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

تم دعم هذه الدراسة من خلال الإعانة SUBZ. A020.22.060 من الجامعة الطبية في فروتسواف ، بولندا ، ومن خلال منح من وزارة العلوم والتعليم العالي N N401 545940 إلى MS و IP2012 0443 72 إلى MMS.

أجريت جميع التجارب المقدمة في هذه المقالة وفقا للمبادئ التوجيهية للجنة الأخلاقية المحلية 1st للتجارب على الحيوانات في كراكوف. تم تنفيذ جميع الإجراءات الموصوفة وفقا للتوصيات المحلية ، خاصة فيما يتعلق باستخدام الكيتامين / الزيلازين كمخدر ، واستخدام جانبي الأذنين لتطبيق المادة / hapten ، وقطع ا...

<ol>
	<li>Nosbaum, A., Vocanson, M., Rozieres, A., Hennino, A., Nicolas, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+and+irritant+contact+dermatitis.">Allergic and irritant contact dermatitis.</a> European Journal of Dermatology. 19 (4), 325-332 (2009).</li><li>Hertl, 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=Predominance+of+epidermal+CD8++T+lymphocytes+in+bullous+cutaneous+reactions+caused+by+beta-lactam+antibiotics.">Predominance of epidermal CD8+ T lymphocytes in bullous cutaneous reactions caused by beta-lactam antibiotics.</a> The Journal of Investigative Dermatology. 101 (6), 794-799 (1993).</li><li>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=T+lymphocyte-mediated+immune+responses+to+chemical+haptens+and+metal+ions:+implications+for+allergic+and+autoimmune+disease.">T lymphocyte-mediated immune responses to chemical haptens and metal ions: implications for allergic and autoimmune disease.</a> International Archives of Allergy and Immunology. 134 (3), 186-198 (2004).</li><li>Takeyoshi, M., Iida, K., Suzuki, K., Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+sensitization+potency+of+isoeugenol+and+its+dimers+evaluated+by+a+non-radioisotopic+modification+of+the+local+lymph+node+assay+and+guinea+pig+maximization+test.">Skin sensitization potency of isoeugenol and its dimers evaluated by a non-radioisotopic modification of the local lymph node assay and guinea pig maximization test.</a> Journal of Applied Toxicology. 28 (4), 530-534 (2008).</li><li>Nakamura, K., Aizawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+genetic+control+of+picryl+chloride+contact+hypersensitivity+reaction+in+inbred+rats.">Studies on the genetic control of picryl chloride contact hypersensitivity reaction in inbred rats.</a> Transplantation Proceedings. 13 (2), 1400-1403 (1981).</li><li>Asherson, G. L., Ptak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+and+delayed+hypersensitivity+in+the+mouse.+I.+Active+sensitization+and+passive+transfer.">Contact and delayed hypersensitivity in the mouse. I. Active sensitization and passive transfer.</a> Immunology. 15 (3), 405-416 (1968).</li><li>Honda, T., Egawa, G., Grabbe, S., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+of+immune+events+in+the+murine+contact+hypersensitivity+model:+toward+the+understanding+of+allergic+contact+dermatitis.">Update of immune events in the murine contact hypersensitivity model: toward the understanding of allergic contact dermatitis.</a> The Journal of Investigative Dermatology. 133 (2), 303-315 (2013).</li><li>Kaplan, D. H., Jenison, M. C., Saeland, S., Shlomchik, W. D., Shlomchik, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+langerhans+cell-deficient+mice+develop+enhanced+contact+hypersensitivity.">Epidermal langerhans cell-deficient mice develop enhanced contact hypersensitivity.</a> Immunity. 23 (6), 611-620 (2005).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerin+expressing+cells+promote+skin+immune+responses+under+defined+conditions.">Langerin expressing cells promote skin immune responses under defined conditions.</a> Journal of Immunology. 180 (7), 4722-4727 (2008).</li><li>Wang, B., 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=CD4++Th1+and+CD8++type+1+cytotoxic+T+cells+both+play+a+crucial+role+in+the+full+development+of+contact+hypersensitivity.">CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity.</a> Journal of Immunology. 165 (12), 6783-6790 (2000).</li><li>Mori, 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=Cutaneous+hypersensitivities+to+hapten+are+controlled+by+IFN-gamma-upregulated+keratinocyte+Th1+chemokines+and+IFN-gamma-downregulated+langerhans+cell+Th2+chemokines.">Cutaneous hypersensitivities to hapten are controlled by IFN-gamma-upregulated keratinocyte Th1 chemokines and IFN-gamma-downregulated langerhans cell Th2 chemokines.</a> The Journal of Investigative Dermatology. 128 (7), 1719-1727 (2008).</li><li>Peszkowski, M. J., Warfvinge, G., Larsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+and+irritant+contact+responses+to+DNFB+in+BN+and+LEW+rat+strains+with+different+TH1/TH2+profiles.">Allergic and irritant contact responses to DNFB in BN and LEW rat strains with different TH1/TH2 profiles.</a> Acta Dermato-Venereologica. 74 (5), 371-374 (1994).</li><li>Henningsen, S. J., Mickell, J., Zachariae, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+kinins+in+dinitrochlorobenzene+contact+dermatitis+of+guinea-pigs.">Plasma kinins in dinitrochlorobenzene contact dermatitis of guinea-pigs.</a> Acta Allergologica. 25 (5), 327-331 (1970).</li><li>Maibach, H. I., Maguire, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elicitation+of+delayed+hypersensitivity+(DNCB+contact+dermatitis)+in+markedly+panleukopenic+guinea+pigs.">Elicitation of delayed hypersensitivity (DNCB contact dermatitis) in markedly panleukopenic guinea pigs.</a> The Journal of Investigative Dermatology. 41, 123-127 (1963).</li><li>Martel, B. C., Lovato, P., B&#228;umer, W., Olivry, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+animal+models+of+atopic+dermatitis+for+preclinical+studies.">Translational animal models of atopic dermatitis for preclinical studies.</a> The Yale Journal of Biology and Medicine. 90 (3), 389-402 (2017).</li><li>Jin, H., He, R., Oyoshi, M., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+atopic+dermatitis.">Animal models of atopic dermatitis.</a> The Journal of Investigative Dermatology. 129 (1), 31-40 (2009).</li><li>Li, Y. Z., Lu, X. Y., Jiang, W., Li, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+effect+of+qingpeng+ointment+in+atopic+dermatitis-like+murine+model.">Anti-inflammatory effect of qingpeng ointment in atopic dermatitis-like murine model.</a> Evidence-Based Complementary and Alternative. 2013, 907016 (2013).</li><li>Hoggatt, J., Hoggatt, A. F., Tate, T. A., Fortman, J., Pelus, L. 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>

يتم تحفيز CHS عن طريق haptens ، والتي ترتبط بمستضدات البروتين الذاتي في الجلد ، مما يخلق مستضدات جديدة. يتم التوسط في CHS من خلال التوظيف المحلي خارج الأوعية الدموية للخلايا التائية المستجيبة ل CHS المستجيبة للمستضد ، مما يؤدي إلى تورم في الأنسجة المتحدية ، ويبلغ ذروته بعد 24 ساعة من تعرض الجل?...

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

التهاب الجلد التماسي التحسسي (ACD) هو مرض التهابي جلدي شائع في البلدان الصناعية ناجم عن تفاعل فرط الحساسية من النوع الرابع الناتج عن التعرض لمواد كيميائية منخفضة الوزن الجزيئي تسمى haptens. تشمل المواد المسببة للتحسس التماسي لدى البشر أيونات المعادن الثقيلة (الكروم والنيكل والحديد والكوبالت) وزيت التربنتين والعطور والأصباغ والمواد الحافظة الموجودة في مستحضرات التجميل (على سبيل المثال ، p-phenylenediamine) ، وبعض الأدوية (مثل النيومايسين والبنزوكايين) والمضادات الحيوية β-lactam (أي البنسلين) والمواد الكيميائية التي تنتجها النباتات (pentadecacatechol ، وهي مادة موجودة في اللبلاب السام) ، وكذلك الهيدروكينون المستخدم في صناعة التصوير الفوتوغرافي 1,2 . العوامل المسببة ل ACD عالية جدا حيث يتم استخدام أكثر من 100000 مادة كيميائية في الصناعة وحدها ، ويتم تصنيع 2000 مادة كيميائية جديدة كل عام. حتى الآن ، تم تحديد أكثر من 3700 جزيء قد يكون ملامسا للهابتنز / المواد المسببة للحساسية3. تفاعل فرط الحساسية التماسية (CHS) هو نموذج تجريبي ل ACD يمكن دراسته في الفئران والخنازير الغينية والجرذان ويمكن أن يسببه التطبيق الجلدي الموضعي للهابتنز الكيميائي التفاعلي المذاب في المذيبات العضوية 4,5,6. تهدف هذه الدراسة إلى وصف طريقة مختبرية موضوعية قد تساعد في دراسة تفاعل CHS في الفئران ، والتي يمكن قياسها وتحديدها كميا من خلال اختبارات مختلفة.
يتكون CHS من مراحل التوعية (الحث) والمستجيب (التحدي). في النماذج الحيوانية ، ترتبط الهابتنز أولا تساهميا بالبروتينات في الجسم لإنشاء مستضدات جديدة. خلال مرحلة التحسس، تعزز الخلايا الكيراتينية المنشطة هجرة ونضج الخلايا المتغصنة الجلدية (sDCs) عن طريق إنتاج عامل نخر السيتوكينات المؤيد للالتهابات α (TNF-α) والإنترلوكين 1β (IL-1β)7. تقدم خلايا لانجرهانز الجلدية (LCs) مستضدات خلال مراحل الحث والمستجيب CHS8. LCs المعرضة للهابتن أثناء التحسس تعزز تحريض كل من الخلايا التنظيمية والمستجيبة9. تشير الأدلة المتزايدة من العديد من الدراسات إلى أن استجابات CHS يمكن أن تتوسط فيها إما خلايا Th1 المقيدة من الفئة الثانية CD4 + MHC ، والتي تطلق محليا γ الإنترفيرون (IFN-γ) لاستخدام تسلل التهابي مميز ، والخلايا الليمفاوية Tc1 المقيدة من الفئة الأولى CD8 + MHC التي يمكنها أيضا إطلاق γ IFN ولكن في الغالب توسط الضرر السام للخلايا للخلايا الكيراتينية ، والآن أيضا خلايا Th17 المنتجة للإنترلوكين 17 (IL-17)10 ، 11.
تم تطوير العديد من نماذج CHS المختلفة التي تستخدم أنواعا مختلفة 1 2,13,14 و haptens (يتم تلخيص مقارنة مفصلة بين مختلف haptens والمذيبات ووقت التطبيق في الجدول 1). يقدم الفأر ، وهو نوع مختبري يستخدم بشكل متكرر ، بعض المزايا في دراسة CHS. هناك المزيد من السلالات والضربات القاضية (KO) والحيوانات المعدلة وراثيا بين الفئران مقارنة بالأنواع الأخرى ، مما يجعلها حيوانا جذابا للغاية15. بالإضافة إلى ذلك ، يتطلب نموذج CHS العديد من الحيوانات ، والفئران أكثر اقتصادا هنا. النماذج الحيوانية لا تحاكي ACD في جميع الجوانب. على وجه الخصوص ، فإنها تظهر التقشر والتقشر ، وهو أمر غير شائع بالنسبة للبشر16,17. من الصعب إعادة إنتاج ميزات الأمراض المزمنة ، ويرجع ذلك أساسا إلى أن النموذج الموصوف لا يفترض تطبيق hapten لفترة طويلة من الزمن. ومع ذلك، فقد تأكد هنا أن العديد من الجوانب الهامة لحوار التعاون الآسيوي مستنسخة. وقد تبين أيضا أنه ، كما هو الحال في البشر ، ترتبط هذه الميزات بالحساسية المحلية. تم إملاء اختيار hapten ومذيبه وتطبيقه الموضح في هذا البروتوكول من خلال حقيقة أن النتائج قد تم تأكيدها من خلال العديد من الاختبارات في المختبر وأنه تم اختبارها وتعديلها في المختبر لسنوات عديدة حتى تم إنشاء الإصدار الحالي. تسمح نماذج المورين بتحليل المجموعات الفرعية للخلايا أو السيتوكينات التي تشارك في تطوير ACD وهي ضرورية للتقييمات قبل السريرية للعلاجات الجديدة.

<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

فرط الحساسية التماسية (CHS) هو نموذج تجريبي لالتهاب الجلد التماسي التحسسي (ACD) يمكن دراسته على الفئران. تهدف هذه الدراسة إلى تقديم طريقة مختبرية موضوعية قد تساعد في دراسة تفاعل CHS في الفئران ، والتي يمكن قياسها وتحديدها كميا من خلال اختبارات مختلفة. وللحث على CHS، في اليوم "0"، تم تحسس الفئران على بقعة كانت محلوقة سابقا عن طريق طلاء جلد البطن باستخدام الهابتن 2،4،6-trinitrochlorobenzene (TNCB) في خليط الأسيتون والإيثانول، في حين تم تحسس الفئران ذات التحكم السلبي بخليط الأسيتون والإيثانول وحده في السيارة. في اليوم "4" ، تم قياس سمك الأذن الأساسي بميكرومتر قبل استنباط CHS (التحدي) عن طريق طلاء كلتا الأذنين ب TNCB المخفف في كل من مجموعات الاختبار والتحكم. بعد 24 ساعة ، تم قياس تورم الأذن باستخدام ميكرومتر. CHS هو مثال على الاستجابة المناعية بوساطة الخلايا التائية التي تسبب تورما في الأنسجة الملتهبة ، وتبلغ ذروتها بعد 24 ساعة من تحدي الجلد بنفس الهابتن. ارتبطت الزيادة في وذمة الأذن بزيادة وزن الأذن ، ونشاط الميلوبيروكسيديز (MPO) ، وتركيز السيتوكين المؤيد للالتهابات في مستخلصات الأذن ، وزيادة سماكة الأدمة الوذمية في الفحص النسيجي ، ونفاذية الأوعية الدموية في الأذن. كانت هناك أيضا زيادة في تركيز الأجسام المضادة IgG1 الخاصة ب TNP في مصال مجموعة الاختبار عند مقارنتها بالفئران الضابطة. وبالإضافة إلى ذلك، يمكن نقل هذه المواد بنجاح باستخدام الخلايا المستجيبة ل CHS التي تم الحصول عليها من الجهات المانحة التي سبق توعيتها باستخدام TNCB. تم إعطاء الخلايا المستجيبة ل CHS عن طريق الوريد في الفئران المتلقية الساذجة ، والتي تم تحديها لاحقا بنفس الهابتن المخفف. تم قياس تورم الأذن مع ميكرومتر 24 ساعة في وقت لاحق.

بالنسبة لتحريض CHS ، تم تحسس الحيوانات عن طريق طلاء الجلد (البطن) مع 150 ميكرولتر من 5٪ TNCB أو تحسس زائف مع السيارة وحدها. في اليوم "4" ، تم تحفيز استجابات تورم الأذن لكلتا الأذنين عن طريق الطلاء بالاتصال (التحدي) مع 10 ميكرولتر من 0.4٪ TNCB في كل من الفئران التي كانت على اتصال سابق مع TNCB (مجموعة اخت...

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

فرط الحساسية التماسية (CHS) هو نموذج تجريبي للفئران لالتهاب الجلد التماسي التحسسي (ACD). يعتمد CHS على التحسس باستخدام hapten التفاعلي عن طريق طلاء الجلد الحليق للصدر والبطن ، مع تحدي جلد الأذن اللاحق باستخدام هابتن مخفف ، مما يسبب رد فعل تورم يتم تقييمه بطرق مختلفة.

فرط الحساسية التماسية كنموذج مورين لالتهاب الجلد التماسي التحسسي

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

قد تساعد الطريقة المختبرية الموضوعية المعروضة هنا في دراسة تفاعل فرط الحساسية التلامسية في الفئران ، والذي يحاكي التهاب الجلد التماسي التحسسي لدى البشر. يمكن قياس تفاعل فرط الحساسية الملامس ليس فقط عن طريق وذمة الأذن ، ولكن أيضا عن طريق الاختبارات المعملية المختلفة التي تسمح بدراسة الآليات الخلوية المشاركة في هذا التفاعل. يمكن لنموذج فرط الحساسية الأكثر ملامسة اختبار العوامل المختلفة والبيئية والمواد الجديدة لإظهار ما إذا كانت العوامل المختبرة تعدل الاستجابات المناعية المعتمدة على الخلايا التائية ، والتي يمكن استخدامها لتنفيذ علاجات جديدة.ابدأ بحلق جلد الفأر بشفرة حلاقة ووضع علامات على المخلب. قبل ست ساعات من تحريض فرط الحساسية للتلامس ، أو CHS ، ضع صابونا رماديا بالماء واحلق مساحة 2 سم مربع على صدر وبطن الماوس. في نفس اليوم ، قم بتوعية الفئران عن طريق تطبيق 150 ميكرولتر من 5٪ hapten الطازجة على المكان المحلوق مسبقا.في ماوس التحكم ، قم بتطبيق السيارة فقط. جفف بقعة hapten لمدة 30 ثانية قبل إعادة الحيوان إلى القفص. في اليوم الرابع ، قم بقياس سمك أذن فأر مخدر باستخدام ميكرومتر عند ساعة الصفر بواسطة مراقب غير مدرك للمجموعات التجريبية.بعد ذلك ، ضع 10 ميكرولتر من 04٪ hapten المحضرة حديثا على جانبي الأذنين في مجموعات الاختبار والتحكم واتركها تجف لمدة 30 ثانية. في اليوم الخامس ، بعد 24 ساعة من تطبيق 0.4٪ hapten السابق ، كرر قياس سمك الأذن للقياس على مدار 24 ساعة. بعد 24 ساعة من قياس سمك الأذن ، عندما لا يزال الفأر في التخدير العميق ، قم بقطع الأذنين بالقرب من الجمجمة قدر الإمكان باستخدام المقص.في الجانب البعيد من الأذنين ، قم بعمل لكمة قطرها ستة ملليمترات باستخدام لكمة خزعة. قياس وزن كل خزعة الأذن على الميزان التحليلي ، والتعبير عنها بالملليغرام. بالنسبة لفحص الميلوبيروكسيديز ، أو MPO ، ضع خزعات الأذن في أنبوب طرد مركزي دقيق سعة 2 ملليلتر مع حبات من الفولاذ المقاوم للصدأ وأضف 500 ميكرولتر من المخزن المؤقت المحضر.تجانس الخزعات لمدة 10 دقائق في الخالط ثم تبريد العينة لمدة 15 دقيقة عند 4 درجات مئوية قبل تجانسها مرة أخرى لمدة 10 دقائق. أخرج الخرزات بمجرد تجانس الخزعات. قم بتجميد التجانسات عند 20 درجة مئوية تحت الصفر لمدة 30 دقيقة.إذابة ودوامة العينات ، وتكرار إجراء التجانس والتجميد ثلاث مرات. بمجرد الانتهاء من ذلك ، يقوم جهاز الطرد المركزي بالتجانس عند 3،000 جم لمدة 30 دقيقة عند 4 درجات مئوية. احصد المادة الطافية باستخدام ماصة لقياس نشاط MPO ، والتعبير عنها بوحدات لكل ملليغرام من البروتين.لإجراء اختبار نفاذية الأوعية الدموية ، قم بتوعية الفئران في اليوم صفر ، وفي اليوم الرابع ، تحدى مباشرة من خلال تطبيق hapten على الأذنين باستخدام الإجراء المذكور سابقا. بعد 23 ساعة ، حقن 8.3 ميكرولتر لكل جرام من وزن الجسم بنسبة 1٪ صبغة إيفانز الزرقاء في محلول ملحي مخزن بالفوسفات من Dulbecco ، أو DPBS ، إلى الفأر المخدر. بعد ساعة واحدة ، قم بتخدير الماوس مرة أخرى لجمع خزعات الأذن ، كما هو موضح سابقا.لاستخراج الصبغة من الأنسجة ، احتضان الخزعات في الأنابيب التي تحتوي على ملليلتر واحد من الفورماميد عند 37 درجة مئوية في جو من 5 ٪ ثاني أكسيد الكربون لمدة 18 ساعة. بعد جمع خزعات الأذن ، عندما لا يزال الماوس تحت التخدير العميق ، قم بإزالة مقلة العين بالملاقط. ضع ضغطا لطيفا على الماوس واجمع الدم من الجيب الحجاجي الخلفي إلى الأنبوب لجمع المصل.اقلب الأنبوب ست مرات وانتظر 30 دقيقة حتى يتجلط الدم. ثم ، الطرد المركزي الأنبوب في 1،300 إلى 2،000 غرام لمدة 10 دقائق في درجة حرارة الغرفة. توعية الفئران المانحة ب TNCB hapten في اليوم صفر باستخدام الإجراء المذكور سابقا في اليوم الرابع ، بعد تطهير الجلد ، قم بعزل الغدد الليمفاوية الإبطية والأربية ، أو ALNs ، من الفأر المخدر باستخدام الملقط ، متبوعا بعزل الطحال.هرس الأنسجة بين الأطراف المتجمدة لشريحتين مجهريتين وقم بتمرير تعليق الخلية من خلال مصفاة خلية بحجم مسام 70 ميكرومتر. بعد ذلك ، اغسل الخلايا باستخدام DPBS المكمل بمصل بقري جنيني 1٪ وطردها مركزيا عند 300 جم لمدة 10 دقائق عند أربع درجات مئوية. صب المادة الطافية وإعادة تعليق حبيبات الخلية المتبقية في واحد إلى خمسة ملليلتر من DPBS.قم بإعداد خليط 1: 1 من ALNs والطحال لتحقيق تركيز يصل إلى 7.0 مرات 10 إلى الخلايا السابعة في 200 ميكرولتر من DBPS قبل حقن خليط الخلايا المستجيبة CHS عن طريق الوريد في فأر مخدر. قم بقياس سمك الأذن عند ساعة الصفر وبعد 24 ساعة من التحدي. أظهرت البيانات أن الفئران التي تحسست ل TNCB وتحدت في الأذنين بعد أربعة أيام طورت زيادة كبيرة في تورم الأذن ، مقارنة بالفئران الضابطة التي تحسست للوهم.أدت الزيادة في وذمة الأذن في مجموعة الاختبار إلى زيادة وزن الأذن ، ونشاط MPO ، وتركيز إنترفيرون جاما في مستخلصات الأذن ، وتغيرات الأدمة الوذمية في الفحص النسيجي ، ونفاذية الأوعية الدموية للأذن كانت الأجسام المضادة IgG1 الخاصة ب TNP مرتفعة في أمصال فئران الاختبار عند مقارنتها بالحيوانات الضابطة. أظهرت الحيوانات التي تلقت خلايا CHS-respondor من المتبرعين الذين تم تحسسسهم سابقا مع TNCB زيادة كبيرة في تورم الأذن ، مقارنة بالحيوانات التي لم تتلق الخلايا. اللحظة الأكثر أهمية هي تحفيز فرط الحساسية للتلامس.حل Hapten متقلب للغاية وحساس للضوء ، لذلك يجب تطبيقه بسرعة على جلد الحيوانات. يمكن إجراء اختبارات إضافية على الخلايا المستجيبة المعزولة. على سبيل المثال ، يمكن إنشاء مزارع الخلايا لتقييم القدرة على التكاثر في وجود المستضدات.

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.

الاعتراف ناقلة الغلوتامين البشرة بواسطة الأجسام المضادة IgA و 2 ناقلة الغلوتامين الأنسجة في حالة نادرة من القرد الجلد

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

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.

إدخال إجهاد القص في دراسة الالتصاق الجرثومي

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.

قعدة اختبار لتنشيط التحقيق في إيج بوساطة آليات في فرط الحساسية للمخدرات

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.

تحريض الطعم ضد المضيف المرض و<em&gt; في الجسم الحي</em&gt; مراقبة الخلايا T باستخدام نموذج MHC المطابقة الفئران

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.

قياس آلام جلدية اللمس في نموذج الفئران التهاب البروستاتا الجرثومي

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.

استخدام<em&gt; غاليريا mellonella</em&gt; كما كائن نموذج لدراسة<em&gt; البكتيريا المستروحة</em&gt; عدوى

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.

Organoids كنموذج للأمراض المعدية: ثقافة الإنسان وOrganoids المعدة الفئران وهيليكوباكتر بيلوري Microinjection من

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.

الزرد نموذجا لتقييم إمكانات المسخية من النطرون

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.

نموذج الفئران من المجموعة الثانية<em&gt; العقدية</em&gt; المهبلية الاستعمار

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.

آثار تعرض فورمالدهايد لنموذج الفئران الاستشرائية الناجمة عن معاملة الأطفال حديثي الولادة كبخاخات

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