Clinical efficacy of sildenafil in primary pulmonary hypertension: a randomized, placebo-controlled, double-blind, crossover study

OBJECTIVES: In a randomized, double-blind, crossover design, we compared the efficacy of sildenafil with placebo in patients with primary pulmonary hypertension (PPH). The primary end point was the change in exercise time on treadmill using the Naughton protocol. Secondary end points were change in cardiac index and pulmonary artery systolic pressure as assessed by Doppler echocardiography and quality of life (QOL) as assessed by a questionnaire. BACKGROUND: Primary pulmonary hypertension is a disorder with limited treatment options. Uncontrolled studies had shown sildenafil to be beneficial in the treatment of PPH. METHODS: After initial clinical evaluation, including Doppler echocardiography and treadmill exercise test, patients were randomized to placebo or sildenafil with dosages ranging from 25 to 100 mg thrice daily on the basis of body weight. The evaluation was repeated after six weeks. Then patients were crossed over to alternate therapy. Final evaluation was performed after another six weeks of treatment. RESULTS: Twenty-two patients completed the study. Exercise time increased by 44% from 475 +/- 168 s at the end of placebo phase to 686 +/- 224 s at the end of sildenafil phase (p < 0.0001). With sildenafil, cardiac index improved from 2.80 +/- 0.9 l/m2 to 3.45 +/- 1.1 l/m(2) (p < 0.0001), whereas pulmonary artery systolic pressure decreased insignificantly from 105.23 +/- 17.82 mm Hg to 98.50 +/- 24.38 mm Hg. There was significant improvement in the dyspnea and fatigue components of the QOL questionnaire. During the placebo phase, one patient died and another had syncope. There were no serious side effects with sildenafil. CONCLUSIONS: Sildenafil significantly improves exercise tolerance, cardiac index, and QOL in patients with PPH.     

Effect of ozone on bronchial mucosal inflammation in asthmatic and healthy subjects

Epidemiological studies suggestthat asthmatics are more affected by ozone than healthy people. This study tested three hypotheses (1) that short-term exposure to ozone induces inflammatory cell increases and up-regulation of vascular adhesion molecules in airway lavages and bronchial tissue 6 h after ozone exposure in healthy subjects; (2) these responses are exaggerated in subjects with mild allergic asthma; (3) ozone exacerbates pre-existent allergic airways inflammation. We exposed 15 mild asthmatic and 15 healthy subjects to 0.2 ppm of ozone or filtered air for 2 h on two separate occasions. Airway lavages and bronchial biopsies were obtained 6 h post-challenge. We found that ozone induced similar increases in bronchial wash neutrophils in both groups, although the neutrophil increase in the asthmatic group was on top of an elevated baseline. In healthy subjects, ozone exposure increased the expression of the vascular endothelial adhesion molecules P-selectin and ICAM- 1, as well as increasing tissue neutrophil and mast cell numbers. The asthmatics showed allergic airways inflammation at baseline but ozone did not aggravate this at the investigated time point. At 6 h post-ozone-exposure, we found no evidence that mild asthmatics were more responsive than healthy to ozone in terms of exaggerated neutrophil recruitment or exacerbation of pre-existing allergic inflammation. Further work is needed to assess the possibility of a difference in time kinetics between healthy and asthmatic subjects in their response to ozone.     

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a γ turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.     

Erythrocyte survival in severe falciparum malaria.

Erythrocyte survival was studied in 17 Thai patients (10 males, 7 females; aged 13-57 years) with severe falciparum malaria. To ensure radioisotopic labelling of cells before bone marrow recovery and survival analysis under near-steady state conditions, 51Cr labelling of autologous erythrocytes was performed at the time of admission (0 h) and calculation of mean cell lifespan (MCL) was based on semilogarithmic plots of corrected counts from 60 h onwards. Five patients received blood transfusions, all within 48 h of admission. The overall mean (+/- S.D.) MCL was short (44.1 +/- 21.7 days). Nontransfused patients had similar MCL values (43.6 +/- 20.4) to those of transfused patients (45.5 +/- 27.3 days, p greater than 0.8). Patients with and without palpable splenomegaly had MCL values which were not significantly different (54.1 +/- 28.8 vs. 37.2 +/- 12.3 days respectively, p greater than 0.1). There was no association between admission haematocrit or peripheral parasitaemia and MCL (p greater than 0.2 in each case), but there was an inverse correlation between total serum bilirubin and MCL (r = -0.49, p less than 0.025). There is accelerated destruction of non-parasitised erythrocytes in severe malaria resulting in a mean MCL that is half that found previously in healthy Thai volunteers (89.6 +/- 13.1 days, p less than 0.001) and significantly shorter than that reported previously in Thai patients with uncomplicated P. falciparum infections studied after parasite clearance (56.8 +/- 10.2 days, p less than 0.05).     

Impact of Obesity on Outcomes of Pregnancy in Women With Heart Disease

BackgroundWomen with heart disease are at risk for complications during pregnancy. This study sought to examine the effect of maternal obesity on pregnancy complications in women with heart disease.ObjectivesThe objective was to determine the incidence of adverse cardiac events (CE) in pregnant women with heart disease and obesity.MethodsAdverse CE during pregnancy were examined in a prospective cohort of women with heart disease. CE were a composite of the following: cardiac death/arrest, arrhythmias, heart failure, myocardial infarction, stroke, aortic dissection, and thromboembolic events. Pre-eclampsia and post-partum hemorrhage were also studied. Outcomes were examined according to body mass index (BMI). To identify additional predictors of CE, a baseline risk score (CARPREG [Canadian Cardiac Disease in Pregnancy Study] II score) for predicting cardiac complications was calculated for all pregnancies and included in a multivariable logistic regression model.ResultsOf 790 pregnancies, 19% occurred in women with BMI ≥30 kg/m² (obesity), 25% in women with BMI 25 to 29.9 kg/m² (overweight), 53% in women with BMI 18.5 to 24.9 kg/m² (normal weight), and 3% in women with BMI <18.5 kg/m² (underweight). Women with obesity were at higher risk of CE when compared with women with normal weight (23% vs. 14%; p = 0.006). In a multivariable model, obesity (odds ratio: 1.7; 95% confidence interval: 1.0 to 2.7) and higher CARPREG II risk scores (odds ratio: 1.7; 95% confidence interval: 1.5 to 1.9) predicted CE. Pre-eclampsia was more frequent in women with obesity compared with those with normal weight (8% vs. 2%; p = 0.001).ConclusionsObesity increases the risk of maternal cardiovascular complications in pregnant women with heart disease. This modifiable risk factor should be addressed at the time of preconception counseling.     

Beta-adrenergic receptor-stimulated apoptosis in cardiac myocytes is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of the mitochondrial pathway

Stimulation of beta-adrenergic receptors (betaARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating betaAR-stimulated apoptosis is not known. Stimulation of betaARs with norepinephrine (10 micromol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6- fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 micromol/L) and Euk-134 decreased betaAR-stimulated apoptosis by 89+/-6% and 76+/-10%, respectively. Infection with an adenovirus expressing catalase decreased betaAR-stimulated apoptosis by 82+/-15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 micromol/L) decreased betaAR-stimulated apoptosis by 76+/-8%, and the caspase inhibitor zVAD-fmk (25 micromol/L) decreased betaAR-stimulated apoptosis by 62+/-11%. betaAR-stimulated cytochrome c release was inhibited by MnTMPyP. betaAR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited betaAR-stimulated apoptosis by 81+/-12%, and the JNK inhibitor SP600125 inhibited both betaAR-stimulated apoptosis and cytochrome c release. Thus, betaAR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.     

Conformational investigation of peptides using solid‐state NMR spectroscopy—A study of polymorphism of β‐turn peptides containing diprolines

The construction of complex protein folds relies on the precise conversion of a linear polypeptide chain into a compact 3‐dimensional structure. In this context, study of isolated secondary structural modules containing short stretches of amino acids assumes significance. Additionally, peptides, both natural and synthetic, play a major role as potential drugs. With a view to understand the local conformations adopted by peptides in the solid state, we propose a multinuclear NMR approach utilizing spectra of nuclei in their natural isotopic abundance. Various solid‐state NMR experiments have been utilized for assignment of the spectra. Additionally, the gauge‐including projector augmented‐wave (GIPAW) calculations were used to confirm the assignments. Particularly, the utility of the double‐quantum–single‐quantum correlation experiments is highlighted for the purpose of assignment and for inferring the conformation across the peptide bond. The methodology is illustrated for the case of designed peptides containing diproline residues occurring at the β‐turns for identifying their cis‐trans conformational polymorphism. The proposed method promises to be of use in the study of conformations of small‐ to medium‐sized peptides such as antimicrobial peptides and in the study of polymorphism leading to applications in drug development protocols.     

The Effect of Standard Versus Longer Intestinal Bypass on GLP-1 Regulation and Glucose Metabolism in Patients With Type 2 Diabetes Undergoing Roux-en-Y Gastric Bypass: The Long-Limb Study

OBJECTIVERoux-en-Y gastric bypass (RYGB) characteristically enhances postprandial levels of glucagon-like peptide 1 (GLP-1), a mechanism that contributes to its profound glucose-lowering effects. This enhancement is thought to be triggered by bypass of food to the distal small intestine with higher densities of neuroendocrine L-cells. We hypothesized that if this is the predominant mechanism behind the enhanced secretion of GLP-1, a longer intestinal bypass would potentiate the postprandial peak in GLP-1, translating into higher insulin secretion and, thus, additional improvements in glucose tolerance. To investigate this, we conducted a mechanistic study comparing two variants of RYGB that differ in the length of intestinal bypass.RESEARCH DESIGN AND METHODSA total of 53 patients with type 2 diabetes (T2D) and obesity were randomized to either standard limb RYGB (50-cm biliopancreatic limb) or long limb RYGB (150-cm biliopancreatic limb). They underwent measurements of GLP-1 and insulin secretion following a mixed meal and insulin sensitivity using euglycemic hyperinsulinemic clamps at baseline and 2 weeks and at 20% weight loss after surgery.RESULTSBoth groups exhibited enhancement in postprandial GLP-1 secretion and improvements in glycemia compared with baseline. There were no significant differences in postprandial peak concentrations of GLP-1, time to peak, insulin secretion, and insulin sensitivity.CONCLUSIONSThe findings of this study demonstrate that lengthening of the intestinal bypass in RYGB does not affect GLP-1 secretion. Thus, the characteristic enhancement of GLP-1 response after RYGB might not depend on delivery of nutrients to more distal intestinal segments.     

The evolving art of hematopoietic stem cell transplantation: translational research in post-transplant immune reconstitution and immunosuppression

Immunologic Research - Allogeneic hematopoietic stem cell transplantation (SCT) offers the best chance for cure and/or long-term survival for a broad range of diseases, including many high-risk...