This work was supported by a grant from the Virginia Tech College of Science Discovery Fund.

All methods described below including the use of human volunteers have been reviewed and approved by the local Institutional Review Board (IRB), and are in accordance with the Declaration of Helsinki and Belmont Report. All participants (N=72) signed informed consent as proscribed by the IRB protocol. Inclusion/exclusion criteria and discontinuation procedures were designed to maximize participant safety, and any deviation from these procedures should be considered in light of their impact on risk and tolerability to human subjects. In the context of the work presented here, the exclusionary criteria restricted participation to individuals with no personal or family history of inflammatory conditions, or any licit or illicit substances. The justification for doing so is that these factors may influence responses to the MED testing procedure.
1. Participant Selection
<ol>
	<li>Use the following inclusion criteria: 18-55 years old; in good general health as determined by Medical Symptom Checklist (MSCL)5; can understand and communicate about the lab safety protocol presented in English; can provide written consent.</li>
	<li>Use the following exclusion criteria: Fitzpatrick skin type I, as determined by self-report; uses commercial tanning equipment regularly; skin wounds or lesions at the planned site of exposure; current skin cancer, or personal history of skin cancer; family history of skin cancer; diabetes; psoriasis or other inflammatory skin condition; peripheral vascular disease, peripheral arterial disease, Raynaud&#39;s disease, or any other diagnosed circulatory disorders; any involuntary motor disorders; allergic to adhesive tape; takes inhaled steroids for asthma (e.g., Fluticasone); takes any corticosteroids; 2 or more of the following (diagnosed hypertension, hyperlipidemia, high cholesterol, smoke cigarettes, family history of coronary or atherosclerotic disease (parents/siblings prior to age 55)); active substance dependence - legal or illicit; people with substance dependence new to recovery (less than one year); use of medications that affects CNS function, including psychotropics, opiate medication or corticosteroids, during the last 3 months; any prescribed psychotropic medications, currently or during the last 3 months (These include medications for anxiety, depression, or other psychological problems).</li>
</ol>
2. Scheduling and Preparation for the MED
<ol>
	<li>Schedule participants for two appointments: the first, the MED exposure event (approximately 45 min), and the second, a follow-up to gather spectrophotometry readings (approximately 10 min). Schedule the follow-up appointment for 24 h subsequent to the first appointment.</li>
	<li>Before participants arrive, lay out and set up essential equipment including two dose testing cuffs and safety equipment. Have a variety of UV-protective clothing (such as UV-protective sports sleeves, UV-protective gloves, long sleeve medical scrubs, UV-protective sheets and tape to affix the sheets) for both the participant and the researcher to cover all skin exposed to UV radiation.</li>
	<li>Calibrate the spectrophotometer according to manufacturer specifications. Do this for each subject and each session.</li>
</ol>
3. Determining Fitzpatrick Skin Type (FST)
<ol>
	<li>When the participant arrives for the MED exposure event (Visit 1), identify FST either through self-report or spectrophotometry. To maximize participant safety, do not conduct MED testing on participants categorized as FST 1. For all other Fitzpatrick Skin Types (2&#8212;6), use the FST score to determine which exposure schedule should be used.</li>
</ol>
4. Cuff 1 Application
<ol>
	<li>Explain to the participant how MED testing works and solicit questions before proceeding.</li>
	<li>Generally, perform the MED procedure on the inside of the non-dominant forearm.</li>
	<li>Place Cuff 1 (with all aperture coverings removed) avoiding freckles, moles, scars, hair (to the extent possible), and any cuts, bruises or lesions on the skin. Remove only the protective wax paper backing from the lateral (not central) portions of Cuff 1. It is important that the wax paper backing from the central portion of Cuff 1 not be removed, as the adhesive has a strong potential to irritate the skin when peeled off after baseline readings, causing reddening of skin proximal to the apertures.</li>
	<li>After situating Cuff 1 at the intended exposure site, place landmarks using a permanent marker to ensure that Cuff 2 will be situated at precisely the same location. Mark the skin at four points outside of the creases of each of the side flaps of Cuff 1, the upper right, upper left, lower right and lower left points.
	<ol>
		<li>Make these marks dark enough to survive approximately 24 h, as they will also be used to place Cuff 3 in precisely the same location at the follow-up appointment 24 h later.</li>
	</ol>
	</li>
</ol>
5. Baseline Reading: Cuff 1 Application
<ol>
	<li>Using a spectrophotometer that has been calibrated according to manufacturer specifications, obtain and permanently record readings at each of the six open apertures in sequence.</li>
	<li>Ensure that the spectrophotometer is placed the center of the cuff apertures while avoiding moles, scars, or other blemishes to the extent possible.</li>
	<li>Permanently record all &#8220;SCI&#8221; values (L, A, B). To ensure consistent readings with the same calibration point, keep the spectrophotometer in the ON for the duration of the MED procedure and do not turn off until the post-exposure readings have been completed.</li>
	<li>After baseline spectrophotometry measurements have been recorded, remove Cuff 1. To minimize participant discomfort, apply medical adhesive solvent to the perimeter of Cuff 1 as it is peeled off, which will prevent painful removal of hair on the arm.</li>
</ol>
6. Pre-exposure Reading: Cuff 2 Application
<ol>
	<li>After removal of Cuff 1, situate Cuff 2 at the same location using the landmarks drawn on the skin for Cuff 1. The full adhesive backing may be exposed and applied to ensure that Cuff 2 is sufficiently sealed to prevent cross exposure among apertures due to insufficient adhesion to the skin.</li>
	<li>Have both the participant and the researcher don UV-protective clothing and safety accessories. At minimum, the participant, the technician administering the procedure and any other parties in the room must don UV-protective glasses. Technicians should wear long sleeves or use a UV-protective sleeve.</li>
	<li>Prior to activating the lamp, have the technician assist the participant in covering all exposed skin, including the arm above the patch, the arm and wrist below the patch, the hand, and parts of the front or back of the arm that may be exposed on the sides of the patch (UV-protective sheets and tape to affix the sheets may be helpful for this). Additionally, some participants wearing shirts with open necklines may want to drape UV-protective cloth over their neck and chest if these areas will be close to the UV source.</li>
	<li>Spread a UV-protective cloth under the participant&#8217;s arm (to reduce reflectance off the table surface).</li>
</ol>
7. MED Procedure: Pre-exposure
NOTE: The rays from the lamp must be perpendicular to the exposure site. In general, physical movement of the lamp is less possible than movement or arrangement of the angle of participants&#8217; arm.
<ol>
	<li>Prior to activating the lamp, arrange the participant&#8217;s arm such that the UV rays from the lamp will be perpendicular to the angle of Cuff 2 on the participant&#8217;s arm.</li>
	<li>Identify the proper distance between the lamp and Cuff 2 on the participant&#8217;s arm. Place the radiometer&#8217;s sensor facing the UV lamp parallel to the surface of the skin and as close as possible to the location of Cuff 2.</li>
	<li>Cover the participants arm with a UV proof cloth to prevent exposure, and briefly activate the lamp to adjust the distance to Cuff 2 until the radiometer&#8217;s sensor reads 270 &#956;W/cm2.
	<ol>
		<li>To achieve this reading, adjust the distance between the lamp and the surface of the skin until the radiometer reads 270 &#956;W/cm2 (&#177; 10 &#956;W). Once the proper distance has been determined, deactivate the lamp. 
		NOTE: It is important to note here that small differences in the angle of the radiometer will greatly affect the reading. Thus, the angle of the radiometer should be as close to parallel to the surface of the skin as possible.</li>
	</ol>
	</li>
	<li>Make further adjustments in distance throughout the exposure session to prevent drift in the location of the arm. At each reading, confirm the distance and readjust as necessary to maintain radiometer readings at approximately 270 &#956;W/cm2 (&#177; 10 &#956;W).</li>
</ol>
8. MED Procedure: Exposure
<ol>
	<li>Use a stopwatch to implement the MED schedule. Remove the first aperture covering before activating the UV source. Activate the source and the stopwatch simultaneously and remove each aperture covering on Cuff 2 according to the schedule specified below, based on FST.</li>
	<li>At the point of removal for each aperture covering, record the radiometer reading when the radiometer is held parallel to the surface of the skin and pointed at the lamp. If the distance has changed, adjust the distance to lamp to ensure that the radiometer once again reads 270 (&#177; 10) &#956;W/cm2.</li>
	<li>Have the technician monitor the participant&#8217;s arm to ensure consistent positioning. In particular, readjust the arm if the arm rotates, as many participants will rotate the arm as they relax. Subsequent to any adjustments, re-confirm that the radiometer reads 270 &#956;W/cm2 (&#177; 10 &#956;W).</li>
	<li>Turn off the lamp at precise time specified by the dosage schedule in Table 1. Do not deactivate the stopwatch, as an additional series of spectrophotometer readings should be gathered exactly 7 minutes subsequent to deactivation of the lamp, as described below.</li>
</ol>
9. 7 Min Post-exposure Reading
<ol>
	<li>Exactly 7 minutes subsequent to the deactivation of the lamp, record the final spectrophotometer readings from each aperture in Cuff 2. The purpose in collecting data immediately subsequent to the exposure procedure is to first confirm no adverse reaction to the UV radiation, and second to evaluate initial responses, which in some cases can be slight, but measurably different from baseline (pre-exposure) values. Any increase in redness after 7 minutes is likely to be a thermal effect and not erythema. 
	NOTE: An adverse reaction to UV radiation exposure after 7 minutes is likely related to the minimal urticarial dose of solar urticaria, an acquired photosensitivity disorder. Photosensitivity disorders are assessed prior to the MED procedure and subjects with these disorders should be ruled out. However, if this is observed at any point during testing the exposure protocol should be discontinued immediately.</li>
	<li>After exposure to UV radiation, Cuff 2 can be particularly difficult to remove. Use medical-grade adhesive solvent, if necessary, to minimize discomfort to participants during removal of Cuff 2. Participants with particularly hirsute or otherwise sensitive skin may find it helpful to apply either olive oil or alcohol-based adhesive remover under the edge of Cuff 2, as they remove the patch slowly. After Cuff 2 is removed participants may have residual adhesive on their skin, which can also be removed with either olive oil or medical adhesive solvent.</li>
	<li>Prior to departure from the exposure session, remind participants not to wash the landmarks and not apply any lotions to the exposure site.</li>
</ol>
10. Follow-up Appointment: Cuff 3 Application
<ol>
	<li>Before the participant arrives, calibrate the spectrophotometer according to manufacturer specifications.</li>
	<li>Prepare Cuff 3 by removing all of the aperture coverings (leaving the white wax paper backing on the central portion of the patch). When placing Cuff 3 on the participant&#8217;s arm, remove the white wax paper backing from the two side flaps of the patch. Using the landmarks on the participant&#8217;s forearm, place Cuff 3 in the same location as the previous two patches.</li>
	<li>Take a reading at each of the six open apertures in sequence. Additionally, visually inspect each aperture and record whether there appears to be visual evidence of an erythema response in each of the six apertures (red or pink skin indicates erythema). After permanently recording spectrophotometer readings, remove Cuff 3, using solvent if necessary.</li>
	<li>To further enhance participant comfort and safety, provide 4-6 single-use burn gel or aloe vera, and indicate to the participant that if the exposure site becomes itchy or uncomfortable, it may be treated like a sunburn with these or similar over the counter products.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermatological+Photobiology:+Clinical+and+Experimental+Aspects.">Dermatological Photobiology: Clinical and Experimental Aspects.</a> Blackwell Scientific Publications. , (1976).</li><li>Grundmann-Kollmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototherapy+for+atopic+eczema+with+narrow-band+UVB.">Phototherapy for atopic eczema with narrow-band UVB.</a> Journal of the American Academy of Dermatology. 40 (6), 995-997 (1999).</li><li>Honigsmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototherapy+for+psoriasis.">Phototherapy for psoriasis.</a> Clinical and Experimental Dermatology. 26 (4), 343-350 (2001).</li><li>Heckman, C. 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The authors on this study declare no conflicts of interest, financial or otherwise.

Precision implementation of MED testing as described here could offer several advantages over other extant lab-based inflammatory challenges that have achieved popular use. For example, suction blister protocols17,18,19 raise a fluid-filled blister on the skin that is subsequently aspirated with a syringe to gain direct access to the cytokine microenvironment. Although skin blistering is a well-known tool for studying skin immun...

Minimal erythema dose (MED) testing is an FDA-approved procedure to evaluate cutaneous sensitivity to radiation typically in the UVB range, although the MED can be determined at other wavelengths in the UV and visible spectrum1. Erythema is defined as superficial reddening on the surface of the skin caused by engorgement of capillaries (later stages of erythema are more commonly known as sunburn). MED testing has been used extensively in the dermatology literature and clinical phototherapy settings to identify the minimal amount of ultraviolet (UV) radiation that will produce the smallest unit of measurable change in the redness of the skin. MED testing can be accomplished with a commercially available UV lamp, equivalent to what is used in most commercial tanning facilities.
MED testing involves continuous dispersal of UV radiation or light from the visible spectrum onto the surface of the skin for a predetermined length of time, with dosage schedules depending primarily on pigmentation of the skin and the intensity and type of radiation. This procedure is commonly used in clinical settings to determine dosage schedules for patients receiving UV radiation therapy for skin conditions such as psoriasis and eczema2,3. Basic procedures for determining the MED in clinical settings have been described elsewhere4, and can be used to adjust the total dosage of UV radiation upward or downward, depending on individual variation in skin sensitivity.
Skin pigmentation is perhaps the most important subject-specific variable in conducting and measuring results from the MED procedure6. This is because the duration of UV exposure required to evoke the minimal erythema response is principally determined by the lightness or darkness of the participant&#8217;s skin, as defined by the participant&#8217;s Fitzpatrick skin type (FST). FST7 is a numerical scheme for classifying human skin color. The Fitzpatrick scale is a recognized tool for dermatological research into human skin pigmentation8,9, and classifies human skin into one of six categories from lightest (FST I) to darkest (FST VI).
Darker FST typologies require longer UV duration, therefore accurate classification of FST is important. There is an extensive literature on methods for accurate assessment of FST, using a wide variety of approaches including self-report, dermatologist interview and instrumentation-based assessment. Observer ratings of FST have been shown to be correlated with current, but not natural skin color10, however FST can be determined subjectively11 using self-report via questionnaire12 and/or objective assessment via spectrophotometry. Fitzpatrick typing by spectrophotometry has been shown to correlate closely to participant self-report in a number of studies10,13,14,15.
Despite the utility and widespread use of MED testing in clinical services, this procedure has not been widely adopted in laboratory settings for measurement of individual variation in response to pro-inflammatory stimulation. The purpose of the methodology outlined here is to provide techniques and step-by-step procedures that increase the precision and reproducibility of the MED testing procedure, in order to facilitate future work in laboratory settings focused on fine-grained quantification of intra-individual variability in inflammatory response. We further provide representative results that illustrate the capability of this standardized protocol to accurately capture person-to-person variation in inflammation.

<table><tbody><tr><td>6-aperture dose testing patch (&amp;ldquo;Cuff&amp;rdquo;)</td><td>Daavlin</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Medical grade adhesive solvent</td><td></td><td></td><td></td></tr><tr><td>Non-reflective UV proof cloth</td><td></td><td></td><td></td></tr><tr><td>Radiometer</td><td>SolarLight</td><td>Model 6.2 UVB Meter</td><td></td></tr><tr><td>Single use aloe or burn gel</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Konika-Minolta</td><td>CM-2600D</td><td></td></tr><tr><td>Stopwatch</td><td></td><td></td><td></td></tr><tr><td>UV lamp &amp;ndash; Fiji Sun</td><td>Sperti</td><td></td><td>Emission spectrum 280 nm-400 nm, approximately 25% UVB</td></tr><tr><td>UV-proof safety glasses (2 pair)</td><td></td><td></td><td></td></tr><tr><td>UV-proof sleeve</td><td></td><td></td><td></td></tr><tr><td>White cotton gloves (2 pair)</td><td></td><td></td><td></td></tr></tbody></table>

precision implementation of minimal erythema dose (med) testing to assess individual variation in human inflammatory response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

The timing schedule presented in Table 1 is a novel dosage schedule that was calculated to capture the MED, on average, at the mid-point of the exposure event (i.e., aperture 3 or 4) for each FST. The basis for the calculated schedule is as follows.
Prior work has established that for individuals with FST 2, the median MED for radiation in the UVB range is 66.9 milliwatts (mW) per cm2, 77.429 mW/cm2 for FST 3 and 85.0 for FST 416. ...

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Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

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Research

JoVE Journal

Medicine

8.6K Views.  Virginia Tech. Minimale erythema dosis testen, of MED testen, is een techniek met een lange geschiedenis in dermatologische en klinische fototherapie instellingen. En het wordt voornamelijk gebruikt om de kleinste hoeveelheid straling te bepalen die nodig is om erytheem te produceren, of zichtbare roodheid op het oppervlak van de huid. In deze specifieke studie, wat we hebben gedaan is het ontwikkelen van verschillende nieuwe technieken en procedures die het mogelijk maken MED-testen worden aangepast voor e...

Video: Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

Behavior

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Biology

Minimal Erythema Dose (MED) Testing

Ultraviolet radiation (UV) therapy is sometimes used as a treatment for various common skin conditions, including psoriasis, acne, and eczema. The dosage of UV light is prescribed according to an individual's skin sensitivity. Thus, to establish the proper dosage of UV light to administer to a patient, the patient is sometimes screened to determine a minimal erythema dose (MED), which is the amount of UV radiation that will produce minimal erythema (sunburn or redness caused by engorgement of capillaries) of an individual's skin within a few hours following exposure. This article describes how to conduct minimal erythema dose (MED) testing. There is currently no easy way to determine an appropriate UV dose for clinical or research purposes without conducting formal MED testing, requiring observation hours after testing, or informal trial and error testing with the risks of under- or over-dosing. However, some alternative methods are discussed.

Minimal Erythema Dose MED Testing

This article describes how to conduct minimal erythema dose (MED) testing in order to determine the lowest dose of ultraviolet radiation that will cause erythema (burning) when administered to an individual.

Ultraviolet radiation (UV) therapy is  sometimes used as a treatment for various common skin conditions, including  psoriasis, acne, and eczema. The ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants (6). The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semi-solids, solid or wax. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37&deg;C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations (7).

An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Bioengineering

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Immunology and Infection

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Source: Amaro-Ortiz, A. et al. <a href="https://www.jove.com/v/50670"> Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model</a>. J. Vis. Exp. (2013)
This video describes a method to measure UV sensitivity in a humanized mouse model&#8212;a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

Source: Amaro-Ortiz, A. et al.  Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model. J. Vis. Exp. ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

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