Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR

Bruña–Romero, Oscar; Hafalla, Julius Clemence R.; González‐Aseguinolaza, Gloria; Sano, Gen Ichiro; Tsuji, Moriya; Zavala, Fidel

doi:10.1016/s0020-7519(01)00265-x

Cited by 201 publications

(206 citation statements)

References 17 publications

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“…injection of 10 4 P. yoelii sporozoites. Real-time PCR was used for quantification of parasite load in the livers [52].…”

Section: Methodsmentioning

confidence: 99%

“…Mice were treated with TGF-b inhibitory peptides or the drugs indomethacin and aspirin for 15 days. After challenge with infective P. yoelii sporozoites, development of parasites in the liver was detected by real time PCR of P. yoelii ribosomal RNA [52]. The group of mice receiving DC preincubated with P. yoelii-infected erythrocytes present a decreased level of protection when compared with mice injected with DC preincubated with uninfected erythrocytes.…”

Section: Pge 2 and Tgf-b Inhibit Cd8 + T Cell Responses Against The Lmentioning

confidence: 99%

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Role of TGF‐β and PGE₂in T cell responses during Plasmodium yoelii infection

et al. 2007

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During an acute blood-stage malaria infection, T cell responses to malaria and other bystander antigens are inhibited. Plasmodium infection induces strong cytokine responses that facilitate parasite clearance but may interfere with T cell functions, as some of the soluble immune mediators induced are also general inhibitors of T cell responses. Using a malaria mouse model, we have analyzed the cytokines produced by dendritic cells in response to P. yoelii infection that have potential T cell inhibitory activity. We found that during acute infection DC migrate to the spleen and secrete TGF-b, prostaglandin E 2 (PGE 2 ) and IL-10. We have analyzed the role of these general T cell inhibitors in a particular T cell response of evident importance in malaria infections: the CD8 + T cells generated against the liver-stage of the disease. During blood-stage infection, inhibition of the activity of TGF-b and PGE 2 restores the CD8 + T cell responses generated by sporozoites, increasing protection against re-infection. Our findings suggest that the strong cytokine response induced by blood-stage P. yoelii infection affects host T cell responses, inhibiting protective CD8 + T cells against the liver-stage of the disease.

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“…injection of 10 4 P. yoelii sporozoites. Real-time PCR was used for quantification of parasite load in the livers [52].…”

Section: Methodsmentioning

confidence: 99%

Section: Pge 2 and Tgf-b Inhibit Cd8 + T Cell Responses Against The Lmentioning

confidence: 99%

Role of TGF‐β and PGE₂in T cell responses during Plasmodium yoelii infection

et al. 2007

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“…This method distinguishes between parasites attached to the cells and invading ones, but it is a time-consuming procedure requiring pre-labelled, parasite-specific antibodies. Another approach that can be employed to determine infection levels in vitro is quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), using primers specific for the parasite in parallel with primers for a housekeeping gene to allow normalization of the results, as described for the quantification of in vivo liver infections (Bruna-Romero et al, 2001). However, quantification by qRT-PCR is not only a time-consuming approach but also an expensive one, rendering it unsuitable for large-scale or routine studies.…”

Section: Quantification Of Invasionmentioning

confidence: 99%

Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry

et al. 2007

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SummaryThe study of the liver stage of malaria has been hampered by limitations in the experimental approaches required to effectively dissect and quantify hepatocyte infection by Plasmodium. Here, we report on the use of flow cytometry, in conjunction with GFP-expressing Plasmodium sporozoites, to assess the various steps that constitute a successful malaria liver infection: cell traversal, hepatocyte invasion and intrahepatocyte parasite development. We show that this rapid, efficient and inexpensive method can be used to overcome current limitations in the independent quantification of those steps, facilitating routine or large-scale studies of host-pathogen molecular interactions.

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“…Forty-two hours later, the livers of these mice were excised for total RNA isolation. Following reverse transcription, we used a real-time PCR assay to quantitatively measure the copies of 18s rRNA of liver stage parasites against known standards [10]. Immunization with IrrSpz conferred *95% reduction in liver stage burden as compared to a non-significant reduction when immunizing with HKSpz (Fig.…”

Section: Induction Of Protective Immunity Following Immunization Withmentioning

confidence: 99%

Priming of CD8⁺ T cell responses following immunization with heat‐killed Plasmodium sporozoites

et al. 2006

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Protective immune responses against malaria are induced by immunization with radiation-attenuated Plasmodium sporozoites. In contrast, non-viable, heat-killed sporozoites do not induce protection, emphasizing the requirement for live parasites to achieve effective immune responses. Using an experimental system with CD8 + T cells from T cell receptor-transgenic mice, we analyzed the primary CD8 + T cell responses elicited by heat-killed inactivated sporozoites. We found that the numbers of specific CD8 + T cells induced were much lower compared to when immunizing with attenuated sporozoites; however, the kinetics of activation and the phenotype of these T cells were similar in both groups. Despite their low frequency after priming, high numbers of specific CD8 + T cells were observed after boosting with a recombinant vaccinia virus. Upon induction of the recall response, the same level of protection was observed when either heat-killed or attenuated sporozoites were used for priming. We propose that live parasites are not critical for the induction of memory T cell populations against the malaria liver stages.

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Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR

Cited by 201 publications

References 17 publications

Role of TGF‐β and PGE₂in T cell responses during Plasmodium yoelii infection

Role of TGF‐β and PGE₂in T cell responses during Plasmodium yoelii infection

Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry

Priming of CD8⁺ T cell responses following immunization with heat‐killed Plasmodium sporozoites

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Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR

Cited by 201 publications

References 17 publications

Role of TGF‐β and PGE2 in T cell responses during Plasmodium yoelii infection

Role of TGF‐β and PGE2 in T cell responses during Plasmodium yoelii infection

Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry

Priming of CD8+ T cell responses following immunization with heat‐killed Plasmodium sporozoites

Contact Info

Product

Resources

About

Role of TGF‐β and PGE₂in T cell responses during Plasmodium yoelii infection

Role of TGF‐β and PGE₂in T cell responses during Plasmodium yoelii infection

Priming of CD8⁺ T cell responses following immunization with heat‐killed Plasmodium sporozoites