Multiphoton High-Resolution 3D Imaging of Langerhans Cells and Keratinocytes in the Mouse Skin Model Adopted for Epidermal Powdered Immunization

Abstract

Langerhans cells (LCs) can be targeted with DNA-coated gold micro-projectiles ("Gene Gun") to induce potent cellular and humoral immune responses. It is likely that the relative volumetric distribution of LCs and keratinocytes within the epidermis impacts on the efficacy of Gene Gun immunization protocols. This study quantified the three-dimensional (3D) distribution of LCs and keratinocytes in the mouse skin model with a near-infrared multiphoton laser-scanning microscope (NIR-MPLSM). Stratum corneum (SC) and viable epidermal thickness measured with MPLSM was found in close agreement with conventional histology. LCs were located in the vertical plane at a mean depth of 14.9 microm, less than 3 mum above the dermo-epidermal boundary and with a normal histogram distribution. This likely corresponds to the fact that LCs reside in the suprabasal layer (stratum germinativum). The nuclear volume of keratinocytes was found to be approximately 1.4 times larger than that of resident LCs (88.6 microm3). Importantly, the ratio of LCs to keratinocytes in mouse ear skin (1:15) is more than three times higher than that reported for human breast skin (1:53). Accordingly, cross-presentation may be more significant in clinical Gene Gun applications than in pre-clinical mouse studies. These interspecies differences should be considered in pre-clinical trials using mouse models.

Improving the reach of vaccines to low-resource regions, with a needle-free vaccine delivery device and long-term thermostabilization

“…In human skin, the density of the characterized APCs is actually comparable with that in mouse skin (e.g. Langerhans cell density is 1052±109 mm -2 in mouse skin [37], and 730±60 mm -2 in human upper arm skin [38]). If the densely packed microprojections can also deliver vaccine to a large population of APCs in human skin, we expect that the vaccine efficacy can be dramatically enhanced.…”

“…However, the human skin is much thicker than mouse skin (e.g. mouse ear epidermis is 17.5±2.1 µm [37]; the human forearm dorsal epidermis is 61.3±11.0 µm [39]), so it is necessary to test if the densely packed microprojections can penetrate human skin. In order to test this, we tested the NP penetration into pig skin, because pig is considered as a good model for human skin penetration studies, because pig skin is physiologically similar to human skin both in skin thicknesses and mechanical properties [40].…”

Notochordal intervertebral disc cells: Sensitivity to nutrient deprivation

“…To determine cell morphology of NCs and MNPCs, noninvasive live imaging was performed by using two-photon laser scanning microscopy (Zeiss, Jena, Germany) (30,31). Threedimensional volume-rendered images were produced by advanced image processing (26).…”

Predicting the optimal geometry of microneedles and their array for dermal vaccination using a computational model

“…the default values for the rate constants, initial receptor concentrations, dose per microneedle, and diffusion coefficients are presented. these were based on values reported in the literature (avraméas et al 1996;mclellan et al 1998;szolnoky et al 2001;metz et al 2003;mulholland et al 2006;ibrahim et al 2012;römgens et al 2015;Vainshtein et al 2015). these values were used in all simulations unless stated otherwise.…”

“…Our previous study revealed that the rate constants had little effect on the output of the model and can therefore be taken from general values from the literature (Römgens et al 2016). The initial receptor concentration was estimated from the cell density (6.2 × 10 4 cells/mm 3 for the dermis, 1.3 × 10 6 cells/mm 3 for the epidermis) and the number of receptors per cell (1 × 10 5 ) for both the epidermis and dermis (Avraméas et al 1996;McLellan et al 1998;Szolnoky et al 2001;Mulholland et al 2006;Römgens et al 2016). The diffusion coefficient in both these layers was based on measurements with 40 kDa dextran (Römgens et al 2015).…”