[Embolic stroke of undetermined source (ESUS) : How much monitoring is necessary?] / ESUS ("embolic stroke of undetermined source") : Wie viel Monitoring ist nötig?

The clinical construct of embolic stroke of unknown source (ESUS) was first described in 2014. It is defined as cryptogenic ischemic stroke after the exclusion of a lacunar infarct, a significant (≥50%) stenosis of extracranial or intracranial arteries and a cardiac source of embolism. Initially, there was hope that these patients would benefit from anticoagulation. This was based on the suspicion that imaging criteria of stroke mimic features of embolism from cardiac sources or the great arteries. In two large randomized trials with 12,600 patients neither rivaroxaban nor dabigatran could reduce the risk of recurrent stroke. Based on these results, current research is focused on paroxysmal atrial fibrillation as a potential cause of stroke in these patients. Several randomized trials could show that by prolongation of monitoring to 30 days atrial fibrillation can be detected in approximately 10% of the patients. Using continuous monitoring (e. g. by implantable loop recorders) atrial fibrillation can even be detected in one quarter of the patients. Not all stroke patients can receive such an intensive monitoring. Therefore, this article summarizes the evidence and presents the resulting recommendations for patient selection and staged rhythm diagnostics and discusses a recently presented algorithm of an expert group for use in daily clinical practice.

Development of a Virosomal RSV Vaccine Containing 3D-PHAD® Adjuvant: Formulation, Composition, and Long-Term Stability.

PURPOSE: Characterization of virosomes, in late stage preclinical development as vaccines for Respiratory Syncytial Virus (RSV), with a membrane-incorporated synthetic monophosphoryl lipid A, 3D-PHAD® adjuvant. METHODS: Virosomes were initially formed by contacting a lipid film containing 3D-PHAD® with viral membranes solubilized with the short chain phospholipid DCPC, followed by dialysis, later by adding solubilized 3D-PHAD to viral membranes, or to preformed virosomes from DMSO. RESULTS: Virosomes formed from lipid films contained the membrane glycoproteins G and F, at similar F to G ratios but lower concentrations than in virus, and the added lipids, but only a fraction of the 3D-PHAD®. By single particle tracking (SPT), the virosome size distribution resembled that seen by cryo-electron microscopy, but dynamic light scattering showed much larger particles. These differences were caused by small virosome aggregates. Measured by SPT, virosomes were stable for 300 days. 3DPHAD ® incorporation in virosomes could be enhanced by providing the adjuvant from DCPC solubilized stock, but also by adding DMSO dissolved adjuvant to pre-formed virosomes. Virosomes with 0.1 mg/mg of 3D-PHAD®/viral protein from DMSO induced antibody titers similar to those by virosomes containing 0.2 mg/mg of DCPC-solubilized 3D-PHAD®. CONCLUSIONS: Stable 3D-PHAD® adjuvanted RSV virosomes can be formulated.

Rational design of an influenza subunit vaccine powder with sugar glass technology: preventing conformational changes of haemagglutinin during freezing and freeze-drying.

The development of a stable influenza subunit vaccine in the dry state was investigated. The influence of various carbohydrates, buffer types and freezing rates on the integrity of haemagglutinin after freeze-thawing or freeze-drying was investigated with a range of analytical and immunological methods. The use of fast freezing, Hepes buffer and carbohydrates (trehalose, inulin or dextran) as cryo- and lyoprotectants resulted in a significant reduction or even absence of conformational changes of HA as revealed by the used methods. The subunit vaccine in the powder was shown to remain immunogenic in an in vivo study in mice, using reconstituted powder. Moreover, the HA potency of the influenza subunit vaccine powder was stable for at least 26 weeks at room temperature.

Fusion of human neutrophil phagosomes with lysosomes in vitro: involvement of tyrosine kinases of the Src family and inhibition by mycobacteria.

The intracellular killing of microorganisms in phagocytes involves the fusion of lysosomes containing bactericidal factors with phagosomes, and several intracellular pathogens are able to inhibit this fusion event. In this study, we report the reconstitution of phagosome-lysosome fusion in vitro, using an assay based on resonance energy transfer between fluorescent phospholipid analogues that were inserted into whole human NB4-neutrophil membranes from liposomes containing positively charged lipids. Cytosol was required for fusion, and fusion was stimulated 3-fold if this cytosol had been prepared from neutrophils activated by using opsonized zymosan or a combination of the calcium ionophore (A23187) and phorbol myristate acetate (PMA). Fusion was inhibited by the addition of PP1, an inhibitor of Src family protein kinases, or GTPgammaS. We have previously reported that the biogenesis of phagolysosomes in human neutrophils is inhibited by mycobacteria. Here we show that cytosol from cells having internalized live (not heat-killed) Mycobacterium smegmatis or cytosol simply incubated with mycobacteria inhibited fusion, indicating that soluble factors are involved in mycobacterial inhibition of phagosome-lysosome fusion.

[Induction of myocardial neoangiogenesis by human growth factors. A new therapeutic approach in coronary heart disease]. / Induktion der myokardialen Neoangiogenese durch humane Wachstumsfaktoren. Ein neuer Therapieansatz bei koronarer Herzkrankheit.

Currently available approaches for treating human coronary heart disease aim to relieve symptoms and the risk of myocardial infarction either by reducing myocardial oxygen demand, preventing further disease progression, restoring coronary blood flow pharmacologically or mechanically, or bypassing the stenotic lesions and obstructed coronary artery segments. Gene therapy, especially using angiogenic growth factors, has emerged recently as a potential new treatment for cardiovascular disease. Following extensive experimental research on angiogenic growth factors, the first clinical studies on patients with coronary heart disease and peripheral vascular lesions have been performed. The polypeptides fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) appear to be particularly effective in initiating neovascularization (neoangiogenesis) in hypoxic or ischemic tissues. The first clinical study on patients with coronary heart disease treated by local intramyocardial injection of FGF-1 showed a 3-fold increase of capillary density mediated by the growth factor. Also, angiogenic growth factor injection intramyocardially as sole therapy for end-stage coronary disease showed an improvement of myocardial perfusion in the target areas as well as a reduction of symptoms and an increase in working capacity. Angiogenic therapy of the human myocardium introduces a new modality of treatment for coronary heart disease in terms of regulation of blood vessel growth. Beyond drug therapy, angioplasty and bypass surgery, this new approach may evolve into a fourth principle of treatment of atherosclerotic cardiovascular disease.

Clinical trials in coronary angiogenesis: issues, problems, consensus: An expert panel summary.

The rapid development of angiogenic growth factor therapy for patients with advanced ischemic heart disease over the last 5 years offers hope of a new treatment strategy based on generation of new blood supply in the diseased heart. However, as the field of therapeutic coronary angiogenesis is maturing from basic and preclinical investigations to clinical trials, many new and presently unresolved issues are coming into focus. These include in-depth understanding of the biology of angiogenesis, selection of appropriate patient populations for clinical trials, choice of therapeutic end points and means of their assessment, choice of therapeutic strategy (gene versus protein delivery), route of administration, and the side effect profile. The present article presents a summary statement of a panel of experts actively working in the field, convened by the Angiogenesis Foundation and the Angiogenesis Research Center during the 72nd meeting of the American Heart Association to define and achieve a consensus on the challenges facing development of therapeutic angiogenesis for coronary disease.

Membrane perturbation and fusion pore formation in influenza hemagglutinin-mediated membrane fusion. A new model for fusion.

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a "fusion peptide" domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion. By using whole virus, purified HA, or HA ectodomains, we found that shortly after insertion, pores of less than 26 A in diameter were formed in liposomal membranes. As measured by a novel assay, these pores stay open, or continue to close and open, for minutes to hours and persist after pH neutralization. With virus and purified HA, larger pores, allowing the leakage of dextrans, were seen at times well after insertion. For virus, dextran leakage was simultaneous with lipid mixing and the formation of "fusion pores," allowing the transfer of dextrans from the liposomal to the viral interior or vice versa. Pores did not form in the viral membrane in the absence of a target membrane. Based on these data, we propose a new model for fusion, in which HA initially forms a proteinaceous pore in the target, but not in the viral membrane, before a lipidic hemifusion intermediate is formed.

Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity.

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.

Membrane fusion mechanisms: the influenza hemagglutinin paradigm and its implications for intracellular fusion.

The mechanism of membrane fusion induced by the influenza virus hemagglutinin (HA) has been extensively characterized. Fusion is triggered by low pH, which induces conformational changes in the protein, leading to insertion of a hydrophobic 'fusion peptide' into the viral membrane and the target membrane for fusion. Insertion perturbs the target membrane, and hour glass-shaped lipidic fusion intermediates, called stalks, fusing the outer monolayers of the two membranes, are formed. Stalk formation is followed by complete fusion of the two membranes. Structures similar to those formed by HA at the pH of fusion are found not only in many other viral fusion proteins, but are also formed by SNAREs, proteins involved in intracellular fusion. Substances that inhibit or promote HA-induced fusion because they affect stalk formation, also inhibit or promote intracellular fusion, cell-cell fusion and even intracellular fission similarly. Therefore, the mechanism of influenza HA-induced fusion may be a paradigm for many intracellular fusion events.

[Papillary muscle rupture during stress ECG after myocardial infarct]. / Papillarmuskelriss während eines Belastungs-EKGs nach Myokardinfarkt.

The case of a 57 year old patient is reported, who suffered from an acute myocardial infarction with maximum CK and CKMB values of 821 and 84 U/l, respectively. The patient underwent bicycle exercise testing 9 days after a myocardial infarction in 25 W steps every 2 min starting with 50 W. The ergometry was interrupted at 125 W because of ST segment depression of 0.28 mV in V6. Systolic blood pressure dropped to 55 mm Hg, combined with severe angina and shock. Volume substitution and catecholamines did not elevate blood pressure. Immediate M-mode and Doppler echo revealed a "stiletto"-shaped mitral regurgitation profile typical of acute mitral valve insufficiency. The transesophageal echocardiogram showed a distinct mass moving between the left ventricle and left atrium, diagnostic of papillary muscle rupture. Despite of shock, mitral valve replacement was performed successfully. To our knowledge, this is the first report of a papillary muscle rupture during exercise testing after myocardial infarction. Papillary muscle rupture can be induced by exercise. This fateful event may not be predicted by the course of the ergometry. In case of hypotension during exercise, papillary muscle rupture should be considered. The diagnosis is to be established by transesophageal echocardiography.

Influenza hemagglutinin mediated fusion of membranes containing poly(ethylene-glycol) grafted lipids: new insights into the fusion mechanism.

The influence of a hydrophilic layer covering the membrane on influenza hemagglutinin (HA) mediated fusion was investigated using membranes containing poly(ethylene-glycol) grafted phosphatidylethanolamine (PEG-2000-PE). Steric inhibition of HA-membrane interactions by these lipids affected virus fusion (half-maximal inhibition at 0.8 mol% for lipids with 114 ethylene glycol residues, or at 3.2 mol% for 45 residues (PEG-2000-PE), concentrations at which the PEG moieties adopt a random coil structure). Reconstituted viral membranes containing 3 mol% PEG-2000-PE retained 40% of their fusion activity. Therefore, efficient fusion is possible with membranes completely covered by a hydrophilic layer of several nanometers, and fusogenic virosomes containing PEG-PE are feasible.

New approaches to coronary heart disease: induction of neovascularisation by growth factors.

Currently available approaches for treating human coronary heart disease aim to relieve symptoms and the risk of myocardial infarction by reducing myocardial oxygen demand (drugs), preventing further disease progression (drugs), restoring coronary blood flow either pharmacologically (thrombolysis) or mechanically (angioplasty), or bypassing the stenotic lesions and obstructed coronary artery segments (surgery). Direct gene therapy, as well as gene-derived therapy, especially by angiogenic growth factors, is emerging as a potential new treatment for cardiovascular disease. After extensive experimental research on angiogenic growth factors, the first clinical studies on patients with coronary heart disease or peripheral vascular lesions are being performed. The polypeptides fibroblast growth factor (FGF) and vascular endothelial growth factor seem to be effective in initiating neovascularisation (neo-angiogenesis) in hypoxic or ischaemic tissues. The first clinical study on patients with coronary heart disease treated by local injection of FGF-1 into the compromised underperfused myocardial tissue showed a 3-fold increase of capillary density mediated by the growth factor. Angiogenic therapy of the human myocardium introduces a new modality of treatment for coronary heart disease in terms of regulation of blood vessel growth. Beyond drug therapy, angioplasty and bypass surgery, this therapy may evolve to be a fourth principle of treatment of atherosclerotic cardiovascular disease.

Induction of neoangiogenesis in ischemic myocardium by human growth factors: first clinical results of a new treatment of coronary heart disease.

BACKGROUND: The present article is a report of our animal experiments and also of the first clinical results of a new treatment for coronary heart disease using the human growth factor FGF-I (basic fibroblast growth factor) to induce neoangiogenesis in the ischemic myocardium. METHODS AND RESULTS: FGF-I was obtained from strains of Escherichia coli by genetic engineering, then isolated and highly purified. Several series of animal experiments demonstrated the apathogenic action and neoangiogenic potency of this factor. After successful conclusion of the animal experiments, it was used clinically for the first time. FGF-I (0.01 mg/kg body weight) was injected close to the vessels after the completion of internal mammary artery (IMA)/left anterior descending coronary artery (LAD) anastomosis in 20 patients with three-vessel coronary disease. All the patients had additional peripheral stenoses of the LAD or one of its diagonal branches. Twelve weeks later, the IMA bypasses were selectively imaged by intra-arterial digital subtraction angiography and quantitatively evaluated. In all the animal experiments, the development of new vessels in the ischemic myocardium could be demonstrated angiographically. The formation of capillaries could also be demonstrated in humans and was found in all cases around the site of injection. A capillary network sprouting from the proximal part of the coronary artery could be shown to have bypassed the stenoses and rejoined the distal parts of the vessel. CONCLUSIONS: We believe that the use of FGF-I for myocardial revascularization is in principle a new concept and that it may be particularly suitable for patients with additional peripheral stenoses that cannot be revascularized surgically.

The stimulation of neoangiogenesis in the ischemic human heart by the growth factor FGF: first clinical results.

BACKGROUND: This paper is a report of our clinical experience with the human growth factor FGF as applied to the ischemic human myocardium. METHODS: After the completion of extensive preliminary animal experiments, the growth factor FGF, obtained from genetically manipulated E. coli bacteria and highly purified, was introduced into aortocoronary bypass surgery as an additional therapeutic agent. A double blind study was carried out on 40 patients with CHD, separated into "growth factor" and control groups, each containing 20 members. All the patients were treated for threefold vascular disease, in each case with an IMA bypass for the LAD and single venous bypasses for the RCX and/or RCA. In order to bridge over additional peripheral stenoses in the LAD or one of its branches, human growth factor FGF was injected into the myocardium of those in the growth factor group. Twelve weeks later, the IMA bypasses were selectively demonstrated by intraarterial DSA. These angiographs were then quantitatively evaluated. RESULTS: In all patients of the growth factor group, the formation of new vessels could be demonstrated in the region where FGF had been administered, in a manner strictly reminiscent of our experimental results. A capillary net sprouting from the coronary artery and making further connection with this vessel could be demonstrated, and the computer-supported evaluation of the angiographs showed a significant increase in the blood supply of the region of the myocardium injected. CONCLUSIONS: It is therefore our opinion that employment of the human growth factor FGF represents a useful extension to bypass surgery, particularly for patients with an additional peripheral stenosis that cannot be operatively revascularized.

FGF-1: a human growth factor in the induction of neoangiogenesis.

Currently available approaches for treating human coronary heart disease aim to relieve symptoms and the risk of myocardial infarction either by reducing myocardial oxygen demand, preventing further disease progression, restoring coronary blood flow pharmacologically or mechanically, or bypassing the stenotic lesions and obstructed coronary artery segments. Gene therapy, especially using angiogenic growth factors, has emerged recently as a potential new treatment for cardiovascular disease. Following extensive experimental research on angiogenic growth factors, the first clinical studies on patients with coronary heart disease and peripheral vascular lesions have been performed. The polypeptides fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) appear to be particularly effective in initiating neovascularisation (neo-angiogenesis) in hypoxic or ischaemic tissues. The first clinical study on patients with coronary heart disease treated by local intramyocardial injection of FGF-1 showed a 3-fold increase of capillary density mediated by the growth factor. Angiogenic therapy of the human myocardium introduces a new modality of treatment for coronary heart disease in terms of regulation of blood vessel growth. Beyond drug therapy, angioplasty and bypass surgery, this new approach may evolve into a fourth principle of treatment of atherosclerotic cardiovascular disease.

Pores formed by influenza hemagglutinin.

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves a conformational change in the protein that leads to the insertion of a "fusion peptide" of the protein into the target membrane. It has been suggested that this insertion, aided by the formation of a complex of multiple HA trimers, would lead to perturbation of the bilayer structure of the membrane, initiating fusion. Here we present data showing that the interaction of the bromelain released ectodomain of the protein (BHA) with liposomal membranes at low pH leads to pore formation, at least at low temperatures. Strongly temperature-dependent low pH-induced inactivation of BHA resulted in a complete lack of activity of BHA above 10 degrees C. Even at 0 degrees C, only about 5% of the BHA participated in pore formation. Viral HA was less rapidly inactivated and still induced pores at 37 degrees C. BHA-induced pore formation showed a sigmoidal time course. Once BHA had formed a pore in one liposome, it did not form a pore in a further liposome. Quantitative analysis of pore formation indicated that one single BHA trimer sufficed to produce a pore. These data indicate that fusion peptide insertion perturbs the membrane and that the formation of a complex of trimers is not a prerequisite for the perturbation.

How lysophosphatidylcholine inhibits cell-cell fusion mediated by the envelope glycoprotein of human immunodeficiency virus.

We have investigated the effect of lysophosphatidylcholine (LPC) on fusion of cells expressing the envelope glycoprotein of HIV-1, gp120/gp41, with cells expressing the receptor for this virus, CD4. Fusion was inhibited by micromolar concentrations of LPC added from an aqueous stock solution, but not by membrane-associated LPC or LPC produced in the membrane by the action of phospholipase A2. No inhibition was found for LPCs with acyl chain lengths shorter than 12 carbon atoms, and the inhibitory effect of longer molecules increased with their length. gp120-CD4 specific cell-cell binding was inhibited with a chain length dependence corresponding to that of fusion, but with palmitoyl-LPC, fusion was more strongly affected than binding. These data indicate that gp120/gp41-induced fusion is inhibited by LPC because LPC affects viral protein-host cell binding and not because LPC prevented the formation of lipid intermediates required for fusion.

Gene transfer mediated by cationic lipids: lack of a correlation between lipid mixing and transfection.

Complexes of DNA with cationic lipids are used to transfect eukaryotic cells. The mechanism of transfection is unknown, but it has been suggested that the complexes are taken up into the cell by endocytosis, after which fusion of the cationic lipids with the membranes of intracellular vesicles would allow the DNA to escape into the cytoplasm. Here, we have compared transfection of CHO-K1 cells with lipid mixing measured by fluorescence assays, using liposomes or complexes with plasmid DNA of the cationic lipids 1,2 dioleolyl-3-N, N, N,-trimethylammonium-propane (DOTAP), N-[2,3-(dioleoyloxy)propyl]-N, N, N,-trimethylammonium (DOTMA), or combinations of these lipids with dioleoylphosphatidylethanolamine (DOPE), at various lipid/DNA charge ratios. Mixing of the lipids of the complexes or liposomes with cellular membranes occurred readily at 37 degrees C, and was more efficient with liposomes than with complexes. Lipid mixing was inhibited at low temperatures (0-17 degrees C), by the presence of NH(4)Cl in the medium, and by low extracellular pH, indicating the involvement of the endocytic pathway in entry. In the absence of DOPE, there was no correlation between the efficiency of lipid mixing and the efficiency of transfection. Moreover, although DOPE, which is thought to promote membrane fusion, enhanced transfection, it did not always enhance lipid mixing. Neither the size nor the zeta potential of the complexes were clearly associated with transfection efficiency. Therefore, although fusion between the lipids of the complexes and cellular membranes takes place, a step at a later stage in the transfection process determines the efficiency of transfection.

Kinetics of fusion between endoplasmic reticulum vesicles in vitro.

The endoplasmic reticulum (ER) is a highly dynamic organelle, continuously undergoing membrane fusion and fission. We have measured homotypic fusion between ER vesicles isolated from Chinese hamster ovary cells kinetically in vitro, using an assay based on the metabolic incorporation of pyrene-labeled fatty acids into the phospholipids of cellular membranes. An increase in pyrene-monomer fluorescence was observed after mixing labeled and unlabeled ER vesicles in the presence of ATP and GTP. The protein, temperature, and nucleotide dependence of the increase indicated that it was caused by membrane fusion rather than molecular transfer of labeled lipids to unlabeled membranes. This assay allowed the first kinetic measurements with virtually nonexchangeable probes of a homotypic membrane fusion event. At 37 degrees C, fusion started off immediately at a rate of 1.14 +/- 0.29%/min and reached a half-maximal level after 56 min. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), or after treatment of the membranes with N-ethylmaleimide, fusion was reduced but not completely inhibited. Addition of GTP during a fusion reaction immediately accelerated, and GTPgammaS immediately slowed down the fusion reaction. Thus, these kinetic measurements indicate that G-proteins might act to rapidly enhance fusion beyond a basic level.