Evaluation of in vitro Assays to Assess the Modulation of Dendritic Cells Functions by Therapeutic Antibodies and Aggregates.

Therapeutic antibodies have the potential to induce immunogenicity leading to the development of anti-drug antibodies (ADA) that consequently may result in reduced serum drug concentrations, a loss of efficacy or potential hypersensitivity reactions. Among other factors, aggregated antibodies have been suggested to promote immunogenicity, thus enhancing ADA production. Dendritic cells (DC) are the most efficient antigen-presenting cell population and are crucial for the initiation of T cell responses and the subsequent generation of an adaptive immune response. This work focuses on the development of predictive in vitro assays that can monitor DC maturation, in order to determine whether drug products have direct DC stimulatory capabilities. To this end, four independent laboratories aligned a common protocol to differentiate human monocyte-derived DC (moDC) that were treated with either native or aggregated preparations of infliximab, natalizumab, adalimumab, or rituximab. These drug products were subjected to different forms of physical stress, heat and shear, resulting in aggregation and the formation of subvisible particles. Each partner developed and optimized assays to monitor diverse end-points of moDC maturation: measuring the upregulation of DC activation markers via flow cytometry, analyzing cytokine, and chemokine production via mRNA and protein quantification and identifying cell signaling pathways via quantification of protein phosphorylation. These study results indicated that infliximab, with the highest propensity to form aggregates when heat-stressed, induced a marked activation of moDC as measured by an increase in CD83 and CD86 surface expression, IL-1ß, IL-6, IL-8, IL-12, TNFα, CCL3, and CCL4 transcript upregulation and release of respective proteins, and phosphorylation of the intracellular signaling proteins Syk, ERK1/2, and Akt. In contrast, natalizumab, which does not aggregate under these stress conditions, induced no DC activation in any assay system, whereas adalimumab or rituximab aggregates induced only slight parameter variation. Importantly, the data generated in the different assay systems by each partner site correlated and supported the use of these assays to monitor drug-intrinsic propensities to drive maturation of DC. This moDC assay is also a valuable tool as an in vitro model to assess the intracellular mechanisms that drive DC activation by aggregated therapeutic proteins.

MiR-210 promotes sensory hair cell formation in the organ of corti.

BACKGROUND: Hearing loss is the most common sensory defect afflicting several hundred million people worldwide. In most cases, regardless of the original cause, hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. Despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration, thereby providing new avenues for the treatment of hearing loss. RESULTS: In order to identify new genetic elements enabling regeneration of inner ear sensory hair cells, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent miRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on these miRSeq results eight most differentially expressed miRNAs were selected for further characterization. In UB/OC-1, miR-210 silencing in vitro resulted in hair cell marker expression, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. Using a lineage tracing mouse model, transdifferentiation of supporting epithelial cells was identified as the likely mechanism for this new hair cell formation. Potential miR-210 targets were predicted in silico and validated experimentally using a miR-trap approach. CONCLUSION: MiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells suggesting that miR-210 might be a potential new factor for hearing loss therapy. In addition, identification of inner ear pathways regulated by miR-210 identified potential new drug targets for the treatment of hearing loss.

Activation of the epidermal growth factor receptor promotes lymphangiogenesis in the skin.

BACKGROUND: The lymphatic vascular system regulates tissue fluid homeostasis and plays important roles in immune surveillance, inflammation and cancer metastasis. However, the molecular mechanisms involved in the regulation of lymphangiogenesis remain incompletely characterized. OBJECTIVE: We aimed to identify new pathways involved in the promotion of skin lymphangiogenesis. METHODS: We used a mouse embryonic stem cell-derived embryoid body vascular differentiation assay to investigate the effects of a selection of pharmacological agents with the potential to inhibit blood and/or lymphatic vessel formation. We also used a subcutaneous Matrigel assay to study candidate lymphangiogenesis factors as well as skin-specific transgenic mice. RESULTS: We found that compounds inhibiting the epidermal growth factor (EGF) receptor (EGFR) led to an impaired formation of lymphatic vessel-like structures. In vitro studies with human dermal lymphatic endothelial cells (LECs), that were found to express EGFR, revealed that EGF promotes lymphatic vessel formation. This effect was inhibited by EGFR-blocking antibodies and by low molecular weight inhibitors of the EGFR associated tyrosine kinase. Incorporation of EGF into a mouse matrigel plug assay showed that EGF promotes enlargement of lymphatic vessels in the skin in vivo. Moreover, transgenic mice with skin-specific overexpression of amphiregulin, another agonistic ligand of the EGFR, displayed an enhanced size and density of lymphatic vessels in the skin. CONCLUSION: These findings reveal that EGFR activation is involved in lymphatic remodeling and suggest that specific EGFR antagonists might be used to inhibit pathological lymphangiogenesis.