Billingham Me

Noninvasive discrimination of rejection in cardiac allograft recipients using gene expression profiling

Rejection diagnosis by endomyocardial biopsy (EMB) is invasive, expensive and variable. We investigated gene expression profiling of peripheral blood mononuclear cells (PBMC) to discriminate ISHLT grade 0 rejection (quiescence) from moderate/severe rejection (ISHLT ≥3A). Patients were followed prospectively with blood sampling at post‐transplant visits. Biopsies were graded by ISHLT criteria locally and by three independent pathologists blinded to clinical data. Known alloimmune pathways and leukocyte microarrays identified 252 candidate genes for which real‐time PCR assays were developed. An 11 gene real‐time PCR test was derived from a training set (n = 145 samples, 107 patients) using linear discriminant analysis (LDA), converted into a score (0–40), and validated prospectively in an independent set (n = 63 samples, 63 patients). The test distinguished biopsy‐defined moderate/severe rejection from quiescence (p = 0.0018) in the validation set, and had agreement of 84% (95% CI 66% C94%) with grade ISHLT ≥3A rejection. Patients >1 year post‐transplant with scores below 30 (approximately 68% of the study population) are very unlikely to have grade ≥3A rejection (NPV = 99.6%). Gene expression testing can detect absence of moderate/severe rejection, thus avoiding biopsy in certain clinical settings. Additional clinical experience is needed to establish the role of molecular testing for clinical event prediction and immunosuppression management.

Detection of coronary atherosclerosis in young adult hearts using intravascular ultrasound.

BackgroundCoronary atherosclerosis has been demonstrated in young adults by postmortem pathology. Angiographic evaluation of coronary disease in young adults is limited by ethical issues and the insensitivity of angiography for detecting early pathology. Catheter-based intracoronary ultrasound has proven useful both in detecting and quantitating coronary disease, but the ultrasound appearance of young, angiographically normal, coronary arteries has not been well defined. Methods and ResultsTwenty-five subjects were examined with intracoronary ultrasound within 1 month of cardiac transplantation. Mean age of the donor hearts was 28 years (range, 14–43 years). Measurements of an index of intimal thickening were obtained at four left anterior descending coronary artery sites in each patient. All study patients had angiographically normal coronary arteries. Ultrasound in 14 subjects demonstrated a three-layered appearance of the coronary vessel wall with a mean intimal index of 0.16±0.07. The other 10 subjects, including all donors under the age of 25 years, had coronary vessel wall layers too thin to be imaged separately at the 30-MHz sound frequency. Five subjects had ultrasound evidence of focal intimal thickening greater than 500 μm. The donors of these hearts each had risk factors for coronary artery disease. Two subjects died within 5 weeks of their ultrasound study. Histological measurements of the vessel wall layers were similar to the corresponding ultrasound values. ConclusionsThis study provides a reference for the intravascular ultrasound appearance ofyoung adult coronary arteries and confirms pathology findings that young subjects with angiographically normal vessels have a range of coronary intimal thickening, which includes occasional evidence of focal, early atheromatous lesions.

Study of the normal and failing isolated human heart: Decreased response of failing heart to isoproterenol

We evaluated the effects of isoproterenol in right ventricular papillary muscles derived from normal and failing isolated human hearts. Basal values for the peak force developed, rate of force development (dF/dt), and time to peak tension (TPT) were similar in both groups. Isoproterenol produced a significantly smaller (p less than 0.05) increase in peak force developed and dF/dt in failing papillary muscles. The half equivalent dose (ED50) of isoproterenol was fivefold higher in failing muscle as compared to normal muscle. We conclude that failing cardiac muscle demonstrates decreased responsiveness to beta-receptor mediated stimulation.

Effects of cyclosporin, FK506, and rapamycin on graft-vessel disease.

Graft-vessel disease (GVD) limits the long-term survival of heart-transplant patients, and this effect has not been altered by use of cyclosporin for immunosuppression. We compared the effects of the immunosuppressants cyclosporin, FK506, and rapamycin on GVD in a rat-heart transplantation model. Allografted hearts from rats treated with 1 mg/kg FK506 for 50 days showed the same degree of myocardial rejection but a significantly worse (p less than 0.05) grade of GVD compared with grafted hearts from rats treated with 1.5 mg/kg cyclosporin for the same time. 2 mg/kg FK506 for 50 days prevented cellular rejection but GVD was as severe as that found with 1 mg/kg FK506. Moderate GVD was present in two of five allografted hearts after treatment with 4 mg/kg FK506. 1.5 mg/kg rapamycin for 50 days was an effective inhibitor of rejection and GVD. Based on our results in rats, the possibility that GVD may occur in human heart-transplant recipients treated with FK506 cannot be excluded.

Sensitivity and specificity of radionuclide ejection fractions in doxorubicin cardiotoxicity

We examined radionuclide-determined left ventricular ejection fractions (LVEF) at rest and during graded exercise in 37 patients receiving doxorubicin (Adriamycin) therapy in whom the risk of developing congestive heart failure (CHF) was precisely defined by endomyocardial biopsy and right heart catheterization. Echocardiographic (Echo %FS) and phonocardiographic (PEP/LVET) measurements of LV function were also determined. An abnormal LVEF at rest (less than or equal to 45%) had a sensitivity of 53% and a specificity of 75% for detecting patients at moderate or high risk of developing CHF. The addition of exercise LVEF increased the sensitivity of detection of moderate or high-risk patients to 89% but lowered the specificity to 41%. Exercise LVEF improved the sensitivity of detection of high-risk patients from 58% to 100%. Echo %FS and PEP/LVET yielded lower sensitivities than rest or exercise LVEF. As a single test, exercise LVEF possesses the sensitivity for use as a screening method for anthracycline cardiotoxicity, but the lack of specificity prevents the use of single values as a definitive test. Single rest LVEF determinations, although more specific than exercise LVEF, do not possess the sensitivity for use as screening or definitive tests.

INDUCTION OF DONOR-SPECIFIC UNRESPONSIVENESS TO CARDIAC ALLOGRAFTS IN RATS BY PRETRANSPLANT ANTI-CD4 MONOCLONAL ANTIBODY THERAPY

In the present report a monoclonal antibody designated OX-38 directed against the rat CD4 molecule was tested for its ability to prolong the survival of heterotopic vascularized rat heart allografts transplanted across major histocompatibility barriers. Fluorescence-activated cell-sorter analysis showed that administration of OX-38 selectively depleted 80–95% of CD4+ cells from peripheral blood of treated rats. The immunosup-pressive effects of OX-38 in vivo were verified by suppression of an antibody response against OX-38 itself as a heterologous protein immunogen. Recipient rats received OX-38 antibody as a single agent given in pretransplant regimens. Nine of 12 treated rats have maintained heterotopic abdominal heart allografts for >175 days. Control rats that did not receive antibody therapy rejected their grafts within 14 days. Rats that maintained heart allografts for >100 days accepted second donor strain hearts but rejected third-party heart grafts transplanted into the femoral space. Anti-CD4-induced allograft unresponsiveness persisted for at least 90 days following surgical removal of donor tissue and retransplantation of a second donor-matched heart. These results indicated that transient, pretransplant therapy with monoclonal antibodies directed against the CD4+ lymphocyte induced specific, long-lasting unre-sponsiveness to fully MHC-mismatched cardiac allografts in rats without additional immunosuppression.

A hemodynamic and Doppler echocardiographic study of ventricular function in long-term cardiac allograft recipients. Etiology and prognosis of restrictive-constrictive physiology.

Conventional hemodynamic measurements and Doppler echocardiography were used to assess ventricular physiology of the human cardiac allograft and to examine the influence of pertinent clinical factors on chronic myocardial performance. Sixty-four patients (18-55 years old; mean, 39 years) undergoing routine annual hemodynamic assessment were studied. Blood-flow velocity properties across the mitral, tricuspid, and aortic valves were analyzed from Doppler ultrasound recordings. Ten of these patients had elevated diastolic pressures associated with a sharp early diastolic dip followed by an exaggerated and abrupt rise in pressure, consistent with restrictive-constrictive ventricular physiology. Left ventricular dP/dt and stroke volume were lower in these patients compared with the other 54 patients. Doppler echocardiographic indexes of left ventricular filling and ejection in these 10 patients differed significantly. Isovolumic relaxation time and pressure half-time were shorter, peak early mitral and tricuspid flow velocities were higher, and mean aortic flow velocity and acceleration were lower. A higher rejection incidence was the only demonstrable clinical factor associated with impaired ventricular function. Doppler echocardiography may, therefore, noninvasively identify patients with hemodynamic evidence of restrictive-constrictive physiology. This abnormality occurs in approximately 15% of allograft recipients, is associated with impaired systolic performance, and may be related to rejection incidence.

Cardiac abnormalities in myotonic dystrophy. Electrophysiologic and histopathologic studies.

Eight young adult male patients with myotonic dystrophy, mean age 26 years, underwent 24-hour Holter electrocardiographic monitoring and intracardiac electrophysiologic study. Right ventricular endomyocardial biopsies were performed at the end of the electrophysiologic study in five of them. The atrial to His[A-H] interval was 155 msec in one case and less than or equal to 55 msec in all patients. Twenty-four hour Holter electrocardiographic monitoring demonstrated more than 4 premature ventricular contractions per minute in two patients and marked cyclical sinus arrhythmia during sleep in two others. Electron microscopic analysis of the endomyocardial biopsy specimens disclosed no prominent sarcoplasmic reticulum abnormalities but prominent I bands compared to previously obtained controls. Myofibrillar degeneration was seen in all cases and was associated with abnormal mitochondria in two. Cardiac abnormalities can be detected very early in the evolution of myotonic dystrophy, even prior to the onset of cardiac symptoms. The reported abnormalities appear closely related to the pathologic process affecting other skeletal muscles.

Mildly dilated congestive cardiomyopathy.

Five patients with only mildly dilated ventricles but other features typical of congestive cardiomyopathy underwent cardiac transplantation for class IV NYHA heart failure. The findings of clinical studies, cardiac catheterization, endomyocardial biopsy, and pathologic examination of the removed hearts in this group with mildly dilated congestive cardiomyopathy (MDCM) were compared with similar data in four patients with idiopathic restrictive cardiomyopathy (IRCM) and in 10 patients with typical dilated congestive cardiomyopathy (DCM). In comparison with the other groups, patients with MDCM had a higher incidence of familial cardiomyopathy (p = .02) and a shorter symptomatic period than patients with IRCM (p less than .02). Patients with both MDCM and DCM had globular hearts (with predominant left ventricular dilatation), congestive hemodynamics and poor left ventricular contractility (ejection fraction 12% to 19%), and high incidence of ventricular thrombi. Patients with IRCM showed normal ventricular size, marked atrial dilatation, restrictive hemodynamics, mild-to-moderate decrease in left ventricular contractility (ejection fraction 29% to 55%), and absence of ventricular thrombi. Cardiac index, ventricular wall thickness, and light microscopic findings were similar in the three groups of patients. Electron microscopy showed no myofibrillar loss in patients with IRCM but mild (partial) or moderate-to-severe (almost total) myofibrillar loss in those with MDCM and DCM, respectively. We conclude that end-stage congestive cardiomyopathy may occur without significant ventricular dilatation and patients with MDCM have heart sizes intermediate between those found in IRCM and DCM but their clinical, hemodynamic, and pathologic findings are virtually identical to those of patients with typical DCM.

Calcification of porcine prosthetic heart valves: a radiographic and light microscopy study.

To determine the incidence and extent of calcification of implanted glutaraldehyde-treated porcine prosthetic heart valves, 82 valves explanted from 73 patients were examined for calcium by radiography and light microscopy. At the time of valve implantation, the patients were 2¼-76 years old. They included 15 children (patients younger than 15 years of age, mean age at time of valve implantation 8.7 ± 4.1 years) and 58 adults (patients older than 15 years, mean age at time of valve implantation 53.5 ± 15.1years). Valves explanted from children (average time implanted 4.6 ± 1.7 years) included four aortic, five mitral, as well as six right ventricle-pulmonary artery conduits and one left ventricle-abdominal aorta conduit. Valves explanted from adults (average time implanted 3.2 ± 2.5 years) included 32 aortic and 32 mitral, as well as one tricuspid valve and one valve from a right ventricle-pulmonary artery conduit. Calcification of explanted valves was graded from 0 to 4± based on radiographs. All 16 valves from children were calcified, with grade 3 ± or 4 ± calcification in each of the aortic and mitral valves. In adult patients, calcification was present in 10 of 33 valves (30%) implanted for less than 3 years (average time implanted 1.0 year), in nine of 11 valves (82%) implanted for 3-5 years (average time implanted 3.7 years) and in 21 of 22 valves (96%) implanted for 5 years or longer (average time implanted 6.2 years). Analysis of variance demonstrated that calcification was strongly related to the duration that valves were implanted (p < 0.001). Age at the time of valve implantation also had a strong effect (p < 0.001) on the amount of valvular calcium. Valves from children showed the most calcification, and the amount did not change when valves were implanted in patients 30 years of age or older. Patient sex and valve position had no effect on the amount of calcification. Calcification occurred at each right- and left-heart valve position, most frequently at sites of commissural attachments.