Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement.

OBJECTIVE Short-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme that is involved in the oxidation of fatty acids and essential amino acids such as valine. Here, we describe the broad phenotypic spectrum and pathobiochemistry of individuals with autosomal-recessive ECHS1 deficiency. METHODS Using exome sequencing, we identified ten unrelated individuals carrying compound heterozygous or homozygous mutations in ECHS1. Functional investigations in patient-derived fibroblast cell lines included immunoblotting, enzyme activity measurement, and a palmitate loading assay. RESULTS Patients showed a heterogeneous phenotype with disease onset in the first year of life and course ranging from neonatal death to survival into adulthood. The most prominent clinical features were encephalopathy (10/10), deafness (9/9), epilepsy (6/9), optic atrophy (6/10), and cardiomyopathy (4/10). Serum lactate was elevated and brain magnetic resonance imaging showed white matter changes or a Leigh-like pattern resembling disorders of mitochondrial energy metabolism. Analysis of patients' fibroblast cell lines (6/10) provided further evidence for the pathogenicity of the respective mutations by showing reduced ECHS1 protein levels and reduced 2-enoyl-CoA hydratase activity. While serum acylcarnitine profiles were largely normal, in vitro palmitate loading of patient fibroblasts revealed increased butyrylcarnitine, unmasking the functional defect in mitochondrial β-oxidation of short-chain fatty acids. Urinary excretion of 2-methyl-2,3-dihydroxybutyrate - a potential derivative of acryloyl-CoA in the valine catabolic pathway - was significantly increased, indicating impaired valine oxidation. INTERPRETATION In conclusion, we define the phenotypic spectrum of a new syndrome caused by ECHS1 deficiency. We speculate that both the β-oxidation defect and the block in l-valine metabolism, with accumulation of toxic methacrylyl-CoA and acryloyl-CoA, contribute to the disorder that may be amenable to metabolic treatment approaches.

Markers of endothelial cell activation and immune activation are increased in patients with severe leptospirosis and associated with disease severity

OBJECTIVES Previous studies concluded that haemorrhage is one of the most accurate prognostic factors of mortality in leptospirosis. Therefore, endothelial cell activation was investigated in relation to disease severity in severe leptospirosis. METHODS Prospective cohort study of severe leptospirosis patients. Plasma levels of sE-selectin and Von Willebrand factor (VWF) were determined. Consequently, an in vitro endothelial cell model was used to assess endothelial activation after exposure to virulent Leptospira. Finally, immune activation, as a potential contributing factor to endothelial cell activation, was determined by soluble IL2-receptor (sIL-2r) and soluble Fas-ligand (sFasL) levels. RESULTS Plasma levels of sE-selectin and VWF strongly increased in patients compared to healthy controls. Furthermore, sE-selectin was significantly elevated (203 ng/ml vs. 157 ng/ml, p < 0.05) in survivors compared to non-survivors. Endothelial cells exposed to virulent Leptospira showed increased VWF expression. E-selectin and ICAM-1 expression did not change. Immunohistochemistry revealed the presence of intracellular Leptospira and qPCR suggested replication. In vivo analysis showed that increased levels of sFasL and sIL-2r were both strongly associated with mortality. Furthermore sIL-2r levels were increased in patients that developed bleeding and significantly correlated to duration of hospital stay. DISCUSSION Markers of endothelial activation and immune activation were associated with disease severity in leptospirosis patients.

Investigation of hemodynamics in an in vitro system simulating left ventricular support through the right subclavian artery using 4-dimensional flow magnetic resonance imaging.

OBJECTIVES Left ventricular assist devices are an important treatment option for patients with heart failure alter the hemodynamics in the heart and great vessels. Because in vivo magnetic resonance studies of patients with ventricular assist devices are not possible, in vitro models represent an important tool to investigate flow alterations caused by these systems. By using an in vitro magnetic resonance-compatible model that mimics physiologic conditions as close as possible, this work investigated the flow characteristics using 4-dimensional flow-sensitive magnetic resonance imaging of a left ventricular assist device with outflow via the right subclavian artery as commonly used in cardiothoracic surgery in the recent past. METHODS An in vitro model was developed consisting of an aorta with its supra-aortic branches connected to a left ventricular assist device simulating the pulsatile flow of the native failing heart. A second left ventricular assist device supplied the aorta with continuous flow via the right subclavian artery. Four-dimensional flow-sensitive magnetic resonance imaging was performed for different flow rates of the left ventricular assist device simulating the native heart and the left ventricular assist device providing the continuous flow. Flow characteristics were qualitatively and quantitatively evaluated in the entire vessel system. RESULTS Flow characteristics inside the aorta and its upper branching vessels revealed that the right subclavian artery and the right carotid artery were solely supported by the continuous-flow left ventricular assist device for all flow rates. The flow rates in the brain-supplying arteries are only marginally affected by different operating conditions. The qualitative analysis revealed only minor effects on the flow characteristics, such as weakly pronounced vortex flow caused by the retrograde flow via the brachiocephalic artery. CONCLUSIONS The results indicate that, despite the massive alterations in natural hemodynamics due to the retrograde flow via the right subclavian and brachiocephalic arteries, there are no drastic consequences on the flow in the brain-feeding arteries and the flow characteristics in the ascending and descending aortas. It may be beneficial to adjust the operating condition of the left ventricular assist device to the residual function of the failing heart.

Conditioned medium from fresh and demineralized bone enhances osteoclastogenesis in murine bone marrow cultures

OBJECTIVES Osteoclasts rapidly form on the surface of bone chips at augmentation sites. The underlying molecular mechanism, however, is unclear. Soluble factors released from bone chips in vitro have a robust impact on mesenchymal cell differentiation. Whether these soluble factors change the differentiation of hematopoietic cells into osteoclasts remains unknown. METHODS Osteoclastogenesis, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells, was studied with murine bone marrow cultures exposed to RANKL and M-CSF, and conditioned medium from fresh (BCM) and demineralized bone matrix (DCM). Histochemical staining, gene and protein expression, as well as viability assays were performed. RESULTS This study shows that BCM had no impact on osteoclastogenesis. However, when BCM was heated to 85°C (BCMh), the number of tartrate-resistant acid phosphatase-positive multinucleated cells that developed in the presence of RANKL and M-CSF approximately doubled. In line with the histochemical observations, there was a trend that BCMh increased expression of osteoclast marker genes, in particular the transcription factor c-fos. The expression of c-fos was significantly reduced by the TGF-β receptor I antagonist SB431542. DCM significantly stimulated osteoclastogenesis, independent of thermal processing. CONCLUSIONS These data demonstrate that activated BCM by heat and DBM are able to stimulate osteoclastogenesis in vitro. These in vitro results support the notion that the resorption of autografts may be supported by as yet less defined paracrine mechanisms.

The use of plant-based bioassays in homeopathic basic research.

OBJECTIVES The objective was to evaluate homeopathic basic research studies that use plant-based bioassays. With this in view, a compilation was made of the findings of three systematic literature reviews covering plant-based bioassays in the three fields of healthy, abiotically, or biotically stressed plants. This compilation focused on investigations using advanced experimental methods and detailed descriptions, also with the aim of supporting the design of future experiments. METHODS Publications included had to report on studies into the effects of homeopathic preparations on whole plants, seeds, plant parts and cells. Outcomes had to be measured by established procedures and statistically evaluated. A Manuscript Information Score (MIS) was applied using predefined criteria to identify publications with sufficient information for adequate interpretation (MIS ≥ 5). Additional evaluation focused on the use of adequate controls to investigate specific effects of homeopathic preparations, and on the use of systematic negative control (SNC) experiments to ensure the stability of the bioassay. Only a fraction of the studies reported here were performed with 'ultra high' dilutions, whereas other studies were performed with moderate or high dilutions. RESULTS A total of 157 publications were identified, describing a total of 167 experimental studies. 84 studies included statistics and 48 had a MIS ≥ 5, thus allowing adequate interpretation. 29 studies had adequate controls to identify specific effects of homeopathic preparations, and reported significant effects of decimal and centesimal homeopathic potencies, including dilution levels beyond Avogadro's number. 10 studies reported use of SNC experiments, yielding evidence for the stability of the experimental set-up. CONCLUSION Plant models appear to be a useful approach for investigating basic research questions relating to homeopathic preparations, but more independent replication trials are needed in order to verify the results found in single experiments. Adequate controls and SNC experiments should be implemented on a routine basis to exclude false-positive results.

Update on food allergy

Food allergies are a global health issue with increasing prevalence. Allergic reactions can range from mild local symptoms to severe anaphylactic reactions. Significant progress has been made in diagnostic tools such as component-resolved diagnostics and its impact on risk stratification as well as in therapeutic approaches including biologicals. However, a cure for food allergy has not yet been achieved and patients and their families are forced to alter eating habits and social engagements, impacting their quality of life. New technologies and improved in vitro and in vivo models will advance our knowledge of the pathogenesis of food allergies and multicenter-multinational cohort studies will elucidate interactions between genetic background, lifestyle, and environmental factors. This review focuses on new insights and developments in the field of food allergy and summarizes recently published articles.

Placental expression of the angiogenic placental growth factor is stimulated by both aldosterone and simulated starvation

Aldosterone is an important factor supporting placental growth and fetal development. Recently, expression of placental growth factor (PlGF) has been observed in response to aldosterone exposure in different models of atherosclerosis. Thus, we hypothesized that aldosterone up-regulates growth-adaptive angiogenesis in pregnancy, via increased placental PlGF expression. We followed normotensive pregnant women (n = 24) throughout pregnancy and confirmed these results in a second independent first trimester cohort (n = 36). Urinary tetrahydroaldosterone was measured by gas chromatography-mass spectrometry and corrected for creatinine. Circulating PlGF concentrations were determined by ELISA. Additionally, cultured cell lines, adrenocortical H295R and choriocarcinoma BeWo cells, as well as primary human third trimester trophoblasts were tested in vitro. PlGF serum concentrations positively correlated with urinary tetrahydroaldosterone corrected for creatinine in these two independent cohorts. This observation was not due to PlGF, which did not induce aldosterone production in cultured H295R cells. On the other hand, PlGF expression was specifically enhanced by aldosterone in the presence of forskolin (p < 0.01) in trophoblasts. A pronounced stimulation of PlGF expression was observed with reduced glucose concentrations simulating starvation (p < 0.001). In conclusion, aldosterone stimulates placental PlGF production, enhancing its availability during human pregnancy, a response amplified by reduced glucose supply. Given the crucial role of PlGF in maintaining a healthy pregnancy, these data support a key role of aldosterone for a healthy pregnancy outcome.