Ludwig-Maximilians-Universität München

Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains.

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains.

Radiochemotherapy with or without cetuximab for unresectable esophageal cancer: final results of a randomized phase 2 trial (LEOPARD-2).

To investigate the efficacy and toxicity of cetuximab when added to radiochemotherapy for unresectable esophageal cancer. This randomized phase 2 trial (clinicaltrials.gov, identifier NCT01787006) compared radiochemotherapy plus cetuximab (arm A) to radiochemotherapy (arm B) for unresectable esophageal cancer. Primary objective was 2‑year overall survival (OS). Arm A was considered insufficiently active if 2‑year OS was ≤40% (null hypothesis = H), and promising if the lower limit of the 95% confidence interval was >45%. If that lower limit was >40%, H was rejected. Secondary objectives included progression-free survival (PFS), locoregional control (LC), metastases-free survival (MFS), response, and toxicity. The study was terminated early after 74 patients; 68 patients were evaluable. Two-year OS was 71% in arm A (95% CI: 55-87%) vs. 53% in arm B (95% CI: 36-71%); H was rejected. Median OS was 49.1 vs. 24.1 months (p = 0.147). Hazard ratio (HR) for death was 0.60 (95% CI: 0.30-1.21). At 2 years, PFS was 56% vs. 44%, LC 84% vs. 72%, and MFS 74% vs. 54%. HRs were 0.51 (0.25-1.04) for progression, 0.43 (0.13-1.40) for locoregional failure, and 0.43 (0.17-1.05) for distant metastasis. Overall response was 81% vs. 69% (p = 0.262). Twenty-six and 27 patients, respectively, experienced at least one toxicity grade ≥3 (p = 0.573). A significant difference was found for grade ≥3 allergic reactions (12.5% vs. 0%, p = 0.044). Given the limitations of this trial, radiochemotherapy plus cetuximab was feasible. There was a trend towards improved PFS and MFS. Larger studies are required to better define the role of cetuximab for unresectable esophageal cancer.

Successful decellularization of thick-walled tissue: Highlighting pitfalls and the need for a multifactorial approach.

Decellularization of thick tissue is challenging and varying. Therefore, we tried to establish a multifactorial approach for reliable aortic wall decellularization. Porcine aortic walls were decellularized according to different procedures. Decellularization was performed for 24 (G1), 48 (G2), and 72 h (G3) with a solution of 0.5% desoxycholate and 0.5% dodecyl sulfate. The procedure was characterized using intermittent washing steps, the inclusion of sonication as well as DNase and α-galactosidase treatment. The decellularization efficiency was measured by the evaluation of 4',6-diamidino-2-phenylindole and hematoxylin and eosin staining and quantitative DNA assays. Pentachrome and picrosirius red staining, scanning electron microscopy as well as glycosaminoglycan assays were performed to evaluate the effect of the procedure on the extracellular matrix. 4',6-Diamidino-2-phenylindole and hematoxylin and eosin staining revealed a large amount of remaining nuclei in all groups. However, consecutive DNase treatment had a significant effect. While the remaining DNA was detected in some samples of G1 and G2, samples of G3 were fully decellularized. Glycosaminoglycan content was significantly reduced to 50% after 24 h (G1) but remained constant for G2 and G3. Picrosirius red staining revealed an intact and stable collagen network without any visible defects. Pentachrome staining substantiated these results. Nonetheless, the fiber network remains intact, which could be confirmed by reflection electron microscopy analysis. In this study, we developed a procedure that grants successful decellularization of porcine aortic wall while maintaining the fibrous microstructure. We highlighted the significant effect of DNase and α-galactosidase treatment. In addition, we could show the need for a multifactorial treatment and comprehensive evaluation protocols for thick tissue decellularization.

Meat allergy associated with galactosyl-α-(1,3)-galactose (α-Gal)-Closing diagnostic gaps by anti-α-Gal IgE immune profiling.

Glycoproteins and glycolipids of some mammalian species contain the disaccharide galactosyl-α-(1,3)-galactose (α-Gal). It is known that α-Gal is immunogenic in humans and causes glycan-specific IgG and also IgE responses with clinical relevance. α-Gal is part of the IgE-reactive monoclonal therapeutic antibody cetuximab (CTX) and is associated with delayed anaphylaxis to red meat. In this study, different α-Gal-containing analytes are examined in singleplex and multiplex assays to resolve individual sensitization patterns with IgE against α-Gal. Three serum groups, α-Gal-associated meat allergy (MA) patients, idiopathic anaphylaxis (IA) patients with suspected MA, and non-meat-allergic healthy control individuals (HC), were analyzed via singleplex allergy diagnostics and a newly established immunoblot diagnostic system. The new dot blot detection system resolved individual IgE sensitization profiles for α-Gal-containing analytes CTX, bovine thyroglobulin (Bos d TG), and human serum albumin (HSA)-conjugated α-Gal. Singleplex allergy diagnostics using the α-Gal analytes CTX and Bos d TG confirms the history of MA patients in 91% and 88% of the cases, respectively. A novel dot blot-based assay system for the detection of IgE against α-Gal reveals individual IgE sensitization profiles for α-Gal-containing analytes. An α-Gal-associated IgE cross-reactivity profile (IgE against CTX, Bos d TG, and HSA-α-Gal) was identified, which is associated with MA. Detection of individual sensitization patterns with different α-Gal-containing analytes provides the basis for an individual allergy diagnosis for α-Gal-sensitized patients. Higher amounts of α-Gal in pork and beef innards compared to muscle meat as indicated by a higher staining intensity are a plausible explanation for the difference in allergic symptom severity.

Feasibility of 6-month maintenance cetuximab after adjuvant concurrent chemoradiation plus cetuximab in squamous cell carcinoma of the head and neck.

Close resection margins < 5 mm (CM) or extra capsular extent at the lymph nodes (ECE) impair the prognosis of patients with squamous cell cancer of the head and neck (SCCHN) scheduled for adjuvant radiochemotherapy. We conducted a multicenter phase II study to investigate toxicity and efficacy of additional cetuximab administered concomitantly and as maintenance for the duration of 6 months following adjuvant radiochemotherapy., Ppreliminary results on feasibility and acute toxicity on skin and mucosa are presented in this article. Patients with SCCHN following CM resection or with ECE were eligible for the study. In all, 61.6 Gy (1.8/2.0/2.2 Gy, days 1-36) were administered using an integrated boost intensity-modulated radiotherapy (IMRT) technique. Cisplatin (20 mg/m(2), days 1-5 and days 29-33) and 5-fluorouracil (5-FU) as continuous infusion (600 mg/m(2), days 1-5 + days 29-33) were given concurrently. Cetuximab was started 7 days prior to radiochemotherapy at 400 mg/m(2) followed by weekly doses of 250 mg/m(2). Maintenance cetuximab began after radiochemotherapy at 500 mg/m(2) every 2 weeks for 6 months. Of the 55 patients (46 male, 9 female, mean age 55.6, range 29-70 years) who finished radiochemotherapy, 50 were evaluable for acute toxicity concerning grade III/IV toxicities of skin and mucosa. Grade 3-4 (CTC 3.0) mucositis, radiation dermatitis, and skin reactions outside the radiation portals were documented for 46, 28, and 14 % of patients, respectively. One toxic death occurred (peritonitis at day 57). Cetuximab was terminated in 5 patients due to allergic reactions after the first application. In addition, 22 % of patients discontinued cetuximab within the last 2 weeks or at the end of radiochemotherapy. Of patients embarking on maintenance treatment, 80 % were still on cetuximab at 3 months and 63 % at 5 months. Concurrent and maintenance treatment with cetuximab could be administered as scheduled in 48 % of patients. Adjuvant radiochemotherapy with concomitant and maintenance cetuximab is feasible and acute toxicities are within the expected range. Compliance within the first 3-5 months is moderate.

α-N-Linked glycopeptides: conformational analysis and bioactivity as lectin ligands.

Natural N-glycosylation involves a β-anomeric linkage connecting the sugar to one asparagine residue of the protein. We herein report NMR- and modelling-based data on glycomimetics containing α-glycosidic linkages. The bioactivity of α-Gal-containing glycopeptides has been documented by revealing binding to two plant lectins, i.e. a potent β-trefoil toxin (Viscum album agglutinin) and β-sandwich lectin (Erythrina corallodendron agglutinin), by NMR protocols. Docking provided insights into the 3D structures of the resulting complexes. These results provide the basis to introduce α-substituted neoglycopeptides to the toolbox of scaffold for the design of potent lectin inhibitors.

Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation.

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.

Suitability of binary mixtures of water with aprotic solvents to turn hydroxyl protons of carbohydrate ligands into conformational sensors in NOE and transferred NOE experiments.

The structural analysis of protein-carbohydrate interactions is essential for the long-range aim to sort out entropic/ enthalpic factors in the binding process. Of conspicuous clinical interest, this work can also offer the perspective to devise new classes of therapeuticals which interfere with disease-related glycan recognition. We have shown that it is possible to use exchangeable hydroxyl protons of carbohydrate ligands as conformational sensors for defining their bound-state topology by measurements in dimethyl sulfoxide(d6) (Siebert et al. (2000) ChemBioChem, 1, 181-195). However, the proteins are required to maintain binding capacity in the aprotic solvent. To define conditions to limit its harmful effect on sensitive protein structures while still being able to pick up solvent-exchangeable hydroxyl signals we systematically tested binary solvent mixtures of dimethyl sulfoxide and acetone with water. These solvent mixtures did not preclude to monitor hydroxyl protons of carbohydrate ligands even at temperatures well above 0 degrees C. Notably, hydrogen bonding of the two tested disaccharides (Galbeta1-4Glcalpha/beta and Galalpha1-3Galalpha/beta or Galalpha1-3Galbeta1-OCH(3)), which are common lectin ligands, resembled the situation under physiological conditions. Also, a refined topological description for hydroxyl positioning could be achieved for Galalpha1-3Gal. At least equally important, this approach worked for elucidation of the mistletoe-lectin-bound topology of lactose in its syn-conformation with indication for formation of a characteristic interresidual hydrogen bond. These measurements were performed in a binary dimethyl sulfoxide(d6):water mixture (6:4 ratio, v/v) at -12 degrees C and encourage to pursue this line of investigation by monitoring in the course of stepwise temperature increases. Our experiments reveal that binary mixtures have favorable properties for the conformational analysis of the free- and bound-state topologies of bioactive ligands.

Antibodies against galactosyl (alpha 1----3) galactose in connective tissue diseases.