Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation.

Suitably brief pulses of selectively absorbed optical radiation can cause selective damage to pigmented structures, cells, and organelles in vivo. Precise aiming is unnecessary in this unique form of radiation injury because inherent optical and thermal properties provide target selectivity. A simple, predictive model is presented. Selective damage to cutaneous microvessels and to melanosomes within melanocytes is shown after 577-nanometer (3 x 10(-7) second) and 351-nanometer (2 x 10(-8) second) pulses, respectively. Hemodynamic, histological, and ultrastructural responses are discussed.

Immunologic inhibition of ultraviolet radiation-induced tumor suppressor cell activity.

Long-term exposure of C3H mice to ultraviolet radiation resulted in the formation of suppressor T cells that recognize ultraviolet radiation-induced regressor skin cancers as a class before the appearance of overt tumors. Administration of monoclonal antibodies to the product of the I-Jk subregion of the major histocompatibility complex or low doses of cyclophosphamide in vivo inhibited the development or activity of these cells. This activity of the monoclonal antibody was eliminated by adsorption on B10.BR (I-Jk) but not B10.D2 (I-Jd) splenocytes. These findings provide evidence that elements expressing the I-J determinant are important in regulating the host response prior to the overt development of ultraviolet radiation-induced skin cancers and suggest novel therapeutic approaches to malignancies or other diseases involving suppressor T cells in their pathogenesis.

Photosynthesis of previtamin D3 in human skin and the physiologic consequences.

Photosynthesis of previtamin D3 can occur throughout the epidermis in the dermis when hypopigmented Caucasian skin is exposed to solar ultraviolet radiation. Once previtamin D3 is formed in the skin, it undergoes a temperature-dependent thermal isomerization that takes at least 3 days to complete. The vitamin D-binding protein preferentially translocates the thermal product, vitamin D3, into the circulation. These processes suggest a unique mechanism for the synthesis, storage, and slow, steady release of vitamin D3 from the skin into the circulation.

Preferential light absorption in atheromas in vitro. Implications for laser angioplasty.

Laser angioplasty, the in situ ablation of arterial obstructions with laser radiation, has been demonstrated in animal models and early clinical trials. A problem with this technique, however, is the possibility of thermal damage to adjacent or underlying normal tissues that also absorb the radiation. Using a spectrophotometer with an integrating sphere and a specially constructed tunable-dye laser-based spectrophotometer, we evaluated the transmittance and remittance of human cadaveric atheromas and adjacent normal aorta from 250 to 1,300 nm to identify wavebands where there is preferential light absorption by atheromas. Data were analyzed by both the Kubelka-Munk formalism and a Beer's law model. Both methods indicate that atheromas absorb more than normal aorta between 420 and 530 nm. At 470 nm the average Kubelka-Munk absorption coefficient of atheromas from 10 cadavers was 54 +/- 9 cm-1 compared with 26 +/- 6 cm-1 for normal aortic specimens from seven cadavers. Yellow chromophores responsible for the atheroma absorbance were extractable with xylenes. Thin-layer chromatography and absorption spectra identified the extracted chromophores as predominantly consisting of a mix of carotenoids, which are known constituents of atheromatous lesions. Preferential absorption of blue light by carotenoids in atheromas may permit selective ablation of atheromatous obstructions with appropriate pulses of laser radiation.

Therapeutic in vivo photochemistry: photochemical toxicity studies in humans.

Information obtained by studies of phototherapy and photochemotherapy is reviewed and some of the advantages and disadvantages of using in vivo photochemistry to treat disease are examined. Selective in vivo photochemistry is used to treat a variety of diseases. Study of the dosimetric aspects of these treatments and quantitative analysis of acute risks are instructive to individuals interested in photochemical toxicity in humans. Results from prospective short-term and long-term clinical trials are especially instructive because the model used is the intact living human. These studies provide important data about toxic, allergic, and carcinogenic aspects of photochemical toxicity, which could not be obtained in routine animal toxicity experiments. Work done to develop, explain, or improve these treatments also provides insight into molecular mechanisms of photon-induced cell injury and the sequelae of that injury.

Induction and transplantation of murine skin cancers induced by methoxsalen plus ultraviolet (320-400 nm) radiation.

Properties of skin tumors resulting from treatment of mice with ip methoxsalen plus UVA (320-400 nm) radiation (PUVA) were compared to those of skin tumors induced by UVB (280-320 nm) radiation. Repeated treatment of C3H/HeN murine mammary tumor virus-negative (C3H-) mice with PUVA produced both fibrosarcomas and squamous carcinomas that were indistinguishable morphologically from UVB-induced tumors. Fragments of 12 primary PUVA-induced tumors were transplanted into normal and immunosuppressed syngeneic recipients, and all grew progressively. In contrast, 19 primary tumors induced by repeated exposure of C3H- mice to UVB grew only in the immunosuppressed mice and not in the normal recipients. The growth of graded doses of cells from 5 PUVA-induced tumors was compared in normal and UVB-irradiated recipients. No preferential growth in UVB-irradiated mice was observed, even though this is characteristic of UVB-induced tumors. Thus PUVA-induced tumors do not appear to have the same antigenic properties as UVB-induced tumors.

Cytotoxicity and mutagenicity of low intensity, 248 and 193 nm excimer laser radiation in mammalian cells.

The cytotoxicity of 193 and 248 nm excimer laser radiation was compared to that produced by a germicidal lamp (predominantly 254 nm) using Chinese hamster ovary cells (CHO), and a human diploid fibroblast line, AG-1522A. Excimer laser radiation at 248 nm (3.5 X 10(2) w/m2) and germicidal radiation (5.3 X 10(-5) w/m2) caused toxicity in both cell lines, with the AG-1522A cells (D37 = 7-8 J/m2) being slightly more sensitive than the CHO cells (D37 = 11 J/m2). Incident 193 nm radiation was less cytotoxic than 248 nm to AG-1522A and CHO cells with D37 values of 18 and 85 J/m2, respectively. The mutagenic potential of UV excimer radiation at 193 and 248 nm was evaluated using the hypoxanthine guanine phosphoribosyl transfer assay system with CHO cells. Excimer laser radiation at 248 nm induced mutation in proportion to dose (1.7 X 10(-5) resistant colonies per survivor per J/m2 incident radiation) up to 14 J/m2, similar to results reported for 254 nm light. However, excimer laser radiation at 193 nm did not cause mutation greater than the dark control. The decreased cytotoxicity and mutagenicity of 193 nm radiation may be due to the shielding of the nucleus by cytoplasmic and membrane components or to the formation of different DNA photoproducts. These differences between 193 and 248 nm radiation may be important in choosing an excimer wavelength for ablation in biological systems.

Phototherapy and photochemotherapy of skin diseases.

One important aspect of photomedicine is the use of nonionizing electromagnetic radiation with and without exogenous photosensitizers to treat diseases. Phototoxicity (cell injury by photons) is a likely mechanism for phototherapy and photochemotherapy of several skin diseases. The mechanism of action for phototherapy of hyperbilirubinemia and of uremic pruritus appears to be photochemical alteration of extracellular metabolites. Psoriasis is an example of a disease benefitted by several forms of phototherapy and photochemotherapy with varying relative effectiveness and safety. Two successful forms of treatment are oral psoralen photochemotherapy and UVB plus topical adjunctive agents. New information about UVB therapy of psoriasis includes data about the therapeutic action spectrum and about the relative roles of various topical agents such as coal tar, mineral oil, "lubricants" and steroids. Although there are many surface similarities, phototherapy and psoralen photochemotherapy have fundamental differences which may alter longterm risks in quantitative and qualitative ways.

New concepts in therapeutic photomedicine: photochemistry, optical targeting and the therapeutic window.

Advances in optics technology, synthetic photochemistry, and the science of photobiology make it possible to think beyond phototherapy and photochemotherapy which is dependent on direct photochemical alteration of metabolites or direct phototoxic insult to cells. This report discusses another gender of photomedicine therapy which includes in vivo photoactivation of medicines, photon-dependent drug delivery, and manipulation of host and exposure source to maximize therapeutic index. These therapeutic manipulations are made possible because the skin is highly overperfused and because non-ionizing electromagnetic radiation that enters skin and blood has adequate photon energy to cause electronic excitation. Radiation of 320-800 nm is not very directly phototoxic, is absorbed by a variety of relatively nontoxic photolabile molecules and has an internal dosimetric depth profile. This radiation can therefore be used to activate, deactivate, bind, release or biotransform medications in vivo in skin or other organs. The photochemist, synthetic chemist and photobiologist can collaborate to significantly increase therapeutic possibilities.

Action spectrum for phototherapy of psoriasis.

Using a monochromator the action spectrum for ultraviolet phototherapy of psoriasis was determined for radiation between 254 and 313 nm and compared to the action spectrum for erythema of uninvolved adjacent skin. Daily exposures of different doses of 254, 280, 290, 296, 300, 304 and 313 nm radiation were observed. Wavelengths of 254, 280, 290 nm were erythemogenic but not therapeutic even at 10 to 50 times the minimal erythema dose. At the other wavelengths studied, the 2 action spectra were similar. In general, fixed daily doses cleared at lower cumulative dose than did incrementally increased daily doses. The small number of suberythemogenic exposure doses required suggests that monochromatic radiation may have advantages over broadband sources.

The interaction of UVA and UVB in the production of threshold erythema.

A study was done to demonstrate quantitatively and graphically the way in which suberythemogenic doses of broadband UVA and UVB interact in producing a visible erythema. On the backs of fair-skinned human volunteers the minimal erythema dose (MED) was determined for polychromatic UVA and UVB. Increasing fractions of the UVA MED were given to sites already exposed to various fractions of the UVB MED resulting in sites exposed to various doses of both UVA and UVB. The same experiment was repeated with the order of wavebands reversed. It was demonstrated that when UVA was followed by UVB an erythema was produced in those sites where the sum of the fractions was equal to one, an interaction termed photoaddition. When the UVA exposure followed the UVB, erythema was again predominantly noted in those sites demonstrating photoaddition. However, in the latter case, numerous sites of threshold erythema were noted where the sum of the fractions was greater than one. This is suggestive of photorecovery. No evidence of photoaugmentation was observed with either order of exposure.

The effect of topical fluocinonide ointment on phototherapy of psoriasis.

The effect of fluocinonide ointment and 5% crude coal tar on clearance of plaque-type psoriasis vulgaris by phototherapy was studied in 25 hospitalized patients using the bilateral comparison technique. All treated areas received ultraviolet radiation from Westinghouse fluorescent FS-40 bulbs (290-400 nm) in doses calculated to produce a minimal delayed erythema. The topically applied compounds (fluocinonide ointment, 5% Crude coal tar, white petrolatum) were applied individually or in combination. In nearly all comparisons clearance of psoriatic plaques was obtained after the same number of ultraviolet exposures, although many (8 or 14) of the areas treated with fluocinonide ointment had an accelerated early response. It thus appears that although the use of topical corticosteroids may enhance the early therapeutic response of psoriatic plaques it does not hasten the clearance of these plaques.

The optics of human skin.

An integrated review of the transfer of optical radiation into human skin is presented, aimed at developing useful models for photomedicine. The component chromophores of epidermis and stratum corneum in general determine the attenuation of radiation in these layers, moreso than does optical scattering. Epidermal thickness and melanization are important factors for UV wavelengths less than 300 nm, whereas the attenuation of UVA (320-400 nm) and visible radiation is primarily via melanin. The selective penetration of all optical wavelengths into psoriatic skin can be maximized by application of clear lipophilic liquids, which decrease regular reflectance by a refractive-index matching mechanism. Sensitivity to wavelengths less than 320 nm can be enhanced by prolonged aqueous bathing, which extracts urocanic acid and other diffusible epidermal chromophores. Optical properties of the dermis are modelled using the Kubelka-Munk approach, and calculations of scattering and absorption coefficients are presented. This simple approach allows estimates of the penetration of radiation in vivo using noninvasive measurements of cutaneous spectral remittance (diffuse reflectance). Although the blood chromophores Hb, HbO2, and bilirubin determine dermal absorption of wavelengths longer than 320 nm, scattering by collagen fibers largely determines the depths to which these wavelengths penetrate the dermis, and profoundly modifies skin colors. An optical "window" exists between 600 and 1300 nm, which offers the possibility of treating large tissue volumes with certain long-wavelength photosensitizers. Moreover, whenever photosensitized action spectra extend across the near UV and/or visible spectrum, judicious choice of wavelengths allows some selection of the tissue layers directly affected.

Prolongation of murine skin allograft survival by the systemic effects of 8-methoxypsoralen and long-wave ultraviolet radiation (PUVA).

Systemic administration of the photoactive drug 8-methoxypsoralen to a group of mice bearing cutaneous allografts, followed by exposure to long-wave ultraviolet radiation (UVA, 320-400 nm) (PUVA) daily for 14 days at a site distant from the allograft, significantly increased the survival time of the allografts. This effect was seen both in donor-recipient combinations that differ at the major histocompatibility complex and in those differing only at minor histocompatibility loci. Treatment with 8-methoxypsoralen or long-wave UV radiation alone was ineffective in prolonging allograft survival, as were doses of mid-wave UV radiation (UVB, 280-320 nm) that produced greater inflammation than the PUVA protocol. Allografted, PUVA-treated animals also demonstrated decreased alloantigen reactivity against donor-strain spleen cells during the period of treatment by cytotoxicity assays. Allografts of skin in the murine system are highly immunogenic and are generally rejected faster than organ allografts; thus PUVA treatment appears to exert a potent effect on prolonging allograft survival. The systemic nature of the effect and the fact that adverse side effects from PUVA are largely limited to the skin suggest that PUVA might have a role in clinical organ transplantation management.

Cumulative effects of repeated subthreshold doses of ultraviolet radiation.

For fair Caucasian skin, the minimal delayed erythema dose (MED) 24 hr after exposure to broadband UVA is about 1200 times greater than the MED of broadband UVB, for both single and multiple daily exposures. Repeated daily exposure to doses less than MED results in cumulative effects manifest by gradual lowering of the daily dose threshold for delayed erythema and pigmentation induced by UVA or UVB. At threshold doses, UVB is more erythemogenic than melanogenic; the opposite is true for UVA. Repeated daily UVA exposure greatly enhances melanogenesis such that markedly suberythemogenic exposure doses of UVA result in true melanogenesis.

Recovery of skin from a single suberythemal dose of ultraviolet radiation.

Previous studies of exposure of normal skin to ultraviolet radiation have demonstrated a cumulative effect lasting greater than 24 h when repeated suberythemal exposures are given. However, the time course of recovery from a single suberythemal dose of ultraviolet A (UVA) or ultraviolet B (UVB) has not been determined. We show here for the first time that the period required for recovery of normal skin (as measured by delayed erythema) following a single suberythemal dose of UVA is between 30 and 48 h, and for UVB is between 24 and 30 h. Photoprotection was noted for both UVA and UVB from the fifth through the ninth day after the single suberythemal exposure, but was only statistically significant on the fourth day after UVB exposure. The curve depicting recovery from a single suberythemal dose of UVA or UVB from the first irradiation time through the fourth day after irradiation may be described as an exponential decay curve. Formulas are given for both UVA and UVB which describe the exponential nature of this curve. These formulas may be used to predict the exact difference in erythema threshold between preirradiated and normal skin. From the fourth day after exposure to the ninth day, the curve is nearly constant. The nature of the recovery curve in the first 4 days after exposure suggests that an exponential decay process occurs in UVA or UVB damage, consistent with unstable photoproduct decay, DNA repair, or constitutive enzymatic processes.

Psoriasis and the risk of cancer.

To test the validity of the common hypothesis that patients with psoriasis have an inherently lower susceptibility to cancer, we prospectively studied 1367 patients with severe psoriasis who enrolled in a clinical trial of oral methoxsalen photochemotherapy for treatment of psoriasis for an average of 3.2 yr. The incidence of non-cutaneous cancers and the number of deaths from such cancers were slightly but not significantly higher than that expected for the general population (relative risk = 1.1; standard mortality ratio = 1.3). Among 318 patients (23%) in this cohort who were over 35 yr of age and who lacked appreciable exposure to suspected cutaneous carcinogens, the observed incidence for cutaneous carcinoma was 1.4 times that expected, based on rates for the general population (90% confidence interval = .8 to 2.7). These data suggest that patients with psoriasis have a risk of systemic and cutaneous cancer that is comparable to the risk for the general population.

Effect of skin temperature on selective vascular injury caused by pulsed laser irradiation.

The effect of skin temperature on vascular-specific injury caused by pulsed laser irradiation was examined. Ten healthy human volunteers were exposed to 1.5 microsecond pulses from a dye laser tuned to 577 nm. Compared to normothermic conditions (33 degrees C skin temperature) significantly more laser energy (p less than 0.01) was required to produce grossly visible purpura when the skin was cooled to 20 degrees C or heated to 40 degrees C. Histologically, laser-induced damage was confined to blood vessels at all three skin temperatures studied. At purpura threshold dose, there was intravascular agglutination without extravasation of red blood cells at 20 degrees C whereas at 33 degrees and 40 degrees C there was extravasation of red blood cells.

UVA-induced erythema, pigmentation, and skin surface temperature changes are irradiance dependent.

Human cutaneous erythemogenic and melanogenic responses to long-wave (UVA) ultraviolet radiation were investigated using irradiances ranging from 5-50 mW/cm2. Skin surface temperature changes resulting from the different irradiances were also compared. In general, threshold doses for erythema and pigmentation were higher when UVA was administered at the lowest irradiance (5 mW/cm2) than at the highest (50 mW/cm2). Erythema was maximal immediately after exposure to UVA. The most intense responses (erythema with edema, or intense pigmentation) were induced more frequently by the highest irradiance. Components of both the erythema and the pigment response to UVA are therefore irradiance-dependent. The greatest increase in skin surface temperature was observed after exposure to the highest irradiance.

Ultrastructural changes in human skin after exposure to a pulsed laser.

Selective vascular injury following irradiation using a pulsed laser source at 577 nm was examined using ultrastructural methods in the skin of 3 fair-skinned healthy human volunteers. This vascular-specific damage was confined to the papillary dermis. Red blood cells were altered in several ways. As well as an increase in the electron density, configurational distortion modified the normal biconcave forms to ameboid structures. The most interesting finding was the appearance within these altered cells of well-defined circular/oval electron-lucent areas of 800 A diameter, possibly representing a heat-fixed record of steam formation within the red blood cell. In addition, considerable degenerative changes were evident in endothelial cells and pericytes, while mast cells, neutrophils, histiocytes, and fibroblasts as well as collagen bundles immediately surrounding most laser-damaged blood vessels appeared normal.