A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity

BACKGROUND: Despite the availability of efficacious drugs, the success of treating hypertension is limited by patients' inconsistent drug intake. Immunization against angiotensin II may offer a valuable alternative to conventional drugs for the treatment of hypertension, because vaccines induce relatively long-lasting effects and do not require daily dosing. Here we describe the preclinical development and the phase I clinical trial testing of a virus-like particle (VLP)-based antihypertensive vaccine. METHODS AND RESULTS: An angiotensin II-derived peptide was conjugated to the VLP Qbeta (AngQb). AngQb was highly immunogenic in mice and rats. To test for efficacy, spontaneously hypertensive rats (SHR) were immunized with 400 microg AngQb or VLP alone. Group mean systolic blood pressure (SBP) was reduced by up to 21 mmHg (159 +/- 2 versus 180 +/- 5 mmHg, P < 0.001), and total angiotensin II levels (antibody-bound and free) were increased ninefold (85 +/- 20 versus 9 +/- 1 pmol/l, P = 0.002) compared with VLP controls. SHR treated with the angiotensin-converting enzyme (ACE) inhibitor ramipril (1 mg/kg per day by mouth) reached an SBP of 155 +/- 2 mmHg. Twelve healthy volunteers of a placebo-controlled randomized phase I trial were injected once with 100 microg AngQb. Angiotensin II-specific antibodies were raised in all subjects (100% responder rate) and AngQb was well tolerated. CONCLUSIONS: AngQb reduces blood pressure in SHR to levels obtained with an ACE inhibitor, and is immunogenic and well tolerated in humans. Therefore, vaccination against angiotensin II has the potential to become a useful antihypertensive treatment providing long-lasting effects and improving patient compliance.     

Advances in musculoskeletal imaging and their clinical utility in the early diagnosis of spondyloarthritis

Interest in imaging ankylosing spondylitis (AS) and related spondyloarthropathies has increased in recent years. MRI is regarded as the most sensitive imaging modality to detect early inflammatory lesions in the sacroiliac joints and in the spine. Standard radiography plays a major role in evaluating potential disease-modifying properties of the recently introduced, symptomatically effective anti-tumor necrosis factor-α agents. Various radiographic scoring methods have been developed to assess and monitor spinal structural damage in AS. The modified stoke ankylosing spondylitis spine score is the method of choice based on its sensitivity to change. MRI is emerging as a useful tool to detect AS early, but this remains a challenge. Recent progress in MR technology with the advent of whole body MRI has expanded the potential role of imaging in AS. Further data are needed before MRI can replace radiographs for earlier diagnosis of AS.     

Consequence of parvalbumin deficiency in the mdx mouse: histological,biochemical and mechanical phenotype of a new double mutant

We tested the hypothesis whether the mild dystrophy in mdx mice could result from the contribution of the cytosolic calcium buffer parvalbumin in maintaining a normal cytosolic [Ca2+]i, in spite of an increased passive Ca2+ influx. By crossing mdx mice with parvalbumin-deficient mice, double mutant mice, lacking both dystrophin and parvalbumin, were obtained. Though resting cytosolic [Ca2+]i and total calcium content were similar to that of mdx muscles, this new animal model presented a slightly more severe phenotype than the mdx mouse. Muscle pseudo-hypertrophy, the density of myotubes and of centronucleated fibres as well as the loss of IIB fibres were all increased in parvalbumin-deficient mdx mice. Many of these deficits were overcome in late adulthood, albeit fibrosis was clearly more pronounced than in mdx muscles. At 90 days, parvalbumin-deficient mdx mice showed higher levels of creatine phosphokinase and lower muscle strength, in vivo, than mdx mice. Isometric tension of isolated muscle was reduced, but the susceptibility to eccentric contraction was not increased. The slight aggravation of muscle dystrophy observed in mdx mice deprived of parvalbumin cannot explain the severity of the affection observed in xmd dogs and Duchenne dystrophy patients where parvalbumin is constitutively not expressed.     

Valvular Heart Disease: Assessment of Valve Morphology and Quantification Using MR

For clinical evaluation and decision-making in patients with valvular heart disease, the diagnostic armamentarium expands steadily. This evolution makes it difficult to choose the most appropriate approach for a specific valvular lesion. It may also reflect our uncertainty of what are the findings that best predict clinical outcome of patients, e. g. after surgery. Accordingly, for each type of valvular lesion, some pathopysiological considerations are stated in order to derive the most important measures that would allow optimal guidance of patients. Based on the considerations the value of an MR study is discussed for each valvular lesion. Newest advances in MR technology allow for highly accurate measurements of regurgitant volumes and hence, MR may be the method of choice of a quantitative evaluation of regurgitant valves. For assessment of stenosis severity, measurement of transvalvular pressure gradient is an appropriate measure and MR may not confer benefits over echocardiography, provided the ultrasound window is adequate (and stroke volume is in the normal range). With respect to surgical treatment, valvular morphology is of pivotal importance, particularly for the mitral valve, and echocardiography still appears to be the first line method. Little data relate lesion severity and/or morphology to clinical outcome. Conversely, the extent of cardiac adaptation to pressure- and/or volume-overload, i. e. ventricular remodeling, is a strong predictor of outcome, ad is therefore most important for final judgement of the patient. For assessment of left and right ventricular remodeling, echocardiography typically provides all the necessary information. However, in special cases with discrepant findings, with inadequate ultrasound window, or in the preoperative work-up, MR may provide important information regarding cardiac adaptation to valvular lesion. Zusammenfassung Das diagnostische Repertoire zur Evaluation von Klappenvitien ist in den letzten Jahren stetig ausgebaut und verfeinert worden. Dennoch bleiben viele Fragen offen, wenn es um die prognostische Wertigkeit der verschiedenen Messparameter geht. Im Folgenden werden deshalb für jede Klappenerkrankung einige pathophysiologische Überlegungen angestellt, aufgrund derer die Bedeutung der verschiedenen Messparameter erläutert wird. In diesem Kontext wird für jede Klappenerkrankung der Stellenwert der Magnetresonanz-(MR-)Diagnostik diskutiert. Neueste MR-Entwicklungen werden vorgestellt, die eine genaue Quantifizierung von Regurgitationsvolumina bei Aorten- und Mitralinsuffizienzen erlauben. Bei der Frage nach dem Schweregrad von Klappeninsuffizienzen dürfte die MR-Untersuchung als Methode der Wahl gelten. Bei einem Klappenvitium ist zwar der Schweregrad von Insuffizienz und/oder Stenose wichtig, das Adaptationsvermögen der Herzkammern an die Volumen-/Druckbelastung ist aber von entscheidender Bedeutung bezüglich Prognose und Operationsindikation. So wird bei Patienten mit schwerer Aorteninsuffizienz der Aortenklappenersatz empfohlen, wenn eine eingeschränkte linksventrikuläre Funktion und/oder eine ausgeprä,gte linksventrikuläre Dilatation nachgewiesen und mittels einer zweiten Untersuchung (Echokardiographie, MR, Radionuklidventrikulographie) bestätigt wird (Richtlinien der American Heart Association/American College of Cardiology [AHA/ACC]). Bei der Aortenstenose mit erhaltener linksventrikulärer Funktion erlaubt die Messung des mittleren transvalvulären Druckgradienten eine zuverlässige Beurteilung des Schweregrades der Stenose. Bei dieser Fragestellung dürfte die MR-Untersuchung gegenü,ber der Echokardiographie kaum Vorteile aufweisen, insbesondere, wenn für die Echokardiographie ein adäquates Schallfenster vorliegt. Während symptomatische Patienten mit schwerer Aortenstenose grundsätzlich einer Klappenoperation zugeführt werden sollten, gilt es bei asymptomatischen Patienten diejenigen zu identifizieren, die eine eingeschränkte Langzeitprognose aufzuweisen, das heißt Patienten mit einer eingeschränkten linksventrikulären Funktion und/oder einer exzessiven linksventrikulären Hypertrophie. Asymtomatische Patienten mit schwerer Aortenstenose sollten deshalb gemäß Richtlinien der AHA/ACC regelmäßig bezüglich linksventrikulärer Funktion und linksventrikulärer Hypertrophie mittels Echokardiographie oder MR kontrolliert werden. Bei der Mitralinsuffizienz zeigt die Klappenrekonstruktion dann besonders günstige Resultate, wenn diese in einem frühen Stadium der Erkrankung durchgeführt wird, das heißt bei asymptomatischen oder nur leicht symptomatischen Patienten. In dieser Situation erlangen die Quantifizierung der Mitralinsuffizienz und die genaue Erfassung der linksventrikulären Funktion und der linksventrikulären Dimensionen eine zentrale Bedeutung, wenn der optimale Zeitpunkt für die operative Sanierung festgelegt werden soll. Die Indikationsstellung basiert dabei auf 1. der Quantifizierung der Mitralinsuffizienz, 2. der Beurteilung der linksventrikulären Adaptation an der Volumenbelastung und 3. der Klappenmorphologie. Die MR-Methode erlaubt eine Quantifizierung der Mitralinsuffizienz und der linksventrikulären Adaptation und ermöglicht dadurch, eine Progression der Erkrankung frühzeitig zu erkennen. Für die Beurteilung der Klappenmorphologie hingegen ist nach wie vor die Echokardiographie die Methode der Wahl. Bei der Mitralstenose vermag die Echokardiographie die wesentlichen Informationen bezüglich Klappenmorphologie und -funktion zu liefern und erfasst ebenfalls die Adaptation des Lungenkreislaufs. Hier kann die MR-Untersuchung allenfalls Zusatzinformationen liefern, wenn es darum geht, eine etwaige Begleitinsuffizienz zu quantifizieren. Zusammenfassend kann gesagt werden, dass die MR-Untersuchung eine exakte Quantifizierung von Klappeninsuffizienzen erlaubt sowie eine umfassende Beurteilung der entsprechenden Adaptationsvorgänge (linksventrikuläre Funktion, linksventrikuläre Hypertrophie, linksventrikuläres Remodeling). Der Schweregrad von Stenosen kann mittels des Druckgradienten bestimmt werden ähnlich der Echokardiographie, während die Klappenmorphologie vorteilhaft mittels Echokardiographie erfasst wird. Bei ungünstigem Schallfenster und/oder bei diskrepanten Befunden in der Echokardiographie kann die MR-Untersuchung wertvolle Zusatzinformationen liefern, insbesondere bei Vorliegen von Klappeninsuffizienzen.     

Do MRI findings correlate with mobility tests? An explorative analysis of the test validity with regard to structure

To find out whether segmental magnetic resonance imaging (MRI) findings such as intervertebral disc degeneration (DD) and facet joint osteoarthritis (FJO) are associated with motion deficiencies as seen in common mobility tests and observed range of motion (ROM). A total of 112 female subjects, nurses and office workers, with and without low back pain, were examined by clinical experts, and lumbar mobility was measured including modified Schober, fingertip-to-floor distance (FTFD) and ZEBRIS motion analysis. An MRI of the lumbar spine was made. Mobility findings were correlated with segmental morphologic changes as seen on MRI at the levels of L1-2 through L5-S1. Only a few statistically significant correlations between MRI findings and the results of the mobility tests could be found. Lateral bending was weakly and negatively correlated to DD and FJO but only on the level of L5-S1. The FTFD showed a weak positive correlation to endplate changes on the level of L4-5. When ROM is observed by clinical experts, there are several significant relationships between MRI findings and the observed motion. There is a highly significant segmental correlation between DD and disc form alteration as seen on MRI on the level of single motion segments. Pain history and current pain level did not moderate any association between MRI and mobility. There is no clear relationship between the structural changes represented by MRI and the measured mobility tests used in this study. Our findings suggest that close observation of spinal motion may provide at least equal information about the influence of spinal structures on motion than the commonly used measured mobility tests do.     

EF-hand protein Ca2+ buffers regulate Ca2+ influx and exocytosis in sensory hair cells

EF-hand Ca²⁺-binding proteins are thought to shape the spatiotemporal properties of cellular Ca²⁺ signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca²⁺ signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca²⁺-dependent inactivation of their Ca²⁺ current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca²⁺ channels and release sites (effective “coupling distance” of 17 nm). Substitution experiments with synthetic Ca²⁺ chelators indicated the presence of endogenous Ca²⁺ buffers equivalent to 1 mM synthetic Ca²⁺-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca²⁺ buffers regulate presynaptic IHC function for metabolically efficient sound coding.Intracellular Ca²⁺ signaling regulates a multitude of cellular processes. In sensory hair cells, Ca²⁺ is crucial for electrical frequency tuning, afferent synaptic transmission, and efferent modulation (reviewed in ref. 1). To separate these signaling pathways and maintain high temporal fidelity of neurotransmission, Ca²⁺ signals must be temporally limited and spatially confined to the site of action. Cells typically achieve this by localizing Ca²⁺ entry and by rapidly removing free Ca²⁺ ions via binding to cytosolic “buffers” and finally Ca²⁺ extrusion (2–4). Of the various EF-hand Ca²⁺-binding proteins, some seem to function primarily as Ca²⁺-dependent signaling proteins (e.g., calmodulin and Ca²⁺-binding proteins 1–8, CaBP1–8), whereas others [parvalbumin-α (PVα), calbindin-D28k (CB), and calretinin (CR)] are thought to mainly serve as mobile Ca²⁺ buffers.Hair cells of various species strongly express the Ca²⁺-binding proteins PV, CB, and, in some cases, CR. This possibly reflects the need for buffers with different biophysical properties to functionally isolate different Ca²⁺ signaling mechanisms, which are spatially not well separated in these compact epithelial cells. Ca²⁺-binding proteins are particularly abundant in frog and chicken hair cells, which contain millimolar concentrations of parvalbumin-3 (5) as well as of CR (6, 7). An immune-EM study in rats indicated hundreds of micromolar of proteinaceous Ca²⁺-binding sites in inner hair cells (IHCs) (8). A patch-clamp study in gerbil IHCs reported endogenous buffers equivalent to approximately 0.4 mM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (9). Genetic deletion of the EF-hand Ca²⁺-binding proteins PVα, CB, and CR in mice has facilitated the analysis of their function (10–13; reviewed in ref. 14), but the combined deletion of these proteins remains to be studied. IHCs provide an experimentally well-accessible presynaptic preparation that uses all three. Here, we studied IHC function and hearing in mice lacking the three buffers [triple buffer KO (TKO); Pv^−/−Cb^−/−Cr^−/−]. By using perforated and ruptured-patch recordings, we analyzed voltage-gated Ca²⁺ currents and exocytosis of Pv^−/−Cb^−/−Cr^−/− IHCs, in which we also substituted the deleted endogenous buffers with the synthetic Ca²⁺ chelators EGTA or BAPTA. Auditory systems function was probed by measuring otoacoustic emissions and auditory brainstem responses (ABRs) as well as by recordings from single spiral ganglion neurons (SGNs). We performed mathematical modeling to estimate concentrations of the endogenous mobile Ca²⁺ buffers and to better understand how these proteins control exocytosis at IHC synapses. We conclude that the endogenous buffer capacity of IHCs is well approximated by 1 mM synthetic Ca²⁺-binding sites with different kinetics. A tight spatial coupling between Ca²⁺ channels and sensors of exocytosis (Ca²⁺ channel-exocytosis coupling) precludes interference of PVα, CB, and CR with fusion of the readily releasable pool of vesicles (RRP). Instead, we suggest that these buffers jointly regulate IHC presynaptic function by restricting neurotransmitter release to active zones (AZs).