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

这项研究得到了亚必胜区补助金的支持。波兰弗罗茨瓦夫医科大学的A020.22.060，以及科学和高等教育部N N401 545940至MS和IP2012 0443 72至MMS的赠款。

本文中介绍的所有实验均根据克拉科夫第一届当地动物试验伦理委员会的指导方针进行。所描述的所有程序都是根据当地的建议进行的，特别是关于使用氯胺酮/甲苯噻嗪作为麻醉剂，使用耳朵的两侧涂抹该物质/半抗原，切断耳朵，并通过去除眼球来收集血液。本研究使用BALB/c（单倍型H-2d）、CBA/J（H-2k）和C57BL/6（H-2b）雄性和雌性小鼠，6-12周龄（见 材料表）?...

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CHS 通过 半抗原诱导，半抗原与皮肤中的自身蛋白抗原结合，产生新抗原。CHS由循环抗原特异性CHS效应T细胞的局部血管外募集介导，这导致受攻击组织肿胀，在次级皮肤暴露于同一半抗原6后24小时达到峰值。组织的肿胀主要是由白细胞浸润和白细胞依赖性纤维蛋白沉积24引起的。这些变化可以用一个千分尺测量半抗原致敏和激发小鼠与假敏和激发小鼠的?...

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

过敏性接触性皮炎（ACD）是工业化国家常见的皮肤炎症性疾病，由暴露于称为半抗原的低分子量化学物质引起的IV型超敏反应引起。在人类中引起接触致敏的物质包括重金属离子（铬、镍、铁、钴）、松节油、香料、染料和化妆品中存在的防腐剂（例如，对苯二胺）、一些药物（例如新霉素、苯佐卡因）、β内酰胺类抗生素（即青霉素）、植物产生的化学物质（十五甲苯甲酚，毒藤中存在的物质）以及照相工业中使用的对苯二酚1，2.ACD的病因剂非常高，因为仅在工业中使用了100，000多种化学品，并且每年合成2，000种新的化学品。迄今为止，已经鉴定出3，700多个分子可能是接触性半抗原/过敏原3。接触超敏反应（CHS）是ACD的实验模型，可以在小鼠，豚鼠和大鼠中研究，并且可以通过局部皮肤应用溶解在有机溶剂中的活性化学半抗原4，5，6来诱导。本研究旨在描述一种客观的实验室方法，该方法可能有助于研究小鼠中的CHS反应，该方法可以通过各种测试进行测量和定量。
CHS由致敏（诱导）和效应器（激发）阶段组成。在动物模型中，半抗原首先与体内的蛋白质共价结合以产生新抗原。在致敏阶段，活化的角质形成细胞通过产生促炎细胞因子 - 肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）7来促进皮肤树突状细胞（sDC）的迁移和成熟。表皮朗格汉斯细胞（LC）在CHS诱导和效应器阶段8期间存在抗原。在致敏过程中暴露于半抗原的LC促进了调节细胞和效应细胞的诱导9。来自几项研究的越来越多的证据表明，CHS反应可以由CD4 + MHC II类限制性Th1细胞介导，局部释放干扰素γ（IFN-γ）以采用特征性炎症浸润，CD8 + MHC I类限制性Tc1淋巴细胞，其也可以释放IFN-γ但主要介导对角质形成细胞的细胞毒性损伤，现在还有白细胞介导素17（IL-17）产生Th17细胞10，11.
已经开发了采用各种物种12，13，14 和半抗原的几种不同的CHS模型（不同半抗原，溶剂和施用时间的详细比较总结在 表1中）。小鼠是一种常用的实验室物种，在研究CHS方面具有一些优势。与其他物种相比，小鼠中有更多的菌株，敲除（KO）和转基因动物，这使它们成为非常有吸引力的动物15。此外，CHS模型需要许多动物，而老鼠在这里更经济。动物模型并非在所有方面都模仿ACD;特别是，它们表现出结痂和脱屑，这在人类中并不常见16，17。慢性病的特征难以重现，主要是因为所描述的模型没有假设半抗原在很长一段时间内的应用。然而，这里已经证实，ACD的许多重要方面都被复制了。还表明，与人类一样，这些特征与局部过敏反应有关。本方案中概述的半抗原，其溶剂及其应用的选择取决于这样一个事实，即结果已被众多 体外 测试证实，并且在实验室中对其进行了多年的测试和修改，直到建立当前版本为止。小鼠模型允许分析参与ACD开发的细胞亚群或细胞因子，并且对于新疗法的临床前评估至关重要。

<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

接触超敏反应（CHS）是过敏性接触性皮炎（ACD）的实验模型，可以在小鼠中研究。本研究旨在提出一种客观的实验室方法，该方法可能有助于研究小鼠的CHS反应，该方法可以通过各种测试进行测量和定量。为了诱导CHS，在第“0”天，小鼠通过用丙酮 - 乙醇混合物中的半抗原2，4，6-三硝基氯苯（TNCB）进行腹部皮肤涂漆，使小鼠在先前剃须的斑点上致敏，而阴性对照小鼠则用载体单独 - 丙酮 - 乙醇混合物进行假敏化。在第“4天”，在诱发CHS（激发）之前用千分尺测量基线耳朵厚度，方法是在测试组和对照组中用稀释的TNCB涂上两只耳朵。24小时后，用千分尺测量耳朵肿胀。CHS是T细胞介导的免疫反应的一个例子，它导致发炎组织肿胀，在皮肤挑战后24小时达到峰值，同时出现半抗原。耳水肿的增加与耳朵重量增加、髓过氧化物酶 （MPO） 活性、耳提取物中的促炎细胞因子浓度、组织学检查中水肿真皮增厚增加以及耳血管通透性相关。与对照小鼠相比，测试组血清中TNP特异性IgG1抗体的浓度也有所增加。此外，CHS可以与先前用TNCB致敏的供体获得的CHS效应细胞成功转移。将CHS效应细胞静脉注射到幼稚的受体小鼠中，随后用相同的稀释半抗原挑战。24小时后用千分尺测量耳朵肿胀。

对于CHS诱导， 通过 皮肤涂漆（腹部）用150μL5%TNCB或假用载体敏化动物。在第“4天”，在先前与TNCB接触的小鼠（测试组）和对照组小鼠（假敏化）中，用10μL0.4%TNCB接触绘画（激发）诱导两只耳朵的耳朵肿胀反应。提供的数据表明，与同样受到挑战的假敏小鼠相比，对TNCB敏感并4天后受到挑战的小鼠的耳朵肿胀显着增加（图3， 表2，测试与对照组）。耳部?...

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

接触性超敏反应（CHS）是过敏性接触性皮炎（ACD）的小鼠实验模型。CHS基于通过涂上胸部和腹部的剃须皮肤对反应性半抗原的致敏，随后用稀释的半抗原进行耳朵皮肤激发，引起肿胀反应，以各种方式评估。

接触超敏反应作为过敏性接触性皮炎的小鼠模型

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

这里介绍的客观实验室方法可能有助于研究小鼠的接触性超敏反应，其类似于人类的过敏性接触性皮炎。接触性超敏反应不仅可以通过耳水肿来测量，还可以通过各种实验室测试来测量，这些测试可以研究参与该反应的细胞机制。接触性更强的超敏反应模型可以测试各种环境因素和新物质，以显示测试因素是否调节T细胞依赖性免疫反应，可用于实施新疗法。首先用剃须刀片刮掉老鼠的皮肤并标记爪子。在诱导接触性超敏反应（CHS）前六小时，用水涂抹灰色肥皂，并在小鼠的胸部和腹部剃掉两厘米见方的区域。在同一天，通过在先前剃光的斑点上施用150微升新鲜制备的5%半抗原来使小鼠敏感。在控制鼠标中，仅应用车辆。将半抗原点干燥 30 秒，然后将动物放回笼子中。在第四天，由不知道实验组的观察者在零小时使用千分尺测量麻醉小鼠的耳朵厚度。然后，在测试组和对照组中，在耳朵两侧涂抹 10 微升新鲜制备的 04%hapten，并使其干燥 30 秒。在第五天，即上一次 0.4% 半抗原应用后 24 小时，重复测量耳朵厚度以进行 24 小时测量。耳朵厚度测量24小时后，当小鼠仍处于深度麻醉状态时，用剪刀尽可能靠近颅骨切断耳朵。在耳朵的远端侧，使用活检打孔器打一个直径六毫米的冲头。在分析天平上测量每个耳活检的重量，并以毫克为单位表示。对于髓过氧化物酶或MPO测定，将耳活检放入带有不锈钢珠的两毫升微量离心管中，并加入500微升制备的缓冲液。在均质器中均质化活检10分钟，然后在4摄氏度下将样品冷却15分钟，然后再次均质10分钟。活检均质化后取出珠子。将匀浆在零下 20 摄氏度下冷冻 30 分钟。解冻并涡旋样品，重复均质和冷冻过程三次。完成后，将匀浆在3， 000 G下在4摄氏度下离心30分钟。用移液管收获上清液以测量MPO活性，并以每毫克蛋白质的单位表达。为了进行血管通透性测试，在第零天和第四天使小鼠敏感，通过使用前面提到的程序在耳朵上涂抹半抗原来直接激发。23小时后，在Dulbecco的磷酸盐缓冲盐水或DPBS中静脉注射每克体重8.3微升的1%Evans蓝色染料给麻醉小鼠。一小时后，如前所述，再次麻醉小鼠以收集耳活检。为了从组织中提取染料，将活检在含有一毫升甲酰胺的管中，在37摄氏度的5%二氧化碳气氛中孵育18小时。收集耳活检后，当小鼠仍处于深度麻醉状态时，用镊子取出眼球。对小鼠施加轻轻的压力，并将血液从眶后窦收集到管中以进行血清收集。将试管倒置六次，等待 30 分钟让血液凝结。然后，在室温下以1， 300至2， 000g离心管10分钟。使用前面提到的程序在第0天用TNCB半抗原使供体小鼠敏感 第四天，在对皮肤进行消毒后，使用镊子从麻醉小鼠中分离腋窝和腹股沟淋巴结或ALN，然后分离脾脏。将组织捣碎在两个显微镜载玻片的磨砂端之间，并将细胞悬液通过孔径为70微米的细胞过滤器。然后，用补充有1%胎牛血清的DPBS洗涤细胞，并在4摄氏度下以300G离心10分钟。倒出上清液并将剩余的细胞沉淀重悬于一至五毫升DPBS中。在麻醉小鼠中静脉注射CHS效应细胞混合物之前，准备ALN和脾脏的1：1混合物，以达到200微升DBPS中7.0乘以10至第七个细胞的浓度。在零小时和挑战24小时后测量耳朵厚度。数据显示，与假致敏然后类似的挑战对照小鼠相比，对TNCB敏感并在四天后受到耳朵攻击的小鼠的耳朵肿胀显着增加。试验组耳水肿增加导致耳重、MPO活性、耳提取物中干扰素γ浓度增加、组织学检查水肿性真皮改变、耳血管通透性 与对照动物相比，试验小鼠血清中TNP特异性IgG1抗体升高。与未接受过TNCB致敏的动物相比，从先前用TNCB致敏的供体那里接受CHS效应细胞的动物的耳朵肿胀显着增加。最关键的时刻是触发接触超敏反应。半抗原溶液非常易挥发且对光敏感，因此必须迅速将其涂抹在动物的皮肤上。可以在分离的效应细胞上进行其他测试。例如，可以建立细胞培养物以评估在抗原存在下增殖的能力。

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.

在极少数情况下，表皮转谷氨酰胺酶IgA和组织转谷氨酰胺酶2抗体的识别恒河猴皮炎

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

科研

免疫与感染

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.

在细菌粘附的研究剪应力

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.

嗜碱粒细胞激活测试IgE介导的药物过敏机制的调查

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.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

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.

细菌性前列腺炎小鼠模型的测量触觉异常

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.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

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.

类器官的模型传染病：人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

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.

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

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组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

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.

甲醛暴露对大鼠新生辣椒素致过敏性皮炎模型的影响

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