Name: inducing meningococcal meningitis serogroup c in mice via intracisternal delivery
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Description: Watch this Scientific Journal Video about Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery at JoVE.com

अध्ययन PRIN द्वारा भाग में समर्थित थे 2012 [अनुदान संख्या 2012WJSX8K]: "होस्ट-माइक्रोब बातचीत मॉडल mucosal संक्रमण में: उपन्यास चिकित्सीय रणनीतियों का विकास" और PRIN द्वारा 2017 [2017SFBFER]: "एक एकीकृत दृष्टिकोण के बीच परस्पर क्रिया से निपटने के लिए अनुकूलन, तनावपूर्ण स्थितियों और चुनौतीपूर्ण रोगजनकों के एंटीमाइक्रोबियल प्रतिरोध"।

यह प्रोटोकॉल पशु पीड़ा को कम करने और 24 नवंबर 1986 (86/609/EEC) के यूरोपीय समुदाय परिषद के निर्देश के अनुसार चूहों की संख्या को कम करने के लिए आयोजित किया गया था। इस अध्ययन में रिपोर्ट किए गए विवो प्रयोगों में एथिक?...

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इस अध्ययन में, हम i.cist द्वारा वयस्क चूहों में मेनिंगोकोकल मेनिनजाइटिस प्रेरित करने के लिए एक प्रयोगात्मक प्रोटोकॉल का वर्णन करते हैं। मेनिंगोकोकल बैक्टीरिया का टीका. हमारी जानकारी के अनुसार, आई.सी.सी. द...

नेसेरिया मेनिंगिटिस एक ग्राम नकारात्मक है - प्रोटीओबैक्टीरियम मानव मेजबान तक ही सीमित है, जो दुनिया भर में मानव आबादी में मेनिनजाइटिस और सेप्सिस के सबसे आम कारणों में से एक होने के लिए जाना जाता है। यह स्वस्थ और स्पर्शोन्मुख वाहक (2-30% आबादी) के ऊपरी श्वसन पथ (नोज और गले) को उपनिवेश करता है, लेकिन जीवाणु कभी-कभी विभिन्न मेजबान प्रतिरक्षा सुरक्षा से बच जाता है और खून से मस्तिष्क तक फैलता है जिससे एक अनियंत्रित स्थानीय सूजन, मेनिंगोकोकल मेनिनजाइटिस के रूप में जाना जाता है। मेजबान और जीवाणु कारकों का एक संयोजन आक्रामक व्यवहार1को commensal से संक्रमण के लिए योगदान करने के लिए प्रकट होता है.
एन मेनिंगटिडिस विशेष रूप से मानव उपनिवेशन और संक्रमण में विशेष है। यह एक संकीर्ण मेजबान रेंज है और, इसलिए, मानव मेनिंगोकोकल रोग को पुन: उत्पन्न करने वाले उपयुक्त पशु मॉडल की कमी के कारण विवो रोगजनन अध्ययन में सीमित है। नतीजतन, यह मेनिंगोकोकस के कारण सेप्टिकेमिया और मेनिनजाइटिस के रोगजनन से संबंधित समझ में बुनियादी अंतराल के लिए नेतृत्व किया था। पिछले दशकों में , कई इन विट्रो प्रणालियों के विकास ने कई मेनिंगोकोकल उग्रता कारकों की पहचान की अनुमति दी2,3,4. हालांकि इन मूल्यवान अध्ययन एक सफल मेनिंगोकोकल संक्रमण के लिए इन कारकों की भूमिका को समझने के लिए महत्वपूर्ण अंतर्दृष्टि प्रदान की, इन मॉडलों हास्य और सेलुलर के साथ जीवाणु बातचीत के परिणामों के आकलन की अनुमति नहीं दी प्रतिरक्षा प्रणाली और पूरे ऊतक के साथ भी कम. विवो पशु में संक्रमण के मॉडल महान प्रासंगिकता के रूप में अच्छी तरह से टीका योगों द्वारा प्रदत्त संरक्षण की डिग्री के मूल्यांकन के लिए कर रहे हैं. एक मानव-उष्णकटिबंधीय रोगजनक के रूप में, मेनिंगोकोकस में सतह संरचनाओं (यानी, टाइप IV पिली और अस्पष्टता प्रोटीन) और मानव रिसेप्टर्स और परिवहन प्रोटीन के लिए लोहे की तेज प्रणाली जैसे सफल संक्रमण के लिए आवश्यक उचित निर्धारक ों का अधिकारी होता है (यानी, transferrin और lactoferrin)5,6,7 ठीक से पालन करने के लिए, जीवित है और मानव मेजबान पर आक्रमण. अंत में, रोगज़नक़ की आनुवंशिक भिन्नता क्षमताओं से बचने के लिए और / या मानव प्रतिरक्षा प्रतिक्रिया ब्लॉक आगे उच्च प्रजातियों tropism8,9के लिए योगदान . इसलिए, विशिष्ट मेजबान कारकों की अनुपस्थिति, बातचीत में शामिल, रोगजनक के जीवन चक्र के चरणों को ब्लॉक कर सकती है, मेनिंगोकोकल जीवन चक्र का सारांश देने वाले छोटे पशु मॉडलों के विकास में महत्वपूर्ण कठिनाइयों को स्थापित कर सकती है।
पिछले दशकों में, मेनिंगोकोकल संक्रामक चक्र की हमारी समझ में सुधार करने के लिए कई दृष्टिकोण विकसित किए गए हैं। दो पशु मॉडल, माउस और चूहे के संक्रमण, या तो इंट्रापेरिटोनली (आई.पी.) या अंत:प्र.) (i.n.), मेनिंगोकोकल रोग10,11,12,13,14 को पुन: उत्पन्न करने के लिए विकसित किए गए थे ,15,16,17. प्रयोगशाला माउस शायद प्रयोगात्मक मेनिंगोकोकल संक्रमण को प्रेरित करने के लिए अधिक बहुमुखी जानवरों में से एक है।
हालांकि, संक्रमण का आई.पी. तरीका गंभीर सेप्सिस के विकास की ओर जाता है, हालांकि यह संक्रमण के प्राकृतिक मार्ग की नकल नहीं करता है, जबकि संक्रमण का आई.एन. मार्ग मेनिंगोकोकल रोगजनन का मूल्यांकन करने के लिए उपयोगी था, भले ही यह फेफड़ों के संक्रमण को प्रेरित कर सकता है सेप्सिस से पहले10,11,12,13,14, 15,16,17.
आई . पी . माउस मॉडल ने मेनिंगोकोकल चैलेंज10,11,12से सुरक्षा का आकलन करने में महत्वपूर्ण भूमिका निभाई थी . संक्रमण के i.n. मार्ग पर आधारित मेनिंगोकोकल उपनिवेशन के माउस मॉडल शिशु चूहों के साथ विकसित किया गया है, क्योंकि वे मेनिंगोकोकी के लिए अतिसंवेदनशील होते हैं, मनुष्यों में मेनिंगोकोकल रोग के पाठ्यक्रम की नकल करने वाले एक आक्रामक संक्रमण को पुन: पेश करने के लिए 13,14,15,16,17. इसके अलावा, murine मेजबान में मेनिंगोकोकल प्रतिकृति को बढ़ावा देने के लिए, संक्रमण में सुधार करने के लिए जानवरों के लिए लोहे के प्रशासन सहित तकनीकी रणनीतियों की बढ़ती संख्या भी लागू की गई, उच्च जीवाणु inoculumके उपयोग , चूहे से गुजरने वाले जीवाणु तनाव के साथ-साथ शिशु या प्रतिरक्षा पशु के रोजगार के लिए10,13,15,18,19की मेजबानी की जाती है । CD4620 या transferrin21 जैसे विशिष्ट मानव कारकों की अभिव्यक्ति इस मानव-tropic जीवाणु के लिए चूहों की संवेदनशीलता में वृद्धि हुई है; संक्रमण के मानव त्वचा xenograft मॉडल का रोजगार भी मानव endothelium22,23के लिए meningococi के आसंजन क्षमता का मूल्यांकन करने के लिए उपयोगी रहा है . सामूहिक रूप से, मानवीकृत ट्रांसजेनिक चूहों के हाल के विकास ने मेनिंगोकोकल रोगजनन और इसके मेजबान बातचीत की समझ में सुधार किया है।
इससे पहले, हमने मेनिंगोकोकल मेनिनजाइटिस का एक मौरीन मॉडल विकसित किया था जहां चूहे-पास ्डजीवाणुओंके साथ वयस्क चूहों के कुंडमें में बैक्टीरिया का टीका लगाया गया था। नैदानिक मानकों और संक्रमित चूहों के जीवित रहने की दर मानव मेजबान में देखा उन लोगों के लिए तुलनीय विशेषताओं के साथ दिमागी पनकी की स्थापना का प्रदर्शन किया, साथ ही, मस्तिष्क के सूक्ष्मजीवी और हिस्टोलॉजिकल विश्लेषण. इन संक्रमित चूहों से, बैक्टीरिया, भी, रक्त, जिगर, और तिल्ली से बरामद किया गया, और परिधीय अंगों से जीवाणु भार संक्रामक खुराक के साथ सहसंबद्ध. विशेष रूप से, इस मॉडल को एल-ग्लूटामेट ट्रांसपोर्टर GltT24में दोषपूर्ण एक isogenic उत्परिवर्ती तनाव की उग्रता का मूल्यांकन करने के लिए नियोजित किया गया था। हाल ही में, आई.सी.टी. के आधार पर मेनिंगोकोकल मेनिनजाइटिस के हमारे माउस मॉडल का उपयोग करना। सीरोग्रुप सी तनाव के साथ मार्ग 93/42862,24 और एक isogenic उत्परिवर्ती यूडीपी के लिए सीएसए जीन एन्कोडिंग में दोषपूर्ण-एन-एसिटिलग्लूकोसामाइन 2-एपिमेरेज़25, हम स्थापना में उजागर sialic एसिड की भूमिका का विश्लेषण किया है चूहों में रोग की.
इस प्रोटोकॉल में, हम i.cist के आधार पर प्रयोगात्मक मेनिंगोकोकल मेनिनजाइटिस प्रेरित करने के लिए एक सरल विधि का वर्णन करते हैं। Balb/c वयस्क चूहों में संक्रमण का मार्ग. यह विधि विशेष रूप से एक murine मेजबान में मेनिंगोकोकल संक्रमण की विशेषता के लिए उपयोगी है, साथ ही जंगली प्रकार संदर्भ उपभेदों और isogenic उत्परिवर्ती के बीच उग्रता के आकलन के लिए. संक्रमण का इंट्रा-सिस्टरल मार्ग सीधे सिस्टर्ना मैग्ना में मेनिंगोकोसी की पूरी डिलीवरी सुनिश्चित करता है, जो बदले में मस्तिष्कमेरु द्रव (सीएसएफ) में जीवाणु प्रतिकृति की सुविधा प्रदान करता है और मेनिनजाइटिस को उन विशेषताओं के साथ प्रेरित करता है जो उन की नकल करते हैं। मनुष्यों में वर्तमान2,24,25,26.

<table>
	<tbody>
		<tr>
			<td>1,8 Skirted Cryovial With external thread</td>
			<td>Starlab</td>
			<td>E3090-6222</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>30 x 115mm</td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>F.S.T.</td>
			<td>11006-12</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Alarm-Thermometer</td>
			<td>TESTO</td>
			<td>9000530</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoTM Proteose Peptone</td>
			<td>BD</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Micro Fine syringe</td>
			<td>BD</td>
			<td>320837</td>
			<td>U-100 Insulin</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 1ml 25GA 5/8in</td>
			<td>BD</td>
			<td>300014</td>
			<td>05x16mm</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 5ml</td>
			<td>BD</td>
			<td>308062</td>
			<td>07 x 30mm</td>
		</tr>
		<tr>
			<td>BIOHAZARD AURA B VERTICAL LAMINAR FLOW CABINET</td>
			<td>Bio Air s.c.r.l.</td>
			<td>Aura B3</td>
			<td></td>
		</tr>
		<tr>
			<td>BioPhotometer</td>
			<td>Eppendorf</td>
			<td>Model #6131</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle D</td>
			<td>Tecniplast</td>
			<td>D</td>
			<td>Graduated up to:400ml, Total Volume 450ml, 72x72x122mm</td>
		</tr>
		<tr>
			<td>C150 CO2 Incubator</td>
			<td>Binder</td>
			<td>9040-0078</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage Body Eurostandard Type II</td>
			<td>Tecniplast</td>
			<td>1264C</td>
			<td>267x207x140mm, Floor area 370cm2</td>
		</tr>
		<tr>
			<td>Cell Culture Petri Dish With Lid</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Working Volume: 5mL</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Microcentrifuge 5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvetta semi-micro L. Form</td>
			<td>Kartell S.p.A.</td>
			<td>01938-00</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Potassium hydrogen phosphate trihydrate</td>
			<td>Carlo erba</td>
			<td>471767</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium hydrogen phosphate anhydrous ACS-for analysis</td>
			<td>Carlo Erba</td>
			<td>480141</td>
			<td>g1000</td>
		</tr>
		<tr>
			<td>Diete Standard Certificate</td>
			<td>Mucedola s.r.l.</td>
			<td>4RF21</td>
			<td>Food pellet for animal</td>
		</tr>
		<tr>
			<td>Dumont Hp Tweezers 5 Stainless Steel</td>
			<td>F.S.T. by DUMONT</td>
			<td>AGT5034</td>
			<td>0,10 x 0,06 mm tip</td>
		</tr>
		<tr>
			<td>Electronic Balance</td>
			<td>Gibertini</td>
			<td>EU-C1200</td>
			<td>Max 1200g, d=0,01g, T=-1200g</td>
		</tr>
		<tr>
			<td>Eppendorf Microcentrifuge tube safe-lock</td>
			<td>Eppendorf</td>
			<td>T3545-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Sigma-Aldrich</td>
			<td>E-6376</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>F.S.T.</td>
			<td>14084-08</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Filter Top (mini- Isolator), H-Temp with lock clamps</td>
			<td>Tecniplast</td>
			<td>1264C400SUC</td>
			<td></td>
		</tr>
		<tr>
			<td>GC agar base</td>
			<td>OXOID</td>
			<td>CM0367</td>
			<td></td>
		</tr>
		<tr>
			<td>Gillies Forceps 1 x2 teeth</td>
			<td>F.S.T.</td>
			<td>11028-15</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Glicerin RPE</td>
			<td>Carlo Erba</td>
			<td>453752</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T.</td>
			<td>11052-10</td>
			<td>Serrated Tip Width: 0.8mm</td>
		</tr>
		<tr>
			<td>Inner lid</td>
			<td>Tecniplast</td>
			<td>1264C116</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron dextran solution</td>
			<td>Sigma-Aldrich</td>
			<td>D8517-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological Safety Cabinet BH-EN and BHG Class II</td>
			<td>Faster</td>
			<td>BH-EN 2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5ml&#160;</td>
			<td>BRAND</td>
			<td>PP780751</td>
			<td>screw cap PP, grad</td>
		</tr>
		<tr>
			<td>Mouse Handling Forceps</td>
			<td>F.S.T.</td>
			<td>11035-20</td>
			<td>Serrated rubber; Gripping surface:15 x 20 mm</td>
		</tr>
		<tr>
			<td>Mucotit-F2000</td>
			<td>MERZ</td>
			<td>61846</td>
			<td>2000ml</td>
		</tr>
		<tr>
			<td>Natural Latex Gloves</td>
			<td>Medica</td>
			<td>M101</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Classic C24 Incubator Shaker</td>
			<td>PBI international</td>
			<td>C-24 Classic Benchtop Incubator Shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri PS Dishes</td>
			<td>VWR</td>
			<td>391-0453</td>
			<td>90X14.2MM</td>
		</tr>
		<tr>
			<td>Pipetman Classic P20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td>2-20microL</td>
		</tr>
		<tr>
			<td>Pipetman Classic P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td>20-200microL</td>
		</tr>
		<tr>
			<td>Pipetman Classim P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td>200-1000microL</td>
		</tr>
		<tr>
			<td>Polyvitox</td>
			<td>OXOID</td>
			<td>SR0090A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>J.T. Baker Chemicals B.V.</td>
			<td>0208</td>
			<td>250g</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>J.T. Baker Chemicals B.V.</td>
			<td>0240</td>
			<td>1Kg</td>
		</tr>
		<tr>
			<td>PS Disposible forceps</td>
			<td>VWR</td>
			<td>232-0191</td>
			<td></td>
		</tr>
		<tr>
			<td>Removable Divider</td>
			<td>Tecniplast</td>
			<td>1264C812</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polypropylene Tubes</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>5ml</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MOLEKULA</td>
			<td>41272436</td>
			<td></td>
		</tr>
		<tr>
			<td>SS retainer and Polyester FilterSheet</td>
			<td>Tecniplast</td>
			<td>1264C</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Pattern Forceps</td>
			<td>F.S.T.</td>
			<td>11000-12</td>
			<td>Stainless</td>
		</tr>
		<tr>
			<td>Stevens Tenotomy Scissors</td>
			<td>F.S.T.</td>
			<td>14066-11</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Surgical Scissor - ToughCut</td>
			<td>F.S.T.</td>
			<td>14130-17</td>
			<td>Stainless</td>
		</tr>
		<tr>
			<td>Touch N Tuff disposible nitrile gloves</td>
			<td>Ansell</td>
			<td>92-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Low Temperature (ULT) Freezer</td>
			<td>Haier</td>
			<td>DW-86L288</td>
			<td>Volume= 288L</td>
		</tr>
		<tr>
			<td>Wagner Scissors</td>
			<td>F.S.T.</td>
			<td>14070-12</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inducing meningococcal meningitis serogroup c in mice via intracisternal delivery

नेसेरिया मेनिंगिटिस (मेनिंगोकोकस) एक संकीर्ण मेजबान-रेंज सूक्ष्मजीव है, जिसे विश्व स्तर पर बैक्टीरियल मैनिंजाइटिस के प्रमुख कारण के रूप में मान्यता प्राप्त है। मेनिंगोकोकस स्वस्थ विषय के लगभग 10% के मानव नासोफैरिक्स का एक क्षणिक कॉलोनाइजर है। विशेष परिस्थितियों में, यह mucosal बाधा घुसना करने के लिए एक आक्रामक क्षमता प्राप्त करता है और सेप्टिकेमिया के कारण खून पर आक्रमण. नवीनतम मामले में, मैनिजिनाइटिस के परिणामी विकास के बिना भी फुलमिनिंग सेप्सिस उत्पन्न हो सकता है। इसके विपरीत, बैक्टीरिया खराब खून में गुणा कर सकता है, रक्त मस्तिष्क बाधा पार, केंद्रीय तंत्रिका तंत्र तक पहुँचने, fulminant दिमागी शोहकरने के लिए अग्रणी. बैक्टीरियल मैनिंजाइटिस के murine मॉडल मेजबान-रोगजनक बातचीत की जांच करने के लिए और इस घातक बीमारी के लिए जिम्मेदार रोगजनक तंत्र का विश्लेषण करने के लिए एक उपयोगी उपकरण का प्रतिनिधित्व करते हैं। हालांकि, कई प्रयोगात्मक मॉडल प्रणालियों पिछले दशकों में मूल्यांकन किया गया है, इनमें से कोई भी मेनिंगोकोकल रोग की विशेषता रोग घटनाओं को पुन: पेश करने में सक्षम थे. इस प्रयोगात्मक प्रोटोकॉल में, हम बैक्टीरिया के intracisternal टीका के आधार पर एक माउस मॉडल में मेनिंगोकोकल मेनिनजाइटिस के प्रेरण के लिए एक विस्तृत प्रक्रिया का वर्णन. मानव मैनिंजाइटिस के अजीब संकेत नैदानिक मापदंडों (जैसे, तापमान, शरीर के वजन), जीवित रहने की दर का मूल्यांकन, सूक्ष्मजीवी विश्लेषण और मस्तिष्क की चोट की हिस्टोलॉजिकल परीक्षा के आकलन के माध्यम से murine मेजबान में दर्ज किए गए थे। intracisternal (i.cist.) inoculum का उपयोग करते समय, मेनिंगोकोसी पूर्ण वितरण सीधे सिस्टर्ना मैग्नामें, मस्तिष्क के ऊतकों में एक बहुत ही कुशल मेनिंगोकोकल प्रतिकृति के लिए अग्रणी। लगभग 18 ज में बैक्टीरिया की व्यवहार्य संख्या में 1,000 गुना वृद्धि देखी गई है। इसके अलावा, मेनिंगोकोसी भी तिल्ली में पाए जाते हैं, और संक्रमित चूहों के जिगर, सुझाव है कि जिगर मेनिंगोकोकल प्रतिकृति के लिए एक लक्ष्य अंग का प्रतिनिधित्व कर सकते हैं.

एन मेनिंगटिडिस जंगली प्रकार और आइसोजेनिक उत्परिवर्ती उपभेदों से संक्रमित चूहों की उत्तरजीविता। इन प्रतिनिधि परिणामों में प्रयुक्त नेसेरिया मेनिंगिटिस उपभेदों सीरोग्रुप सी संदर्भ ?...

Watch this Scientific Journal Video about Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery at JoVE.com

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

यहां, हम वयस्क चूहों में संक्रमण के एक intracisternal मार्ग के माध्यम से मेनिंगोकोकल मेनिनजाइटिस प्रेरित करने के लिए एक विधि का वर्णन. हम intracisternal संक्रमण के लिए inoculum की तैयारी से मेनिंगोकोकल संक्रमण के कदम प्रोटोकॉल द्वारा एक कदम पेश; फिर पशु अस्तित्व रिकॉर्ड और murine ऊतकों में जीवाणु भार का मूल्यांकन.

Intracisternal वितरण के माध्यम से चूहे में मेनिंगोकोकल मेनिनजाइटिस सेरोग्रुप सी को प्रेरित करना

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

inducing-meningococcal-meningitis-serogroup-c-mice-via-intracisternal

Research

JoVE Journal

Immunology and Infection

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Transnuclear चूहे के खिलाफ पूर्व परिभाषित टी सेल रिसेप्टर specificities के साथ Toxoplasma gondii SCNT के माध्यम से प्राप्त

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3 के एचआईवी -1 tropism के genotypic निष्कर्ष का प्रयोग जनसंख्या आधारित अनुक्रमण

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

भ्रूण चूहों में vasculature लेबल के लिए एक विधि

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

की मौखिक संचरण<em&gt; लिस्टेरिया monocytogenes</em&gt; दूषित भोजन की घूस के माध्यम से चूहों में

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

नवजात और वयस्क केन्द्रीय तंत्रिका तंत्र से संवर्धन Microglia

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Effector के सीडी 4 की दत्तक हस्तांतरण द्वारा Murine आंतों में सूजन की प्रेरण<sup&gt; +</sup&gt; CD45RB<sup&gt; उच्च</sup&gt; Immunodeficient चूहों में टी कोशिकाओं

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

एक<em&gt; विट्रो में</em&gt; ट्यूबरकुलोसिस घावों में ड्रग एन्डेन्टेशन की भविष्यवाणी करने वाली केसम बाइंडिंग परख

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Intracranial Subarachnoidal एक माउस संक्रमण मॉडल में दिमागी बुखार में स्ट्रेप्टोकोकस सुईस की भूमिकाओं की जांच के लिए संक्रमण का मार्ग

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

T7 भोजी की मात्रात्मक पीसीआर panning से

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

नवजात चूहों में प्रोबायोटिक अध्ययन Gavage का उपयोग

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...