Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

<p>Abstract</p> <p>Background</p> <p>Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.</p> <p>Methods</p> <p>Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.</p> <p>Results</p> <p>Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.</p> <p>Conclusions</p> <p>The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy.</p

Stereotypical Chronic Lymphocytic Leukemia B-Cell Receptors Recognize Survival Promoting Antigens on Stromal Cells

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as “subset 1”) recognize antigens highly expressed in stromal cells – vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20–45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease

PKG1α oxidation negatively regulates food seeking behaviour and reward

Genes that are highly conserved in food seeking behaviour, such as protein kinase G (PKG), are of interest because of their potential role in the global obesity epidemic. PKG1α can be activated by binding of cyclic guanosine monophosphate (cGMP) or oxidant-induced interprotein disulfide bond formation between the two subunits of this homodimeric kinase. PKG1α activation by cGMP plays a role in reward and addiction through its actions in the ventral tegmental area (VTA) of the brain. ‘Redox dead’ C42S PKG1α knock-in (KI) mice, which are fully deficient in oxidant- induced disulfide-PKG1α formation, display increased food seeking and reward behaviour compared to wild-type (WT) littermates. Rewarding monoamines such as dopamine, which are released during feeding, are metabolised by monoamine oxidase to generate hydrogen peroxide that was shown to mediate PKG1α oxidation. Indeed, inhibition of monoamine oxidase, which prevents it producing hydrogen peroxide, attenuated PKG1α oxidation and increased sucrose preference in WT, but not KI mice. The deficient reward phenotype of the KI mice was rescued by expressing WT kinase that can form the disulfide state in the VTA using an adeno-associated virus, consistent with PKG1α oxidation providing a break on feeding behaviour. In conclusion, disulfide-PKG1α in VTA neurons acts as a negative regulator of feeding and therefore may provide a novel therapeutic target for obesity

Quantitation of serum IgE by using chimeras of human IgE receptor and avian immunoglobulin domains

Anti-IgE therapeutics represent an efficient approach in the management of IgE-mediated allergic asthma. However, monitoring the reduction of IgE levels into a therapeutically efficient range requires the determination of residual serum IgE. We established an analytical approach to distinguish free and anti-IgE complexed serum IgE based on soluble derivatives of the human high-affinity IgE receptor. Soluble receptor derivatives represent an ideal means to analyze receptor antagonism by any ligand or blocking antibody. Therefore, the FcRI ectodomain was fused with avian IgY constant domains that circumvent susceptibility to interference phenomena and improve assay performance. After production in HEK293 cells, subsequent characterization by enzyme-linked immunosorbent assay and immunoblotting confirmed the suitability of avian IgY constant domains for immobilization and detection purposes. To provide further insights into the different IgE reactivities, free allergen-specific IgE was also determined. Monitoring of sera from omalizumab-treated patients during the course of therapy revealed the applicability for assessment of omalizumab-complexed versus noncomplexed serum IgE. These parameters may allow correlation to clinical responses during anti-IgE therapy with the perspective of biomonitoring. © 2011 Elsevier Inc. All rights reserved

An improved model of moderate sleep apnoea for investigating its effect as a comorbidity on neurodegenerative disease

Sleep apnoea is a highly prevalent disease that often goes undetected and is associated with poor clinical prognosis, especially as it exacerbates many different disease states. However, most animal models of sleep apnoea (e.g., intermittent hypoxia) have recently been dispelled as physiologically unrealistic and are often unduly severe. Owing to a lack of appropriate models, little is known about the causative link between sleep apnoea and its comorbidities. To overcome these problems, we have created a more realistic animal model of moderate sleep apnoea by reducing the excitability of the respiratory network. This has been achieved through controlled genetically mediated lesions of the preBötzinger complex (preBötC), the inspiratory oscillator. This novel model shows increases in sleep disordered breathing with alterations in breathing during wakefulness (decreased frequency and increased tidal volume) as observed clinically. The increase in dyspnoeic episodes leads to reduction in REM sleep, with all lost active sleep being spent in the awake state. The increase in hypoxic and hypercapnic insults induces both systemic and neural inflammation. Alterations in neurophysiology, an inhibition of hippocampal long-term potentiation (LTP), is reflected in deficits in both long- and short-term spatial memory. This improved model of moderate sleep apnoea may be the key to understanding why this disorder has such far-reaching and often fatal effects on end-organ function

Nanobody-Enhanced Targeting of AAV Gene Therapy Vectors

International audienc

Repair of Mybpc3 mRNA by 5′-trans-splicing in a Mouse Model of Hypertrophic Cardiomyopathy

International audienceRNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C) is frequently mutated. We evaluated the 5′-trans-splicing strategy in a mouse model of HCM carrying a Mybpc3 mutation. 5′-trans-splicing was induced between two independently transcribed molecules, the mutant endogenous Mypbc3 pre-mRNA and an engineered pre-trans-splicing molecule (PTM) carrying a FLAG-tagged wild-type (WT) Mybpc3 cDNA sequence. PTMs were packaged into adeno-associated virus (AAV) for transduction of cultured cardiac myocytes and the heart in vivo. Full-length repaired Mybpc3 mRNA represented up to 66% of total Mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. Repaired cMyBP-C protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. This study provides (i) the first evidence of successful 5′-trans-splicing in vivo and (ii) proof-of-concept of mRNA repair in the most prevalent cardiac genetic disease. Since current therapeutic options for HCM only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease

Phosphomimetic cardiac myosin-binding protein C partially rescues a cardiomyopathy phenotype in murine engineered heart tissue

Phosphorylation of cardiac myosin-binding protein C (cMyBP-C), encoded by MYBPC3, increases the availability of myosin heads for interaction with actin thus enhancing contraction. cMyBP-C phosphorylation level is lower in septal myectomies of patients with hypertrophic cardiomyopathy (HCM) than in non-failing hearts. Here we compared the effect of phosphomimetic (D282) and wild-type (S282) cMyBP-C gene transfer on the HCM phenotype of engineered heart tissues (EHTs) generated from a mouse model carrying a Mybpc3 mutation (KI). KI EHTs showed lower levels of mutant Mybpc3 mRNA and protein, and altered gene expression compared with wild-type (WT) EHTs. Furthermore, KI EHTs exhibited faster spontaneous contractions and higher maximal force and sensitivity to external [Ca2+] under pacing. Adeno-associated virus-mediated gene transfer of D282 and S282 similarly restored Mybpc3 mRNA and protein levels and suppressed mutant Mybpc3 transcripts. Moreover, both exogenous cMyBP-C proteins were properly incorporated in the sarcomere. KI EHTs hypercontractility was similarly prevented by both treatments, but S282 had a stronger effect than D282 to normalize the force-Ca2+-relationship and the expression of dysregulated genes. These findings in an in vitro model indicate that S282 is a better choice than D282 to restore the HCM EHT phenotype. To which extent the results apply to human HCM remains to be seen