Tackling agricultural diffuse pollution: What might uptake of farmer-preferred measures deliver for emissions to water and air?

Mitigation of agricultural diffuse pollution poses a significant policy challenge across Europe and particularly in the UK. Existing combined regulatory and voluntary approaches applied in the UK continue to fail to deliver the necessary environmental outcomes for a variety of reasons including failure to achieve high adoption rates. It is therefore logical to identify specific on-farm mitigation measures towards which farmers express positive attitudes for higher future uptake rates. Accordingly, a farmer attitudinal survey was undertaken during phase one of the Demonstration Test Catchment programme in England to understand those measures towards which surveyed farmers are most receptive to increasing implementation in the future. A total of 29 on-farm measures were shortlisted by this baseline farm survey. This shortlist comprised many low cost or cost-neutral measures suggesting that costs continue to represent a principal selection criterion for many farmers. The 29 measures were mapped onto relevant major farm types and input, assuming 95% uptake, to a national scale multi-pollutant modelling framework to predict the technically feasible impact on annual agricultural emissions to water and air, relative to business as usual. Simulated median emission reductions, relative to current practise, for water management catchments across England and Wales, were estimated to be in the order sediment (20%)>ammonia (16%)>total phosphorus (15%) ≫ nitrate/methane (11%)>nitrous oxide (7%). The corresponding median annual total cost of the modelled scenario to farmers was £3 ha(-1)yr(-1), with a corresponding range of -£84 ha(-1)yr(-1) (i.e. a net saving) to £33 ha(-1)yr(-1). The results suggest that those mitigation measures which surveyed farmers are most inclined to implement in the future would improve the environmental performance of agriculture in England and Wales at minimum to low cost per hectare.

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.

Limiting Effects of Low Temperature on Growth and Spore Germination in Gibberella circinata, the Cause of Pitch Canker in Pine Species.

Pitch canker, caused by Gibberella circinata (anamorph = Fusarium circinatum), causes canopy dieback and mortality in susceptible pine species in many parts of the world. Pitch canker is most problematic in areas with a relatively warm climate, suggesting a possible limitation on disease development imposed by low temperatures. To test this hypothesis, the effect of temperature on radial growth was examined in isolates of G. circinata of diverse geographic origin. All isolates grew most rapidly at 25°C and progressively more slowly at 20, 15, and 10°C. Spore germination occurred most rapidly at 20°C and was slowest at 10°C. To determine if the time required for spore germination might influence the likelihood of infection, the duration of wound susceptibility was examined by inoculating branches of susceptible Monterey pines (Pinus radiata). In each of six field trials, branches were wounded and then inoculated immediately or at 2, 6, or 9 days after wounding. The results indicated that wounds inoculated immediately became infected at a significantly higher rate than those inoculated 2 days later. Thus, if low temperatures extend the time required for germination beyond this period, a reduced infection frequency would be expected. Such a limiting effect of temperature could help to explain the current distribution of pitch canker.

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.

Effects of short-term high-dose and low-dose dermal exposure to Jet A, JP-8 and JP-8 + 100 jet fuels.

Occupational and environmental exposures to jet fuel recently have become a source of public and regulatory concern. This study investigates the cutaneous toxicity of three fuels used in both civilian and military aircraft. Pigs, an accepted animal model for human skin, were exposed to low-dose (25 microl or 7.96 microl cm(-2)) or high-dose (335 microl or 67 microl cm(-2)) Jet A, JP-8 and JP-8 + 100 under occluded (Hill Top) chamber or cotton fabric) and non-occluded conditions for 5 h, 24 h and 5 days. To mimic occupational exposure, fuel-soaked fabric (high dose) was used. Erythema, edema, transepidermal water loss (TEWL) and epidermal thickness were quantified. High-dose fabric occluded sites had slight erythema at 5 h with increased erythema at 5 days. No erythema was noted in any of the occluded (Hill Top) or non-occluded sites at any of the time points. Morphological assessments depicted slight intracellular epidermal edema at all time points. An increase in change in TEWL (DeltaTEWL) was observed at the 5-h and 24-h fabric and Hill Top occluded treatments and a decrease at the 5-day fabric and Hill Top occluded sites. In all 5-day JP-8 + 100 fabric sites, intracorneal microabscesses filled with inflammatory cells were observed. Epidermal thickening was significant (P < 0.05) in all three jet fuels at the high-dose fabric sites, with JP-8 + 100 being the thickest. The epidermal rete peg depth increased significantly (P < 0.05) at 24 h and 5 days with Jet A, JP-8, and JP-8 + 100 in the fabric sites. No significant differences were noted in the 5-day non-occluded fabric and Hill Top occluded and non-occluded sites. Jet fuel JP-8 + 100 tended to have the greatest proliferative response. In conclusion, the high-dose fabric-soaked exposure at 5 days to Jet A, JP-8 and JP-8 + 100 fuels caused the greatest increase in cutaneous erythema, edema, epidermal thickness and rete peg depth compared with high-dose non-occluded or low-dose exposure under Hill Top occluded and non-occluded conditions.

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.

Evaluation of Internet-based oncologic teaching for medical students.

BACKGROUND: Electronic tools with substantial educational applications are now widely available. METHODS: In a prospective, randomized study, the value of Web-based educational tools for teaching second-year medical students was evaluated. The 35-hour, image-intensive multifaculty neoplasia course was selected for the experiment, with 103 students assigned to the control group (C) and 61 to the experimental group (E). Representative password-controlled multimedia course modules, accessible via the Internet, were developed. The E cohort was exposed to both classroom and Web-aided materials, whereas the C group had access to the Web modules only after the experiment was concluded (but before the final examination). Pre- and post-exposure questionnaires assessed computer knowledge, familiarity with the Internet, availability of computer access, and the value of Web-based education for both cohorts. Additionally, pre-and post-exposure tests were administered to both cohorts based on educational materials presented in the Web modules. RESULTS: The overall participation rate was 64% (E = 69%; C = 60%). The post-test showed no major performance difference between the two groups. The questionnaires revealed that: less than 1% of the students had not accessed the Internet previously; less than 5% had not used the Internet for medical education before; 34% felt that computer resources on campus were inadequate; and over 75% found Web-based education to be an important additional educational resource. The major negative aspect was the slow pace of data transfer for modem-based home access. Only 1% of students felt that Web-based education could completely replace traditional teaching. CONCLUSION: The potential for incorporating Web-based education in the medical curriculum is considerable.

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.

[Quality assurance in amputation of the lower extremities due to vascular insufficiency]. / Kvalitetssikring ved amputationer på underekstremiteterne grundet vasculaer insufficiens.

In this retrospective study we reviewed the clinical charts for 82 patients who underwent lower limb amputation at our department during 1990 and 1991. Our results are compared with the literature, and different aspects of the treatment are discussed. Subsequently a model for future quality assurance is presented.

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.