Heart and combined heart-kidney transplantation in patients with concomitant renal insufficiency and end-stage heart failure.

In patients with end-stage heart failure (ESHF) who are candidates for isolated heart transplant (HRT), dialysis dependence (DD) is considered an indication for combined heart-kidney transplantation (HKT). HKT remains controversial in ESHF transplant candidates with nondialysis-dependent renal insufficiency (NDDRI). Using United Network for Organ Sharing data, we examined the cumulative incidences of transplant and mortality in patients with DD and NDDRI waitlisted for HKT or HRT. In all groups, 3-month waitlist mortality was dismal: 31% and 21% for HRT- and HKT-listed patients with DD and 12% and 7% for HRT- and HKT-listed patients with NDDRI. Five-year posttransplant survival was improved in HKT recipients compared with HRT recipients for both patients with DD (73% vs. 51%, p<0.001) and NDDRI (80% vs. 69%, p<0.001). Likewise, multivariable analysis associated HKT with better outcomes than HRT in HKT-listed patients, although both improved survival. These data argue strongly for HKT in ESHF transplant candidates with DD. However, in patients with NDDRI, HKT must be weighed against the possibility of renal recovery with isolated HRT. Whether HRT (followed by a staged kidney transplant in patients who do not recover renal function after HRT), as opposed to HKT, maximizes organ benefit for patients with NDDRI and ESHF requires assessment. Nevertheless, given their dismal waitlist outcomes and excellent posttransplant results, we suggest that patients with DD and NDDRI with ESHF be considered for early listing and transplant.

Combined heart-liver transplantation in the MELD era: do waitlisted patients require exception status?

Combined heart-liver transplant (HLT) is a viable therapy for patients with concomitant end-stage heart and liver failure. Using data from the United Network for Organ Sharing database, we examined the cumulative incidences of transplant and mortality in waitlisted candidates for HLT, isolated heart transplant (HRT) and isolated liver transplant (LIV) in the Model for End-Stage Liver Disease era. The incidence of waitlist mortality was higher in HLT candidates than in HRT candidates (p = 0.001, 26% vs. 12% at 1 year) or LIV candidates (p = 0.005, 26% vs. 14% at 1 year). These differences persisted after stratifying by disease severity. Posttransplant survival was not significantly different between HLT and HRT recipients or between HLT and LIV recipients. In a multivariable model, undergoing HLT was associated with enhanced survival for HLT candidates (hazard ratio, 0.41; confidence interval, 0.21-0.79; p = 0.008), but undergoing HRT alone was not. Interestingly, 90% of HLT recipients were allocated an organ locally, compared to 60% of HRT candidates and 73% of LIV candidates (both p < 0.001). These data suggest that the current cardiac and liver allocation systems may underestimate the risk of death for patients with concomitant end-stage heart and liver failure on the HLT waitlist.

Trends in invasive disease due to Candida species following heart and lung transplantation.

Although invasive candidiasis (IC) causes significant morbidity and mortality in patients who undergo heart, lung, or heart-lung transplantation, a systematic study in a large cohort of thoracic organ transplant recipients has not been reported to date. Clinical and microbiological data were reviewed for 1305 patients who underwent thoracic organ transplantation at Stanford University Medical Center between 1980 and 2004. We identified and analyzed 76 episodes of IC in 68 patients (overall incidence 5.2% per patient).The incidence of IC was higher in lung (LTx) and heart-lung transplant (HLTx) recipients as compared with heart transplant (HTx) recipients (risk ratio [RR] 1.7, 95% confidence interval [CI] 1.1-2.7).The incidence of IC decreased over time in all thoracic organ transplant recipients, decreasing from 6.1% in the 1980-1986 time period to 2.1% in the 2001-2004 era in the HTx recipients, and from 20% in the 1980-1986 period to 1.8% in the 2001-2004 period in the LTx and HLTx recipients.The most common site of infection differed between the HTx and LTx cohorts, with bloodstream or disseminated disease in the former and tracheobronchitis in the latter. IC in the first year after transplant was significantly associated with death in both HTx (RR 2.9, 95% CI 1.8-4.6, P=0.001) and LTx and HLTx patients (RR 3.0, 95% CI 1.9-4.6, P<0.001). The attributable mortality from IC decreased during the 25-year period of observation, from 36% to 20% in the HTx recipients and from 39% to 15% in the LTx and HLTx recipients. There were a significant number of cases caused by non-albicans Candida species in all patients, with a trend toward higher mortality in the HTx group. In conclusion, the incidence and attributable mortality of IC in thoracic organ transplant recipients has significantly declined over the past 25 years.The use of newer antifungal agents for prophylaxis and treatment, the decrease in the incidence of cytomegalovirus disease, and the use of more selective immunosuppression, among other factors, may have been responsible for this change.

Twenty-year survivors of heart transplantation at Stanford University.

Human heart transplantation started 40 years ago. Medical records of all cardiac transplants performed at Stanford were reviewed. A total of 1446 heart transplantations have been performed between January 1968 and December 2007 with an increase of 1-year survival from 43.1% to 90.2%. Sixty patients who were transplanted between 1968 and 1987 were identified who survived at least 20 years. Twenty-year survivors had a mean age at transplant of 29.4 +/- 13.6 years. Rejection-free and infection-free 1-year survivals were 14.3% and 18.8%, respectively. At their last follow-up, 86.7% of long-term survivors were treated for hypertension, 28.3% showed chronic renal dysfunction, 6.7% required hemodialysis, 10% were status postkidney transplantation, 13.3% were treated for diabetes mellitus, 36.7% had a history of malignancy and 43.3% had evidence of allograft vasculopathy. The half-life conditional on survival to 20 years was 28.1 years. Eleven patients received a second heart transplant after 11.9 +/- 8.0 years. The most common causes of death were allograft vasculopathy (56.3%) and nonlymphoid malignancy (25.0%). Twenty-year survival was achieved in 12.5% of patients transplanted before 1988. Although still associated with considerable morbidity, long-term survival is expected to occur at much higher rates in the future due to major advances in the field over the past decade.

Sequential orthotopic heart transplantation in man.

Between January 1968 and March 1980, 202 hearts had been transplanted into 185 patients at Stanford University Medical Center. Occasionally, patients after transplantation develop myocardial failure which is amenable only to retransplantation. Sixteen patients underwent initial orthotopic allograft using standard techniques. Eight patients developed accelerated arteriosclerotic coronary disease, six had unrelenting rejection, and two had donor heart dysrrhythmia or right ventricular failure requiring retransplantation. One patient required a third transplant because of donor left ventricular ischemia. All sequential transplants were managed similarly to the primary transplant. Of the initial transplant hearts at risk, 60% survived for more than 1 year, and 57% survived for more than 2 years. These results are similar to those of patients not requiring retransplantation. Of the secondary transplant hearts at risk, 31% survived for more than 1 year and 29% survived for more than 2 years. The severity of infection and/or rejection contributed most significantly to secondary heart transplant mortality. Sequential orthotopic cardiac transplantation offers an acceptable alternative to patients with allograft failure. Survival is not as favorable as with initial transplantation because of the prolonged immunosuppression during sequential transplantation.

Use of rabbit antithymocyte globulin in cardiac transplantation. Relationship of serum clearance rates to clinical outcome.

Serum rabbit globulin (RG) clearance rates were determined in 30 consecutive cardiac transplant recipients by radioimmune assay of serum RG levels after completion of an initial postoperative course of rabbit anti-human antithymocyte globulin (RATG). Twenty patients, who exhibited rapid RG elimination rates (average half-life, 1.6 days), had a rejection onset time of 16.2 days rejection frequency of 3.9 episodes/100 patient days, and a 1-year survival rate of 59%, respectively, as compared with 28.3 days, 1.9 episodes/100 patient days, and 80%, respectively, for the 10 patients with more prolonged initial RG elimination rates (average half-life, 11.4 days); Nineteen patients received one or more repeat courses of RATG. In 16 of these a progressive increase in RG half-life during subsequent RATG administration could be demonstrated. A close correlation was observed between total RATG doses given in the initial course and peak serum levels of RG obtained (r = 0.82) and between onset of rejection and initial t1/2 RG (r = 0.69). This latter correlation was improved by the elimination of one of the 30 patients (r = 0.81) or by considering only those patients treated from a single RATG batch (r = 0.85; n = 15). No significant relationship was detected between any of the parameters assayed and (1) total RATG dose, or (2) rosette inhibition titers of RATG administered. Survival and rejection parameters of the first 30 patients receiving RATG were compared with the previous 20 receiving equine antithymocyte globulin; these 50 comprising the entire population in which rejection was confirmed by cardiac biopsy. Rejection onset was 20 versus 12 days, rejection frequency was 3.1 versus 5.0 episodes/100 patient days, and graft survival at 1 year was 66 versus 41% for the RATG-equine antithymocyte globulin-treated patients, respectively. From these data it was concluded that (1) RATG administration favorably affects transplantation outcome; (2) RATG half-life, as reflected by RG clearance rates, was the most important variable governing RATG effectiveness, (3) variation in rosette inhibition titers within RATG batches made in the same fashion from large rabbit pools were of minimal clinical importance; and (4) monitoring of serum RG levels provided a necessary and rational basis for effective modulation of immunosuppressive therapy.

Radioimmune assay of heterologous serum gamma-globulin in patients receiving rabbit antihuman thymocyte globulin.

A radioimmune assay (RIA) method for detecting heterologous serum rabbit gamma-globulin (RG) and antibody to this protein is described. The methodology is used for monitoring serum levels of rabbit globulin in patients receiving rabbit ATG (RATG). In 7 cardiac recipients receiving RATG, maximum serum levels of RG were achieved 1-3 days after administration of final dose. RG half-life subsequent to peak serum levels was rapid (X = 36 hr) in 4 patients and prolonged (X = 18 days) in 3 patients. Patient antibody to rabbit gamma-globulin was detectable only in those patients with short RG half-life. Antirabbit antibody titers in these patients were extremely low and barely detectable by RIA.

Clinical heart-lung transplantation.

Combined heart and lung transplantation was carried out in thirteen patients at Stanford University between March 1981 and May 1983. The recipients were between 22 and 45 years old. All patients were suffering from end-stage pulmonary hypertension; nine patients had Eisenmenger's syndrome; the remaining four were transplanted for primary pulmonary hypertension. Three patients died within one month of surgery. The remainder are well at between 22 months and three weeks from operation. The duration of stay in the hospital for the surviving patients ranged from 38 to 85 days. The immunosuppressive protocol has been essentially the same for all recipients, and has consisted of cyclosporine with an initial course of rabbit antithymocyte globulin (RATG) with azathioprine given for the first two weeks, and then replaced with prednisone. Rejection, as diagnosed by cardiac biopsy, was treated with pulses of methylprednisolone. Early complications included bleeding that necessitated reexploration (five patients); damage to the vagus, recurrent laryngeal, or phrenic nerves (three patients); and failure of the donor lungs (one patient). Modifications of technique that have developed include removal of the recipient heart and lungs separately, and preservation of the lungs with a modified Collins' solution instead of a cardioplegic solution. The results of this operation are considerably superior to clinical efforts in lung transplantation. The combined operation may be preferable for the following reasons: All diseased tissue is removed, thus eliminating recurrent infection, and also perfusion/ventilation disparity. Transplantation of the entire heart and lung block preserves coronary-tracheal vascular anastomoses and makes airway dehiscence less likely. Diagnosis of rejection by cardiac biopsy seems to be a satisfactory method of diagnosis and treatment of pulmonary rejection.

Survival of primates following orthotopic cardiac transplantation treated with total lymphoid irradiation and chemical immune suppression.

Fractionated total lymphoid irradiation (TLI) has been used for attempts at induction of a donor-specific tolerant-like state in allograft recipients and for immunosuppressive effects. Cyclosporin A (Cy A) has been shown to suppress rejection of organ grafts in many species including man. The present study was designed to test the effectiveness of TLI in combination with either CY A or rabbit anticynomolgus thymocyte globulin (ATG) and azathioprine. Thirty-one orthotopic cardiac allografts were performed using surface cooling and total circulatory arrest in outbred cynomolgus monkeys. TLI was administered preoperatively in fractions of 100 rad until a total of 600 or 1800 rad was achieved. Cy A was administered 17 mg/kg/day. All treatment groups demonstrated extended survival. Myocardial biopsies as early as 4 weeks were consistent with mild rejection in all treatment groups. No significant synergistic effect upon survival could be demonstrated utilizing TLI plus Cy A when compared with using Cy A alone. TLI (1800 rad) plus ATG and azathioprine was associated with a high incidence of early death attributable to leukopenia and infection. Cy A alone or in combination with TLI was associated with the development of lymphoid malignancy.

Vulnerability of patients with obstructive hypertrophic cardiomyopathy to ventricular arrhythmia induction in the operating room. Analysis of 17 patients.

To evaluate vulnerability to ventricular arrhythmia induction, programmed electrical stimulation was performed in the operating room in 17 consecutive patients undergoing myotomy-myectomy for obstructive hypertrophic cardiomyopathy (HC). A control group of 5 patients undergoing coronary artery bypass grafting with normal left ventricular function and no previous myocardial infarction also was tested. Of the 17 patients with HC, 14 had inducible sustained ventricular tachycardia (VT) or ventricular fibrillation (VF), 1 had inducible unsustained VT and the remaining 2 had less than 6 ventricular beats. In contrast, none of the 5 control patients had an inducible sustained ventricular arrhythmia, 1 had inducible unsustained VT, and the remaining 4 had less than 3 ventricular beats. The difference between the 2 groups with respect to induction of a sustained ventricular arrhythmia, unsustained VT or less than 6 ventricular beats was significant (p less than 0.001). It is concluded that patients with severe obstructive HC are unusually vulnerable to ventricular arrhythmia induction. This suggests that spontaneous ventricular tachyarrhythmias may be an important cause of sudden death in patients with HC.

Relative efficacy of blind left ventricular aneurysm resection for the treatment of recurrent ventricular tachycardia.

Coronary arterial bypass grafting and left ventricular aneurysm resection and the two combined have been reported effective in control of refractory ventricular tachyarrhythmias; 82 percent of a pool of 127 patients (from 22 reports) survived after surgery. However, the follow-up period in this group is short and the extent of medical therapy is not well defined. Actuarial analysis of results of conventional left ventricular aneurysm resection in 32 Stanford patients with well documented ventricular tachyarrhythmias shows an arrhythmia recurrence rate of 50 +/- 9 percent (mean +/- standard error of the mean) during the postoperative hospitalization. In contrast, after 10 months only 11 +/- 9 percent of 18 patients who underwent myocardial resection guided by intraoperative electrical activation sequence mapping experienced arrhythmia recurrence. These data demonstrate that simple left ventricular aneurysm resection is less effective in preventing ventricular tachyarrhythmias than originally believed. Preliminary data suggest that surgery of the left ventricle guided by intraoperative mapping may provide more effective control of ventricular tachyarrhythmias. However, intraoperative mapping has many technical and interpretive problems. Investigations are needed to determine the roles of conventional and new operative approaches to treatment of medically refractory ventricular tachyarrhythmias.

The automatic implantable defibrillator: local ventricular bipolar sensing to detect ventricular tachycardia and fibrillation.

The first-generation automatic implantable defibrillator implanted in man sensed arrhythmias by monitoring a transcardiac electrocardiographic signal. This sensing system reliably detected ventricular fibrillation and sinusoidal ventricular tachycardia but failed to sense all nonsinusoidal ventricular tachycardias. To solve this problem, a new ventricular tachycardia detection scheme was developed using a local ventricular bipolar electrogram and electronic circuits using rate averaging and automatic gain control to permit sensing of electrograms down to 0.1 mV. This detection scheme was tested during electrophysiologic studies in 11 patients with ventricular tachycardia and fibrillation. All 22 episodes of induced ventricular tachycardia with a rate above the selected cutoff were detected after an average of 5.1 +/- 1.8 seconds. No episodes below the rate cutoff were detected. The bipolar circuits also reliably detected ventricular fibrillation. Arrhythmia detection and signal quality in 9 patients receiving automatic defibrillators using the new bipolar rate detection circuit were compared with the findings in 5 patients previously receiving units that sensed arrhythmias using the transcardiac electrocardiographic signal. Compared with the transcardiac monitoring units the newer bipolar units had shorter and more uniform sense times (5.5 +/- 1.4 versus 12.2 +/- 7.1 seconds). It is concluded that malignant ventricular tachyarrhythmias can be sensed accurately using bipolar rate detection and that this system has numerous advantages over the previously used transcardiac electrocardiographic signal.

Surgical treatment of aneurysms of the inferior left ventricular wall.

Experience with surgical treatment of 10 patients with aneurysms of the inferior wall of the left ventricle is presented. Six of the 10 aneurysms were false (pseudoaneurysms), and four were classified as true aneurysms. All except one resulted from myocardial infarction. Combined procedures, performed at the time of aneurysm resection, included mitral valve replacement (five patients), coronary artery bypass grafting (four patients), and closure of an interventricular septal defect (one patient). Three of four patients with true inferior aneurysms had mitral valve dysfunction, whereas only two of six patients with false aneurysms required mitral valve replacement (one because of infective endocarditis). Nine of the 10 patients survived operation, and all are functionally improved except one. On the basis of this and previously reported experience, it is concluded that a substantial proportion of inferior left ventricular aneurysms exhibit the pathological features of false aneurysms. Because of the associated propensity toward rupture of such lesions, an aggressive surgical approach is recommended.

Long-term hemodynamic results after cardiac transplantation.

Although survival after cardiac transplantation has improved since the introduction of cyclosporine to clinical practice in 1980, the long-term hemodynamic results of transplantation in cyclosporine-treated recipients has not been reported. Annual cardiac catheterization data for 109 cyclosporine-treated recipients were analyzed and compared to those of a nonconcurrent group of 65 recipients treated with azathioprine and corticosteroids. Recipient age, donor age, sex, and human leukocyte antigen mismatch were comparable for the two groups. Satisfactory left ventricular function of the cyclosporine-treated heart was characterized on the first annual study by a normal ejection fraction (60% +/- 10%), cardiac index (3.0 +/- 0.8 L/min/m2) and stroke work index (53 +/- 15 gm-m/m2) associated with moderately increased left ventricular end-diastolic pressures (12 +/- 6 mm Hg) and significantly increased mean aortic pressures (116 +/- 8 mm Hg). With the exception of aortic diastolic pressure, which tended to increase with time, the mean values of each variable analyzed did not change significantly over the period of study. In comparison to the azathioprine group, the cyclosporine cohort displayed higher aortic, left ventricular end-diastolic, and pulmonary artery pressures and produced more stroke work at each annual study. Analysis of the azathioprine group over extended (8 year) follow-up suggested excellent preservation of graft function. In summary, the long-term hemodynamic function of the transplanted heart treated with cyclosporine was satisfactory, demonstrated no deterioration over 5 year follow-up, but manifested substantially greater hypertension than hearts from the pre-cyclosporine era.

Long-term experience with porcine aortic valve xenografts.

Between 1971 and 1975, glutaraldehyde-preserved porcine aortic valve xenografts were employed for isolated replacement of the mitral valve (MVR) in 243 patients, replacement of the aortic valve (AVR) in 167 patients, and double valve replacement (AVR and MVR) in 51 patients. Postoperatively, long-term anticoagulation was not routinely given. Operative mortality rates for AVR, MVR, and double valve groups were 7.8, 6.0, and 11.8 per cent, respectively; the majority of early postoperative deaths were associated with concomitant coronary artery disease. No death was attributable to xenograft dysfunction. Follow-up of all patients was obtained. The total duration of follow-up for the MVR group was 347 patient-years, for the AVR GROUP 148 148 patient-years, and for double valve replacement 37 patient-years; maximum follow-up for these three groups was 4.4, 4.0, and 2.4 years, respectively. Actuarial analysis of postoperative survival rates at a common interval of 3 years showed 78 per cent for MVR patients, 91 per cent for AVR patients, and 80 per cent (projected) for patients with double valve replacement (85, 96, and 91 per cent for operative survivors, respectively. At this same interval 92 per cent of MVR patients, 99 per cent of AVR patients, and 93 per cent (projected) of patients with double valve replacement were free of thromboembolic episodes. Altogether, 12 of the total 512 valves implanted exhibited some evidence of dysfunction during the entire period of follow-up evaluation, but in only 2 instances (both mitral) was intrinsic pathological involvement of the xenograft tissue documented. Actuarial analysis of xenograft dysfunction at a common interval of 3 years after operation showed 95 per cent of MVR patients, 98 per cent of AVR patients, and 97 per cent (projected) of patients with double valve replacement to be free of this complication. These data support the use of glutaraldehyde-preserved porcine xenografts as superior bioprostheses that pose a low risk of thromboembolism without anticoagulation. The over-all durability of such valves, within the restriction of a maximum current follow-up interval of 4.4 years, appears comparable to that of currently available mechanical prostheses and justifies continued clinical use.

The superiority of aortic cross-clamping with profound local hypothermia for myocardial protection during aorta-coronary bypass grafting.

Two different operative techniques for aorta-coronary bypass grafting were utilized in two comparable groups of patients. In one group (155 patients) distal anastomoses were carried out with the aorta cross-clamped and myocardial protection provided by profound local hypothermia (clamped group). In a second group (149 patients) distal anastomoses were carried out with the aorta unclamped and the left ventricle fibrillating and vented (unclamped group). Mortality rates were similar in the two groups (0.6 per cent in clamped group versus 1.3 per cent in unclamped group). The incidence of perioperative infarction was 15 per cent in the unclamped group and 8 per cent in the clamped group (p less than 0.05). Postoperative serum glutamic oxaloacetic transaminase (SGOT) and lactic dehydrogenase (LDH) levels were significantly higher for the first 4 postoperative days in the unclamped group than in the clamped group. Hemodynamic studies in a subset of each group revealed no important differences in left ventricular function in the immediate postoperative period. The data demonstrate that in patients undergoing aorta coronary bypass grafting, performance of distal anastomoses with aortic cross-clamping and profound local hypothermia results in less intraoperative myocardial injury than performance of distal anastomoses in the perfused, fibrillating, and vented left ventricle.

Left ventricular aneurysm. Preoperative risk factors and long-term postoperative results.

The results of operative treatment of postinfarction left ventricular aneurysm in 169 patients undergoing operation since 1970 are analyzed in this report. Maximum follow-up extended to 7 year (average 2.9 years). Average patient age was 56 years (range 34 to 82 years). Nearly all patients (94%) had left anterior descending coronary artery disease with anterior aneurysm formation and 73% had multivessel disease. Sixty-eight percent of patients underwent aorta-coronary bypass grafting (ACBG) and/or mitral valve replacement (MVR) concomitantly with aneurysmectomy. The over-all operative mortality rate was 17.8%. Preoperative factors that correlated significantly (p less than 0.05) with increased operative risk reflected primarily the quality of left ventricular function, and included functional classification, cardiac index, contractile function of residual myocardium not involved by aneurysm, and mitral regurgitation. Patients whose primary preoperative disability consisted of angina pectoris (42 patients) exhibited significantly higher over-all survival rates (actuarial 5 year survival 75%) than those undergoing operation because of congestive heart failure (86 patients) or ventricular tachyarrhythmias (38 patients), whose 5 year survival rates were 52 and 57%, respectively. Concomitant ACBG (+/- MVR) was associated with a higher operative mortality rate than aneurysmectomy alone (21.1 versus 10.9%), but late postoperative attrition was similar. The over-all 5 year survival rate, including operative death, was 60%, and 90% of surviving patients were in Functional Class I or II at follow-up evaluation. We conclude from this analysis that the long-term prognosis of patients with symptomatic postinfection left ventricular aneurysms, although determined importantly by preoperative left ventricular function, is enhanced by surgical treatment.

Operative technique for heart-lung transplantation.

Combined heart and lung transplantation has now been carried out in 17 patients at Stanford University Hospital. The emphasis on the management of donors is upon thorough tracheobronchial toilet and ensuring an absence of significant cardiopulmonary disease. The most important aspects of the recipient operation are to remove the heart and lungs without injury to the phrenic, vagus, or recurrent laryngeal nerves and to ensure hemostasis. This is best effected if the heart and lungs are removed separately. The operative technique for both the donor and recipient is described.

Hemodynamic observations in the early period after human heart transplantation.

Serial hemodynamic measurements, including determination of cardiac output by the Fick technique, were obtained in 10 human cardiac recipients for intervals up to 38 days after transplantation. Immediately postoperatively, donor cardiac output was severely depressed because of limitation of stroke volume. Spontaneous recovery of cardiac output and stroke volume then occurred gradually over the first 4 postoperative days to normal or nearly normal levels. Rate augmentation by arterial and/or ventricular pacing early after transplantation had little effect on donor heart performance, but isoproterenol caused significant enhancement of graft function and is now used routinely in postoperative management. Serial hemodynamic monitoring proved to be of little use in the prediction or confirmation of acute graft rejection episodes.