WO2006119972A1

WO2006119972A1 - Methods for treating skin with high-fluency, intense pulsed light

Info

Publication number: WO2006119972A1
Application number: PCT/EP2006/004340
Authority: WO
Inventors: Careen A. Schroeter
Original assignee: Schroeter Careen A
Priority date: 2005-05-09
Filing date: 2006-05-09
Publication date: 2006-11-16
Also published as: EP1904177A1

Abstract

The present invention relates to the use of a high- fluency, intense pulsed light source for inducing the expression of TGF-β in skin cells, resulting in the release of extracellular matrix proteins such as tenascin, and an increase in the concentration of fibroblasts and plasma cells. As the high-fluency, intense pulsed light source methodology disclosed herein produces an artificial wound which stimulates the immune system and extracellular matrix formation, the present invention relates to the use of a high- fluency intense pulsed light source alone, or in combination with laser or radiofrequency, for dermatologic treatment and plastic surgery treatment.

Description

METHODS FOR TREATING SKIN WITH HIGH-FLUENCY, INTENSE PULSED LIGHT Background of the Invention

Transforming growth factor-beta (TGF-β) is a protein, which plays a principal role in the intracellular signaling (Lawrence (1996) Eur. Cytokine Netw. 7:363-74).

The most important function is its ability to stimulate synthesis of extracellular matrix that is responsible for wound formation and tissue reconstruction (Grande (1997) Proc. Soc. Exp. Biol. Med. 214:27-40). TGF-β acts via autocrine and paracrine mechanisms to regulate the interactions between cells and between cells and matrix in wound healing, involving inflammation, re- epithelialization, angiogenesis, and the production of extracellular matrix (Roberts and Sporn (1996) The Molecular and Cellular Biology of Wound Repair. Clark RAF eds . pp 275-308 Plenum Press, New York; O'Kane and Ferguson (1997) Int. J. Biochem. Cell Biol. 29:63-78).

TGF-β stimulates the synthesis of multiple extracellular matrix components, including collagens, fibronectin, vitronectin, tenascin, and proteoglycans (Pearson, et al .

(1988) EMBO J. 7:2977-82; Ignotz and Massague (1986) J. Biol. Chem. 261:4337-4). Further, it suppresses matrix degradation by down regulating the expression of proteases, such as plasminogen activators (Edwards, et al.

(1987) EMBO J. 6:1899-904; Laiho, et al . (1986) J^". Cell

Biol. 103:2403-10) and stromelysin (Kerr, et al . (1990) Cell 61:267-7) and by inducing protease inhibitors, such as plasminogen activator inhibitor-1 (Laiho, et al . (1986) supra; Wahl , et al . (1989) J. Cell. Biochem. 40:193-9) and tissue inhibitor of metalloproteases-1 (Edwards, et al.

(1987) supra) . The presence of extracellular matrix has been found to down regulate the expression of the TGF- βl gene (Streuli, et al. (1993) J. Cell Biol. 120:253-60). Thus, TGF-β may act as a feedback for extracellular matrix formation. TGF-β is also involved in scar formation as neutralization of TGF-βl and TGF-β2 in adult wounds reduces scarring in rat dermal wounds (Shah, et al . (1994) J. Cell Sci. 107:1137-57). By contrast, exogenous addition of TGF-β3 to dermal rat wounds results in reduced scar formation (Shah, et al. (1995) J^". Cell Sci. 108:985-1002). Therefore, it is clear that different isoforms of TGF-β can have opposite effects in wound repair although the mechanisms behind these differences are still under investigation (Cowin, et al. (2001) Bur. J. Dermatol. 11:424-31). TGF-β is also found to be involved in ' lymphopoiesis and is required for the development of plasma cells secreting all secondary isotypes (Lebman and Edmiston (1999) Microbes Infect. 1:1297-304).

It has been documented that TGF-β plays important roles in the induction of catagen phase of the human hair cycle (Tsuji, et al . (2003) J^". Investig. Dermatol. Symp. Proc. 8:65-8; Soma, et al . (2002) J^". Invest. Dermatol. 118: 993-7). An isoform of TGF-β acts as an inducer of hair follicle morphogenesis and is both required and sufficient to promote this process (Foitzik, et al . (1999) Dev. Biol. 2122:278-89).

During embryological development, specialized cells of developing multicellular organisms are surrounded by extracellular matrix, composed largely of different collagens, proteoglycans, and glycoproteins. This extracellular matrix is a substrate for tissue morphogenesis, which lends support and flexibility to mature tissues and acts as an epigenetic informational unit in the sense that it transduces and integrates intracellular signals via distinct cell surface receptors. Consequently, extracellular matrix-receptor interactions have a profound influence on major cellular programs including growth, differentiation, migration, and survival. In contrast to many other extracellular matrix proteins, the tenascin (TN) family of glycoproteins (TN-C, TN-R, TN-W, and TN-Y) displays highly restricted and dynamic patterns of expression in the embryo, particularly during neural development, skeletogenesis, and vasculogenesis . These molecules are re-expressed in the adult during normal processes such as wound healing, nerve regeneration, and tissue involution, and in the pathological states including vascular disease, tumorogenesis, and metastasis. Expression of tenascins is known to be regulated by a variety of growth factors, cytokines, vasoactive peptides, extracellular matrix proteins, and biomechanical factors (Jones and Jones (2000) Dev. Dyn. 218:235-59).

The roles of tenascin and transforming growth factors have been analyzed in the diagnosis and prognosis of various diseases and pathological conditions including breast cancer, wherein it has been demonstrated that tenascin limits tumor spread (Kaya, et al . (2002) Eur. J. Gynaecol. Oncol. 23:261-3). Immunohistochemical detection of molecules involved in inflammatory reaction can be useful for the diagnosis of vitality in skin wounds. The expression of fibronectin

(FN) and TN in 58 human skin wounds has been analyzed, wherein both vital and postmortem hemorrhages showed an enhanced positivity for FN and TN, thus impeding the diagnosis (Ortiz-Rey, et al . (2002) Forensic Sci . Int. 126:118-22). The presence and distribution of tenascin in the human intervertebral disc has been studied. It was found that in young, healthy disc, tenascin was abundant throughout the annulus, while degenerating discs in adults showed localization restricted to the pericellular, and rarely, more restricted intra territorial matrix (Kaya, et al . (2002) supra). These observations indicated that changes in the amount and distribution of tenascin might have a role in disc aging and degeneration (Gruber, et al . (2002) Biotech. Histochem. 77:37-41).

The role of TGF-β in the pathogenesis of liver diseases and its possible use as an indicator of disease progression has been suggested (Flisiak, et al. (2000) Wiad. Lek. 53:530-7). The effect of tropically applied TGF-βl on the rat gingival wound healing process after flap surgery was evaluated by immuno-histochemistry for extracellular matrix molecules, such as tenascin, heparan sulfate proteoglycan and type IV collagen, and for proliferating cell nuclear antigen in fibroblasts. It was found that TGF-βl application appeared to promote granulation tissue formation in periodontal wound healing (Okuda, et al. (1998) J. Oral Pathol. Med. 27:463-9). A study on wound healing proposed that the frozen cultures of human keratinocytes promote faster re-epitheliazation through the release of growth factors such as TGF-α and through the stimulation of murine subepithelial cells, by TGF-β, to secrete basement membrane proteins such as collagen IV, laminin, and tenascin, which provide a provisional substrate that improves migration of the murine epidermal cells (Okuda, et al. (1998) supra).

There have been very few studies probing into the changes brought about by radiation on TN and TGF. One early response to radiation exposure is the induction of TGF-β, which mediates numerous events during tissue repair, growth, and extracellular matrix production (Tamariz- Dominguez, et al . (2002) Cell Tissue Res. 307:79-89). Radiation-induced activation of TGF-β may have profound implications for understanding tissue effects caused by radiation therapy (Barcellos-Hoff (1993) Cancer Res. 53:3880-6). TGF-β has been shown to be rapidly activated after exposure of mice to ⁶⁰Co-γ radiation, which also generates reactive oxygen species. It was postulated that oxidation of specific amino acids in the latency-conferring peptides leads to a conformation change in the latent complex that allows release of TGF-β (Barcellos-Hoff, et al. (1994) J. Clin. Invest. 93:892-9).

Light therapy is an emerging field, wherein light emitting diodes and other emitters of electromagnetic radiation are used to treat various medical conditions such as acne, hair growth stimulation, hair growth inhibition, scar reduction and removal, wrinkle reduction, etc. For example, U.S. Patent Application Serial No. 10/119,772 describes the manipulation of collagen, fibroblast, and fibroblast-derived cell levels in mammalian tissue using a plurality of pulses from at least one source of narrowband, multichromatic electromagnetic radiation (e.g., from a light emitting diode, a laser, a fluorescent light source, an organic light emitting diode, a light emitting polymer, a xenon arc lamp, a metal halide lamp, a filamentous light source, an intense pulsed light source, a sulfur lamp, and combinations thereof) having a dominant emissive wavelength of from about 300 nm to about 1600 run, and wherein said pulses have a duration of from about 0.1 femtoseconds to about 100 seconds, the interpulse delay between said pulses is from about 0.1 to about 1000 milliseconds, and the energy fluence received by said tissue is less than about 10 joule per square centimeter. Light therapy has also been used employed in the treatment of skin diseases such as psoriasis. For example, psoralen, administered orally or topically before ultraviolet-A exposure, selectively induces T-lymphocytes to undergo apoptosis (Coven, et al . (1999) Photodermatol . Photoimmunol. Photomed. 15:22-7); however, this treatment also suppresses DNA-synthesis by cross-linking DNA-strands and conjugating proteins thereby causing cell cycle arrest. Ultraviolet-B light (UVB) exposure has also been used in the treatment of psoriasis to diminish the hyperkeratosis of the epidermal cells by up-regulating the tumor suppressor gene product p53, which may also play a role in induced apoptosis (Honigsmann (2001) Clin. Exp. Dermatol. 26:343-50). Another treatment using UV-light is the 308-nm Excimer laser, wherein psoriasis lesions are irradiated with UVB laser light (Gerber, et al . (2003) Br. J. Dermatol. 149:1250-8; Trehan and Taylor (2002) J. Am. Acad. Dermatol. 47:701-8; Feldman, et al . (2002) J. Am. Acad. Dermatol. 46:900-6; Asawanonda, et al . (2000) Arch. Dermatol. 136:619-24).

Summary of the Invention

The present invention relates to a method for increasing expression of TGF- β by exposing tissue to a high-fluency, intense pulsed light source.

The present invention also relates to a method for modulating an immune response and a method for treating a disease or condition of the skin by exposing skin to a high-fluency, intense pulsed light source.

Brief Description of the Drawings

Figure 1 illustrates the relative expression of tenascin and TGF-β in the follicular bulb (Figure IA) , germinative epidermis (Figure IB) and in plasma cells (Figure 1C) up to approximately 4 months after treatment with an intense pulsed light source.

Detailed Description of the Invention

It has now been shown that a high-fluency, intense pulsed light source can induce the expression of TGF-β in skin cells, resulting in the release of extracellular matrix proteins such as tenascin, and an increase in the concentration of fibroblasts and plasma cells. As the high-fluency, intense pulsed light source methodology disclosed herein produces an artificial wound which stimulates the immune system and extracellular matrix formation, the present invention relates to the use of a high-fluency intense pulsed light source for dermatologic treatment and plastic surgery treatment. Such dermatologic treatment includes, e.g., accelerating wound and burn healing and the treatment of skin diseases such as psoriasis and eczema. To demonstrate the utility of high-fluency intense pulsed light in dermatologic treatment, pigs were treated with 40 flashes (6.5-21 ms) of light having a wavelength ranging from 565 to 695 run and a fluency of 35-60 J/cm². With higher fluency, erythema was observed. Using a wavelength of 640 nm, pulse time between 4.2 to 4.8 milliseconds (ms) and energy of 35 J/cm², negligible erythema was observed. While no blisters were identified, hair on the pigs was burnt and transient depigmentation was observed, but disappeared after some time. Biopsy samples taken from the irradiated pigs were stained for the presence of TGF-βl and tenascin. One day after radiation, the appearance of TGF-βl in the basal epidermal layer and the follicular bulb was detected. Fibroblasts and plasma cells were also strongly positive. After 1 week, the outermost epidermis, endothelial vessels, follicular bulb, fibroblasts and plasma cells were intensely stained (Figure 1). TGF-βl immunoreactivity decreased after 2 weeks, reaching a level similar to that found on the first day of treatment. After one month, the expression of TGF-β reached a peak value and then began to slowly fall in the second and third month. Subsequently, TGF-βl levels began to increase and on day 120 elevated levels were observed.

Tenascin was visible below the epidermal germ layer and around the hair follicles. At about 7-10 days after radiation, the biopsies showed a strong increase in tenascin expression (Figure 1) . Tenascin was found to be localized in the papular dermis, the epidermal layer and also in the depths of the dermis, while in the endothelial vessels and the follicular bulb there was a sharp decrease. Biopsies showed a sharp decrease in tenascin immunoreactivity after one month, but this gradually recovered in the germinative epidermis, papular dermis and the follicular bulb in the second month, but not in the endothelial vessels and plasma cells, where it was absent. In the third month, tenascin levels gradually increased. The results of the present analysis indicate that high-fluency, intense pulsed light can induce the expression of TGF-β in skin cells, resulting in the release of extracellular matrix proteins such as tenascin. Accordingly, the present invention is a method for increasing TGF-β levels, in particular TGF-βl, in a tissue (e.g., human or mammalian skin) using an effective amount of high-fluency, intense pulsed light. An effective amount of said light is an amount which elevates or increases the absolute or relative amounts of TGF-β in the tissue being treated by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the surrounding tissue or a similar tissue which has not been treated with the high-fluency, intense pulsed light source (e.g., the same tissue before treatment) . TGF-β levels can be monitored in accordance with the immunohistochemical methods disclosed herein or with more quantitative methods such as ELISA, western blot analysis, northern blot analysis or RT-PCR. As used herein, fluency is defined as the power level of the radiation reaching target tissue. In the context of the instant methods, a high- fluency radiation source produces about 3 to 90 joule per square centimeter (J/cm²) of tissue. In one embodiment of the present invention, high-fluency of about 10 to 50 J/cm² is employed. In particular embodiments, high-fluency of about 35 J/cm² is employed.

For purposes of the present invention, any device that emits intense pulsed light in a bandwidth of ± about 100 nanometers around a dominant wavelength can be can be used in accordance with the methods disclosed herein. Generally, any intense pulsed light source capable of exposing the target tissue with from about 3 to 90 J/cm² of energy in the desired wavelength (generally within the range of from about 435 ran to about 1900 nm) will be able to effect an increase in TGF-β. It is contemplated that the tissue penetration depth for intact skin may be different than the tissue penetration depth for ulcerated or burned skin and may also be different for skin that has been abraded or enzymatically peeled or that has had at least a portion of the stratum corneum removed. Thus, the wavelength used for any particular application can vary. By way of illustration, light having a dominant wavelength emission in the range of about 500 nm may be more suitable for epidermal layers of skin as such a short wavelength has limited depth of penetration, whereas light having a wavelength of about 600 nm to about 660 nm can more easily penetrate to a greater depth, if treatment of the lower dermal layers or even deeper is desired. Accordingly, the selection of the dominant wavelength of the radiation emitter is also dependent on the depth of treatment desired. Suitable wavelengths for inducing TGF-β are in the range of 590 to 690 nm. As a wavelength of 640 nm was found to minimize erythema, a dominant wavelength of 640 nm is particularly suitable for use in the methods of the instant invention. Exposure time is another aspect of intense pulsed light which can vary with the desired effect and the target cell, tissue or organ. In general, pulse lengths can vary from less than one picosecond to several seconds with interpulse intervals of a few picoseconds to a few hundred milliseconds. In particular embodiments, the pulse length is in the range of 0.1 to 500 milliseconds and the interpulse interval in the range of 20 to 60 milliseconds. To maximize TGF-β production, while minimizing erythema, pulse durations of from about 4.2 to 4.8 milliseconds with an interpulse interval of about 20 milliseconds are particularly suitable.

Similarly, the number of pulses (also referred to as repetitions or flashes) per treatment can also be varied. For example, large numbers of pulses may be less effective at inducing TGF-β than a smaller numbers of pulses. In general, a treatment regime can include between 10 and 100 pulses. In particular embodiments, the number of pulses per treatment is in the range of 40. Further, a treatment can be repeated multiple times over a given period of time (e.g., weeks or months) to achieve the desired result.

Further, light penetration into the target tissue can be optimized by varying the treatment head of the intense pulsed light source. For example, a large spot size diminishes the scattering of the light beam. Treatment head size can vary from 8-10 mm x 20-45 mm. A particularly suitable treatment head is in the range of 8 mm x 34-35 mm. By changing the different parameters of the flashlamp, namely energy, wavelength, pulse duration etc., efficient treatment for a variety of diseases or conditions can be achieved with a high degree of selectivity and safety. In a particular embodiment of the present invention, a high-fluency, intense pulsed light source used in accordance with the instant methods has an emissive wavelength of from about 435 run to about 1900 nm, wherein the pulses have a duration of from about 2 ms to about 30 ms, and the energy fluence received by the tissue is about 3 to 90 joule per square centimeter.

Suitable flashlamp systems which can be used for carrying out the methods disclosed herein include, but are not limited to, the IPL™ Quantum, VASCULIGHT™, and LUMENIS ONE™, PHOTODERM™ and EPILIGHT™ systems as well as those disclosed in GB 2,293,648; EP 0 565 331; U.S. Patent No. 5,405,368; and WO 91/15264. For example, the VASCULIGHT™ (ESC Medical Systems Ltd. , Yokneam, Israel) and IPL™ Quantum (ESC Sharplan, Yokneam, Israel) systems are based on the principal of selective photothermolysis . In contrast to laser systems, these flashlamps use noncoherent light of a broad-band spectrum in the range of 435-1900 nm. By applying different cut-off filters (e.g., 515, 550, 570, 590, 615, 645, 695, and 795 nm) , a specific part of the shorter wavelengths can be cut off. The pulse duration ranges between 2-5 ms (VASCULIGHT™) and 6-26 ms

(.IPL™ Quantum) . Further, fluence ranges between 3-90 J/cm²

(VASCULIGHT™) and 5-45 J/cm² (IPL™ Quantum) can be achieved. Other suitable intense pulsed light sources or flashlamps which can be used in accordance with the parameters disclosed herein are well-known to those of skill in the art.

In particular embodiments, a water-containing gel or solution is used between the skin and the intense pulsed light source probe during treatment to facilitate optical coupling of the light and facilitate heat transfer from the epidermis to the gel.

Inflammation is the normal acute reaction of the tissues after any wound or injury. Macrophages and neutrophils enter the wounded area and start to clean up foreign material, bacteria and dead cells (Clark, et al.

(1996) Am. J. Pathol. 148:1407-21). When inflammation has subsided, proliferation of fibroblasts begins, which also stimulates the synthesis of extracellular matrix (Lee, et al. (1993) J. Burn Care Rehabil. 14:319-35). Eventually, fibroblast proliferation stops and the extracellular matrix matures to provide a connective tissue structure

(Lee, et al . (1993) supra). Several growth factors including TGF-β are responsible for this process. Moreover, TGF-βl and matrix metalloproteinase levels in the medium of cultured cells have been found to be significantly elevated after near-infrared irradiation and repeated exposure has been associated with a significant acceleration in the rate of wound closure (Danno, et al. (2001) Photodermatol . Photoimmunol . Photomed. 17:261-5). Studies in humans have shown that exposure to short-wave ultraviolet light significantly decreases the time to healing of superficial pressure ulcers (Wills, et al .

(1983) J. Amer. Geriatr. Soc. 31:131-3; Nussbaum, et al.

(1994) Phys. Ther. 74:812-25). Further, application of exogenous TGF-β, either locally or systemically, has been found to accelerate healing, particularly in chronic or impaired wounds (Mustoe, et al . (1997) Science 237:1333-6;

Beck, et al. (1991) Growth Factors 5:295-304). Increased tenascin expression in radiated connective tissue of human skin has also been associated with activation of cytokines (Riekki, et al . (2001) Acta Derm. Venereol . 81:329-33). Moreover, within 24 hours of wounding, tenascin appears in the basement membranes beneath epidermis and hair follicles adjacent to the wound edges and in the wounded edges of cutaneous muscle layer (Murakami, et al . (1989) Int. J. Dev. Biol. 33:439-44). At about 5-7 days, granulation tissue intensely stains with anti-tenascin serum and decreases as granulation tissue is replaced with reconstructed dermal tissue at 7-14 days (Betz, et al. (1993) Int. J. Legal Med. 105:325-8). Further, tenascin appears first in the wound area pericellularIy around fibroblastic cells approximately 2 days after wounding and after 5 days or more, intensive reactivity for tenascin is observed in the lesional area (dermal-epidermal junction, wound edge, areas of bleeding) and decreases to undetectable levels in the scar tissue (-1.5 months; Betz, et al. (1993) supra) .

In view of role of TGF-β in immunomodulation and the instant findings which demonstrate that high-fluency, intense pulsed light produces an artificial wound (i.e., not occurring by natural means such as by accident) and increases TGF-β levels in the tissue of a subject, the present invention embraces the use of a high fluency intense pulsed light source to modulate (e.g., increase or decrease) an immune response and to treat diseases or conditions of the skin. The skin of a subject in need of such immunomodulation or treatment of a disease or condition is exposed to an effective amount of high- fluency, intense pulsed light. An effective amount of said light is an amount which causes an increase in TGF-β (e.g., as discussed supra), or in the context of clinical outcome is an amount which decreases or attenuates the signs or symptoms of the disease or condition being treated or abbreviates the duration of the disease or condition in the subject being treated (e.g., accelerates wound healing) when compared to a subject who has not received such treatment. Suitable effective amounts or parameters associated with high-fluency, intense pulsed light which achieve the desired effect are disclosed herein and can vary with the disease or condition and the patient being treated. In one embodiment, an immune response is stimulated. In another embodiment, an immune system is suppressed. In particular embodiments, such methods produce little or no permanent injury or damage to nearby skin tissue.

Diseases or conditions of the skin which can be treated using a high-fluency, intense pulsed light source include, but are not limited to wounds, burns, psoriasis, eczema (neurodermitis) , viral warts, precancerous solar keratosis or skin lesions, skin ulcers (diabetic, pressure, venous stasis), and the like.

In particular embodiments, the high-fluency, intense pulsed light source as disclosed herein is combined, either serially or simultaneously with another light or wavelength source (e.g., laser or radiofrequency) to carry out the therapeutic methods disclosed herein. The high- fluency, intense pulsed light source and other light or wavelength source can be used together to work synergistically, or combined, wherein each has its own effect. Suitable parameters for radiofrequency include a fluence in the range of about 0.5 J/cm² to about 500 J/cm²; a wavelength in the range of about 300 nm to about 1200 nm; a frequency in the range of 500 kHz to 300 MHz; and a spot size in the range of 1 mm² to 10 cm².

The invention is described in greater detail by the following non-limiting examples.

Example 1 : Animal Model

Male, dark-haired pigs were randomly selected for analysis. An intra muscular injection of Azopyrone (0.5 mg/kg) was administered to initiate anesthesia. The anesthesia was maintained with halothane (3-4%) and nitrous oxide. The back region of the pigs, i.e., between their forelegs and hind legs, and the abdomen was shaved to achieve a maximum penetration depth. The area was washed with hibiscub. Treatment sites were outlined by West Indian ink applied to the skin with hypodermic needles. Four marks outlined a 40 x 20cm² treatment area. No ointment was applied pre- or post-operatively in the treatment area. No chemical or mechanical debridement was performed during follow up examinations. Buprenorphine, 0.05-0.1 mg/kg i.p., was given soon after the treatment to minimize pain. The animals were euthanized 120-360 days after treatment using intravenous nembutal (100-150 mg/kg) .

Example 2: High-Fluency, Intense Pulsed Light Method A stencil was placed on the back of the pigs to treat the same surface every time. The pigs underwent general anesthesia for 15-30 minutes (5 times for the treatment and 5 times for the biopsies) . In total there were 40 flashes per pig, per treatment. A VASCULIGHT™ system with 590 and 695 nm filters and a QUANTUM™ system with 565 and 640 nm filters were used. Pulse times were 6.5 to 21 milliseconds and the fluence was 35 to 60 J/cm². An entire surface of 40 x 2.8 cm² was treated per pig. The treatment took 4 to 5 minutes with an interval of 4 weeks between treatments. Treatment regimes are listed in Table 1.

TABLE 1

Biopsies were performed immediately following light treatment and again on the second, seventh, fourteenth and twenty-eighth post-operative day. In the second month, one week after the last biopsy, the pigs were radiated again and after 3 weeks biopsies were performed. This was continued for four months. A total amount of 240 biopsies were thus taken. Full-thickness, 3-mm punch biopsies were obtained from each treated site. Tissue specimens were fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin for light microscopic examination.

Pigs were followed daily during the first week. After that they were seen and evaluated after 1, 2, 3, 6, 12, and 30 weeks. They were examined for presence of crust formation, swelling, bleeding, erythema, wound healing, scar formation, depigmentation and loss of hair. Photographs were taken before and after treatment.

Example 3 : Immunohistochemistry Tissue blocks of pig skin biopsy were fixed in 4% neutral-buffered paraformaldehyde at 4°C for 4 to 12 hours. The specimens were then routinely processed for paraffin embedding at 56⁰C. Paraffin sections measuring 4 μm were used for light microscopy and immunohistochemical analysis. Paraffin sections were deparaffinized and rehydrated. Immunohistochemical demonstration of tenascin and TGF-β antigens was performed on these sections with the avidin- biotin peroxidase (ABC) method. Briefly, these sections were processed through the following incubation steps: hydrogen peroxide 3% in methanol for 30 minutes to block endogenous peroxidase; normal goat serum for TGF-βl and normal horse serum for tenascin, diluted 1:75 v/v, for 30 minutes to 1 hour to reduce non-specific background staining; incubation with primary antibody anti-TGF-βl (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and with primary antibody anti-tenascin (Sigma, St Louis, MO) overnight at 4⁰C; biotinylated secondary goat anti-rabbit IgG (for TGF- βl ), and horse anti-mouse IgG (for tenascin), diluted 1:200 v/v, for 30 minutes to 1 hour (Vector Labs, Burlingame, CA) ; ABC complex for 1 hour (VECTASTAIN® ABC kit, Vector Labs); and histochemical visualization of peroxidase using 3 ' , 3 ' -diaminobenzidine hydrochloride as chromogen (Sigma, St. Louis, MO) for 5 minutes in a dark room. Sections were then rinsed in tap water, counterstained with hematoxylin, dehydrated and mounted with EUKITT® (Kindler GmbH & Co . , Freiburg, Germany) .

For the above immunohistochemical procedures controls included omission of the primary or secondary antibody which resulted in negative staining and the use of human placenta as positive control for tenascin and TGF-βl antibodies .

Example 4: Statistical Analysis

The statistical analysis of the data was descriptive.

The data is presented as contingency tables to detect association between technical and clinical parameters.

Exact Fisher tests were used to evaluate the homogeneity of the tables, p-values below 0.05 are considered significant.

Claims

What is claimed is;

1. A method for increasing expression of TGF-β comprising exposing tissue to a high-fluency, intense pulsed light source thereby increasing expression of TGF-β.

2. A method for modulating an immune response in skin comprising exposing skin to a high-fluency, intense pulsed light source thereby modulating an immune response in the skin.

3. The method of claim 2, further comprising exposing the skin to laser or radiofrequency.

4. A method for treating a disease or condition of the skin comprising exposing skin in need of treatment to a high-fluency, intense pulsed light source thereby treating the disease or condition of the skin.

5. The method of claim 4, further comprising exposing the skin to laser or radiofrequency.

6. An apparatus for carrying out the method of any of claims 1 to 5.