetectable IL-4 production by PBMC. These results suggest that, after stimulation donor-specific "primed" GIL, and not "naive" PBMC, rapidly produce abundant levels of IL-2 (Th1) and IL-4 (Th2) at both the transcriptional and protein level.

Acute rejection (AR) and graft vascular disease (GDV) are processes mediated, at least in part, by cellular processes. Therefore, we cultured graft-infiltrating lymphocytes (GIL) from endomyocardial biopsies (EMB) taken during the first year after transplantation, determined their phenotypic composition, and correlated it to AR and GVD. We observed more often GIL growth from EMB with AR than from non-rejection EMB (P = 0.02), but no difference was found between patients with and without GVD 1 year after transplantation. CD4+ cells were always more numerous than CD8+ cells, and no difference in phenotypic composition was found between AR and non-rejection EMB nor between EMB derived from patients with or without signs of GVD. In conclusion, AR is correlated with cell growth of EMB, but the development of GVD is not associated with AR, GIL growth from EMB, or their phenotypic composition.

Monitoring for the responses to alloantigens presented either by the direct or the indirect presentation pathway have been reported to be of clinical value after kidney transplantation. Amongst others, the level of these responses may be dependent on the immunosuppressive treatment. We studied both presentation routes in peripheral blood mononuclear cells (PBMC) of cardiac transplant patients, who experienced episodes of rejection, and related them to the in vivo cyclosporin A (CsA) levels in plasma. PBMC of the recipients were stimulated with irradiated donor cells to determine the direct presentation pathway. As a method for the activation of the immune response via the indirect pathway, PBMC were stimulated with tetanus toxoid. Both immune responses increased when CsA levels inadvertently decreased to inadequate concentrations and histological rejection was diagnosed. After clinical heart transplantation, CsA may prevent rejection by blocking both the direct and the indirect antigen presentation pathway.

Both fresh and cryopreserved human cardiac valve allografts are transplanted without matching donor and recipient for blood group or human leukocyte antigens (HLA) and without the usual immunosuppressive therapy that follows organ transplantation. Calcification occurs in almost all transplanted valves, and in children acute valve failure is frequently seen. We hypothesized that failure of the human valve allografts could have an immunologic basis. This hypothesis was tested in a functional way by performing lymphocyte stimulation assays using fresh and cryopreserved valve pieces and endothelial cells derived from valve leaflets as stimulator. Human peripheral blood lymphocytes, both matched and mismatched for HLA antigens, were used as responder cells. The results were expressed as the stimulation index. Fresh valve pieces induced a significantly higher stimulation index (median, 9; range, 4 to 117) compared with the cryopreserved pieces (median, 2; range, 0 to 9; p = 0.002 by Wilcoxon test). The stimulation index was significantly reduced when lymphocytes matched for HLA-DR with the valve pieces were used (median, 1; range, 0 to 5) as compared with the HLA-DR-mismatched combination (median, 4; range, 2 to 117; p = 0.006, Wilcoxon test). Valve leaflet-derived endothelial cells were able to induce a median stimulation index of 8 (range, 3 to 15) when incubated with lymphocytes mismatched for HLA-A, -B, and -DR. In conclusion, stimulation of immune-competent cells in vitro is induced by both fresh and cryopreserved human valve pieces and by endothelial cells derived from fresh valve leaflets. The immune response can be reduced by using cryopreserved valves or by matching valve donor and responder lymphocytes for HLA-DR.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Anti-interleukin-2 receptor monoclonal antibodies have been used successfully in the prevention of rejection in cardiac allografts in several animal models., Methods: In an open randomized study murine monoclonal CD3 antibody and BT563, a murine anti-interleukin-2 receptor monoclonal antibody, were given as rejection prophylaxis during the first week after heart transplantation. Cyclosporine therapy was initiated at the third postoperative day., Results: In half the BT563-treated patients an early rejection was histologically shown at week 1, whereas heart transplant recipients treated with murine monoclonal CD3 antibody had a rejection incidence at week 1 of only 9%. During BT563 treatment CD25-positive cells (i.e., cells bearing the interleukin-2 receptor) were not detectable in peripheral blood. However, immunohistologic studies of endomyocardial biopsy specimens taken 1 week after transplantation showed the presence of CD25-positive cells within these specimens in 8 of 10 (80%) of patients with rejection. In patients without rejection CD25-positive cells were present in the biopsy specimens of only two of nine patients (22%). Reverse-transcriptase polymerase chain reaction studies on biopsy material showed the presence of messenger RNA for the interleukin-2 receptor in all and for interleukin-2 in three of five (60%) of biopsy specimens of rejecting grafts., Conclusions: Although CD25-positive cells were not detectable in peripheral blood during BT563 treatment, these cells were at the same time found to be present within 80% of the endomyocardial biopsy specimens from the rejecting grafts. By initiating cyclosporine treatment at day 0, the synergistic effect of combining cyclosporine and anti-interleukin-2 receptor monoclonal antibodies may result in a lower rejection incidence.

The cellular immunogenicity of fresh and cryopreserved human cardiac valve leaflets was measured in a lymphocyte proliferation assay. One fresh leaflet and a cryopreserved leaflet derived from the same valve were cut into 2 mm diameter pieces and incubated with responder peripheral blood mononuclear cells, matched or mismatched for HLA-DR. The tritiated thymidine incorporation into the lymphocytes measured after 7 days, was expressed as stimulation index. Fresh, HLA-mismatched valve pieces induced high stimulation index in all cases (median 9, range 4 to 117). The cryopreservation procedure resulted in a significantly lower stimulation index (p = 0.002, Wilcoxon), with a median stimulation index of 2 (range 0 to 9). In the instances where HLA-DR matched combinations were studied, cryopreservation was also associated with lower stimulation index. HLA matching itself was able to reduce the stimulation index both with cryopreserved and fresh valve pieces as stimulator, resulting in a median stimulation index of 4 (range 2 to 117) for the HLA-DR-mismatched and 1 (range 0 to 5) for the matched lymphocytes (p = 0.006, Wilcoxon). In conclusion, human cardiac valves are able to stimulate immune competent cells in vitro, even after cryopreservation. The cellular allogeneic response in vitro could be an explanation for valve allograft degeneration observed in the clinic. Matching for HLA-DR may reduce these effects.

Long-term survival of heart transplant recipients is limited by the development of transplant coronary artery disease (TCAD). We analysed whether the development of TCAD is correlated with the incidence of acute rejection episodes, with the formation of anti-HLA antibodies or with the composition and function of T lymphocyte cultures derived from endomyocardial biopsies. TCAD was assessed by visual analysis of annually performed coronary angiograms and defined as the presence of all vascular changes, including minor wall irregularities. One year after transplantation, 31 of the 77 patients studied had TCAD (40%). The median age and mean number of HLA mismatches in patients with or without TCAD were highly comparable. The patient groups did not differ in incidence of acute rejection episodes, nor in percentage of endomyocardial biopsies yielding T cell cultures. At 1 year after transplantation, lymphocyte cultures from 18/31 TCAD+ patients (58%) and 27/46 TCAD- patients (57%) were analysed. The TCAD+ patients had, compared with the TCAD- patients, a higher median percentage of CD8+ T cells (71% versus 25%, P = 0.06) and a lower median percentage of CD4+ T cells (4% versus 40%, P = 0.04). Similar differences were found in a longitudinal analysis of the culture results of endomyocardial biopsies (EMBs) obtained during the first year. The cytotoxic reactivity of the cultures against donor HLA class I or class II antigens was comparable in the two groups, although a difference in recognition of heart specific antigens remains possible. The fact that EMB-derived cultures from TCAD+ and TCAD- patients differed in T cell phenotype populations gives some support to the hypothesis that cellular immunological processes are involved in the development of TCAD. However, while the median values differed, the overlap of the percentages of CD8+ cells in cultures from TCAD- and TCAD+ patients shows that other factors besides CD8+ T cells also play a role.

We have used limiting dilution analysis to study the qualitative and quantitative differences between graft-infiltrating cytotoxic T cell populations propagated from endomyocardial biopsies of heart transplant patients who experienced one or more acute rejection episodes and patients who never showed signs of rejection. Limiting dilution cultures were stimulated with autologous or donor cells both in the absence or in presence of cyclosporine and of CD8 in the cytotoxic phase. Almost all antigen-primed, committed cytotoxic T cells (cCTL) present in the graft of patients with rejections were CsA resistant. In contrast, in most patients of the nonrejector group, a substantial part of the cCTL could be inhibited by CsA. The CTL precursors (pCTL) in both groups were predominantly CsA sensitive. Addition of CD8 mAb during the cytotoxicity phase of the limiting dilution analysis was used to differentiate between CTL populations with high avidity for donor antigens and populations with low avidity. The predominant subpopulation in the graft of rejectors was a CsA-resistant cCTL with high avidity, while in the graft of most nonrejectors, cCTL with low avidity dominated. In most rejectors, CD8 mAb had only a minor influence on the pCTL frequency estimates, and thus on T cells with high avidity. CsA-sensitive pCTL with high avidity might represent an intermediate stage between the naive pCTL and mature, functional, CsA-insensitive cCTL with high avidity for donor antigens.

Objective: To evaluate the experiences of aortic valve replacement with cryopreserved aortic allografts in the subcoronary position., Design: Descriptive clinical study., Setting: Thorax Centre of Rotterdam University Hospital., Method: In the period July 1988-March 1993, 68 patients (45 men and 23 women) with a mean age of 45 years underwent 69 operations. One woman was operated twice. Main criteria for aortic valve replacement by an allograft were age and endocarditis. The pathology leading to aortic valve dysfunction had been: endocarditis (38%), congenital lesions (32%), rheumatic lesions (19%), senile degeneration (7%), paravalvular leakage (3%) and an active autoimmune disease (1%)., Results: Hospital mortality was 4%. The mean follow-up was 22 months, with a cumulative follow-up of 126 patient years. One patient died of lung cancer during this period. Six patients needed a second operation (9%); 4 of these were among the first 15 patients operated on. One of these reoperations was necessary to treat a recurrent endocarditis. Only one other late valve-related event took place: a transient ischaemic attack 2 years postoperatively. Ninety-one per cent of the patients with an allograft in situ were in NYHA class I at their last outpatient clinic visit. The other patients were in class II. Pre-cordial color Doppler echocardiography showed no or mild aortic regurgitation in 95% of these patients. The remaining 3 patients had moderate aortic regurgitation., Conclusion: Aortic valve replacement with an allograft was performed with low hospital mortality. The expected advantages are greatest in young patients and patients with endocarditis; these patients, therefore, should have priority in allograft allocation.

To monitor their immunological status we determined donor and third-party-specific cytotoxic T-cell precursor frequencies (CTLpf) in the peripheral blood of 15 heart transplant recipients. PBL samples were obtained at different time points before and after transplantation. Donor-specific CTLpf and third-party-specific CTLpf were within the same range for all samples (1-1489/10(6) cells). The donor-specific CTLpf were not different between patients who had never had an acute rejection (AR) and patients who had an acute rejection as diagnosed by endomyocardial biopsy (EMB). No difference was observed between donor-specific CTLpf of samples taken on the day of transplantation and those obtained between 3 months and 3 years after transplantation. There was also no relationship between the donor-specific CTLpf in the PBL and the culturing success of lymphocytes from EMB taken at the same time. CTLpf were in the same range both when cultures could be propagated from the graft and when no cells grew out. We conclude that long-term graft survival is possible in the presence of CTLpf in peripheral blood.

BT 563, a murine anti-IL-2R MoAb, was found to be more potent than anti-Tac in inhibiting proliferation in the mixed lymphocyte reaction. Results obtained with 33B3.1 in these experiments were similar to those with BT 563. The anti-IL-2R MoAb 2A3 was shown to be a suitable agent for monitoring the effect of BT 563 on peripheral blood. IL-2R-positive cells were not detected in peripheral blood samples from 1 h after the first dose until 8 days after the last dose. Plasma trough levels were measured in patients receiving 5 or 10 mg daily. The administration of BT 563 to allograft recipients did not lead to clinically significant side effects.

We analyzed the results of passive immunization against CMV in 146 heart transplant recipients. The 65 seronegative recipients were prophylactically treated with anti-CMV immunoglobulins during and after the operation. Twenty-nine of these 65 patients received a seropositive donor heart. CMV infection occurred in 21/65 seronegative and in 40/81 seropositive recipients (difference not significant). The incidence of CMV infection in seronegative recipients of a CMV-matched donor heart (3/34) was significantly lower than in seronegative recipients of a positive donor heart and lower than in seropositive recipients, but no significant difference in infection rate was found between the two latter groups (18/29 vs. 40/81). Although primary infection more frequently resulted in CMV disease than secondary infection (11/21 vs. 10/40) no difference in incidence of disease was noted between seronegative and seropositive patients (11/65 vs. 10/81), nor was there a difference in the severity of symptoms following primary or secondary infection. There was a higher incidence of CMV disease in all patients who received a heart from a seropositive donor versus a seronegative donor. However, after transplantation of a heart from a seropositive donor the incidence (27%) of CMV disease observed in our passively immunized seronegative patients was the same as in the patients with naturally acquired seropositivity. There was no difference in the prevalence of coronary artery disease between patients with and without CMV infection or disease. We conclude that using the current passive immunization scheme the occurrence of CMV infection and disease is largely dependent on the serostatus of the donor.

The value of pulsed-wave transmitral Doppler for the diagnosis of moderate acute rejection was examined in a total of 347 Doppler recordings obtained in 32 consecutive cardiac allograft recipients. Serial Doppler examinations (median, 11 per patient; range, 1 to 23) were performed simultaneously with endomyocardial biopsies from the first week after heart transplantation to a follow-up of 186 days (median; range, 10 to 395 days after transplantation). Pulsed-wave transmitral Doppler did not allow noninvasive diagnosis of moderate acute rejection in individual patients. Peak filling rate normalized for mitral stroke volume, early diastolic velocity, and mean diastolic velocity were significantly increased, whereas diastolic filling period was decreased during moderate acute rejection compared to other biopsy classes. The wide overlap of measurements in individual recipients with or without rejection may be due, however, to a variety of hemodynamic factors after transplantation affecting diastolic function, which are superimposed on the restrictive left ventricular filling pattern caused by persistent mild acute rejection and left ventricular hypertrophy. These hemodynamic factors include pulmonary hypertension, perioperative ischemia, reperfusion injury, and changes in both blood pressure and loading conditions caused by hypertension and its treatment. Differences between studies with regard to the detection of moderate acute rejection by transmitral Doppler may be caused by chance, because most studies were relatively small. Differences in methods, patient selection, duration of follow-up, prevalence of hypertension and left ventricular hypertrophy, and differences in antihypertensive drug regimens may also play a role. Furthermore differences in the incidence of mild acute rejection, its treatment, and the type of maintenance immunosuppressive regimen used may have influenced the outcome of these studies considerably.

The increasing numbers of long-term survivors after heart transplantation make yearly coronary arteriography, used by most centers to study the development of transplant coronary artery disease, less practical. Therefore the prevalence and clinical relevance of coronary artery disease in 119 one-year survivors of heart transplantation were studied. Visual analysis revealed two main patterns of vascular changes: abnormalities of the epicardial vessels and their major branches and abnormalities of the tertiary branches. The prevalence of all abnormalities in the coronary vascular tree increased from 34% after 1 year to 79% after 5 years. The prevalence of anatomically significant lesions (more than 50% stenosis in the epicardial branches or abrupt ending/proximal occlusion of tertiary branches) was only 11% after 5 years. During follow-up of 25 to 87 (median, 43) months, no significant coronary artery disease developed in the 101 patients who showed normal epicardial vessels or abnormal tertiary branches only at their first year angiography, and none of the patients died of ischemic heart disease. Of the 18 patients with abnormal epicardial vessels, three patients died of ischemic heart disease; one of these patients was treated with atherectomy and is alive at the moment of this report, and two patients showed progression of discrete lesions without evidence of ischemia until now. Based on these findings, a schedule for timing of arteriography was developed depending on the first-year coronary findings.

Endothelial cells may be involved in the acute rejection of allografts. In the present study, graft infiltrating lymphoid cell lines were propagated from a heart graft at the time of histological diagnosis of rejection. The cell lines containing CD8+ cells lysed donor-derived BLCL and endothelial cells (EC) but not third party BLCL or random EC, suggesting that HLA antigens were recognized. The cell lines containing CD4+ cells only did not lyse any target cells. The lysis of EC without preincubation with gamma interferon (gIFN) indicated that the HLA antigens recognized were class I antigens. These results suggested that lysis of donor EC may be one of the mechanisms involved in rejection.

Chronic rejection (CR) is a major problem in long-term survival in heart transplantation. We analysed whether the occurrence of CR correlates with the incidence of acute rejections (AR) or with characteristics of endomyocardial biopsy-derived cell cultures. CR was diagnosed by annual angiography and defined as all coronary vascular changes. One year after transplantation 24 of the 63 patients had CR (38%). The incidence of AR in CR+ and CR- patients was comparable. The patients in both groups had similar individual median percentages of EMB-yielding cell cultures. During the first year the CR- patients had more cultures in which at least 60% of the cells were CD4+ T cells (50% vs 37%, P = 0.05), due to a stronger CD4 predominance in the first 6 months. In the second year the CD4 predominance in the patients diagnosed as CR+ after 1 year tended to be higher (P = 0.08). The patients had comparable percentages of cultures predominated by CD8+ T cells, gammadelta T cells or NK cells, irrespective of the time interval. These results might indicate that CD4+ T lymphocytes play a dual role in the aetiology of CR.

Recent studies comparing the effects of induction therapy with polyclonal antilymphocyte globulins (ALG) or with monoclonal T-cell-specific antibodies are not unanimous. Therefore, 55 heart recipients were allocated to either 7-day courses of polyclonal ALG (n = 28) or of monoclonal OKT3 (n = 27). Additionally, azathioprine and low dose steroids were given. There were no severe side effects after OKT3; the course of ALG, however, had to be discontinued in 20 patients because of extensive flares. No differences between the two groups were found in freedom from rejection or in the incidence of infection. The 1- and 2-year survival was 96% in both groups. Although monoclonal and polyclonal induction therapies are equally effective for rejection prophylaxis, OKT3 may be preferred because of a lack of important side effects. However, the fact that a shorter course of ALG is equally effective may be in favour of ALG.