Functional sorting of actin isoforms in microvascular pericytes.

We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber-containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea-polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.

Low levels of high-density lipoprotein cholesterol are associated with vascular remodeling in cardiac transplant recipients.

BACKGROUND: Early atherosclerosis may be associated with compensatory vessel enlargement, termed positive remodeling. Enlarged brachial artery diameter has been reported in patients with risk factors for atherosclerosis and in individuals with coronary atherosclerosis, indicating that brachial artery enlargement is a marker for the presence of atherosclerotic changes. Cardiac transplant recipients often have abnormal lipid levels, but the effect of specific lipid abnormalities on vascular remodeling in this population has not been evaluated. This study examined the relationship between lipid levels and brachial artery diameter in cardiac transplant recipients. METHODS: Thirty-five stable cardiac transplant recipients underwent high-resolution brachial artery ultrasound to evaluate resting brachial artery diameter. Levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides were determined and the presence of other cardiac risk factors was assessed. RESULTS: Brachial artery diameter was larger (4.3 +/- 0.1 mm) in subjects with low levels of HDL-C (< 40 mg/dL, n = 11) compared to subjects with high HDL-C (> or = 40 mg/dL, n = 24), who had a mean brachial artery diameter of 3.7 +/- 0.1 mm (P = .006). Neither high LDL-C (> or = 100 mg/dL) nor high triglycerides (> or = 200 mg/dL) were associated with differences in brachial artery diameter. Multivariate analysis demonstrated that the relationship between low HDL-C and increased brachial artery diameter was independent of body surface area or statin use. CONCLUSIONS: Low levels of HDL-C are an independent predictor of brachial artery enlargement in stable cardiac transplant recipients. These findings suggest that suboptimal HDL-C levels may be associated with the development of vascular remodeling and atherosclerosis in this population.

Detection of anti-HLA antibody by flow cytometry in patients with a left ventricular assist device is associated with early rejection following heart transplantation.

BACKGROUND: Patients with a left ventricular assist device (LVAD) as a bridge to heart transplantation (HT) often have elevated levels of panel reactive antibodies (PRA). The clinical significance of anti-human histocompatibility leukocyte antigen (HLA) antibodies detected by flow cytometry in PRA negative patients remains unclear. METHODS: Eighteen patients who underwent LVAD placement as a successful bridge to HT had standard anti-human globulin complement-dependent cytotoxicity and retrospective flow cytometry assays performed to detect class I anti-HLA antibodies. A positive flow result was defined as a fluorescent ratio of 23:1 versus a negative control. RESULTS: Six patients had anti-HLA antibodies detected by flow cytometry. Univariate analysis demonstrated more moderate-severe rejection episodes (ISHLT > or = IIIA) at 2 months (0.83+/-0.75 vs. 0; P=0.04) and a trend toward decreased time to first rejection (61+/-17 vs. 225+/-62 days; P=0.06) in these patients. No differences were observed in donor-recipient HLA mismatch or 1 year Kaplan-Meier survival between patients with or without anti-HLA antibodies. CONCLUSION: Despite a negative PRA, LVAD patients with class I anti-HLA antibodies detected by flow cytometry have a greater incidence of moderate-severe rejection in the first 2 months after HT. Flow cytometry may be a useful clinical tool in screening PRA negative LVAD patients before transplantation. Patients with positive anti-HLA antibody screening by flow cytometry may require more intensive immunosuppression in the early post-HT period.

Prognostic significance of atrial fibrillation in patients at a tertiary medical center referred for heart transplantation because of severe heart failure.

Atrial fibrillation (AF) occurs frequently in advanced heart failure. The prognostic significance of AF remains controversial. To determine the relation of AF to survival in patients with advanced heart failure, 234 consecutive patients referred for heart transplantation evaluation from January 1993 to June 1996 were studied to determine the effect of AF on event-free survival (freedom from death, heart transplantation, or placement of a left ventricular assist device). Clinical characteristics of the study population included: age, 51 +/- 17 years; maximum exercise oxygen consumption, 14.2 +/- 5.3 ml/kg/min; left ventricular ejection fraction, 24 +/- 11%; pulmonary capillary wedge pressure, 23 +/- 9 mm Hg; and ischemic etiology, 52%. Medical therapy included: diuretics (86%), angiotensin-converting enzyme inhibitors (80%), digoxin (80%), and anticoagulation therapy (72%). Mean duration of follow-up was 1.1 +/- 1.0 years. Sixty-two patients (27.4%) had AF. One-year event-free survival of the study population was 48%. No difference in event-free survival between patients with and without AF was observed. Univariate predictors of decreased event-free survival included: (1) advanced New York Heart Association class; (2) higher pulmonary capillary wedge pressure; (3) lower cardiac index; (4) lower maximum exercise oxygen consumption; (5) use of inotropic therapy; and (6) greater pulmonary artery systolic pressure. By multivariate analysis, independent predictors of decreased event-free survival included advanced New York Heart Association class (p <0.002) and higher pulmonary capillary wedge pressure (p = 0.02). Thus, AF in patients with advanced heart failure is not associated with decreased event-free survival.

Extrinsic compression of the left main coronary artery by the pulmonary artery in patients with long-standing pulmonary hypertension.

Left main coronary artery compression by the pulmonary artery may be seen in patients with pulmonary hypertension who are undergoing cardiac catheterization. Cardiac magnetic resonance imaging is useful in these patients to document extrinsic compression, which might otherwise be mistaken for intrinsic atherosclerotic disease.

CD3 delta and epsilon gene expression in CD3-CD16+ natural killer cell clones derived from thymic precursors.

To better understand the maturational stages during T-cell development, we studied the expression of CD3 delta and CD3 epsilon genes, as well as the presence of TCR gene rearrangements, within CD3-CD16+ NK clones derived from thymic precursors in vitro. Northern blot analysis revealed that CD3-CD16+ clones derived from CD7+CD3-CD4-CD8- (TN) thymocytes expressed transcripts for the CD3 epsilon gene; however, no transcripts for the CD3 delta gene were detected. Importantly, both the CD3 epsilon and CD3 delta genes were expressed in TN thymocytes examined prior to cloning. A CD7+CD8+CD3-CD4- thymocyte population that makes up only 0.4% of the total thymocyte pool was also isolated from human thymus. We determined the maturation potential of this CD7+CD8+CD3-CD4- population by limiting dilution cloning and found that 67% of the clones generated in vitro had a CD3-CD16+CD8+ phenotype. In contrast to the NK clones derived from TN precursors, most CD3-CD16+ clones derived from CD7+CD8+CD3-CD4- thymocytes expressed transcripts for both CD3 epsilon and CD3 delta genes. Southern blot analysis of the NK clones derived from either thymic precursor population revealed no rearrangement of the TCR beta or gamma genes. These results demonstrate that the TN progenitor population and their CD3-CD16+ progeny differ in their expression of the CD3 delta transcript and during in vitro culture there is loss of CD3 delta expression and acquisition of surface CD16 within these NK clones. Furthermore, the CD3-CD16+ clones derived from TN versus CD7+CD8+CD3-CD4- thymocytes differed in their expression of the CD3 delta gene. The signaling events regulating the expression of the CD3 invariant chain genes within immature lymphoid progenitor cells may be important in determining their eventual maturation into T-cell and NK-cell lineages in vivo.

Desmin myopathy involving cardiac, skeletal, and vascular smooth muscle: report of a case with immunoelectron microscopy.

Desmin myopathy is a rare idiopathic disorder characterized by abnormal aggregates of desmin-type intermediate filaments, which affects cardiac and skeletal muscle, and rarely the intestinal smooth muscle. We report a 42-year-old woman with atrial fibrillation and progressive restrictive cardiomyopathy. Left ventricular biopsy, cardiac explant, and subsequent autopsy study of skeletal muscle revealed cytoplasmic granulo-filamentous inclusions that were continuous with Z-lines and were immunoreactive for desmin filaments both at the light immunohistochemical and electron microscopic level. In addition, we report the presence of characteristic inclusions within the smooth muscle of intramural coronary blood vessels. This is the first description of desmin inclusions within vascular smooth muscle, and underscores the systemic nature of this rare myopathy.

Successful reversal of severe refractory cardiac allograft rejection by photopheresis.

We treated 4 patients with refractory International Society of Heart and Lung Transplantation Grades IIIA to IV cardiac allograft rejection with extracorporeal photopheresis. Following treatment on 2 consecutive days, 3 patients demonstrated complete histologic reversal of rejection. The remaining patient improved more gradually, but manifested complete cessation of rejection following three 2-day treatments. We conclude that photopheresis is a safe and effective modality for the treatment of severe refractory cardiac allograft rejection and that these results support the use of photopheresis in this clinical setting.

Cortical blindness secondary to cyclosporine after orthotopic heart transplantation: a case report and review of the literature.

Cyclosporine neurotoxicity has been described after liver, kidney, and bone marrow transplantation and has been associated with a number of risk factors, including hypomagnesemia and low serum cholesterol levels. Reports in heart transplant recipients are less common. We present a patient with cortical blindness secondary to cyclosporine after orthotopic heart transplantation. The patient had confusion, focal visual field defects, and bilateral occipital lobe lesions shown on magnetic resonance imaging. Although he had significant clinical improvement with decreasing cyclosporine levels, residual computerized visual field defects and magnetic resonance imaging abnormalities were documented several months later.

Early post-transplant lymphoproliferative disease following heart transplantation in the absence of lymphocytolytic induction therapy.

We report a case of post-transplant lymphoproliferative disease presenting as a disseminated polymorphous B-cell lymphoma involving the cardiac allograft 3 months following transplantation in a recipient who did not receive anti-lymphocyte induction immunosuppression. In situ hybridization for the lytic Epstein-Barr virus marker NOT I was positive within a lymphocytic infiltrate on endomyocardial biopsy. Our case is the third of early post-transplant lymphoproliferative disease (within 6 months of transplantation) involving the heart allograft in the absence of anti-lymphocyte induction immunosuppression. Post-transplant lymphoproliferative disease of the heart allograft should be considered in the presence of an atypical cardiac lymphocytic infiltrate, with possible differentiation from allograft rejection using in situ hybridization for Epstein-Barr virus.

Hepatitis C transmission and infection by orthotopic heart transplantation.

BACKGROUND: The transmission and clinical consequences of hepatitis C viral (HCV) infection acquired by orthotopic heart transplantation (OHT) from an HCV-infected donor to an HCV-naive recipient have not been well described. We report our experience in 5 HCV-naive patients who were transplanted with hearts from HCV-positive donors. All transplants occurred within a 1-year period. METHODS: After cardiac transplantation we retrospectively examined the recipients' clinical course, liver-associated enzymes, HCV-antibody serology, quantitative HCV RNA level, and HCV genotype. RESULTS: Five subjects with rapidly deteriorating heart failure and negative serum antibodies to HCV received an emergent OHT from a donor known to be infected with HCV. Liver-associated enzymes peaked at 2 to 6 weeks post-transplant: mean peak alanine aminotransferase was 180 U/L (normal, 9 to 52) and aspartate aminotransferase was 111 U/L (normal, 14 to 36). Liver enzymes had returned to normal limits by 6 and 12 months post-OHT. At a mean 15 months after transplantation, only 1 of 5 patients has developed antibodies to HCV, but 4 of 5 have evidence of infection, as shown by serum HCV RNA. No patient has developed evidence of liver failure. CONCLUSIONS: (1) Transmission of HCV from an HCV-positive donor to an HCV-naive recipient at the time of OHT is likely. (2) Antibodies to HCV post-OHT may remain negative for more than 1 year in these patients. (3) Hepatitis C viral RNA using polymerase chain reaction should be the test of choice for diagnosis of HCV infection post-OHT. (4) Hepatitis C viral donor hearts should be limited to critically ill patients in extremis until the long-term consequences of acquisition of HCV by an OHT recipient are known.

Mycophenolic acid concentrations are associated with cardiac allograft rejection.

BACKGROUND: Mycophenolate mofetil (MMF) therapy decreases the incidence of allograft rejection following solid-organ transplantation. Current dosing strategies of MMF are not routinely adjusted based on mycophenolic acid (MPA) area under the concentration-time curve (AUC), MPA trough, or free MPA (fMPA) AUC values. METHODS: To determine the clinical significance of MPA concentrations following orthotopic heart transplantation (OHT), we measured pre-dose MPA trough, MPA free fraction, an estimated MPA AUC using an abbreviated sampling schedule, and fMPA AUC in 38 consecutive patients. We measured MPA concentrations using a validated high-performance liquid chromatography method and graded endomyocardial biopsies based on the International Society for Heart and Lung Transplantation (ISHLT) grading system. RESULTS: The MPA values for the study group were as follows: MPA trough of 1.2 +/- 0.6 microg/ml; MPA free fraction of 1.9 +/- 0.4%; MPA AUC of 44.5 +/- 16. 1 microg/hour/ml; and fMPA AUC of 0.83 +/- 0.30 microg/hour/ml. We compared patients with Grade 0 (n = 22), Grade 1 (n = 13), or Grade 2/3 (n = 3). The MPA AUC values were lower in patients with Grade 2/3 than in patients with Grade 0 (26.1 +/- 6.6 vs 42.8 +/- 14.0 microg/hour/ml, p < 0.08) or Grade 1 rejection (26.1 +/- 6.6 vs 51.7 +/- 17.5 microg/hour/ml, p < 0.05). The fMPA AUC values were lower in patients with Grade 2/3 than with patients with Grade 0 (0.49 +/- 0.11 vs 0.81 +/- 0.25 microg/hour/ml, p < 0.05) or Grade 1 (0.49 +/- 0.25 vs 0.95 +/- 0.34 microg/hour/ml, p < 0.05) rejection. We noted a trend in MPA trough concentrations between patients with Grade 2/3 vs 0 (0.65 +/- 0.15 vs 1.20 +/- 0.58 microg/ml, p = 0.15) and Grade 1 (0.65 +/- 0.15 vs 1.24 +/- 0.72 microg/ml, p = 0.14) rejection. CONCLUSION: These preliminary results suggest that lower MPA AUC and fMPA AUC values are associated with cardiac allograft rejection in heart transplant recipients. Individualizing MMF dosing based on MPA determinations may minimize the risk of rejection following OHT.

Pharmacokinetic, pharmacodynamic, and outcome investigations as the basis for mycophenolic acid therapeutic drug monitoring in renal and heart transplant patients.

Mycophenolate mofetil is widely used in combination with either cyclosporine or tacrolimus for rejection prophylaxis in renal and heart transplant patients. Although not monitored routinely nearly to the degree that other agents such as cyclosporine or tacrolimus, there is an expanding body of experimental evidence for the utility of monitoring mycophenolic acid, the primary active metabolite of mycophenolate mofetil, plasma concentration as an index of risk for the development of acute rejection. The following are important experimentally-based reasons for recommending the incorporation of target therapeutic concentration monitoring of mycophenolic acid: (1) the MPA dose-interval area-under-the-concentration-time curve, and less precisely, MPA predose concentrations predict the risk for development of acute rejection; (2) the strong correlation between mycophenolic acid plasma concentrations and expression of important cell surface activation antigens, whole blood pharmacodynamic assays of lymphocyte proliferation and median graft rejection scores in a heart transplant animal model; (3) the greater than 10-fold interindividual variation of MPA area under the concentration time curve values in heart and renal transplant patients receiving a fixed dose of the parent drug; (4) drug-drug interactions involving other immunosuppressives are such that when switching from one to another (eg, from cyclosporine to tacrolimus or vice-versa) substantial changes in MPA concentrations can occur in patients receiving a fixed dose of the parent drug; (5) significant effects of liver and kidney diseases on the steady-state total and free mycophenolic acid area under the concentration time curve values; (6) the need to closely monitor mycophenolic acid when a major change in immunosuppression is planned such as steroid withdrawal. Current investigations are focused on determination of the most optimal sampling time and for mycophenolic acid target therapeutic concentration monitoring. Further investigations are needed to evaluate the pharmacologic activity of the newly described acyl glucuronide metabolite of mycophenolic acid which has been shown to inhibit, in vitro, inosine monophosphate dehydrogenase.

Prolonged quinidine half-life with associated toxicity in a patient with hepatic failure.

A markedly prolonged quinidine elimination half-life due to hepatic failure and resultant quinidine toxicity occurred in a 57-year-old man with a history of atrial fibrillation. A prolonged QT interval, development of torsades de pointes, and a serum quinidine concentration of 3.1 micrograms/ml contributed to a decision favoring permanent pacemaker implantation. The apparent quinidine half-life ranged from 66-99 hours and was associated with QT prolongation and persistent U waves. On discontinuing quinidine, all signs associated with toxicity resolved as serum quinidine concentrations decreased, which resulted in reversal of the decision to implant a permanent pacemaker. This case reports an extremely long quinidine elimination half-life and reillustrates the importance of drug pharmacokinetics in patient care.

Accelerated TPA for treatment of prosthetic valve thrombosis.

The first case of prosthetic valve thrombosis treated with an accelerated regimen of TPA is reported. The experience suggests that this therapeutic approach is feasible and may produce higher rates of patency for thrombosed prosthetic valves than other thrombolytic therapies currently available. This therapeutic strategy is worthy of further evaluation as a treatment for this disorder.

Myocardial viability in patients with coronary artery disease and left ventricular dysfunction: transplantation or revascularization?

Coronary artery bypass surgery performed in patients with coronary artery disease and left ventricular dysfunction improves survival compared with antianginal therapy alone. The mechanisms for this survival advantage with revascularization therapy have not been systematically elucidated. Many of these patients have "hibernating" myocardium secondary to chronic ischemia with the potential for substantial improvement in left ventricular function and heart failure symptoms following revascularization therapy. Nevertheless, as survival with cardiac transplantation continues to improve, a significantly larger number of patients with coronary artery disease and left ventricular dysfunction are being referred for cardiac transplantation in lieu of revascularization surgery. Recently developed imaging modalities, which include positron emission tomography, thallium imaging, and dobutamine echocardiography, can reliably predict recovery of regional myocardial dysfunction after revascularization in these areas of hibernating heart. New modalities to detect hibernating myocardium include 99mTc-sestamibi, contrast echocardiography, nuclear magnetic resonance spectroscopic imaging, and ultrasonic tissue characterization. In an era of medicine characterized by increased concern for cost containment and the judicious application of expensive technology, the choice of the most appropriate tests to detect viability is a growing challenge and is essential in the choice between transplantation and revascularization.

Cardiomyopathy and embolization: risks and benefits of anticoagulation in sinus rhythm.

Systemic embolic complications in patients with cardiomyopathy are associated with significant morbidity and mortality. Despite the lack of prospective, randomized control data, the literature supports the use of left ventricular ejection fraction as an important determinant of the need for systemic anticoagulation therapy in patients with systolic dysfunction. This review discusses the risks and benefits of systemic anticoagulation for patients with cardiomyopathy and proposes a treatment algorithm for its initiation.

Amlodipine overdose.

OBJECTIVE: To report a nonfatal intentional overdose of amlodipine. CASE SUMMARY: A 42-year-old woman with a history of hypertension reported ingesting 50-100 mg amlodipine besylate and at least 40 ounces of beer in a suicide attempt. The patient's symptoms were mild; BP ranged from 79/50 to 113/76 mm Hg and HR from 92 to 129 beats/min (sinus tachycardia). Laboratory studies revealed normoglycemia, mild metabolic acidosis, mild hypocalcemia, blood ethanol concentration of 263 mmol/L, and a serum amlodipine concentration of 88 ng/mL (normal 3-11) 2.5 hours after ingestion. Therapy included activated charcoal, whole bowel irrigation, and intravenous NaCl 0.9%. After receiving 1.5 L of NaCl 0.9%, the patient developed signs of mild pulmonary edema that resolved over several hours without intervention. A serum amlodipine concentration obtained 35 hours later was 79 mg/mL. The patient was discharged on day 2 in good condition. DISCUSSION: In this case, an amlodipine overdose was associated with sustained hypotension and sinus tachycardia, as well as transient pulmonary edema following relatively low-volume fluid replacement. A previously published report described an amlodipine overdose that was fatal due to refractory hypotension and was complicated by concomitant oxazepam overdose. CONCLUSIONS: Amlodipine overdose produces prolonged hemodynamic effects and may lead to pulmonary edema. Due to a long elimination half-life and delayed onset of effects, patients with amlodipine overdose should receive aggressive decontamination therapy and may require extended clinical monitoring and supportive care if they are hemodynamically unstable.