Two-dimensional echocardiographic assessment of right ventricular function as a predictor of outcome in hypoplastic left heart syndrome.

This study was undertaken to assess the importance of right ventricular function at the time of initial presentation on early and intermediate outcome in patients with hypoplastic left heart syndrome (HLHS). Several studies have attempted to define physiologic risk factors for poor early outcome following the Norwood palliation for HLHS. No clinical or hemodynamic factors including right ventricular function have been found to reliably predict Norwood I operative survival. The relation between initial ventricular function and later survival has not been investigated. To assess the importance of right ventricular (RV) function at the time of initial presentation on outcome in patients with HLHS, systolic function was determined by qualitative and quantitative methods in 60 consecutive patients before surgical intervention. The effects on stage I operative survival, survival to stage II, and overall survival were analyzed. Initial RV function did not impact on stage I survival. However, analysis of later outcome of the stage I survivors showed that those with prestage I RV dysfunction had significantly greater mortality before stage II. Actuarial survival 18 months after Norwood surgery was 93% for patients with initially normal RV function compared with 47% for those with abnormal function (p = <0.005). The relative risk for later mortality was approximately 11 times greater for patients with initial RV dysfunction. Thus, RV dysfunction identifiable soon after initial presentation does not impact on early survival after Norwood I operation for HLHS. Intermediate and overall survival, however, is significantly decreased in patients with initially diminished RV function.

Noninvasive prediction of pulmonary artery pressure in patients with isolated ventricular septal defect.

Management of patients with isolated ventricular septal defect (VSD) requires information regarding pulmonary artery pressure (PAP). The purpose of this study was to evaluate the individual predictive value of noninvasive methods for assessment of PAP and to determine if any combination of techniques significantly improved their predictive power. We reviewed the clinical history, electrocardiogram, and echocardiogram of 31 patients (age 1.9 +/- 1. 73 years) who underwent catheterization for isolated VSD. Noninvasive data were compared for patients with mean PAP <20 mmHg (group 1) and those with mean PAP > or =20 (group 2) at catheterization. Fourteen (45%) patients were in group 1 and 17 (55%) in group 2. Doppler estimation of VSD gradient, right ventricular hypertrophy by echocardiogram, interventricular septal orientation, and VSD size had predictive value for elevated mean PAP (p < 0.01). All patients (n = 6) with normal findings in all four variables had normal PAP. All patients (n = 12) with at least three of four abnormal findings had elevated PAP. Six patients in group 1 had at least one variable that incorrectly predicted high PAP, whereas 3 patients with normal findings on three of the four variables nevertheless had elevated PAP. No single noninvasive variable accurately predicted PAP in all cases. However, normal findings for all four significant variables did predict normal PAP and suggest that cardiac catheterization is unnecessary in that setting. However, any other combination of normal and abnormal findings for the four significant variables did not reliably predict PAP and such patients may require catheterization to directly measure PAP.

Serial echocardiographic measurements of the pulmonary autograft in the aortic valve position after the Ross operation in a pediatric population using normal pulmonary artery dimensions as the reference standard.

Serial echocardiographic measurements of the annulus and sinus were obtained in children before the Ross operation, and early and late postoperatively. Values were compared with normal standards for the aorta and pulmonary artery (PA). There was no significant difference between PA annulus measurements before surgery and the corresponding autograft immediately afterward (1.73 +/- 0.60 cm preoperatively; 1. 63 +/- 0.58 cm postoperatively, p = NS). Late after surgery the mean annulus diameter was enlarged compared with the normal aorta (DeltaZ 1.9 +/- 2.4), but remained relatively unchanged compared with the normal PA (DeltaZ 0.7 +/- 1.1, p <0.01). In contrast, the autograft sinus was dilated early after surgery (1.83 +/- 0.58 cm preoperatively; 2.18 +/- 0.73 cm postoperatively, p <0.01). Mean sinus Z score further increased compared with both the aorta (DeltaZ 1.3 +/- 1.7) and PA (DeltaZ 1.3 +/- 1.6). Use of standard PA measurements may be important in the assessment of autograft enlargement. Minimal change in autograft Z scores over time suggests that annulus enlargement is mainly due to somatic growth. In contrast, the autograft sinus showed an immediate and continued disproportionate increase in size over time, suggesting that sinus enlargement is largely due to passive dilation.

Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein.

ES-62 is an abundant phosphorylcholine-containing secreted glycoprotein of the filarial nematode Acanthocheilonema viteae. Using an antiserum directed against the parasite molecule, 3 cDNAs of size, approximately 1.5-1.6 kbp were isolated from an A. viteae expression library. Sequence analysis in combination with N-terminal amino acid sequencing of purified ES-62 revealed that each clone contained a full-length cDNA for ES-62 corresponding to 474 amino acid residues but differed in their 5' and 3' untranslated regions. Characterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of cDNA ends showed that it coded for a signal sequence. Several tryptic peptides were independently sequenced using quadruple-time-of-flight mass spectrometry and used to confirm the cDNA sequence. The mature protein was found to contain three potential N-linked glycosylation sites. Comparison of the derived amino acid sequence of ES-62 with the SwissProt database identified a sequence (between amino acid residues approximately 250 and 350 of mature ES-62) with significant similarity to several bacterial/fungal aminopeptidases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrate confirmed that ES-62 possessed aminopeptidase activity.

Up-regulation of extracellular copper/zinc superoxide dismutase mRNA after transmission of the filarial parasite Acanthocheilonema viteae in the vertebrate host Meriones unguiculatus.

The gene encoding the cytoplasmic copper/zinc superoxide dismutase (AVSOD1) from the filarial parasite Acanthocheilonema viteae was isolated from a genomic DNA library using a degenerate oligonucleotide probe. Additionally, cDNAs of the AVSOD1 and the secreted extracellular SOD (AVSOD2) were both cloned by RT-PCR, and the AVSOD2 was expressed at high levels in E. coli. The amino acid sequence of the AVSOD1 is 89.5 and 87.5% identical to that of the corresponding enzymes of Brugia pahangi and Onchocerca volvulus, respectively. In contrast, the AVSOD2 shows a lower degree of identity to the other filarial SODs and is extensively glycosylated. RT-PCR studies demonstrate the expression of both SOD subtypes in all developmental stages of A. viteae and indicate up-regulation of the AVSOD2 expression after transmission from the vector to the definitive host. This suggests an enhanced requirement for SOD activity in post-infective larval stages and adults of A. viteae. ELISAs performed with purified recombinant AVSOD2 show that the AVSOD2 is not a major target for the immune system in naturally infected jirds.

Immunogenicity of the extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae delivered by a two-phase vaccine strain of Salmonella typhimurium.

The recombinant extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae (AVSOD2) was cloned in an expression vector under control of the bacteriophage T7 promoter and the resulting plasmid pLAT7 was introduced in tha aroA attenuated Salmonella typhimurium vaccine strain SL3261:pYZ84. This vaccine strain carries a chromosomally integrated two phase expression system containing inducible T7 RNA polymerase. The recombinant AVSOD2 was efficiently expressed, constituting up to 5% of the total bacterial protein. Furthermore, the plasmid vector containing the AVSOD2 cDNA was shown to be stable over a long period of time in the vaccine strain without antibiotic selection in vitro and in vivo. Jirds which were immunised orally with the recombinant vaccine strain expressing the A. viteae EC-SOD produced a strong humoral immune response.

Pattern of pulmonary venous blood flow in the hypoplastic left heart syndrome in the fetus.

OBJECTIVE: To determine whether restriction at the atrial septum in the newborn with hypoplastic left heart syndrome can be predicted accurately by examining the pattern of pulmonary venous flow in the fetus. A restrictive atrial septum can contribute to haemodynamic instability before surgery for this lesion and has been associated with an increased mortality. DESIGN: Pulmonary venous pulsed Doppler tracings were compared between fetuses with hypoplastic left heart syndrome and controls. The size of the atrial septal defect on the postnatal echocardiogram was graded according to the degree of restriction. Pulsed Doppler tracings of pulmonary venous blood flow were obtained in 18 fetuses with left atrial outflow atresia and compared with 77 controls, adjusted for gestational age. Postnatal echocardiograms were available for analysis in 13 of 18 neonates. SETTING: A tertiary referral centre for fetal cardiology and paediatric cardiac surgery. RESULTS: Fetuses with hypoplastic left heart syndrome were different from controls in all pulmonary vein indices measured. As assessed from the postnatal echocardiogram, there were seven fetuses with a restrictive atrial septum. In these fetuses, the systolic flow velocity (p < 0.01), S/D ratio (p < 0.01), and peak reversal wave (p < 0.001) in the pulmonary vein tracing showed a good correlation with the degree of restriction. CONCLUSIONS: The Doppler pattern of pulmonary venous flow in the fetus with hypoplastic left heart syndrome appears to be a reliable predictor of restriction of the atrial septum in the neonate. This may help in the immediate post-delivery management of these infants before surgery.

Usefulness of preoperative echocardiography in predicting left ventricular outflow obstruction after primary repair of interrupted aortic arch with ventricular septal defect.

Residual left ventricular outflow tract (LVOT) obstruction is a significant problem after repair of interrupted aortic arch (IAA) and ventricular septal defect. Resection of subaortic tissue at the time of primary repair, however, is associated with increased morbidity and mortality. We reviewed the preoperative echocardiograms and the postoperative clinical course and echocardiograms of 23 consecutive patients who underwent primary repair of IAA without widening of the subaortic region. Nine patients (39%) developed significant LVOT obstruction (pressure gradient >40 mm Hg). LVOT obstruction was noted postoperatively in 7 of 9 patients by 1 month, 8 of 9 by 2 months, and 9 of 9 by 1 year. On retrospective analysis of the preoperative echocardiograms, the indexed cross-sectional area of the LVOT, the subaortic diameter index, and the subaortic diameter Z score were all significantly smaller in those requiring reintervention (p <0.04, p <0.05, p <0.05, respectively). Of these, indexed cross-sectional area had the least reproducibility and subaortic diameter index the most (coefficient of variation of 26.3% vs 11.2%). In conclusion, most patients who develop significant LVOT obstruction after repair of IAA do so within 1 month of operation. Although subaortic indexed cross-sectional area is the most sensitive predictor of LVOT obstruction after primary repair of IAA, other more simple standardized measurements of the subaortic diameter were comparably predictive and had better reproducibility.

Outcome after prenatal diagnosis of the hypoplastic left heart syndrome.

OBJECTIVE: To derive accurate survival figures in the current surgical era for counselling in early pregnancy after the diagnosis of fetal hypoplastic left heart syndrome. SETTING: A tertiary referral centre for paediatric cardiology and cardiac surgery. DESIGN: A retrospective study of the outcome in all cases of hypoplastic left heart syndrome presenting in fetal life between mid-1993 and the end of 1996. PATIENTS: The diagnosis was made in 30 fetuses. In four of 12 identified before 24 weeks' gestation the mothers chose to terminate the pregnancy. There was an intention to treat in 24 of the remaining fetuses. MAIN OUTCOME MEASURE: Survival to six months of postnatal life. RESULTS: Of the 24 infants, five were not offered Norwood stage 1 because of trisomy 18 (n = 2), unfavourable cardiac anatomy (n = 2), or neurological impairment (n = 1). One further infant did not survive to cardiac surgery after gastrointestinal surgery. Of the remaining 18 patients, eight had features that were considered to increase the risk of surgical repair. Of the 18 patients who underwent Norwood stage 1, there were nine survivors. There was a survival rate of 70% in infants undergoing surgery with no complicating features, a 50% survival of the all surgical candidates, and 37.5% survival from an intention to treat position. CONCLUSIONS: At the initial diagnosis of fetal hypoplastic left heart syndrome, the overall survival appears to be less than 40%. Evaluation must include detailed extracardiac and intracardiac assessment to predict the risks of surgical treatment. Prenatal counselling can be modified as pregnancy advances, depending on the detection or exclusion of complicating factors.

Regulation of ion channels in rat hepatocytes.

Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K+ channel with a mean inward conductance of 55 +/- 6.5 pS (n = 20) and a mean outward conductance of 25 +/- 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl- channel with a mean outward conductance of 30 +/- 4.5 pS (n = 8) and a mean inward conductance of 10 +/- 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca2+ activity on the intracellular side. Accordingly, the Ca2+ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K+ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K+ channel is inhibited by progesterone, while the outwardly rectifying Cl- channel is insensitive to progesterone.

Comparison of three-dimensional echocardiographic assessment of volume, mass, and function in children with functionally single left ventricles with two-dimensional echocardiography and magnetic resonance imaging.

Diminished systolic function or inappropriate hypertrophy are considered risk factors for outcome following the Fontan procedure. These parameters are difficult to assess in univentricular hearts that do not conform to the uniform shapes prescribed by conventional 2-dimensional imaging volume algorithms. Three-dimensional echocardiography requires no geometric assumptions and has been validated in both normal and distorted left ventricles. To assess the feasibility and accuracy of this technique in patients with univentricular hearts, we compared 2- and 3-dimensional echocardiographic estimates of ventricular volume, ejection fraction, and mass in patients with functionally single left ventricles with results obtained by magnetic resonance imaging (MRI). Twelve patients with functionally single left ventricles (6 months to 22 years) underwent examination by all 3 modalities. Correlation and agreement with MRI were calculated for volumes, ejection fraction, and mass. Three-dimensional echocardiographic comparison with MRI yielded a bias of 3.4 +/- 5.5 ml and 14.2 +/- 8.3 ml for systolic and diastolic volumes, respectively. Agreement analysis for mass showed a bias of 5.8 +/- 8.4 grams. Two-dimensional echocardiography showed less agreement for both volumes and mass (bias of -2.9 +/- 8.1, 2.9 +/- 10.4 ml and -8.3 +/- 12.0 g for volume and mass, respectively, p >0.05). Ejection fraction by 3-dimensional echocardiography showed significantly closer agreement with MRI (bias of 4.4 +/- 5.3%) than 2-dimensional echocardiography (bias of 8.5 +/- 10.3%, p = 0.04). Thus, 3-dimensional echocardiography provides estimates of ventricular volumes, ejection fraction, and mass that are comparable to MRI in this select group of patients with single ventricles of left ventricular morphology.

PCR-based amplification of total cDNA with high fidelity and high yield from minute amounts of parasite RNA.

cDNA, synthesised from total RNA from Acanthocheilonema viteae, was amplified by PCR with a primer derived from the spliced leader 1 sequence of nematodes and oligo-dT. Due to the great number of side products observed in the reaction, a biotinylated oligo-dT primer was used for cDNA-synthesis and the first cycles of PCR. After binding of the PCR products to streptavidin/paramagnetic particles, the (+)strands of the cDNAs were recovered and reamplified. Analysis of the PCR products obtained revealed the presence of full-length cDNAs of at least 1.7 kbp in size in amplified total cDNA from microfilariae, postinfective L3, and adult worms. The total cDNA, from only 20 ex vivo recovered postinfective L3, was efficiently amplified.

Late left ventricular function after surgery for children with chronic symptomatic mitral regurgitation.

BACKGROUND: The use of quantitative echocardiography has been emphasized in optimizing timing of surgery in adult patients with mitral regurgitation to avoid irreversible left ventricular dysfunction. In contrast, surgery for infants and children is often delayed until the appearance of severe symptoms because of the patient's size and anticoagulation requirements and the possible need for early reoperation. The purpose of this study was to determine long-term ventricular function after mitral valve surgery in symptomatic children and to analyze risk factors for adverse outcome. METHODS AND RESULTS: Thirty-three patients (0.5 to 19 years old) operated on for mitral regurgitation as a single hemodynamically significant lesion were studied. All but 3 had medically refractory symptoms. One patient died during surgery, and 32 were followed for 0.3 to 17.1 years (mean, 4.5 years). The mean preoperative left ventricular shortening fraction was 0.38+/-0.09. Successful mitral valvuloplasty or replacement was documented by long-term normalization of end-diastolic dimensions. Early postoperative shortening fraction was significantly reduced (0.28+/-0.1, P<.01), but it improved to 0.40+/-0.07 (P<.01) on late follow-up, at which time only 1 patient had ventricular dysfunction. Preoperative shortening fractions did not correlate well with early or late postoperative values (r=.18 and r=.31, respectively). Seven of 32 surviving patients had preoperative shortening fractions <0.33 (mean, 0.26+/-0.05) and 25 >0.33 (mean, 0.39+/-0.08). Analysis of these subgroups showed no significant differences between the groups in early or late postoperative function. Duration of mitral insufficiency appeared to be associated with the development of atrial arrhythmias. CONCLUSIONS: Late left ventricular function normalizes in children after surgical correction of mitral insufficiency. In contrast to adults, delay of surgery in children with significant mitral regurgitation until the onset of severe symptoms does not increase the risk for long-term ventricular dysfunction, although late atrial arrhythmias are more likely to be encountered.

Quantitative three dimensional echocardiography in patients with pulmonary hypertension and compressed left ventricles: comparison with cross sectional echocardiography and magnetic resonance imaging.

OBJECTIVE: To evaluate the accuracy of quantitative three dimensional echocardiography in patients with deformed left ventricles. DESIGN: Three dimensional and cross sectional echocardiographic estimates of left ventricular volume and ejection fraction were prospectively compared to those obtained from magnetic resonance imaging. SETTING: Echocardiography laboratory of a university hospital. PATIENTS: 26 patients (9 months to 42 years, median age 11 years) with pulmonary hypertension and fixed reversal of normal interventricular septal curvature. MAIN OUTCOME MEASURES: Left ventricular end diastolic and end systolic volumes and ejection fraction. RESULTS: Three dimensional echocardiographic comparison to magnetic resonance imaging (MRI) yielded r values of 0.94 and 0.87 with a bias of -6.9 (SD 6.9) ml and -16 (11.2) ml for systolic and diastolic volumes respectively. Inter-observer variability was minimal (8.3% and 7.6% respectively). Cross sectional echocardiography gave correlation coefficients of 0.62 and 0.80 and bias of 3.1 (14.1) ml and 16.3 (18.3) ml for systolic and diastolic volumes respectively. Ejection fraction by three dimensional echocardiography also had closer agreement with MRI (bias = 1.1 (7.7)%) than cross sectional echocardiography (bias = 4.4 (13.9)%). CONCLUSIONS: Three dimensional echocardiography provides reliable estimates of left ventricular volumes and ejection fraction, comparable to magnetic resonance imaging in pulmonary hypertension patients with compressed ventricular geometry. Because it eliminates the need for geometric assumptions it shows closer agreement with magnetic resonance imaging in that setting than cross sectional echocardiography.

Feasibility of a two-dimensional echocardiographic method for the clinical assessment of right ventricular volume and function in children.

The relative ease of acquisition and safety of two-dimensional echocardiography has established it as the mainstay for routine cardiac imaging. Translation of imaging data into useful quantitative information, however, requires fitting the ventricle to a specific geometric model. Because of its complex shape and anterior position, many attempts at right ventricular quantitation by two-dimensional echocardiography have been criticized as impractical and not reproducible. A simple method incorporating subcostal and apical imaging was introduced in 1984. This approach appeared to combine accuracy and practicability but was never validated in a clinical setting because of the difficulties of subcostal imaging in adults. This study assessed the feasibility and accuracy of this technique in the pediatric population. Results of volume comparison to values derived by magnetic resonance imaging were r = 0.96, standard error of the estimate (SEE) = 19.3 ml, and mean difference = 15 +/- 19.4 ml and r = 0.97, SEE = 12.3 ml, and bias = 5 +/- 11.8 ml for diastolic and systolic volumes, respectively. Comparison of estimates of ejection fraction with magnetic resonance imaging demonstrated r = 0.90, SEE = 5.9%, and bias = 3% +/- 5.7%. Interobserver and intraobserver variability was 9.9% and 8.2%, respectively, for systolic volumes and 11.5% and 8.9%, respectively, for diastolic volumes. Evaluation of right ventricular size and function by this approach is comparable to determinations by magnetic resonance imaging and may be clinically useful in the management of pediatric patients.

Tyrosine phosphorylation-dependent suppression of a voltage-gated K+ channel in T lymphocytes upon Fas stimulation.

Selective cell death plays a critical role in the development of the immune system and in the elimination of target cells expressing foreign antigens. Most of programmed cell death occurs by apoptosis. Apoptotic cell death of lymphocytes can be triggered by ligation of APO-1/Fas (CD95) antigen (Suda, T., and Nagata, S. (1994) J. Exp. Med. 179, 873-879; Nagata, S., and Golstein, P. (1995) Science 267, 1449-1456). We find that activation of Fas leads to the inhibition of the voltage-dependent n-type K+ channels (Kv1.3) studied by patch clamp technique in Jurkat T lymphocytes. Tyrosine kinases have been shown to be crucial in Fas-induced cell death (Eischen, C. M., Dick, C. J., and Leibson, P. J. (1994) J. Immunol. 153, 1947-1954). The inhibition of the current is correlated with the tyrosine phosphorylation of immunoprecipitated and blotted K+ channel protein. We show, that the Src-like protein-tyrosine kinase inhibitor herbimycin A and the deficiency of the p56(lck) tyrosine kinase in mutant Jurkat cells abolished the channel inhibition and phosphorylation by anti-Fas antibody, while reconstitution of the p56(lck) kinase partly restored these effects of Fas receptor triggering. These results suggest a regulation of n-type K+ channels by tyrosine kinases upon Fas receptor triggering, which might be important for apoptosis.

Ca2+ entry and vasoconstriction during osmotic swelling of vascular smooth muscle cells.

Exposure of aortic strips from guinea-pigs to hypotonic extracellular fluid is followed by marked vasoconstriction, which is inhibited by D-600 (3 microM), a blocker of voltage-sensitive Ca2+ channels. Conventional electrophysiology, patch-clamp studies, pH determination with 2',7' bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and Ca2+ measurements with Fura-2 have been performed on smooth muscle cells cultured either from rat or human aorta to further elucidate the underlying mechanisms. Exposure of the cells to a 25% hypotonic extracellular fluid leads to a rapid and fully reversible depolarization, paralleled by an increase of the selectivity and conductance of the cell membrane to Cl-, an acidification of the cytoplasm and an increase of intracellular Ca2+ concentration ([Ca2+]i). The latter is inhibited by the Ca2+ channel blocker D-600 (1-3 microM). It is concluded that osmotic cell swelling leads to the activation of an anion channel. The subsequent depolarization of the cell membrane activates voltage-sensitive Ca2+ channels which increases [Ca2+]i, thus stimulating the contraction of vascular smooth muscle cells.

Electrophysiological effects of progesterone on hepatocytes.

The addition of progesterone (1-100 mumol/l) to the extracellular fluid bathing rat hepatocytes led to a rapid and fully reversible depolarization of the cell membrane. The progesterone-induced depolarization was paralleled by a decrease of potassium selectivity and an increase of cell membrane resistance and was abolished in the presence of the potassium channel blocker barium. Accordingly, in whole cell recordings, progesterone led to a decrease of the cell membrane conductance. 17 alpha-Hydroxyprogesterone and beta-estradiol were less effective by a factor of 10, whereas cholesterol, corticosterone and hydrocortisone did not significantly alter the potential difference across the cell membrane. In conclusion, acute administration of progesterone depolarized rat hepatocytes by decreasing the potassium conductance of the cell membrane.