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The multityrosine kinase inhibitor sorafenib remains an important systemic treatment option for hepatocellular carcinoma (HCC). Signaling pathways, which are targeted by sorafenib, are involved in checkpoint and DNA repair response, RAD51 being a candidate protein. Here, we aim to evaluate the effect of the human RAD51 inhibitor B02 in combination with sorafenib in human HCC cells. Impact of RAD51 expression on HCC patient survival was evaluated by an in silico approach using Human Protein Atlas dataset. Cell viability of HUH7, AKH12, AKH13, and 3P was assessed by neutral red assay. To measure the cytotoxicity, we quantified loss of membrane integrity by lactate dehydrogenase release. We also employed colony formation assay and hanging drop method to assess clonogenic and invasive ability of HCC cell lines upon sorafenib and B02 treatment. Cell cycle distribution and characterization of apoptosis was evaluated by flow cytometry. In silico approach revealed that HCC patients with higher expression of RAD51 messenger RNA had a significantly shorter overall survival. The RAD51 inhibitor B02 alone and in combination with sorafenib significantly reduced viability, colony formation ability, and invasion capacity of HCC cells. Cell cycle analysis revealed that the combination of both agents reduces the proportion of cells in the G2/M phase while leading to an accumulating in the subG1 phase. The RAD51 inhibitor B02 seems to be a promising agent for HCC treatment and enhances the antitumor effects of sorafenib in vitro.

Supplemental Figure Legends for Supplemental Figures 1 to 5

SF1: Phenotypic and functional changes of 451Lu, WM9 and WM164 following N4ICD overexpression SF2: Immunoblots and immunohistochemistry of EMT markers and Notch4 SF3: Details of CSL binding to Snail2 and Twist1 promoter regions SF4: Immunoblots of overexpression and silencing experiments confirming the Notch4-Hey1/2-Snail2/Twist1 axis (SF4) SF5: Immunoblots of pLNCX-N4ICD and siHey1/2 transduced cells and details of Snail2 and Twist1 promoter regions ST1: Plasmids and siRNAs ST2: Antibodies ST3: Primers for qPCR and ChIP ST4: Sequence of EMSA fragments ST5: Sequence of luciferase fragments ST6: Fold regulation of EMT markers determined by PCR-array ST7: Details of immunohistochemical analyses of melanoma samples Supplementary references

Supplemental Materials and Methods used to create Supplemental Figures only in addition to Materials and Methods listed in the main manuscript

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 reexpression 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. Cancer Res; 76(7); 1690–7. ©2016 AACR.

Alergia; Anafilaxia; Asma Allergy; Anaphylaxis; Asthma Al·lèrgia; Anafilaxi; Asma Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the “EAACI Molecular Allergology User's Guide” (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

The WHO/IUIS Allergen Nomenclature Database (http://allergen.org) provides up-to-date expert-reviewed data on newly discovered allergens and their unambiguous nomenclature to allergen researchers worldwide. This review discusses the 106 allergens that were accepted by the Allergen Nomenclature Sub-Committee between 01/2019 and 03/2021. Information about protein family membership, patient cohorts, and assays used for allergen characterization is summarized. A first allergenic fungal triosephosphate isomerase, Asp t 36, was discovered in Aspergillus terreus. Plant allergens contained 1 contact, 38 respiratory, and 16 food allergens. Can s 4 from Indian hemp was identified as the first allergenic oxygen-evolving enhancer protein 2 and Cic a 1 from chickpeas as the first allergenic group 4 late embryogenesis abundant protein. Among the animal allergens were 19 respiratory, 28 food, and 3 venom allergens. Important discoveries include Rap v 2, an allergenic paramyosin in molluscs, and Sal s 4 and Pan h 4, allergenic fish tropomyosins. Paramyosins and tropomyosins were previously known mainly as arthropod allergens. Collagens from barramundi, Lat c 6, and salmon, Sal s 6, were the first members from the collagen superfamily added to the database. In summary, the addition of 106 new allergens to the previously listed 930 allergens reflects the continuous linear growth of the allergen database. In addition, 17 newly described allergen sources were included.

Vegetables provide important nutrients but can also induce allergic symptoms. Celery tuber allergy frequently occurs in Central European countries and can cause allergic reactions including fatal anaphylactic shocks. There is little information about allergen content in seeds. Therefore, we analyzed 2 patients with allergic reaction after remoulade sauce consumption who entered the clinic for a diagnostic work-up. The routine diagnostic included serum derived specific IgE testing by ImmunoCAP, ImmunoCAP ISAC, and skin prick tests (SPTs). Furthermore, protein extracts were prepared from both celery tuber and celery seeds and IgE binding capacity of these extracts was assessed by immunoblots, ELISA, and rat basophil leukemia (RBL) assay. We also determined role of cross-reactive carbohydrate determinants (CCDs) by IgE inhibition ELISA. Results revealed distinct protein patterns from celery tuber and seed extracts, suggesting differences in content and quantity of allergenic proteins. IgE antibodies from both sera bound to high molecular weight (HMW) proteins on immunoblots and caused high basophil response, which was also observed upon addition of glycosylated proteins as horseradish peroxidase and Api g 5, respectively. Our results indicate that it is worth considering CCDs from plant foods as a possible allergenic factor and their contribution to the mugwort-celery syndrome.

Although recent studies indicated that many fish-allergic patients may safely consume certain fish species, no clinical guidelines are available for identification of the exact species tolerated by specific patients.To investigate whether multiplex immunoglobulin E (IgE) testing reveals potentially tolerated fish through absence of IgE to parvalbumin (PV) and extracts from specific species.Sera from 263 clinically well-defined fish-allergic patients from Austria, China, Denmark, Luxembourg, Norway, and Spain were used in a research version of the ALEXUp to 38% of the patients were negative to cod PV, the most commonly used molecule in fish allergy diagnosis. Forty-five patients (17%) tested negative to PVs but positive to the respective fish extracts, underlining the requirement for extracts for accurate diagnosis. Between 60% (Spain) and 90% (Luxembourg) of the patients were negative to PV and extracts from ray, a cartilaginous fish, indicating its potential tolerance. Up to 21% of the patients were negative to at least 1 bony fish species. Of the species analyzed, negativity to mackerel emerged as the best predictive marker of negativity to additional bony fish, such as herring and swordfish.Parvalbumins and extracts from multiple fish species relevant for consumption should be used in fish-allergy diagnosis, which may help identify potentially tolerated species for individual patients.

Background Oral food challenges have demonstrated that diagnosis of almond allergy based on extract‐sIgE tests displays low specificity. Molecular allergy diagnosis is expected to improve accuracy, but its value in diagnosing almond allergy remains unknown. The aim of this study was to identify relevant almond allergens and examine their ability to improve almond allergy diagnosis. Methods IgE‐reactive proteins were purified from almond kernels. IgE binding to almond extract and the allergens was analyzed by quantitative ELISA using sera from 18 subjects with a proven almond allergy. The control group consisted of sera from 18 subjects allergic to peanut and/or tree nuts but tolerant to almond. Results Three IgE‐binding proteins were identified: legumin (Pru du 6), alpha‐hairpinin (Pru du 8), and mandelonitrile lyase (Pru du 10). Positive IgE (≥0.35 kU/L) to almond extract showed 94% sensitivity but only 33% specificity. IgE to Pru du 6 maintained high sensitivity (83%) and provided superior specificity (78%). Sera from almond‐allergic subjects had significantly higher IgE levels to almond extract (P, Three allergens were identified in almond kernels: Pru du 6, Pru du 8, and a new allergen, Pru du 10, belonging to a novel family of allergenic proteins. Sensitization profiles of almond‐allergic individuals were dominated by almond‐specific allergens, particularly Pru du 6. The use of almond allergens for IgE tests markedly increased the diagnostic specificity compared to the extract. Abbreviations: FPLC, fast protein liquid chromatography; LC‐MS/MS: liquid chromatography tandem mass spectrometry; sIgE: specific IgE

The clinical impact of 5-aminolevulinic acid (5-ALA) fluorescence during resection of brain metastases is not yet clear.. Recent data demonstrated significantly lower incidence of visible fluorescence in cerebral melanoma metastases (CMM) compared to other brain metastases (BM). The aim of this study was to investigate if characteristic melanoma features such as pigmentation, intratumoural hemosiderin and bleeding have an influence on visible fluorescence in CMM.A retrospective study of two neurosurgical centers was performed including adult patients with resection of CMM after preoperative administration of 5-ALA. Data on the fluorescence status (visible or no fluorescence), the fluorescence quality (strong, vague, none) and fluorescence homogeneity (homogeneous or heterogeneous) of each CMM were collected. The amount of melanin, hemosiderin and intratumoural bleeding was semi-quantitatively determined and automated computer-based calculation of the relative pigmented area was performed in fluorescing and non-fluorescing CMM samples.Altogether, 29 CMM were surgically removed after 5-ALA administration. Visible fluorescence was detected in 8 CMM (28%), whereas no fluorescence was detected in 21 CMM (72%). In detail, 3 tumors (10%) showed strong fluorescence, 5 tumors (17%) revealed vague fluorescence and in 21 tumors (72%) no fluorescence was found. In total, 8 fluorescing and 25 non-fluorescing CMM samples were investigated. According to the semi-quantitatively calculated fluorescence status, no statistically significant difference in the median amount of melanin (p = 0.242), hemosiderin (p = 0.603) and bleeding (p = 0.762) between CMM samples with and without visible fluorescence was found. Moreover, the automatically assessed relative pigmented area did not show a statistically significant difference between samples with visible and no fluorescence (p = 0.966).Our data indicate that 5-ALA fluorescence is not dependent on the amount of pigmentation, intratumoural hemosiderin and bleeding in CMM. We thus assume that other factors are responsible for the low rate of visible fluorescence in CMM.

The overexpression of chondroitin sulfate proteoglycan 4 (CSPG4) is associated with several tumor types, including malignant melanoma, squamous cell carcinoma, triple‑negative breast carcinoma, oligodendrocytomas or gliomas. Due to its restricted distribution in normal tissues, CSPG4 has been considered a potential target for several antitumor approaches, including monoclonal antibody (mAb) therapies. The aim of the present study was to characterize the impact of the CSPG4‑specific mAb clone 9.2.27 on its own or in combination with the commonly used BRAF‑selective inhibitor, PLX4032, on different functions of melanoma cells to assess the potential synergistic effects. The BRAF V600‑mutant human melanoma cell lines, M14 (CSPG4‑negative) and WM164 (CSPG4‑positive), were exposed to the CSPG4‑specific 9.2.27 mAb and/or PLX4032. Cell viability and colony formation capacity were evaluated. A 3D‑cell culture spheroid model was used to assess the invasive properties of the treated cells. In addition, flow cytometric analysis of apoptosis and cell cycle analyses were performed. Incubation of the WM164 cell line with CSPG4‑specific 9.2.27 mAb decreased viability, colony formation ability and the invasive capacity of CSPG4‑positive tumor cells, which was not the case for the CSPG4‑negative M14 cell line. Combined treatment of the WM164 cells with 9.2.27 mAb plus PLX4032 did not exert any significant additional effect in comparison to treatment with PLX4032 alone in the clonogenic and invasion assays. M14 cell cycle distribution was not influenced by the CSPG4‑specific 9.2.27 mAb. By contrast, the exposure of WM164 cells to the mAb resulted in an arrest of the cells in the S phase. Moreover, combined treatment of the WM164 cells led to a significantly increased accumulation of cells in the subG1 phase, combined with a decrease of cells in the G2/M phase. On the whole, findings of the present study indicate that the CSPG4‑specific 9.2.27 mAb exerts an anti‑invasive effect on CSPG4‑positive melanoma spheroids, which is not enhanced by BRAF inhibition. These findings provide the basis for further investigations on the effects of anti‑CSPG4‑based treatments of CSPG4‑positive tumors.

9528 Background: Cutaneous squamous cell carcinoma (cSCC) is the second most frequent skin cancer and considered as a tumor with strong immunogenicity. Consistently, immune checkpoint-inhibition with programmed death (PD)-1 antibodies has become the novel standard of care in the treatment of advanced cSCC. In this study, we evaluated efficacy and safety of the PD-1 antibody nivolumab in patients with locally advanced or metastatic cSCC, including individuals with concomitant hematological malignancies (CHM) – a highly vulnerable subgroup of cSCC patients typically excluded from clinical trials. Methods: This phase II, open-label, single-arm multicentre study included patients aged ≥ 18 years with histologically confirmed locally advanced and/or metastatic cSCC and at least one measurable lesion according to RECIST v1.1. Enrolled patients received nivolumab 240 mg intravenously over 30 min every 2 weeks for up to 2 years. A sample size of 31 patients was needed to provide 90% power to detect an objective response rate (ORR) of at least 12.6% after 24 weeks with a type I error of 5% assuming a dropout-rate of 15%. The primary endpoint was investigator assessed ORR as per RECIST v1.1. Secondary endpoints included disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). Results: Between July 2017 and October 2020, 31 patients with advanced cSCC, including 11 patients with CHM, were enrolled and received at least one dose of nivolumab. The median age was 80 years (range 66-92) and the majority of patients was male (71%). Upon enrollment, 19.4% of patients had locally advanced, 51.6% loco-regional metastatic and 25.8% distant metastasic disease. Seven patients (22.6%) had received one prior systemic therapy for cSCC. At data-cut-off (March 2021; week-24 RECIST assessment completed in all available patients), five patients (16.1%) were still on treatment, one patient completed treatment per protocol, whereas 25 patients had discontinued therapy. Of 29 patients who were evaluable for response assessment, 12 patients achieved a partial and 7 a complete response, resulting in a best ORR of 65.2% (95% CI: 45.7% - 82.1%), a DCR of 68.9% (95% CI: 50.8%-82.7%) and a median PFS of 11.1 months (95% CI: 3.7 - 12.9). Treatment related adverse events occurred in 18 patients (58.1%) and led to nivolumab discontinuation in two patients (6.5%). Subgroup analysis of patients with CHM revealed a best ORR of 55.6% (95% CI: 21.2% - 86.3%), a DCR of 66.7% (95% CI: 35.4% -87.9%) and a median PFS of 10.9 months (95% CI: 0.6 - 21.4). Median OS in this subgroup was 20.7 months (95% CI: 6.5 - 35.0), whereas overall median OS was not reached. Conclusions: Nivolumab showed a robust antitumor-activity similar to other anti-PD-1 agents in advanced cSCC. Although ORR and OS were slightly reduced in patients with CHM, nivolumab proved effectiveness also in this subgroup while no new safety signals occurred. Clinical trial information: NCT04204837.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth-factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF-mutant CSPG4-positive or -negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient-derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4-positive cells as compared to pre-therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug-resistance in melanoma cells.

BackgroundTalimogene laherparepvec (T-VEC) is a licensed therapy for use in melanoma patients of stage IIIB-IVM1a with injectable, unresectable metastatic lesions in Europe. Approval was based on the Oncovex Pivotal Trial in Melanoma study, which also included patients with distant metastases and demonstrated an overall response rate (ORR) of 40.5% and a complete response (CR) rate of 16.6%.ObjectivesThe aim of this study was to assess the outcome of melanoma patients treated with T-VEC in a real-life clinical setting.MethodsBased on data from 10 melanoma centers in Austria, Switzerland and southern Germany, we conducted a retrospective chart review, which included 88 patients (44 male, 44 female) with a median age of 72 years (range 36–95 years) treated with T-VEC during the period from May 2016 to January 2020.Results88 patients fulfilled the inclusion criteria for analysis. The ORR was 63.7%. 38 patients (43.2%) showed a CR, 18 (20.5%) had a partial response, 8 (9.1%) had stable disease and 24 (27.3%) patients had a progressive disease. The median treatment period was 19 weeks (range: 1–65), an average of 11 doses (range: 1–36) were applied. 39 (45.3%) patients developed adverse events, mostly mild, grade I (64.1%).ConclusionThis real-life cohort treatment with T-VEC showed a high ORR and a large number of durable CRs.

In this study, we designed and realized a sensor platform using deuterium oxide (D2O) based microfluidics to allow in vitro infrared (IR) analysis of living cells. Using D2O instead of H2O based medium successfully resolved the high IR absorption issue of H2O at the 3.3–3.6 μm wavelength region. The spectra in this region contain the symmetric and antisymmetric CH2 and CH3 absorption, which can be used to monitor changes in the cells or the cell membranes. To demonstrate the performance and possible use of the system, IR analyses were conducted on three mammalian cell lines: Madin-Darby canine kidney (MDCK), rat basophilic cells (RS-ATL8), and aneuploid immortalized keratinocyte cells of adult human skin (HaCaT). The determination of the CH2 and CH3 absorption peaks allowed in vitro differentiation of the mammalian cell types. Successive viability tests showed normal cell proliferation in culture medium after incubation in D2O-PBS for 30 min. The IR spectra of fifteen separate measurements showed only small absorbance tolerances, which proved the high level of repeatability of the sensor system. This measurement system opens up the way to perform infrared cell analysis in liquid environments.

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 reexpression 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. Cancer Res; 76(7); 1690–7. ©2016 AACR.

A hypoxic microenvironment is one of the predominant reasons for incomplete response to melanoma treatment. Vemurafenib, which targets the mutated BRAF-V600 kinase, improves melanoma patient survival, however, resistance invariably develops. The present study evaluated the effect of hypoxia on three BRAF-V600E mutant melanoma cell lines, M14, A375 and 518A2, treated with vemurafenib. Compared with the other two cell lines, hypoxic vemurafenib-treated A375 cells exhibited an enhanced cell proliferation rate and migratory capacity compared with normoxic vemurafenib-treated A375 cells. Immunoblotting analyses revealed that the expression levels of hypoxia inducible factor (HIF)1α and carbonic anhydrase IX were reduced in vemurafenib‑treated M14 and 518A2 cells, however, not in A375 cells. The expression levels of the mitogen‑activated protein kinase, Janus kinase-signal transducer and activator of transcription, and phosphatidylinositol-4,5-bisphosphate 3‑kinase signaling pathway proteins revealed a cell‑type specific response to vemurafenib and hypoxia. Knockdown experiments of HIF1α performed in hypoxic A375 cells decreased the expression of phosphorylated (p‑)protein kinase B, which was restored following vemurafenib treatment, and increased the expression of p‑extracellular‑signal‑regulated kinases. Therefore, three melanoma cell lines responded to vemurafenib under hypoxia in a cell type‑specific manner, suggesting that a subset of cells provides a treatment-resistant pool, from which disease relapse may originate. These data confirmed that vemurafenib may be successful in treating the proliferating cells, whereas the non‑proliferating subpopulation must be addressed by a combination of vemurafenib with other treatment strategies.

Background Clinical reactions to bony fish species are common in patients with allergy to fish and are caused by parvalbumins of the β-lineage. Cartilaginous fish such as rays and sharks contain mainly α-parvalbumins and their allergenicity is not well understood. Objective To investigate the allergenicity of cartilaginous fish and their α-parvalbumins in individuals allergic to bony fish. Methods Sensitization to cod, salmon, and ray among patients allergic to cod, salmon, or both (n = 18) was explored by prick-to-prick testing. Clinical reactivity to ray was assessed in 11 patients by food challenges or clinical workup. IgE-binding to β-parvalbumins (cod, carp, salmon, barramundi, tilapia) and α-parvalbumins (ray, shark) was determined by IgE-ELISA. Basophil activation tests and skin prick tests were performed with β-parvalbumins from cod, carp, and salmon and α-parvalbumins from ray and shark. Results Tolerance of ray was observed in 10 of 11 patients. Prick-to-prick test reactions to ray were markedly lower than to bony fish (median wheal diameter 2 mm with ray vs 11 mm with cod and salmon). IgE to α-parvalbumins was lower (median, 0.1 kU/L for ray and shark) than to β-parvalbumins (median, ≥1.65 kU/L). Furthermore, α-parvalbumins demonstrated a significantly reduced basophil activation capacity compared with β-parvalbumins (eg, ray vs cod, P < .001; n = 18). Skin prick test further demonstrated lower reactivity to α-parvalbumins compared with β-parvalbumins. Conclusions Most patients allergic to bony fish tolerated ray, a cartilaginous fish, because of low allergenicity of its α-parvalbumin. A careful clinical workup and in vitro IgE-testing for cartilaginous fish will improve patient management and may introduce an alternative to bony fish into patients’ diet.

Peanut allergy is considered to be the most common cause for food-induced anaphylaxis. Currently, no approved treatment is available. Avoidance is the only measure to prevent anaphylactic reactions to peanuts. T-helper cells are of special importance for the sensitization process and the maintenance of allergic inflammation. Identifying markers of allergen-specific T-cell responses may help to develop novel treatment approaches. Therefore, we aimed to define new T-cell target genes in Ara h 2-specific T cells and to investigate the possibility of using them as biomarkers of peanut allergy in peripheral blood mononuclear cells (PBMCs). We performed whole mRNA array analysis (whole human genome oligo microarray) of in vitro expanded Ara h 2-specific T cells (CFSElowCD3+CD4+) from 5 peanut-allergic (PA) and 5 non-peanut-sensitized individuals. Expression of selected genes as a result of a two-step bioinformatic approach was confirmed in a second cohort by quantitative PCR. TGF-β- activated kinase 1 and MAP3K7 binding protein 3 (TAB3), calcium/calmodulin-dependent protein kinase type IV (CAMK4) and HemK methyltransferase family member 1 (HEMK1) were significantly upregulated in Ara h 2-specific T cells of PA patients. In addition, the expression of these genes was also assessed in unstimulated PBMCs from a cohort (n = 43) of PA, atopic non-PA, and nonatopic controls. Interestingly, in unstimulated PBMCs, TAB3 expression was significantly downregulated in PA patients compared to atopic non-PA individuals. Thus, TAB3 may play a significant role at the level of T-cell activation and may also be a candidate biomarker for PA.

Background Talimogene Laherparepvec (TVEC), a genetically modified GM-CSF expressing HSV1 Virus that preferentially replicates in tumor cells is approved in Europe for use in melanoma patients with injectable metastatic lesions in stage III-IVM1a. Approval was based on the OPTIM study which did also include patients with distant metastases and demonstrated a ORR of 40.5% and a CR rate of 16.6%. The aim of this study was to assess the outcome of melanoma patients treated with TVEC in a real life clinical setting outside of clinical studies. Methods To this aim a retrospective chart review in 7 melanoma centers in Austria and 1 center in Switzerland was conducted and anonymized data on disease stage, treatment duration, treatment response by investigator assessment following RECIST 1.1, tolerability as well as data on follow up therapies was collected. Results A total of 62 patients received TVEC since December of 2016. The majority of patients were AJCC stage IIIB and IIIC. Two patients with stage IV M1b and M1d who had complete control of their distant metastases and a locoregional progression were treated in parallel with a PD-1 antibody in one case and in parallel with a BRAF/MEK inhibitor combination in the other. In 3 other cases TVEC was used in combination with a PD-1 inhibitor as first-line of therapy. The median number of intralesional injection cycles was 11. The ORR was 67,7%; 50% of patients achieved a complete remission. 7 of 31 patients with a CR had a subsequent progression, 4 with distant and 3 with locoregional metastases. The main side effects observed were fever and chills. Conclusions In this real life cohort treatment of TVEC shows a high overall and complete remission rate with the majority of complete responses being durable. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure C. Hoeller: Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen. J.M. Ressler: Speaker Bureau / Expert testimony: Amgen. H. Kehrer: Advisory / Consultancy: Amgen. P. Koelblinger: Advisory / Consultancy: Amgen. F. Weihsengruber: Advisory / Consultancy: Amgen. J. Kofler: Advisory / Consultancy: Amgen. E. Richtig: Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen. O.A. Michielin: Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen. C. Hafner: Advisory / Consultancy: Amgen. All other authors have declared no conflicts of interest.

Walnuts are ranked high in the list of the culprit foods inducing severe allergic reactions. Jug r 2 has been identified as a major allergen in common walnut by cDNA cloning from a somatic cell line. So far, studies were performed on the allergenic activity of recombinant Jug r 2, yet there is still no evidence about the physicochemical characteristics of the natural allergen. Therefore, we aimed to purify and deeply characterize natural Jug r 2 and to assess IgE cross-reactivity among vicilins from different tree nuts. Extensive mass spectrometry analysis of the obtained purified vicilin allowed identification of the protein sequence that displayed only 44% identity to Jug r 2. The newly identified vicilin (Jug r 6) was recognized by IgE of 26% in walnut allergic patients’ sera tested. In contrast to Jug r 2, Jug r 6 displayed a remarkable level of cross-reactivity when tested with homologues from hazelnut, sesame and pistachio. It is the first report showing the necessity of proteomic studies to improve allergy component resolved diagnosis.

In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications., Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

Background Neutrophils and allergen-specific T cells accumulate in patients with allergic late-phase reactions (LPRs). Their presence is associated with severe inflammation. Cytokines, such as GM-CSF, IFN-γ, and IL-3, which are typically found in patients with allergic LPRs, have been proposed to convert neutrophils into antigen-presenting cells (APCs). Objective We sought to assess the antigen-processing and antigen-presenting capacities of neutrophils from allergic patients. Methods Neutrophils were isolated from peripheral blood of donors with birch pollen allergy and stimulated with GM-CSF, IFN-γ, and IL-3. The viability and expression of HLA-DR, CD80, and CD86 were assessed by using flow cytometry. HLA-DM expression was analyzed by means of immunoblotting. Allergen uptake was studied after fluorescence labeling of the major birch pollen allergen Bet v 1. Bet v 1 was digested with neutrophilic endolysosomal extracts, and the resulting fragments were sequenced by using mass spectrometry. Neutrophils were used as APCs in coculture experiments with autologous HLA-DR–restricted and Bet v 1–specific T-cell clones reactive with epitopes in different regions of the allergen. In all experiments monocytes were used for comparison. Fluids from suction blisters formed on top of LPRs induced by using intradermal allergen injection were assessed for HLA-DR+ neutrophils by using flow cytometry. Results The cytokines significantly enhanced the survival, allergen uptake, and expression of HLA-DM and HLA-DR on neutrophils. Neutrophils rapidly degraded Bet v 1 into fragments containing all relevant T-cell epitopes. Cytokine-activated, allergen-pulsed neutrophils induced proliferative and cytokine responses of Bet v 1–specific T cells irrespective of epitope specificity, confirming that they fully processed and presented the allergen. HLA-DR+ neutrophils were detected in patients with cutaneous allergic LPRs. Conclusion Neutrophils can serve as APCs for local allergen-specific effector T cells in patients with allergic LPRs.

In this paper, we report on the CH 2 -symmetric to CH 2 -antisymmetric stretch ratio as a method to determine membrane morphology alterations in mammalian cells. Cell membrane morphology is an important parameter for tumour development studies and for determining the lowest effective dose of antitumour agents, reducing the occurrence of side effects. For this study a normal epithelial kidney cell line and three human melanoma cell lines were investigated. The epithelial kidney cell line Madin-Darby Canine Kidney was exposed to the cholesterol reduction agent methyl- β -cyclodextrin. The cholesterol reduction changes the cell membrane morphology and the plasma membrane of the cells become less rigid. A thin layer chromatography analysis was performed into test the efficiency of the cholesterol reduction method. The antitumour agent cisplatin is known to cause the opposite effect and makes the cell membrane more rigid. In order to investigate the cell membrane morphology change three human melanoma cell lines were treated with cisplatin. For the CH 2 -stretch ratio measurements we used a self-designed infrared sensor system, which has the potential to be further developed in a point-of-care instrument. With the sensor system the morphology changes due to cholesterol reduction and cisplatin treatments were established. This study proves that the CH 2 -symmetric to CH 2 -antisymmetric stretch ratio can be successfully applied for label-free cell membrane morphology analysis.

Peanut allergy is an IgE-mediated severe hypersensitivity disorder. The lack of a treatment of this potentially fatal allergy has led to intensive research on vaccine development. Here, we describe the design and initial characterization of a carrier-bound peptide derived from the most potent peanut allergen, Ara h 2, as a candidate vaccine. Based on the adjuvant capability of bacterial surface (S-) layers, a fusion protein of the S-layer protein SlpB from Lactobacillus buchneri CD034 and the Ara h 2-derived peptide AH3a42 was produced. This peptide comprised immunodominant B-cell epitopes as well as one T cell epitope. The fusion protein SlpB-AH3a42 was expressed in E. coli, purified, and tested for its IgE binding capacity as well as for its ability to activate sensitized rat basophil leukemia (RBL) cells. The capacity of Ara h 2-specific IgG rabbit-antibodies raised against SlpB-AH3a42 or Ara h 2 to inhibit IgE-binding was determined by ELISA inhibition assays using sera of peanut allergic patients sensitized to Ara h 2. IgE specific to the SlpB-AH3a42 fusion protein was detected in 69% (25 of 36) of the sera. Despite the recognition by IgE, the SlpB-AH3a42 fusion protein was unable to induce β-hexosaminidase release from sensitized RBL cells at concentrations up to 100 ng per ml. The inhibition of IgE-binding to the natural allergen observed after pre-incubation of the 20 sera with rabbit anti-SlpB-AH3a42 IgG was more than 30% for four sera, more than 20% for eight sera, and below 10% for eight sera. In comparison, anti-Ara h 2 rabbit IgG antibodies inhibited binding to Ara h 2 by 48% ±13.5%. Our data provide evidence for the feasibility of this novel approach towards the development of a peanut allergen peptide-based carrier-bound vaccine. Our experiments further indicate that more than one allergen-peptide will be needed to induce a broader protection of patients allergic to Ara h 2.

An infrared (IR) absorbance sensor has been designed, realized and tested with the aim of detecting malignant melanomas in human skin biopsies. The sensor has been designed to obtain fast measurements (80 s) of a biopsy using a small light spot (0.5 mm in diameter, typically five to 10 times smaller than the biopsy size) to investigate different biopsy areas. The sensor has been equipped with a monochromator to record the whole IR spectrum in the 3330–3570 nm wavelength range (where methylene and methyl stretching vibrations occur) for a qualitative spectral investigation. From the collected spectra, the CH2 stretch ratio values (ratio of the absorption intensities of the symmetric to asymmetric CH2 stretching peaks) are determined and studied as a cancer indicator. Melanoma areas exhibit different spectral shapes and significantly higher CH2 stretch ratios when compared to healthy skin. The results of the infrared investigation are compared with standard histology. This study shows that the IR sensor is a promising supportive tool to improve the diagnosis of melanoma during histopathological analysis, decreasing the risk of misdiagnosis.

Allergies to certain fruits such as banana, avocado, chestnut and kiwi are described in 30–70% of latex-allergic patients. This association is attributed to the cross-reactivity between the major latex allergen hevein and hevein-like domains (HLDs) from fruit class I chitinases. We aimed to assess the extent of cross-reactivity between hevein and HLDs using sera from latex-allergic patients with and without plant food allergy. Hevein and HLDs of latex, banana, and avocado chitinases were expressed in Escherichia coli as fusion proteins with the maltose-binding protein and purified by affinity chromatography. IgE binding to these proteins was studied in sera from 59 latex-allergic patients and 20 banana-allergic patients without latex allergy by ELISA and ELISA inhibition. Additionally, 16,408 allergic patients’ sera were tested for IgE binding to hevein, latex chitinase, and wheat germ agglutinin using an allergen microarray. Hevein-specific IgE was detected in 34/59 (58%) latex-allergic patients’ sera. HLDs of latex, banana, and avocado chitinases were recognized by 21 (36%), 20 (34%), and 9 (15%) sera, respectively. In contrast, only one of 20 banana-allergic patients without latex allergy was sensitized to chitinase HLDs. In most tested latex-allergic patients’ sera, IgE binding to hevein was only partially reduced by preincubation with HLDs. Among hevein-sensitized, latex-allergic patients, the percentage of plant food allergy (15/34 = 44%) was equal to latex-allergic patients without hevein sensitization (11/25 = 44%). In the general allergic population, 230 of 16,408 sera (1.4%) reacted to hevein and/or a hevein-like allergen. Of these, 128 sera showed an isolated sensitization to hevein, whereas only 17 bound to latex chitinase or wheat germ agglutinin without hevein sensitization. In conclusion, the IgE response to HLDs is elicited by hevein as sensitizing allergen in most cases. Despite considerable cross-reactivity between these allergens, no correlation between latex-associated plant food allergy and sensitization to hevein or HLDs was found.

Summary Background Allergy to kiwifruit is increasingly reported across Europe. Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying. Objective To evaluate the usefulness of a component-based allergen microarray for the diagnosis of kiwifruit allergy in a large group of patients. Methods With an allergen microarray, we measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. Sera from 198 allergic patients without kiwifruit allergy served as controls. Furthermore, we determined the extent of sensitization to latex. Results The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Sera from kiwifruit-allergic patients contained significantly more frequently Act d 1-specific IgE than sera from control patients. Furthermore, 51% of the positive sera contained IgE directed to a single allergen, namely Act d 1 (45%), Act d 9 (27%) or Act d 7 (13%). Within the control group, 36% sera recognized a single allergen. Out of those, 48% were positive to the cross-reactive glycoallergen Act d 7, 43% to the profilin Act d 9 and only 5% to Act d 1. Allergen-specific IgG4 levels did not differ between kiwifruit-allergic and -tolerant patients. Kiwifruit- and latex-allergic patients contained Hev b 11-specific IgE significantly more frequently than latex-allergic patients without kiwifruit allergy. Conclusions Act d 1 can be considered a marker allergen for genuine sensitization to kiwifruit. We demonstrated that a component-based kiwifruit allergen microarray would improve the prognostic value of in vitro diagnostic tests. Cite this as: M. Bublin, S. Dennstedt, M. Buchegger, M. Antonietta Ciardiello, M. L. Bernardi, L. Tuppo, C. Harwanegg, C. Hafner, C. Ebner, B. K. Ballmer-Weber, A. Knulst, K. Hoffmann-Sommergruber, C. Radauer, A. Mari and H. Breiteneder, Clinical & Experimental Allergy, 2011 (41) 129–136.

The major birch pollen allergen Bet v 1 is present in pollen as a mixture of at least 14 isoforms that share high sequence and structural identities. These isoforms possess either a high or a low IgE-binding capacity which defines them as allergenic or hypoallergenic. Recently, we could demonstrate that only the allergenic isoform Bet v 1.0101 was able to induce an IgE response in birch pollen allergic individuals. The hypoallergenic isoforms Bet v 1.0401 and Bet v 1.1001 were unable to induce IgE synthesis. T-helper cell responses against allergens are characterised by increased levels of Th2 cytokines. Therefore, we examined extent and polarisation of the Th cell response and the kinetics of the allergen uptake after exposure of dendritic cells (DCs) to these isoforms. Monocyte-derived DCs (MDDCs) from birch pollen allergic and non-atopic individuals stimulated with Bet v 1.0101, Bet v 1.0401 or Bet v 1.1001 in combination with the maturation factors TNF-alpha and IL-1 beta resulted in a mature DC phenotype as measured by costimulatory molecule up-regulation. Only Bet v 1.0101-stimulated MDDCs from allergic subjects enhanced proliferation of autologous Th cells and the expression of the Th2 cytokines IL-5 and IL-13. Immature MDDCs of allergic individuals internalised equivalent amounts of the allergenic Bet v 1.0101 and the hypoallergenic Bet v 1.0401. In contrast, the uptake of the hypoallergenic Bet v 1.0401 by immature MDDCs of non-atopic individuals was significantly higher. These results provide evidence that DCs discriminate between allergens and highly related hypoallergens. This process may have an impact on the early phase of sensitisation. (C) 2009 Elsevier GmbH. All rights reserved

Purpose: The high molecular weight melanoma-associated antigen (HMW-MAA) is an attractive target for immunotherapy of malignant melanoma. We have recently generated a vaccine based on the HMW-MAA mimotope 225D9.2+ that was able to induce anti-HMW-MAA antibodies with antitumor activity in vitro. Here, we investigated the antitumor activity of these antibodies in a human melanoma xenotransplant severe combined immunodeficient (SCID) mouse model. Experimental Design: Tumors were established by injecting the human melanoma 518A2 cells into C.B.17 SCID/SCID mice. In tumor prevention experiments, 200 μg purified total IgG antibodies were injected intravenously the same day or on day 5 in therapeutic experiments. Antibody administration was repeated every fourth day and tumor volumes were measured. Antibody specificity and tumor infiltration by macrophages were investigated by immunohistochemistry. Results: Within 35 days after cell inoculation, antibody treatment reduced tumor growth up to 40% in the therapeutic and up to 62% in the tumor prevention experiments compared with the control mice. In tumors of all groups, a similar distribution of the HMW-MAA and no differences in infiltration of macrophages were detected by immunohistochemistry. Conclusions: Here, we showed that antibodies induced by the 225D9.2+ mimotope effectively inhibited melanoma tumor growth. Additional mechanisms besides antibody-dependent cell cytotoxicity like disruption of interactions of melanoma cells mediated by extracellular matrix components seem to be involved in tumor growth inhibition. Based on our findings, we suggest that active immunization with this mimotope might be a promising strategy for treatment of melanoma.

Background: The latex of Hevea brasiliensis trees contains a complex proteome that includes a range of allergenic proteins. Current latex extracts that are used for the diagnosis of latex allergy still lack important allergens. We aimed to devise a production process for an improved reagent that would ideally contain the complete latex allergome. Methods: Latex C-serum was fractionated by ammonium sulfate precipitation, and B- and C-serum proteins were then separated by anion exchange chromatography. Proteins eluting within defined salt concentration ranges were pooled into six final fractions. Fractions were evaluated by two-dimensional electrophoresis and subsequent IgE immunoblot for their spectrum of allergens. The presence of the most important latex allergens in the fractions was checked by Western blot analyses. Each fraction was further evaluated by skin prick test (SPT). Results: Reproducibility of the preparation method was demonstrated with two batches of latex. Comparison of latex B- and C-serum to the six fractions showed a remarkable increase in the number of detectable allergens in the fractions. The presence of the latex allergens Hev b 1–8 and Hev b 13 in the fractions was demonstrated. In SPTs, the fractions produced wheal-and-flare reactions comparable to commercial latex extracts. Conclusions: This method provides reproducible latex protein fractions of high allergen content for the diagnosis of latex allergy.

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.