Bu çalışma tarafından finanse edildi hibe SORUN-009 CS ve Secretaria Nacional de Ciencia y Tecnología (SENACYT), Panama LC doktora bursu için.

Protokolünün tüm adımları, centrifugations hariç, örnek steril tutmak için bir başlık içinde yapılmalıdır. 1. P. Geç Evre İzolasyonu falciparum-enfekte Eritrositler Parazit paramanyetizma bahşeder hemozoin, cins için ortak bir metabolit olduğundan Plasmodium enfekte eritrositlerin tüm evrelerinde, bu metodoloji ile ayrılabilir. Tipik bir kültür% 2-4 hematokrit (kırmızı kan hücrelerinin hacminin yüzdesi) ve% 1-8 parazitemi (enfekte yüzdesi içerecektir ancak kültürde yüksek parazitemi (% 3-10), bu protokol ile daha iyi verim almak için tavsiye edilir kırmızı kan hücreleri). Trager ve Jansen 1 tarafından açıklandığı gibi parazitler yetiştirilebilir. <ol><li> Basit, çok parçalı kurulum her schizont izolasyon prosedürü için monte edilmelidir. Bunun için, LS kolonlar, bir manyetik MidiMACS Ayırıcı ve Miltenyi Biotec bir MACS MultiStand (Şekil 1 </strong&gt;) kullanılır. İlk olarak, metal stand için manyetik ayırıcı bağlamak ve ardından ayırıcı üzerinde kolon manyetik yerleştirin. </li><li> Ayrı bir tüp içinde (genelinde 50 ml conicals kullanın), RPMI 1640 23.2 ml ve iş çözümü oluşturmak için% 7.5 sodyum bikarbonat 750 ul karıştırın. Atılması için ise ek borular var ve akan toplama parazit kültür. </li><li> Bunu dengelemek için sütun çalışma çözeltisinin 3 ml ekleyin ve akan bir tüp içine atın. </li><li> P. 8 mL sütun falciparum kültürü, en kısa sürede çalışma çözeltisinin son bölümü olarak kültür ile sütun dışarı itilmiş olan emin, alıcı tüp bağsız kültür iyileşme başlatmak için bir toplama ile değiştirilir. Bu, daha fazla kültür seyreltilmesi önlemek için gereklidir. </li><li> Sütunu boyunca kültür akış bittikten sonra, sütunun geri kalan eritrositler dışarı itmek için çalışma çözeltisinin 1 ml eklemek. </li><li> Adım Yıkama: kolon için çalışma çözeltisi başka bir 3 ml ilave edilir ve diğer tüp içinde akan olarak kaydedilmiş olan kültür seyreltilmesi önlemek için &quot;ıskarta&quot; tüp içine akan sıvı toplamak. </li><li> Elüsyon: 3 ml kolonundan geçirilir sonra, stand ikinci çıkarın ve 15 ml tüp üstüne yerleştirin. </li><li> Yine, sütun için çalışma çözelti 3 ml eklenir. Bu adım sütun boncuk mahsur kalmışlardı geç evre parazit ile enfekte eritrositler elutes. </li></ol> Not: bu noktada, ve gereken parazitlerin miktarına bağlı olarak, orijinal kültür içinde parazitemi sınırları belli tabi toplama başka yuvarlak / lar, başlar. Akışını istikrarlı bir devlet korur gibi sütun sürece yeniden kullanılabilir. <ol start="9"><li> Toplanan parazitlerin konsantrasyonu: Oda sıcaklığında 150 x g&#39;de 5 dakika boyunca elüsyon içeren 15 ml&#39;lik santrifüjparazitlenmiş eritrositten karanlık bir pelet oluşturur. </li><li> Çok küçük bir örnek almak ve (aşağıya bakınız adım 2) mikroskopi ile konsantrasyonu ve toplam verim belirlemek için yeterli sıvı bırakarak Süpernatantı. </li><li> Geç evre parazit izolasyonu ve yakalama biçimde gerçekleştirilir, bu noktada, taze medya ile tekrar toplanan kültür inkübe edin. </li><li> Müteakip deneyler için gerekli hacim ve çözelti içinde toplanmış parazit seyreltilir. </li></ol> 2. Saflık ve Verim Analizi <ol><li> Hemasitometre kullanarak ve bir ışık mikroskobu altında sayılarak elde edilen parazitlerin konsantrasyonu doğrulayın. Saymak için, hemasitometre bir kapak kayma altında toplanan parazit ve medya karışımı 10 ul ekleyin. Hemasitometre dört kadran parazit sayısını sayın ve 4 ile toplam sayısını bölün. Enfekte e konsantrasyonu elde etmek için 10 4 ile bu sayı çarpmaml başına rythrocytes. </li></ol> Notlar: karışımı a) konsantrasyon daha fazla veya daha az orta ekleyerek, pelet resuspending yapıldığı andaki kullanıcı tarafından belirlenecektir. Önerilen bir çalışma konsantrasyonu ul başına 5,5 X 10 4 schizontes (bu genellikle toplanan parazitler 100-300 ul ekleyerek elde edilir) &#39;dir. Bu konsantrasyon 20 ul ortam ve eritrositler, tipik bir% 4 hematokrit kültür içinde yaklaşık% 1 arasında bir parazitemi 100 ul karışım nihai hacmi eklenmiş ise elde edilecektir. b) hemasitometre altında Sayma eritrositler odasının içindeki örnek kurudukça parçalama başından beri, hızlı olmak zorunda. Çoğunluk olmalı Enfekte eritrositler, non-enfekte olanlardan onları ayıran bir karanlık nokta içinde gösterecektir. <ol start="2"><li> İnce tabaka Giemsa,% 10 metanol içinde çok hızlı bir şekilde slaytlar sabitleme onları kurutma, ve t daldırarak toplama saflığını belirlemek için leke gerçekleştirmeGiemsa bir% 20 çözelti halinde kenar damıtık su ile seyreltilmiştir leke. Onlar hava ile kurutulur ve mikroskop altında incelenebilir sonra 10 dakika için slaytlar Leke. De% 70 üzerinde bir parazitemi ulaşılabilir olmalıdır. </li></ol>

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D., Kanjananggulpan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+ferriprotoporphyrin+IX+in+malaria+pigment.">The state of ferriprotoporphyrin IX in malaria pigment.</a> J. Biol. Chem. 262 (32), 15552-15555 (1987).</li><li>Moore, L. R., Fujioka, H., Williams, P. S., Chalmers, J. J., Grimberg, B., Zimmerman, P. A., Zborowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemoglobin+degradation+in+malaria-infected+erythrocytes+determined+from+live+cell+magnetophoresis.">Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis.</a> FASEB J. 20 (6), 747-749 (2006).</li><li>Spadafora, C., Gerena, L., Kopydlowski, K. 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L., Gilberger, T., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+permeabilization+of+the+host+cell+membrane+of+Plasmodium+falciparum-infected+red+blood+cells+with+streptolysin+O+and+equinatoxin+II.">Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II.</a> Biochem. J. 403, 167-175 (2007).</li><li>Goodyer, I. D., Johnson, J., Eisenthal, R., Hayes, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+mature-stage+Plasmodium+falciparum+by+gelatine+flotation.">Purification of mature-stage Plasmodium falciparum by gelatine flotation.</a> Ann. Trop. Med. Parasitol. 88 (2), 209-211 (1994).</li><li>Pasvol, G., Wilson, R. J., Smalley, M. E., Brown, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+viable+schizont-infected+red+cells+of+Plasmodium+falciparum+from+human+blood.">Separation of viable schizont-infected red cells of Plasmodium falciparum from human blood.</a> Ann. Trop. Med. Parasitol. 72, 87-88 (1978).</li><li>Pertoft, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+cells+and+subcellular+particles+with+Percoll.">Fractionation of cells and subcellular particles with Percoll.</a> J. Biochem. Biophys. Methods. 44 (1-2), 1-30 (2000).</li><li>Paul, F., Roath, S., Melville, D., Warhurst, D. C., Osisanya, J. O. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+separation+of+uninfected+erythrocytes+from+those+infected+with+Plasmodium+berghei+and+for+isolation+of+artificially+released+parasites.">A simple method for separation of uninfected erythrocytes from those infected with Plasmodium berghei and for isolation of artificially released parasites.</a> Z. Parasitenkd. 64, 279-284 (1981).</li></ol>

Biz ifşa etmek başka bir şey var.

Sıtma paraziti P. in vitro kültürlerde falciparum kültürü daha fazla olan en yüksek çoğalma noktada enfekte olmamış kırmızı kan hücrelerinin yarısından, sınırlı parazitemi sergiler. En çok araştırma deneyleri için, enfekte olmuş eritrositler ile birlikte çalışmak için sadece arzu edilir. Bu amaçla, bir ayırma tekniği, enfeksiyon göre kültür bölmek için gereklidir. Yararlı yöntemler enfekte olmamış eritrositler 11 ve iki grupların farklı yoğunluk ya...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> RPMI 1640 Hepes Modifiye </td><td> Sigma-Aldrich </td><td> R4130 </td><td> % 10 insan serumu,% 2 glükoz, ve% 0.2 sodyum bikarbonat ile desteklenmiş </td></tr><tr><td> MidiMACS Ayırıcı </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> LS Sütunlar </td><td> MACS Miltenyi BioTec </td><td> 130-042-401 </td><td></td></tr><tr><td> Hemasitometre </td><td> Grafco </td><td> Grafco Neubauer Odası </td><td> Diğer birçok tedarikçileri yoluyla bulunabilir </td></tr></tbody></table>

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

Diğer Plasmodium türlerin aksine, P. falciparum onun çalışma 1 kolaylaştırır laboratuar, kültüre edilebilir. Elde parazitemi ≈% 40 sınırına ulaşabilir olsa da, araştırmacı genellikle yaklaşık% 10 oranda tutar. Pek çok durumda, kültür zenginleştirmek ve belirli bir deney ile devam etmek için enfekte olmamış olanlardan parazit içeren kırmızı kan hücrelerinin (eritrositler), izole etmek için gereklidir. Zaman P. falciparum eritrosit bozar, parazit düşürür ve hemoglobin 2, 3 beslenir. Ancak, parazitin bir çok toksik demir içeren haem parçası 4, 5 ile ilgilenmek zorundadır. Parazit haemozoin 6, 7 olarak adlandırılan bir inert kristal polimer içine haem dönüştürerek kendi toksisite eludes. Bu demir içeren moleküller, gıda vakuol içinde depolanır ve bir metal haem 8 biri farklı bir oksidasyon durumuna sahiptir. Haem demir ferrik durumuozoin bu bulaşmamış eritrosit eksik olan paramanyetik özellikte bahşeder. Işgalci parazit olgunluğa ulaşır gibi haemozoin içeriği de P. son aşamalarında daha paramanyetizma ihsan, 9 da artar eritrosit içindeki falciparum. Bu paramanyetik özelliği, P. son aşamalarında dayanarak falciparum enfekte-kırmızı kan hücreleri manyetik boncuklar ihtiva eden bir sütun içinden geçen kültür tarafından ayrılabilir. Bunları içeren sütunlar bir mıknatıs tutucu üzerine konulduğunda Bu boncuklar manyetik hale gelir. Flow-through içerecektir iken onların paramanyetizma nedeniyle Enfekte RBCs, sonra çoğunlukla, enfekte olmamış eritrosit ve parazit içerenler erken aşamalarında için, sütun içine hapsolmuş edilecektir. Burada, biz iyi parazit canlılığı 10 korur manyetik sütunlar ile geç evre parazitlerin nüfusu zenginleştirmek için metodoloji açıklanmaktadır.Bu yordamı gerçekleştirdikten sonra, bekâr kültürü kalan parazitler büyümeye devam sağlamak için bir inkübatör iade edilebilir.

Şekil 2&#39;de, manyetik sütun içinden geçen kültür önce gösterilmiştir (A) ve işlemden sonra (B). Şekil 2A oklar ile enfekte eritrositleri işaret ile, Şekil 2 &#39;de gösterildiği gibi bir veya iki enfekte eritrositler genellikle bir 100X büyütme alan üzerinde görülür. Tipik bir prosedürde asitleştirme,% 5 parazitemi (Şekil 2A) bir kültür ile başlayarak, bu işlemin performansı genellikle% 97 ile% 100 <stron...

Watch this Scientific Journal Video about Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means at JoVE.com

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

Hemozoin bir paramanyetik özelliklerinin geç aşamalarında izole etmek için kullanılırlar<em&gt; Plasmodium falciparum</emKültüründe büyüyen&gt;-enfekte kırmızı kan hücreleri. Bu yöntem çok kolay ve hızlı ve parazitlerin sonraki invazif yetenekleri etkilemez.

Ayrılması<em&gt; Plasmodium falciparum</emManyetik Yoluyla&gt; Geç Evre-enfekte Eritrositler

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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Immunology and Infection

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

13.8K Görüntüleme.  Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). Hemozoin bir paramanyetik özelliklerinin geç aşamalarında izole etmek için kullanılırlar Plasmodium falciparum-enfekte kırmızı kan hücreleri. Bu yöntem çok kolay ve hızlı ve parazitlerin sonraki invazif yetenekleri etkilemez.

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.

Bryostatin 1/Ionomycin Yoluyla Tümör-reaktif T hücrelerinin ex vivo Genişleme ve Ortak Gama Zinciri Sitokinler Formülasyon

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

İmmünoloji ve Enfeksiyon

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.

Seçimi Plasmodium falciparum İnsan Beyninin endotel hücreleri Cytoadhesion için Parazitler

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.

Bir<em&gt; In vitro</emOkumak&gt; Co-enfeksiyon Modeli<em&gt; Plasmodium falciparum</emİnsan İlköğretim Monosit kökenli Bağışıklık Hücrelerde&gt;-HIV-1 Etkileşimleri

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.

Trombosit-aracılı kesekleşme için Basit Protokolü<em&gt; Plasmodium falciparum</emBir kaynak Poor Setting&gt; ile enfekte Eritrosit

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.

Den Beyin sekestre Lökositler İzolasyonu ve Analizi<em&gt; Plasmodium berghei</em&gt; ANKA-enfekte Fare

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.

Otomatik Ayırma<em&gt; C. elegans</em&gt; Değişken derecelerde Bakteriyel Patojen tarafından kolonileştirildiğini

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.

Yüksek Kapasiteli saflaştırılması<em&gt; Plasmodium falciparum</emOpsonizing Antikor Analizlerde kullanım için&gt; merozoitler

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.

Akış Cytometry tarafından Kemirgen Plasmodium parazitemi Vivo Değerlendirilmesi ve Merozoit Invasion

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.

Yöntemler Küçük RNA&#39;ların ve Ribosomal doluluk Düzenleyici Rolü Araştırma<em&gt; Plasmodium falciparum</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.

Biyoparlaklık Görüntüleme Afrika Trypanosomiasis Geç Evre Enfeksiyon Algılama için

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 ...