Pre-Existing Humoral Immunity Enhances Epicutaneously-Administered Allergen Capture by Skin DC and Their Migration to Local Lymph Nodes.

Due to its richness in antigen presenting cells, e.g., dendritic cells (DC), the skin has been identified as a promising route for immunotherapy and vaccination. Several years ago, a skin delivery system was developed based on epicutaneous patches allowing the administration of antigen through intact skin. Using mouse models, we have shown that epicutaneous allergen application leads to a rapid uptake and transport of allergen-positive cells to skin-draining lymph nodes (LN). This occurred primarily in animals previously sensitized to the same allergen. In that context, we sought to better understand the role of the specific preexisting immunity in allergen capture by skin DC and their subsequent migration to LN. Specifically, we investigated the role of humoral immunity induced by sensitization and the involvement of IgG Fc receptors (FcγR). Epicutaneous patches containing fluorescently-labeled ovalbumin (OVA) were applied to naïve mice that had previously received either sera or purified IgG isolated from OVA-sensitized mice. To investigate the involvement of FcγR, animals received 2.4G2 (anti-FcγRII/RIII) blocking antibody, 24 hours before patch application. Mice that received sera or purified IgG originating from OVA-sensitized mice showed an increase in the quantity of OVA-positive DC in skin and LN. Moreover, the blockade of FcγR reduced the number of OVA-positive DC in LN to a level similar to that observed in naïve animals. Overall, these results demonstrate that preexisting specific-IgG antibodies are involved in allergen capture by skin DC following EPIT through the involvement of antigen-specific IgG-FcγR.

Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis.

BACKGROUND: The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children. OBJECTIVE: We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model. METHODS: The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified. RESULTS: Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis. CONCLUSION: We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

In-Vitro and In-Vivo Establishment and Characterization of Bioluminescent Orthotopic Chemotherapy-Resistant Human Osteosarcoma Models in NSG Mice.

Osteosarcoma, the most common bone malignancy with a peak incidence at adolescence, had no survival improvement since decades. Persistent problems are chemo-resistance and metastatic spread. We developed in-vitro osteosarcoma models resistant to chemotherapy and in-vivo bioluminescent orthotopic cell-derived-xenografts (CDX). Continuous increasing drug concentration cultures in-vitro resulted in five methotrexate (MTX)-resistant and one doxorubicin (DOXO)-resistant cell lines. Resistance persisted after drug removal except for MG-63. Different resistance mechanisms were identified, affecting drug transport and action mechanisms specific to methotrexate (RFC/SCL19A1 decrease, DHFR up-regulation) for MTX-resistant lines, or a multi-drug phenomenon (PgP up-regulation) for HOS-R/DOXO. Differential analysis of copy number abnormalities (aCGH) and gene expression (RNAseq) revealed changes of several chromosomic regions translated at transcriptomic level depending on drug and cell line, as well as different pathways implicated in invasive and metastatic potential (e.g., Fas, Metalloproteinases) and immunity (enrichment in HLA cluster genes in 6p21.3) in HOS-R/DOXO. Resistant-CDX models (HOS-R/MTX, HOS-R/DOXO and Saos-2-B-R/MTX) injected intratibially into NSG mice behaved as their parental counterpart at primary tumor site; however, they exhibited a slower growth rate and lower metastatic spread, although they retained resistance and CGH main characteristics without drug pressure. These models represent valuable tools to explore resistance mechanisms and new therapies in osteosarcoma.