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

מחקר זה נתמך על ידי SUBZ subvention. A020.22.060 של האוניברסיטה הרפואית בוורוצלב, פולין, ועל ידי מענקים ממשרד המדע וההשכלה הגבוהה N N401 545940 לטרשת נפוצה ו- IP2012 0443 72 ל- MMS.

כל הניסויים שהוצגו במאמר זה נערכו על פי הנחיות הוועדה האתית המקומית הראשונה לניסויים בבעלי חיים בקרקוב. כל ההליכים שתוארו בוצעו על פי ההמלצות המקומיות, במיוחד לגבי שימוש בקטמין/קסילאזין כחומר הרדמה, שימוש בשני צידי האוזניים למריחת החומר/הפטן, חיתוך האוזן ואיסוף דם על ידי הסרת גלגל העין. BAL...

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CHS מושרה באמצעות haptens, אשר נקשרים לאנטיגנים של חלבון עצמי בעור, ויוצרים ניאו-אנטיגנים. CHS מתווך על ידי גיוס חוץ-וסקולרי מקומי של תאי T ספציפיים לאנטיגן, מה שגורם לנפיחות ברקמה המאותגרת, המגיעה לשיא של 24 שעות לאחר חשיפת העור המשני לאותו הפטן6. הנפיחות של הרקמה נגרמת בעיקר על י?...

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

דרמטיטיס במגע אלרגי (ACD) היא מחלה דלקתית נפוצה בעור במדינות מתועשות הנגרמת על ידי תגובת רגישות יתר מסוג IV הנובעת מחשיפה לכימיקלים במשקל מולקולרי נמוך הנקראים הפטנס. החומרים הגורמים לרגישות למגע בבני אדם כוללים יוני מתכות כבדות (כרום, ניקל, ברזל, קובלט), טרפנטין, חומרי ריח, צבעים וחומרים משמרים הנמצאים במוצרי קוסמטיקה (למשל, p-פנילנדיאמין), תרופות מסוימות (למשל, ניאומיצין, בנזוקאין), אנטיביוטיקה β-לקטם (כלומר, פניצילין), כימיקלים המיוצרים על ידי צמחים (pentadecacatechol, חומר הנמצא בקיסוס רעיל), כמו גם הידרוקינון המשמש בתעשיית הצילום 1,2 . חומרים אטיולוגיים ACD גבוהים מאוד מכיוון שיותר מ-100,000 כימיקלים משמשים בתעשייה בלבד, ו-2,000 כימיקלים חדשים מסונתזים מדי שנה. עד כה זוהו יותר מ-3,700 מולקולות שעשויות להיות מגע עם הפטנים/אלרגנים3. תגובת רגישות יתר למגע (CHS) היא מודל ניסיוני של ACD שניתן לחקור בעכברים, שרקנים וחולדות וניתן לגרום לה על ידי יישום עור מקומי של הפטנים כימיים תגובתיים המומסים בממסים אורגניים 4,5,6. מחקר זה נועד לתאר שיטת מעבדה אובייקטיבית שעשויה לסייע בחקר תגובת CHS בעכברים, אותה ניתן למדוד ולכמת על ידי בדיקות שונות.
ה-CHS מורכב משלבי רגישות (אינדוקציה) ואפקטור (אתגר). במודלים של בעלי חיים, הפטנים נקשרים תחילה באופן קוולנטי לחלבונים בגוף כדי ליצור ניאו-אנטיגנים. במהלך שלב הרגישות, קרטינוציטים מופעלים מקדמים את הנדידה וההבשלה של תאים דנדריטיים בעור (sDCs) על ידי ייצור ציטוקים פרו-דלקתיים-נמק סרטניים α (TNF-α) ואינטרלוקין 1β (IL-1β)7. תאי לנגרהנס אפידרמליים (LCs) מציגים אנטיגנים במהלך אינדוקציה CHS ושלבי אפקטור8. LCs שנחשפו להפטן במהלך הרגישות מקדמים את האינדוקציה של תאי הוויסות והאפקט9. עדויות הולכות וגוברות ממספר מחקרים מצביעות על כך שתגובות CHS ניתנות לתיווך על ידי תאי Th1 מוגבלים מסוג CD4+ MHC Class II, המשחררים באופן מקומי אינטרפרון-γ (IFN-γ) כדי להשתמש בהחדרה דלקתית אופיינית, לימפוציטים מוגבלים ב-Tc1 מסוג CD8+ MHC מסוג I שיכולים גם לשחרר Γ IFN אך בעיקר לתווך נזק ציטוטוקסי לקרטינוציטים, וכעת גם אינטרלוקין 17 (IL-17)-המייצרים תאי Th1710, 11.
פותחו מספר מודלים שונים של CHS המשתמשיםבמינים שונים 1 2,13,14 והפטנים (השוואה מפורטת של הפטנים, ממסים שונים וזמן היישום מסוכמת בטבלה 1). העכבר, מין מעבדה הנפוץ, מציע כמה יתרונות בחקר CHS. ישנם יותר זנים, נוקאאוטים (KO) ובעלי חיים מהונדסים בקרב עכברים בהשוואה למינים אחרים, מה שהופך אותם לחיה מושכת מאוד15. בנוסף, מודל CHS דורש בעלי חיים רבים, ועכברים הם חסכוניים יותר כאן. מודלים של בעלי חיים אינם מחקים ACD בכל ההיבטים; בפרט, הם מראים קרום ו desquamation, אשר אינו נפוץ עבור בני אדם 16,17. המאפיינים של מחלה כרונית הם מאתגרים להתרבות, בעיקר משום המודל המתואר אינו מניח את היישום של hapten במשך תקופה ארוכה של זמן. עם זאת, אושר כאן כי רבים מההיבטים המשמעותיים של ACD משוחזרים. כמו כן הוכח כי, כמו בבני אדם, תכונות אלה קשורות לתגובות אלרגיות מקומיות. הבחירה של hapten, הממס שלה, ואת היישום שלה המתואר בפרוטוקול זה הוכתבו על ידי העובדה כי התוצאות אושרו על ידי בדיקות מבחנה רבות וכי הוא נבדק ושונה במעבדה במשך שנים רבות עד הגרסה הנוכחית הוקמה. מודלים של מורין מאפשרים ניתוח של תת-קבוצות תאים או ציטוקינים המעורבים בפיתוח ACD וחיוניים להערכות פרה-קליניות של טיפולים חדשים.

<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

רגישות יתר למגע (CHS) היא מודל ניסיוני של דרמטיטיס מגע אלרגי (ACD) שניתן לחקור בעכברים. מחקר זה נועד להציג שיטת מעבדה אובייקטיבית שעשויה לסייע בחקר תגובת CHS בעכברים, אותה ניתן למדוד ולכמת על ידי בדיקות שונות. כדי לגרום ל-CHS, ביום "0", עכברים היו רגישים לנקודה מגולחת קודם לכן על ידי צביעת עור בטן עם תערובת הפטן 2,4,6-טריניטרוכלורובנזן (TNCB) בתערובת אצטון-אתנול, בעוד שעכברי בקרה שלילית היו רגישים לתערובת אצטון-אתנול לבדה ברכב. ביום "4", עובי האוזן הבסיסי נמדד במיקרומטר לפני ההתעוררות של CHS (אתגר) על ידי צביעת שתי האוזניים עם TNCB מדולל הן בקבוצות הבדיקה והן בקבוצות הביקורת. לאחר 24 שעות נמדדה נפיחות האוזן במיקרומטר. CHS הוא דוגמה לתגובה חיסונית בתיווך תאי T הגורמת לנפיחות ברקמה מודלקת, המגיעה לשיא של 24 שעות לאחר אתגר העור עם אותו הפטן. עלייה בצקת אוזניים מתואמת עם משקל אוזניים מוגבר, פעילות מיאלופרוקסידאז (MPO), ריכוז ציטוקינים פרו-דלקתיים בתמציות האוזניים, עיבוי מוגבר של הדרמיס האדמטי בבדיקה ההיסטולוגית וחדירות כלי הדם באוזן. כמו כן, נרשמה עלייה בריכוז נוגדני IgG1 ספציפיים ל-TNP בסרה של קבוצת הבדיקה בהשוואה לעכברי הביקורת. בנוסף, ניתן להעביר CHS בהצלחה עם תאי CHS-effector המתקבלים מתורמים שהיו רגישים בעבר ל-TNCB. התאים המשפיעים על CHS ניתנו תוך ורידי לעכברים נמענים תמימים, אשר לאחר מכן אותגרו עם אותו הפטן מדולל. נפיחות באוזן נמדדה במיקרומטר 24 שעות לאחר מכן.

עבור אינדוקציה CHS, החיות היו רגישות באמצעות צביעת עור (בטן) עם 150 μL של 5% TNCB או sham רגיש עם הרכב בלבד. ביום "4", תגובות הנפיחות באוזן של שתי האוזניים הושרו על ידי צביעת מגע (אתגר) עם 10 μL של 0.4% TNCB בשני העכברים שהיו בעבר במגע עם TNCB (קבוצת בדיקה) והן בעכברים של קבוצת הביקורת (רגישות ל-sham). הנתונים שה...

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

רגישות יתר למגע (CHS) היא מודל ניסיוני של מורין של דרמטיטיס מגע אלרגי (ACD). CHS מבוסס על רגישות עם hapten תגובתי על ידי צביעת העור המגולח של החזה והבטן, עם אתגר עור אוזן לאחר מכן עם hapten מדולל, הגורם לתגובת נפיחות כי הוא מוערך בדרכים שונות.

צור רגישות יתר כמודל מורין של דרמטיטיס מגע אלרגי

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

שיטת מעבדה אובייקטיבית המוצגת כאן עשויה לסייע בחקר תגובת רגישות יתר במגע בעכברים, המחקה דרמטיטיס מגע אלרגי בבני אדם. תגובת רגישות היתר במגע יכולה להימדד לא רק על ידי בצקת אוזניים, אלא גם על ידי בדיקות מעבדה שונות המאפשרות לחקור מנגנונים תאיים המעורבים בתגובה זו. מודל רגישות היתר במגע יכול לבדוק גורמים שונים וסביבתיים וחומרים חדשים כדי להראות אם גורמים שנבדקו מווסתים תגובות חיסוניות תלויות תאי T, אשר ניתן להשתמש בהם כדי ליישם טיפולים חדשים.התחל בגילוח עור העכבר עם סכין גילוח ותיוג הכפה. שש שעות לפני השראת רגישות יתר למגע, או CHS, יש למרוח סבון אפור עם מים ולגלח שטח של שני סנטימטרים רבועים על החזה והבטן של העכבר. באותו יום, רגיש את העכברים על ידי מריחת 150 מיקרוליטר של 5% hapten מוכן טרי על המקום שגולח בעבר.בעכבר הבקרה, החל רק את הרכב. יבשו את נקודת ההפטן למשך 30 שניות לפני שתחזירו את בעל החיים לכלוב. ביום הרביעי, מדדו את עובי האוזן של עכבר מורדם באמצעות מיקרומטר בשעה אפס על ידי צופה שאינו מודע לקבוצות הניסוי.לאחר מכן, יש למרוח 10 מיקרוליטר של 04% הפטן טרי משני צידי האוזניים בקבוצות בדיקה וביקורת ולאפשר לו להתייבש במשך 30 שניות. ביום החמישי, 24 שעות לאחר היישום הקודם של 0.4% hapten, חזור על מדידת עובי האוזן עבור המדידה של 24 שעות. 24 שעות לאחר מדידת עובי האוזן, כאשר העכבר עדיין בהרדמה עמוקה, חתכו את האוזניים קרוב ככל האפשר לגולגולת באמצעות מספריים.בצד הדיסטלי של האוזניים, בצע ניקוב בקוטר שישה מילימטר באמצעות ניקוב ביופסיה. למדוד את המשקל של כל ביופסיית אוזן על האיזון האנליטי, ולבטא אותו במיליגרם. עבור myeloperoxidase, או MPO, assay, לשים את ביופסיות האוזן בתוך צינור מיקרוצנטריפוגה שני מיליליטר עם חרוזי נירוסטה ולהוסיף 500 מיקרוליטר של חיץ מוכן.הומוגניזציה של הביופסיות במשך 10 דקות בהומוגנייזר ולאחר מכן קירור הדגימה למשך 15 דקות ב -4 מעלות צלזיוס לפני הומוגניזציה שלה שוב למשך 10 דקות. מוציאים את החרוזים ברגע שהביופסיות הומוגניות. מקפיאים את ההומוגנטים במינוס 20 מעלות צלזיוס למשך 30 דקות.מפשירים ומערבלים את הדגימות, וחוזרים על הליך ההומוגניזציה וההקפאה שלוש פעמים. לאחר שנעשה, צנטריפוגה הומוגנט ב 3, 000 G במשך 30 דקות ב 4 מעלות צלזיוס. קצרו את הסופרנאטנט עם פיפטה כדי למדוד את פעילות ה-MPO, ובטאו אותה ביחידות לחלבון של מיליגרם אחד.כדי לבצע בדיקת חדירות כלי דם, יש לחוש עכברים ביום האפס, וביום הרביעי, לאתגר ישירות על ידי מריחת הפטן על האוזניים באמצעות ההליך שהוזכר לעיל. 23 שעות לאחר מכן, הזריקו תוך ורידי 8.3 מיקרוליטר לגרם משקל גוף של 1% צבע כחול אוונס במי מלח חוצצים פוספט של דולבקו, או DPBS, לעכבר המורדם. לאחר שעה, שוב להרדים את העכבר כדי לאסוף את ביופסיות האוזן, כפי שתואר קודם לכן.כדי לחלץ את הצבע מהרקמה, יש לדגור על הביופסיות בצינורות המכילים מיליליטר אחד של פורממיד בטמפרטורה של 37 מעלות צלזיוס באטמוספירה של 5% פחמן דו חמצני למשך 18 שעות. לאחר איסוף ביופסיות אוזניים, כאשר העכבר עדיין תחת הרדמה עמוקה, להסיר את גלגל העין עם פינצטה. הפעילו לחץ עדין על העכבר ואספו דם מהסינוס הרטרו-אורביטלי לתוך הצינור לאיסוף הסרום.הפוך את הצינור שש פעמים והמתן 30 דקות עד שהדם יקרש. לאחר מכן, צנטריפוגה את הצינור ב 1, 300 ל 2, 000 G במשך 10 דקות בטמפרטורת החדר. רגיש את העכברים התורמים עם TNCB hapten ביום אפס באמצעות ההליך שהוזכר לעיל ביום הרביעי, לאחר חיטוי העור, בודד את בלוטות הלימפה בבית השחי ובמפשעה, או ALNs, מהעכבר המרדים באמצעות מלקחיים, ולאחר מכן בודד את הטחול.מועכים את הרקמה בין הקצוות החלביים של שתי שקופיות מיקרוסקופ ומעבירים את תרחיף התא דרך מסננת תאים בגודל נקבוביות של 70 מיקרומטר. לאחר מכן, שטפו את התאים עם DPBS בתוספת סרום בקר עוברי 1% וצנטריפוגו אותם ב 300 גרם למשך 10 דקות בארבע מעלות צלזיוס. דקנו את הסופרנאטנט והשהו מחדש את גלולת התא הנותרת באחד עד חמישה מיליליטר של DPBS.הכינו תערובת של 1:1 של ALN וטחול כדי להשיג ריכוז של עד 7.0 כפול 10 לתאים השביעיים ב -200 מיקרוליטר של DBPS לפני הזרקה תוך ורידית של תערובת של תאי אפקט CHS בעכבר מורדם. מדדו את עובי האוזן בשעה אפס ולאחר 24 שעות של האתגר. הנתונים הראו כי עכברים שהיו רגישים ל-TNCB ואותגרו באוזניים ארבעה ימים לאחר מכן פיתחו נפיחות מוגברת משמעותית באוזניים, בהשוואה לעכברי ביקורת שסבלו מרגישות דמה ולאחר מכן אותגרו באופן דומה.העלייה בבצקת אוזניים בקבוצת הבדיקה הביאה לעלייה במשקל האוזן, פעילות MPO, ריכוז אינטרפרון גמא בתמציות האוזן, שינויים בדרמיס בצקתי בבדיקה ההיסטולוגית וחדירות כלי הדם באוזן נוגדני IgG1 ספציפיים ל-TNP היו גבוהים בסרה של עכברי הבדיקה בהשוואה לחיות הביקורת. בעלי החיים שקיבלו את תאי אפקט CHS מתורמים שהיו רגישים בעבר ל- TNCB הראו נפיחות אוזניים מוגברת באופן משמעותי, בהשוואה לבעלי חיים שלא קיבלו את התאים. הרגע הקריטי ביותר הוא לעורר רגישות יתר למגע.פתרון Hapten הוא נדיף מאוד ורגיש לאור, ולכן זה חייב להיות מיושם במהירות על העור של בעלי החיים. ניתן לבצע בדיקות נוספות על תאי האפקט המבודדים. לדוגמה, תרביות תאים ניתן להקים כדי להעריך את היכולת להתרבות בנוכחות אנטיגנים.

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.

הכרה Transglutaminase עוריות על ידי IgA ו Transglutaminase רקמות 2 נוגדנים במקרה נדיר של רזוס דרמטיטיס

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.

היכרות עם מתח שאר בחקר הדבקה חיידקית

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.

Basophil מבחן הפעלה לחקירות IgE בתיווך מנגנונים של תרופות רגישות יתר

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.

אינדוקציה של מחלת שתל נגד מארח ו<em&gt; In Vivo</em&gt; ניטור תא T תוך שימוש במודל Murine MHC התאמה

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.

מדידה של Allodynia משושים בדגם Murine של ערמונית קטריאלי

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.

שימוש<em&gt; Mellonella Galleria</em&gt; כאורגניזם מודל לחקר<em&gt; הלגיונלה pneumophila</em&gt; זיהום

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 כמודל למחלות זיהומיות: התרבות של האדם וOrganoids הקיבה Murine וMicroinjection של הליקובקטר פילורי

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.

דג הזברה כמודל להעריך את הפוטנציאל טרטוגניות של ניטריט

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.

מודל Murine של הקבוצה B<em&gt; סטרפטוקוקוס</em&gt; נרתיקי Colonization

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.

ההשפעות של חשיפה של פורמלדהיד מודל עכברוש של אטופיק דרמטיטיס המושרה על ידי קפסאיצין Neonatal טיפול

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