Apparatus and method for the treatment of Epidermal Dysplasias

Abstract

Described herein are methods and apparatus for treating or preventing epidermal dysplasias, including, for example, dysplastic epidermal lesions and dermatological pre-cancerous disease.

Claims

1. A method of treating at least one of: (i) dysplastic epidermal lesions; and/or (ii) a dermatological pre-cancerous disease; said method comprising administering to a subject having said at least one of a dysplastic epidermal lesion and/or dermatological disease, a therapeutically effective and/or immunostimulatory amount or dose of microwave energy.

2. The method of claim 1, wherein the treatment comprises repeated rounds of treatment with microwave energy.

3. The method of claim 1, wherein the dysplastic epidermal lesion and/or dermatological disease is at least one of: a. Actinic keratosis; b. Solar keratosis; c. Actinic cheilitis; d. Arsenical keratosis; e. PUVA keratosis; f. Dysplastic lesion g. Pre-cancerous dermatological disease

4. The method of claim 1, wherein the microwave energy has a frequency selected from the group consisting of: (i) between about 500 MHz and about 200 GHz; (ii) between about 900 MHz and about 100 GHz; (iii) between about 5 GHz to about 15 GHz.

5. The method of claim 1, wherein the microwave energy has a frequency of about 8 GHz.

6. The method of claim 1 wherein the dose or amount of microwave energy produces, induces or elevates levels of heat shock factor (HSF) to stimulate production of a heat shock protein within a tissue or lesion being treated.

7. The method of claim 6, wherein the heat shock protein is selected from the group consisting of HSP90, HSP72, HSP70, HSP65. HSP60, HSP27, HSP16 and any another heat shock protein(s).

8. The method of claim 8, wherein the microwave energy promotes an association between the elevated heat shock protein and the dysplastic tissue so as to elicit an immune response against the disease.

9. The method of claim 1, wherein the subject is suffering from a viral lesion and the method is in part applied for the treatment or prevention of a viral lesion, said method comprising delivering a therapeutically effective amount or dose of microwave energy to the lesion, wherein the microwave energy causes the denaturing of viral particles within the lesion thus exposing antigenic sites stimulating an immune response.

10. The method of claim 9, wherein the viral lesion is a viral skin lesion comprising at least one of an actinic keratosis, solar keratosis or dysplastic lesion.

11. An apparatus for use in treating a dysplastic epidermal lesion, said apparatus comprising a microwave source for providing microwave energy and a delivery system for delivering the microwave energy to a subject to be treated.

12. The apparatus of claim 11, further comprising at least one of: (i) a controller for controlling at least one property of the microwave energy produced by the microwave source; and/or (ii) a monitor for monitoring the microwave energy produced by the microwave source.

13. The apparatus of claim 11, wherein the delivery system for delivering microwave energy electrically matches the range of epsilon relative values of the tissue affected by said at least one dysplastic epidermal lesion.

14. The apparatus of claim 11, wherein the delivery system for delivering the microwave energy to a subject comprises a component for contact with a subject to be treated.

15. The apparatus of claim 14, wherein the component is removable such that it can be discarded or sterilised after use.

16. A method of treating or preventing dysplastic epidermal lesions and/or a dermatological pre-cancerous disease, said method comprising administering to a subject having said dysplastic epidermal lesion and/or a dermatological pre-cancerous disease, or susceptible/predisposed to developing the same, a therapeutically effective amount or dose of microwave energy to the dysplastic epidermal lesion and/or dermatological pre-cancerous disease; or to a site vulnerable to the development of the same, wherein: the administering of the therapeutically effective amount or dose of microwave energy comprises using a delivery system to administer the therapeutically effective amount or dose of microwave energy via a microwave applicator to tissue of the subject that has dysplastic epidermal lesions and/or a dermatological pre-cancerous disease or is susceptible/predisposed to the development of the same.

17. The method of claim 16, wherein the method comprises electrically matching the microwave applicator to the tissue that has said dysplastic epidermal lesion and/or a dermatological pre-cancerous disease, based on the tissue that has said dysplastic epidermal lesion and/or a dermatological pre-cancerous disease having a lower dielectric constant than a dielectric constant that said tissue would have if said tissue did not have said dysplastic epidermal lesion and/or a dermatological pre-cancerous disease, such that the microwave applicator is better matched to the tissue that has said dysplastic epidermal lesion and/or a dermatological pre-cancerous disease than it would have been if said tissue did not have said dysplastic epidermal lesions and/or a dermatological pre-cancerous disease

18. The method of claim 17, wherein the microwave energy is selected such as to stimulate a localised immune response at the dysplastic epidermal lesion and/or dermatological pre-cancerous disease and the administering of the therapeutically effective amount or dose of microwave energy to the dysplastic epidermal lesion and/or a dermatological pre-cancerous disease comprises repeatedly applying the microwave energy in a pulsed manner thus providing repeated rounds of localised hyperthermia at the dysplastic epidermal lesions and/or a dermatological pre-cancerous disease and repeated localised stimulation of the immune response at the dysplastic epidermal lesions and/or a dermatological pre-cancerous disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Embodiments of the invention are now described, by way of non-limiting example, and are illustrated in the following figures, in which:

[0086] FIG. 1 is a schematic illustration of an embodiment of a microwave treatment system.

[0087] FIG. 2(a) is a schematic illustration of a Papilloma caused by HPV infection.

[0088] FIG. 2(b) is a schematic illustration of the terminal differentiation pathway of epidermal cells infected by the HPV virus.

[0089] FIG. 3 is a schematic illustration of functional representation of a microwave treatment system for application to treat Papilloma or other dermatological lesions.

[0090] FIG. 4 is a schematic illustration of a microwave treatment system applicator according to an alternative embodiment.

[0091] FIG. 5 is a schematic illustration of the microwave activation of the heat shock response.

[0092] FIG. 6(a) is a schematic illustration of measurement results of Er vs. Frequency for verrucae tissue for a sample population.

[0093] FIG. 6(b) is a schematic illustration of results of Loss tangent vs. Frequency for verrucae tissue for a sample population.

[0094] FIG. 7 is a schematic illustration of Er vs. Frequency for various plantar tissues for a sample population.

[0095] FIG. 8 is a schematic illustration of the statistical analysis of the sample median of verrucae tissue for a sample population.

[0096] FIG. 9 is a schematic illustration of a comparison of the statistical analysis of sample medians of various plantar tissues for a sample population.

[0097] FIG. 10(A) is a Clinical image of plantar wart pre-microwave treatment (left), after one treatment (middle) and after two treatments (right). FIG. 10(B) is a clinical image of plantar wart pre-microwave treatment (left), after one treatment (right). FIG. 10(C) is a graphical representation of data from an intention to treat analysis, in which 32 patients with 54 HPV foot warts were treated by microwave therapy over 5 visits: baseline, 1 week, 1 month, 3 months, and 12 months. Resolved warts were enumerated.

[0098] FIG. 11(A) shows histological analysis of human skin treated with microwave visualised at the epidermis/papillary dermis (top and bottom panels), or deep dermis (middle panels). Skin was subject to microwave therapy (0-200 J), before punch excision. Tissue was cultured for 1 hour before fixation and paraffin embedding. H&E or TUNEL staining. Original magnifications, 20. FIG. 11(B) shows histological analysis of human skin treated with microwave visualised at the epidermis/papillary dermis (upper panels), or deep dermis (lower panels). Skin was subject to various liquid nitrogen therapy for 5, 10, or 30 seconds, before punch excision. Tissue was cultured for 1 hour before fixation and paraffin embedding. H&E or TUNEL staining. Original magnifications, 20. FIG. 11(C) is a graphical representation of data obtained following microwave therapy (top panel) or cryotherapy (bottom panel), skin samples (in triplicate) were excised and cultured in media for 1 or 16 hours before harvesting supernatant for measurement of lactate dehydrogenase (LDH) by ELISA. FIG. 11(D) shows images in which skin was subject to microwave therapy (150 J), before punch excision at the margin of the treated zone. Tissue was cultured for 1 hour before fixation and paraffin embedding. H&E stain. Original magnifications, 10. FIG. 11(E) shows images in which skin was subject to microwave therapy (150 J), before punch excision. Tissue was cultured for 1 hour before fixation and paraffin embedding. H&E stain showing deep dermis. Original magnifications, 100. Representative of 3 independent experiments.

[0099] FIG. 12(A), Left panel shows 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/IFNg treatment. Keratinocytes were treated, then kept in culture for 24, 48 or 72 hours before analysis. FIG. 12(A), Right panel shows flow cytometric analysis of keratinocyte viability after microwave therapy or control depicted as a histogram. X-axis: MFI LIVE/DEAD stain; y-axis: cell count. FIG. 12(B) shows 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/IFNg, or nil (control), rested in culture for 24, 48 or 72 hours, before analysis of the viable population. FIG. 12(C) shows flow cytometric analysis of CD86, CD80, and CD40 expression on viable monocyte derived dendritic cells (moDCs). Keratinocytes were treated with microwave therapy (5-150 J), LPS/IFNg, or nil (control), rested in culture for 8 hours then washed. They were left in culture for the remaining time until 24 or 48 hours, before transfer of supernatant onto moDCs. MoDCs were incubated for 24 hours before harvesting for analysis. Data representative of 3 independent experiments. Mean+SD.

[0100] FIG. 13(A) shows ELISpot assay of IFN- 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 keratinocytes. moDCs were primed with supernatant for 24 hours prior to pulsing with control or HPV peptide for 2 hours. Pulsed moDCs were then cocultured with an HLA-matched CD8+ HPV-specific T cell line before assay with IFN- ELISpot. Statistical significance determined using the Holm-Sidak method, with alpha=5%. Data representative of 3 independent experiments. Mean+SD. FIG. 13(B) shows ELISpot assay of IFN- 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 keratinocytes. moDCs were primed with supernatant for 24 hours prior to pulsing with control or HPV peptide for 2 hours. Pulsed moDCs were then co-cultured with an HLA-matched CD8+ HPV-specific T cell line before assay with IFN- ELISpot. Statistical significance determined using the Holm-Sidak method, with alpha=5%. Data representative of 3 independent experiments. Mean+SD. FIG. 13(C) shows 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: MFI anti-HSP-70; y-axis: cell count. FIG. 13(D) shows ELISA of IL-6, TNF, IL-1 production by primary human keratinocytes 24 h after treatment with microwave therapy (150 J), LPS/IFNg, cryotherapy or nil (untreated). FIG. 13(E) shows expression changes of IRF1 and IRF4 in human skin with microwave therapy (25 J and 150 J) by qPCR.

DETAILED DESCRIPTION OF EMBODIMENTS

[0101] An embodiment of a microwave power generator system for medical applications is illustrated in FIG. 1 The apparatus comprising:a microwave source for providing microwave energy 1, connectable to a system controller 2 for controlling at least one property of the microwave radiation provided by the microwave source; and a monitoring system 3 for monitoring the delivery of energy and an interconnecting cable 4 and an applicator hand piece 5 and a removable applicator means 6, for example an applicator device, for delivering microwave energy, wherein:the applicator is configured to deliver precise amounts of microwave energy provided by the source at a single frequency or across a range of frequencies.

[0102] A typical HPV infection is illustrated in FIG. 2(a), this comprises the normal tissue 7, the papilloma surface 8, the capillary feed network 9. The terminal differentiation pathway of epidermal cells infected by the HPV virus is illustrated in FIG. 2(b) basal cells 10 become infected with the HPV virus leading to viral replication in the stratum spinosum 11 followed by assembly of virus particles in the stratum granulosum and release of virus particles in the in the stratum corneum (papilloma surface).

[0103] FIG. 3 shows the components of an apparatus according to an embodiment of the present invention, the components shown separately for ease of reference. The apparatus comprises a generator system 14 with a locking microwave connection 15 to a flexible microwave cable 16 connected to a hand piece 17 (which may have the same type of locking connection) which accepts an applicator component 19. The applicator component is designed to match to the tissue properties of the papilloma 20 and not match to the normal tissue 18. The cable 16 may include both microwave and signal data cables and may be reversible to enable connection to either port.

[0104] An alternative embodiment of an applicator 21 is illustrated in FIG. 4 with the component having a domed or enclosing surface compatible with raised or curved lesions.

[0105] The method of inducing a microwave heat shock responses is illustrated in FIG. 5. In this illustration microwave radiation creates a thermal stress which results in protein denaturation 22. Heat shock proteins (HSP) are normally bound to heat shock factors (HSF) (23), but dissociate in the presence of denatured proteins (PD). Once dissociated, HSPs bind to the denatured proteins by rapid release. This requires Adenosine Triphosphate (ATP) 24. Further HSPs are generated when HSFs phosphorylate (PO4) (25) and trimerize (26). These trimers bind to heat shock elements (HSE) 27 that are contained within the promoters of the HSPs and generate more protein 28. Newly generated HSPs can then free to bind more denatured proteins 29.

[0106] The measured dielectric properties for the sample population median versus frequency of plantar verrucae are reported in FIGS. 6(a) and 6(b). Measured results for ER and loss tangent versus frequency from 2 GHz to 20 GHz are reported. The measurements were made using an Agilent PNA-L Network Analyzer connected to an Agilent 85070E dielectric measurement system with the 85070E Performance Probe Kit (Option 050) measuring from 300 kHz to 20 GHz. Deionised water and air were used as dielectric references for calibration.

[0107] The measured dielectric properties for the sample population (median taken across the population) versus frequency for various plantar tissues are reported in FIG. 7. Measured results for Er versus frequency from 2 GHz to 20 GHz are reported illustrating demarcation between each tissue type.

[0108] Statistically analysed dielectric property values taken over a sample population (using the median of the measurement range 7.5-8.5 GHz taken from each sample) for Verrucae tissue is presented in FIG. 8. The median Er value was measured at 4.93 for this dataset.

[0109] With reference to FIG. 9 a comparison of statistically analysed measurements of the dielectric properties of various plantar tissues over a sample population (using the median of the measurement range 7.5-8.5 GHz taken from each sample) is illustrated.

Microwave Therapy for Cutaneous Human Papilloma Virus Infection

Methods and Materials

Patients and In Vivo Microwave Treatment

[0110] Patients with treatment-refractory plantar warts were excluded if they had a pacemaker fitted, were pregnant or breast feeding, had any metal implants within the foot or ankle, suffered any known disease or condition affecting their immune function or their capacity to heal. Adverse events were categorised as being specifically associated with the microwave procedure, or unrelated. A complete examination of the affected area was undertaken at each study visit. At the conclusion of the treatment session all patients were given an advice information sheet advised to report any complications. No post-operative dressing was required and patients were advised to subsequently undertake normal everyday activities as usual with no restrictions.

[0111] A total of 32 patients with 54 foot warts were enrolled into the study. Of the 32, 17 were males and 15 females. Ages ranged from 22-71 years with a mean age of 44.79 years (sd 13.019]. Of the 54 lesions, 16 were reported as single lesions, and 38 as multiple type lesions (including mosaic verrucae). The average lesion duration was 63 months (5.25 years) with a range of 2-252 months (<1-21 years). The mean lesion diameter was 7.43 mm (sd 6.021), ranging from just 2 mm to 38 mm in diameter.

[0112] The procedure was performed in an out-patient setting, with standard podiatric facilities. The Swift device settings were titrated up as tolerated to 50 J over a 7 mm.sup.2 application area (7.14 j/mm.sup.2). The microwave energy was delivered to the affected area over 5 s duration (50 J delivered as 10 watts for 5 s). Lesions which were <7 mm in diameter were treated with one application of the probe at a single treatment session whilst lesions >7 mm were underwent multiple applications until the entire surface of the wart had been treated.

[0113] Clinical assessments were performed at baseline and at 1 week, 1 month, 3 months, and 12 months after treatment by a podiatrist experienced in the management of plantar warts. Response to treatment was assessed by the same investigator as completely resolved or unresolved. Complete resolution was indicated by fulfilling three criteria: i. lesion no longer visible, ii. return of dermatoglyphics to the affected area, iii. no pain on lateral compression. Pain was assessed using a 10 point visual analogue scale.

Human Skin and Ex Vivo Microwave Treatment

[0114] Normal skin samples were acquired from healthy individuals after obtaining informed written consent with approval by the Southampton and South West Hampshire Research Ethics Committee in adherence to Helsinki Guidelines. Skin samples were treated immediately ex-vivo with microwave (Swift s800; Emblation Ltd., UK) or liquid nitrogen therapy and treated skin excised. Excised skin was sent for histological analysis or placed in culture media.

[0115] Histological analysis with hematoxylin and eosin (H&E) tissue sections were undertaken following fixation and embedded in paraffin wax. DNA damage was assessed by staining for single stranded and double stranded DNA breaks by TUNEL assay using the ApopTag In Situ Apoptosis Detection Kit (Millipore, UK). Following culture, supernatants were collected and analysed for lactate dehydrogenase release using the Cytotoxicity Detection Kit (Roche applied science) as a measure of apoptosis.

Culture and In Vitro Microwave Treatment.

[0116] Human skin and HaCaT keratinocytes were cultured in calcium-free DMEM (ThermoFisher Scientific) with 100 U/mL penicillin, 100 g/mL streptomycin, 1 mM sodium pyruvate, 10% fetal bovine serum (FBS) and supplemented with calcium chloride at 70 M final concentration.

[0117] Lymphocytes were cultured in RPMI-1640 media with 100 U/mL penicillin, 100 g/mL streptomycin, 1 mM sodium pyruvate, 292 g/mL L-glutamine, supplemented with 10% FBS or 10% heat inactivated human serum (HS). HaCaT cells were cultured at sub-confluency to avoid cell differentiation and used in assays at passage 60-70. Cells were plated at 2.5103 cells/well in 96-well flat plate (Corning Costar) and cultured overnight to reach confluence. HaCaTs were washed once with PBS before treatment with 150 J microwave, liquid nitrogen (10 s), heat (42 C. preheated media) or with LPS+IFN- (1 ng/mL+1000 U/mL). Cells were cultured for 24 h before supernatants were harvested.

[0118] For HPV-specific T cell lines, PBMCs were isolated from HLA-A2 individuals as previously described 11. PBMCs were seeded at 2-4106 cells/well in 24-well culture plate and 10 g/mL of 9mer HLA-A2 restricted HPV16 epitope LLM (LLMGTLGIV) 12 was added, cells were cultured in 1 mL RPMI+10% HS. On day 3, cells were fed with RPMI+10% HS+IL-2 (200 IU/mL), and then fed again on day 7 or when needed. After day 10, HPV-specific T cells were harvested for cryopreservation before testing against HPV in ELISpot assays.

[0119] Monocyte derived dendritic cells (moDCs), CD14+ cells were positively isolated from PBMCs by magnetic separation using CD14 microbeads (Milentyi Biotec), according to manufacturer's protocol. Cells were washed and resuspended in RPMI+10% FBS+250 U/mL IL-4 and 500 U/mL GM-CSF. At day 3, cells were fed with RPMI+10% FBS+IL-4 and GM-CSF, and then harvested on day 5 for use in functional assays.

[0120] In vitro, microwave therapy of cell cultures was delivered through the base of the plastic culture dish and showed a linear dose response between the energy delivered and thermal induction (not shown). Utilising the equation E=mc (E=energy transferred, J; m=mass, kg; c=specific heat capacity, J/kg C.; =temperature change, C.), we calculated that in our system the 150 J Swift programme delivered 15.58 J (s.d. 0.921) through the plastic to the culture.

ELISpot, Flow Cytometry and qPCR

[0121] Keratinocytes were treated with microwave at various energy settings before removal of supernatant at various time points. MoDCs were treated overnight with keratinocyte supernatant, then washed twice before incubation with 10 g/mL LLM peptide for 2 hours before a further wash.

[0122] Human IFN- ELISpot (Mabtech, Sweden) was undertaken as per manufacturer's protocol and as reported previously 11. 1103 moDCs were plated with autologous HPV peptide-specific T cells at 1:25 ratio. Spot forming units (sfu) were enumerated with ELISpot 3.5 reader (AID, Germany). MoDCs were treated with HaCaT supernatant and harvested at 24 hours for flow cytometric analysis of cell phenotype. Cells were stained with violet LIVE/DEAD stain (Invitrogen) for 30 min at 4 C., then washed with PBS+1% BSA and stained with antibodies PerCP-Cy5.5 anti-HLA-DR, FITC anti-CD80, FITC anti-CD86, PE anti-CD40, all purchased from BD, for 45 min at 4 C. Cells were washed then resuspended in PBS+1% BSA and analysed using the BD FACSAria and the FlowJo v10.0.08 analysis software.

[0123] The expression of chosen genes was validated with quantitative PCR, using the TaqMan gene expression assays for target genes: YWHAZ (HS03044281_g1), IRF1 (Hs00971960_m1), IRF4 (Hs00543439_CE) (Applied Biosystems, Life Technologies, Paisley, UK) in human skin treated as indicated. RNA extraction (RNeasy micro kit, Qiagen) and reverse transcription (NanoScript kit; Primer Design, Southampton, UK) were carried out accordingly to the manufacturer's protocol.

Results:

[0124] Treatment of Human Papilloma Virus Infection in Humans with Microwave Therapy

[0125] From January 2015 to September 2015 at the University of Southampton, we enrolled 32 patients with severe, treatment-refractory plantar warts. The diagnosis of plantar wart was confirmed by a podiatrist experienced in management of such lesions. A clinically significant wart was defined as >1 year duration, which had failed at least two previous treatments (salicylic acid, laser, cryotherapy, needling and surgical excision). In each patient, the most prominent plantar wart (most severely affected) was targeted for treatment (FIG. 10 a,b).

[0126] At the end of the study period, of the 54 warts treated, 41 had resolved (75.9%), 9 remained unresolved (16.7%), 3 warts (n=2 patients) had withdrawn from the study (5.6%) and 1 patient (with 1 wart) was lost to follow up (1.9%). The mean number of days to resolution 79.49 days (sd 34.561; 15-151 days). 94% of resolving lesions had cleared after 3 treatments (FIG. 10c). No significant difference in resolution rates were observed between males and females (p=0.693) was observed.

Microwave Treatment of Human Skin

[0127] Human skin has not been previously treated with microwave therapy, therefore, we proceeded to undertake a full histological analysis. Skin removed during routine surgery was sectioned 1 hour after treatment ex vivo. Neither macroscopic, nor histological changes were noted with the lowest energy setting (5 J). At 50 J, mild macroscopic epidermal changes only were noted, and microscopically minor architectural changes, and slight elongation of keratinocytes were seen without evidence of dermal collagen sclerosis. At higher energies (100, 200 J) gross tissue contraction was visible macroscopically. Microscopic changes in the epidermis were prominent, showing spindled keratinocytes with linear nuclear architectural changes and subepidermal clefting (FIG. 11a).

[0128] Dermal changes were prominent at energies of 100 J and above and showed a homogenous zone of papillary dermal collagen, thickened collagenous substances, accentuation of basophilic tinctorial staining of the dermal collagen with necrotic features (FIG. 11a). These features are similar to electrocautery artefacts and suggest the potential to induce scarring at >100 J. Histological analysis both at 16 h and 45 h showed similar changes (not shown).

[0129] In clinical practice, cryotherapy is delivered to the skin by cryospray, which is time-regulated by the operator. In contrast to microwave therapy, minimal epidermal or dermal architectural change was identified with cryotherapy at standard treatment duration times (5-30 s), but did show a dose dependent clumping of red blood cells in vessels (FIG. 11b).

[0130] Tissue release of LDH acts as a biomarker for cellular cytotoxicity and cytolysis. To examine the extent of cell death induced by microwave irradiation, human skin was treated with 0, 50, 100 or 200 J before punch excision of the treated area and incubation in medium for 1 hour or 16 hours. Measurement of LDH revealed a dose dependent induction of tissue cytotoxicity with increasing microwave energies (FIG. 11c). In line with the lack of histological evidence of cellular damage, at 5 J, cytotoxicity of microwave application was equivalent to control. Early cytotoxicity was not prominent at 50 J, but became more evident after 16 hours. Higher energy levels induced more prominent cytotoxic damage. In contrast to microwave therapy, liquid nitrogen treatment of skin induced cytotoxicity at the lowest dose both at 1 hour and 16 hours.

[0131] Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) identifies cells in the late stage of apoptosis. Analysis at 0, 5, 50, 100 and 200 J identified increased cellular apoptosis in the epidermis above 100 J (FIG. 11a). In contrast, cryotherapy with a liquid nitrogen spray applicator directly to the skin as used in clinical practice, even at a very short treatment time (5 s) induced significant epidermal and dermal DNA fragmentation (FIG. 11b).

[0132] The physics of microwave therapy suggests a tight boundary between treated and untreated tissue with minimal spreading of the treated field. This was borne out histologically by a clear demarcation between treated areas extending vertically from the epidermis through the dermis (FIG. 11d). Examination of the dermis showed that microwave therapy modified skin adnexae inducing linear nuclear architectural changes in glandular apparatus, microthrombi, fragmented fibroblasts and endothelial cells (FIG. 11e).

Microwave Induction of Immune Responses in Skin

[0133] We first examined the response of keratinocytes to microwave therapy in vitro. Keratinocyte apoptosis was induced by microwave therapies above 100 J in vitro (FIG. 12a). Only above the apoptotic threshold were surface phenotypic changes of cellular activation noted in viable cells with increased expression of HLA-DR, CD40 and CD80 (FIG. 12b). Next, we utilised a model of skin cross talk of keratinocyte signalling to dermal dendritic cells. Keratinocytes were treated with microwave therapy as above, and washed after 8 hours to remove dead or apoptotic cells. Treated keratinocytes were then incubated for a further 16 hours before supernatant collection to prime monocyte derived dendritic cells (moDCs) which had not been directly exposed to microwave therapy. This showed a potent induction of moDC activation with increased expression of CD86, CD80 and to a lesser extent CD40 (FIG. 12c).

[0134] We next set out to test the functional outcome on skin dendritic cells following microwave treatment of keratinocytes. Keratinocytes were untreated, microwave, or cryotherapy treated before supernatant harvesting. Supernatant primed DCs were pulsed with a 9 amino acid HLA-A2 epitope (LLM) from human papilloma virus (HPV) E16 protein and cultured with an autologous HPV specific CD8+ T cell line. As expected, in all conditions, the DCs efficiently presented HPV peptide to CD8+ T cells inducing IFN- (FIG. 13a). However, dendritic cell presentation of HPV virus is dependent upon cross-presentation to the MHC class I pathway. Therefore we also tested the ability of untreated, microwave treated or cryotherapy KC-primed DCs to present human papilloma virus (HPV) E16 protein to an HLA matched HPV-specific CD8+ T cell line. Strikingly, only microwave treated KCs were able to prime DCs to enhance cross-presentation (FIG. 13b). To explore the potential mechanism of keratinocyte response to microwave therapy we confirmed up regulation of HSP-70 in response to microwave therapy of keratinocytes (FIG. 13c) and showed significant IL-6 induction in keratinocytes above that seen in cryotherapy treated cells (FIG. 13d). IRF1 and IRF4 are key regulators of dendritic cell activation and we confirmed that microwave therapy induced down regulation of IRF1 and up-regulation of IRF4 (FIG. 13e).

Discussion

[0135] This is the first study to investigate the potential efficacy of locally delivered microwaves in the treatment of cutaneous viral warts in vivo. We report a complete resolution rate of 75.9% recalcitrant plantar warts (average lesion duration of over 5 years). This compares very well with previous reports of plantar wart resolution for salicylic acid and or cryotherapy (23-33%).sup.13. Whilst this study was a pilot phase, and did not include a control untreated arm, we believe the treatment effect to be significant.

[0136] For all novel therapies, adverse events are critical. In this study we did not identify a strong signal for adverse events with microwave therapy of cutaneous warts. As with current physical treatments for warts discomfort is expected for the patient. During the study patients typically reported that for a typical 5 second treatment that they endured moderate discomfort for approximately 2 seconds, which immediately diminished after the treatment had completed. In addition, it was commonly noted that discomfort was less with subsequent treatments. One male patient, withdrew from the study after one treatment, citing the pain of treatment as the reason. In the study design phase, preoperative use of topical anaesthetic cream was tested, but appeared to do little to mitigate the pain (unpublished data) and it was felt that the pain of local anaesthetic injection would exceed that normally experienced during a microwave treatment. Following microwave therapy, patients did not require dressings or special advice as microwave therapy utilised in this study did not cause a wound or ulcer in the skin, allowing the patient continue normal activity. The short microwave treatment time (5 s) offers a significant clinical advantage over current wart therapies such as cryotherapy and electro-surgery. Within 5 s, microwaves penetrate to a depth of over 3.5 mm at the energy levels adopted for the study.sup.14possibly a greater depth than can be attained by cryosurgery or laser energy devices. Moreover, as microwaves travel in straight lines energy is deposited in alignment the device tip with little collateral spread, meaning minimal damage to surrounding tissue, as observed in this study. Microwaves induce dielectric heating. When water, as a polar molecule, is exposed to microwave energy, the molecule is excited and rotates attempting to align with the alternating electro-magnetic field. At microwave frequencies the molecule is unable to align fully with the continuously shifting field resulting in heat generation. Within tissues, this acts to rapidly elevate temperatures. This process rapidly changes cellular heat because it does not depend on tissue conduction. Microwave treatment produces no vapour or smoke unlike ablative lasers and electro-surgery, eliminating the need for air extraction systems due to the risk of spreading viral particles within the plume.sup.15.

[0137] Although, microwave therapy has been considered a tissue ablation tool, we saw minimal skin damage after treatment with 50 J, yet apparent good clinical response. Therefore we investigated whether there was evidence to support an induction of immunity by microwave therapy. The critical nature of CD8+ T cell immunity for host defence against HPV skin infection is well established and supported by the observation of increased prevalence of infection in immunosuppressed organ transplant recipients.sup.16, and that induction of protection from HPV vaccines is mediated by CD8+ T cells.sup.17. We show here, that microwave therapy of skin induces keratinocyte activation and cell death through apoptosis. However, at sub-apoptotic doses, microwave primed keratinocytes are able to signal to dendritic cells and enhance cross-presentation of HPV antigens to CD8+ lymphocytes which offers a potential explanation for the observed response rate in our clinical study. In vitro evidence suggests that this is likely to be mediated by cross-talk between microwave treated skin keratinocytes and dendritic cells, with resultant enhanced cross-presentation of HPV protein to CD8+ T cells. Microwave therapy also induced enhanced IL-6 synthesis from keratinocytes.

[0138] IL-6, is a pro-inflammatory mediator, important in anti-viral immunity which has been recently shown to induce rapid effector function in CD8+ cells.sup.18. Thus, IL-6 up-regulation may provide an important additional mechanism for microwave anti-viral immunity. IRFs have been shown to be central to the regulation of immune responses.sup.19-21. IRF4 is essential for differentiation of cytotoxic CD8+ T cells.sup.2223, but up-regulation in dendritic cells has also been shown to enhance CD4+ differentiation, thereby potentially enhancing both CD8+ immunity and T cell help following microwave treatment. IRF1 expression has been previously reported to be modulated by HPV infection, but different models have shown opposite outcomes.sup.24,25. We show down-regulation of IRF1 in human skin in association with a microwave therapy which supports the proposal of IRF-1 as a therapeutic target in HPV infection.sup.25.

[0139] This study is the first of its kind studying microwaves in the treatment of plantar warts in vivo. However, the authors acknowledge the limitations of the uncontrolled, non-randomised design. Despite the promising results shown here, studies with larger sample sizes are needed to assess the efficacy of this treatment and for infrequent but serious adverse events.

[0140] It will be understood that embodiments of the present invention have been described above purely by way of example, and modifications of detail can be made within the scope of the invention.

[0141] Each feature disclosed in the description, and (where appropriate) the claims and drawings may be provided independently or in any appropriate combination.

REFERENCES

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ADDITIONAL REFERENCES

[0167] 1. https://emedicine.medscape.com/article/1099775-treatment [0168] 2. https://www-clinical key-com.rsm.idm.ocic.org/#!/content/book/3-s2.0-B978070205183800031X?scrollTo=%23hl0001716 [0169] 3. https://www.ncbi.nlmnih.gov/pmc/articles/PMC4065271/ [0170] 4. http://www.bad.org.uk/for-the-public/patient-information-leaflets/actinic-keratoses/?showmore=1&returnlink=http%3A%2F%2Fwww.bad.org.uk%2Ffor-the-public%2Fpatient-information-leaflets#.Wn2AyiXFKHt [0171] 5. http://www.pcds.org.uk/clinical-guidance/actinic-keratosis-syn.-solar-keratosis