John Libbey Eurotext

European Journal of Dermatology

Microwave therapy for cutaneous human papilloma virus infection Volume 27, numéro 5, September-October 2017

Figure 1

Response of recalcitrant warts to microwave therapy. A) Clinical image of plantar wart pre-microwave treatment (left), after one treatment (middle), and after two treatments (right). B) Clinical image of plantar wart pre-microwave treatment (left) and after one treatment (right). C) Intention to treat analysis of 32 patients with 54 HPV foot warts treated by microwave therapy over five visits: baseline, one week, one month, three months, and 12 months. Resolved warts were enumerated. D) Pain scores were assessed using a 10-point visual analogue score at each visit. Statistical test: one-way ANOVA.

Figure 2

Microwave effects on human skin. A) Histological analysis of normal human skin treated with microwave stimulation visualised in the epidermis/papillary dermis (upper and lower panels), or deep dermis (middle panels). Skin was subjected to microwave therapy (0-200 J) before punch excision. Tissue was cultured for one hour before fixation and paraffin embedding (H&E or TUNEL staining; original magnification: ×20). B) Histological analysis of human skin treated with liquid nitrogen therapy for 5, 10, or 30 seconds, before punch excision. Tissue was cultured for one hour before fixation and paraffin embedding (H&E or TUNEL staining; original magnification: ×20). C) Following microwave therapy (upper panel) or cryotherapy (lower panel), skin samples (in triplicate) were excised and cultured in media for one or 16 hours before measurement of cytotoxicity, assessed by harvesting supernatant to measure supernatant lactate dehydrogenase (LDH) release by ELISA. D) Skin was subjected to microwave therapy (150 J) before punch excision at the margin of the treated zone. Tissue was cultured for one hour before fixation and paraffin embedding (H&E stain; original magnification: ×10 [upper panel], x100 [lower panel]. E) Skin was subjected to microwave therapy (150 J) before punch excision. Tissue was cultured for one hour before fixation and paraffin embedding (H&E stain showing deep dermis adnexae: glandular [left] and vascular [right]; original magnification: ×100). Data are representative of three independent experiments.

Figure 3

Microwave activation of keratinocytes and dendritic cells. A) Left: flow cytometric analysis of viable keratinocytes (% of total cells) indicated by negative staining with the amine reactive viability dye LIVE/DEAD after control, microwave (5-150 J), or LPS/IFN-γ treatment. Keratinocytes were treated then kept in culture for 24 (black bars), 48 (light grey) or 72 (dark grey) hours before analysis. Right: flow cytometric analysis of keratinocyte viability after microwave therapy or control, depicted as a histogram. X-axis: LIVE/DEAD stain; y-axis: cell count. B) Flow cytometric analysis of HLA-DR, ICAM-1, CD40 or CD80 expression on viable keratinocytes. Keratinocytes were treated with microwave therapy (5-150 J), LPS/IFN-γ, or nil (control), rested in culture for 24 (black bars), 48 (light grey) or 72 (dark grey) hours, before analysis of the viable population. C) Flow cytometric analysis of CD86, CD80, and CD40 expression on viable monocyte-derived dendritic cells (moDCs). Keratinocytes were treated with microwaves (5-150 J), LPS/IFN-γ, or untreated (control), rested in culture for eight hours, then washed. They were left in culture for the remaining time until 24 (black bars) or 48 (light grey) hours, before transfer of supernatant onto moDCs. MoDCs were incubated for 24 hours before harvesting for analysis. Data are representative of three independent experiments. Mean+SD; * p<0.05; ** p<0.01; *** p<0.001.

Figure 4

Microwave induction of HPV antigen cross-presentation. A) ELISpot assay of IFN-γ production by HPV-specific CD8+ cells following co-culture with HPV peptide-pulsed moDCs primed by supernatant from untreated, microwave-treated (150 J) or cryotherapy (cryo)-treated HaCaT keratinocytes. MoDCs were primed with supernatant for 24 hours prior to pulsing with control (open squares) or HPV peptide (closed squares) for two hours. Pulsed moDCs were then co-cultured with an HLA-matched CD8+ HPV-specific T cell line before assay with IFN-γ ELISpot. Statistical significance was determined using the Holm-Sidak method, with alpha = 5%. Data are representative of three independent experiments (mean+SD). B) ELISpot assay of IFN-γ production by HPV-specific CD8+ cells following co-culture with HPV E16 protein-pulsed moDCs primed by supernatant from untreated, microwave-treated (150 J) or cryotherapy (cryo)-treated HaCaT keratinocytes. MoDCs were primed with supernatant for 24 hours prior to pulsing with control (open squares) or HPV protein (closed squares) for two hours. Pulsed moDCs were then co-cultured with an HLA-matched CD8+ HPV-specific T cell line before assay with IFN-γ ELISpot. Statistical significance was determined using the Holm-Sidak method, with alpha = 5%. Data are representative of three independent experiments (mean+SD). C) Flow cytometric analysis of intracellular HSP-70 expression on viable keratinocytes after microwave therapy or control, depicted as a histogram. Primary human keratinocytes were treated with microwave therapy (150 J), or nil (untreated), rested in culture for 24 hours, before analysis. X-axis: anti-HSP-70; y-axis: cell count. D) ELISA of IL-6, TNFα, and IL-1β production by primary human keratinocytes 24 hours after treatment with microwave therapy (150 J), LPS/IFNg, cryotherapy or control (untreated). E) Fold expression of change of IRF1 and IRF4 over a housekeeping gene in normal human skin with microwave therapy (25 J and 150 J) by qPCR.