Project description:Purpose: The goals of this study are to compare parasite transcriptomes in sickle cell trait infected red blood cells during the intraerythrocytic developmental cycle (IDC) from in vitro time series and in vivo blood samples to identify new therapeutic targets in the treatment of malaria Methods: In vitro time series: Parasites were synchronized to early ring-stage parasites and sampled every three hours for 48 hours to capture every stage of the IDC, generating 16 RNA-seq libraries per replicate (128 total). In vivo blood samples: Samples were collected in an observational study of malaria in children in Kenieroba, Mali (ClinicalTrials.gov Identifier: NCT02645604). From children presenting with uncomplicated falciparum malaria, we selected all available samples from children with HbAS and matched each of these to a sample from a child with HbAA on: month of episode, parasite density, ethnic background, and, if possible, ABO blood type.
Project description:Whole blood transcriptomes from a longitudinal study of 5 Malawian children who first present with severe Plasmodium falciparum malaria, and return in one month with mild malaria We used microarrays to identify transcripts that were associated with each clinical presentation. A blood sample was taken upon presentation during the severe and mild malaria episodes in 5 Malawian children (total n=5 pairs) followed by RNA extraction and hybridization on Affymetrix GeneChip Human Gene 1.0 ST Array, using a paired analysis
Project description:Comaprision of P.falciparum clinical isolates showing Uncomplicated disease with that shwoing complicated disease(Cerebral malaria) The experiment was designed to try and identify differences if any, at the genome level between P.falciparum isolates from patients with uncomplicated malaria vs. patients with complicated malaria (Cerebral malaria). The emphasis was to highlight possible amplifications/deletions in different regions of the parasite genome.
Project description:Whole blood transcriptomes from a longitudinal study of 5 Malawian children who first present with severe Plasmodium falciparum malaria, and return in one month with mild malaria We used microarrays to identify transcripts that were associated with each clinical presentation.
Project description:This study examined the differences in human and parasite gene expression pattern between children with severe malaria and those with uncomplicated malaria through a dual RNA-seq approach. Peripheral blood samples were collected which contained substantial numbers of parasites that required no RNA enrichment prior to library preparation and sequencing.
Project description:During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes M-bM-^@M-^S sexual stage precursor cells M-bM-^@M-^S likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the blood stream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and ~30 hours post invasion and mature gametocytes after around 7 days post invasion. We used 164/TdTom, a transgenic parasite line expressing a red fluorescent protein reporter under a gametocyte-specific promoter to generate schizont samples. Schizonts were subsequently isolated from both the fluorescent and non-fluorescent population by FACS and prepared for microarray analysis. Two biological replicates were produced for both the fluorescent and the non-fluorescent samples.
Project description:Objectives: Malaria, caused by Plasmodium infection, remains a major global health problem. Monocytes are integral to the immune response yet, their transcriptional and functional responses in primary Plasmodium falciparum infection and in clinical malaria are poorly understood. Methods: The transcriptional and functional profile of monocytes were examined in controlled human malaria infection with P. falciparum blood-stages and in children and adults with acute malaria. Monocyte gene expression and functional phenotypes were examined by RNA-sequencing and flow cytometry at peak-infection and compared to pre-infection or at convalescence in acute malaria. Results: In subpatent primary infection, the monocyte transcriptional profile was dominated by an interferon (IFN) molecular signature. Pathways enriched included type I IFN signalling, innate immune response, cytokine-mediated signalling. Monocytes increased TNF and IL-12 production upon in vitro toll-like receptor stimulation, and increased IL-10 production upon in vitro parasite restimulation. Longitudinal phenotypic analyses revealed sustained significant changes in the composition of monocytes following infection, with increased CD14+CD16- and decreased CD14-CD16+ subsets. In acute malaria, monocyte CD64/FcγRI expression was significantly increased in children and adults, while HLA-DR remained stable. Although children and adults showed a similar pattern of differentially expressed genes, the number and magnitude of gene expression change was greater in children. Conclusions: Monocyte activation during subpatent malaria is driven by an IFN molecular signature with robust activation of genes enriched in pathogen detection, phagocytosis, antimicrobial activity and antigen presentation. The greater magnitude of transcriptional changes in children with acute malaria suggest monocyte phenotypes may change with age or exposure.
Project description:Cerebral malaria is a severe complication of Plasmodium falciparum infection characterized by the loss of blood-brain barrier (BBB) integrity, which is associated with brain swelling and mortality in patients. P. falciparum-infected red blood cells and in!ammatory cytokines, like tumor necrosis factor alpha (TNF-a), have been implicated in the development of cerebral malaria, but it is still unclear how they contribute to the loss of BBB integrity. Here, a combination of transcriptomic analysis and cellular assays detecting changes in barrier integrity and endothelial activation were used to distinguish between the effects of P. falciparum and TNF-a on a human brain microvascular endothelial cell (HBMEC) line and in primary human brain microvascular endothelial cells. We observed that while TNF-a induced high levels of endothelial activation, it only caused a small increase in HBMEC permeability. Conversely, P. falciparum-infected red blood cells (iRBCLs) led to a strong increase in HBMEC permeability that was not mediated by cell death. Distinct transcriptomic pro"les of TNF-a and P. falciparum in HBMECs con"rm the differential effects of these stimuli, with the parasite preferentially inducing an endoplasmic reticulum stress response. Our results establish that there are fundamental differences in the responses induced by TNF-a and P. falciparum on brain endothelial cells and suggest that parasite-induced signaling is a major component driving the disruption of the BBB during cerebral malaria, proposing a potential target for much needed therapeutics.
Project description:During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes – sexual stage precursor cells – likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the blood stream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and ~30 hours post invasion and mature gametocytes after around 7 days post invasion.