Effective detection of proteins following electrophoresis using extracts of locally available food species

Procedures in life sciences research laboratories often require chemicals and plasticware that are costly, toxic or pose a risk to the environment. Therefore, sustainable alternatives would be of interest, provided that they generate suitable data quality. Coomassie blue and silver staining are the most widely used methods for detecting proteins following electrophoresis in the laboratory. However, their use presents challenges in terms of safety and waste management. In the current study, aqueous extracts were prepared from a series of common food species and evaluated as alternative stains for protein detection. Beets, blueberries, purple cabbage, raspberries and strawberries were employed to stain identical proteins separated under the same conditions in electrophoresis gels. Extracts of the first two species resulted in protein bands that were detectable through visible light transillumination, whereas extracts from all five species generated specific protein bands under ultraviolet light. The raspberry-derived extract was selected for further study based on the brightness of the fluorescent protein bands and minimal background staining. For both bovine serum albumin and lysozyme at 2.5 μg and 0.5 μg protein per band, the mean signal intensities obtained with raspberry extract staining were just below half of those obtained with Coomassie blue. Furthermore, the mean intensities using raspberry extract were equivalent to those obtained using Coomassie blue in the detection of 0.1 μg protein. Therefore, raspberry could be used to produce an effective stain for the routine laboratory analysis of proteins

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival