Name: precision implementation of minimal erythema dose (med) testing to assess individual variation in human inflammatory response
Uploaded: 2019-10-03T13:30:00
Description: Watch this Scientific Journal Video about Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response at JoVE.com

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 hum...

<ol>
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J., 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=Minimal+Erythema+Dose+(MED)+testing.">Minimal Erythema Dose (MED) testing.</a> Journal of Visualized Experiments. (75), e50175 (2013).</li><li>Kroenke, K., 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=Physical+symptoms+in+primary+care.+Predictors+of+psychiatric+disorders+and+functional+impairment.">Physical symptoms in primary care. Predictors of psychiatric disorders and functional impairment.</a> Archives of Family Medicine. 3 (9), 774-779 (1994).</li><li>Coelho, S. G., 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=Non-invasive+diffuse+reflectance+measurements+of+cutaneous+melanin+content+can+predict+human+sensitivity+to+ultraviolet+radiation.">Non-invasive diffuse reflectance measurements of cutaneous melanin content can predict human sensitivity to ultraviolet radiation.</a> Experimental Dermatology. 22 (4), 266-271 (2013).</li><li>Fitzpatrick, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+validity+and+practicality+of+sun-reactive+skin+types+I+through+VI.">The validity and practicality of sun-reactive skin types I through VI.</a> Archives of Dermatology. 124 (6), 869-871 (1988).</li><li>Matts, P. J., Dykes, P. J., Marks, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+melanin+in+skin+determined+in+vivo.">The distribution of melanin in skin determined in vivo.</a> British Journal of Dermatology. 156 (4), 620-628 (2007).</li><li>Eilers, S., 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=Accuracy+of+self-report+in+assessing+Fitzpatrick+skin+phototypes+I+through+VI.">Accuracy of self-report in assessing Fitzpatrick skin phototypes I through VI.</a> JAMA Dermatology. 149 (11), 1289-1294 (2013).</li><li>Daniel, L. C., Heckman, C. J., Kloss, J. D., Manne, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+alternative+methods+of+measuring+skin+color+and+damage.">Comparing alternative methods of measuring skin color and damage.</a> Cancer Causes, Control. 20 (3), 313-321 (2009).</li><li>Ravnbak, M. H., Philipsen, P. A., Wulf, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimal+melanogenesis+dose/minimal+erythema+dose+ratio+declines+with+increasing+skin+pigmentation+using+solar+simulator+and+narrowband+ultraviolet+B+exposure.">The minimal melanogenesis dose/minimal erythema dose ratio declines with increasing skin pigmentation using solar simulator and narrowband ultraviolet B exposure.</a> Photodermatology Photoimmunology &amp; Photomedicine. 26 (3), 133-137 (2010).</li><li>Miller, S. A., 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=Evidence+for+a+new+paradigm+for+ultraviolet+exposure:+a+universal+schedule+that+is+skin+phototype+independent.">Evidence for a new paradigm for ultraviolet exposure: a universal schedule that is skin phototype independent.</a> Photodermatology Photoimmunology &amp; Photomedicine. 28 (4), 187-195 (2012).</li><li>Pershing, L. K., 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=Reflectance+spectrophotometer:+The+dermatologists'+sphygmomanometer+for+skin+phototyping.">Reflectance spectrophotometer: The dermatologists' sphygmomanometer for skin phototyping.</a> Journal of Investigative Dermatology. 128 (7), 1633-1640 (2008).</li><li>Kollias, N., Baqer, A., Sadiq, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimum+Erythema+Dose+Determination+in+Individuals+of+Skin+Type-V+and+Type-Vi+with+Diffuse-Reflectance+Spectroscopy.">Minimum Erythema Dose Determination in Individuals of Skin Type-V and Type-Vi with Diffuse-Reflectance Spectroscopy.</a> Photodermatology Photoimmunology &amp; Photomedicine. 10 (6), 249-254 (1994).</li><li>Treesirichod, A., Chansakulporn, S., Wattanapan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+Between+Skin+Color+Evaluation+by+Skin+Color+Scale+Chart+and+Narrowband+Reflectance+Spectrophotometer.">Correlation Between Skin Color Evaluation by Skin Color Scale Chart and Narrowband Reflectance Spectrophotometer.</a> Indian Journal of Dermatology. 59 (4), 339-342 (2014).</li><li>Gambichler, T., 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=Reference+limits+for+erythema-effective+UV+doses.">Reference limits for erythema-effective UV doses.</a> Photochemistry and Photobiology. 82 (4), 1097-1102 (2006).</li><li>Kool, J., 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=Suction+blister+fluid+as+potential+body+fluid+for+biomarker+proteins.">Suction blister fluid as potential body fluid for biomarker proteins.</a> Proteomics. 7 (20), 3638-3650 (2007).</li><li>Clark, K. E., Lopez, H., Abdi, B. A., 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=Multiplex+cytokine+analysis+of+dermal+interstitial+blister+fluid+defines+local+disease+mechanisms+in+systemic+sclerosis.">Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis.</a> Arthritis Research &amp; Therapy. 17, 73 (2015).</li><li>Rosenkranz, M. A., 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=A+comparison+of+mindfulness-based+stress+reduction+and+an+active+control+in+modulation+of+neurogenic+inflammation.">A comparison of mindfulness-based stress reduction and an active control in modulation of neurogenic inflammation.</a> Brain Behavior and Immunity. 27 (1), 174-184 (2013).</li><li>Smith, T. J., Wilson, M. A., Young, A. J., Montain, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suction+blister+model+reliably+assesses+skin+barrier+restoration+and+immune+response.">A suction blister model reliably assesses skin barrier restoration and immune response.</a> Journal of Immunological Methods. 417, 124-130 (2015).</li><li>Holm, L. L., 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=A+Suction+Blister+Protocol+to+Study+Human+T-cell+Recall+Responses+In+Vivo.">A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo.</a> Journal of Visualized Experiments. (138), 57554 (2018).</li><li>Seitz, J. C., Whitmore, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+erythema+and+tanning+responses+in+human+skin+using+a+tri-stimulus+colorimeter.">Measurement of erythema and tanning responses in human skin using a tri-stimulus colorimeter.</a> Dermatologica. 177 (2), 70-75 (1988).</li><li>Henriksen, M., Na, R., Agren, M. S., Wulf, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+erythema+dose+after+multiple+UV+exposures+depends+on+pre-exposure+skin+pigmentation.">Minimal erythema dose after multiple UV exposures depends on pre-exposure skin pigmentation.</a> Photodermatology Photoimmunology &amp; Photomedicine. 20 (4), 163-169 (2004).</li><li>Stamatas, G. N., Zmudzka, B. Z., Kollias, N., Beer, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+skin+erythema+and+pigmentation:+new+means+of+implementation+of+diffuse+reflectance+spectroscopy+with+a+commercial+instrument.">In vivo measurement of skin erythema and pigmentation: new means of implementation of diffuse reflectance spectroscopy with a commercial instrument.</a> British Journal of Dermatology. 159 (3), 683-690 (2008).</li><li>Latreille, J., 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=Influence+of+skin+colour+on+the+detection+of+cutaneous+erythema+and+tanning+phenomena+using+reflectance+spectrophotometry.">Influence of skin colour on the detection of cutaneous erythema and tanning phenomena using reflectance spectrophotometry.</a> Skin Research and Technology. 13 (3), 236-241 (2007).</li></ol>

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 (&#8220;Cuff&#8221;)</td>
			<td>Daavlin</td>
			<td>&#160;</td>
			<td>&#160;</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 &#8211; Fiji Sun</td>
			<td>Sperti</td>
			<td></td>
			<td>Emission spectrum 280nm-400nm, 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. ...

Watch this Scientific Journal Video about Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response at JoVE.com

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 ...

Minimal erythema dose testing, or MED testing, is a technique with a long history in dermatological and clinical phototherapy settings. And it's primarily used to determine the smallest amount of radiation required to produce erythema, or visible reddening on the surface of the skin. In this particular study, what we've done is develop several new techniques and procedures that allow MED testing to be adapted for somewhat different purpose, which is to precisely quantify variation in human inflammatory activation.The main advantage of precision dosing and measurement in MED testing pertains to the reproducibility of results, which then opens up new possibilities for systematically examining other factors that could be related to inflammation. Demonstrating the procedure will be two PhD students from my lab, Holly Sullivan-Toole and Corinne Carlton. Begin by escorting the participant to the testing room.Explain the MED testing procedure to the participant and confirm understanding. Place cuff one on the non-dominant forearm. Remove only the protective wax paper backing from the lateral, not central, portions of the cuff.After situating cuff one at the intended exposure site, place landmarks using a permanent marker to ensure that cuff two will be situated at precisely the same location. Then, mark the skin at four points outside of the creases of each of the side flaps of cuff one, the upper right, upper left, lower right, and lower left points. Ensure the marks are dark enough to last for 24 hours, so they may be used to place cuff three in precisely the same location at the follow-up appointment.Next, use a calibrated spectrophotometer to record readings at each of the six open apertures in sequence. Ensure that the spectrophotometer is placed in the center of the cuff apertures, while avoiding moles, scars, or other blemishes to the extent possible. Finally, permanently record all SCI values.To ensure consistent readings with the same calibration point, keep the spectrophotometer in the on position for the duration of the MED procedure and do not turn off until the post-exposure readings have been completed. After removal of cuff one, situate cuff two at the same location using the landmarks drawn on the skin for cuff one. Put on the necessary safety equipment, including UV-proof glasses, sleeves, and gloves, and make sure the participant does the same.Prior to activating the lamp, arrange the participant's arm such that the UV rays from the lamp will be perpendicular to the angle of cuff two on the participant's arm. Briefly activate the lamp to adjust the distance to cuff two until the radiometer sensor reads 270 microwatts per centimeter squared. Next, place the radiometer sensor facing the UV lamp, parallel to the surface of the skin, and as close as possible to the location of cuff two.Then, remove the first aperture covering before activating the UV source. Remove each window in the dose testing patch according to the schedule displayed here, which illustrates separate dosage schedules depending on the participant's Fitzpatrick Skin Type. Activate the UV source and the stopwatch simultaneously, and remove each aperture covering on cuff two according to the correct dosage schedule, which is based on Fitzpatrick Skin Type.Next, turn off the lamp at the precise time specified by the dosage schedule. Do not deactivate the stopwatch, as an additional series of spectrophotometer readings should be gathered exactly seven minutes subsequent to deactivation of the lamp, as described below. Lastly, after exactly seven minutes have passed since the deactivation of the lamp, record the final spectrophotometer readings from each aperture in cuff two.Begin by calibrating the spectrophotometer in preparation for the follow-up appointment, 24 hours later. Prepare cuff three by removing all of the aperture coverings and leaving the white wax paper backing on the central portion of the patch. When placing cuff three on the participant's arm, remove the white wax paper backing from the two side flaps of the patch.Use the landmarks on the participant's forearm to place cuff three in the same location as the previous two patches. Finally, take a reading at each of the six open apertures in sequence. These results show an accurate skin response to MED testing.Here are complete data for a single representative subject, broken down by assessment period. Pre-exposure, seven minutes post exposure, and 24 hours follow-up. Each aperture, two through six, receives 25%more energy than the one before it.Further, all 72 subjects are shown, indicating the variation that are likely to be encountered, across different FSTs. Perhaps the most important aspects of precision MED testing relate to the adherence to the proper dosage schedule for the identified Fitzpatrick Skin Type and also consistently maintaining the correct distance between the surface of the skin and the UV light source. Part of our motivation in developing these techniques is to facilitate future research examining how inflammatory responses could relate to any number of potentially interesting variables, such as psychological, pharmacological, or health-related factors.

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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 ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Technique and Patient Selection Criteria of Right Anterior Mini-Thoracotomy for Minimal Access Aortic Valve Replacement

Aortic valve stenosis has become the most prevalent valvular heart disease in developed countries, and is due to the aging of these populations. The incidence of the pathology increases with growing age after 65 years. Conventional surgical aortic valve replacement through median sternotomy has been the gold standard of patient care for symptomatic aortic valve stenosis. However, as the risk profile of patients worsens, other therapeutic strategies have been introduced in an attempt to maintain the excellent results obtained by the established surgical treatment. One of these approaches is represented by transcatheter aortic valve implantation. Although the outcomes of high-risk patients undergoing treatment for symptomatic aortic valve stenosis have improved with transcatheter aortic valve replacement, many patients with this condition remain candidates for surgical aortic valve replacement. In order to reduce the surgical trauma in patients who are candidates for surgical aortic valve replacement, minimally invasive approaches have garnered interest during the past decade. Since the introduction of right anterior thoracotomy for aortic valve replacement in 1993, right anterior mini-thoracotomy and upper hemi-sternotomy have become the predominant incisional approaches among cardiac surgeons performing minimal access aortic valve replacement. Beside the location of the incision, the arterial cannulation site represents the second major landmark of minimal access techniques for aortic valve replacement. The two most frequently used arterial cannulation sites include central aortic and peripheral femoral approaches. With the purpose of reducing surgical trauma in these patients, we have opted for a right anterior mini-thoracotomy approach with a central aortic cannulation site. This protocol describes in detail a technique for minimally invasive aortic valve replacement and provides recommendations for patient selection criteria, including cardiac computer tomography measurements. The indications and limitations of this technique, as well as its alternatives, are discussed.

The goal of this protocol is to describe in detail the technique of minimally invasive aortic valve replacement through a right anterior mini-thoracotomy and central aortic cannulation. This technique can potentially enhance patients&#39; comfort and, by reducing post-operative morbidity, promote lowering the length of stay and global costs.

Aortic valve stenosis has become the most prevalent valvular heart disease in developed countries, and is due to the aging of these populations. The ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...