Daniel N. Sauder

Antifungal agents: An overview. Part II

The recent introduction of a new generation of antifungal drugs promises to alter significantly therapy for both systemic and superficial mycoses, in particular, onychomycosis. This article presents an in-depth review of the azoles (the triazoles itraconazole and fluconazole), the allylamines (naftifine and terbinafine), and the morpholine derivative amorolfine.

A 40-100 MHz B-SCAN ULTRASOUND BACKSCATTER MICROSCOPE FOR SKIN IMAGING

There is a growing interest in high resolution, subsurface imaging of cutaneous tissues using higher frequency ultrasound, and several commercial systems have been developed recently which operate at 20 MHz. Some of the possible applications of higher frequency skin imaging include tumour staging, boundary definition, and studies of the response of tumours to therapy, investigations of inflammatory skin conditions such as psoriasis and eczema, and basic studies of skin aging, sun damage and the effects of irritants. Investigation of these areas is quite new, and the role of ultrasound skin imaging is continuing to evolve. Lateral resolution in the 20 MHz imaging systems ranges from 200 to 300 microns, which limits imaging applications to cutaneous structures which are relatively large in size. In this paper, a real-time ultrasound backscatter microscope (UBM) for skin imaging is described which operates in the 40-100 MHz range, providing axial resolution between 17 and 30 microns and lateral resolution between 33 and 94 microns. This improvement in resolution over current skin ultrasound systems should prove useful in determining the margins of small skin lesions, and in obtaining more precise, in vivo skin thickness measurements to characterize nonmalignant skin disease. Example images of normal skin, seborrhoeic keratosis and malignant melanoma illustrate the imaging potential of this system.

Role of cytokines in epidermal Langerhans cell migration.

In the epidermal compartment of skin, keratinocytes (KC), Langerhans cells (LC), and their soluble products, i.e. cytokines, constitute a unique immunologic microenvironment. KC participate in cutaneous immune responses by producing various cytokines. LC, a member of the dendritic cell (DC) family, represent the professional antigen‐presenting cells in the epidermis. Although it has been demonstrated that migration of LC from skin to lymph nodes is a critical step for the antigen presentation, molecular mechanisms for such an event remain unclear. Recent studies suggest that cytokines are able to modulate LC/DC migration. There is accumulating evidence that proinflammatory cytokines including interleukin (IL)‐1 and tumor necrosis factor a promote LC emigration from the skin, whereas the anti‐inflammatory cytokine IL‐10 is a counter‐regulator. LC/DC express chemokine receptors. Chemokines generated from lymphatic endothelial cells and lymph node cells play a role in the directional migration of LC/DC into lymph nodes. This article reviews current studies on the role of cytokines in LC/DC migration. J. Leukoc. Biol. 66: 33–39; 1999.

Ultrasound backscatter microscope analysis of mouse melanoma progression

The incidence and mortality rate of cutaneous melanoma continue to increase throughout the world, making the study of melanoma biology an important area of current research. While recent breakthroughs in transgenic mouse technology have led to promising mouse skin models of melanoma, there is presently no technique available for quantitatively studying subsurface melanoma progression, in vivo. We demonstrate the first application of an imaging method called ultrasound backscatter microscopy (UBM) for imaging early murine melanomas with spatial resolution of 30 microns axial and 60 microns lateral. Murine B16 F10 melanomas have been imaged from their earliest detection, over several days, until they are 2 to 5 mm in diameter. Melanoma dimensions measured by UBM were found to be in excellent agreement with those determined histopathologically on the excised tumours. The relative rms errors in UBM-determined melanoma height and width were found to be 8.7% and 4.2%, respectively. The mean rate of increase in tumour height of early murine melanoma was found to be 0.37 +/- 0.06 mm/day. Computer-generated volumetric renderings of melanomas have been produced from three-dimensional image data, allowing quantitative comparisons of tumour volumes to be made. Using a priori assumptions of ellipsoid tumour shape, the relative error in UBM-determined volume was shown to be less than 17%. These results should be of considerable interest to investigators studying melanoma biology using mouse skin models, and have implications in the use of high frequency ultrasound imaging for the clinical assessment of cutaneous melanoma.

Contribution of Langerhans Cell-Derived IL-18 to Contact Hypersensitivity

The epidermal Langerhans cells (LC), a member of the dendritic cell family, and the LC-derived cytokine IL-12 play a pivotal role in the initiation of contact hypersensitivity (CHS), a Th1 immune response in the skin. Because IL-18, another LC-derived cytokine, shares functional and biological properties with IL-12, we examined a potential role for IL-18 in CHS initiation. Our studies demonstrated that during the induction phase of murine CHS, IL-18 mRNA was significantly up-regulated in the skin-draining lymph nodes (LN). Migratory hapten-modified LC in LN expressed high levels of IL-18 mRNA and secreted functional IL-18 protein. LN cells produced significant amounts of IFN-γ following in vitro IL-12 stimulation, which could be partially blocked by anti-IL-18 Ab, suggesting a synergistic role for endogenous IL-18 in IFN-γ production by LN cells. Because mature IL-18 requires cleavage of immature precursors by caspase-1, we further examined IL-12-induced IFN-γ production in caspase-1−/− LN cells. An impaired IFN-γ production was seen in caspase-1−/− LN cells, which could be restored by addition of exogenous IL-18, supporting a role for caspase-1-cleaved, mature IL-18 in IFN-γ production. Finally, in vivo studies showed that CHS responses were significantly inhibited in mice treated with neutralizing IL-18 Ab as well as in caspase-1−/− mice deficient in mature IL-18, indicating functional relevance for IL-18 in CHS. Taken together, our studies demonstrate that LC-derived IL-18 significantly contributes to CHS initiation.

Antiangiogenic properties of a novel shark cartilage extract: potential role in the treatment of psoriasis.

Background: A number of inflammatory and immune diseases are associated with vascular changes. Psoriasis, as an example, is a common inflammatory skin disease with dilation of capillaries as an early histological change. In more developed psoriatic lesions there is proliferation of blood vessels and neovascularization. The use of agents that target these vascular changes represents a novel therapeutic strategy in the treatment of inflammatory diseases. Since cartilage is an avascular tissue, it has been hypothesized that there may be factors found in cartilage that inhibit blood vessel formation. Objective: The objectives of this study were 1) to determine whether extracts of cartilage could inhibit angiogenesis, and 2) since altered angiogenesis is associated with certain diseases, including psoriasis, to examine whether inhibition of angiogenesis could potentially contribute to the treatment of psoriasis. Methods: Extracts of shark cartilage were prepared by homogenization and ultrafiltration to derive the active agent termed Æ-941. This agent was tested for antiangiogenesis activity using the embryonic vascularization test, which is a modification of the ex vivo chick embryo culture (CAM). Since one of the first steps in angiogenesis is degradation by metalloproteinases of the basement membrane of capillaries, Æ-941 was tested for collagenase activity using a fluorogenic peptide substrate. Anti-inflammatory properties were tested using a cutaneous irritation model in humans. Results: A dose dependent inhibition in embryonic neovascularization as well as in collagenase activity by Æ-941 was demonstrated. When test compounds were applied on the forearms of test subjects, Æ-941 was shown to have anti-inflammatory properties. Anecdotal data suggested that topical Æ-941 had a beneficial effect in psoriasis. Conclusion: Our results show that Æ-941 has anti-angiogenic and anti-inflammatory properties. Antiangiogenesis agents such as Æ-941 provide an entirely new class of agents to treat cutaneous and systemic diseases associated with altered vascularity.

Differential modulation of interleukin‐1α (IL‐1α) and interleukin‐1β(IL‐1β in human epidermal keratinocytes by UVB

Abstract Conflicting reports exist concerning ultraviolet‐B (UVB) effects on keralinocyte (KC) interleukin‐l (IL‐1) expression. To clarify the modulatery effects of UVB on IL‐1, the following study was undertaken. Normal human epidermal KCs cultured in a standard low Ca2+ and serum‐free medium were irradiated in quiescent phase with UVB. In this study, we used semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) to determine the mRNA level of interleukin‐1α (IL‐1α) and interleukin‐1β (IL‐β). After exposure to 100 or 300 J/m2 UVB, a transient increase in mRNA levels was observed within I hour for IL‐1α and 3 to 6 h for IL‐β Following this transient induction, mRNA levels for both IL‐1α and IL‐1β returned to steady‐state levels after 100 J/m2. After 300 J/m2 irradiation, IL‐1α and IL‐1β levels were downregulated compared to unirradiated cultures at 24‐h post‐irradiation. The half‐life for IL‐1α and IL‐1β was estimated using actinomycin D treatment. Both IL‐1α and IL‐1β mRNAs half‐lives (t1/2) decreased faster in irradiated cells (t1/2 = 30 minutes for IL‐1α and 2 h for IL‐1β) compared to unirradiated cells (t1/2= 1 h and 4 h, respectively). These results suggest that IL‐1α and IL‐1β mRNA expression are differentially regulated by UVB. In contrast to down‐regulation of mRNA levels, a significant increase in IL‐1α protein levels, measured by ELISA. was observed in culture supernatant from 6 h to 24 h after 300 J/m2 UVB irradiation. Cycloheximide treatment did not abrogate this increase in IL‐1α protein level. Since this dose of UVB irradiation decreased the stability of IL‐1α and IL‐1β mRNA, this suggests that the release of IL‐1α after UVB irradiation was due to leakage from UVB‐damaged cells and not from de novo protein synthesis.

Cytokine knockouts in contact hypersensitivity research.

Contact hypersensitivity (CHS) is a Langerhans cell (LC)-dependent, T cell-mediated cutaneous immune response. CHS reflects a culmination of LC activities in vivo: uptake of epicutaneous antigens, migration into lymph nodes, and presentation of antigens to naïve T cells. Although studies have suggested involvement of the cytokine network in LC migration and CHS initiation, the in vivo function of individual cytokines remains largely unknown. Gene targeting technology has made it possible to study in vivo functions of cytokines through gene-targeted knockout (KO) mice deficient in a given cytokine or its receptor. A variety of cytokine knockouts have been used to assign biological functions to specific cytokines in CHS. These studies have contributed significantly to our understanding of molecular mechanisms underlying CHS.

Combined treatment with oral etretinate and electron beam therapy in patients with cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome)

BACKGROUND Total skin irradiation for early-stage mycosis fungoides produces clinical remission in 90% of patients, but the median duration of remission is only 2 to 4 years. Etretinate has proven efficacy in advanced mycosis fungoides. Its potential as adjuvant therapy to radiation could be limited by toxicity from the combination. OBJECTIVE Our purpose was to determine whether oral etretinate at 1 mg/kg can be combined with 35 Gy of radiation without intensifying or prolonging the radiation reaction and to determine the relapse-free survival rate. METHODS Twenty-three patients began etretinate on day one of radiation and the dose was reduced for nose bleeds, dry skin, or hyperlipidemia. During the reaction skin toxicity questionnaires were completed weekly. Nine concurrent patients receiving only radiation completed identical forms. RESULTS Etretinate did not intensify the skin reaction but did prolong it by 2 weeks. At a median follow-up of 2 years the relapse-free survival rate in complete responders was similar to stage-matched concurrent and historic control subjects. CONCLUSIONS Both radiation and etretinate can be given together with acceptable toxicity and without compromising either therapy.

Penetration of keratinocyte-derived cytokines into basement membrane

Keratinocytes are known to produce a wide variety of cytokines which are believed to play a significant role in cutaneous inflammatory and immunologic reactions. Considering the array of proteolytic enzymes present in the skin and the transient nature of cytokines produced from keratinocytes, it is unclear whether cytokines released by keratinocytes cross the basement membrane and contribute to distal inflammatory and immune reactions. To investigate the ability of cytokines released from human keratinocytes to cross basement membrane, we used a two chamber culture model. Keratinocytes were plated in the upper chamber coated with a reconstituted basement membrane matrix (matrigel) on a microporous membrane. To augment cytokine production, we exposed keratinocytes to 300 J/m2 UVB; 24 h later the supernatants were collected, and the levels of cytokine were measured by ELISA. IL‐1α, IL‐6, and TNF‐α were found to be increased after UVB irradiation in the upper chamber, and significant amounts (70–80%) of each cytokine were detected in the lower chamber. Our results indicate that keratinocyte‐derived cytokines are available for interactions below the basement membrane and present circumstantial evidence that the production of those cytokines from keratinocytes contributes to the elevation of circulation after the UVB exposure. J. Cell. Physiol. 171:190–195, 1997.