Antibody Profiling and In Silico Functional Analysis of Differentially Reactive Antibody Signatures of Glioblastomas and Meningiomas.

Studies on tumor-associated antigens in brain tumors are sparse. There is scope for enhancing our understanding of molecular pathology, in order to improve on existing forms, and discover new forms, of treatment, which could be particularly relevant to immuno-oncological strategies. To elucidate immunological differences, and to provide another level of biological information, we performed antibody profiling, based on a high-density protein array (containing 8173 human transcripts), using IgG isolated from the sera of n = 12 preoperative and n = 16 postoperative glioblastomas, n = 26 preoperative and n = 29 postoperative meningiomas, and n = 27 healthy, cancer-free controls. Differentially reactive antigens were compared to gene expression data from an alternate public GBM data set from OncoDB, and were analyzed using the Reactome pathway browser. Protein array analysis identified approximately 350-800 differentially reactive antigens, and revealed different antigen profiles in the glioblastomas and meningiomas, with approximately 20-30%-similar and 10-15%-similar antigens in preoperative and postoperative sera, respectively. Seroreactivity did not correlate with OncoDB-derived gene expression. Antigens in the preoperative glioblastoma sera were enriched for signaling pathways, such as signaling by Rho-GTPases, COPI-mediated anterograde transport and vesicle-mediated transport, while the infectious disease, SRP-dependent membrane targeting cotranslational proteins were enriched in the meningiomas. The pre-vs. postoperative seroreactivity in the glioblastomas was enriched for antigens, e.g., platelet degranulation and metabolism of lipid pathways; in the meningiomas, the antigens were enriched in infectious diseases, metabolism of amino acids and derivatives, and cell cycle. Antibody profiling in both tumor entities elucidated several hundred antigens and characteristic signaling pathways that may provide new insights into molecular pathology and may be of interest for the development of new treatment strategies.

Defending Dynepo detection.

Doping analysis as the detection of prohibited substances in an athlete's bodily specimen is not solely a scientific task; anti-doping scientists are challenged by the athlete's experts and have to defend their results in front of arbitration panels such as the Court of Arbitration in Sports (CAS). Compliance with the internationally accepted standards issued by the World Anti-Doping Agency (WADA) is commonly the main aspect to prove and demonstrate. Taking the example of four cases of doping with Epoetin delta (Dynepo) in endurance disciplines like marathon, triathlon, and cycling, the challenges and experiences in court and the argumentation lines of the defence experts are discussed. In all cases, doping with Epoetin delta was detected by two methods: isoelectric focusing (IEF) and SDS-PAGE. Epoetin delta is known to be produced in a human fibrosoma cell line. The slightly more abundant bands alpha and beta result in a short detection window using IEF analysis alone. With the additional complementary information obtained by SDS-PAGE analysis, data interpretation and defence in court is facilitated.

SDS-PAGE of recombinant and endogenous erythropoietins: benefits and limitations of the method for application in doping control.

Doping of athletes with recombinant and genetically modified erythropoietins (EPO) is currently detected by isoelectric focusing (IEF). The application of these drugs leads to a significant change in the isoform profile of endogenous urinary erythropoietin (uhEPO). Dynepo, MIRCERA, biosimilars with variable IEF-profiles as well as active urines and effort urines have made additional testing strategies necessary. The new generation of small molecule EPO-receptor stimulating agents like Hematide will also challenge the analytical concept of detecting the abuse of erythropoiesis stimulating agents (ESA). By determining their apparent molecular masses with SDS-PAGE a clear differentiation between endogenous and exogenous substances also concerning new EPO modifications is possible. Due to the orthogonal character of IEF- and SDS-PAGE both methods complement each other. The additional benefits of SDS-PAGE especially in relation to active and effort urines as well as the detection of Dynepo were investigated. Due to significant differences between the apparent molecular masses of uhEPO/serum EPO (shEPO) and recombinant, genetically or chemically modified erythropoietins the presence of active or effort urines was easily revealed. The characteristic band shape and apparent molecular mass of Dynepo on SDS-PAGE additionally evidenced the presence of this substance in urine. A protocol for the detection of EPO-doping in serum and plasma by SDS-PAGE was developed. Blood appears to be the ideal matrix for detecting all forms ESA-doping in the future.