Bone consists of a complex mineralised matrix that is maintained by a controlled equilibrium of synthesis and resorption by different cell types. Hyaluronan (HA) is an important glycosaminoglycan in many tissues including bone.Previously, the importance of HA synthesis for bone development during embryogenesis has been shown. We therefore investigated whether HA synthesis is involved in adult bone turnover and whether abrogation of HA synthesis in adult mice would alter bone quality.To achieve complete abrogation of HA synthesis in adult mice, we generated a novel Has-total knockout (Has-tKO) mouse model in which a constitutive knockout of Has1 and Has3 was combined with an inducible, Ubc-Cre-driven Has2 knockout.By comparing bone tissue from wild-type, Has1,3 double knockout and Has-tKO mice, we demonstrate that Has2-derived HA mainly contributes to the HA content in bone. Furthermore, Has-tKO mice show a significant decrease of bone integrity in trabecular and cortical bone, as shown by µ-CT analysis. These effects are detectable as early as five weeks after induced Has2 deletion, irrespective of sex and progress with age.Mesenchymal stem cells (MSC) during osteogenic differentiation in vitro showed that Has2 expression is increased while Has3 expression is decreased during differentiation. Furthermore, the complete abrogation of HA synthesis results in significantly reduced osteogenic differentiation as indicated by reduced marker gene expression (Runx-2, Tnalp, Osterix) as well as alizarin red staining. RNAseq analysis revealed that MSC from Has-tKO are characterised by decreased expression of genes annotated for bone and organ development, whereas expression of genes associated with chemokine related interactions and cytokine signalling is increased.Taken together, we present a novel mouse model with complete deletion of HA synthases in adult mice which has the potential to study HA function in different organs and during age-related HA reduction. With respect to bone, HA synthesis is important for maintaining bone integrity, presumably based on the strong effect of HA on osteogenic differentiation.

Zusammenfassung Die Allergologie ist ein wesentlicher Bestandteil der dermatologischen Praxis. In dieser Arbeit wird ein Überblick gegeben zu ausgewählten Entwicklungen auf den Gebieten Pathophysiologie, Diagnostik und Therapie allergischer Erkrankungen vom Soforttyp. Die Typ‐2‐Inflammation spielt eine Rolle bei vielen allergischen Erkrankungen wie allergischer Rhinitis und Asthma. Bezüglich der bei diesen Krankheitsbildern therapeutisch wichtigen Allergen‐Immuntherapie (AIT) regelt in Deutschland die Therapieallergeneverordnung den Einsatz von Präparaten. Therapeutisch stehen bereits verschiedene Biologika zur Beeinflussung von Interleukin (IL)‐4, ‐5, ‐13, ‐33 oder TSLP (thymic stromal lymphopoietin) zur Verfügung. Im Sinne einer „kollateralen“ Effektivität können gleichzeitig auftretende allergische Erkrankungen heute vielfach durch ein einzelnes Therapeutikum behandelt werden. Bei Mastzell‐abhängigen Erkrankungen (Urtikaria, Anaphylaxie) hat sich das Verständnis für die Aktivierung über eine Vielzahl neu identifizierter Oberflächenrezeptoren wie MRGPRX2 (mas‐related G protein‐coupled receptor‐X2) und Siglec‐8 (Sialinsäure‐bindendes Ig‐ähnliches Lektin‐8) sowie für die intrazelluläre Signaltransduktion verbessert. In Studien werden Medikamente zur Beeinflussung der Oberflächenrezeptoren und auch der intrazellulären Signaltransduktion wie die Bruton‐Tyrosinkinase‐Inhibitoren untersucht. Beim hereditären Angioödem gehen die therapeutischen Entwicklungen in Richtung einer Langzeitprophylaxe mit Kallikrein‐Inhibitoren. Darüber hinaus werden weitere absehbare Perspektiven für potenzielle Biomarker, neue Therapeutika und Forschungsbedarf aufgezeigt.

Allergology is a key part of dermatological care. This paper reviews current pathophysiological, diagnostic and therapeutic developments in immediate-type allergies. Type-2 inflammation is involved in several allergological diseases such as allergic rhinitis and asthma. Allergen immunotherapy as an important therapeutic procedure is regulated in Germany by an official legal directive (Therapieallergene-Verordnung). Therapeutically, several biologics are already available that target interleukin (IL)-4, -5, -13, -33, or TSLP (thymic stromal lymphopoietin). Collateral efficacy may result in simultaneous treatment of allergological comorbidities. In mast cell mediated diseases (urticaria, anaphylaxis), there is increasing understanding of mast cell activation pathways. Several mast cell receptors such as MRGPRX2 (mas-related G protein coupled receptor X2) and Siglec-8 (sialinic acid binding Ig like lectin-8) as well as intracellular signaling pathways have recently been identified. Clinical trials are underway with drugs acting on mast cell receptors and intracellular signaling, i.e., Bruton's tyrosine kinase inhibitors. Further perspectives on biomarkers, novel therapeutics and unmet needs for future research activities are presented.

Background: The therapy of severe food allergy so far consists mainly of allergen ab-stinence , emergency treatment. The use of anti-IgE antibodies is reported to be prom-ising.Case report: We report on a 22-year -old male with severe cow's milk allergy with multiple anaphylactic reactions, known since infancy and persisting into adulthood with sometimes severe immediate type re-actions on unintended consumption. The prick test for native whole milk was positive, the CAP-FEIA was also positive for milk pro-tein, mare's milk, whey, sheep's milk whey as well as Bos d4, Bos d5 and Bos d8 and blue cheese, total IgE was 1,265 kU/l. The patient's history included well controlled bronchial asthma. An off-label therapy with omalizumab (3 x 150 mg/month s. c.) and cetirizine 10 mg once daily was initiated. Under this therapy, we performed a double-blind oral exposure test to cow's milk in the patient after long term. Thereby, 14 ml could be tolerated. After consumption of 30 ml of cow's milk, urticaria, dyspnea and angioede-ma occurred.".Conclusion: Under therapy with omalizumab, an increase of the toler-ance threshold to cow's milk was shown in our patient. As a consequence, reactions during accidental consumption could be prevented.

The green-lipped mussel (Perna canaliculus) originates from New Zealand. To preserve the health benefits of green-lipped mussel meat, it is freeze-dried to make a long-lasting powder. The powder is used to treat arthritis because of its potential anti-inflammatory properties. The report describes a 54-year-old woman who developed immediate rhinoconjunctival and respiratory symptoms after inhaling green-lipped mussel powder she gave to her dog for arthritis. A skin prick test with green-lipped mussel powder was performed. Protein extracts from P canaliculus were separated by sodium dodecyl-sulfate polyacrylamide (SDS) gel electrophoresis and probed with serum from patients and serum preincubated with green-lipped mussel extract. Bound immunoglobulin E (IgE) was detected by specific anti-human-IgE antibodies, and IgE-binding proteins were subsequently identified by liquid chromatography and mass spectrometry. The skin prick test was positive for green-lipped mussel. Specific IgE against green-lipped mussel extract was detected using Western immunoblotting. These potential allergenic proteins were identified by mass spectrometry as actin, tropomyosin, and paramyosin. All three allergens are reported for the first time for P canaliculus. Actin is a major allergen in Paphia textile, paramyosin in Sarcoptes scarbiei, and tropomyosin in Haliotis discus. For all IgE-binding proteins, the software AllCatPro predicted high allergenicity, supporting our conclusion that these proteins from P canaliculus may also be allergenic. The identification of allergens from P canaliculus provides the opportunity for specific tests to assess the frequency of allergic reactions to P canaliculus.

The phase 2 study IMMUNED demonstrated significantly longer relapse-free survival (RFS) of NIVO alone or in combination with IPI compared to placebo in stage IV melanoma patients (pts) with NED after surgery or radiotherapy (Zimmer L, et al; Lancet 2020; 395:1558-68). Final RFS and first and final OS data are presented. Pts aged ≥18 y with stage IV cutaneous or unknown primary melanoma with NED were randomly assigned 1:1:1 (stratified by trial site, site of metastasis and PD-L1 status) to either NIVO 1 mg/kg + IPI 3 mg/kg Q3W for 4 doses followed by NIVO 3 mg/kg Q2W (56 patients) or NIVO 3 mg/kg Q2W (n=59) or matching placebo (n=52) for up to 1 year, or until disease recurrence, unacceptable toxicity, or withdrawal of consent. RFS (ITT-population) was the primary endpoint, time to progression, OS and safety secondary endpoints. At a median follow-up of 49.2 months, NIVO and NIVO+IPI continued to demonstrate superior RFS vs placebo (Table). Overall, 36 OS events had occurred. Median OS was not reached in either group, risk of death was significantly lower for NIVO+IPI vs placebo (HR 0.41, 95% CI, 0.17-0.99) but not for NIVO alone vs placebo (HR 0.75, 95% CI, 0.36-1.56). Most patients of the placebo group with progression (32/42) received anti-PD-1 antibody containing treatment as first subsequent systemic therapy either as crossover or outside the trial. Treatment-related adverse events of grade 3/4 remained largely unchanged (70.9% for NIVO+IPI, 28.6% for NIVO).Table: 784ORFSOSNIVO+IPINIVOPlaceboNIVO+IPINIVOPlaceboNo. Events183842713161-year rate75.3%51.7%32.2%95.7%92.2%93.9%2-year rate66.5%36.9%15.0%83.8%75.3%68.0%3-year rate64.2%31.4%15.0%83.8%75.3%68.0%4-year rate64.2%31.4%15.0%83.8%72.6%63.1%Median (months)NR12.36.3NRNRNRHR (97.5% CI for RFS, 95% CI for OS) vs. PLA0.25 (0.13-0.48)0.60 (0.36-1.00)0.41 (0.17-0.99)0.75 (0.36-1.56)Log-rank p-value (vs. PLA)<0.00010.02360.03960.4423HR (95% CI) NIVO+IPI vs. NIVO0.41 (0.23-0.72)0.55 (0.22-1.38)Log-rank p-value (vs. NIVO)0.00130.1969HR: hazard ratio; CI: Confidence interval; NR: not reached. Open table in a new tab HR: hazard ratio; CI: Confidence interval; NR: not reached. NIVO and NIVO+IPI continued to demonstrate improved RFS in stage IV patients at high risk of recurrence. OS was markedly improved for patients receiving NIVO+IPI compared with placebo. Use of subsequent anti-PD-1 based therapy was high in placebo pts and most likely impacted the OS comparison of NIVO mono vs placebo.

ZusammenfassungZiel Darstellung der aktuellen Literatur und neuer Erkenntnisse zum Zusammenhang der chronisch spontanen Urtikaria (CSU) mit Adipositas-assoziierter Inflammation und metabolischen Erkrankungen.Methodik Eine englischsprachige, Pubmed-basierte Literaturrecherche mit den Stichwörtern „chronic urticaria“ und „hypertension“, „hyperlipidemia“, „metabolic syndrome“, „metabolic diseases“, „obesity“, „overweight“, „glucose intolerance“, „diabetes“ sowie zu „cytokines“, „pro-inflammatory“, „adipokines“, „immunological dysregulation“.Ergebnisse In der aktuellen Literatur zeigen sich erhöhte Prävalenzen für Adipositas, Diabetes mellitus, Hyperlipidämie und Hypertonie bei CSU-Patienten. Zudem zeigen sich signifikante Assoziationen zwischen metabolischen Komorbiditäten und der klinischen Ausprägung, Krankheitsdauer oder dem therapeutischen Ansprechen bei der CSU. Darüber hinaus lassen sich gemeinsame immunologische Merkmale zwischen der CSU und Adipositas erkennen, da eine Dysbalance pro- und anti-inflammatorisch wirksamer Zytokine sowie Adipokine zugunsten eines pro-inflammatorischen Zustandes bei beiden Erkrankungen vorliegt. Die Ableitung klinischer Implikationen in Hinblick auf Screening-, Präventions- oder Interventionsmaßnahmen metabolischer Erkrankungen bei der CSU sollte diskutiert werden.

This project focuses on elucidating the pathogenic effects of iron overload due to erythrocyte extravasation in the skin on the immune-tissue cell crosstalk leading to lipodermatosclerosis and leg ulcer in patients with chronic venous insufficiency. We generated a new mice model with local iron-overload in the skin, via ID-injection of iron-dextran (Fedx). Prussian blue staining proved that iron accumulates in the dermis and in the dWAT of these mice, like in patients' skin. Skin of Fedx mice shows signs of lipodermatosclerosis; an increase of total cell count (p=0.001) in the dermal layer, particularly of F4/80+ macrophages (p=0.019) and PDGFRa+ fibroblasts (p<0.001), accompanied with less collagen in the deeper dermis and a reduction in the size of the dWAT (p<0.001) and of the adipocytes (p<0.001). Microscopic and gene expression analysis highlight lipolysis, the consequent reduction of lipids droplets (p=0.04), and loss of plin-1 (p=0.004) on adipocytes membrane, associated with downregulation of pro-adipogenic genes in the dWAT of Fedx mice. FACS analysis reveals a shift in F4/80+ Macrophage subtypes with the induction of TNFa (p=0.0163) and reduction of CD301b and Relm-a (both p<0.001). Upregulation of CCL2 (p=0.02), IL1ß, HMOX-1 (p=0.0001 dermis, p=0.01 dWAT) and CCR2 (p=0.01) genes, indicate for a pro-inflammatory activation in the dermal compartment. Mechanistic analysis with human cells in vitro shows that erythrocyte-uptake induces ROS and a pro-inflammatory phenotype in Macrophages. Fibroblasts in response to erythrocyte-fed macrophages resemble those phenotypes found in Fedx mice. The impact of erythrocytes on the differentiation and activation of adipocytes and their crosstalk to Macrophages and Fibroblasts is ongoing. Our data suggest that local iron-overload causes a complex skin phenotype with a maintained inflammatory status in the dermis, proliferative and less fibrogenic fibroblasts, impaired adipogenesis, and increased lipolysis.

Wound healing of acute full-thickness injuries and chronic non-healing ulcers leads to delayed wound closure, prolonged recovery period and hypertrophic scarring, generating a demand for an autologous cell therapy and a relevant pre-clinical research models for wound healing. In this study, an immunocompetent model for wound healing was employed using a syngeneic murine cell line of mesenchymal stem cells cultured from the mouse whisker hair follicle outer root sheath (named moMSCORS). moMSCORS were isolated using an air-liquid interface method, expanded in vitro and characterized according to the MSC definition criteria - cell viability, in vitro proliferation, MSC phenotype and multi-lineage differentiations. Moreover, upon applying moMSCORS in an in vivo full-thickness wound model in the syngeneic C57BL/6 mice, the treated wounds displayed different morphology to that of the untreated wound beds. Quantitative evaluation of angiogenesis, granulation and wound closure involving clinical scoring and software-based quantification indicated a lower degree of inflammation in the treated wounds. Histological staining of treated wounds by the means of H&E, Alcian Blue, PicroSirius Red and αSMA immune labelling showed lower cellularity, less collagen filaments as well as thinner dermal and epidermal layers compared with the untreated wounds, indicating a general reduction of hypertrophic scars. The decreased inflammation, accelerated wound closure and non-hypertrophic scarring, which were facilitated by moMSCORS, hereby address a common problem of hypertrophic scars and non-physiological tissue properties upon wound closure, and additionally offer an in vivo model for the autologous cell-based wound healing.

9548 Background: Immunotherapies (ICI) and targeted therapies (TT) have improved PFS and OS in BRAFV600-mutated advanced melanoma pts, but evidence regarding their optimal sequence is limited. The randomized phase 2 ImmunoCobiVem study evaluated efficacy and safety of an early switch to Atezo after initial treatment with V + C. Interim results are reported. Methods: Pts with previously untreated BRAFV600-mutated advanced melanoma received a 3-mo run-in with V (960 mg twice daily) + C (60 mg once daily for 21/28 days). Pts without PD/treatment interruption due to AEs during run-in were then randomized 1:1 to continue V + C (Arm A) or switch to Atezo (1200 mg every 3 wks; Arm B) until first documented PD (PD1), followed by crossover to the alternate treatment until second documented PD (PD2). End points were PFS1 (time from start of run-in until PD1 or death from any cause), PFS2 (time from start of run-in until PD2 or death from any cause), PFS3 (time from PD1 until PD2 or death from any cause), DCR, ORR, OS, and safety. Results: 185 pts were enrolled between Nov 2016 and Dec 2019 (63% male; median age 58 y); 135 pts completed run-in and were randomized to Arm A (n=69) or Arm B (n=66). At data cutoff, median follow-up for all pts was 19.0 mo. In Arm A, 36/69 pts (52%) discontinued V + C due to PD and 21/36 (58%) crossed over to Atezo; in Arm B, 49/66 pts (74%) discontinued Atezo due to PD and 35/49 (71%) crossed over to V + C. Median PFS1 was significantly longer in Arm A vs Arm B (HR 0.55; 95% CI 0.37–0.84; P=0.001), while median PFS3 was significantly shorter in Arm A vs Arm B (HR 2.24; 95% CI 1.17–4.30; P=0.013); median PFS2 was not significantly different between arms (HR 1.57; 95% CI 0.83–2.96; P=0.163) (Table). During the randomized phase, ORR and DCR were higher in Arm A before crossover and in Arm B after crossover (Table). OS was similar between arms (HR 1.22; 95% CI 0.69–2.16; P=0.389). Median (range) treatment duration across treatment phases was 11.2 mo (2.3–56.1) for Arm A and 10.7 mo (2.8–56.7) for Arm B. Grade 3/4 AEs occurred in 55% of pts in Arm A and 64% in Arm B; AEs led to discontinuation in 10% and 12%, respectively. Conclusions: Early switch from V + C to Atezo is feasible and safe, but tumor control achieved in run-in is maintained in only a subset of pts on subsequent ICI monotherapy. Crossover to ICI monotherapy at PD results in low response, while response to TT re-exposure is frequent. Clinical trial information: NCT02902029. [Table: see text]