A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL

Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.Varicella-zoster virus (VZV) is an enveloped, double-stranded DNA virus of the family Alphaherpesvirinae. Primary VZV infection initiates at the respiratory mucosa. T cells are infected in regional lymph nodes and disseminate virions to the skin, causing varicella (chickenpox) (1). VZV reaches sensory ganglia by the hematogenous route or by anterograde axonal transfer from skin lesions and establishes latency in neurons (2). VZV reactivation can lead to herpes zoster (shingles), which is particularly prevalent in the elderly and in immunocompromised individuals (1, 3). An estimated one third of people in the United States will develop herpes zoster and are at risk for the chronic pain syndrome referred to as postherpetic neuralgia.Live attenuated vaccines derived from the Oka strain are safe and effective against varicella and herpes zoster in healthy individuals but are contraindicated in most immunocompromised patients (4, 5). Subunit vaccines have the potential to be safe in these populations (6). High-titer VZV immunoglobulin given shortly after exposure decreases the incidence of varicella (7), indicating that antibodies against the VZV envelope glycoproteins can interfere with the initiation of infection and cell-to-cell spread. Thus, subunit vaccines made from one or more of these proteins could be an alternative to live attenuated VZV vaccines. Indeed in a phase 1/2 clinical trial, AS01_B-adjuvanted envelope glycoprotein gE has been reported to be more immunogenic than a live attenuated VZV vaccine in healthy individuals in the age groups of 18–30 or 50–70 y. Advances are occurring in the development of this gE-based vaccine candidate against herpes zoster (6).Similar to other herpesviruses, VZV entry into host cells is presumed to be initiated by virion attachment to the cell surface, followed by fusion between the virus envelope and the plasma membrane of the target cell. The envelope glycoproteins gB and the gHgL heterodimer (8) are highly conserved components of the herpesvirus entry machinery. VZV gB and gHgL are necessary and sufficient to induce cell fusion, which is considered a surrogate for virion envelope fusion (9, 10).The crystal structures of the gHgL heterodimer ectodomain for herpes simplex virus 2 (HSV-2) and Epstein–Barr virus (EBV) showed that gH and gL cofold to form a tightly packed complex with no similarity to any known viral fusion proteins (11, 12). In contrast, the structures of HSV-1 and EBV gB are highly similar to the vesicular stomatitis virus membrane fusion glycoprotein G, indicating that gB is the herpesvirus fusion protein (13–15). Fluorescence bimolecular complementation studies with HSV-1 envelope glycoproteins imply that gHgL interacts with gB during membrane fusion (16, 17). This interaction was inhibited by the anti-gHgL neutralizing monoclonal antibody (mAb) LP11 (11). How gHgL facilitates the conformational changes in gB required for membrane fusion and viral entry remains elusive.A homology model of VZV gH based on the HSV-2 gHgL structure was used to identify gH residues involved in membrane fusion (9). Incorporating gH mutations into recombinant VZV showed the importance of the gH ectodomain and cytoplasmic domain for viral replication and regulation of cell fusion in vitro and for infection of human skin xenografts in the SCID mouse model of VZV pathogenesis, as are gB mutations (9, 18, 19).The contribution of VZV gHgL to viral entry and spread was also demonstrated by the capacity of an anti-gH mouse mAb, mAb206, to neutralize VZV in vitro and to severely impair infection of human skin xenografts (20–22). Human neutralizing mAbs generated from a library of pooled B cells obtained from naturally immune individuals also target gHgL (23). Two of these, designated IgG-24 and IgG-94, and their respective fragment of antigen-binding (Fab), had the most VZV-neutralizing activity. Recently, a VZV-neutralizing human mAb (rec-RC IgG), which recognized the gHgL heterodimer in VZV infected cells, was isolated from plasmablasts recovered from an individual who was immunized with a zoster vaccine (24).The present study provides the first report, to our knowledge, on the structure of a herpesvirus gHgL heterodimer in complex with human mAbs and delineates the location of clinically relevant neutralizing epitopes on VZV gHgL. Structural and binding studies demonstrated that Fab-RC and Fab-94 target the same epitope formed by gH and gL residues, whereas Fab-24 binds to a distal site on gH. Immunization studies proved that the gHgL Fab-RC/-94 epitope is a critical target for neutralizing Abs against VZV. Compared with postfusion gB, gHgL elicited Abs in mice that more potently inhibited cell fusion and neutralized VZV. Vulnerability to Ab-mediated inhibition suggests the gHgL complex could be a component of an effective subunit vaccine.     

Interstitial and plasma adenosine stimulate nitric oxide and prostacyclin formation in human skeletal muscle

One major unresolved issue in muscle blood flow regulation is that of the role of circulating versus interstitial vasodilatory compounds. The present study determined adenosine-induced formation of NO and prostacyclin in the human muscle interstitium versus in femoral venous plasma to elucidate the interaction and importance of these vasodilators in the 2 compartments. To this end, we performed experiments on humans using microdialysis technique in skeletal muscle tissue, as well as the femoral vein, combined with experiments on cultures of microvascular endothelial versus skeletal muscle cells. In young healthy humans, microdialysate was collected at rest, during arterial infusion of adenosine, and during interstitial infusion of adenosine through microdialysis probes inserted into musculus vastus lateralis. Muscle interstitial NO and prostacyclin increased with arterial and interstitial infusion of adenosine. The addition of adenosine to skeletal muscle cells increased NO formation (fluorochrome 4-amino-5-methylamino-2',7-difluorescein fluorescence), whereas prostacyclin levels remained unchanged. The addition of adenosine to microvascular endothelial cells induced an increase in NO and prostacyclin levels. These findings provide novel insight into the role of adenosine in skeletal muscle blood flow regulation and vascular function by revealing that both interstitial and plasma adenosine have a stimulatory effect on NO and prostacyclin formation. In addition, both skeletal muscle and microvascular endothelial cells are potential mediators of adenosine-induced formation of NO in vivo, whereas only endothelial cells appear to play a role in adenosine-induced formation of prostacyclin.     

Should a new tuberculosis vaccine be administered intranasally?

Most of the world's population is vaccinated with the only available vaccine against tuberculosis (TB), the Bacillus Calmette-Guérin (BCG) vaccine that was developed almost a century ago. Despite the wide coverage of the BCG vaccine, there are great variations in protective efficacy among different study populations. BCG vaccination protects against childhood forms of TB, but this immunity wanes with age, resulting in none, or insufficient, protection against adult pulmonary TB (PTB). PTB is the major disease manifestation of TB in adults and it causes death at the most productive age, further adding to poverty in already impoverished countries. Therefore, new more effective vaccines and novel immunisation strategies are urgently needed. The most common route of TB is by inhalation of tubercle bacilli leading to the establishment of a primary infection in the lung. Immunising through the nasal mucosal surface should therefore have advantage over other routes, as such vaccine administration elicits protective immune responses also in the lung, i.e. at the site of primary infection. Several new TB-vaccine candidates have been evaluated for their protective efficacy in animal models using the mucosal route of immunisation. In formulating such vaccines, the adjuvants and delivery systems are crucially important.     

Free fatty acids are independently associated with all-cause and cardiovascular mortality in subjects with coronary artery disease

CONTEXT: Free fatty acids (FFAs) are associated with several cardiovascular risk factors and exert harmful effects on the myocardium. OBJECTIVE: The aim of our study was to elucidate the relationship between FFAs and mortality in subjects who underwent coronary angiography. DESIGN, SETTING, AND PARTICIPANTS: Ludwigshafen Risk and Cardiovascular Health is a prospective cohort study of Caucasians who had undergone coronary angiography at baseline (1997-2000). During a median time of follow-up of 5.38 yr, 513 deaths had occurred among 3315 study participants with measured FFAs. MAIN OUTCOME MEASURE: Hazard ratios for mortality according to FFA levels were measured. RESULTS: At the fourth quartile of FFAs, fully adjusted hazard ratios for death from any cause and cardiovascular causes were 1.58 (P = 0.002) and 1.83 (P = 0.001), respectively. In persons with angiographic coronary artery disease (CAD), stable CAD, and unstable CAD, the predictive value of FFAs was similar to that in the entire cohort, but the association did not attain statistical significance in persons without CAD analyzed separately. FFA levels were not related to the presence of angiographic CAD but were elevated in subjects with unstable CAD, compared with probands with stable CAD. Furthermore, FFAs increased with the severity of heart failure and were positively correlated with N-terminal pro-B-type natriuretic peptide (P < 0.001). CONCLUSIONS: FFA levels independently predict all-cause and cardiovascular mortality in subjects with angiographic CAD. A possible diagnostic use of FFAs warrants further studies, but our results may underline the importance of therapeutic approaches to influence FFA metabolism.     

The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs

Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. Because of their bone-selective pharmacokinetics, nitrogen-containing bisphosphonates (N-BPs), currently used as clinical inhibitors of bone-resorption diseases, target osteoclast farnesyl pyrophosphate synthase (FPPS) and inhibit protein prenylation. FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. To understand the molecular events involved in inhibition of FPPS by N-BPs, we used protein crystallography, enzyme kinetics, and isothermal titration calorimetry. We report here high-resolution x-ray structures of the human enzyme in complexes with risedronate and zoledronate, two of the leading N-BPs in clinical use. These agents bind to the dimethylallyl/geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-BP cyclic nitrogen with Thr-201 and Lys-200 suggest that these inhibitors achieve potency by positioning their nitrogen in the proposed carbocation-binding site. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight binding character, indicating that isomerization of an initial enzyme-inhibitor complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N-BPs to the apoenzyme is entropy-driven, presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of these important drugs.     

Staging of portal hypertension and portosystemic shunts using dynamic nuclear medicine investigations

AIM： To explore portal hypertension and portosystemic shunts and to stage chronic liver disease （CLD） based on the pathophysiology of portal hemodynamics. METHODS： Per-rectal portal scintigraphy （PRPS） was performed on 312 patients with CLD and liver angioscintigraphy （LAS） on 231 of them. The control group included 25 healthy subjects. We developed a new model of PRPS interpretation by introducing two new parameters, the liver transit time （LTT） and the circu-lation time between right heart and liver （RHLT）. LTT for each lobe was used to evaluate the early portal hypertension. RHLT is useful in cirrhosis to detect liver areas missing portal inflow. We calculated the classical per-rectal portal shunt index （PRSI） at PRPS and the hepatic perfusion index （HPI） at LAS. RESULTS： The normal LTT value was 24 ± 1 s. Abnormal LTT had PPV = 100% for CLD. Twenty-seven noncirrhotic patients had LTT increased up to 35 s （median 27 s）. RHLT （42 ± 1 s） was not related to liver disease. Cirrhosis could be excluded in all patients with PRSI 〈 5% （P 〈 0.01）. PRSI 〉 30% had PPV = 100% for cirrhosis. Based on PRPS and LAS we propose the classification of CLD in 5 hemodynamic stages. Stage 0 is normal （LTT = 24 s, PRSI 〈 5%）. In stage 1, LTT is increased, while PRSI remains normal. In stage 2, LTT is decreased between 16 s and 23 s, whereas PRSI is increased between 5% and 10%. In stage 3, PRSI is increased to 10%-30%, and LTT becomes undetectable by PRPS due to the portosystemic shunts. Stage 4 includes the patients with PRSI 〉 30%. RHLT and HPI were used to subtype stage 4. In our study stage 0 had NPV = 100% for CLD, stage 1 had PPV = 100% for non-cirrhotic CLD, stages 2 and 3 represented the transition from chronic hepatitis to cirrhosis, stage 4 had PPV = 100% for cirrhosis. CONCLUSION： LTT allows the detection of early portal hypertension and of opening of transhepatic shunts. PRSI is useful in CLD with extrahepatic portosystemic shunts. Our hemodynamic model stag     

Anemia in chronic congestive heart failure: frequency,prognosis, and treatment

Chronic heart failure (CHF) is a leading cause of morbidity and mortality. Although a precise definition for a cut-off value of hemoglobin level for anemia is still lacking, it has recently been found to be a common complication in CHF, occurring in 10-20% of patients. There are several possible pathogenetic mechanisms for anemia in CHF, and a precise underlying cause is found in only a minority of patients. In CHF, more than 50% of anemia cases are considered to be 'anemia in chronic illness'. In CHF patients, low hemoglobin values directly relate to poor peak oxygen consumption, disabling symptoms, and impaired survival. Recent pilot studies showed that correction of anemia with erythropoietin and iron may lead to improvement in symptoms and exercise capacity. These issues need to be tested in larger, double-blind, randomized, placebo-controlled trials before anemia treatment becomes routine in patients with CHF.     

Sustained monomorphic ventricular tachycardia ablation from the aortic sinus of valsalva

INTRODUCTION: The aim of this study was to delineate the electrophysiologic mechanisms of a novel type of ventricular tachycardia (VT) originating from the aortic sinus of Valsalva. METHODS AND RESULTS: Endocardial mapping was performed in four patients with symptomatic VT originating from the aortic sinus of Valsalva. Two patients suffered from dilative cardiomyopathy; the other two patients had no structural heart disease. Five VTs could be induced and terminated by programmed ventricular stimulation. Successful ablation was performed in the noncoronary sinus of Valsalva in three VTs and in the left aortic sinus in two. Abnormal (diastolic or presystolic) potentials were recorded during sinus rhythm (mean interval from the end of QRS complex to the potential 121+/-98 msec) and during VT (mean interval from the potential to QRS complex 64+/-45 msec) at effective sites in the aortic sinuses of Valsalva. Concealed entrainment was demonstrated at all successful sites. VT recurred in one patient after 1 month, whereas no recurrences were observed during follow-up of 8+/-6 months in the other three patients. CONCLUSION: Reentry constitutes one mechanism of VT originating from the aortic sinus of Valsalva. Entrainment mapping is useful to characterize the reentrant circuit of these VTs and to guide ablation.