Effect of glucocorticosteroids on epidermal Langerhans cells.

The effects of topical and systemic administration of various glucocorticoids on the density of epidermal Langerhans cells (LC) were studied in guinea pigs. Glucocorticoids, such as betamethasone dipropionate and valerate, caused a marked decrease in LC demonstrable by staining for cell membrane ATPase activity and Ia antigens. By electronmicroscopy, LC also showed morphologic alterations. The observed decrements in LC density correlated with the concentration and known vasoconstrictive potency of the glucocorticoids administered. The anti-inflammatory action of glucocorticoids in skin disorders may, at least in part, be through their ability to alter epidermal LC, thus interfering with the antigen-presenting functions of these cells.

Expression of murine CD1 on gastrointestinal epithelium.

Cluster of differentiation 1 (CD1) in humans is a family of major histocompatibility complex (MHC) class I-like molecules expressed on the surface of immature thymocytes, Langerhans cells, and a subpopulation of B cells. The only function identified for human CD1 is as a ligand recognized by a subpopulation of T lymphocytes. In order to study the distribution and function of these molecules in the mouse, a murine CD1 complementary DNA was expressed in mouse fibroblasts and used to produce monoclonal antibodies. These antibodies revealed prominent expression of murine CD1 only on gastrointestinal tract epithelium and in the cytoplasm of hepatocytes. Low levels of expression were also detected on thymocytes and peripheral lymphocytes. The gastrointestinal distribution of murine CD1 suggests that this molecule may be important in epithelial immunity.

Metastatic malignant melanoma.

Metastatic malignant melanoma is an incurable malignancy with extremely poor prognosis. Patients bearing this diagnosis face a median survival time of approximately 9 months with a probability of surviving 5 years after initial presentation at less than 5%. This is contrasted by the curative nature of surgical resection of early melanoma detected in the skin. To date, no systemic therapy has consistently and predictably impacted the overall survival of patients with metastatic melanoma. However, in recent years, a resurgence of innovative diagnostic and therapeutic developments have broadened our understanding of the natural history of melanoma and identified rational therapeutic targets/strategies that seem poised to significantly change the clinical outcomes in these patients. Herein we review the state-of-the-art in metastatic melanoma diagnostics and therapeutics with particular emphasis on multi-disciplinary clinical management.

Pharmacokinetics of a fluorescent drug using laser-induced fluorescence.

Laser-induced fluorescence has been used to measure tissue levels of chloroaluminum sulfonated phthalocyanine in vivo in an implanted hamster cheek pouch carcinoma tumor model. The drug was excited at 610 nm via a pulsed nitrogen laser-pumped dye laser, and fluorescence intensity was monitored at 684 nm for up to 30 days after drug administration. Data were acquired noninvasively with high temporal and spatial resolution using the laser-induced fluorescence apparatus and were analyzed with a multicompartment pharmacokinetic model. In addition, our published data on a C6-BAG glioma rat brain tumor model were analyzed to illustrate the effect of different tumor models on the rates. The rates extracted from the pharmacokinetic model elucidate the mechanisms of drug uptake and retention in the cheek pouch and brain tumor models. The laser-induced fluorescence approach should lead to better drug dosimetry for photochemotherapy and allow quick characterization of the pharmacokinetics of new photosensitizers in tissue.

Rhodamine 123 phototoxicity in laser-irradiated MGH-U1 human carcinoma cells studied in vitro by electron microscopy and confocal laser scanning microscopy.

Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that localizes preferentially within mitochondria of living carcinoma cells. MGH-U1 human bladder carcinoma cells incubated in vitro with 10 microM R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser underwent selective, phototoxic injury to mitochondria. Ultrastructurally, treatment with R123 plus irradiation with 10 J/cm2 caused selective, progressive mitochondrial alterations consisting of disruption of cristae, vacuolization, swelling, increasing numbers of ring-shaped and angulated mitochondria at 4 to 8 h after irradiation, and obliteration of many mitochondria at 24 to 48 h. Confocal laser scanning microscopy after treatment with R123 plus irradiation with 10 to 30 J/cm2 demonstrated altered uptake and localization of subsequently administered R123, accompanied by striking mitochondrial fragmentation. Irradiation caused a dose-dependent depletion of extractable R123, due to a photosensitized efflux that began immediately and progressed by 4 h after irradiation with 10 to 30 J/cm2; further uptake after reincubation in the presence of R123 was also quantitatively impaired in cells previously irradiated with 30 J/cm2.

Putative photoacoustic damage in skin induced by pulsed ArF excimer laser.

Argon-fluoride excimer laser ablation of guinea pig stratum corneum causes deeper tissue damage than expected for thermal or photochemical mechanisms, suggesting that photoacoustic waves have a role in tissue damage. Laser irradiation (193 nm, 14-ns pulse) at two different radiant exposures, 62 and 156 mJ/cm2 per pulse, was used to ablate the 15-microns-thick stratum corneum of the skin. Light and electron microscopy of immediate biopsies demonstrated damage to fibroblasts as deep as 88 and 220 microns, respectively, below the ablation site. These depths are far in excess of the optical penetration depth of 193-nm light (1/e depth = 1.5 micron). The damage is unlikely to be due to a photochemical mechanism because (a) the photons will not penetrate to these depths, (b) it is a long distance for toxic photoproducts to diffuse, and (c) damage is proportional to laser pulse intensity and not the total dose that accumulates in the residual tissue; therefore, reciprocity does not hold. Damage due to a thermal mechanism is not expected because there is not sufficient energy deposited in the tissue to cause significant heating at such depths. The damage is most likely due to a photoacoustic mechanism because (a) photoacoustic waves can propagate deep into tissue, (b) the depth of damage increases with increasing laser pulse intensity rather than with increasing total residual energy, and (c) the effects are immediate. These effects should be considered in the evaluation of short pulse, high peak power laser-tissue interactions.

Langerhans cells as macrophages in skin and lymphoid organs.

Properties of epidermal Langerhans cell were compared with those of a number of other dendritic cells in lymphoid organs and of mononuclear phagocytes. Among the dendritic "reticulum" cells included were indeterminate cells from the epidermis, interdigitating "reticulum" cells from T-dependent areas of lymphoid tissue and thymus, follicular dendritic cells of Nossal, and the dendritic cells described by Steinman and Cohn. Interdigitating cells with typical Birbeck granules, in the thymus and in the paracortices of lymph nodes, which are morphologically indistinguishable from Langerhans cells and indeterminate dendritic cells in the epidermis, appear to belong to the same system and possibly represent a subpopulation of "macrophages." On the basis of their similarity to these other dendritic cells, we believe Langerhans cells may function in antigen presentation, lymphokine production, provision of a microenvironment for T lymphocytes, and prostaglandin secretion.

Demonstration of T200 on human Langerhans cell surface membranes.

We have demonstrated the presence of the glycoprotein T200 on the surface of human epidermal Langerhans and indeterminate cells by immunoelectron microscopy with the use of a monoclonal antibody. No other epidermal cells were positive. The presence of T200 on Langerhans cells confirms their hematopoietic origin because T200 is limited to hematopoietic cells and neoplasms. Although the function of T200 is not known, antibodies directed against T200 have been previously shown to inhibit accessory cell and natural killer cell activities. While the existence of a possible relationship between Langerhans cells and natural killer cells is unclear, Langerhans cells are important in cutaneous accessory cell functions; therefore. T200 may be important in understanding the biology of Langerhans cells and their relationship to other cells in the immune response.

Interferons and collagen production.

The immunoregulatory, antiviral, and antiproliferative agents known as the interferons have profound effects on collagen synthesis. Interferons alpha, beta, and gamma suppress collagen synthesis by dermal fibroblasts. In addition, interferon gamma (IFN-gamma) inhibits the constitutively increased collagen synthesis characteristic of fibroblasts derived from lesions of patients with scleroderma. IFN-gamma also inhibits collagen synthesis by myofibroblasts and synovial fibroblast-like cells. Inhibition of collagen synthesis by IFN-gamma is associated with a coordinate inhibition of transcription for types I and III collagen. In addition, IFN-gamma suppresses levels of procollagen mRNA and type II collagen synthesis in human articular chondrocytes. In vivo studies in mice have demonstrated that IFN-gamma inhibits the collagen synthesis associated with the fibrotic response to an implanted foreign body, bleomycin-induced pulmonary fibrosis, and the healing response to cutaneous thermal burns. In the latter case, while collagen content of the wound scar was decreased, hyaluronic acid was increased in mice receiving IFN-gamma compared to controls. This is in accord with in vitro studies showing that, while interferons alpha and beta decrease production of glycosaminoglycans, IFN-gamma increases production of glycosaminoglycans. Of interest, acute inflammation at sites of thermal injury, or when elicited by proinflammatory agents in separate experiments, also was suppressed in mice treated with IFN-gamma. The means by which IFN-gamma inhibits collagen synthesis involves transcriptional regulation. There is a single report that interferon alpha can decrease the size of a keloid of recent onset in a human patient. Because the interferons can inhibit collagen synthesis in vivo, further studies may be warranted to evaluate the usefulness of these agents in the treatment of disease states characterized by abnormal fibrotic responses as well as their potential for altering the healing response associated with particular therapeutic interventions.

Light and electron microscopic analysis of tattoos treated by Q-switched ruby laser.

Short-pulse laser exposures can be used to alter pigmented structures in tissue by selective photothermolysis. Potential mechanisms of human tattoo pigment lightening with Q-switched ruby laser were explored by light and electron microscopy. Significant variation existed between and within tattoos. Electron microscopy of untreated tattoos revealed membrane-bound pigment granules, predominantly within fibroblasts and macrophages, and occasionally in mast cells. These granules contained pigment particles ranging from 2-in diameter. Immediately after exposure, dose-related injury was observed in cells containing pigment. Some pigment particles were smaller and lamellated. At fluences greater than or equal to 3 J/cm2, dermal vacuoles and homogenization of collagen bundles immediately adjacent to extracellular pigment were occasionally observed. A brisk neutrophilic infiltrate was apparent by 24 h. Eleven days later, the pigment was again intracellular. Half of the biopsies at 150 d revealed a mild persistent lymphocytic infiltrate. There was no fibrosis except for one case of clinical scarring. These findings confirm that short-pulse radiation can be used to selectively disrupt cells containing tattoo pigments. The physial alteration of pigment granules, redistribution, and elimination appear to account for clinical lightening of the tattoos.

Wavelength and fluence effect on vascular damage with photodynamic therapy on skin.

Normal skin phototoxicity is clinically predictable during photodynamic therapy with light at 690 and 458 nm wavelengths, in the first 5 h after intravenous bolus infusion of benzoporphyrin derivative mono-acid ring A. This study goal was to determine histologic milestones that lead to tissue necrosis with exposure to red (690 nm) and blue (458 nm) light. The threshold doses for skin necrosis on rabbits were equal at both wavelengths. Lower, equal to, and higher than threshold fluences were delivered in duplicates at hourly intervals, with 40% increments, at constant irradiance. Pathology specimens from irradiated and control sites, were collected at 0, 2, 7, 24, 48 h, and 2 wk after treatment and were paired to equivalent treated sites for clinical evaluation. Immediately after irradiation, at 690 and 458 nm thresholds, light microscopy showed stasis and inflammatory infiltrate in the papillary dermis, respectively; electron microscopy demonstrated pericyte and endothelial cell damage - greater at 690 than 458 nm. At day 1, vascular stasis in the dermis showed a steeper dose-response with red than blue light, and led to necrosis of skin appendages (day 1) and epidermis (days 1-2) at both wavelengths. Sub-threshold fluences induced similar, but significantly milder (p < 0.05) changes and epidermis recovered. Skin necrosis, at threshold fluences in photodynamic therapy with benzoporphyrin derivative mono-acid ring A, was primarily due to vascular compromise to a depth potentially reaching the subcutaneous muscle at 690 nm, whereas at 458 nm vascular damage was confined to upper dermis. This system facilitates selective destruction of skin vasculature, sparing normal epidermis.

Photomechanical transcutaneous delivery of macromolecules.

Transcutaneous drug delivery has been the subject of intensive research. In certain situations, rapid transcutaneous delivery is very desirable. A mechanical (stress) pulse generated by a single laser pulse was shown to transiently increase the permeability of the stratum corneum in vivo. The barrier function of the stratum corneum recovers within minutes. The increased permeability during these few minutes allows macromolecules to diffuse through the stratum corneum into the viable epidermis and dermis. Macromolecules (40 kDa dextran and 20 nm latex particles) were deposited into the skin using a photomechanical pulse generated by a single 23 ns laser pulse. This treatment can potentially be utilized in therapies that currently require occlusive dressings for hours or day(s).

A computerized image analysis method for measuring elastic tissue.

A computerized image analysis technique for quantifying area fractions of elastic tissue in light microscopic sections is presented that considers the nonuniform distribution of the elastic tissue. The method uses Verhoeff-van Gieson stained sections. The analysis is stratified vertically in three uniform layers starting at the dermo-epidermal junction and horizontally by two schemes. A standard method consists of five equally spaced measurements. The densest method uses the three areas that contain the most elastic tissue. The area fractions are determined by counting the positive and total pixels (thresholding). A validity test utilizing independent physical measurements demonstrated differences of no more than 1.7%. Reliability tests for reading the same section on different days and adjacent sections showed no significant differences (p less than 0.05) between the readings. Reliability tests of sections using different stain lots and adjacent biopsy sites also did not have significant differences. This method may be particularly useful for studies in which the distribution of the material to be measured may be very uneven, such as in solar elastosis.

The systemic administration of gamma interferon inhibits collagen synthesis and acute inflammation in a murine skin wounding model.

The ability of gamma interferon (IFN-gamma) to affect cutaneous collagen synthesis in vivo was examined in a murine wounding model. Reproducible areas of full-thickness skin necrosis were produced by argon laser radiation. Mice received recombinant murine IFN-gamma (rMuIFN-gamma) (8.7 X 10(3) units/hr) over 14 d via osmotic pumps implanted subcutaneously or intraperitoneally. At 14 and 21 d after wounding, there was less fibrous tissue in healing scars of treated animals as determined by light and transmission electron microscopy. Associated with the decrease in connective tissue was an increase in the acid mucopolysaccharide content of healing scars, which was largely hyaluronate. Quantitative image analysis of electron micrographs confirmed that less collagen was present in healing scars of animals receiving rMuIFN-gamma. The mean cross-sectional area of collagen fibers was smaller in specimens from treated mice, but no difference was seen in the size of collagen fibrils. The time required to obtain full skin closure was also delayed 23%-27% in treated animals. Using this injury model, we also found that rMuIFN-gamma significantly reduced the degree of perilesional erythema surrounding the laser injury sites and, in the first 6 d after wounding, the degree of polymorphonuclear infiltrate present histologically at lesional sites. Indeed, rMuIFN-gamma also decreased the cutaneous accumulation of neutrophils induced by known proinflammatory mediators, such as interleukin 1 and activated serum. Thus, systemically administered IFN-gamma not only down-regulates collagen synthesis in the skin but also modulates in a previously unrecognized manner: neutrophil accumulation at sites of tissue injury in vivo.

Erythema induratum and active pulmonary tuberculosis.

The etiology of erythema induratum, a rare disease of the skin in the United States but occasionally seen in natives of Asian countries, remains a source of debate. Its association with tuberculosis, although strongly suspected for more than one century, has not been clearly defined. We report a case of erythema induratum occurring in a young Chinese woman in the setting of active pulmonary tuberculosis. Both diseases promptly responded to antituberculous therapy. The diagnosis of erythema induratum should urge the clinician to search for a source of active tuberculosis, and treatment should be initiated accordingly.

Tubular carcinoma and sclerosing adenosis: the use of basal lamina as a differential feature.

Recent ultrastructural studies have shown that tubular carcinoma of the breast lacks basal lamina. This study was undertaken to determine the usefulness of light-microscopic examination of PAS staining for basal lamina in the differentiation of tubular carcinoma and sclerosing adenosis. Ten cases of tubular carcinoma show no basal lamina except for an occasional short segment. Ten cases of sclerosing adenosis show intact basal lamina except for short focal discontinuities. It is concluded that the lack of basal lamina in tubular carcinoma and its presence in sclerosing adenosis is a useful differential feature.

Photodynamic therapy of pigmented choroidal melanomas.

PURPOSE: To evaluate the effectiveness of photodynamic therapy with chloroaluminum sulfonated phthalocyanine in the treatment of pigmented choroidal melanomas in a rabbit model. METHODS: Pigment containing B16F10 murine melanoma cells were implanted transclerally into the subchoroidal space of 28 immunosuppressed New Zealand albino rabbits. The animals were treated with daily injections of cyclosporine and were followed up until tumors at least 2 mm in height were detected by ultrasonography. Twenty-four hours after the intravenous injection of chloroaluminum sulfonated phthalocyanine (CASPc, 5 mg/kg), tumors were irradiated at 675 nm through an argon-pumped dye laser at estimated total light doses of 25 to 70 J/cm2. Control animals were treated with light only or photosensitizer only. The animals were followed up for 4 1/2 to 8 weeks with regular fundus examinations. RESULTS: Twenty tumor-bearing rabbits were treated with light and dye. The tumor regressed in 12 animals. Five of these animals were followed up for at least 4 1/2 weeks and the other seven for 8 weeks after treatment. At light doses under 40 J/cm2, tumor regrowth was observed in five animals within 10 days of treatment. In all control groups, the tumor-bearing eyes were filled with tumor cells by the third week after implantation. Histologic examination of tumors treated with photosensitizer and light revealed prominent vascular damage early after treatment that resulted in vascular occlusion. Tumor necrosis was evident within 24 hours of treatment. CONCLUSIONS: Results suggest that photodynamic therapy may have a role in the treatment of pigmented choroidal melanomas.

Morphologic and ultrastructural examination of I-A+ cells in the murine iris.

The surface membrane expression of major histocompatibility (MHC) class II antigens is an important prerequisite for presentation of foreign antigens to the immune system. Because particular antigens that are placed within the anterior chamber of the eye elicit a deviant form of immunity in which effector delayed-type hypersensitivity responses are suppressed, it has been proposed that novel MHC class II antigen-bearing cells exist in the tissues that line the anterior chamber. Class II MHC antigen expression has been identified within the iris, but the detailed morphologic description of these cells is incomplete. With the use of in situ immunoperoxidase and immunoelectron microscopic techniques, we examined the morphologic and ultrastructural characteristics of resident MHC-positive class II (I-A+) cells in murine irises. A significant number of these cells was found in the connective tissue of BALB/c irises. The majority showed extensive dendritic morphologic characteristics and formed a network throughout the iris that did not overlap. Ultrastructurally, I-A+ cells had an indented nucleus, some vacuoles, lysosomes, mitochondria, and an occasional phagosome within their cytoplasm and an absence of desmosomes or other intercellular junctions. Based on these features, it is unlikely that these cells are epithelial or endothelial in origin, but rather are similar to cells of the monocyte/macrophage/dendritic cell lineage. These results show the presence of an I-A+ dendritic cell population, within the murine iris, distributed in a pattern that is similar to that of Langerhans cells in the skin. Due to their compartmentalization within the eye, this cell population may represent a novel antigen-presenting cell that contributes to the immunologic privilege of the anterior chamber.

Effects of photodynamic therapy using verteporfin on experimental choroidal neovascularization and normal retina and choroid up to 7 weeks after treatment.

PURPOSE: To study the long-term effects of photodynamic therapy (PDT), using liposomal benzoporphyrin derivative (BPD) or Verteporfin, on experimental choroidal neovascularization (CNV) and on normal retina and choroid (with no CNV) in the cynomolgus monkey eye. METHODS: Photodynamic therapy was performed in 8 cynomolgus monkey eyes with experimental CNV induced by laser injury. The effect of PDT on normal retina and choroid (with no CNV) was studied in 9 monkey eyes. Liposomal BPD was administered intravenously (0.375 mg/kg) either as a bolus, as a slow infusion over 32 minutes, or as a fast infusion over 10 minutes. Photodynamic therapy was performed using light at a wavelength of 689 or 692 nm, with an irradiance of 600 mW/cm2 and fluence of 150 J/cm2. Follow-up studies, including fundus photography and FA, were performed at 24 hours after PDT and then weekly. Indocyanine green and BPD angiography were performed in selected cases. Tissues were examined with light and electron microscopy at the end of follow-up. RESULTS: Twenty-three of the 32 areas of CNV treated with PDT showed absence of angiographic leakage at 24 hours. Twenty-eight areas of CNV were followed for 4 weeks; 22 of 28 showed absence of angiographic leakage at 2 weeks; and 20 of 28 at 4 weeks of follow-up. Forty spots on the normal retina and choroid were treated with PDT and were followed for 4 to 7 weeks. These spots showed pigment-laden cells in the outer retina, variably pigmented retinal pigment epithelium (RPE) in the treated area, intact neurosensory retina, and reperfusion of the choriocapillaris. CONCLUSIONS: Photodynamic therapy leads to absence of angiographic leakage for at least 4 weeks in experimental CNV in the monkey model. In the normal monkey eye the RPE and choriocapillaris show generalized recovery with preservation of the neurosensory retina 7 weeks after PDT.