Strong evidence exists for a central role of amyloid β-protein (Aβ) oligomers in the pathogenesis of Alzheimer’s disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aβ aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAβ3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aβ aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease.

Background and PurposeTransient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation.Experimental ApproachWhole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances.Key ResultsPregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels.Conclusions and ImplicationsOur data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels.

Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.

Canine mammary carcinoma is a highly metastatic tumor that is poorly responsive to available treatment. Therefore, there is an urgent need to identify novel agents for therapy of this disease. Recently, we reported that the oncolytic vaccinia virus GLV-1h68 could be a useful tool for therapy of canine mammary adenoma in vivo. In this study we analyzed the therapeutic effect of GLV-1h68 against canine mammary carcinoma. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the mammary carcinoma cell line MTH52c. Furthermore, after systemic administration, this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. Finally, infection with GLV-1h68 led to strong inflammatory and oncolytic effects resulting in significant growth inhibition of the tumors. In summary, the data showed that the GLV-1h68 virus strain has promising potential for effective treatment of canine mammary carcinoma.

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ1–42 species, reduced Aβ1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.