Degradation of Airway Secretory Cell Mucin Granules Is Dependent on Lysosome Activity

Inflammatory airway diseases (e.g. COPD and asthma) are associated with mucous cell metaplasia and mucin hypersecretion, resulting in symptoms such as shortness of breath and cough. However, how secretory cells remove excess mucin granules is poorly understood. Previous research suggests that intracellular degradation pathways, such as autophagy, are involved in the degradation of mucin granules during resolution of mucous cell metaplasia. We thus hypothesized that the elimination of excess mucin granules is dependent on lysosome-mediated degradation in airway secretory cells. Calu-3 cells, an airway epithelial cell line containing abundant mucin granules, were treated with inhibitors of lysosome acidification (Bafilomycin A1) and lysosome enzyme activity (Pepstatin E64d). We found statistically significant increases in the levels of secretory mucin, MUC5AC, by mucin blot, suggesting that the lysosome mediates the elimination of mucin granules. In addition, by immunoblot we observed an increase in the autophagosome markers, LC3-II and SQSTM1, with lysosome inhibition using Bafilomycin A1, indicating an accumulation of autophagosomes and a role for autophagy in the degradation of mucin granules. However, after transfecting Calu-3 cells with a ubiquitin-hemagglutinin tag plasmid to examine the role of the proteasome in the degradation of mucin granules, we observed that our transfection efficiency was low, making it difficult to detect the hemagglutinin epitope by immunoblots. Nevertheless, we found that MUC5AC levels preliminarily increase with the inhibition of the proteasome using MG-132, suggesting a potential role for the proteasome in the degradation of mucin granules. Thus, we can conclude that inhibition of the lysosome increases MUC5AC levels, demonstrating that the lysosome mediates the degradation of mucin granules in airway secretory cells. In addition, while we were not able to conclude that the proteasome is involved in the degradation of mucin granules with certainty, our preliminary data suggests that it is possible that the ubiquitin-proteasome system is involved in the degradation of mucin due to the observed increase in MUC5AC levels with MG-132.https://digitalcommons.unmc.edu/surp2022/1039/thumbnail.jp

The Pudding of Trust

Trust - "reliance on the integrity, ability, or character of a person or thing" - is pervasive in social systems. We constantly apply it in interactions between people, organizations, animals, and even artifacts. We use it instinctively and implicitly in closed and static systems, or consciously and explicitly in open or dynamic systems. An epitome for the former case is a small village, where everybody knows everybody, and the villagers instinctively use their knowledge or stereotypes to trust or distrust their neighbors. A big city exemplifies the latter case, where people use explicit rules of behavior in diverse trust relationships. We already use trust in computing systems extensively, although usually subconsciously. The challenge for exploiting trust in computing lies in extending the use of trust-based solutions, first to artificial entities such as software agents or subsystems, then to human users' subconscious choices

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

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