Limits...
Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporation.

Mendoza JM, Amante DH, Kichaev G, Knott CL, Kiosses WB, Smith TR, Sardesai NY, Broderick KE - Vaccines (Basel) (2013)

Bottom Line: H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment.Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen.These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

View Article: PubMed Central - PubMed

Affiliation: Inovio Pharmaceuticals Inc., 1787 Sentry Parkway West, Building 18, Suite 400, Blue Bell, PA 19422, USA.

ABSTRACT
The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP) device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21) of enhanced gene delivery with electroporation (EP) was performed to observe the localization of green fluorescent protein (GFP) expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

No MeSH data available.


Related in: MedlinePlus

Histological analysis reveals reporter gene expression localized to cells in the epidermis. Histological analysis of GFP and red fluorescent protein (RFP) expression after ID plasmid administration followed by SEP in guinea pig skin. (A) GFP treated skin biopsies were removed 4 h post treatment, cryosectioned, DAPI stained and visualized using fluorescence microscopy (20× and 40×). An injection only control (no EP) is also shown; (B) RFP treated skin biopsies were removed, cryosectioned, stained with an antibody against K10 (a keratinocyte cell surface marker), Hoechst stained and visualized using confocal imaging.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494224&req=5

vaccines-01-00384-f003: Histological analysis reveals reporter gene expression localized to cells in the epidermis. Histological analysis of GFP and red fluorescent protein (RFP) expression after ID plasmid administration followed by SEP in guinea pig skin. (A) GFP treated skin biopsies were removed 4 h post treatment, cryosectioned, DAPI stained and visualized using fluorescence microscopy (20× and 40×). An injection only control (no EP) is also shown; (B) RFP treated skin biopsies were removed, cryosectioned, stained with an antibody against K10 (a keratinocyte cell surface marker), Hoechst stained and visualized using confocal imaging.

Mentions: Magnified DAPI stained images from the 4 h time point plus and minus electroporation (Figure 3A) demonstrate the morphology of the transfected cells as well as the enhancing effect of electroporation. Using a keratinocyte specific antibody, we were able to identify reporter gene positive cells (in this case, red fluorescent protein (RFP)) which also co-stained for K10, the keratinocyte marker (Figure 3B) visualized with an Alexa 488 secondary antibody. In addition to the positive antibody staining, the morphology of the cells depicted through the reporter gene expression is indicative of keratinocytes.


Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporation.

Mendoza JM, Amante DH, Kichaev G, Knott CL, Kiosses WB, Smith TR, Sardesai NY, Broderick KE - Vaccines (Basel) (2013)

Histological analysis reveals reporter gene expression localized to cells in the epidermis. Histological analysis of GFP and red fluorescent protein (RFP) expression after ID plasmid administration followed by SEP in guinea pig skin. (A) GFP treated skin biopsies were removed 4 h post treatment, cryosectioned, DAPI stained and visualized using fluorescence microscopy (20× and 40×). An injection only control (no EP) is also shown; (B) RFP treated skin biopsies were removed, cryosectioned, stained with an antibody against K10 (a keratinocyte cell surface marker), Hoechst stained and visualized using confocal imaging.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494224&req=5

vaccines-01-00384-f003: Histological analysis reveals reporter gene expression localized to cells in the epidermis. Histological analysis of GFP and red fluorescent protein (RFP) expression after ID plasmid administration followed by SEP in guinea pig skin. (A) GFP treated skin biopsies were removed 4 h post treatment, cryosectioned, DAPI stained and visualized using fluorescence microscopy (20× and 40×). An injection only control (no EP) is also shown; (B) RFP treated skin biopsies were removed, cryosectioned, stained with an antibody against K10 (a keratinocyte cell surface marker), Hoechst stained and visualized using confocal imaging.
Mentions: Magnified DAPI stained images from the 4 h time point plus and minus electroporation (Figure 3A) demonstrate the morphology of the transfected cells as well as the enhancing effect of electroporation. Using a keratinocyte specific antibody, we were able to identify reporter gene positive cells (in this case, red fluorescent protein (RFP)) which also co-stained for K10, the keratinocyte marker (Figure 3B) visualized with an Alexa 488 secondary antibody. In addition to the positive antibody staining, the morphology of the cells depicted through the reporter gene expression is indicative of keratinocytes.

Bottom Line: H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment.Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen.These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

View Article: PubMed Central - PubMed

Affiliation: Inovio Pharmaceuticals Inc., 1787 Sentry Parkway West, Building 18, Suite 400, Blue Bell, PA 19422, USA.

ABSTRACT
The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP) device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21) of enhanced gene delivery with electroporation (EP) was performed to observe the localization of green fluorescent protein (GFP) expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

No MeSH data available.


Related in: MedlinePlus