Name: contact hypersensitivity as a murine model of allergic contact dermatitis
Uploaded: 2022-09-26T15:00:00
Description: Watch this Scientific Journal Video about Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis at JoVE.com

Este estudio fue apoyado por la subvención SUBZ. A020.22.060 de la Universidad de Medicina de Wroclaw, Polonia, y por subvenciones del Ministerio de Ciencia y Educación Superior N N401 545940 a MS e IP2012 0443 72 a MMS.

Todos los experimentos presentados en este artículo se llevaron a cabo de acuerdo con las directrices del 1er Comité Ético Local de Pruebas con Animales en Cracovia. Todos los procedimientos descritos se realizaron de acuerdo con las recomendaciones locales, especialmente con respecto al uso de ketamina / xilazina como anestésico, el uso de ambos lados de las orejas para aplicar la sustancia / hapteno, el corte de la oreja y la recolección de sangre mediante la extracción del globo ocular. Para el presente estudio ...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleeding+the+laboratory+mouse:+Not+all+methods+are+equal.">Bleeding the laboratory mouse: Not all methods are equal.</a> Experimental Hematology. 44 (2), 132-137 (2016).</li><li>Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+th17+differentiation+of+na&#239;ve+CD4+T+lymphocytes.">Isolation and th17 differentiation of na&#239;ve CD4 T lymphocytes.</a> Journal of Visualized Experiments. (79), e50765 (2013).</li><li>Hemocytometer - Counting of Cells. Amrita University Available from: <a target="_blank" href="https://www.youtube.com/watch?v=MKS0KM3lr90">https://www.youtube.com/watch?v=MKS0KM3lr90</a> (2011)</li><li>Majewska-Szczepanik, M., Strzepa, A., Marci&#324;ska, K., Wen, L., Szczepanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicutaneous+immunization+with+TNP-Ig+and+Zymosan+induces+TCR&#945;&#946;++CD4++contrasuppressor+cells+that+reverse+skin-induced+suppression+via+IL-17A.">Epicutaneous immunization with TNP-Ig and Zymosan induces TCR&#945;&#946;+ CD4+ contrasuppressor cells that reverse skin-induced suppression via IL-17A.</a> International Archives of Allergy and Immunology. 164 (2), 122-136 (2014).</li><li>Majewska-Szczepanik, M., Zemelka-Wi&#261;cek, M., Ptak, W., Wen, L., Szczepanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicutaneous+immunization+with+DNP-BSA+induces+CD4++CD25++Treg+cells+that+inhibit+Tc1-mediated+CS.">Epicutaneous immunization with DNP-BSA induces CD4+ CD25+ Treg cells that inhibit Tc1-mediated CS.</a> Immunology and Cell Biology. 90 (8), 784-795 (2012).</li><li>Directive 2010/63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union Available from: <a target="_blank" href="https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:En:PDF">https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:En:PDF</a> (2010)</li><li>Colvin, R. B., Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+clotting+system+in+cell-mediated+hypersensitivity.+II.+Kinetics+of+fibrinogen/fibrin+accumulation+and+vascular+permeability+changes+in+tuberculin+and+cutaneous+basophil+hypersensitivity+reactions.">Role of the clotting system in cell-mediated hypersensitivity. II. Kinetics of fibrinogen/fibrin accumulation and vascular permeability changes in tuberculin and cutaneous basophil hypersensitivity reactions.</a> Journal of Immunology. 114, 377-387 (1975).</li><li>Szczepanik, 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=Regulation+of+contact+sensitivity+in+non-obese+diabetic+(NOD)+mice+by+innate+immunity.">Regulation of contact sensitivity in non-obese diabetic (NOD) mice by innate immunity.</a> Contact Dermatitis. 79 (4), 197-207 (2018).</li><li>Askenase, P. W., Majewska-Szczepanik, M., Kerfoot, S., Szczepanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+iNKT+cells+in+the+early+and+late+components+of+Tc1-mediated+DNFB+contact+sensitivity:+Cooperative+role+of+&#947;&#948;-T+cells.">Participation of iNKT cells in the early and late components of Tc1-mediated DNFB contact sensitivity: Cooperative role of &#947;&#948;-T cells.</a> Scandinavian Journal of Immunology. 73 (5), 465-477 (2011).</li><li>Zemelka-Wi&#261;cek, M., Majewska-Szczepanik, M., Ptak, W., Szczepanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicutaneous+immunization+with+protein+antigen+induces+antigen-non-specific+suppression+of+CD8+T+cell+mediated+contact+sensitivity.">Epicutaneous immunization with protein antigen induces antigen-non-specific suppression of CD8 T cell mediated contact sensitivity.</a> Pharmacological Reports. 64 (6), 1485-1496 (2012).</li><li>Van Loveren, H., 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=Use+of+micrometers+and+calipers+to+measure+various+components+of+delayed-type+hypersensitivity+ear+swelling+reactions+in+mice.">Use of micrometers and calipers to measure various components of delayed-type hypersensitivity ear swelling reactions in mice.</a> Journal of Immunological Methods. 67 (2), 311-319 (1984).</li><li>Tsuji, R. F., 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=B+cell-dependent+T+cell+responses:+IgM+antibodies+are+required+to+elicit+contact+sensitivity.">B cell-dependent T cell responses: IgM antibodies are required to elicit contact sensitivity.</a> The Journal of Experimental Medicine. 196 (10), 1277-1290 (2002).</li><li>Campos, R. 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=Cutaneous+immunization+rapidly+activates+liver+invariant+Valpha14+NKT+cells+stimulating+B-1+B+cells+to+initiate+T+cell+recruitment+for+elicitation+of+contact+sensitivity.">Cutaneous immunization rapidly activates liver invariant Valpha14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity.</a> The Journal of Experimental Medicine. 198 (12), 1785-1796 (2003).</li><li>R&#252;hl-Muth, A. C., Maler, M. D., Esser, P. R., Martin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+of+a+fat-enriched+diet+causes+the+loss+of+resistance+to+contact+hypersensitivity.">Feeding of a fat-enriched diet causes the loss of resistance to contact hypersensitivity.</a> Contact Dermatitis. 85 (4), 398-406 (2021).</li><li>Bour, H., 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=histocompatibility+complex+class+I-restricted+CD8++T+cells+and+class+II-restricted+CD4++T+cells,+respectively,+mediate+and+regulate+contact+sensitivity+to+dinitrofluorobenzene.">histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene.</a> European Journal of Immunology. 25 (11), 3006-3010 (1995).</li><li>Majewska-Szczepanik, 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=Obesity+aggravates+contact+hypersensitivity+reaction+in+mice.">Obesity aggravates contact hypersensitivity reaction in mice.</a> Contact Dermatitis. 87 (1), 28-39 (2022).</li><li>Katagiri, K., Arakawa, S., Kurahashi, R., Hatano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+contact+hypersensitivity+in+diet-induced+obese+mice.">Impaired contact hypersensitivity in diet-induced obese mice.</a> Journal of Dermatological Science. 46 (2), 117-126 (2007).</li><li>Bouloc, A., Cavani, A., Katz, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+hypersensitivity+in+MHC+class+II-deficient+mice+depends+on+CD8+T+lymphocytes+primed+by+immunostimulating+Langerhans+cells.">Contact hypersensitivity in MHC class II-deficient mice depends on CD8 T lymphocytes primed by immunostimulating Langerhans cells.</a> The Journal of Investigative Dermatology. 111 (1), 44-49 (1998).</li><li>Martin, 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=Peptide+immunization+indicates+that+CD8++T+cells+are+the+dominant+effector+cells+in+trinitrophenyl-specific+contact+hypersensitivity.">Peptide immunization indicates that CD8+ T cells are the dominant effector cells in trinitrophenyl-specific contact hypersensitivity.</a> The Journal of Investigative Dermatology. 115 (2), 260-266 (2000).</li><li>Vennegaard, M. 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=Epicutaneous+exposure+to+nickel+induces+nickel+allergy+in+mice+via+a+MyD88-dependent+and+interleukin-1-dependent+pathway.">Epicutaneous exposure to nickel induces nickel allergy in mice via a MyD88-dependent and interleukin-1-dependent pathway.</a> Contact Dermatitis. 71 (4), 224-232 (2014).</li></ol>

El CHS se induce a través de haptenos, que se unen a antígenos autoproteicos en la piel, creando neoantígenos. El CHS está mediado por el reclutamiento extravascular local de células T efectoras chS específicas del antígeno circulante, lo que resulta en hinchazón en el tejido desafiado, alcanzando su punto máximo 24 h después de la exposición de la piel secundaria al mismohapteno 6. La hinchazón del tejido es causada principalmente por la infiltración de leucocitos y la depos...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64329">Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis</a>. The Protocol&#160;and Representative Results sections were updated.
Step 3.2.1 of the Protocol has been updated from:
On day &#34;4&#34;, prepare 0.4% hapten: TNCB in an acetone-ethanol mixture (ratio 1:1). Prepare the solution just before use in a glass vial and protect it from light by covering the vial with aluminum foil.
to:
On day &#34;4&#34;, prepare 0.4% hapten: TNCB in an acetone: olive oil&#160;mixture (ratio 1:1). Prepare the solution just before use in a glass vial and protect it from light by covering the vial with aluminum foil.
Figure 1 of the Representative Results has been updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64329/64329fig01.jpg" /> 
Figure 1: Induction of CHS. Sensitization, challenge, and ear measurement. Abbreviations: CHS = contact hypersensitivity reaction; TNCB = 2,4,6-trinitrochlorobenzene. <a href="https://www.jove.com/files/ftp_upload/64329/64329fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64329/64329fig1v2.jpg" /> 
Figure 1: Induction of CHS. Sensitization, challenge, and ear measurement. Abbreviations: CHS = contact hypersensitivity reaction; TNCB = 2,4,6-trinitrochlorobenzene. <a href="https://www.jove.com/files/ftp_upload/64329/64329fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2 of the Representative Results has been updated from:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64329/64329fig02.jpg" /> 
Figure 2: Adoptive transfer of the CHS-effector cells. Abbreviations: ALNs =axillary and inguinal lymph nodes; CHS = contact hypersensitivity reaction; i.v. = intravenously; SPLs = spleens; TNCB = 2,4,6-trinitrochlorobenzene. <a href="https://www.jove.com/files/ftp_upload/64329/64329fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64329/64329fig2v2.jpg" /> 
Figure 2: Adoptive transfer of the CHS-effector cells. Abbreviations: ALNs =axillary and inguinal lymph nodes; CHS = contact hypersensitivity reaction; i.v. = intravenously; SPLs = spleens; TNCB = 2,4,6-trinitrochlorobenzene. <a href="https://www.jove.com/files/ftp_upload/64329/64329fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64329">Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis</a>. The Protocol&#160;and Representative Results sections were updated.

Erratum: Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

La dermatitis alérgica de contacto (ACD) es una enfermedad inflamatoria común de la piel en los países industrializados causada por una reacción de hipersensibilidad tipo IV resultante de la exposición a sustancias químicas de bajo peso molecular llamadas haptenos. Las sustancias que causan sensibilización por contacto en los seres humanos incluyen iones de metales pesados (cromo, níquel, hierro, cobalto), trementina, fragancias, colorantes y conservantes presentes en cosméticos (por ejemplo, p-fenilendiamina), algunos medicamentos (por ejemplo, neomicina, benzocaína), antibióticos β-lactámicos (es decir, penicilina), productos químicos producidos por las plantas (pentadecacacecol, una sustancia presente en la hiedra venenosa), así como hidroquinona utilizada en la industria fotográfica 1,2 . Los agentes etiológicos acD son muy altos, ya que solo en la industria se utilizan más de 100,000 productos químicos, y se sintetizan 2,000 nuevos cada año. Hasta la fecha, se han identificado más de 3.700 moléculas que pueden ser haptenos/alérgenos de contacto3. La reacción de hipersensibilidad de contacto (CHS) es un modelo experimental de ACD que puede estudiarse en ratones, cobayas y ratas y puede ser inducida por la aplicación tópica en la piel de haptenos químicos reactivos disueltos en disolventes orgánicos 4,5,6. Este estudio tiene como objetivo describir un método de laboratorio objetivo que puede ayudar a estudiar la reacción chS en ratones, que puede medirse y cuantificarse mediante diversas pruebas.
El CHS consiste en fases de sensibilización (inducción) y efector (desafío). En modelos animales, los haptenos primero se unen covalentemente a las proteínas del cuerpo para crear neoantígenos. Durante la fase de sensibilización, los queratinocitos activados promueven la migración y maduración de las células dendríticas de la piel (sDC) mediante la producción de citoquinas proinflamatorias-factor de necrosis tumoral α (TNF-α) e interleucina 1β (IL-1β)7. Las células de Langerhans epidérmicas (LC) presentan antígenos durante las fases de inducción y efector del CHS8. Los LC expuestos al hapteno durante la sensibilización promueven la inducción de células reguladoras y efectoras9. La creciente evidencia de varios estudios sugiere que las respuestas de CHS pueden estar mediadas por células Th1 restringidas a CD4 + MHC clase II, liberando localmente interferón-γ (IFN-γ) para emplear un infiltrado inflamatorio característico, linfocitos Tc1 restringidos por CD8 + MHC clase I que también pueden liberar IFN-γ pero en su mayoría median daño citotóxico a los queratinocitos, y ahora también células Th17 productoras de interleucina 17 (IL-17)10, 11.
Se han desarrollado varios modelos diferentes de CHS que emplean variasespecies 1 2,13,14 y haptenos (una comparación detallada de diferentes haptenos, disolventes y tiempo de aplicación se resume en la Tabla 1). El ratón, una especie de laboratorio de uso frecuente, ofrece algunas ventajas en el estudio de CHS. Hay más cepas, knockouts (KO) y animales transgénicos entre los ratones en comparación con otras especies, lo que los convierte en un animal muy atractivo15. Además, el modelo CHS requiere muchos animales, y los ratones son más económicos aquí. Los modelos animales no imitan el ACD en todos los aspectos; en particular, muestran costras y descamación, lo que no es común para los humanos16,17. Las características de la enfermedad crónica son difíciles de reproducir, principalmente porque el modelo descrito no asume la aplicación del hapteno durante un largo período de tiempo. Sin embargo, se ha confirmado aquí que muchos de los aspectos significativos de ACD se reproducen. También se ha demostrado que, al igual que en los seres humanos, estas características están asociadas con reacciones alérgicas locales. La elección del hapteno, su disolvente y su aplicación descrita en este protocolo fueron dictadas por el hecho de que los resultados han sido confirmados por numerosas pruebas in vitro y que fue probado y modificado en el laboratorio durante muchos años hasta que se estableció la versión actual. Los modelos murinos permiten el análisis de los subconjuntos celulares o citoquinas que están involucradas en el desarrollo de ACD y son esenciales para las evaluaciones preclínicas de nuevos tratamientos.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70% ethanol</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>65350-M</td>
			<td>for surface disinfection</td>
		</tr>
		<tr>
			<td>96-well flat-bottom plates, polypropylene</td>
			<td>Greiner Bio-One GmbH, Kremsmunster, Austria</td>
			<td>655101</td>
			<td>for MPO and Evans blue measurement - plates should be made of polypropylene, that has a lower binding capacity so proteins or DNA will not bind</td>
		</tr>
		<tr>
			<td>Acetone (ACS reagent, &#8805;99.5%)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>Z185140</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius Weighing Technology GmbH, Goettingen, Germany</td>
			<td>PRACTUM224-1s, 29105177</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated tissue processor</td>
			<td>MediMeas Instruments, Sarsehri, Haryana, India</td>
			<td>MTP-E-212</td>
			<td>automatically prepare tissue samples by fixing, dehydrating, clearing, and infiltrating them with paraffin</td>
		</tr>
		<tr>
			<td>BD Vacutainer SST II Advance (tube with gel for obtaining serum)</td>
			<td>Becton Dickinson (BD), Franklin Lakes, NJ, USA</td>
			<td>BD 366882</td>
			<td></td>
		</tr>
		<tr>
			<td>Bicinchoninic acid kit for protein determination</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>BCA1-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse IgG1</td>
			<td>Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA</td>
			<td>553441</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumine)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>A9418</td>
			<td>protein assays &#38; analysis, 2 mg/mL</td>
		</tr>
		<tr>
			<td>Cell strainer, pore size 70 &#956;m</td>
			<td>BIOLOGIX, China</td>
			<td>15-1070</td>
			<td>suitable for 50 mL tubes</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>VWR, Radnor, Pennsylvania, United States</td>
			<td>631-1583</td>
			<td>24 mm, but it possible to use different size</td>
		</tr>
		<tr>
			<td>Disposable pipettes capacity: 5 mL, 10 mL, 25 mL</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>Z740301, Z740302, Z740303</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#8242;s phosphate buffered saline)</td>
			<td>ThermoFisher Scientific,&#160; Waltham, Massachusetts, USA</td>
			<td>14190144</td>
			<td>no calcium, no magnesium, mammalian cell culture</td>
		</tr>
		<tr>
			<td>DPX Mountant for histology</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>6522</td>
			<td>mounting media for H-E, might be used some other e.g, Canada balsam</td>
		</tr>
		<tr>
			<td>Dumont 5 tweezers - straight</td>
			<td>Animalab, Poznan, Poland</td>
			<td>11251-10FST</td>
			<td>surgical instruments for procedures on mice (should be steriled)</td>
		</tr>
		<tr>
			<td>Dumont 7 tweezers - bent</td>
			<td>Animalab, Poznan, Poland</td>
			<td>11272-50FST</td>
			<td>surgical instruments for procedures on mice (should be steriled)</td>
		</tr>
		<tr>
			<td>Eosin Y solution, alcoholic</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 1.5 mL</td>
			<td>Eppenforf, Germany</td>
			<td>3,01,20,086</td>
			<td>polypropylene</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 2.0 mL</td>
			<td>Eppenforf, Germany</td>
			<td>3,01,20,094</td>
			<td>polypropylene, round bottom (the homogenization beads can easily move)</td>
		</tr>
		<tr>
			<td>Ethanol 100% (absolute alkohol)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>1.07017</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>1.59010</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans blue</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (fetal bovine serum)</td>
			<td>ThermoFisher Scientific, Waltham, Massachusetts, USA</td>
			<td>A3160802</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10%</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide 99.5% (GC)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>F7503</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer -20 &#176;C</td>
			<td>Bosch, Germany</td>
			<td>GSN54AW30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fridge +4 &#176;C / freezer -20 &#176;C</td>
			<td>Bosch, Germany</td>
			<td>KGV36V10</td>
			<td>mammalian Cell Culture, qualified, Brazil, 10 x 50 mL</td>
		</tr>
		<tr>
			<td>Glass microskope slides, SuperFrost Plus</td>
			<td>VWR, Radnor, Pennsylvania, United States</td>
			<td>631-0108, 631-0446, 631-0447, 631-0448, 631-0449</td>
			<td>Slides that eliminates the need to apply adhesive materials or protein coatings, to preventing any tissue sections loss during staining.</td>
		</tr>
		<tr>
			<td>Graph Pad Prism</td>
			<td>GraphPad Software Inc.</td>
			<td>v. 9.4.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Grey soap</td>
			<td>Pollena Ostrzesz&#243;w, Producent Chemii Gospodarczej Sp. Z.o.o. , Sp. K., Poland</td>
			<td>Bialy jelen soap bar</td>
			<td>grey Soap Bar Natural Hypoallergenic. Generally available product</td>
		</tr>
		<tr>
			<td>H2SO4 (sulfuric acid) 1 mol/l (1 M)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>1.60313</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris hematoxylin solution</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>HHS16</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR, Avantor, U.S.A</td>
			<td>612-5719</td>
			<td>manual counting chamber is recommend, which is more accurate</td>
		</tr>
		<tr>
			<td>Hexadecyltrimethylammonium bromide</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>H5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>QIAGEN Hilden, Germany</td>
			<td>Tissue Lyser LT, SN 23.1001/05234</td>
			<td>homogenizer with stainless steel beads (diameter 5 mm) for 2 mL centrifuge tubes</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase streptavidin (HRP streptavidin)</td>
			<td>Vector Laboratories, Inc., Burlingame, CA, USA</td>
			<td>SA-5004-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution (H202)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Heracell 150i</td>
			<td>Thermo Electron LED Gmbh, Germany</td>
			<td>41071068</td>
			<td>37 oC in the atmosphere of 5 % CO2, and 65 0C for deparaffinization the sections for histology</td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL, solution for injection</td>
			<td>Biowet Pulawy Sp. z o.o., Pulawy, Poland</td>
			<td>cat.# not avaliable</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4 (potassium dihydrogen phosphate) 99.995% anhydrous basis</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>1.05108</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Centrifuge</td>
			<td>Heraeus Megafuge 1.0R, Thermo Scientific, Germany</td>
			<td>B00013899</td>
			<td>speed to 300 x g, with cooling to 4 0C</td>
		</tr>
		<tr>
			<td>Laboratory Centrifuge</td>
			<td>Heraeus Fresco 21, Thermo Scientific, Germany</td>
			<td>75002425</td>
			<td>speed to 3,000 x g, with cooling to 4 0C</td>
		</tr>
		<tr>
			<td>Mask (FFP2)</td>
			<td>VWR, Radnor, Pennsylvania, United States</td>
			<td>111-0917</td>
			<td>for working with ortho-dianisine dihydrochloride</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Breeding unit of the Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College, Krakow, Poland</td>
			<td>CBA/J, C57BL/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Micrometer</td>
			<td>Mitutoyo, Tokyo, Japan</td>
			<td>193-111</td>
			<td>digit Outside Micrometer, Ratchet Stop, 0-25mm Range, 0.001mm Graduation, +/-0.002mm Accuracy, https://shop.mitutoyo.pl/web/mitutoyo/pl_PL/all/all/Mikrometr%20analogowy%20/PR/193-111/index.xhtml&#160;&#160;</td>
		</tr>
		<tr>
			<td>microplate, 96 well, microlon, high binding (for ELISA test)</td>
			<td>Greiner Bio-One GmbH, Kremsmunster, Austria</td>
			<td>655061</td>
			<td>with maxi-sorp binding surfaces for reliable and reproducible results in colormetric assays</td>
		</tr>
		<tr>
			<td>Microscope with objectives</td>
			<td>Leica Microsystems CMS GmbH, Germany</td>
			<td>DM1000, 294011-082007</td>
			<td>histology presented in the paper was performed under ThermoFisher Scientific EVOS M5000 Imaging System, with objectives: FL 20X LWDPH, 0.40NA/3.1WD and FL 40X LWDPH 0.65NA/1.79WD</td>
		</tr>
		<tr>
			<td>Myeloperoxidase from human leukocytes (MPO standard)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>M6908</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 x 7 H2O (sodium phosphate dibasic heptahydrate)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>S9390</td>
			<td></td>
		</tr>
		<tr>
			<td>Olive-oil</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>75343</td>
			<td>pure, natural</td>
		</tr>
		<tr>
			<td>OptEIA Mouse IFN-&#947; ELISA Set</td>
			<td>Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA</td>
			<td>555138</td>
			<td></td>
		</tr>
		<tr>
			<td>Ortho-dianisine dihydrochloride</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>D3252</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>76242</td>
			<td>beads, waxy solid</td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline)</td>
			<td>ThermoFisher Scientific,&#160; Waltham, Massachusetts, USA</td>
			<td>20012027</td>
			<td>pH 7.2, mammalian cell culture</td>
		</tr>
		<tr>
			<td>ph meter</td>
			<td>Elmetron, Poland</td>
			<td>CP-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes, variable volume with tips</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>EP3123000900-1EA</td>
			<td>3-pack, Option 1, 0.5-10 uL/10-100 uL/100-1000 uL, includes epT.I.P.S.</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>VWR, Radnor, Pennsylvania, United States</td>
			<td>PERS94-0462</td>
			<td>scraper and cutter blades, single edge, aluminium spine, 100 blades per box, individually wrapped</td>
		</tr>
		<tr>
			<td>Rotary microtome</td>
			<td>MRC Laboratory-Instruments, Essex, CM20 2HU UK</td>
			<td>HIS-202A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors - straight, sharp / sharp</td>
			<td>Animalab, Poznan, Poland</td>
			<td>14060-10FST</td>
			<td>Surgical instruments for procedures on mice (should be steriled)</td>
		</tr>
		<tr>
			<td>Screw cap (open top)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>27056</td>
			<td>black polypropylene hole cap, for use with 22&#160;mL vial with 20-400 thread</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BioTek Instruments, U.S.A</td>
			<td>201446</td>
			<td>universal microplate spectrophotometer: &#955; range: 200 - 999 nm, absorbance measurement range: 0.000 - 4.000 Abs</td>
		</tr>
		<tr>
			<td>Staining dish 20 slides with rack</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>S6141</td>
			<td>e.g. 20 slide staining dishes complete with covers, slide rack and handle</td>
		</tr>
		<tr>
			<td>Sterile Disposable Biopsy Punch 6mm</td>
			<td>Integra LifeSciences, Princeton, NJ, USA</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Animalab, Poznan, Poland</td>
			<td>52138-46</td>
			<td>surgical instruments for procedures on mice (should be steriled)</td>
		</tr>
		<tr>
			<td>Tissue processing cassettes</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>Z672122</td>
			<td>tissue processing/ embedding cassettes with lid</td>
		</tr>
		<tr>
			<td>TMB Substrate Reagent Set</td>
			<td>Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA</td>
			<td>555214</td>
			<td></td>
		</tr>
		<tr>
			<td>TNCB (2,4,6-trinitrochlorobenzene)</td>
			<td>Tokyo Chemical Industry CO., LTD, Japan</td>
			<td>C0307</td>
			<td></td>
		</tr>
		<tr>
			<td>TNP-BSA (bovine serum albumin conjugated with 2,4,6-trinitrophenyl)</td>
			<td>Biosearch Technologies LGC, Petaluma, CA, USA</td>
			<td>T-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>T-PER (tissue protein extration reagent)</td>
			<td>ThermoFisher Scientific, Waltham, Massachusetts, USA</td>
			<td>78510</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 15 mL sterile</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>CLS430055 (Corning)</td>
			<td>polypropylene, conical bottom</td>
		</tr>
		<tr>
			<td>Tubes 50 mL, sterile</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>CLS430290 (Corning)</td>
			<td>polypropylene, conical bottom</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Vials, screw top, clear glass (vial only) 22 mL</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>27173</td>
			<td>for the preparation of hapten, screwed on so that it does not evaporate</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>AJL Electronic, Poland</td>
			<td>LW102</td>
			<td></td>
		</tr>
		<tr>
			<td>Wax (paraffin) dispenser</td>
			<td>VWR, Radnor, Pennsylvania, United States</td>
			<td>114-8737</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (xylapan 20 mg/mL) solution for injection</td>
			<td>Vetoquinol Biowet Sp. z o.o., Gorzow Wielkopolski, Poland</td>
			<td>cat.# not avaliable</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene (histological grade)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

contact hypersensitivity as a murine model of allergic contact dermatitis

La hipersensibilidad de contacto (CHS) es un modelo experimental de dermatitis alérgica de contacto (ACD) que se puede estudiar en ratones. Este estudio tiene como objetivo presentar un método de laboratorio objetivo que pueda ayudar a estudiar la reacción chS en ratones, que puede medirse y cuantificarse mediante diversas pruebas. Para inducir CHS, en el día "0", los ratones fueron sensibilizados en un punto previamente afeitado por pintura de piel abdominal con el hapteno 2,4,6-trinitroclorobenceno (TNCB) en una mezcla de acetona-etanol, mientras que los ratones de control negativo fueron sensibilizados simuladamente con la mezcla de vehículo solo de acetona-etanol. En el día "4", el grosor basal del oído se midió con un micrómetro antes de la elicitación de CHS (desafío) pintando ambos oídos con TNCB diluido tanto en el grupo de prueba como en el de control. Después de 24 h, la hinchazón del oído se midió con un micrómetro. CHS es un ejemplo de una respuesta inmune mediada por células T que causa hinchazón en el tejido inflamado, alcanzando su punto máximo 24 horas después del desafío de la piel con el mismo hapteno. Un aumento en el edema del oído se correlacionó con el aumento del peso del oído, la actividad de la mieloperoxidasa (MPO), la concentración de citoquinas proinflamatorias en los extractos de oído, el aumento del engrosamiento de la dermis edematosa en el examen histológico y la permeabilidad vascular del oído. También hubo un aumento en la concentración de anticuerpos IgG1 específicos de TNP en los sueros del grupo de prueba en comparación con los ratones de control. Además, el CHS se puede transferir con éxito con las células efectoras de CHS obtenidas de donantes previamente sensibilizados con TNCB. Las células efectoras de CHS se administraron por vía intravenosa en ratones receptores ingenuos, que posteriormente fueron desafiados con el mismo hapteno diluido. La hinchazón del oído se midió con un micrómetro 24 h más tarde.

Para la inducción de CHS, los animales fueron sensibilizados mediante pintura cutánea (abdominal) con 150 μL de TNCB al 5% o sensibilizados simuladamente con el vehículo solo. En el día "4", las respuestas de hinchazón del oído de ambos oídos fueron inducidas por pintura de contacto (desafío) con 10 μL de TNCB al 0,4% en ambos ratones previamente sensibilizados en contacto con TNCB (grupo de prueba) y en ratones del grupo control (sensibilizados simuladamente). Los datos presentados muestran que los ra...

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Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

La hipersensibilidad de contacto (CHS) es un modelo experimental murino de dermatitis alérgica de contacto (ACD). El CHS se basa en la sensibilización con hapteno reactivo mediante la pintura de la piel afeitada del pecho y el abdomen, con un posterior desafío de la piel del oído con un hapteno diluido, causando una reacción de hinchazón que se evalúa de varias maneras.

Hipersensibilidad de contacto como modelo murino de dermatitis alérgica de contacto

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...

Un método de laboratorio objetivo presentado aquí puede ayudar a estudiar la reacción de hipersensibilidad de contacto en ratones, que imita la dermatitis alérgica de contacto en humanos. La reacción de hipersensibilidad de contacto se puede medir no solo por edema de oído, sino también por diversas pruebas de laboratorio que permiten el estudio de los mecanismos celulares involucrados en esta reacción. El modelo de hipersensibilidad más en contacto puede probar varios factores ambientales y nuevas sustancias para mostrar si los factores probados modulan las respuestas inmunes dependientes de células T, que pueden usarse para implementar nuevas terapias.Comience afeitando la piel del ratón con una hoja de afeitar y etiquetando la pata. Seis horas antes de la inducción de la hipersensibilidad de contacto, o CHS, aplique jabón gris con agua y afeite un área de dos centímetros cuadrados en el pecho y el abdomen del ratón. El mismo día, sensibilizar a los ratones aplicando 150 microlitros de 5% recién preparado en el lugar previamente afeitado.En el ratón de control, aplique únicamente el vehículo. Seque el punto de hapteno durante 30 segundos antes de volver a colocar al animal en la jaula. En el cuarto día, medir el grosor de la oreja de un ratón anestesiado usando un micrómetro a la hora cero por un observador que desconoce los grupos experimentales.Luego, aplique 10 microlitros de 04% hapteno recién preparado en ambos lados de las orejas en grupos de prueba y control y deje que se seque durante 30 segundos. En el día cinco, 24 horas después de la aplicación anterior de 0,4% de hapteno, repita la medición del grosor de la oreja para la medición de 24 horas. 24 horas después de la medición del grosor de la oreja, cuando el ratón todavía esté en anestesia profunda, corte las orejas lo más cerca posible del cráneo con tijeras.En el lado distal de las orejas, haga un punzón de seis milímetros de diámetro con un punzón de biopsia. Mida el peso de cada biopsia de oído en la balanza analítica y explíquelo en miligramos. Para el ensayo de mieloperoxidasa, o MPO, coloque las biopsias de oído en un tubo de microcentrífuga de dos mililitros con perlas de acero inoxidable y agregue 500 microlitros del tampón preparado.Homogeneizar las biopsias durante 10 minutos en un homogeneizador y luego enfriar la muestra durante 15 minutos a 4 grados centígrados antes de homogeneizarla nuevamente durante 10 minutos. Saque las perlas una vez que las biopsias estén homogeneizadas. Congele los homogeneizados a menos 20 grados centígrados durante 30 minutos.Descongele y vortex las muestras, y repita el procedimiento de homogeneización y congelación tres veces. Una vez hecho esto, centrifugar el homogeneizado a 3, 000 G durante 30 minutos a 4 grados centígrados. Coseche el sobrenadante con una pipeta para medir la actividad MPO y explíquelo en unidades por miligramo de proteína.Para realizar una prueba de permeabilidad vascular, sensibilizar a los ratones en el día cero y en el día cuatro, desafie directamente aplicando hapteno en las orejas utilizando el procedimiento mencionado anteriormente. 23 horas más tarde, inyecte por vía intravenosa 8,3 microlitros por gramo de peso corporal de colorante azul Evans al 1% en solución salina tamponada con fosfato de Dulbecco, o DPBS, al ratón anestesiado. Después de una hora, vuelva a anestesiar al ratón para recoger las biopsias de oído, como se describió anteriormente.Para extraer el tinte del tejido, incubar las biopsias en los tubos que contienen un mililitro de formamida a 37 grados centígrados en una atmósfera de 5% de dióxido de carbono durante 18 horas. Después de recolectar biopsias de oído, cuando el ratón todavía esté bajo anestesia profunda, retire el globo ocular con pinzas. Aplique una presión suave sobre el ratón y recoja sangre del seno retroorbitario en el tubo para la recolección de suero.Invierta el tubo seis veces y espere 30 minutos para que la sangre se coagule. Luego, centrifugar el tubo a 1, 300 a 2, 000 G durante 10 minutos a temperatura ambiente. Sensibilizar a los ratones donantes con TNCB hapteno en el día cero utilizando el procedimiento mencionado anteriormente En el cuarto día, después de desinfectar la piel, aislar los ganglios linfáticos axilares e inguinales, o ALN, del ratón anestesiado con fórceps, seguido de aislar el bazo.Triture el tejido entre los extremos esmerilados de dos portaobjetos de microscopio y pase la suspensión celular a través de un colador celular con un tamaño de poro de 70 micrómetros. Luego, lave las células con DPBS suplementado con suero bovino fetal al 1% y centrifugarlas a 300 G durante 10 minutos a cuatro grados centígrados. Decantar el sobrenadante y resuspender el pellet celular restante en uno a cinco mililitros de DPBS.Prepare una mezcla 1: 1 de ALN y bazo para lograr una concentración de hasta 7.0 veces 10 a la séptima célula en 200 microlitros de DBPS antes de inyectar por vía intravenosa la mezcla de células efectoras de CHS en un ratón anestesiado. Mide el grosor de la oreja a la hora cero y después de 24 horas del desafío. Los datos mostraron que los ratones sensibilizados a TNCB y desafiados en los oídos cuatro días después desarrollaron un aumento significativo de la hinchazón del oído, en comparación con los ratones de control sensibilizados simuladamente y luego desafiados de manera similar.El aumento del edema del oído en el grupo de prueba resultó en un aumento del peso del oído, la actividad de MPO, la concentración de interferón gamma en los extractos del oído, cambios edematosos en la dermis en el examen histológico y permeabilidad vascular del oído Los anticuerpos IgG1 específicos de TNP se elevaron en los sueros de los ratones de prueba en comparación con los animales de control. Los animales que recibieron las células efectoras de CHS de donantes previamente sensibilizados con TNCB mostraron un aumento significativo de la hinchazón del oído, en comparación con los animales que no recibieron las células. El momento más crítico es desencadenar la hipersensibilidad de contacto.La solución de Hapten es muy volátil y sensible a la luz, por lo que debe aplicarse rápidamente a la piel de los animales. Se pueden realizar pruebas adicionales en las células efectoras aisladas. Por ejemplo, se pueden establecer cultivos celulares para evaluar la capacidad de proliferar en presencia de antígenos.

contact-hypersensitivity-as-murine-model-allergic-contact

Research

JoVE Journal

Immunology and Infection

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

Reconocimiento de la transglutaminasa epidérmica por IgA y anticuerpos transglutaminasa tisular 2 en un raro caso de Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

Investigación

Inmunología e Infección

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Test de activación de basófilos para la investigación de mecanismos IgE mediados de Hipersensibilidad a las Drogas

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

La inducción de la enfermedad de injerto contra huésped y<em&gt; En Vivo</em&gt; T Monitoreo Celular Utilizando un modelo murino MHC de concordancia

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Medida de la alodinia táctil en un modelo murino de la prostatitis bacteriana

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

El uso de<em&gt; Galleria mellonella</em&gt; Como organismo modelo para estudiar<em&gt; Legionella pneumophila</em&gt; Infección

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Organoides como Modelo para Enfermedades Infecciosas: Cultura de Derechos Humanos y organoides estomacales murinos y microinyección de Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

El pez cebra como modelo para evaluar el potencial teratogénico de nitrito

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonización vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Efectos de la exposición de formaldehído a un modelo de rata de la Dermatitis atópica inducida por el tratamiento Neonatal de capsaicina

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...