Urban trench fever presenting as culture-negative endocarditis.

A young Russian man presented with increasing shortness of breath and signs of worsening aortic regurgitation. A diagnosis of infective endocarditis was made before emergency valve replacement. The infective cause was not discovered by routine culture but was suggested by electron microscopy and confirmed by serology and PCR testing.

Chronic treatment with tetrahydrobiopterin reverses endothelial dysfunction and oxidative stress in hypercholesterolaemia.

BACKGROUND: Reduced availability of tetrahydrobiopterin (BH(4)), an essential cofactor of nitric oxide (NO) synthase (NOS), decreases NO production and increases reactive oxygen species. Both mechanisms contribute to atherosclerotic vascular disease. Although acute supplementation of BH(4) improves endothelial dysfunction, the effect of chronic BH(4) in humans is unknown. OBJECTIVE: To investigate the effect of chronic BH(4) supplementation on endothelial function and oxidative stress in hypercholesterolaemia. DESIGN: Randomised double-blind, placebo-controlled trial. SETTING: University Hospital. PATIENTS: 22 hypercholesterolaemic patients (low-density lipoprotein (LDL) >4.5 mmol/l) were randomised to 4 weeks of oral BH(4) (400 mg twice daily) or placebo. Age-matched healthy volunteers served as controls. MAIN OUTCOME MEASURES: Endothelium-dependent and -independent vasodilatation was assessed by venous occlusion plethysmography. To elucidate the mechanisms of BH(4) effect, NO release and superoxide anion (O(2)(-)) production were measured in human aortic endothelial cells exposed to native LDL (2.6 mmol cholesterol/l). RESULTS: BH(4) plasma levels were significantly increased by oral supplementation. NO-mediated vasodilatation to acetylcholine was reduced in patients compared with controls and restored by BH(4). No effect of BH(4) on endothelium-independent vasodilatation was seen. Furthermore, 8-F(2 )isoprostane plasma levels, a marker of vascular oxidative stress, were reduced by BH(4). In LDL-treated endothelial cells, BH(4) levels and NO release were reduced and O(2)(-) production increased compared with control cells. Exogenous BH(4) normalised NO and O(2)(-) production. CONCLUSIONS: In hypercholesterolaemia, endothelial dysfunction and oxidative stress can be reversed by chronic oral treatment with BH(4). Thus, BH(4) availability is essential for maintaining NO synthesis and low O(2)(-) production by endothelial NOS in vivo, and may provide a rational therapeutic approach to prevent cardiovascular disease.

Severe endothelial dysfunction in the aorta of a mouse model of Fabry disease; partial prevention by N-butyldeoxynojirimycin treatment.

OBJECTIVE: Fabry disease results from alpha-gala-ctosidase A deficiency and is characterized by the lysosomal accumulation of globotriaosylceramide. Globotriaosylceramide storage predominantly affects endothelial cells, altering vascular wall morphology and vasomotor function. Our objective was to investigate aortic globotriaosylceramide levels, morphology and function in a mouse model of Fabry disease, and the effect of substrate reduction therapy, using the glycosphingolipid biosynthesis inhibitor N-butyldeoxynojirimycin. METHODS AND RESULTS: Mice used were C57BL/6J and alpha-galactosidase A knockout (Fabry). We show progressive accumulation of aortic globotriaosylceramide throughout the lifespan of untreated Fabry mice (55-fold elevation at 2 months increasing to 187-fold by 19 months), localized to endothelial and vascular smooth-muscle cells; there was no effect on vascular wall morphology in young Fabry mice. In old mice, storage resulted in intimal thickening. Endothelial function declined with age in Fabry mouse aorta. Aortae from N-butyldeoxynojirimycin-treated Fabry mice at 19 months of age had reduced endothelial globotriaosylceramide storage, fewer morphological abnormalities and less severe vasomotor dysfunction compared with untreated littermates. CONCLUSION: We provide evidence of a novel vascular phenotype in the Fabry mouse that has relevance to vascular disease in Fabry patients. N-Butyldeoxynojirimycin treatment partially prevented the phenotype in the Fabry mouse by reducing endothelial globotriaosylceramide storage.

Relationships between nitric oxide-mediated endothelial function, eNOS coupling and blood pressure revealed by eNOS-GTP cyclohydrolase 1 double transgenic mice.

Endothelium-dependent relaxation in conduit vessels is mediated largely by nitric oxide (NO), produced by the enzyme endothelial nitric oxide synthase (eNOS) in the presence of the cofactor tetrahydrobiopterin (BH4) and mediated through a cGMP-dependent downstream signalling cascade. Endothelial NOS regulates blood pressure in vivo, and impaired endothelial NO bioactivity in vascular disease states may contribute to systemic hypertension. In the absence of sufficient levels of the cofactor BH4, NO becomes uncoupled from arginine oxidation and eNOS produces superoxide rather than NO. The enzymatic uncoupling of eNOS is an important feature of vascular disease states associated with increased oxidative stress. However, whether eNOS coupling, rather than overall eNOS activity, has specific effects on endothelium-dependent vasorelaxation in vitro, or on blood pressure regulation in vivo, remains unclear. In this study, we evaluate the relationships between blood pressure and endothelial function in models of eNOS uncoupling, using mice with endothelium-targeted transgenic eNOS overexpression (eNOS-Tg), in comparison with littermates in which eNOS coupling was rescued by additional endothelium-targeted overexpression of GTP cyclohydrolase 1 (eNOS/GCH-Tg) to increase endothelial BH4 levels. Despite the previously characterized differences in eNOS-dependent superoxide production between these animals, we find that blood pressure is equally reduced in both genotypes, compared with wild-type animals. Furthermore, both eNOS-Tg and eNOS/GCH-Tg mice exhibit similarly impaired endothelium-dependent vasorelaxation. We show that reduced vasorelaxation responses result from desensitization of cGMP-mediated signalling and are associated with increased NO production rather than changes in superoxide production.

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation.

AIMS/HYPOTHESIS: Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice. METHODS: Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting. RESULTS: In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged. CONCLUSIONS/INTERPRETATION: Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.

Candida prosthetic valve endocarditis cured by caspofungin therapy without valve replacement.

A 64-year-old woman with a mechanical mitral valve prosthesis developed late-onset Candida endocarditis. Blood cultures grew Candida glabrata and Candida krusei. Transesophageal echocardiography demonstrated vegetations on the valve. The patient was not medically fit for valve replacement, but her condition was successfully treated with 6 weeks of intravenous caspofungin therapy.

A rapid troponin-I-based protocol for assessing acute chest pain.

In a prospective randomized open trial with 30-day follow-up, we compared a troponin-I-based protocol to 'standard management' for the diagnosis and risk stratification of patients with acute non-ST-elevation chest pain. Patients with acute chest pain (n=400) were randomized to standard diagnostic tests and management, or a protocol based on the admission ECG and the troponin-I result 6 h after onset of chest pain. Low-risk patients were discharged early from CCU; high-risk patients were treated with medical therapy or referred for in-patient angiography as appropriate. We measured length of CCU stay, and followed all patients for major adverse cardiac events (MACE) of death, non-fatal myocardial infarction (MI), or urgent revascularization during the admission and for 30 days post-discharge. The troponin protocol allowed earlier discharge in the low-risk group (10 vs. 30 h, p<0.001) with no excess of adverse events compared to standard management (3% vs. 5%, p=0.32). It identified a group of patients at moderate risk of cardiac events (15% MACE rate during admission and 30-day follow-up), and a high-risk group (75% MACE rate) more accurately than did standard management. The prognostic power of troponin testing in combination with the admission ECG was higher than with either test used alone. The protocol improved the efficiency of low-risk patient management, and improved patient risk stratification. This study adds to the evidence favouring troponin evaluation as part of the management of acute coronary syndromes.

Randomised comparison of antero-lateral versus antero-posterior paddle positions for DC cardioversion of persistent atrial fibrillation.

We designed a prospective, randomised, single-blind trial to compare the relative efficacy of antero-lateral versus antero-posterior paddle positions for DC cardioversion of persistent atrial fibrillation. A total of 59 patients were randomised to cardioversion using standard gel pads placed either in the antero-lateral (AL) or antero-posterior (AP) positions. The first synchronised shock was given at 360 J; if this was unsuccessful, a second shock of 360 J was given in the alternative position. We compared cardioversion success rate and energy requirements with each strategy. With the first 360 J DC shock, a significantly greater proportion of patients were restored to sinus rhythm from the antero-lateral position (18/30) compared to the antero-posterior position (10/29) (P=0.048). For those patients remaining in atrial fibrillation, there was no difference in the proportions cardioverted from the antero-lateral position (4/19) compared to the antero-posterior position (5/12) with the second 360 J DC shock (P=0. 22). The total cardioversion success rate was 23/30 (77%) for antero-lateral followed by antero-posterior shocks compared to a success rate of 14/29 (48%) for antero-posterior followed by antero-lateral shocks, and this difference was significant (P=0.024). There was no significant difference in the mean energy delivered for patients randomised to an initial antero-lateral shock (504 J), compared to patients given an initial antero-posterior shock (583 J) (P=0.1). We conclude that the antero-lateral paddle position appears more effective for DC cardioversion of persistent atrial fibrillation.

Randomised double blind trial of oral versus intravenous flecainide for the cardioversion of acute atrial fibrillation.

OBJECTIVE: To investigate whether an oral loading dose of flecainide is as safe and effective as intravenous flecainide for the cardioversion of acute atrial fibrillation. DESIGN: Prospective, randomised, double blind, double placebo study. SETTING: Cardiac care unit of a large district general hospital in the UK. PATIENTS AND METHODS: 79 patients presenting with symptomatic acute atrial fibrillation: patients were given either intravenous flecainide (n = 39) or a solution of oral flecainide (n = 40), with appropriate placebos. All patients were heparinised during the study. PRIMARY OUTCOME MEASURES: Safety; mean time to cardioversion; proportion of patients restored to sinus rhythm at two hours and eight hours after treatment. Analysis was by intention to treat. RESULTS: There were no differences in baseline characteristics between the oral and intravenous groups. Both forms of flecainide were well tolerated, with no adverse clinical events during the study. The mean time to cardioversion was 110 minutes in the oral group and 52 minutes in the intravenous group (p = 0.002). Two hours after treatment, 27 of the 40 patients in the oral group (68%) and 25 of the 39 in the intravenous group (64%) had reverted to sinus rhythm (p = 0.74). Eight hours after treatment, 30 patients in the oral group (75%) and 28 in the intravenous group (72%) had reverted to sinus rhythm (p = 0.76). CONCLUSIONS: Intravenous flecainide restored sinus rhythm more rapidly than oral flecainide, but at two hours and eight hours after treatment there was no difference in the proportion of patients cardioverted by the two approaches. These results suggest a role for oral loading doses of flecainide in the treatment of acute or symptomatic paroxysmal atrial fibrillation.

Vertical transmission of human immunodeficiency virus.

We discuss the interactions between mother and infant that influence the transmission of HIV. Potential routes of infection are identified, and maternal and infant risk factors for transmission are explored. The role of the immune system in controlling HIV infection in the infant is discussed. Finally, preventive measures for reducing vertical transmission are proposed and evaluated.

Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein.

Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IE1. Responding cells were predominantly CD3+ CD4+. IE1 antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IE1-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IE1-specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IE1 molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa IE1 protein would be required to induce specific T-cell responses in humans.

Automation of limiting dilution cytotoxicity assays.

Limiting dilution analysis is a valuable technique for the quantitation and clonal analysis of immunocompetent cells. However, manual processing of the large number of samples necessary for satisfactory statistical analysis is laborious, and consequently results in inaccuracies reflected as increased standard errors. We describe the application of an automated robotic liquid handling tool to process samples in limiting dilution cytotoxicity assays. Our studies have shown that automated liquid handling is more accurate than manual methods, and that errors are limited. This results in savings of both time and resources. Furthermore, the process may be adapted for the safe, remote handling of sterile cell cultures, and human pathogens.