Treatment of pulmonary arterial hypertension in congenital heart disease in Singapore versus the Netherlands: age exceeds ethnicity in influencing clinical outcome.

BACKGROUND: Advanced treatment of pulmonary arterial hypertension (PAH) in congenital heart disease (CHD) is increasingly applied worldwide following the-mainly Western world based-international PAH-CHD guidelines. However, studies comparing clinical presentation and outcome after the initiation of PAH-specific treatment are lacking. We aimed to analyse this in a Singaporean and Dutch cohort of PAH-CHD patients. METHODS: Adult CHD patients starting PAH-specific therapy, enrolled in two nationwide registries, were analysed. Patients received phosphodiesterase-type-5 inhibitors, endothelin receptor antagonists, or a combination. Change in six-minute walk test (6MWT) during follow-up was analysed using linear mixed model analysis. Determinants for mortality were assessed using Cox proportional hazard analyses. RESULTS: A total of 74 patients, 45 Dutch (mean age 47 ± 14 years) and 29 Singaporean (mean age 41 ± 14 years) were analysed. Despite a lower 6MWT (312 versus 395 metres, p = 0.01) and peak VO2 (35 versus 49 % of predicted, p = 0.01) at baseline in Singaporean patients, the treatment effect was similar in the two populations. Age at initiation of therapy (per 5 year lower age, ß = + 4.5, p = 0.017) was the strongest predictor of improvement in exercise capacity, corrected for ethnicity, baseline 6MWT, sex and CHD defect. CONCLUSIONS: Patients from Singapore had a worse clinical performance at baseline compared with the PAH-CHD patients from the Netherlands. No relation between ethnicity and improvement in 6MWT after PAH-specific therapy was found. Age at initiation of PAH-specific therapy was the strongest predictor of treatment efficacy and mortality, emphasising the need for early initiation of treatment in these patients.

Effect of culture media and nutrients on biofilm growth kinetics of laboratory and clinical strains of Enterococcus faecalis.

OBJECTIVE: Enterococcus faecalis is a bacterial pathogen that is often associated with endodontic infections. Biofilm formation is a key virulence attribute in the pathogenicity of E. faecalis. In the present study, we comprehensively examined the effect of various culture media and nutrients on the development of E. faecalis biofilms. DESIGN: A reference strain and a clinical isolate of E. faecalis were used in all experiments for comparison. Commonly used liquid culture media with different nutrient compositions were used to support the development of E. faecalis biofilms in a time-dependent assay. E. faecalis biofilms were quantified by colony forming unit (CFU) and crystal violet (CV) assays. Biofilm architecture and cellular viability were evaluated by scanning electron microscopy and confocal laser scanning microscopy. RESULTS: Growth kinetics evaluated by CFU and CV assays and by microscopy showed that E. faecalis biofilms reached maturity at 72h. "Pg broth" (Tryptic Soy Broth with yeast extract, hemen and vitamin K) promoted E. faecalis biofilm formation more than Brain Heart Infusion broth or Tryptic Soy Broth. Addition of 2% glucose enhanced biofilm formation. Thus, it seems that nutrients such as hemen, vitamin K and glucose are important for E. faecalis for the formation of biofilms. CONCLUSION: The present study demonstrated that nutrient-rich media containing glucose enhances the formation of E. faecalis biofilms, which exhibit maturation at 72h.

Electrocardiography Series. ECGs of structural heart disease: Part 1.

Electrocardiogram (ECG) is a useful but imperfect investigation in the diagnosis and possible follow-up of structural heart disease such as ventricular hypertrophy. Different ECG criteria with different sensitivity and specificity are available to aid the detection of left or right ventricular hypertrophy. Subsequent echocardiography can help in the quantification of ventricular mass and identification of the aetiology.

ReoPro Observational Registry (RAPOR): insights from the multicentre use of abciximab in Asia.

INTRODUCTION: The pattern of use of abciximab in real-life clinical patients undergoing percutaneous coronary intervention (PCI) in 11 high-volume centres in Singapore, Malaysia, Thailand, Philippines, India, Pakistan and Korea was prospectively examined. METHODS: These centres enrolled 224 consecutive patients over eight months to receive abciximab during PCI for the study. The cohort consisted of 82.1 percent males, with mean age of 55 (+/- 11) years and mean weight of 67 (+/- 17) kg. RESULTS: The use of abciximab during PCI ranged between 6.2 percent and 21.6 percent. The indications for the use of abciximab were: acute coronary syndromes (34.3 percent), complex coronary lesions (17.9 percent) and multivessel PCI (17.7 percent). Based on a risk scoring system devised for this registry, majority (60.0 percent) of the patients was considered high risk when abciximab was used. Among the patients enrolled, 36.6 percent received abciximab as a "bail-out". The overall in-hospital ischaemic event rates were low at 4.0 percent. The complication rates included major bleeding 0.7 percent, thrombocytopenia 2.7 percent and need for blood transfusion 2.8 percent. There was a trend towards a higher incidence of in-hospital non-Q myocardial infarction in the "bail-out" group (2.1 percent versus 7.3 percent, p-value equals 0.07). CONCLUSION: Abxicimab was uncommonly used among patients (9.4 percent) undergoing PCI in this Asian region, with the operators reserving it mainly for high-risk patients.

Ectopic sympathetic preganglionic neurons maintain proper connectivity in the reeler mutant mouse.

The location of sympathetic preganglionic neurons (SPN) in the spinal cord of the reeler mouse mutant is abnormal. Instead of their normal location in the intermediolateral column, the majority of SPN in the reeler cluster around the central canal. To determine whether ectopically located SPN in the reeler form appropriate synaptic connections with their pre- and postsynaptic partners, we examined 1). whether the axons of descending neural pathways that normally terminate on SPN follow them to their ectopic location, and 2). whether the central autonomic neural circuit that controls sympathetic output to the kidney is organized normally in the reeler. Using antibodies against tyrosine hydroxylase, serotonin, neuropeptide Y, substance P and calcitonin gene-related peptide as markers for adrenergic, serotonergic and peptidergic terminals, we found that axons which normally innervate SPN follow these neurons to their ectopic spinal location in the reeler. Injection of pseudorabies virus into the kidney of wild type and reeler mutant mice revealed similar patterns of renal sympathetic and pre-sympathetic control circuits in the spinal cord, brainstem and forebrain. These results indicate that the presynaptic inputs and postsynaptic targets of SPN in the reeler are normal, despite the ectopic spinal location of their cell bodies.

Usefulness of ST elevation II/III ratio and ST deviation in lead I for identifying the culprit artery in inferior wall acute myocardial infarction.

In a study of 92 patients presenting with inferior wall acute myocardial infarction, the infarct-related artery was the right coronary artery in 72 patients (78%) and the left circumflex artery in 20 (22%). An ST II/III ratio of 1 or an isoelectric ST in lead I are sensitive and specific markers of left circumflex artery occlusion, whereas an ST II/III ratio <1 (ST elevation in lead III >II) or ST depression in lead I are sensitive and specific markers of right coronary artery occlusion.

Reelin controls position of autonomic neurons in the spinal cord.

Mutation of the reeler gene (Reln) disrupts neuronal migration in several brain regions and gives rise to functional deficits such as ataxic gait and trembling in the reeler mutant mouse. Thus, the Reln product, reelin, is thought to control cell-cell interactions critical for cell positioning in the brain. Although an abundance of reelin transcript is found in the embryonic spinal cord [Ikeda, Y. & Terashima, T. (1997) Dev. Dyn. 210, 157-172; Schiffmann, S. N., Bernier, B. & Goffinet, A. M. (1997) Eur. J. Neurosci. 9, 1055-1071], it is generally thought that neuronal migration in the spinal cord is not affected by reelin. Here, however, we show that migration of sympathetic preganglionic neurons in the spinal cord is affected by reelin. This study thus indicates that reelin affects neuronal migration outside of the brain. Moreover, the relationship between reelin and migrating preganglionic neurons suggests that reelin acts as a barrier to neuronal migration.

Electrocardiographic patterns in posterior chest leads (V7, V8, V9) in normal subjects.

The electrocardiographic patterns in leads V7, V8, and V9 were studied in 225 young, normal men (age range 17 to 21 years). The prevalence of 0.5- to 1.0-mm ST-segment elevation in leads V7, V8, and Vg 0.08 second after the J point was 8.9%, 5.8%, and 3.1%, respectively; the ST-segment elevation was not >1.0 mm in any subject.

The ECG "lead I sign" in cardiac disease--an indicator of coexisting obstructive pulmonary disease.

Two patients with co-existing cardiac disease and chronic obstructive pulmonary disease are described. The first patient had Wolff-Parkinson-White syndrome and the second patient had extensive anterior Q wave myocardial infarction. In addition to the distinctive ECG patterns of their cardiac abnormalities, both patients also showed the "lead I sign" which is a highly specific marker of chronic obstructive pulmonary disease. These two patients suggest that even in the presence of cardiac disease, the diagnosis of chronic obstructive pulmonary disease should be strongly suspected when the "lead I sign" is present.

Blood pressure, sodium intake, insulin resistance, and urinary nitrate excretion.

The objective of this study was to investigate the relationships among various humoral factors thought to be involved in the regulation of blood pressure during high NaCl intake. Nineteen healthy subjects underwent sequential 5-day periods ingesting a low-sodium (25 mmol/d) or high-sodium (200 mmol/d) diet. Insulin resistance was assessed by the steady-state plasma glucose concentration at the end of a 3-hour insulin suppression test. Insulin resistance correlated inversely with natriuresis (P=0.04) and directly with increase in weight (P=0.03). The increase in mean arterial pressure associated with the high-sodium diet correlated directly with the gain in weight (P<0.05) and inversely with the increase in urinary nitrate excretion (P<0.0001). In a multiple regression model, more than 2/3 of the variance in mean arterial pressure was accounted for by the gain in weight and change in urinary nitrate excretion. The steady-state plasma glucose concentrations obtained with the 2 diets were similar, indicating that insulin resistance was unaffected by sodium intake. During high sodium intake, plasma renin activity and aldosterone decreased and plasma atrial natriuretic peptide increased; these changes did not correlate with the change in mean arterial pressure, insulin resistance, or change in urinary nitrate excretion. To the extent that urinary nitrate excretion reflects activity of the endogenous nitric oxide system, these results suggest that the salt sensitivity of mean arterial pressure may be related to blunted generation of endogenous nitric oxide. The results also demonstrate that insulin-resistant individuals have an impaired natriuretic response to high sodium intake.

Segmental specificity of chick sympathetic preganglionic projections is influenced by preganglionic neurons from neighboring spinal cord segments.

Sympathetic preganglionic neurons of the chick are located between the brachial and lumbosacral enlargements of the spinal cord. Their axons exit the spinal cord via their adjacent ventral roots and project rostrally or caudally along the sympathetic trunk to innervate sympathetic ganglia. The projections of sympathetic preganglionic neurons are segmentally specific. Neurons from the 16th cervical (C16) and the first thoracic (T1) spinal cord segments project predominantly in the rostral direction, whereas those from the fifth thoracic (T5) to the first lumbar (L1) spinal segments project predominantly in the caudal direction. Neurons from intervening spinal cord segments (T2-T4) project in rostral and caudal directions. In the present study, neural tube manipulations show that the direction of preganglionic projections is altered by both the elimination and addition of preganglionic neurons projecting into the sympathetic trunk from neighboring segments. The present study also compares the projections of preganglionic neurons from transplants of multiple neural tube segments with those from transplants of single neural tube segments reported in a previous study (Yip, 1987). In the previous study when single thoracic neural tube segments were transplanted to the cervical level, preganglionic neurons did not maintain their original projection patterns. The present study found that, when contiguous neighboring segments were transplanted to the cervical level, preganglionic neurons maintained projection patterns characteristic of their original segmental levels. These results indicate that the direction of preganglionic projections can be influenced by neurons from neighboring segments, suggesting that the formation of segmentally specific preganglionic projections during embryogenesis may involve the interactions of preganglionic neurons with those from neighboring spinal cord segments.

Specific projections of sympathetic preganglionic neurons are not intrinsically determined by segmental origins of their cell bodies.

Sympathetic preganglionic projections of the chick are segmentally specific. Neurons from the 16th cervical (C16) and the first thoracic (T1) spinal cord segments project almost exclusively in the rostral direction, while those from the fifth thoracic (T5) to the first lumbar (L1) spinal segments project almost exclusively in the caudal direction. Neurons from the intervening spinal cord segments (T2-4) project in rostral and caudal directions. There is also a tendency for rostrally located neurons in each segment to project rostrally and caudally located neurons to project caudally. To investigate whether specific projections of preganglionic neurons are intrinsically determined by segmental origins of their cell bodies, neural tube segments were transplanted or rotated in embryos at stages 19-26; these stages include times during and after preganglionic cell birth and just prior to axon outgrowth. When the T1 neural tube segment was replaced with the T5 or T7 neural tube segment the transplanted T5 or T7 preganglionic neurons, now in the T1 position, projected rostrally. Conversely, when the T5 or T7 neural tube segment was replaced with the T1 neural tube, the transplanted T1 preganglionic neurons projected caudally. In addition, when individual T3 spinal cord segments were rotated 180 degrees along the A-P axis, neurons which were originally in the caudal part of the segment projected rostrally, whereas neurons originally from the rostral part of the segment projected caudally. These results show that specific projections of preganglionic neurons are not intrinsically determined by segmental origins of their cell bodies.

Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger.

The epithelial brush border Na+/H+ exchanger isoform 3 (NHE3) is regulated by growth factors and protein kinases. When stably expressed in PS120 fibroblasts, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by phorbol esters. To examine the role of phosphorylation of NHE3 in growth factor/protein kinase regulation, NHE3 was C-terminally tagged with an 11-amino acid epitope of the vesicular stomatitis virus glycoprotein (VSVG) and stably expressed in Na+/H+ exchanger null PS120 fibroblasts (PS120/NHE3V). NHE3V was regulated by serum, FGF, and phorbol ester in a manner identical to wild type non-VSVG-tagged NHE3. Phosphorylation of NHE3V was evaluated via immunoprecipitation with anti-VSVG antibody after in vivo labeling of PS120/NHE3V cells with [32P]orthophosphate. NHE3V was phosphorylated under basal conditions. However, FGF and PMA, under conditions in which these agonists regulate NHE3V, altered neither the amount of phosphorylation of NHE3V as analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography nor two-dimensional phosphopeptide maps of tryptic digests of NHE3V. In contrast, while changes in NHE3V phosphorylation were not observed with serum exposure by one-dimensional SDS-polyacrylamide gel electrophoresis, two-dimensional studies showed increases in two phosphopeptides. Under all these conditions, phosphoamino acid analysis showed that NHE3V was phosphorylated only on serine residues. By cell surface protein biotinylation studies under basal conditions, at least 27% of the NHE3V was expressed on the cell surface. To further analyze the phosphorylation status of the surface and intracellular forms of NHE3V under basal conditions and determine whether the amount of phosphorylation of the surface form changes upon serum, FGF, and PMA regulation, the surface form of NHE3V was separated from intracellular form by biotinylation/avidin-agarose precipitation. Under basal conditions, both intracellular and surface forms of NHE3V were phosphorylated. However, the amount of phosphorylation of the surface form of NHE3V did not change upon stimulation by serum and FGF and inhibition by PMA based on one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography. Thus, we conclude that when expressed in PS120 cells, while NHE3 is a phosphoprotein under basal conditions, its regulation by FGF and PMA is not by changes in the phosphorylation of NHE3, while regulation by serum may involve changes in its phosphorylation. Regulation of NHE3 probably involves intermediate associated regulatory proteins. The function of basal phosphorylation of NHE3 is not known.

Glomerular hyperfiltration in the prediction of nephropathy in IDDM: a 10-year follow-up study.

Glomerular hyperfiltration has been proposed as an independent risk factor for the development of diabetic nephropathy in patients with IDDM. In a case-controlled prospective study of IDDM patients without albuminuria, serial glomerular filtration rate (GFR) measurements were performed over an observation period of 10 years. A group of 25 IDDM patients (20 men, 5 women; initial age, 29 [17-49] years) with glomerular hyperfiltration (GFR >135 ml x min(-1) x 1.73 m(-2)) were matched for age, sex, and duration of diabetes with 25 IDDM patients (20 men, 5 women; initial age, 30 [17-48] years) with glomerular normofiltration (GFR 83-135 ml x min(-1) x 1.73 m(-2)). GFR, urinary albumin excretion rate (AER), blood pressure, and glycated hemoglobin were measured at baseline and at 5, 8, and 10 years. The two groups had similar entry levels of blood pressure, AER, and glycated hemoglobin. Metabolic control was similar in the two groups during follow-up. The final GFR remained higher in the group with hyperfiltration (122 [109-135] vs. 103 [95-111] ml x min(-1) x 1.73 m(-2); P = 0.02) despite a nonsignificantly faster rate of fall of GFR compared with that of the control group (2.54 [1.20-3.88] vs. 1.50 [1.01-1.99] ml x min(-1) x year(-1); P = 0.14). A similar number of patients in each group progressed to either microalbuminuria or macroalbuminuria (n = 4 vs. n = 3) or developed hypertension (blood pressure, >160/95 mmHg; n = 3 vs. n = 4). End-of-study AER was, however, higher in the group with hyperfiltration (geometric mean [95% CI]: 18.9 [11.3-31.6] vs. 11.0 [8.1-15.0]; P = 0.05), and baseline glomerular hyperfiltration was an independent determinant of end-of-study blood pressure (P = 0.04). The strongest predictors of end-of-study AER and blood pressure were their baseline values (P < 0.04 and P < 0.01, respectively). In conclusion, levels of AER and blood pressure are the main risk factors for renal outcome, while glomerular hyperfiltration appears to play a lesser role.

Insulin resistance in patients with essential hypertension can occur in the absence of microalbuminuria.

This study was initiated to see if the presence of resistance to insulin-mediated glucose disposal, glucose intolerance, and hyperinsulinemia in healthy patients with hypertension was dependent upon the coexistence of microalbuminuria. For this purpose we compared these variables in 68 individuals: 34 patients with hypertension and 34 normal volunteers. The two groups were similar in terms of age, gender distribution, body mass index, and ratio of waist to hip girth. Furthermore, although four patients with hypertension satisfied the criteria for microalbuminuria, as compared to one normal volunteer, the urinary albumin excretion (UAE) rates were similar in the two groups (8.07 +/- 1.08 v 7.67 +/- 1.12 micrograms/min). Despite the similarities, both the plasma glucose and insulin responses to a 75 g oral glucose challenge were significantly higher (P < .01) in those with high blood pressure. In addition, the steady-state plasma glucose (SSPG) concentrations at the end of a 180 min continuous infusion of somatostatin, insulin, and glucose was significantly higher in those with hypertension (156 +/- 13 v 107 +/- 10 mg/dL, P < .01). Since the steady-state plasma insulin levels were also somewhat higher in those with hypertension, the higher SSPG values indicate that these individuals were relatively insulin resistant as compared to the control population. Finally, UAE rates were not correlated with either the plasma glucose or insulin responses to oral glucose or to the SSPG concentrations--either in the entire group of 68, or when the 34 patients in each group were considered separately. These results demonstrate that insulin resistance, glucose intolerance, and hyperinsulinemia can occur independently of microalbuminuria in patients with hypertension.

NHE2 and NHE3 are human and rabbit intestinal brush-border proteins.

Rabbit NHE2 and NHE3 are two epithelial isoform Na+/H+ exchangers (NHE), the messages for which are found predominantly and entirely, respectively, in renal, intestinal, and gastric mucosa. The current studies used Western analysis and immunohistochemistry to identify and characterize the apical vs. basolateral membrane distribution of NHE2 and NHE3 in intestinal epithelial cells. Based on Western analysis, NHE2 and NHE3 both are present in brush-border but not basolateral membranes of small intestine. Both NHE2 and NHE3 are 85-kDa proteins. Consistent with Western analysis, NHE2 and NHE3 are immunolocalired to the brush-border but not basolateral membranes of villus epithelial cells, but not goblet cells, in human jejunum and ileum and in surface epithelial cells in the ascending and descending colon and rectum. In addition, NHE2 and NHE3 are present in small amounts in the crypt cell brush border of human jejunum, ileum, ascending and descending colon, and rectum. In rabbit jejunum, ileum, and ascending colon, NHE2 and NHE3 are present in the brush border of epithelial and not goblet cells, again much more in the villus (small intestine)/ surface cells (colon) than the crypt. NHE2 but not NHE3 is present in the brush border of rabbit descending colon surface cells and in small amounts in crypt cells. NHE2 and NHE3 are both human and rabbit small intestinal and colonic epithelial cell brush-border Na+/H+ exchanger isoforms that colocalize in all intestinal segments except rabbit descending colon, which lacks NHE3.

Specificity of sympathetic preganglionic projections in the chick is influenced by the somitic mesoderm.

It has been shown that the development of segmentally specific sympathetic preganglionic projections in the chick is influenced by the tissue environment along the pathway of the preganglionic axons. The cellular origin of this influence, however, is not known. In the present study, transplantation of quail somites into chick hosts showed that the cells in the local environment of the sympathetic trunk are derived from the somite. Surgical manipulations of chick somites then were performed to investigate whether somites play a role in the establishment of preganglionic projection patterns. When cervical somites were transplanted to the thoracic region, preganglionic neurons adjacent to the transplanted somites projected aberrantly. In addition, when somites were removed, the pattern of preganglionic axonal projections to their target ganglia was altered. These results indicate that the specificity of sympathetic preganglionic projections is influenced by the somitic mesoderm.

Effects of MR exposure on axonal outgrowth in the sympathetic nervous system of the chick.

The effects of MR exposure on the rate and specificity of sympathetic preganglionic axonal outgrowth were examined in the chick embryo. Embryos were exposed to a static magnetic field of 1.5 T for 6 hours, 64 MHz RF field pulses, and a switched magnetic field gradient of amplitude 0.6 G/cm for 4 hours. No significant difference in axonal outgrowth was observed between MR-exposed and control embryos. In addition, the distributions of several major extracellular matrix (ECM) molecules, laminin, fibronectin, and collagen IV, were examined. Immunostaining patterns of these ECM molecules during axonal outgrowth showed no difference between MR-exposed and control embryos. Our results suggest that the MR exposure conditions used in this study do not affect axonal outgrowth in the sympathetic nervous system of the chick.

Separate C-terminal domains of the epithelial specific brush border Na+/H+ exchanger isoform NHE3 are involved in stimulation and inhibition by protein kinases/growth factors.

NHE3, a cloned intestinal and renal brush border Na+/H+ exchanger, has previously been shown to be both stimulated and inhibited by different protein kinases/growth factors. For instance, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by protein kinase C. In the present study, we used a series of NHE3 C terminus truncation mutants to identify separate regions of the C-terminal cytoplasmic tail responsible for stimulation and inhibition by protein kinases/growth factors. Five NHE3 C terminus truncation mutant stable cell lines were generated by stably transfecting NHE3 deletion cDNAs into PS120 fibroblasts, which lack any endogenous Na+/H+ exchanger. Using fluorometric techniques, the effects of the calcium/calmodulin (CaM) inhibitor W13, calcium/CaM kinase inhibitor KN-62, phorbol myristate acetate, okadaic acid, FGF, and fetal bovine serum on Na+/H+ exchange were studied in these transfected cells. Inhibition of basal activity of full-length NHE3 is mediated by CaM at a site C-terminal to amino acid 756; this CaM effect occurs through both kinase dependent and independent mechanisms. There is another independent inhibitory domain for protein kinase C between amino acids 585 and 689. In addition, there are at least three stimulatory regions in the C-terminal domain of NHE3, corresponding to amino acids 509-543 for okadaic acid, 475-509 for FGF, and a region N-terminal to amino acid 475 for fetal bovine serum. We conclude that separate regions of the C terminus of NHE3 are involved with stimulation or inhibition of Na+/H+ exchange activity, with both stimulatory and inhibitory domains having several discrete subdomains. A conservative model to explain the way these multiple domains in the C terminus of NHE3 regulate Na+/H+ exchange is via an effect on associated regulatory proteins.