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

Bu çalışma subvention SUBZ tarafından desteklenmiştir. Polonya, Wroclaw'daki Tıp Üniversitesi'nin A020.22.060 ve Bilim ve Yüksek Öğretim Bakanlığı'ndan N N401 545940'tan MS'ye ve IP2012 0443 72'den MMS'ye hibelerle.

Bu makalede sunulan tüm deneyler, Krakow'daki 1. Hayvan Deneyleri Yerel Etik Komitesi'nin yönergelerine göre yürütülmüştür. Açıklanan tüm prosedürler, özellikle ketamin / ksilazinin anestezik olarak kullanılması, madde / hapten uygulamak için kulakların her iki tarafının kullanılması, kulağın kesilmesi ve göz küresinin çıkarılmasıyla kan toplanması ile ilgili olarak yerel önerilere göre gerçekleştirilmiştir. Bu çalışmada 6-12 haftalık BALB/c (haplotip H-2d), CBA/J (H-2<s...

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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. 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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>

CHS, derideki kendi kendine protein antijenlerine bağlanan ve neoantijenler yaratan haptenler yoluyla indüklenir. CHS, dolaşımdaki antijene özgü CHS-efektör T hücrelerinin lokal ekstravasküler alımı ile aracılık eder, bu da meydan okunan dokuda şişmeye neden olur ve sekonder cildin aynı hapten6'ya maruz kalmasından 24 saat sonra zirveye ulaşır. Dokunun şişmesi esas olarak lökositlerin infiltrasyonundan ve lökosit bağımlı fibrin birikiminden kaynaklanır<sup class...

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

Alerjik kontakt dermatit (AKB), sanayileşmiş ülkelerde, hapten adı verilen düşük moleküler ağırlıklı kimyasallara maruz kalmaktan kaynaklanan tip IV aşırı duyarlılık reaksiyonunun neden olduğu yaygın bir deri enflamatuar hastalığıdır. İnsanlarda temas duyarlılaşmasına neden olan maddeler arasında ağır metal iyonları (krom, nikel, demir, kobalt), terebentin, kokular, boyalar ve kozmetiklerde bulunan koruyucular (örneğin, p-fenilendiamin), bazı ilaçlar (örneğin, neomisin, benzokain), β-laktam antibiyotikler (yani penisilin), bitkiler tarafından üretilen kimyasallar (pentadekakatekol, zehirli sarmaşıklarda bulunan bir madde) ve fotoğraf endüstrisinde kullanılan hidrokinonbulunur 1,2 . ACD etiyolojik ajanları çok yüksektir, çünkü sadece endüstride 100.000'den fazla kimyasal kullanılmaktadır ve her yıl 2.000 yenisi sentezlenmektedir. Bugüne kadar, temas haptenleri / alerjenleri olabilecek 3.700'den fazla molekül tanımlanmıştır3. Temas aşırı duyarlılık reaksiyonu (CHS), farelerde, kobaylarda ve sıçanlarda çalışılabilen deneysel bir ACD modelidir ve organik çözücülerde çözünmüş reaktif kimyasal haptenlerin topikal cilt uygulaması ile indüklenebilir 4,5,6. Bu çalışma, farelerde CHS reaksiyonunu incelemeye yardımcı olabilecek ve çeşitli testlerle ölçülebilen ve ölçülebilen objektif bir laboratuvar yöntemini tanımlamayı amaçlamaktadır.
CHS, sensitizasyon (indüksiyon) ve efektör (meydan okuma) aşamalarından oluşur. Hayvan modellerinde, haptenler ilk önce neoantijenler oluşturmak için vücuttaki proteinlere kovalent olarak bağlanır. Sensitizasyon aşamasında, aktive keratinositler, pro-inflamatuar sitokinler-tümör nekroz faktör α (TNF-α) ve interlökin 1β (IL-1β)7 üreterek deri dendritik hücrelerinin (sDC'ler) göçünü ve olgunlaşmasını teşvik eder. Epidermal Langerhans hücreleri (LC'ler) CHS indüksiyonu ve efektör fazları8 sırasında antijenler sunar. Duyarlılaşma sırasında hapten'e maruz kalan LC'ler hem düzenleyici hem de efektör hücrelerin indüksiyonunu teşvik eder9. Birkaç çalışmadan elde edilen kanıtların artması, CHS yanıtlarının CD4 + MHC sınıf II ile kısıtlanmış Th1 hücreleri tarafından aracılık edilebildiğini, karakteristik bir enflamatuar infiltrasyon kullanmak için lokal olarak interferon-γ (IFN-γ), IFN-γ serbest bırakabilen, ancak çoğunlukla keratinositlere sitotoksik hasara aracılık edebilen CD8 + MHC sınıf I-kısıtlı Tc1 lenfositleri ve şimdi de interlökin 17 (IL-17) üreten Th17 hücreleri10, 11.
Çeşitli türler 12,13,14 ve haptenleri kullanan birkaç farklı CHS modeli geliştirilmiştir (farklı haptenlerin, çözücülerin ve uygulama zamanlarının ayrıntılı bir karşılaştırması Tablo 1'de özetlenmiştir). Sık kullanılan bir laboratuvar türü olan fare, CHS'yi incelemede birkaç avantaj sunar. Fareler arasında diğer türlere kıyasla daha fazla suş, nakavt (KO) ve transgenik hayvan vardır, bu da onları çok çekici bir hayvan yapar15. Ek olarak, CHS modeli birçok hayvan gerektirir ve fareler burada daha ekonomiktir. Hayvan modelleri ACD'yi her açıdan taklit etmez; özellikle, insanlar için yaygın olmayan kabuklanma ve deskuamasyon gösterirler16,17. Kronik hastalığın özelliklerinin çoğaltılması zordur, çünkü tarif edilen model haptenin uzun süre uygulanmasını üstlenmez. Bununla birlikte, burada ACD'nin önemli yönlerinin çoğunun çoğaltıldığı doğrulanmıştır. İnsanlarda olduğu gibi, bu özelliklerin lokal alerjik reaksiyonlarla ilişkili olduğu da gösterilmiştir. Bu protokolde özetlenen hapten seçimi, çözücüsü ve uygulaması, sonuçların çok sayıda in vitro testle doğrulanması ve mevcut versiyon kurulana kadar uzun yıllar laboratuvarda test edilmesi ve modifiye edilmesi gerçeğiyle belirlendi. Murin modelleri, ACD'nin gelişiminde rol oynayan ve yeni tedavilerin klinik öncesi değerlendirmeleri için gerekli olan hücre alt kümelerinin veya sitokinlerin analizine izin verir.

<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

Kontakt aşırı duyarlılık (CHS), farelerde incelenebilen deneysel bir alerjik kontakt dermatit (ACD) modelidir. Bu çalışma, farelerde CHS reaksiyonunu incelemeye yardımcı olabilecek, çeşitli testlerle ölçülebilen ve ölçülebilen objektif bir laboratuvar yöntemi sunmayı amaçlamaktadır. CHS'yi indüklemek için, "0" gününde, fareler bir aseton-etanol karışımında hapten 2,4,6-trinitroklorobenzen (TNCB) ile abdominal cilt boyaması ile daha önce traş edilmiş bir noktaya duyarlılaştırılırken, negatif kontrol fareleri tek başına araç-aseton-etanol karışımı ile düzmece duyarlılaştırıldı. "4" gününde, temel kulak kalınlığı, CHS'nin (meydan okuma) ortaya çıkmasından önce, her iki kulağın hem test hem de kontrol gruplarında seyreltilmiş TNCB ile boyanmasıyla bir mikrometre ile ölçüldü. 24 saat sonra, kulak şişmesi bir mikrometre ile ölçüldü. CHS, iltihaplı dokuda şişmeye neden olan ve aynı hapten ile cilt mücadelesinden 24 saat sonra zirveye ulaşan T hücresi aracılı bir bağışıklık tepkisinin bir örneğidir. Kulak ödeminde artış, artmış kulak ağırlığı, miyeloperoksidaz (MPO) aktivitesi, kulak ekstrelerinde pro-inflamatuar sitokin konsantrasyonu, histolojik incelemede ödemli dermisin kalınlaşmasının artması ve kulak vasküler geçirgenliği ile korelasyon gösterdi. Test grubunun serumlarında TNP-spesifik IgG1 antikorlarının konsantrasyonunda, kontrol fareleri ile karşılaştırıldığında bir artış vardı. Ek olarak, CHS, daha önce TNCB ile duyarlılaştırılmış donörlerden elde edilen CHS-efektör hücrelerle başarılı bir şekilde aktarılabilir. CHS-efektör hücreler, daha sonra aynı seyreltilmiş hapten ile zorlanan naif alıcı farelere intravenöz olarak uygulandı. Kulak şişmesi 24 saat sonra mikrometre ile ölçüldü.

CHS indüksiyonu için, hayvanlar cilt boyama (karın) yoluyla 150 μL% 5 TNCB ile hassaslaştırıldı veya sadece araçla hassaslaştırıldı. "4" gününde, her iki kulağın kulak şişmesi tepkileri, daha önce TNCB (test grubu) ve kontrol grubu fareleri (sahte duyarlılaştırılmış) ile daha önce temas halinde olan her iki farede de% 0.4 TNCB'nin 10 μL'si ile temas boyama (meydan okuma) ile indüklendi. Sunulan veriler, TNCB ile duyarlılaşan ve 4 gün sonra meydan okuyan farelerin, benzer şekilde ...

Watch this Scientific Journal Video about Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis at JoVE.com

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Kontakt aşırı duyarlılık (CHS), alerjik kontakt dermatitin (AKB) murin deneysel bir modelidir. CHS, göğüs ve karın bölgesinin traş edilmiş cildini boyayarak reaktif hapten ile hassaslaşmaya dayanır, daha sonra seyreltilmiş bir hapten ile kulak derisi meydan okuması ile çeşitli şekillerde değerlendirilen bir şişlik reaksiyonuna neden olur.

Alerjik Kontakt Dermatitin Murin Modeli Olarak Kontakt Aşırı Duyarlılık

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

Burada sunulan objektif bir laboratuvar yöntemi, insanlarda alerjik kontakt dermatiti taklit eden farelerde kontakt aşırı duyarlılık reaksiyonunun incelenmesine yardımcı olabilir. Kontakt aşırı duyarlılık reaksiyonu sadece kulak ödemi ile değil, aynı zamanda bu reaksiyonda yer alan hücresel mekanizmaların incelenmesine izin veren çeşitli laboratuvar testleri ile de ölçülebilir. Daha fazla temas içi aşırı duyarlılık modeli, test edilen faktörlerin yeni tedavileri uygulamak için kullanılabilecek T hücresine bağımlı bağışıklık tepkilerini modüle edip etmediğini göstermek için çeşitli ve çevresel faktörleri ve yeni maddeleri test edebilir.Fare derisini bir tıraş bıçağı ile tıraş ederek ve pençeyi etiketleyerek başlayın. Kontakt aşırı duyarlılığın veya CHS'nin indüksiyonundan altı saat önce, gri sabunu suyla uygulayın ve farenin göğsünde ve karnında iki santimetre karelik bir alanı tıraş edin. Aynı gün, daha önce tıraş edilen noktaya 150 mikrolitre taze hazırlanmış% 5hapten uygulayarak fareleri hassaslaştırın.Kontrol faresinde, sadece aracı uygulayın. Hayvanı tekrar kafese koymadan önce hapten noktasını 30 saniye kurutun. Dördüncü günde, anestezi uygulanan bir farenin kulak kalınlığını, deney gruplarından habersiz bir gözlemci tarafından sıfır saatte bir mikrometre kullanarak ölçün.Daha sonra test ve kontrol gruplarında kulakların her iki tarafına taze hazırlanmış %04 hapten 10 mikrolitre uygulayın ve 30 saniye kurumasını bekleyin. Beşinci günde, önceki %0,4 hapten uygulamasından 24 saat sonra, 24 saatlik ölçüm için kulak kalınlığı ölçümünü tekrarlayın. Kulak kalınlığı ölçümünden 24 saat sonra, fare hala derin anestezi altındayken, makas kullanarak kulakları kafatasına mümkün olduğunca yakın kesin.Kulakların distal tarafında, biyopsi zımba kullanarak altı milimetre çapında bir yumruk yapın. Her kulak biyopsisinin ağırlığını analitik terazide ölçün ve miligram cinsinden ifade edin. Miyeloperoksidaz veya MPO için, tahlil olarak, kulak biyopsilerini paslanmaz çelik boncuklu iki mililitrelik bir mikrosantrifüj tüpüne koyun ve hazırlanan tamponun 500 mikrolitresini ekleyin.Biyopsileri bir homojenizatörde 10 dakika homojenize edin ve ardından numuneyi 10 dakika boyunca tekrar homojenize etmeden önce 4 santigrat derecede 15 dakika soğutun. Biyopsiler homojenize edildikten sonra boncukları çıkarın. Homojenatları eksi 20 santigrat derecede 30 dakika dondurun.Numuneleri çözdürün ve vorteksleyin ve homojenizasyon ve dondurma prosedürünü üç kez tekrarlayın. Tamamlandığında, homojenatı 3.000 G'de 4 santigrat derecede 30 dakika boyunca santrifüj edin. MPO aktivitesini ölçmek için süpernatantı bir pipetle hasat edin ve bir miligram protein başına birimler halinde ifade edin.Bir vasküler geçirgenlik testi yapmak için, sıfır gününde fareleri hassaslaştırın ve dördüncü günde, daha önce belirtilen prosedürü kullanarak kulaklara hapten uygulayarak doğrudan meydan okuyun. 23 saat sonra, anestezi uygulanan fareye intravenöz olarak Dulbecco'nun fosfat tamponlu salininde veya DPBS'de% 1 Evans mavi boyasının gram vücut ağırlığı başına 8.3 mikrolitre enjekte edin. Bir saat sonra, daha önce açıklandığı gibi, kulak biyopsilerini toplamak için fareyi tekrar uyuşturun.Boyayı dokudan çıkarmak için, biyopsileri 18 saat boyunca% 5 karbondioksit atmosferinde 37 santigrat derecede bir mililitre formamid içeren tüplerde inkübe edin. Kulak biyopsilerini topladıktan sonra, fare hala derin anestezi altındayken, göz küresini cımbızla çıkarın. Fareye hafif bir baskı uygulayın ve serum toplanması için retro-orbital sinüsten tüpe kan toplayın.Tüpü altı kez ters çevirin ve kanın pıhtılaşması için 30 dakika bekleyin. Ardından, tüpü oda sıcaklığında 10 dakika boyunca 1.300 ila 2.000 G'de santrifüj edin. Donör fareleri daha önce bahsedilen prosedürü kullanarak sıfır gününde TNCB hapten ile hassaslaştırın Dördüncü günde, cildi dezenfekte ettikten sonra, aksiller ve inguinal lenf düğümlerini veya ALN'leri forseps kullanarak anestezi uygulanan fareden izole edin, ardından dalağı izole edin.İki mikroskop slaytının buzlu uçları arasındaki dokuyu ezin ve hücre süspansiyonunu 70 mikrometre gözenek boyutuna sahip bir hücre süzgecinden geçirin. Daha sonra, hücreleri% 1 fetal sığır serumu ile desteklenmiş DPBS ile yıkayın ve dört santigrat derecede 10 dakika boyunca 300 G'de santrifüj edin. Süpernatantı boşaltın ve kalan hücre peletini bir ila beş mililitre DPBS içinde yeniden askıya alın.CHS-efektör hücrelerin karışımını anestezi uygulanmış bir fareye intravenöz olarak enjekte etmeden önce, 200 mikrolitre DBPS'deki yedinci hücrelere 7.0 kat 10'a kadar bir konsantrasyon elde etmek için 1: 1'lik bir ALN ve dalak karışımı hazırlayın. Kulak kalınlığını sıfır saatte ve meydan okumadan 24 saat sonra ölçün. Veriler, TNCB'ye duyarlı hale gelen ve dört gün sonra kulaklara meydan okuyan farelerin, sahte duyarlı ve daha sonra benzer şekilde zorlanan kontrol farelerine kıyasla, önemli ölçüde artmış kulak şişmesi geliştirdiğini gösterdi.Test grubunda kulak ödemindeki artış, kulak ağırlığının artmasına, MPO aktivitesine, kulak ekstraktlarında interferon gama konsantrasyonuna, histolojik incelemede ödemli dermis değişikliklerine ve kulak vasküler geçirgenliğine neden oldu TNP-spesifik IgG1 antikorları, kontrol hayvanlarıyla karşılaştırıldığında test farelerinin serumlarında yükselmiştir. CHS-efektör hücrelerini daha önce TNCB ile hassaslaştırılmış donörlerden alan hayvanlar, hücreleri almayan hayvanlara kıyasla önemli ölçüde artmış kulak şişmesi göstermiştir. En kritik an, temas aşırı duyarlılığını tetiklemektir.Hapten çözeltisi çok uçucu ve ışığa duyarlıdır, bu nedenle hayvanların derisine hızlı bir şekilde uygulanmalıdır. İzole efektör hücreler üzerinde ek testler yapılabilir. Örneğin, antijenlerin varlığında çoğalma yeteneğini değerlendirmek için hücre kültürleri kurulabilir.

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.

Nadir bir Case IgA ve Doku Transglutaminaz 2 Antikorlar Epidermal Transglutaminaz tanınması Rhesus Dermatit

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

İmmünoloji ve Enfeksiyon

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.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

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.

İlaç Aşırı duyarlılık IgE aracılığı ile Mekanizmalarının Araştırılması bazofil Etkinleştirme Test

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.

Graft-versus-host hastalığı indüksiyonu ve<em&gt; In Vivo</emBir MHC-eşlemeli murin Model Kullanma&gt; T Hücre İzleme

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.

Bakteriyel Prostatit bir fare modelinde Dokunsal allodini Ölçümü

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.

Kullanımı<em&gt; Galleria mellonella</em&gt; Bir Model Organizma olarak Eğitim için<em&gt; Legionella pneumophila</em&gt; Enfeksiyon

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.

Enfeksiyon Hastalıkları Modeli Olarak Organoids: Helicobacter Pylori ve İnsan Kültür ve kemirgen Mide Organoids ve Mikroenjeksiyon

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.

Bir Model Olarak Zebra balığı Nitrit teratojenik potansiyelini değerlendirmek için

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.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

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.

Formaldehit maruz bir fare modeli Atopik dermatit yenidoğan kapsaisin tedavi tarafından indüklenen etkileri

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