In vitro toxicity assessment of three hydroxylated fullerenes in human skin cells.

Carbon fullerenes possess unique properties and their interactions with biomolecules have widespread applications. Functionalization of fullerenes with hydroxyl groups (fullerenols) can increase the solubility and potential for cellular interaction, but the health and safety effects of varying degrees of fullerene hydroxylation in biological systems is poorly understood. Existing reports regarding the toxicity and inflammatory potential of fullerenols give conflicting conclusions. To further elucidate the potential for toxicity of fullerenols, human epidermal keratinocytes (HEK) were exposed to fullerenols (low (C60(OH)20), medium (C60(OH)24), and high (C60(OH)32)) at concentrations ranging from 0.000544-42.5 µg/ml for 24 and 48 h. A statistically significant (p<0.05) decrease in viability with alamar Blue (aB) was noted only with C60(OH)32 at 42.5 µg/ml after 24 h. Nanoparticle (NP) controls showed minimal NP/assay interference of the three fullerenols with the aB viability assay. Normalized IL-8 concentration for C60(OH)20 was not significantly different from control, while C60(OH)24 and C60(OH)32 showed a significant decrease at 24 and 48 h. These results suggest that different hydroxylation of fullerenes caused no cytotoxicity or inflammation up to 8.55 µg/ml. These findings suggest that extrapolation across similar NP will be dependent upon surface chemistry and concentration which may affect the degree of agglomeration and thus biological effects.

Safety evaluation of sunscreen formulations containing titanium dioxide and zinc oxide nanoparticles in UVB sunburned skin: an in vitro and in vivo study.

Sunscreens containing titanium dioxide (TiO(2)) and zinc oxide (ZnO) nanoparticles (NP) are effective barriers against ultraviolet B (UVB) damage to skin, although little is known about their disposition in UVB-damaged skin. Pigs were exposed to UVB that resulted in moderate sunburn. For in vitro studies, skin in flow-through diffusion cells were treated 24 h with four sunscreen formulations as follows: 10% coated TiO(2) in oil/water (o/w), 10% coated TiO(2) in water/oil (w/o), 5% coated ZnO in o/w, and 5% uncoated ZnO in o/w. TiO(2) (rutile, crystallite) primary particle size was 10 × 50 nm with mean agglomerates of 200 nm (range ca. 90 nm--460 nm); mean for ZnO was 140 nm (range ca. 60--200 nm). Skin was processed for light microscopy, scanning (SEM) and transmission electron microscopy (TEM), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). UVB-exposed skin had typical sunburn histology. TEM showed TiO(2) NP 17 layers into stratum corneum (SC), whereas ZnO remained on the surface. TOF-SIMS showed TiO(2) and ZnO epidermal penetration in both treatments. Perfusate analyzed by TEM/energy dispersive x-ray spectroscopy or inductively coupled plasma mass spectrometry detected no Ti or Zn, indicating minimal transdermal absorption. In vivo, skin was dosed at 24 h occluded with formulations and at 48 h. TiO(2) NP in o/w formulation penetrated 13 layers into UVB-damaged SC, whereas only 7 layers in normal skin; TiO(2) in w/o penetrated deeper in UVB-damaged SC. Coated and uncoated Zn NP in o/w were localized to the upper one to two SC layers in all skin. By SEM, NP were localized as agglomerates in formulation on the skin surface and base of hair. TOF-SIMS showed Ti within epidermis and superficial dermis, whereas Zn was limited to SC and upper epidermis in both treatments. In summary, UVB-damaged skin slightly enhanced TiO(2) NP or ZnO NP penetration in sunscreen formulations but no transdermal absorption was detected.

Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line.

Single-walled carbon nanotubes (SWCNT), fullerenes (C(60)), carbon black (CB), nC(60), and quantum dots (QD) have been studied in vitro to determine their toxicity in a number of cell types. Here, we report that classical dye-based assays such as MTT and neutral red (NR) that determine cell viability produce invalid results with some NM (nanomaterials) due to NM/dye interactions and/or NM adsorption of the dye/dye products. In this study, human epidermal keratinocytes (HEK) were exposed in vitro to CB, SWCNT, C(60), nC(60), and QD to assess viability with calcein AM (CAM), Live/Dead (LD), NR, MTT, Celltiter 96 AQueous One (96 AQ), alamar Blue (aB), Celltiter-Blue (CTB), CytoTox Onetrade mark (CTO), and flow cytometry. In addition, trypan blue (TB) was quantitated by light microscopy. Assay linearity (R(2) value) was determined with HEK plated at concentrations from 0 to 25,000 cells per well in 96-well plates. HEK were treated with serial dilutions of each NM for 24 h and assessed with each of the viability assays. TB, CAM and LD assays, which depend on direct staining of living and/or dead cells, were difficult to interpret due to physical interference of the NM with cells. Results of the dye-based assays varied a great deal, depending on the interactions of the dye/dye product with the carbon nanomaterials (CNM). Results show the optimal high throughput assay for use with carbon and noncarbon NM was 96 AQ. This study shows that, unlike small molecules, CNM interact with assay markers to cause variable results with classical toxicology assays and may not be suitable for assessing nanoparticle cytotoxicity. Therefore, more than one assay may be required when determining nanoparticle toxicity for risk assessment.

Inhibition of jet fuel aliphatic hydrocarbon induced toxicity in human epidermal keratinocytes.

Jet propellant (JP)-8, the primary jet fuel used by the U.S. military, consists of hydrocarbon-rich kerosene base commercial jet fuel (Jet-A) plus additives DC1-4A, Stadis 450 and diethylene glycol monomethyl ether. Human epidermal keratinocytes (HEK) were exposed to JP-8, aliphatic hydrocarbon (HC) fuel S-8 and aliphatic HC pentadecane (penta), tetradecane (tetra), tridecane (tri) and undecane (un) for 5 min. Additional studies were conducted with signal transduction pathway blockers parthenolide (P; 3.0 microm), isohelenin (I; 3.0 microm), SB 203580 (SB; 13.3 microm), substance P (SP; 3.0 microm) and recombinant human IL-10 (rHIL-10; 10 ng ml(-1)). In the absence of inhibitors, JP-8 and to a lesser extent un and S-8, had the greatest toxic effect on cell viability and inflammation suggesting, as least in vitro, that synthetic S-8 fuel is less irritating than the currently used JP-8. Each inhibitor significantly (P < 0.05) decreased HEK viability. DMSO, the vehicle for P, I and SB, had a minimal effect on viability. Overall, IL-8 production was suppressed at least 30% after treatment with each inhibitor. Normalizing data relative to control indicate which inhibitors suppress HC-mediated IL-8 to control levels. P was the most effective inhibitor of IL-8 release; IL-8 was significantly decreased after exposure to un, tri, tetra and penta but significantly increased after JP-8 exposure compared with controls. Inhibitors were not effective in suppressing IL-8 release in JP-8 exposures to control levels. This study shows that inhibiting NF-kappa B, which appears to play a role in cytokine production in HC-exposed HEK in vitro, may reduce the inflammatory effect of HC in vivo.

Effect of JP-8 jet fuel exposure on protein expression in human keratinocyte cells in culture.

Dermal exposure to jet fuel is a significant occupational hazard. Previous studies have investigated its absorption and disposition in skin, and the systemic biochemical and immunotoxicological sequelae to exposure. Despite studies of JP-8 jet fuel components in murine, porcine or human keratinocyte cell cultures, proteomic analysis of JP-8 exposure has not been investigated. This study was conducted to examine the effect of JP-8 administration on the human epidermal keratinocyte (HEK) proteome. Using a two-dimensional electrophoretic approach combined with mass spectrometric-based protein identification, we analyzed protein expression in HEK exposed to 0.1% JP-8 in culture medium for 24 h. JP-8 exposure resulted in significant expression differences (p<0.02) in 35 of the 929 proteins matched and analyzed. Approximately, a third of these alterations were increased in protein expression, two-thirds declined with JP-8 exposure. Peptide mass fingerprint identification of effected proteins revealed a variety of functional implications. In general, altered proteins involved endocytotic/exocytotic mechanisms and their cytoskeletal components, cell stress, and those involved in vesicular function.

Electron microscopic observations of stratum corneum intercellular lipids in normal and atopic dogs.

The barrier function of mammalian skin is maintained by intercellular stratum corneum lipids. In human patients with atopic dermatitis, an abnormal lipid barrier results in dry skin and increased transepidermal water loss. At this time, it is not known if a defective lipid barrier is present in atopic dogs. Normal and atopic canine skin were postfixed in ruthenium tetroxide and studied using transmission electron microscopy to determine structural differences within stratum corneum lipids. Intercellular lipid lamellae were graded on a semiquantitative scale. The deposition of stratum corneum lipid lamellae in atopic canine skin appeared markedly heterogeneous compared with that seen in normal canine skin. When present, the lamellae often exhibited an abnormal structure. The continuity and thickness of the intercellular lipid lamellae were significantly less in nonlesional atopic than in normal canine skin. These preliminary observations suggest that the epidermal lipid barrier is defective in atopic canine skin. Additional studies are needed to further characterize the biochemical defect and to possibly correct it with nutritional and/or pharmacologic intervention.

Effect of selective lipid extraction from different body regions on epidermal barrier function.

PURPOSE: To assess the effects of selective lipid extraction and tape stripping on transepidermal water loss (TEWL) at three body regions in the pig. METHODS: Lipids were extracted from the abdominal, inguinal. and back regions using three different solvent extraction procedures or cellophane tape stripping (15x) on Yorkshire pigs. Three solvent extraction stages were I, cyclohexane (5 ml for three, 1-min extractions): II, cyclohexane/ethanol (4:1) (5 ml for three, 1-min extractions): and III, cyclohexane/ethanol (1:4) (5 ml for three, 3-min extractions) extracted as follows: Site A, Stage I: Site B, Stage I and II; Site C, Stage I, II and III. Erythema, edema, and TEWL were assessed in control, tape-stripped, and extracted sites at 0, 6, and 24 h. The extracted lipids were analyzed by thin layer chromatography and quantified by densitometry for ceramide, cholesterol, cholesterol esters, fatty acids, and triglycerides. RESULTS: The change in TEWL (delta TEWL) in 14 of the 15 sites was the highest at 24 h and generally increased with each additional extraction. The greatest changes were present in the back. Each extraction stage removed specific lipids in reproducible quantities that caused the delta TEWL to increase from 0 to 24 h. Lipid removal was verified by transmission electron microscopy. The mean total lipid concentration depended on extraction solvents and body region, and was reproducible across sites and regions at equivalent stages of lipid extraction. Relative proportions of individual lipids extracted were similar across all body regions. Higher concentrations of total lipids were extracted from the back. CONCLUSIONS. These studies demonstrate that extraction of lipids increased the delta TEWL to a level similar to repeated tape stripping at all body sites in the pig. This study suggested that strategies that could biochemically alter epidermal lipid composition may increase absorption of simultaneously administered topical compounds and may be useful to enhance drug delivery.

Efficacy of topical phenol decontamination strategies on severity of acute phenol chemical burns and dermal absorption: in vitro and in vivo studies in pig skin.

Pure phenol is colorless and used in the manufacture of phenolic resins, plastics, explosives, fertilizers, paints, rubber, textiles, adhesives, pharmaceuticals, paper, soap, and wood preservatives. The purpose of this study was to compare the efficacy of several phenol decontamination strategies following dermal exposure using the pig as a model for human exposure, and then assess the effect of the two best treatments on phenol absorption in the isolated perfused porcine skin flap (IPPSF). Six anesthetized Yorkshire pigs were exposed to 89% aqueous phenol for 1 min using Hilltop chambers (10 skin sites/pig; 400 microl/site). Exposure to phenol was followed by one of 10 different decontamination procedures: 1-, 5-, 15-, and 30-min water wash; Ivory soap solution; polyethylene glycol (PEG 400); PEG 400/industrial methylated spirits (IMS); PEG 400/ethanol (EtOH); polyvinyl pyrrolidone (PVP)/70% isopropanol (IPA); and 70% IPA. For each of the last five strategies, 1-min treatment washes were repeatedly alternated with 1-min water washes for a total of 15 min. Evaluation was based on scoring of erythema, edema, and histological parameters such as intracellular and intercellular epidermal edema, papillary dermal edema, perivascular infiltrates, pyknotic stratum basale cells, and epidermal-dermal separation. It was concluded that PEG 400 and 70% IPA were superior to the other treatments investigated and equally efficacious in the reduction of phenol-induced skin damage. In addition, phenol absorption was assessed utilizing the two most effective in vivo treatments in the IPPSF. The assessment of percutaneous absorption of phenol found the PEG 400, 70% IPA, and 15-min water treatments significantly (P < 0.05) reduced phenol absorption relative to no treatment.

Immunohistochemical characterization of the basement membrane epitopes in bis(2-chloroethyl) sulfide-induced toxicity in mouse ear skin.

Sulfur mustard (bis(2-chloroethyl) sulfide (HD)), a potent cutaneous vesicant and bifunctional alkylating agent, produces significant time-dependent histopathological changes in the skin of the mouse. The right ears of male CD1 mice were exposed topically to 5.0 microl of 195 mM (0.16 mg) HD in dichloromethane and harvested at 6, 12, 18 and 24 h. The left ear control was dosed with 5.0 microl of dichloromethane. In all controls and HD-treated mouse ear, moderate immunofluorescence staining was seen at the epidermal-dermal junction with bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA) and laminin (Lam), and light staining was observed with bullous pemphigoid 180 (BP180), fibronectin (Fn) and type IV collagen (Coll IV). Mouse anti-human monoclonal antibodies for GB3, L3d and 19-DEJ-1 (Uncein) did not cross-react. In microvesicles, BP, BP180 and Fn showed areas of light focal epidermal staining and homogeneous dermal staining, and EBA, Lam and Coll IV showed moderate dermal staining. Both BP and Fn exhibited weak, inconsistent staining with time. Immunoelectron microscopy (IEM) revealed similar results, with an increase in cell damage from 6 to 24 h, which corresponded to a decrease in staining intensity. Cell proliferation, expressed as the growth fraction of proliferating cell nuclear antigen (PCNA), showed an increase in cell damage. The growth fraction was lower in the inner ear and showed time-dependent differences. The immunofluorescence and IEM results indicate that HD causes an undulating inconsistent separation in the uppermost lamina lucida with focal cleavage into the lower portion of the basal keratinocytes just above the plasma membrane. Although this pattern of separation differs from other in vivo models in which the split occurs exclusively within the lamina lucida, this should not preclude its role as a screening model to study the effects and development of specific prophylactic and therapeutic strategies.

Comparison of an in vitro skin model to normal human skin for dermatological research.

EpiDerm, an in vitro human skin equivalent (HSE), was compared to normal human breast skin (NHS) to morphologically and biochemically assess its feasibility for dermatological research. Intralot and interlot variability was studied in day 0, 1, 2, and 3 in vitro cultures and in day 0, 3, 5, and 7 NHS. For NHS, light microscopy (LM) at day 0 showed stratified epidermis which exhibited an increase in vacuoles and dark basal cells as storage increased to 3, 5, and 7 days. Transmission electron microscopy (TEM) revealed typical organelles in the epidermis and a convoluted basement membrane at day 0. With increased storage, vacuoles and paranuclear clefts became numerous, necrosis increased, tonofilaments became less organized, and overall cellular integrity decreased. Biochemical data showed consistent MTT and glucose utilization (GU) through day 5, while lactate production decreased to 75% by day 3. By LM, day 0 HSE consisted of a thick, compact, stratum corneum that sent projections between the stratum granulosum cells. By TEM, the configuration organization, differentiation, distribution, and frequency of the organelles differed slightly from NHS. In addition, the basement membrane of the HSE was not completely differentiated, and the dermis was thin and acellular. Although day 1 and 2 cultures showed little change, day 3 exhibited an overall degeneration. Biochemical analysis showed GU and the lactate production decreased through day 3. In conclusion, the EpiDerm HSE, although exhibiting slight differences, was morphologically and biochemically similar to normal human epidermis and may be a valuable model in assessing the toxicology, metabolism, or pharmacology of nonvesicating compounds.

Ultrastructural characterization of sulfur mustard-induced vesication in isolated perfused porcine skin.

The isolated perfused porcine skin flap (IPPSF) is a novel alternative, humane in vitro model consisting of a viable epidermis and dermis with a functional microvasculature. For this study, 200 microliters of either 10.0, 5.0, 2.5, 1.25, 0.50, or 0.20 mg/ml of bis (2-chloroethyl) sulfide (HD) in ethanol or ethanol control was topically applied to a 5.0 cm2 dosing area of the IPPSF and perfused for 8 h with recirculating media. HD dermatotoxicity was assessed in the flap by cumulative glucose utilization (CGU), vascular resistance (VR), light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). HD produced a statistically significant dose relationship for gross blisters and microvesicles. The HD-treated IPPSFs were also characterized by a decrease in CGU and an increase in VR. Light microscopic changes included mild intracellular and slight intracellular epidermal edema, multifocal epidermal-dermal separation, and dark basal cells. Ultrastructural alterations consisted of cytoplasmic vacuoles, pyknotic basal cells, nucleolar segregation, and epidermal-dermal separation occurring between the lamina lucida and lamina densa of the basement membrane. The severity of these changes increased in a dose-dependent manner. Morphologically, the IPPSF appeared similar to human skin exposed to HD with the formation of macroscopic blisters and microscopic vesicles. In conclusion, the IPPSF appears to be an appropriate in vitro model with which to study the pathogenesis of vesicant-induced toxicity.

Indirect immunohistochemistry and immunoelectron microscopy distribution of eight epidermal-dermal junction epitopes in the pig and in isolated perfused skin treated with bis (2-chloroethyl) sulfide.

Sulfur mustard (bis [2-chloroethyl] sulfide, HD) is a potent cutaneous vesicant that causes gross blisters by separation of the epidermal-dermal junction (EDJ). The EDJ of the skin is a highly specialized and complex structure composed of several components and plays a major role in the integrity of the skin. The isolated perfused porcine skin flap (IPPSF) was dosed with 0.2 mg/ml (n = 4), 5.0 mg/ml (n = 4), and 10.0 mg/ml (n = 5) HD or ethanol (n = 4) for 8 hr (dose-response study) and 10.0 mg/ml HD or ethanol for 1, 3, 5, and 8 hr (n = 4/treatment) (time-response study). Successful EDJ mapping was carried out in normal pig skin (NPS), ethanol-treated IPPSFs, and HD-treated IPPSFs using the following antibodies: laminin, type IV collagen, fibronectin, GB3 (Nicein), bullous pemphigoid (BP), and epidermolysis bullosa acquisita (EBA). Two mouse anti-human monoclonal antibodies, L3d and 19-DEJ-1 (Uncein), did not cross-react with the EDJ of the pig. Antibody staining in NPS, ranging from very intense for laminin and type IV collagen to weak for fibronectin, was generally more discrete than in the IPPSF. No differences in staining were noted between the ethanol and nonblistered areas of the HD-treated IPPSFs. In HD-blistered areas, BP stained only the epidermal hemidesmosomes, and laminin, fibronectin, and GB3 stained primarily the dermis with fragments attached to the basal pole of the stratum base cells, while type IV collagen and EBA stained only the dermis. Mapping of these epitopes determined that the precise plane of EDJ separation in the HD-treated skin occurred beneath the hemidesmosomes within the upper portion of the lamina lucida. The conservation of human epitopes in the EDJ of the pig further emphasizes the similarities between human skin and pig skin. Therefore, pig skin and the IPPSF may be used to study HD-induced vesication and blistering diseases.

Development and characterization of a novel skin model for cutaneous phototoxicology.

The biological consequences of exposure to ultraviolet radiation (UV) has been receiving increased attention. Most known biological effects (such as sunburn and skin cancer) are attributed to mid-wave UVB (290-320 nm) exposure. Phototoxicity, a nonimmunological UV-induced response, has been studied using in vivo (human and animal) and in vitro models. Ethical considerations and model limitations underscore the need for a reliable in vitro model to assess cutaneous phototoxicity that would ideally possess viable cells and have a normal anatomical structure with an intact and functional vasculature. This would allow therapeutic or preventive drugs to be tested in a system in which their disposition (cutaneous concentration-time profile) has been shown to be similar to the in vivo setting. In addition, morphological, biochemical and physiological changes should be easily monitored within the same system. The purpose of this study was to characterize the isolated perfused porcine skin flap (IPPSF) developed in our laboratory as a model for UVB exposure. IPPSFs (n > or = 4/treatment) were irradiated with UVB doses of 1260 mJ/cm2, 630 mJ/cm2, 315 mJ/cm2 or 0 mJ/cm2 both in vitro and in situ. Biomarkers used to assess phototoxicity demonstrated a decrease in glucose utilization, an increase in vascular resistance (pressure/flow) and an increase in the release of PGE2. Morphologically, intracellular and intercellular epidermal edema and sunburn (pyknotic) cells (SBC) increased with dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the pathway of iontophoretic drug delivery: light and ultrastructural studies using mercuric chloride in pigs.

Although electrically assisted transdermal drug delivery has recently achieved a great deal of research attention, the precise anatomical pathway followed by these drugs through the stratum corneum has not been clearly defined. Pigs are an accepted model for studying iontophoretic drug delivery in humans. The purpose of this investigation was to visualize the pathway of ion transport by iontophoresing mercuric chloride. Weanling Yorkshire swine were dosed with 7.4% mercuric chloride in the positive electrode at a current density of 200 microAmp/cm2 applied for 1 hr. Biopsies were immediately taken, exposed to 25% ammonium sulfide vapor to precipitate and localize the mercury, fixed, and processed for light and transmission electron microscopy. The presence of mercury, which appeared as a black precipitate, was confirmed using energy-dispersive X-ray microanalysis. Although some compound penetrated the skin through appendageal pathways, the electron micrographs clearly revealed that mercuric chloride traversed the intact stratum corneum via an intercellular route. Precipitate was also localized in the outer membrane of the mitochondria in the viable epidermal cells, dermal fibroblasts, and capillaries, demonstrating transdermal delivery and systemic exposure to the mercury. These findings have implications for iontophoretic drug delivery, since they allow visualization of the functional "pores" predicted by mathematical models.

Determination of physicochemical properties of phenol, p-nitrophenol, acetone and ethanol relevant to quantitating their percutaneous absorption in porcine skin.

A knowledge of the rate and extent of chemical absorption across the skin is central to both transdermal drug delivery and cutaneous toxicology. Toward gaining sufficient insight into the relevant mechanisms involved in percutaneous absorption of topically applied agents in solution to validate a predictive model, we have 1) estimated porcine stratum corneum/water partition coefficients of two 14C-labeled compounds of interest (phenol and p-nitrophenol), and 2) measured dynamic surface evaporation from dosed excised porcine skin of these two radiolabeled compounds and two 14C-labeled commonly employed vehicles (acetone and ethanol). The surface evaporation profiles were fit to a kinetic model designed to estimate the liquid/vapor parameters for application to a general biophysically-based model of percutaneous absorption. In an effort to obtain more robust estimates of model parameters, corresponding evaporation experiments were effected on the isolated perfused porcine skin flap (IPPSF) under the same experimental conditions. Stratum corneum/water partition coefficients were determined for phenol and p-nitrophenol using a stratum corneum preparation from excised porcine integument.

Topical penetration of piroxicam is dependent on the distribution of the local cutaneous vasculature.

The mechanism of the topical delivery of piroxicam, a nonsteroidal antiinflammatory drug, has been controversial as to whether systemic absorption is required for topical efficacy. This study, using in vivo pigs treated with topical 3H-piroxicam gel, was designed to assess the role of systemic absorption on its delivery to deep tissues. Further, the role of the structure of the cutaneous vasculature (e.g., direct cutaneous or musculocutaneous) was studied. Finally, piroxicam delivery was measured using in vitro diffusion cells with pig skin obtained from the same sites to determine inherent permeability independent of vascular anatomy. These studies showed that penetration of the radiolabel occurred in subcutaneous and muscle tissue only under the dosed sites and not at the remote sites, ruling out systemic absorption as a prerequisite for local delivery. Tissue penetration in vivo was enhanced at the musculocutaneous compared to the direct cutaneous sites. In contrast, in vitro flux was identical in skin harvested from the two vascular sites, suggesting that the vasculature plays a pivotal role in deep tissue penetration of piroxicam. In conclusion, local delivery of topical drugs occurs independent of systemic absorption and the nature of the cutaneous vasculature at different sites must be taken into consideration for optimal delivery.

Determination of lidocaine concentrations in skin after transdermal iontophoresis: effects of vasoactive drugs.

The purpose of this study was to investigate the effect of vasoactive drugs on transdermal lidocaine inotophoresis by measuring the concentrations of radiolabeled lidocaine which has penetrated the skin. Previous studies had demonstrated that coinotophoresis of vasoactive drugs could modulate the transcutaneous flux of lidocaine and suggested that a dermal depot of lidocaine was involved. To address this, lidocaine hydrochloride (14C) was iontophoresed in vivo in anesthetized weanling pigs either alone or with the vasodilator tolazoline or the vasoconstrictor norepinephrine. Tissue cores under the active electrode were then collected, quick-frozen, and sectioned on a cryostat, and then the radioactivity was determined in each 40-microns section. Coiontophoresis with norepinephrine resulted in increased concentrations of lidocaine in skin up to a depth of 3 mm. These concentrations decreased to lidocaine-alone levels after a 4-hr washout. Tolazoline decreased tissue concentrations of lidocaine. Concentrations were intermediate when lidocaine alone was administered. These studies support the hypothesis that coiontophoresis of vasoactive drugs modulates the transdermal delivery of lidocaine, in part by altering the cutaneous "depot."

Differences in complement-dependent chemotactic activity generated by bullous pemphigoid and epidermolysis bullosa acquisita immune complexes: demonstration by leukocytic attachment and organ culture methods.

Bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA) are chronic blistering diseases associated with circulating complement (C)-binding anti-basement membrane zone (BMZ) antibodies and tissue-deposited immune complexes at the BMZ. Experimental evidence supporting a role for C-activating immune complexes in the pathogenesis of dermal inflammation and blisters has been reported in BP but not in EBA. In this study tissue-deposited immune complexes composed of EBA or BP antibodies were tested for generation of C-dependent chemotactic activity and the capacity to cause dermal leukocyte infiltration and dermal-epidermal separation (DES). Chemotactic activity was measured by the leukocyte attachment (LA) method. The capacity of complexes to mediate leukocyte infiltration and DES was examined in vitro using a newly described organ culture method. The results of LA showed immune complexes formed in vivo in EBA skin or in vitro by treating normal human skin with EBA antibodies were significantly more active in mediating C-dependent chemotaxis than complexes in BP skin or those formed with BP antibodies of equivalent or higher C-binding titers. Furthermore EBA antibodies and C caused leukocyte infiltration and DES in organ culture while BP antibodies did not. These results support a role for C-binding anti-BMZ antibodies in the pathogenesis of EBA lesions and demonstrate differences in the capacity of BP and EBA immune complexes to generate C-dependent chemotactic activity. These results suggest factors in addition to C-binding titers are important in the activation of C by BP and EBA immune complexes and suggest chemotactic factors other than those derived from C activation may be important in the recruitment of leukocytes in BP.

Identification of the skin basement-membrane autoantigen in epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita is an acquired chronic blistering disease of the skin, in which separation of the skin occurs in the basement-membrane zone between the epidermis and the dermis. There is evidence that blistering is initiated by an immune process. Using serum samples from nine patients as a source of antibodies, we have identified a major protein of the basement membrane of human skin that serves as the antigen (or target) for autoantibodies in this disorder. This previously unrecognized protein, which consists of two components of 290,000 and 145,000 daltons, is distinct from other known components of the basement membrane. These studies provide evidence that epidermolysis bullosa acquisita is a specific disease that is different from other primary bullous diseases, such as bullous pemphigoid and pemphigus vulgaris, and suggest that the basement-membrane component that has been identified may have a role in normal epidermal-dermal adherence.

Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin.

Sixty-one bullous disease sera containing IgG anti-BMZ antibodies were examined by indirect immunofluorescence on intact skin and skin separated through the lamina lucida by incubation in 1.0 M NaCl. All sera produced an indistinguishable pattern of linear immunofluorescence on intact skin at dilutions of 1:10 or higher. On separated skin, antibodies bound to either the epidermal (epidermal pattern), dermal (dermal pattern), or epidermal and dermal (combined pattern) sides of the separation. The binding patterns were consistent on separated skin from several donors and titers of anti-basement membrane zone antibodies on separated skin were comparable to those on intact skin. Sera from 3 patients with herpes gestationis (HG), 36 patients with bullous pemphigoid (BP), and 1 patient with clinical and histologic features of epidermolysis bullosa acquisita (EBA) showed an epidermal pattern. Sera from 9 patients with BP showed a combined pattern and sera from 6 patients with EBA and 6 patients with clinical and histologic features of BP showed a dermal pattern. Indirect immunoelectron microscopy of selected sera showed antibodies producing the epidermal and combined patterns were anti-lamina lucida antibodies and those producing the dermal pattern were anti-sublamina densa antibodies. These results show indirect immunofluorescence on separated skin is a dependable method for differentiating bullous disease anti-lamina lucida and anti-sublamina densa antibodies and that differentiating between the antibodies is essential for accurate diagnosis in some patients. The results also suggest BP anti-lamina lucida antibodies may have more than one antigenic specificity.