[Show abstract][Hide abstract] ABSTRACT: The effects of Notch signaling are context-dependent and both oncogenic and tumor suppressive functions have been described. Notch signaling in melanoma is considered oncogenic, but clinical trials testing Notch inhibition in this malignancy have not proved successful. Here, we report that expression of the constitutively active intracellular domain of Notch4 (N4ICD) in melanoma cells triggered a switch from a mesenchymal-like parental phenotype to an epithelial-like phenotype. The epithelial-like morphology was accompanied by strongly reduced invasive, migratory, and proliferative properties concomitant with the downregulation of epithelial-mesenchymal transition markers Snail2 (SNAI2), Twist1, vimentin (VIM), and MMP2 and the re-expression of E-cadherin (CDH1). The N4ICD-induced phenotypic switch also resulted in significantly reduced tumor growth in vivo. Immunohistochemical analysis of primary human melanomas and cutaneous metastases revealed a significant correlation between Notch4 and E-cadherin expression. Mechanistically, we demonstrate that N4ICD induced the expression of the transcription factors Hey1 and Hey2, which bound directly to the promoter regions of Snail2 and Twist1 and repressed gene transcription, as determined by EMSA and luciferase assays. Taken together, our findings indicate a role for Notch4 as a tumor suppressor in melanoma, uncovering a potential explanation for the poor clinical efficacy of Notch inhibitors observed in this setting.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome.
Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined.
Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope.
The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.
Preview · Article · Jan 2016 · Allergy, asthma & immunology research
[Show abstract][Hide abstract] ABSTRACT: Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.
[Show abstract][Hide abstract] ABSTRACT: Objective
To develop a reliable method to assess the stability of Xinyue Capsules (心悦胶囊) containing Panax quinquefolius saponins according to European quality standards.
An efficient high-performance liquid chromatography ultraviolet (HPLC-UV) method was established to analyse six main ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in six different batches (120 capsules/batch) from the same lot of Xinyue Capsules and in one batch measured six times within one day. The six ginsenosides were separated on a Hypersil BDS-C18 column (3 μm, 100 mm×3 mm) at a flow rate of 0.5 mL/min. Gradient elution was performed using a mobile phase gradient of acetonitrile-water modified with 0.01% formic acid. The HPLC chromatograms were analyzed with "LC data comparison" using Lab Solutions software.
The HPLC peaks were identified by comparing their retention times (Rg1: 23.44 min, Re: 23.77 min, Rb1: 35.24 min, Rc: 36.18 min, Rb2: 38.55 min and Rd: 40.88 min) with those of the standards under the same chromatographic conditions, which showed similar results among the samples of six different batches and among the samples from one batch detected six times within one day.
Xinyue Capsules have good drug intra-day consistency at room temperature and exhibit a consistent quality between different batches. This study established a reliable method to assess the stability of Xinyue Capsules, which is suitable for further qualitative analysis and may assist in promoting the safe and effective use of Chinese herbal medicine.
No preview · Article · Oct 2015 · Chinese Journal of Integrative Medicine
[Show abstract][Hide abstract] ABSTRACT: Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment.
Preview · Article · May 2015 · International Journal of Oncology
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals.
[Show abstract][Hide abstract] ABSTRACT: We present an infrared biopsymeter to assist pathologists in the diagnosis of melanoma presence in skin biopsies. The designed and realized system combines the features of visual inspection and physical sensing to reduce false positives and false negatives occurring during standard histopathological analyses. The biopsymeter determines the CH2-stretch ratio by infrared absorbance measurements of skin biopsies. Investigations conducted with the biopsymeter shows that malignant melanomas and melanoma metastases have higher CH2-stretch ratio values compared to healthy skin tissues.
[Show abstract][Hide abstract] ABSTRACT: Background
Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail.Methods
Bet v 1-sensitised birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardised interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA.ResultsBet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in less than 65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific for most Bet v 1-related allergens. Api g 1-specific IgE was significantly (p = 0.01) elevated in celeriac-allergic compared to celeriac-tolerant patients. Likewise, frequencies of IgE (71% versus 15%; p = 0.01) and IgA (86% versus 38%; p = 0.04) binding to Api g 1.01 were increased.Conclusion
Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac.This article is protected by copyright. All rights reserved.