Normally and abnormally functioning left-sided porcine bioprosthetic valves after long-term implantation in patients: distinct spectra of histologic and histochemical changes.

This morphologic study (X-ray examination of gross specimens, histologic study and histochemical staining) compares two groups of explanted left-sided bioprosthetic valves: group I, 6 valves with normal cusp function and group II, 10 valves with significant dysfunction. Implantation periods ranged from 26 to 79 months. A computerized descriptive statistical method (principal component analysis) is used to analyze the qualitative results. Although qualitatively identical alterations are observed in both groups, the findings in the deep layers of the cusps of severe collagen breakdown, intensive fibrin penetration and various degrees of calcification are restricted to group II. Other findings of interest in both groups include amyloid deposits (four cases) and layering of fusiform host cells on the cusp surface (three cases). The computerized study shows that individuals of one clinical group are morphologically different from those of the other. Mechanical stress may contribute to surface alterations early after implantation, while further collagen breakdown and macrophagic activity result in deep penetration of plasma components and fibrin. Subsequent calcification is likely to be dystrophic rather than metabolic. Colonization of the cuspal surface by endothelial cells after long-term implantation of bioprosthetic valves expresses a new type of relation between host and bioprosthesis.

Effects of serotonin on coronary arteries of cardiac transplant recipients.

Serotonin constricts coronary arteries with endothelial dysfunction. To detect early graft artery disease, the responses to intracoronary serotonin were studied 1 month (group A, 14 patients) and 1 year (group B, 13 patients) after orthotopic cardiac transplantation. No patient had evidence of rejection and all had angiographically normal coronary arteries. Serotonin in increasing doses (1, 10 and 20 micrograms/min for 2.5 minutes each) was infused into the coronary circulation. Diameters of proximal, middle and distal segments were measured by quantitative angiography. At the maximal concentration of serotonin, the diameters of the proximal segments decreased to 73 +/- 4% (percentage of the baseline) in group A; the diameters of the middle and distal segments decreased to 67 +/- 5 and 63 +/- 4%, whereas in group B, the diameters of the proximal, middle and distal segments were 90 +/- 6% (p < 0.02 vs group A value), 88 +/- 5% (p < 0.01 vs group A value) and 84 +/- 4% (p < 0.01 vs group A value), respectively. These changes were significantly (p < 0.02) different from those observed in 6 control patients in whom no constriction was induced by intracoronary serotonin. Moreover, coronary plasma endothelin levels were significantly higher in group A than in group B and control patients (5.6 +/- 0.3 vs 4.3 +/- 0.2 fmol/ml in group B and 3.9 +/- 0.3 fmol/ml in control patients). Thus, an abnormal response to intracoronary serotonin seems to occur often in transplant patients, and this abnormality is unexpectedly more pronounced in the early weeks after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of L-arginine effect on coronary hypersensitivity to serotonin in cardiac transplant recipients.

Coronary hypersensitivity to serotonin promotes platelet aggregation and, therefore, the progression of the atherosclerotic process. This abnormality occurs in the early stages of coronary atherosclerosis when the responses to bradykinin are still preserved. To determine whether such changes also occur early after cardiac transplantation, intracoronary injections of bradykinin and serotonin were performed in 7 control patients, in 19 patients with dyslipidemia, and in 15 cardiac transplant recipients (1 year after operation). Coronary angiography was normal in the 3 groups. In the segments where serotonin effects were the most pronounced, the diameter changes were measured by quantitative angiography. Bradykinin (60, 200, and 600 ng) increased in the same way as the coronary diameters in the 3 groups; in contrast, serotonin elicited vasodilation only in the control group (7+/-3%, percentage of baseline) and vasoconstriction in the hyperlipidemic group (-9+/-2%) and in transplant recipients (-15+/-3%). After intracoronary infusion of L-arginine (40 mg/min for 14 minutes), serotonin-induced constriction was attenuated in the hyperlipidemic group but not in transplant recipients. Thus, the response to bradykinin is preserved in the early stages of graft vasculopathy. However, in contrast to patients with hyperlipidemia, the absence of an L-arginine effect on the responses to serotonin suggests the involvement of mechanisms other than a decrease in endothelium-derived nitric oxide availability. Immune processes promoting the release of endothelium-derived contracting factors such as endothelin and/or superoxide anion may play a role.

Inflammatory response to cardiopulmonary bypass: mechanisms involved and possible therapeutic strategies.

BACKGROUND: Recent study of the inflammatory reactions occurring during and after cardiopulmonary bypass (CPB) has improved our understanding of the involvement of the inflammatory cascade in perioperative injury. However, the exact mechanisms of this complex response remain to be fully determined. METHODS: Literature on the inflammatory response to CPB was reviewed to define current knowledge on the possible pathways and mediators involved, and to discuss recent developments of therapeutic interventions aimed at attenuating the inflammatory response to CPB. RESULTS: CPB has been shown to induce complement activation, endotoxin release, leukocyte activation, the expression of adhesion molecules, and the release of many inflammatory mediators including oxygen-free radicals, arachidonic acid metabolites, cytokines, platelet-activating factor, nitric oxide, and endothelins. Therapies aimed at interfering with the inflammatory response include the administration of pharmacologic agents such as corticosteroids, aprotinin, and antioxidants, as well as modification of techniques and equipment by the use of heparin-coated CPB circuits, intraoperative leukocyte depletion, and ultrafiltration. CONCLUSIONS: Improved understanding of the inflammatory reactions to CPB can lead to improved patient outcome by enabling the development of novel therapies aimed at limiting this response.

Combined heart-lung transplantation for terminal pulmonary lymphangioleiomyomatosis.

Combined heart-lung transplantation with cyclosporine is reported in a 26-year-old patient who presented with end-stage pulmonary lymphangioleiomyomatosis. The operation was successful and the patient's rehabilitation excellent over the first 7 postoperative months. She then developed obliterative bronchiolitis of unknown origin. To our knowledge, this is the first published report of an out-hospital survival after heart-lung transplantation for terminal nonvascular lung disease.

Dextroposition of the left lower lobe after heart-lung transplantation.

Immediately after heart-lung transplantation for cystic fibrosis, a patient had development of a right lower lobe retrocardiac density that persisted on all postoperative chest radiographs. A computed tomographic examination of the thorax performed 3 weeks after surgery showed that there was partial collapse of the left lower lobe in the right hemithorax. The patient required a posterolateral thoracotomy for cure.

Myocardium is a major source of proinflammatory cytokines in patients undergoing cardiopulmonary bypass.

Proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-6, and interleukin-8, and anti unflammatory cytokines, such as interleukin-10, may play an important role in patient responses to cardiopulmonary bypass. We sought to define whether the myocardium and the lungs serve as important sources of these cytokines under conditions of cardiopulmonary bypass. Ten patients (age 64 +/- 3 years, mean +/- standard error of the mean) undergoing elective coronary artery bypass grafting were monitored with an arterial catheter, a coronary sinus catheter, and pulmonary artery catheter. Plasma levels of tumor necrosis factor-alpha, interleukin-6, interleukin-8, and interleukin-10 were measured simultaneously in peripheral arterial blood, coronary sinus blood, and mixed venous blood before heparin administration, 1 minute before aortic crossclamping, 5 minutes after aortic declamping, and at 0.5, 1, 1.5 and 2 hours after aortic declamping. The durations of cardiopulmonary bypass and aortic crossclamping were 114 +/- 9 and 64 +/- 5 minutes, respectively. Levels of tumor necrosis factor-alpha and interleukin-6 were significantly higher in coronary sinus blood than in arterial blood after aortic declamping. Tumor necrosis factor-alpha and interleukin-6 levels were also higher in mixed venous blood than in arterial blood within 1 hour after declamping. There were no significant differences among the three sampling sites with respect to interleukin-8 and interleukin-10 levels. In one patient who had postoperative myocardial infarction, however, interleukin-8 levels were three times as high as in coronary sinus blood than in arterial blood. These data indicate that the myocardium is a major source of tumor necrosis factor-alpha and interleukin-6 in patients undergoing cardiopulmonary bypass. The lungs may consume rather than release proinflammatory cytokines in the early phase of reperfusion. The source under these conditions of the antünflammatory cytokine interleukin-10 remains to be determined.

Oxygenation improvement with nitric oxide in right-to-left shunt without significant effects on pulmonary arterial pressure.

Following surgical closure of an interventricular communication complicating an anterior myocardial infarction, a 74-year-old woman developed severe right ventricular failure and hypoxemia due to the opening of a patent foramen ovale (PFO). Mean pulmonary artery pressure was 24 mm Hg. Treatment with inhaled nitric oxide (5 ppm) increased PaO2 from 47 to 90 mm Hg (FIo(2)1). The present observation points out that nitric oxide inhalation could be useful in the management of severe hypoxemia from a right-to-left shunt due to a PFO even when there is no significant pulmonary hypertension present.

Human cytokine responses to cardiac transplantation and coronary artery bypass grafting.

Cardiac surgery with cardiopulmonary bypass triggers an inflammatory response involving proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-6, and interleukin-8. To elucidate the pathophysiology of this cytokine response, we explored the possible differences in cytokine responses between patients undergoing heart transplantation and those undergoing coronary artery bypass grafting. Plasma levels of tumor necrosis factor-alpha, interleukin-6, interleukin-8, and interleukin-10 were measured in eight patients undergoing heart transplantation (mean age 44 years) and eight patients undergoing coronary artery bypass grafting (mean age 61 years). Duration of cardiopulmonary bypass and ischemic time were both longer in the heart transplantation group than in the coronary artery bypass grafting group (133 +/- 26 min vs 100 +/- 31 min, p < 0.05, and 130 +/- 47 min vs 58 +/- 21 min, p < 0.005, respectively). Samples were collected before heparin administration, at aortic crossclamping and declamping, and at 0.5, 1, 1.5, 2, 4, 12, and 24 hours after declamping. Tumor necrosis factor-alpha levels were significantly higher 30 minutes after aortic declamping in the heart transplantation group than in the coronary artery bypass grafting group (68 +/- 30 vs 18 +/- 5 pg/ml, p < 0.05). Interleukin-6 and interleukin-8 levels were also significantly higher 90 minutes after declamping in patients undergoing heart transplantation than in those undergoing coronary artery bypass grafting (310 +/- 63 vs 169 +/- 24 pg/ml, p < 0.05, and 73 +/- 17 vs 24 +/- 5 pg/ml, p < 0.01, respectively). Furthermore, interleukin-6 and interleukin-8 values 90 minutes after declamping were significantly correlated with the ischemic time (r = 0.72 and r = 0.82, respectively, both p < 0.05). Interleukin-10 levels in both groups rose to reach a peak value of around 115 pg/ml 1 hour after declamping. Patients undergoing heart transplantation exhibited a second peak of tumor necrosis factor-alpha, interleukin-8, and interleukin-10 levels 12 hours after declamping, probably related to the administration of rabbit antihuman thymocyte immunoglobulin (Thymoglobuline) 3 hours after declamping. Interleukin-6 levels decreased more significantly 12 and 24 hours after declamping in patients undergoing heart transplantation, probably related to methylprednisolone therapy. In conclusion, cardiopulmonary bypass is associated with the production of both proinflammatory and antiinflammatory cytokines. The production of proinflammatory cytokines in patients undergoing heart transplantation is higher than that in patients undergoing coronary artery bypass grafting, and this increase could be related to the longer duration of ischemia in the former group. The later course of cytokine levels after heart transplantation may be further influenced by immunosuppressive therapy.

Prostaglandin E1 infusion for right ventricular failure after cardiac transplantation.

The infusion of prostaglandin E1, a vasodilating substance with predominant effects on the pulmonary vasculature, has been found effective in the management of pulmonary hypertension associated with various diseases. The reported experience with prostaglandin E1 after cardiac transplantation is, however, limited. We used prostaglandin E1 in 18 patients in whom acute right ventricular failure developed after orthotopic cardiac transplantation. The infusion was started within 24 hours after operation in 16 patients and was continued for up to 7 days. Maximal doses of prostaglandin E1, administered via a central venous catheter, ranged from 30 to 120 ng/kg/min. Norepinephrine was simultaneously infused via a left atrial catheter in 10 patients to prevent a reduction in systemic arterial pressure. The prostaglandin E1 infusion resulted in significant reductions in mean arterial pressure and pulmonary vascular resistance and simultaneous increases in cardiac index and stroke index. Mean arterial pressure was stable and left ventricular stroke work increased. The alveolar oxygen tension/forced inspiratory oxygen index tended to decrease during the infusion. Three patients died, two of right heart failure and one of multiple organ failure associated with cardiac allograft rejection. In patients in whom right ventricular failure associated with pulmonary hypertension develops after cardiac transplantation, prostaglandin E1, combined with norepinephrine whenever the arterial pressure declines, can effectively reduce pulmonary artery pressures and improve global cardiac function without compromising systemic perfusion.

Does steroid pretreatment increase endotoxin release during clinical cardiopulmonary bypass?

OBJECTIVE: The mechanism involved in the endotoxemia frequently recognized during cardiopulmonary bypass remains unclear. It has also been suggested that endotoxin levels were higher in steroid-pretreated patients undergoing cardiopulmonary bypass. METHODS: Twenty patients undergoing cardiopulmonary bypass were randomly pretreated with steroids (methylprednisolone, 30 mg/kg) or placebo. Blood samples for endotoxin measurement were drawn simultaneously from the superior and inferior venae cavae before heparin administration, 5 and 50 minutes after the onset of bypass, 5 minutes after aortic declamping, at the end of bypass, and 1, 2, and 20 hours after the end of cardiopulmonary bypass. RESULTS: The perioperative variables in the two groups were similar. Blood endotoxin levels were higher in the inferior vena cava than in the superior vena cava immediately after the onset of bypass. Endotoxin levels in inferior vena cava blood were significantly lower in steroid-pretreated patients than those in patients not receiving steroids. CONCLUSIONS: Endotoxin is released during cardiopulmonary bypass from the region drained by the inferior vena cava. Steroid pretreatment may actually reduce endotoxin release during bypass.

Hypophosphatemia after cardiothoracic surgery.

The incidence of hypophosphatemia during the first 48 h following cardiothoracic surgery was prospectively studied in 74 patients. Hypophosphatemia, defined by a serum phosphate below 2.50 mg/dl, was observed in 19 of 34 (56%) patients after thoracic surgery and in 20 of 40 (50%) patients after cardiac surgery. As a whole, hypophosphatemia occurred earlier after thoracic than after cardiac surgery. After thoracic surgery, hypophosphatemia was milder for patients in whom bleeding was more severe. The anticoagulant solution CPD used in stored blood was identified as an important source of phosphate. These results indicate hypophosphatemia is a common finding after cardiothoracic surgery. Since severe hypophosphatemia can be related to phosphate depletion, phosphate supplements could be warranted especially during thoracic surgery when blood transfusions are less than 1000 ml.

Corticosteroids increase blood interleukin-10 levels during cardiopulmonary bypass in men.

BACKGROUND: Interleukin (IL)-10 is a potent antiinflammatory cytokine inhibiting the release of tumor necrosis factor--alpha (TNF-alpha) and IL-8 by activated macrophages and polymorphonuclear leukocytes. Cardiopulmonary bypass (CPB) represents a unique situation where an inflammatory reaction is predictably induced. The present study examined the influence of CPB on the release of TNF-alpha, IL-1 beta, IL-8, and IL-10 and also defined the effects of pretreatment with corticosteroids on the release of these cytokines. METHODS: The study included 22 patients undergoing coronary artery bypass graft operations, including eight control patients and seven patients who received dexamethasone, and seven patients who received methylprednisolone 4 hours before the operation. Cytokines were measured with the enzyme-linked immunosorbent assay technique before treatment, before anesthesia induction, immediately before heparin administration, before aorta declamping, 10 minutes and 90 minutes after aorta declamping, and 4 hours after the end of CPB. RESULTS: In the control patients the TNF-alpha levels and especially the IL-8 levels increased during CPB and reached their maximal levels 4 hours after CPB. IL-10 levels rose moderately and transiently, reaching peak values 90 minutes after aorta declamping. Notably, administration of corticosteroids prevented IL-8 release but increased IL-10 levels, which were tenfold higher than in the control group 90 minutes after aorta declamping (dexamethasone, 271 +/- 128 pg/ml; methylprednisolone, 312 +/- 213 pg/ml; control, 17 +/- 12 pg/ml, p < 0.05). IL-1 beta was not detected in any group of patients. CONCLUSIONS: The present data indicate that IL-10 is released together with proinflammatory cytokines during and after CPB and that pretreatment with corticosteroids markedly enhances this release. The release of IL-10 may play an important role in the antiinflammatory effects of corticosteroids.

Cytokine responses to cardiopulmonary bypass: lessons learned from cardiac transplantation.

BACKGROUND: A growing body of evidence relates the release during cardiopulmonary bypass (CPB) of proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8, to the postoperative systemic inflammatory response syndrome. Antiinflammatory cytokines, such as IL-10, however, may also play an important role in limiting these complications. METHODS: The English-language literature was reviewed. Emphasis was placed on cytokine responses during clinical CPB for cardiac operations and, in particular, for heart and heart-lung transplantation. RESULTS: The recent data indicate that (1) although cytokine release can be triggered by many factors during CPB, ischemia-reperfusion may play the most important role; (2) the levels of tumor necrosis factor-alpha, IL-6, and IL-8 are correlated with the duration of cardiac ischemia and the myocardium is a major source of these three cytokines during CPB; (3) IL-10 levels are correlated with the duration of CPB and the liver is a major source of IL-10 during CPB; and (4) steroid pretreatment is an effective intervention to inhibit the release of proinflammatory cytokines and enhance IL-10 production. CONCLUSIONS: The improved knowledge of cytokine responses to CPB may help to develop interventions aimed at reducing postoperative morbidity and mortality.

Thrombus formation on a calcific and severely stenotic bicuspid aortic valve.

We report on a case of thrombus formation on a native bicuspid aortic valve, which was found during an elective operation for aortic valve replacement. Although no apparent predisposing cause of thrombosis could be ascertained, severe calcific stenosis of the bicuspid valve and cardiac catheterization may have played a role. The patient is in excellent condition 9 months after the operation.

Cardiac tamponade due to spontaneous rupture of right coronary artery aneurysm.

A case of acute cardiac tamponade caused by spontaneous rupture of a right coronary artery aneurysm is reported. The aneurysm, which was present distally, was ligated during operation. Postoperative angiography suggested the aneurysm was congenital. The patient is doing well 5 months after operation.

Steroid administration in heart and heart-lung transplantation: is the timing adequate?

BACKGROUND: The release of cytokines after cardiopulmonary bypass may play an important role in postoperative morbidity. The release of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin (IL)-6 and IL-8, is even greater in patients undergoing heart transplantation (HTx) than coronary artery grafting. We tested the hypothesis that in HTx patients the earlier administration of steroids, before rather than after cardiopulmonary bypass as usual, can reduce the inflammatory response. METHODS: In 20 consecutive patients who underwent HTx or heart-lung transplantation (HLTx), plasma levels of tumor necrosis factor alpha, IL-6, IL-8, and anti-inflammatory cytokine IL-10 were measured before heparin administration, at aortic cross-clamping and declamping, and 0.5, 1, 1.5, 2, 4, 12, and 24 hours after aortic declamping. In 10 patients (group I, 6 HTx and 4 HLTx), 500 mg of methylprednisolone was first given as usual at 1.5 hours after aortic declamping (at the end of cardiopulmonary bypass). In the next 10 patients (group II, 6 HTx and 4 HLTx), the first doses of methylprednisolone were given 1 hour before operation. In both groups, 125 mg of methylprednisolone were given every 8 hours thereafter during the first postoperative day. RESULTS: The ischemic time and cardiopulmonary bypass time were similar in the two groups (166 +/- 16 minutes versus 157 +/- 13 minutes, and 192 +/- 21 minutes versus 186 +/- 20 minutes, respectively, mean +/- standard error of the mean). At 30 minutes after aortic declamping and throughout the next 4 hours, tumor necrosis factor alpha levels were significantly lower in group II than in group I (all p < 0.03). Interleukin-8 values 1 hour after declamping were also lower in group II than in group I (49 +/- 15 pg/mL versus 130 +/- 38 pg/mL, p < 0.02). Interleukin-10 levels were significantly higher in group II than in group I from 30 minutes after declamping through 2 hours after (all p < 0.03). Interleukin-6 levels were similar in the two groups. CONCLUSIONS: Earlier steroid administration in the immunosuppressive protocol for HTx or HLTx may be preferable to reduce the inflammatory response to cardiopulmonary bypass, as reflected by a lower production of tumor necrosis factor alpha and IL-8, and a greater release of IL-10.

Heparin-coated circuits reduce myocardial injury in heart or heart-lung transplantation: a prospective, randomized study.

BACKGROUND: The effects of heparin-coated (HC) circuits have been primarily investigated in routine cardiac operations with limited duration of cardiopulmonary bypass (CPB) and ischemia. Their benefits have not been conclusively proven but could be more significant when CPB and ischemic times are longer, such as during heart transplantation (HTx) or heart-lung transplantation (HLTx). METHODS: In a 22-month period, 29 patients undergoing HTx and HLTx were randomly divided into two groups using HC (Duraflo II, n = 14, 10 HTx and 4 HLTx) or uncoated but identical circuits (NHC group, n = 15, 10 HTx and 5 HLTx). All patients received full systemic heparinization (3 mg/kg) during CPB. Plasma endotoxin, interleukin (IL)-6, IL-8, IL-10, IL-12, and cardiac troponin-I were measured before heparin administration, immediately after aortic cross-clamping, 5, 30, 60, 90, 120 minutes, and 12 and 24 hours after aortic declamping. The intensive care unit (ICU) staff and the laboratory technologists were blinded as to the use of HC circuits. RESULTS: No statistically significant differences between groups were found with respect to all baseline values, duration of CPB and aortic cross-clamping, graft ischemic time, doses of heparin, postoperative blood loss and transfusion, peak lactate and creatine kinase-MB isoenzyme values, duration of mechanical ventilation, or length of ICU stay. One patient in each group died during the hospital stay. Patients in the HC group needed more protamine sulfate after CPB. Although endotoxin levels were similar in the two groups, significantly lower IL-6, IL-8, and IL-10 levels were observed 1 hour after aortic declamping in the HC group. The release of cardiac troponin-I was also significantly reduced in the HC group 12 and 24 hours after reperfusion. CONCLUSIONS: The use of HC circuit limits both pro- and anti-inflammatory responses to CPB. It may also reduce myocardial injury after prolonged duration of CPB and ischemia.

Histopathology of the aortic valve in patients with a previous history of acute rheumatic fever. An analysis of 63 surgical specimens.

In a series of 63 surgical patients with a positive history of rheumatic fever and whose aortic valve was removed either singly or in combination with other valves, the histological examination of the aortic valves showed functional lesions in 22 cases (35%). Organic lesions of inflammatory origin, destruction of the architecture with scarring and presence of hypertrophic vessels, were observed in 19 valves (30%). The other cases were doubtful. This ratio was identical in uni- and plurivalvular involvement. Anatomic aspects and significance of microscopic lesions of the aortic valves were discussed. The role of microthrombi and turbulent flow in the pathogenesis of the functional lesions is stressed. The authors conclude that there is no correlation between the anatomic aspects of the aortic valve deformity and the presence of histologically proven organic lesions of inflammatory origin. This provides support to the current opinion that rheumatic carditis is mainly a disease of the mitral valve.

Determinants of right ventricular failure after heart transplantation.

Predictive factors of right ventricular failure after heart transplantation are not well identified. Clinical and hemodynamic data from 20 patients who developed right heart failure were compared to those of 20 matched patients who did not experience this complication after cardiac transplantation. Preoperative systemic and pulmonary hemodynamics were comparable in the two groups. Patients with posttransplant right ventricular failure had longer waiting time (27 +/- 6 vs 16 +/- 3 weeks, mean +/- SE, P < 0.05), no regression of pulmonary hypertension (0 +/- 0.1 vs 2.3 +/- 0.3 Wood units reduction in pulmonary vascular resistance after transplantation, P < 0.01), and had been ventilated with higher levels of positive end-expiratory pressure (5 +/- 1 vs 1.5 +/- 0.5 cm H2O, P < 0.05). One-month postoperative evolution (mortality, hospital stay, radionuclide ejection fractions) was similar in the two groups. These results suggest that a lesser reversibility of pulmonary hypertension (possibly due to a longer evolution of the cardiac disease, as indicated by the longer waiting time) is the main determinant of right ventricular failure after heart transplantation.