VEGF released from a fibrin biomatrix increases VEGFR-2 expression and improves early outcome after ischaemia-reperfusion injury.

Skeletal ischaemia-reperfusion (I-R) injury may influence patient outcome after severe vascular trauma or clamping of major vessels. The aim of this study was to observe whether locally applied vascular endothelial growth factor (VEGF) in fibrin could induce the expression of VEGF-receptor-2 (VEGFR-2) and improve the outcome after I-R injury. Transgenic mice expressing VEGFR-2 promoter-controlled luciferase were used for the assessment of VEGFR-2 expression. Ischaemia was induced for 2 h by a tension-controlled tourniquet to the hind limb, followed by 24 h of reperfusion. The animals were locally injected subcutaneously with fibrin sealant containing 20 or 200 ng VEGF; control animals received no treatment or fibrin sealant application. In vivo VEGFR-2 expression was quantified upon administration of luciferin at several observation times. For oedema and inflammation quantification, wet:dry ratio measurements and a myeloperoxidase assay of the muscle tissue were performed. Laser Doppler imaging showed that ischaemia was present and that the blood flow had returned to baseline levels after 24 h of reperfusion. VEGFR-2 expression levels in the fibrin + 200 ng VEGF were significantly higher than in all other groups. Granulocyte infiltration was reduced in both treatment groups, as well as reduced oedema formation. These results showed that VEGF released from fibrin had a positive effect on early I-R outcome in a mouse model, possibly via VEGFR-2. Copyright © 2016 John Wiley & Sons, Ltd.

Structural and Functional Characterization of the Hazelnut Allergen Cor a 8.

Nonspecific lipid transfer proteins (nsLTPs) are basic proteins, stabilized by four disulfide bonds, and are expressed throughout the plant kingdom. These proteins are also known as important allergens in fruits and tree nuts. In this study, the nsLTP from hazelnuts, Cor a 8, was purified and its crystal structure determined. The protein is stable at low pH and refolds after thermal denaturation. Molecular dynamics simulations were used to provide an insight into conformational changes of Cor a 8 upon ligand binding. When known epitope areas from Pru p 3 were compared to those of Cor a 8, differences were obvious, which may contribute to limited cross-reactivity between peach and hazelnut allergens. Differences in epitope regions may contribute to limited cross-reactivity between Cor a 8 and nsLTPs from other plant sources. The structure of Cor a 8 represents the first resolved structure of a hazelnut allergen.

Applications of Molecular Diagnostic Testing in Food Allergy.

IgE-mediated food allergy is a relevant health problem inducing symptoms ranging from mild local reactions up to severe life-threatening situations. Currently, no immunotherapy is available and avoidance of the incriminating food is the method of choice. Therefore, reliable diagnostic tools to formulate dietary recommendations and to avoid unnecessary exclusion diets for the individual patient are urgently needed. This review provides an update on the current knowledge on food allergens and their application in various diagnostic approaches such as skin prick test, basophil activation test, and serum IgE testing. Furthermore, these new approaches are discussed and compared to conventional extract-based assays and correlated to the gold standard of food allergy diagnosis, the double-blind placebo-controlled food challenge. Finally, the application of food allergens for preventive measurements such as allergen detection assays and the determination of threshold levels for allergen levels are discussed.

Cor a 14, the allergenic 2S albumin from hazelnut, is highly thermostable and resistant to gastrointestinal digestion.

SCOPE: Allergens from nuts frequently induce severe allergic reactions in sensitive individuals. The aim of this study was to elucidate the physicochemical characteristics of natural Cor a 14, the 2S albumin from hazelnut. METHODS AND RESULTS: Cor a 14 was purified from raw hazelnuts using a combination of precipitation and chromatographic techniques. The protein was analyzed using gel electrophoresis, MS, and far-UV circular dichroism (CD) analyses. The immunoglobulin E (IgE) binding of native, heat-treated, and in vitro digested Cor a 14 was studied. We identified two different Cor a 14 isoforms and showed microclipping at the C-terminus. CD spectra at room temperature showed the typical characteristics of 2S albumins, and temperatures of more than 80°C were required to start unfolding of Cor a 14 demonstrating its high stability to heat treatment. In vitro digestion experiments revealed that Cor a 14 is resistant to proteolytic degradation. Native and heat-treated protein was recognized by sera from hazelnut allergic patients. However, denaturation of the allergen led to significantly reduced IgE binding. CONCLUSION: We identified two different isoforms of Cor a 14 displaying high stability under heating and gastric and duodenal conditions. Data from IgE-binding experiments revealed the existence of both, linear and conformational epitopes.

Thrombin as important factor for cutaneous wound healing: comparison of fibrin biomatrices in vitro and in a rat excisional wound healing model.

Fibrin biomatrices have been used for many years for hemostasis and sealing and are a well-established surgical tool. The objective of the present study was to compare two commercially available fibrin biomatrices regarding the effect of their thrombin concentration on keratinocytes and wound healing in vitro and in vivo. Keratinocytes showed significant differences in adhesion, viability, and morphology in the presence of the fibrin matrices in vitro. A high thrombin concentration (800-1,200 IU/mL) caused deteriorated cell compatibility. By using a thrombin inhibitor, those differences could be reversed. In a rat excisional wound healing model, we observed more rapid wound closure and less wound severity in wounds treated with a fibrin matrix containing a lower concentration of thrombin (4 IU/mL). Furthermore, fewer new functional vessels and a lower level of vascular endothelial growth factor were measured in wounds after 7 days treated with the matrix with higher thrombin concentration. These in vivo results may be partially explained by the in vitro biocompatibility data. Additionally, results show that low thrombin biomatrices were degraded faster than the high thrombin material. Hence, we conclude that the composition of fibrin biomatrices influences keratinocytes and therefore has an impact on wound healing.

Immunomodulatory drugs thalidomide and lenalidomide affect osteoblast differentiation of human bone marrow stromal cells in vitro.

Osteoblastic activity is severely impaired in active myeloma, contributing to the development of myeloma bone disease. Although several drugs reducing osteoclast-mediated bone degradation are in clinical use, approaches to specifically augment bone formation are at an early stage of development. Novel antimyeloma drugs not only directly act on myeloma cells, but impact on the microenvironment as well. Proteasome inhibitors were previously shown to have bone anabolic properties. Here we investigated the impact of immunomodulatory drugs (IMiDs) on bone formation. Treatment with thalidomide and lenalidomide significantly inhibited osteoblast development in vitro, as reflected by a reduction of alkaline phosphatase activity and matrix mineralization. The effects were upheld in combination with bortezomib. The IMiDs upregulated Dickkopf-1 (DKK1) and inhibin beta A, but blocking these molecules was not able to restore regular osteoblast development. We therefore performed gene expression profiling to reveal other osteoblast regulatory factors that might be involved in the IMiD-mediated effect on osteoblast development. Our data indicate that osteoblast inhibition is possibly an IMiD-class effect mediated by downregulation of major osteoblast regulators (e.g., runt-related transcription factor 2, distal-less homeobox 5, pleiotrophin) and concurrent induction of secreted inhibitors of osteoblast formation (e.g. DKK1, activin A, gremlin 1). Our results highlight the need for bone anabolic therapeutics in myeloma, counteracting the negative impact of prolonged IMiD exposure on bone metabolism.

High expression of cereblon (CRBN) is associated with improved clinical response in patients with multiple myeloma treated with lenalidomide and dexamethasone.

Cereblon (CRBN) has recently been identified as a target for immunomodulatory drugs (IMiDs) and its downregulation has been linked to resistance to lenalidomide. Here, we studied CRBN expression by real time polymerase chain reaction in 49 bone marrow samples of newly diagnosed patients with multiple myeloma treated with lenalidomide and dexamethasone. Median CRBN expression was 3·45 in patients who achieved complete response, and 3·75, 2·01, 0·78, and 0·70 in those with very good partial response, partial response, stable disease and progressive disease respectively. CRBN expression levels correlated significantly with response to lenalidomide treatment (r = 0·48; P < 0·001). Among established prognostic parameters, only beta-2-microglobulin correlated with cereblon (r = 0·66; P < 0·001). A close association of CRBN with interferon regulatory factor 4 (IRF4) (P < 0·001) and with CTNNB1 (P < 0·001) was found. Overall, a statistically significant association between baseline CRBN expression and response in MM patients treated with lenalidomide is shown. CRBN expression is closely associated with IRF4, which is an important target of IMiD therapy.

Vascular endothelial growth factor and fibroblast growth factor-2 incorporation in starch-based bone tissue-engineered constructs promote the in vivo expression of neovascularization mediators.

The ideal bone tissue-engineered (TE) construct remains to be found, although daily discoveries significantly contribute to improvements in the field and certainly have valuable long-term outcomes. In this work, different TE elements, aiming at bone TE applications, were assembled and its effect on the expression of several vascularization/angiogenesis mediators analyzed. Starch/polycaprolactone (SPCL) scaffolds, obtained by two different methodologies, were combined with fibrin sealant (Baxter(®)), human adipose-derived stem cells (hASCs), and growth factors (vascular endothelial growth factor [VEGF] or fibroblast growth factor-2 [FGF-2]), and implanted in vascular endothelial growth factor receptor-2 (VEGFR2)-luc transgenic mice. The expression of VEGFR2 along the implantation of the designed constructs was followed using a luminescence device (Xenogen(®)) and after 2 weeks, the explants were retrieved to perform histological analysis and reverse transcriptase-polymerase chain reaction for vascularization (VEGF and VEGFR1) and inflammatory (tumor necrosis factor-alpha, interleukin-4, and interferon-gamma) markers. It was showed that SPCL scaffolds obtained by wet spinning and by fiber bonding constitute an adequate support for hASCs. The assembled TE constructs composed by fibrin sealant, hASCs, VEGF, and FGF-2 induce only a mild inflammatory reaction after 2 weeks of implantation. Additionally, the release of VEGF and FGF-2 from the constructs enhanced the expression of VEGFR2 and other important mediators in neovascularization (VEGF and VEGFR1). These results indicate the potential of VEGF or FGF-2 within a bone TE construct composed by wet-spun SPCL, fibrin sealant, and hASCs in promoting the vascularization of newly formed tissue.

Induction of indoleamine-2,3 dioxygenase in bone marrow stromal cells inhibits myeloma cell growth.

PURPOSE: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme with immunoregulatory properties in cancer. By focusing on multiple myeloma cells and its microenvironment as potential sources of IDO, we aimed to delineate its influence on myeloma cell growth and survival and examine effector mechanisms. METHODS: IDO expression was assessed in myeloma cells and in a coculture system with mesenchymal stromal cells (MSCs), including prior cytokine priming to induce IDO in MSCs. IDO expression was correlated with induction of apoptosis in myeloma cells and coupled with tryptophan depletion as well as rescue using IDO inhibitors. RESULTS: We report low levels of expression of IDO in myeloma cell lines (MMCLs) and primary myeloma cells (MMCs), despite priming with interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or hepatocyte growth factor (HGF). In MSCs, however, IDO could be functionally induced by IFN-γ, mediating apoptosis in myeloma cells following coculture. Addition of IDO-specific inhibitors, as well as addition of tryptophan, was shown to abrogate these effects. CONCLUSIONS: IDO is expressed in primary MMCs to a low degree and is unlikely to play a direct major role in vivo in dampening antitumor immunity. However, cytokine stimulation of MSCs specifically induced IDO, which mediated a marked sensitivity of proximal myeloma cells to tryptophan depletion in the microenvironment, suggesting that selective measures to regulate its availability could be a useful strategy to achieve myeloma growth inhibition and apoptosis.

Extracorporeal shock wave therapy (ESWT) minimizes ischemic tissue necrosis irrespective of application time and promotes tissue revascularization by stimulating angiogenesis.

OBJECTIVE: To assess the time-dependent treatment effects of extracorporeal shock wave therapy (ESWT) in a standard rodent ischemic epigastric flap model. BACKGROUND: ESWT has been shown to accelerate tissue repair in acute and chronic wounds and improve graft survival, but the mechanism remains incompletely understood. METHODS: Shock waves at 0.1 mJ/mm and 5 impulses/s (total 300 impulses) were applied to the epigastric flap ischemic region at various times pre-, immediately and 24 hours postischemic insult. Flap survival; vascular perfusion; vessel number; von Willebrand factor and smooth muscle actin protein expression as well as in vivo vascular endothelial growth factor receptor 2 expression were evaluated at 1, 3, and 7 days postoperatively in ESWT-treated and untreated controls. RESULTS: Flap perfusion, microvessel number, and survival (through reduced flap contraction and necrosis) were significantly enhanced in the treated groups compared with controls, irrespective of timing of shock wave treatment (preischemia vs. postischemia). Vascular endothelial growth factor receptor 2 expression was dynamically upregulated in response to ESWT. CONCLUSION: Shock wave preconditioning and treatment postischemic insult improves skin flap survival through neovascularization and early upregulation of angiogenesis-related growth factors.

Interaction of sperm with purified native chicken ZP1 and ZPC proteins.

The avian perivitelline membrane (PVM) is the site of initial contact between sperm and egg. It consists of only two major components, which are both homologues of the mammalian zona pellucida (ZP) proteins, and belong to the ZP1 and ZPC families, respectively. We have established a method to isolate large quantities of both native avian ZP proteins and have used these preparations to investigate their sperm-binding capacities. Chicken ZPC forms multimeric structures of defined size and binds to an approximately 180-kDa protein complex present in rooster sperm extracts. Based on experiments using both PVM and isolated proteins, we show that chicken ZP1 is proteolytically degraded by a sperm-associated protease but that chicken ZPC remains intact. An antiserum directed against chicken ZP1 is capable of inhibiting sperm binding to the PVM. Taken together, these data suggest that ZP1, in addition to ZPC, plays a major role in the initial interactions between sperm and egg.