עבודה זו מומנה על ידי מענק PRB-009 לCS ומלגת דוקטורט לLC, מSecretaría Nacional de Ciencia y Tecnología (SENACYT), פנמה.

כל הצעדים של הפרוטוקול, למעט centrifugations, צריכים להתבצע בתוך מכסה מנוע כדי לשמור על מדגם סטרילי. 1. בידוד שלב מאוחר של פ אריתרוציטים falciparum נגועים כל השלבים המאוחרים של אריתרוציטים Plasmodium הנגועים ניתן להפריד במתודולוגיה זו, מאז hemozoin, שמעניק paramagnetism על הטפיל, הוא מטבוליט משותף לסוג. Parasitemia גבוה (3-10%) בתרבות שמומלץ להגיע תשואות טובות יותר עם פרוטוקול זה, למרות שתרבות טיפוסית תכיל 2-4% המטוקריט (אחוז הנפח של תאי דם אדומים) ו1-8% parasitemia (אחוז נגוע תאי דם אדומים). טפילים יכולים להיות מתורבתים כפי שתואר על ידי טרייגר וינסן 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> התקנה פשוטה, חלק רב צריכה להרכיב אותו על כל הליך בידוד schizont. לשם כך, עמודות LS, מפריד MidiMACS מגנטי וMultiStand MACS מMiltenyi BIOTEC (איור 1 </חזק&gt;) משמשים. ראשית, חבור את המפריד המגנטי למעמד המתכת ולאחר מכן הנח את העמודה המגנטית על המפריד. </li><li style=";text-align:right;direction:rtl"> בצינור נפרד (שימוש 50 מ&quot;ל conicals לאורך), לערבב 23.2 מ&quot;ל של RPMI 1640 ו 750 μl של 7.5% סודיום ביקרבונט כדי ליצור את פתרון העבודה. יש צינורות נוספים על ידו עבור שלכת ואיסוף הזרימה דרך מהתרבות הטפילה. </li><li style=";text-align:right;direction:rtl"> הוסף 3 מ&quot;ל של תמיסת העבודה לעמודה לאזן אותו, והשלך את הזרימה דרך לתוך צינור. </li><li style=";text-align:right;direction:rtl"> הוסף 8 מ&quot;ל של פ falciparum התרבות לעמודה, ולוודא כי ברגע שהחלק האחרון של פתרון העבודה נדחקה אל מחוץ לטור על ידי התרבות, הצינור שקבל הוא הוחלף על ידי אחד האיסוף להתחיל התאוששות של התרבות לא המאוגדת. זה הכרחי כדי להימנע מדילול התרבות יותר מזה. </li><li style=";text-align:right;direction:rtl"> לאחר הזרימה של התרבות דרך העמודה חדל, להוסיף 1 מ&quot;ל של פתרון העבודה כדי לדחוף החוצה את RBCs הנותר של העמודה. </li><li style=";text-align:right;direction:rtl"> כביסת צעד: הוסף עוד מ&quot;ל 3 לפתרון העבודה לעמודה ואוסף את הנוזל זורם לתוך הצינור &quot;נזרק&quot; על מנת להימנע מדילול התרבות שנשמרה כזרימה דרך בצינור האחר. </li><li style=";text-align:right;direction:rtl"> Elution: לאחר 3 המ&quot;ל עבר דרך העמודה, לנתק את האחרון מהדוכן ולמקם אותו על גבי צינור מ&quot;ל 15. </li><li style=";text-align:right;direction:rtl"> שוב, להוסיף 3 מ&quot;ל של תמיסת עבודה לעמודה. צעד זה elutes את אריתרוציטים נגועים בטפילים בשלב מאוחרים שהיו לכודים בחרוזים של העמודה. </li></ol> הערה: בשלב זה, ובהתאם לכמות הטפילים הנדרשים, מתחיל סיבוב נוסף / ים של אוסף, כמובן בכפוף למגבלות של parasitemia בתרבות המקורית. העמודה ניתן לעשות שימוש חוזרת כל עוד הזרימה שומרת על מצב יציב. <ol start="9" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ריכוז של הטפילים שנאספו: צנטריפוגה שפופרת המכילה 15 המ&quot;ל eluate למשך 5 דקות ב 150 XG בטמפרטורת חדרכדי ליצור גלולה כהה מאריתרוציטים parasitized. </li><li style=";text-align:right;direction:rtl"> הסר את supernatant משאיר מספיק נוזלים לקחת דגימה קטנה מאוד ולקבוע את הריכוז ואת התשואה כוללת באמצעות מיקרוסקופיה (ראה שלב 2 להלן). </li><li style=";text-align:right;direction:rtl"> ברגע זה, אם הבידוד ולכידתו של טפילים בשלב מאוחרים נעשה משביע רצון, דגירת התרבות נאספה שוב עם תקשורת טריה. </li><li style=";text-align:right;direction:rtl"> לדלל את הטפילים שנאספו בהיקף והפתרון הנדרש לניסויים באים. </li></ol> 2. טוהר וניתוח תשואה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא הריכוז של טפילים המתקבלים באמצעות hemocytometer וספירה תחת מיקרוסקופ אור. כדי לספור, להוסיף 10 μl של התמהיל של טפילים שנאספו והתקשורת תחת תלוש השער של hemocytometer. לספור את מספר הטפילים בארבעת הרביעים של hemocytometer ומחלק את המספר הכולל של 4. להכפיל מספר זה ב 10 4 לקבל הריכוז של דואר נגועrythrocytes למ&quot;ל. </li></ol> הערות: א) ריכוז של התמהיל שיוחלט על ידי המשתמש ברגע resuspending הגלולה, על ידי הוספה פחות או יותר בינוני. ריכוז עבודה הציעה הוא 5.5 X 10 4 schizontes לμl (זו מושגת בדרך כלל על ידי הוספת 100-300 μl לטפילים שנאספו). אם 20 μl ריכוז זה מתווסף לנפח סופי של תערובת μl 100 של תקשורת ואריתרוציטים, parasitemia של כ 1% ב4% המטוקריט תרבות טיפוסית יושג. ב) ספירה תחת hemocytometer צריך להיות מהיר, מאז אריתרוציטים להתחיל lysing עם התייבשות המדגם בתוך החדר. אריתרוציטים נגועים, אשר אמור להיות הרוב, יציגו בתוך כתם כהה, שמבדיל אותם מאלה שלא נדבקו. <ol start="2" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בצע Giemsa דק שכבת הכתם להעריך את הטוהר של האוסף על ידי תיקון את השקופיות בקצב מהיר מאוד במתנול 10%, מייבש אותם, וטבילה לאמכפלת בפתרון 20% מGiemsa כתם מדולל במים מזוקקים. להכתים את השקופיות עבור 10 דקות, לאחר שהם יכולים להיות מיובשים באוויר ונבדקים תחת המיקרוסקופ. Parasitemia הרבה מעל 70% צריך להיות בר השגה. </li></ol>

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אין לנו מה למסור.

בתרבויות מבחנה של המלריה הטפילה פ falciparum תערוכת parasitemia מוגבל, עם יותר ממחצית תאי הדם האדומים הנגועים בנקודה הגבוהה ביותר של התפשטות התרבות. עבור רוב ניסויי מחקר, רצוי לעבוד רק עם אריתרוציטים הנגועים. לשם כך, טכניקת הפרדה היא הכרחית כדי לחלק את התרבות בהתאם לז?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות (אופציונלי) </td></tr><tr><td> RPMI 1640 Hepes השתנה </td><td> סיגמה אולדריץ </td><td> R4130 </td><td> בתוספת סרום 10% אדם, 2% גלוקוז, וסודה לשתיית 0.2% </td></tr><tr><td> מפריד MidiMACS </td><td> MACS Miltenyi BIOTEC </td><td> 130-042-302 </td><td></td></tr><tr><td> MACS MultiStand </td><td> MACS Miltenyi BIOTEC </td><td> 130-042-303 </td><td></td></tr><tr><td> עמודות LS </td><td> MACS Miltenyi BIOTEC </td><td> 130-042-401 </td><td></td></tr><tr><td> Hemacytometer </td><td> Grafco </td><td> Grafco נויבאואר קאמרי </td><td> ניתן למצוא דרך ספקים רבים אחרים </td></tr></tbody></table>

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

בניגוד למינים אחרים Plasmodium, פ falciparum יכול להיות מתורבת במעבדה, המאפשרת המחקר שלה 1. בעוד parasitemia הושג יכול להגיע לגבול ≈ 40%, החוקר בדרך כלל שומר על אחוז בסביבות 10%. במקרים רבים יש צורך לבודד את תאי טפיל המכילים דם האדומים (RBCs) מאלה הנגועים, כדי להעשיר את התרבות ולהמשיך בניסוי נתון. כאשר פ falciparum מדביק הכדורי האדום, הטפיל מפרק ונזון מהמוגלובין 2, 3. עם זאת, הטפיל צריך לטפל במחצית רעילה מאוד המכיל ברזל haem 4, 5. הטפיל חומק רעילותו על ידי הפיכת haem לתוך פולימר אינרטי גביש קרא 6 haemozoin, 7. מולקולה המכיל ברזל זה מאוחסן בvacuole המזון והמתכת שביש מצב חמצונים שונים מהאחד בhaem 8. מצב הברזל של ברזל בhaemozoin מעניק לו רכוש פאראמגנטיים נעדר באריתרוציטים הנגועים. כטפיל הפולש מגיע לפדיון, תוכן haemozoin גם מגדיל 9, שמעניק עוד יותר paramagnetism בשלבים האחרונים של פ falciparum בתוך כדורי האדום. בהתבסס על נכס זה פאראמגנטיים, בשלבים האחרונים של פ תאי דם אדומים נגועים falciparum יכולים להיות מופרדים על ידי עובר דרך תרבות עמודה המכילה חרוזים מגנטיים. חרוזים אלו הפכו מגנטיים כאשר העמודות שמכילות אותם מונחות על בעל מגנט. RBCs הנגוע, בשל paramagnetism, אז יהיה לכוד בתוך הטור, ואילו זרימה דרך יכיל, על פי רוב, אריתרוציטים נגועים ואלו שלבים מוקדמים של המכילים את הטפיל. כאן, אנו מתארים את המתודולוגיה להעשרת האוכלוסייה של טפילים בשלב מאוחר עם עמודות מגנטיות, אשר שומרת על יכולת קיום טפיל טובה 10.לאחר ביצוע הליך זה, התרבות הפנויה יכולה להיות מוחזרת לאינקובטור על מנת לאפשר לטפילים שנותרו להמשיך ולצמוח.

באיור 2, התרבות עוברת דרך העמודה המגנטית מוצגת, לפני () ואחרי ההליך (B). אחת עד שניים אריתרוציטים נגועים בדרך כלל ראו בתחום הגדלת 100X כפי שמוצגים באיור 2, עם החצים המצביעים על אריתרוציטים הנגועים באיור 2A. בהליך טיפוסי, החל בתרב...

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

את המאפיינים של פאראמגנטיים hemozoin משמשים כדי לבודד שלבים מאוחרים של<em&gt; Plasmodium falciparum</emתאים&gt; נגועים-דם אדומים הגדלים בתרבות. השיטה היא פשוטה ומהירה ואינו משפיעה על היכולות פולשניות הבאות של הטפילים.

הפרדה<em&gt; Plasmodium falciparum</emאריתרוציטים&gt; שלב מאוחר נגועים באמצעי מגנטי

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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JoVE Journal

Immunology and Infection

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). את המאפיינים של פאראמגנטיים hemozoin משמשים כדי לבודד שלבים מאוחרים של Plasmodium falciparum נגועים-דם אדומים הגדלים בתרבות. השיטה היא פשוטה ומהירה ואינו משפיעה על היכולות פולשניות הבאות של הטפילים.

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.

אקס הרחבה vivo של גידול תאים T-reactive באמצעות 1/Ionomycin Bryostatin ואת גמא נפוצים שרשרת גיבוש ציטוקינים

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.

מבחר Plasmodium falciparum טפילים עבור Cytoadhesion לתאי המוח האנושי האנדותל

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.

<em&gt; במבחנה</em&gt; שיתוף זיהום דגם ללמוד<em&gt; Plasmodium falciparum</em&gt;-HIV-1 אינטראקציות אדם מונוציטים הנגזרות תאים ראשוניים החיסון

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.

פרוטוקול פשוט לclumping טסיות דם בתיווך של<em&gt; Plasmodium falciparum</emאריתרוציטים&gt; נגועים בסביבה מסכנה משאבים

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.

בידוד ואנליזה של Leukocytes המוחי מוחרם מ<em&gt; Plasmodium berghei</em&gt; עכברי אנקה נגוע

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.

הפרדה אוטומטית של<em&gt; ג elegans</em&gt; יישבו variably ידי פתוגן חיידקים

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.

טיהור תשואה גבוהה של<em&gt; Falciparum Plasmodium</em&gt; Merozoites לשימוש בOpsonizing נוגדן מבחני

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.

בVivo ההערכה של מכרסמים Plasmodium Parasitemia וMerozoite פלישה על ידי cytometry הזרימה

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.

שיטות כדי לחקור את תפקיד הרגולציה של RNA הקטן וריבוזומלי תפוסה של<em&gt; Falciparum Plasmodium</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.

הדמיה פליטת אור כדי לזהות זיהום בשלב מאוחר של מחלת השינה

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 ...