وقد تم تمويل هذا العمل من قبل منحة PRB CS-009 لومنحة دراسية لشهادة الدكتوراه LC، من الاتحاد الوطني للبحوث والتكنولوجيا ذ Secretaría دي Ciencia (SENACYT)، بنما.

جميع الخطوات من البروتوكول، باستثناء centrifugations، ينبغي القيام بها داخل غطاء للحفاظ على عينة معقمة. 1. في وقت متأخر من مرحلة عزل P. المنجلية المصابة تبدي الكريات الحمر ويمكن فصل جميع المراحل المتأخرة من الكريات الحمراء المنجلية المصابة مع هذه المنهجية، منذ هيموزوين، الذي يمنح المغطسة على الطفيلي، هو المستقلب المشتركة بين جنس. ينصح تطفلن الدم عالية (3-10٪) في الثقافة للحصول على أفضل غلة مع هذا البروتوكول، على الرغم من أن الثقافة التقليدية وتحتوي على 2-4٪ الهيماتوكريت (نسبة حجم خلايا الدم الحمراء) وتطفلن الدم 1-8٪ (النسبة المئوية من المصابين خلايا الدم الحمراء). يمكن تربيتها الطفيليات كما وصفها Trager ويانسن 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يجب أن يتم تجميعها A البسيطة، متعددة الأجزاء الإعداد لإجراء العزل كل المتقسمة. لهذا، والأعمدة LS، فاصل المغناطيسي MidiMACS وMultiStand MACS من بيوتيك Miltenyi (الشكل 1 </STRONG&gt;) يتم استخدامها. أولا، نعلق الفاصل المغناطيسي للحامل معدني ومن ثم وضع العمود المغناطيسي على الفاصل. </li><li style=";text-align:right;direction:rtl"> في أنبوب منفصل (استخدام 50 مل في جميع أنحاء conicals)، مزيج 23،2 مل من RPMI 1640 و 750 ميكرولتر من بيكربونات الصوديوم 7.5٪ لإنشاء حل العمل. لديك أنابيب إضافية على يد لنبذ وجمع التدفق من خلال ثقافة الطفيلي. </li><li style=";text-align:right;direction:rtl"> أضف 3 مل من الحل إلى العمود العمل لتتوازن فيه، وتجاهل التدفق من خلال أنبوب إلى. </li><li style=";text-align:right;direction:rtl"> إضافة 8 مل من P. ثقافة المنجلية إلى العمود، والتأكد من أن بمجرد أن الجزء الأخير من الحل تم عمل طردوا من العمود من قبل الثقافة، ويتم استبدال الأنبوب تلقي من جانب واحد لبدء جمع انتعاش ثقافة غير منضم. هذا ضروري لتجنب إضعاف الثقافة أكثر من ذلك. </li><li style=";text-align:right;direction:rtl"> مرة واحدة في تدفق الثقافة من خلال العمود قد توقف، إضافة 1 مل من محلول العمل لدفع كرات الدم الحمراء المتبقية من العمود. </li><li style=";text-align:right;direction:rtl"> غسل الخطوة: إضافة آخر مل 3 من محلول العمل إلى العمود وجمع السائل التي تصب في أنبوب &quot;تجاهل&quot; من أجل تجنب إضعاف الثقافة يتم حفظ والتدفق من خلال أنبوب في أخرى. </li><li style=";text-align:right;direction:rtl"> شطف: مرة واحدة في 3 مل مرت من خلال العمود، فصل الأخير من الموقف ووضعه على رأس أنبوب 15 مل. </li><li style=";text-align:right;direction:rtl"> مرة أخرى، أضف 3 مل من محلول العمل إلى العمود. هذه الخطوة elutes والكريات الحمراء المصابة بالطفيليات المرحلة المتأخرة التي تم المحاصرين في حبات من العمود. </li></ol> ملاحظة: عند هذه النقطة، وهذا يتوقف على مقدار من الطفيليات المطلوبة، تبدأ جولة أخرى / ثانية من جمع وتخضع بشكل مباشر للحدود تطفلن الدم في الثقافة الأصلية. يمكن إعادة استخدامها العمود طالما تدفق يحافظ على حالة مستقرة. <ol start="9" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تركيز الطفيليات التي تم جمعها: الطرد المركزي في أنبوب 15 مل تحتوي على شطافة لمدة 5 دقائق في 150 XG في درجة حرارة الغرفةلتشكيل بيليه الظلام من الكريات الحمراء الطفيلي. </li><li style=";text-align:right;direction:rtl"> إزالة طاف ترك ما يكفي من السوائل لأخذ عينة صغيرة جدا وتحديد تركيز والمحصول الكلي من خلال المجهر (راجع الخطوة 2 أدناه). </li><li style=";text-align:right;direction:rtl"> في هذه اللحظة، إذا تم إنجاز العزلة والقبض على الطفيليات مرحلة متأخرة مرض، احتضان الثقافة جمعها مرة أخرى مع وسائل الإعلام الجديدة. </li><li style=";text-align:right;direction:rtl"> يخفف من الطفيليات التي تم جمعها في حجم والحل اللازم لتجارب لاحقة. </li></ol> 2. النقاء وتحليل الربح الموزع <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحقق من تركيز الطفيليات التي تم الحصول عليها باستخدام عدادة الكريات والفرز تحت المجهر الخفيفة. لحساب، إضافة 10 ميكرولتر من مزيج من الطفيليات التي تم جمعها وسائل الإعلام تحت غطاء زلة لعدادة الكريات. حساب عدد الطفيليات في الأرباع الأربعة من عدادة الكريات وتقسيم العدد الإجمالي بنسبة 4. مضاعفة هذا العدد بمقدار 10 4 للحصول على تركيز ه المصابةrythrocytes لكل مل. </li></ol> ملاحظات: سيتم تحديد أ) تركيز المزيج من قبل المستخدم في لحظة resuspending بيليه، وذلك بإضافة أكثر أو أقل المتوسطة. A تركيز اقترح العمل هو 5.5 X 10 4 schizontes في ميكرولتر (ويتحقق هذا عادة عن طريق إضافة 100-300 ميكرولتر إلى جمع الطفيليات). إذا تم إضافة 20 ميكرولتر من هذا التركيز إلى وحدة تخزين النهائي من 100 ميكرولتر مزيج من وسائل الاعلام والكريات الحمراء، وتطفلن الدم من حوالي 1٪ في ثقافة نموذجية الهيماتوكريت 4٪ يمكن الحصول عليها. ب) في إطار العد عدادة الكريات يجب أن يكون سريعا، منذ بدء lysing الكريات الحمراء كما أن يجف عينة داخل الغرفة. والكريات الحمراء المصابة، التي ينبغي أن تكون الأغلبية، تظهر داخل بقعة داكنة، والتي تميزهم عن غير المصابة منها. <ol start="2" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنفيذ طبقة رقيقة بالغيمزا وصمة عار لتقييم نقاء جمع عن طريق تحديد الشرائح بسرعة كبيرة في الميثانول بنسبة 10٪، والتجفيف لهم، وتخبط روصمة عار المخفف تنحنح في حل 20٪ من بالغيمزا مع الماء المقطر. وصمة عار الشرائح لمدة 10 دقيقة، وبعد ذلك يمكن أن تجفف الهواء من قبل وفحصها تحت المجهر. وينبغي أن يكون تطفلن الدم أعلى بكثير من 70٪ يمكن تحقيقه. </li></ol>

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ليس لدينا شيء في الكشف عنها.

في الثقافات المختبر من الملاريا الطفيلية P. يحمل المنجلية تطفلن الدم محدودة، مع أكثر من نصف خلايا الدم الحمراء غير مصاب في أعلى نقطة انتشار الثقافة. معظم البحوث للتجارب، من المستحسن أن تعمل فقط مع الكريات الحمراء المصابة. تحقيقا لهذه الغاية، وهي ت?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> التعليقات (اختياري) </td></tr><tr><td> RPMI 1640 HEPES تم التعديل </td><td> سيغما الدريخ </td><td> R4130 </td><td> تستكمل مع مصل الإنسان 10٪ والجلوكوز 2٪، و 0.2٪ بيكربونات الصوديوم </td></tr><tr><td> MidiMACS فاصل </td><td> MACS Miltenyi بيوتيك </td><td> 130-042-302 </td><td></td></tr><tr><td> MACS MultiStand </td><td> MACS Miltenyi بيوتيك </td><td> 130-042-303 </td><td></td></tr><tr><td> LS أعمدة </td><td> MACS Miltenyi بيوتيك </td><td> 130-042-401 </td><td></td></tr><tr><td> عدادة الكريات </td><td> Grafco </td><td> Grafco نويباور غرفة </td><td> ويمكن الاطلاع من خلال العديد من الموردين الآخرين </td></tr></tbody></table>

separation of plasmodium falciparum late stage-infected erythrocytes by magnetic means

على عكس الأنواع الأخرى المتصورة، P. يمكن زرعها في المختبر المنجلية، مما يسهل دراستها 1. في حين أن تحقيق تطفلن الدم يمكن أن تصل إلى حد 40٪ ≈، المحقق يحتفظ عادة نسبة حوالي 10٪. في كثير من الحالات يكون من الضروري عزل الطفيلي التي تحتوي على خلايا الدم الحمراء (كرات الدم الحمراء) عن تلك غير مصاب، لإثراء الثقافة والمضي قدما في تجربة معينة. عندما P. يصيب كرات الدم الحمراء المنجلية، ويحط الطفيلي يتغذى من الهيموغلوبين 2، 3. ومع ذلك، يجب أن الطفيلي التعامل مع شاردة الحديد سامة جدا هيم التي تحتوي على 4 و 5. الطفيلي يتملص سميته عن طريق تحويل هيم إلى البوليمر الكريستال خاملة دعا haemozoin 6 و 7. يتم تخزين هذا الجزيء التي تحتوي على الحديد في فجوة الغذائية والمعدنية في دولة لديها الأكسدة التي تختلف عن تلك الموجودة في هيم 8. الدولة الحديديك الحديد في هيمozoin يضفي عليه خاصية ممغطس غائبة في كرات الدم الحمراء غير المصابة. كما الطفيلي الغازية تصل إلى مرحلة النضج، ومضمون haemozoin يزيد أيضا 9، الذي يمنح المزيد من المغطسة على آخر مراحل P. داخل كرات الدم الحمراء المنجلية. استنادا إلى هذه الخاصية ممغطس، أحدث مراحل P. ويمكن فصل الخلايا المنجلية المصابة الدم الحمراء عن طريق تمرير الثقافة من خلال عمود تحتوي على الخرز المغناطيسي. هذه الخرز المغناطيسي تصبح عندما يتم وضع الأعمدة التي تحتوي عليها على حامل المغناطيس. وبعد ذلك كرات الدم الحمراء المصابة، وذلك بسبب المغطسة بهم، أن المحاصرين داخل العمود، في حين أن التدفق من خلال ستتضمن، بالنسبة للجزء الاكبر، وكرات الدم الحمراء غير مصاب تلك المراحل المبكرة من المحتوية على الطفيلي. هنا، نحن تصف منهجية لإثراء السكان من الطفيليات مرحلة متأخرة مع الأعمدة المغناطيسي، والذي يحافظ على بقاء الطفيليات جيدة 10.بعد تنفيذ هذا الإجراء، يمكن إرجاعها إلى ثقافة غير مرتبط حاضنة للسماح للطفيليات المتبقية لمواصلة النمو.

في الشكل 2، يظهر الثقافة التي تمر عبر العمود المغناطيسي، قبل (A) وبعد العملية. (B) وينظر عادة واحد إلى اثنين الكريات الحمراء المصابة في حقل التكبير 100X كما هو مبين في الشكل 2، ويتجهان إلى الكريات الحمراء المصابة في الشكل 2A. في ...

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Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

وتستخدم خصائص ممغطس من هيموزوين لعزل المراحل المتأخرة من<em&gt; المتصورة المنجلية</em&gt; المصابة خلايا الدم الحمراء نموا في الثقافة. هذه الطريقة بسيطة وسريعة ولا يؤثر على قدرات اللاحقة الغازية من الطفيليات.

فصل<em&gt; المتصورة المنجلية</em&gt; في وقت متأخر مرحلة المصابين تبدي الكريات الحمر بوسائل المغناطيسي

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

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Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

13.8K Views.  Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). وتستخدم خصائص ممغطس من هيموزوين لعزل المراحل المتأخرة من المتصورة المنجلية المصابة خلايا الدم الحمراء نموا في الثقافة. هذه الطريقة بسيطة وسريعة ولا يؤثر على قدرات اللاحقة الغازية من الطفيليات.

Video: Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

توسيع فيفو السابقين الخلايا T ورم التفاعل من قبل وسائل 1/Ionomycin Bryostatin وصياغة سلسلة غاما المشتركة السيتوكينات

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

اختيار المتصورة المنجلية الطفيليات لCytoadhesion لخلايا المخ البشري غشائي

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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 ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

بروتوكول بسيط لتجمع الصفائح الدموية بوساطة<em&gt; المتصورة المنجلية</emتبدي الكريات الحمر&gt; بالفيروس في أجواء فقيرة الموارد

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

العزلة وتحليل الدماغ المعزول من الكريات البيضاء<em&gt; المتصورة berghei</em&gt; ANKA الفئران المصابة

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

الفصل الآلي لل<em&gt; C. ايليجانس</em&gt; المستعمرة بنسب مختلفة من قبل الممرض الجرثومي

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

ارتفاع العائد تنقية<em&gt; المتصورة المنجلية</em&gt; Merozoites للاستخدام في فحوصات ضد طهائي

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

في تقييم فيفو من القوارض المتصورة الطفيل وأقسومة الغزو من قبل التدفق الخلوي

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

طرق بالتحقيق في الدور التنظيمي للالرنا الصغيرة وخاص بالريبوسوم إشغال<em&gt; المتصورة المنجلية</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

تلألؤ بيولوجي التصوير لكشف العدوى المرحلة المتأخرة من داء المثقبيات الأفريقي

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...