The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

1) إعداد الخط الخلوي: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تطوير خط الخلية ربل-2H3.1 معربا عن α FcεRI الفروسية: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام تقنيات زراعة الأنسجة الأساسية للخطوط الخلايا أحادي الطبقة، بالنقل خلايا RBL-2H3.1 الأبوية استخدام البلازميد pEE6، تحمل الجين FcεRIα الخيول (بنك الجينات: Y18204.1) 28. إضافة 2 ميكروغرام ميكرولتر -1 من DNA البلازميد إلى 0.8 مل من الخلايا في مناطق ذات كثافة من 1.2 X10 7 خلايا مل -1. Electroporate الخلايا في 250 V 960 μF باستخدام 0.4 سم electrocuvette ثم احتضان فورا على الجليد لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> تحديد الخلايا تحولت باستخدام وسائل الإعلام التي تحتوي على 0.4 غرام من كبريتات geneticin G418، ثم فرز ما تبقى الخلايا الحية من خلال نظام مراقبة الأصول الميدانية من خلال وضع علامات عليها مع فريق الخبراء الحكومي الدولي الأجسام المضادة الفلورسنت. استخدام الناتج ربل-2H3.1 معربا عن الخيول خط الخلية FcεRIα للتحقيق 2، 3. </li></ol></li><li style=";text-align:right;direction:rtl"> قبل فحص الأجسام المضادة للتوعية: <ol style=";text-align:right;direction:rtl"><lط&gt; الحصاد وربل-2H3.1 معربا عن الخلايا FcεRIα الخيول من طبق بتري متموجة. يغسل ثم اعادة تعليق الخلايا في وسائل الإعلام ثقافة لكثافة خلية من الخلايا 5X10 5 مل -1. </li><li style=";text-align:right;direction:rtl"> إضافة إيج تهم الخلايا علقت لتركيز النهائي من 1 نانوغرام مل -1، ثم لوحة 100 ميكرولتر من الخلايا على 96 لوحة جيدا في الأعمدة 1-6 واحتضان عند 37 درجة مئوية + 5٪ CO 2 + 90٪ الرطوبة النسبية لمدة 16 ساعة. بعد فترة الحضانة، وقبل إجراء الفحص الإفراج، والتحقق من الآبار تحت المجهر لconfluency جيدا والتقيد الخلية. </li></ol></li></ol> 2) بيان الفحص: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسل الخلايا: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الإفراج الدافئ عازلة (25 ملم أنابيب، 120 ملي كلوريد الصوديوم، كلوريد البوتاسيوم 5 ملم، 0.04 ملم كلوريد المغنيسيوم وكلوريد الكالسيوم 1 ملم) عند 37 درجة مئوية للسماح للغسيل خلية لطيف. </li><li style=";text-align:right;direction:rtl"> غسل الخلايا عن طريق التحريك لوحة لإزالة خلية الإعلام وإضافة 100 ميكرولتر الحارة 37 درجة مئوية، عازلة الإصدار. كرر مرتين. </li></ol></li><li style=";text-align:right;direction:rtl"> التحدي مستضد: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد التخفيف التسلسلي للمستضد (NIP-HSA أو إدارة التخطيط الوطني-HSA) 0 نانوغرام مل -1، 0.1 نانوغرام مل -1، 1 نانوغرام مل -1، 10 نانوغرام مل -1، 100 نانوغرام مل -1، 1000 نانوغرام مل -1، 10000 نانوغرام مل -1 في المخزن الإفراج ودافئة عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> بعد غسل الخلية الثانية، تجاهل وسائل الإعلام واستبدالها مع 100 ميكرولتر من الحلول مستضد. ضمان آبار في نفس الصف (A1-6 على سبيل المثال) لديها تركيز المستضد نفسه إضافتها. </li><li style=";text-align:right;direction:rtl"> إنشاء سيطرة سلبية في الصف وذلك بإضافة 0 نانوغرام مستضد -1 مل. إضافة زيادة تركيز المستضد أسفل الصفوف (BG)، يليه تريتون X-عازلة (5٪ تريتون X-100) في H خلايا الصف لليز الخلايا لاستخدامها كعنصر تحكم إيجابية. احتضان عند 37 درجة مئوية لمدة 20 دقيقة للسماح للخلايا لاطلاق سراح الوسطاء لها. </li></ol></li><li style=";text-align:right;direction:rtl"> وضع الضوابط جيدا الفردية: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد الحضانة، ونقل 50 ميكرولتر من طاف الخلية إلى النصف الآخر من لوحة (الآبار A1-6 A7-12 إلى الآبار، وغيرها). تجاهل 50 ميكرولتر المتبقية من طاف واستبدالها مع 50 ميكرولتر من تريتون X-عازلة للسماح بقياس كمية من الوسطاء أفرج عنه في كل بئر في الأعمدة 7-12 كنسبة مئوية من إجمالي الوسطاء داخل الخلايا في العمود 1-6 . </li></ol></li><li style=";text-align:right;direction:rtl"> الركيزة انزيم: </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر من الركيزة β-هيكسوزامينيداز (50 ملي 4-nitrophenyl N-أسيتيل-β-D-glucosaminide أعد DMSO المخفف الى 2 مم عن طريق إضافته إلى عازلة سترات 0.2 M حمض الستريك و 0.2 M خلات الصوديوم، ودرجة الحموضة 4.5) لجميع الآبار لتسهيل تحويل الركيزة ل4-نيتروفينول بواسطة انزيم β-هيكسوزامينيداز. احتضان لوحات عند 37 درجة مئوية لمدة 2 ساعة. </li></ol><img alt="الرقم 2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" widtح = &quot;400&quot; /&gt; <ol start="5" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنهاء رد فعل: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وقف رد فعل من قبل مضيفا 150 ميكرولتر تريس العازلة (1 M تريس، حمض الهيدروكلوريك، ودرجة الحموضة 9) إلى كل جانب من درجة الحموضة العالية العازلة يتوقف رد الفعل، ويحول نيتروفينول 4 إلى اللون الأصفر. </li></ol></li><li style=";text-align:right;direction:rtl"> قراءة وتحليل النتائج: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قراءة لوحة باستخدام لوحة معمل في 405 نانومتر لقياس الامتصاصية من اللون الأصفر. تحسب النسبة المئوية للصدر β-هيكسوزامينيداز باستخدام الصيغة التالية: </li></ol></li></ol><img alt="الرقم 2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تطبيق هذه الصيغة على كل بئر، وبعد ذلك يؤخذ في المتوسط ​​لكل صف. A1 و A7 يمثل الموقع من الآبار في 96 لوحة جيدا. رسم بياني لنسبة الإفراج β-هيكسوزامينيداز (وهو ما يعادل إجمالي الإفراج الوسيط) ضد هبوطNST تركيز المستضد 2، 3. </li></ol>

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The authors declare that they have no competing financial interests in this paper.

في ملخص أظهرت نتائج هذا التحقيق أنه عندما يتم توعية خلايا RBL-2H3.1 معربا عن FcεRIα مع فرسي فرسي إيج وتحدى من قبل المستضد، فإنها تعطي بيان الذروة سيط 36.68٪ ± 4.88٪ من المبلغ الإجمالي الوسيط داخل الخلايا، مقارنة مع ربل-2H3.1 الخلايا الأبوية لا تعبر عن فرسي FcεRIα. <p class="jove_content" style="...

وقد عرفت الحساسية لآلاف السنين. ووصف علاج الربو في النص الطبية المصرية القديمة المعروفة باسم بردية إبيرس (~ 1550 قبل الميلاد) وناقش العلاجات العشبية لعلاج النتيجة 7. وتصنف الحساسية اليوم كرد فعل فرط الحساسية من النوع الأول، حيث T نوع من الخلايا المساعد 2 (TH 2) ذراع الجهاز المناعي يرسم إنتاج الغلوبولين المناعي E (IgE و) الأضداد استجابة لمستضدات البيئية تسمى المواد المسببة للحساسية. هذه هي المواد المتنوعة التي تتفاعل عادة مع الخلايا في جهاز المناعة، وتحفيز تخليق وإفراز السيتوكينات الموالية للالتهابات، بما في ذلك انترلوكين 4 وانترلوكين 13 8، 9 كما لم الجسيمات في دخان السجائر أو جزيئات عادم الديزل التي تعزز إيج التوليف 10. ويعزى الارتفاع في مظاهر الحساسية في البلدان الصناعية في السنوات ال 50 الماضية لمزيج من تأثيرالملوثات البيئية والاتجاه إلى بيئة أكثر مطهرة، والتي تتضافر لتحول الاستجابة المناعية تجاه لمحة سادت قبل TH 2 السيتوكينات، على النحو الذي اقترحه &quot;فرضية النظافة&quot; 11. كما ذكر أعلاه، والبشر ليسوا الثدييات الوحيدة التي تعاني من الحساسية. وخاصة الخيول والكلاب ويمكن أيضا تطوير استجابات حساسية الكلاسيكية وأظهرت دراسة أجرتها أن 12، كما هو الحال في البشر، ويعزى الحساسية الخيول إلى العوامل الوراثية والبيئية. ونتيجة لذلك، فإن هذه الحيوانات تمثل نماذج جيدة لدراسة التفاعل بين الأسباب الوراثية والبيئية للحساسية، تقدمه من توعية لمرض، واستراتيجيات التدخل الممكنة مرة واحدة وضعت في المظاهر السريرية في عام 1887، كان أول شخص Stömmer لوصف التشابه بين الإنسان والربو الخيلي 13، وتأثير الهستامين على نظام القلب والأوعية الدموية الخيول هومشابهة جدا لتلك التي للبشر 14. الخيول هي أيضا حجر الزاوية في صناعة سباقات الخيل، الذي يستحق 72 مليار دولار مع الرهان دوران من 115 مليار دولار سنويا 15. معظم خيول السباق المعاصرة هي من نسل عدد قليل من الخيول العربية ولدت من قبل السيدة آن بلنت من عام 1878 فصاعدا. والفطرية السباق الحديثة عادة لتحديد لقدرات الأداء. هم عرضة للاضطرابات وراثية، واحدة منها هي قابليتها لتركيب استجابات حساسية. لديهم أيضا 1000 مرة أعلى من المصل مستويات إيج حتى الأكثر حساسية شديدة البشر 16. وعادة ما تتجلى الحصان استجابات حساسية مثل فرط الحساسية لدغة حشرة (IBH) 17، 18. IBH النتائج في التهاب الجلد بسبب لدغات الحشرات تشكل في جنس البعضوضيات. شكل آخر من أمراض الحساسية الخيول هو انسداد لمجرى الهواء المتكررة (راو)، وهذا يتجلى في الرئتين والشعب الهوائية. ويتميز هذا الصفير ومختبرored التنفس. راو يحدث عادة استجابة لجراثيم العفن، وسجلت حساسية عالية خاصة بمستويات IgE في الخيول التي تعاني من راو في دراسة واحدة 19 على الرغم من تحقيق آخر لم يؤكد هذا 20. تدور دراسات عن حساسية الخيول حول محاولة لرصد وتحييد الخيول إيج من خلال تطوير إيج الأجسام المضادة وحيدة النسيلة المضادة للالفروسية (MABS) 21، 22. وعلاوة على ذلك الدراسة بنسبة 23 تناقش إنتاج المجالات خارج الخلية من α من الخيول عالية تقارب-FC مستقبلات ل سلسلة (FcεRIα) مستقبلات في محاولة لكشف وquantitate مصل الخيول إيج. تناقش دراسة ذلك من قبل لدين 24 نهج جديد يهدف إلى تحييد إيج المصل بواسطة فتيلة الجهاز المناعي باستخدام مستمنع الذات / غير المتمتعة بالحكم الذاتي. كل هذه الدراسات، ومع ذلك، تفتقر مقايسة فعالة لاختبار سلامة وفعالية بروتوكولاتها. في هذه المقالة، ونحن الآن تقديم مثل هذا الفحص SYSتيم ينطبق على دراسة الاستراتيجيات التشخيصية والعلاجية ذات الصلة لنظام الفروسية، حيث الإفراج β-هيكسوزامينيداز، كمؤشر الوسيط خلية تحبب، تم تقييمها على الخلايا ربل-2H3.1 معربا عن فرسي FcεRIα. ويعتمد هذا البروتوكول على المنشورات السابقة 25، 4، 5، 2، 3 اصفا الهندسة خلايا RBL transfected مع الجينات ترميز المجال الجلوبيولين ملزمة لمستقبلات عالية تقارب لفريق الخبراء الحكومي الدولي من مختلف الأنواع. ويوضح البروتوكول كيفية تنفيذ بيان فحص β-هيكسوزامينيداز، وتعرض نتائج التي كما يعني ± الانحراف المعياري للتجارب ثلاث نسخ. وقد تم تطوير هذا الفحص من خلال إطلاق أول Siraganian وهوك 25 لدراسة الحساسية الإنسان. كما وضعت المجموعة المختبر بقيادة الدكتور روبن Siraganian خط الخلية ربل. وقد وضعت هذه الخلايا ربل للتعبير عن FcεRIα البشرية ونشر بروتوكول بنسبة 4. الجزء الاخيروجاء الفحص مع وضع البلازميد ايندهوفن في الورقة التي نويبيرغر 26 التي وصفت إنتاج من الأجسام المضادة عن طريق الاستنساخ في سلسلة الجينات الثقيلة الخارجة من الجين الماوس لمنطقة متغيرة إيج التي تستهدف ناشبة 4-هيدروكسي-3 -nitro-phenacetyl (NP)، فإن الأجسام المضادة الناتجة خيالية وظيفية بالكامل. القدرة على تطوير أي فريق الخبراء الحكومي الدولي التي تستهدف نفس ناشبة، بينما الاستنساخ أيضا مستقبلات على سطح الخلايا ربل أدى إلى توحيد الفحص مما يجعل من بروتوكول مفيد لقياس تحبب الخلايا قعدة. مقايسة لديه إيجابيات وسلبيات. الايجابيات من الفحص هو قدرتها على التكيف لاستخدامها في أي نظام الثدييات، وبالتالي تستخدم المختبر لاختبار للتحبب في النظم البشرية والكلاب والخيول، وهذا أمر يمكن تحقيقه ببساطة عن طريق تجميع فريق الخبراء الحكومي الدولي الكائن الحي واستنساخ مستقبله على سطح الخلايا ربل. من ناحية أخرى،سلبيات الفحص هو أن خلايا RBL حساسة جدا للتغيرات الحرارية والميكانيكية ودرجة الحموضة، مما يجعلها تعطي تباين مستويات تحبب داخل نفس الفحص. وبالتالي ينصح بشدة أن المقايسات وتتكرر دائما في يثلث ثم يؤخذ بمعدل منها. وعلاوة على ذلك، فإن الخلايا ربل تميل إلى التحول نحو النمط الظاهري غير الإفراج إذا ما تركت في زراعة الأنسجة لمرات طويلة (&gt; 10 أسبوعا) 27، مما يجعل صيانتها مرهقة. هم أيضا عرضة للعدوى من البكتيريا الميكوبلازما، والتي هي غير مرئية من الضوء والمجهر لا تغيير مورفولوجيا الخلية، ولكن من شأنه أن يغير بشكل جذري مستويات تحبب لهم. وبالتالي هناك حاجة إلى اختبارات الميكوبلازما العادية.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>Equine IgE anti NIP-HSA</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Sigma</td>
			<td>CLS3595</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Multi Channel Pipette</td>
			<td>Anachem</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Galaxy R</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td>
			<td>Cambridge Research Biochemicals</td>
			<td>N-1070-1</td>
			<td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td>
		</tr>
		<tr>
			<td>Dinitrophenyl Conjugated to Human Serum Albumin</td>
			<td>Sigma</td>
			<td>A6661</td>
			<td>Abbreviated DNP-HSA</td>
		</tr>
		<tr>
			<td>Plate Spectrophotometer</td>
			<td>Anthos Labtec HT2</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triton x100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4-nitrophenyl N-acetyl-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-hexosaminidase substrate was 50mM prepared in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma</td>
			<td>251275</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma</td>
			<td>S7670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

تمت توعية الخلايا ربل-2H3.1 الأبوية وتلك transfected مع والخيول FcεRIα مستقبلات الجينات أولا مع الماوس فريق الخبراء الحكومي الدولي لمكافحة DNP-HSA وتحدى مع المستضد إدارة التخطيط الوطني، هائل سعيد أنعم. الماوس إيج تربط لمستقبلات الفئران الذاتية في كل من خطوط الخلايا وبالتالي تقو...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÎµRI at JoVE.com

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

تطوير<em&gt; في المختبر</em&gt; نظام نموذجي لدراسة تفاعل<em&gt; ايكوس</em&gt;<em&gt; الحصانية</em&gt; إيج مع مستقبلات لها عالية تقارب FcεRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

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Immunology and Infection

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

12.3K Views.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Video: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

وضع علامة سلبية لإختيار a المتحولة الحالة للنسج</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

تشكيل بيوفيلم في المختبر في غرفة الشرائح 8 جيد

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

رواية<em&gt; في المختبر</em&gt; نموذج لدراسة التفاعلات بين الدم الكامل الإنسان والبطانة Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

في تحليل المختبر من الاستجابة المناعية الخلوية بوساطة Myd88 لفيروس غرب النيل المسخ سلالة العدوى

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

ل<em&gt; في المختبر</em&gt; نموذج لقياس الاستجابات المناعية لمكافحة الملاريا في سياق الفيروس المسبب للإيدز

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

دراسة المجتمعات الميكروبية<em&gt; في فيفو</em&gt;: نموذج من التفاعل بوساطة بين المضيف<em&gt; المبيضات البيض</em&gt; و<em&gt; الزائفة الزنجارية</em&gt; في الخطوط الجوية

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

ل<em&gt; في المختبر</em&gt; نموذج من حاجز الدم في الدماغ عن طريق التحليل الطيفي المعاوقة: التركيز على التفاعل خلية تي خلية البطانية

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

تصور آثار البلغم للتنمية بيوفيلم باستخدام نموذج غرف ساترة

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

تطوير نظام تسليم الاقتصادية DNA من قبل &quot;Acufection&quot; وتطبيقه لبحوث الجلد

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...