Name: selection of plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells
Uploaded: 2012-01-03T12:00:00
Duration: 10 min 9 s
Description: Watch this Scientific Journal Video about Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells at JoVE.com

Мы благодарим Франциско Candal, CDC трансфера технологий Office, Атланте Грузии HBEC-5i клеток. Эта работа финансировалась Wellcome Trust (4 года кандидат студенчества к сети переменного и старший стипендий в основных биомедицинских наук в JAR, номер гранта 084 226).

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Отличительной чертой церебральной малярии является секвестрация П. тропической iRBC в мозге 2 микрососудов, 3. Тем не менее, в пробирке культур P. тропической только плохо cytoadhere к HBEC-5i, модель для человеческого эндотелия капилляров мозга. Здесь мы разработали простой анализ для обогащения населения за связывание с HBEC-5i, более «в естественных условиях, как« фенотип. Три из 4 P. тропической штаммы были успешно выбраны с помощью этого метода. Кроме того, HB3 был также выбран на HDMEC и HPMEC, указывая, что этот протокол может быть использован для различных паразитов и эндотелиальных типов клеток. Различные гипотезы могут объяснить отсутствие связывания с DD2 напряжение. Скорее всего, является тот факт, что эта линия паразит knobless, который глубоко препятствует cytoadherence 13, 14. Мы рекомендуем культивирования невыбранные паразитов вместе с выбором пропроцессом обеспечить контроль для сравнения. Это позволит, например, сравнение транскриптом обязательных и необязательных паразитов, с надеждой на обнаружение паразитов кандидатов лиганда. ФНО цитокинов найти на высоком уровне в церебральной малярии и пациентов было показать индуцируют экспрессию многих белков поверхности HBEC-5i (Claessens и др., в подготовке и 8, 15, 16). В этом случае количество связанного iRBC была одинаковой в нормальном HBEC-5i по сравнению с активированным HBEC-5i. Этот «секвестр модель&quot; также может быть использован для изучения молекулярного взаимодействия между iRBC и эндотелиальных клеток, а также влияние предполагаемых анти-cytoadherence наркотиков. В этом случае, мы рекомендуем покрытие HBEC-5i в небольших скважин, такие как &quot;8-а камера скользит» (BD 354 628) или &quot;CultureWell&quot; (Sigma C7735-20EA).

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.

Watch this Scientific Journal Video about Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells at JoVE.com

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

<em&gt; В пробирке</em&gt; Модели церебральной малярии поглощения описывается<sup&gt; 1</sup&gt;.<em&gt; Plasmodium тропической</em&gt; Инфицированных красных кровяных клеток, выбранных для привязки к увековечен человеческий мозг микрососудистых эндотелиальных клеток. Отдельных паразитов шоу различных фенотипа. Процесс отбора может быть применен с использованием различных<em&gt; П. тропической</em&gt; Штаммов и эндотелиальных клеточных линий.

Выбор Plasmodium тропической Паразиты для Cytoadhesion для мозга человека эндотелиальных клеток

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

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Immunology and Infection

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

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