نشكر Wambsganss نادين للحصول على المساعدة التقنية الممتازة. وأيد هذا العمل من خلال منحة من جمعية الألمانية للبحوث (GL599/1-1) وجزئيا من خلال منحة من صندوق الابتكار الحدودي (جامعة هايدلبرغ) إلى CAG

1. بروتوكول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد المخازن المؤقتة على النحو التالي: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد العازلة للعزلة PBMC: &quot;غسل العازلة&quot; = 0.02٪ EDTA في برنامج تلفزيوني (استخدام 0.5 M EDTA). </li><li style=";text-align:right;direction:rtl"> إعداد عازلة لعزل الوحيدات: &quot;MACS الشطف العازلة&quot; = 0.5٪ BSA (250 ملغ) + 2 مم EDTA (200 ميكرولتر) + برنامج تلفزيوني (50 مل). ديغا المخزن المؤقت. </li><li style=";text-align:right;direction:rtl"> إعداد العازلة للFACS تلطيخ وتخزين الخلايا: &quot;نظام مراقبة الأصول الميدانية العازلة&quot; = 10٪ FCS في برنامج تلفزيوني و&quot;العازلة التثبيت&quot; = 1٪ PFA في برنامج تلفزيوني. </li></ol></li><li style=";text-align:right;direction:rtl"> رسم 30 مل من الدم الكامل من الوريد الساعد. استخدام EDTA كما تخثر. </li><li style=";text-align:right;direction:rtl"> عزل PBMC (histopaque) على النحو التالي: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد اثنين العقيمة 50 مل أنابيب (البولي ستايرين لا). </li><li style=";text-align:right;direction:rtl"> تمييع الدم من الخطوة 2 مع برنامج تلفزيوني 1:1. </li><li style=";text-align:right;direction:rtl"> إضافة 25 مل + 25 مل histopaque الدم الكامل / برنامج تلفزيوني في أنبوب. تأكد من أن histopaque والدم لا يختلطان. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي في 400 x ج لمدة 30 دقيقة في RT، لا الفرامل. </li><li style=";text-align:right;direction:rtl"> نضح البلازما وتجاهل. </li><li style=";text-align:right;direction:rtl"> نضح واجهة غير واضحة ونظيفة ماصة في 50 مل حوض استحمامه. </li><li style=";text-align:right;direction:rtl"> إضافة 20 مل من EDTA 0.02٪ في برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي في 250 x ج لمدة 5 دقائق في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> تجاهل طاف. </li><li style=";text-align:right;direction:rtl"> إضافة 10 مل من EDTA 0.02٪ في برنامج تلفزيوني. </li></ol></li><li style=";text-align:right;direction:rtl"> عد الخلايا على النحو التالي: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مزيج جيد من قبل vortexing لمدة 10 ثانية. </li><li style=";text-align:right;direction:rtl"> تأخذ 200 ميكرولتر تعليق خلية + 300 ميكرولتر EDTA 0.02٪ في برنامج تلفزيوني + 500 ميكرولتر من التريبان الأزرق (200-500 cells/10 الساحات، وتغيير وإلا عامل التخفيف). </li></ol></li><li style=";text-align:right;direction:rtl"> أداء العزلة السلبية للحيدات كما يلي: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الخلايا التي تم الحصول عليها في الخطوة 3.10 لمدة 10 دقيقة، 120 ز س. تجاهل طاف. </li><li style=";text-align:right;direction:rtl"> غسل بيليه الخلية 2X بإضافة 10 مل من برنامج تلفزيوني + 0.02٪ EDTA وأجهزة الطرد المركزي لمدة 10 دقيقة، 120 ز س. </li><li style=";text-align:right;direction:rtl"> إضافة 9 مل ماء معقم لمدة 3 ثوانى، إضافة 1 مل 10X PBS. </li><li style=";text-align:right;direction:rtl"> يغسل مرة واحدة مع أكثر PBS + 0.02٪ EDTA وأجهزة الطرد المركزي 10 دقيقة، 120 ز س. </li><li style=";text-align:right;direction:rtl"> الاعتماد خلال الطرد المركزي. </li><li style=";text-align:right;direction:rtl"> تمييع في المخزن EasySep في 5 × 10 7 / مل. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر / مل الديك تخصيب الوحيداتالذيل. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 10 دقيقة في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> حبات دوامة لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر الخرز / مل. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 5 دقائق في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> تملأ مع EasySep عازلة لإكمال حجم 2.5 مل. ويبدو أن الحل الآن البني. </li><li style=";text-align:right;direction:rtl"> وضعت في المغناطيس. </li><li style=";text-align:right;direction:rtl"> الانتظار 2.5 دقيقة في RT. خلال هذا الوقت الخلايا غير منضمة إلى الوحيدات حبة المغناطيسي سينتقل إلى الجدار أنبوب والعصا هناك. </li><li style=";text-align:right;direction:rtl"> صب العازلة مع حيدات غير ملزم في أنبوب العقيمة الطازجة. هذا الحل يبدو بيضاء. </li><li style=";text-align:right;direction:rtl"> يغسل مرة واحدة مع برنامج تلفزيوني + 0.02٪ EDTA وأجهزة الطرد المركزي لمدة 10 دقيقة، 120 ز س. </li></ol></li><li style=";text-align:right;direction:rtl"> لوحة الخلايا في طبق من البلاستيك أو لوحة متعدد الطبقات في مناطق ذات كثافة من 0.5 × 10 6 / سم 2. إضافة 1 مل من وسائل الإعلام ثقافة / 1 × 10 6 خلايا. </li><li style=";text-align:right;direction:rtl"> خلايا الثقافة في ظل ظروف من الفائدة ل6-14 أيام. </li><li style=";text-align:right;direction:rtl"> تغيير وسائل الإعلام بعد 3 أيام عن طريق استبدال 50٪ من وسائل الإعلام مع وسائل الإعلام الجديدة (انظر الجدول رقم 1 للحصول على التفاصيل). </li><li style=";text-align:right;direction:rtl">بعد ستة أيام حصاد الخلايا لعزل مرنا، التدفق الخلوي، النشاف الغربية، الخ. </li></ol>

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+intraplaque+hemorrhage+evokes+a+novel+atheroprotective+macrophage+phenotype.">Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype.</a> American Journal of Pathology. 174, 1097-1108 (2009).</li><li>Cho, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+dendritic+cell-like+phenotype+in+macrophages+during+foam+cell+formation.">Induction of dendritic cell-like phenotype in macrophages during foam cell formation.</a> Physiol Genomics. 29, 149-160 (2007).</li><li>Vogt, G., Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+human+macrophages+with+enhanced+antimycobacterial+activity.">In vitro differentiation of human macrophages with enhanced antimycobacterial activity.</a> The Journal of Clinical Investigation. 121, 3889-3901 (2011).</li><li>Mikita, T., Porter, G., Lawn, R. M., Shiffman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+low+density+lipoprotein+exposure+alters+the+transcriptional+response+of+macrophages+to+inflammatory+stimulus.">Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus.</a> Journal of Biological Chemistry. 276, 45729-45739 (2001).</li><li>Qin, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+THP-1+cells+as+a+model+for+mimicking+the+function+and+regulation+of+monocytes+and+macrophages+in+the+vasculature.">The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature.</a> Atherosclerosis. 221, 2-11 (2012).</li><li>Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K. B., Dockrell, D. 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الكتاب لم يكن لديك للكشف عن أي تضارب في المصالح.

الضامة الوحيدات المستمدة تمثل نوع من الخلايا الرئيسية لنظام المناعة الفطرية. أنها تلعب دورا هاما في كثير من الأمراض الالتهابية بما في ذلك تصلب الشرايين أو السرطان 2. وهكذا، ودراسة علم الأحياء بلعم أمر ضروري لزيادة المعرفة لدينا في الفيزيولوجيا المرضية لكثير م?...

تمثل الضامة الوحيدات المستمدة عنصرا الخلوية مهم من نظام المناعة الفطرية والمساهمة في العديد من العمليات الالتهابية الحادة أو المزمنة 1. الضامة تلعب دورا هاما في كثير من الأمراض الالتهابية مثل تصلب الشرايين أو السرطان 2. تظهر الضامة درجة عالية من المرونة وقادرة على تحمل الظواهر مختلفة اعتمادا على micromilieu المحلية 3. وهكذا، ودراسة التمايز بلعم وعدم التجانس أمر ضروري لزيادة معرفتنا الفيزيولوجيا المرضية لكثير من الأمراض وحتى يمكن التعرف على أهداف علاجية جديدة وتطوير علاجات جديدة. في كثير من الحالات، يتم استخدام نماذج الفئران للتحقيق في الفيزيولوجيا المرضية لأمراض معينة. ومع ذلك، ودراسة علم الأحياء بلعم باستخدام نماذج الماوس يرافقه العديد من أوجه القصور: (1) نسبة أعداد فرعية الكريات البيض (أي حيدات والمحببة) في peripهيرال الدم من الفئران أو البشر تختلف بشكل ملحوظ مما يدل الأدوار المختلفة للحيدات في الفيزيولوجيا المرضية الفئران والإنسان. (2) هناك اختلافات جوهرية في التعبير الجيني بين الفئران والإنسان حيدات الدم المحيطي مما يوحي اختلافات جوهرية في وظيفتها أثناء الصحة والمرض 4. (3) وهناك عدد من العلامات التي تستخدم لتحديد حيدات الفئران والضامة (F4/80، LYC، الخ.) غير موجود في خلايا الدم النخاعي الإنسان، مما يجعل نقل النتائج في نماذج الماوس إلى الوضع الإنساني الصعب إلى حد ما. وبالتالي، من أجل زيادة فهمنا للتمايز بلعم وعدم التجانس في الأمراض التي تصيب البشر، ونحن بحاجة إلى الاستفادة من نماذج العمل مع الضامة الإنسان. ولذلك، فإننا هنا تصف نموذجا للجيل بلعم الإنسان الأساسية التي هي سهلة الاستخدام وتتيح دراسة الضامة الوحيدات المستمدة البشرية في المختبر تحت ظروف مختلفة مما يؤدي إلى مختلف بلعم ص.أنواع larization. في العديد من الدراسات، ونحن قد استخدمت في نموذج المختبر من الوحيدات المستمدة الضامة الإنسان الأساسية لتحليل البيولوجيا بلعم وصلته المحتملة للتصلب الشرايين الإنسان 5-7. حتى وإن لم يكن تماما لتحل محل التجارب على الحيوانات أو الأنسجة البشرية التي تم الحصول عليها بعد الوفاة، ونموذج الموصوفة هنا يسمح تحديد الهوية والتحقق من آليات المرض والأهداف العلاجية التي قد تكون ذات أهمية كبيرة لمختلف الأمراض التي تصيب الإنسان.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube (sterile)</td>
			<td>Fisher</td>
			<td>055398</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS (1X), liquid (no calcium or magnesium)</td>
			<td>Invitrogen</td>
			<td>14190-250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>T9285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-8806</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Human Monocyte Enrichment Kit</td>
			<td>StemCell Technologies</td>
			<td>19059</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fisher</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Macrophage-SFM (1X)</td>
			<td>Invitrogen</td>
			<td>12065-074</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P-4458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nutridoma-SP</td>
			<td>Roche</td>
			<td>11011375001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>human M-CSF 10 &mu;g</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Culture Plates 6-well</td>
			<td>Fisher</td>
			<td>07-200-80</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an in vitro model to study heterogeneity of human macrophage differentiation and polarization

الضامة الوحيدات المستمدة تمثل نوع من الخلايا مهم من نظام المناعة الفطرية. نماذج الماوس دراسة البيولوجيا بلعم يعانون من الاختلافات المظهرية والوظيفية بين الضامة الوحيدات المستمدة الفئران والإنسان. ولذلك، فإننا هنا وصف نموذج في المختبر لتوليد ودراسة الضامة الإنسان الأساسية. لفترة وجيزة، بعد الطرد المركزي المتدرج الكثافة من الدم المحيطي مستمدة من وريد الساعد، يتم عزل حيدات الخلايا وحيدات النوى من الدم المحيطي باستخدام السلبية العزلة حبة المغناطيسي. ثم يتم استزراع هذه حيدات لمدة ستة أيام في ظل ظروف معينة للحث على أنواع مختلفة من التمايز بلعم أو الاستقطاب. هذا النموذج هو سهل الاستخدام وتلتف المشاكل الناجمة عن الاختلافات بين أنواع محددة الماوس والرجل. وعلاوة على ذلك، فمن أقرب إلى الجسم الحي في ظروف من استخدام خطوط الخلايا خلده. في الختام، النموذج الموصوف هنا هو مناسبة لدراسة macrophagه علم الأحياء، وتحديد آليات المرض وأهداف علاجية جديدة. حتى وإن لم يكن تماما لتحل محل التجارب على الحيوانات أو الأنسجة البشرية التي تم الحصول عليها بعد الوفاة، ونموذج الموصوفة هنا يسمح تحديد الهوية والتحقق من آليات المرض والأهداف العلاجية التي قد تكون ذات أهمية كبيرة لمختلف الأمراض التي تصيب الإنسان.

باستخدام بروتوكول الموصوفة أعلاه، ونحن بشكل روتيني الحصول على 25.1 الدم × 10 6 ± 2.2 × 10 6 monocytes/100 مل (متوسط ​​± الخطأ المعياري من 26 تجارب مستقلة، الشكل 1A). نقاء الوحيدات على النحو الذي يحدده تلطيخ تدفق cytometric لCD14 هو روتيني أكبر من 95٪ (97.1 ± 0.4٪، ومتوسط ​​?...

Watch this Scientific Journal Video about An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization at JoVE.com

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

الضامة الوحيدات المستمدة هي خلايا مهم من نظام المناعة الفطرية. هنا، نحن تصف وسيلة سهلة لاستخدام<em&gt; في المختبر</em&gt; نموذج لتوليد هذه الخلايا. باستخدام الطرد المركزي المتدرج، والعزلة حبة السلبية والظروف والثقافة خلية معينة، يمكن أن تتولد الضامة الوحيدات المستمدة للدراسات المظهرية والوظيفية.

على<em&gt; في المختبر</em&gt; نموذج لدراسة عدم تجانس التمايز بلعم الإنسان والاستقطاب

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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JoVE Journal

Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

18.6K Views.  University of Heidelberg. الضامة الوحيدات المستمدة هي خلايا مهم من نظام المناعة الفطرية. هنا، نحن تصف وسيلة سهلة لاستخدام في المختبر نموذج لتوليد هذه الخلايا. باستخدام الطرد المركزي المتدرج، والعزلة حبة السلبية والظروف والثقافة خلية معينة، يمكن أن تتولد الضامة الوحيدات المستم?...

Video: An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

و<em&gt; في المختبر</em&gt; المشارك عدوى نموذج لدراسة<em&gt; المتصورة المنجلية</em&gt; HIV-1-التفاعلات في الإنسان خلايا الوحيدات المشتقة الابتدائية المناعي

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

التحقيق في الاستقطاب البلاعم عن طريق نخاع العظم الضامة المشتقة

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

في المختبر والمجراة نموذج لدراسة التصاق البكتيريا إلى سفينة ستريت تحت ظروف التدفق

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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. ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

تحليل الخلايا الليمفاوية التسرب باستخدام<em&gt; في المختبر</em&gt; نموذج من الدم في الدماغ الإنسان الحاجز

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

اختﻻق الواجهات الزجاجية الجودة البصرية لدراسة الانصهار بلعم

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

في المختبر التفريق بين الماوس المحببات-بلعم-مستعمرة-حفز عامل (GM-CSF)-إنتاج خلايا مساعد (GMحتي) T

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.
Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.
Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.
Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.
This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

دراسة إيمونوهيستوباثولوجيك الشخصية بلعم بيتا مستقبلات حمض الفوليك والأوعية الدموية المكروية محصنة في الخلية العملاقة الدموية

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...