8 results on '"Stefan H. Kappe"'
Search Results
2. Multiplex, DNase-free one-step reverse transcription PCR for Plasmodium 18S rRNA and spliced gametocyte-specific mRNAs
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Amelia E. Hanron, Zachary P. Billman, Annette M. Seilie, Tayla M. Olsen, Matthew Fishbaugher, Ming Chang, Thomas Rueckle, Nicole Andenmatten, Bryan Greenhouse, Emmanuel Arinaitwe, John Rek, Smita Das, Gonzalo J. Domingo, Kelly Shipman, Stefan H. Kappe, James G. Kublin, and Sean C. Murphy
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Gametocyte ,mRNA ,Spliced ,Antisense ,Plasmodium ,DNase ,Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Plasmodium gametocytes are sexual stages transmitted to female Anopheles mosquitoes. While Plasmodium parasites can be differentiated microscopically on Giemsa-stained blood smears, molecular methods are increasingly used because of their increased sensitivity. Molecular detection of gametocytes requires methods that discriminate between asexual and sexual stage parasites. Commonly tested gametocyte-specific mRNAs are pfs25 and pfs230 detected by reverse transcription polymerase chain reaction (RT-PCR). However, detection of these unspliced mRNA targets requires preceding DNase treatment of nucleic acids to eliminate co-purified genomic DNA. If gametocyte-specific, spliced mRNAs could be identified, DNase treatment could be eliminated and one-step multiplexed molecular methods utilized. Results Expression data was used to identify highly-expressed mRNAs in mature gametocytes that were also low in antisense RNA expression in non-gametocyte stages. After testing numerous candidate mRNAs, the spliced female Pf3D7_0630000 mRNA was selected as a Plasmodium falciparum gametocyte-specific biomarker compatible with Plasmodium 18S rRNA RT-PCR. This mRNA was only detected in samples containing mature gametocytes and was absent in those containing only asexual stage parasites or uninfected human blood. PF3D7_0630000 RT-PCR detected gametocytes across a wide range of parasite densities in both spiked and clinical samples and agreed with pfs25 RT-PCR, the gold standard for RT-PCR-based gametocyte detection. PF3D7_0630000 multiplexed with Plasmodium 18S rRNA RT-PCR was more sensitive than other spliced mRNA targets for one-step RT-PCR gametocyte detection. Conclusions Because the spliced target does not require DNase treatment, the PF3D7_0630000 assay can be multiplexed with Plasmodium 18S rRNA for direct one-step detection of gametocytes from whole human blood.
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- 2017
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3. The Plasmodium liver-specific protein 2 (LISP2) is an early marker of liver stage development
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Devendra Kumar Gupta, Laurent Dembele, Annemarie Voorberg-van der Wel, Guglielmo Roma, Andy Yip, Vorada Chuenchob, Niwat Kangwanrangsan, Tomoko Ishino, Ashley M Vaughan, Stefan H Kappe, Erika L Flannery, Jetsumon Sattabongkot, Sebastian Mikolajczak, Pablo Bifani, Clemens HM Kocken, and Thierry Tidiane Diagana
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Plasmodium vivax ,Plasmodium cynomolgi ,hypnozoite ,liver stage ,Malaria relapse ,molecular marker ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.
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- 2019
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4. P. falciparum enhances HIV replication in an experimental malaria challenge system.
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Marika Orlov, Florin Vaida, Olivia C Finney, David M Smith, Angela K Talley, Ruobing Wang, Stefan H Kappe, Qianqian Deng, Robert T Schooley, and Patrick E Duffy
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Medicine ,Science - Abstract
Co-infection with HIV and P. falciparum worsens the prognosis of both infections; however, the mechanisms driving this adverse interaction are not fully delineated. To evaluate this, we studied HIV-1 and P. falciparum interactions in vitro using peripheral blood mononuclear cells (PBMCs) from human malaria naïve volunteers experimentally infected with P. falciparum in a malaria challenge trial. PBMCs collected before the malaria challenge and at several time points post-infection were infected with HIV-1 and co-cultured with either P. falciparum infected (iRBCs) or uninfected (uRBCs) red blood cells. HIV p24Ag and TNF-α, IFN-γ, IL-4, IL-6, IL-10, IL-17, and MIP-1α were quantified in the co-culture supernatants. In general, iRBCs stimulated more HIV p24Ag production by PBMCs than did uRBCs. HIV p24Ag production by PBMCs in the presence of iRBCs (but not uRBCs) further increased during convalescence (days 35, 56, and 90 post-challenge). In parallel, iRBCs induced higher secretion of pro-inflammatory cytokines (TNF-α, IFN-γ, and MIP-1α) than uRBCs, and production increased further during convalescence. Because the increase in p24Ag production occurred after parasitemia and generalized immune activation had resolved, our results suggest that enhanced HIV production is related to the development of anti-malaria immunity and may be mediated by pro-inflammatory cytokines.
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- 2012
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5. The
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Devendra Kumar, Gupta, Laurent, Dembele, Annemarie, Voorberg-van der Wel, Guglielmo, Roma, Andy, Yip, Vorada, Chuenchob, Niwat, Kangwanrangsan, Tomoko, Ishino, Ashley M, Vaughan, Stefan H, Kappe, Erika L, Flannery, Jetsumon, Sattabongkot, Sebastian, Mikolajczak, Pablo, Bifani, Clemens Hm, Kocken, and Thierry Tidiane, Diagana
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molecular marker ,Microbiology and Infectious Disease ,liver stage ,Protozoan Proteins ,Malaria relapse ,Macaca mulatta ,Biomarkers, Pharmacological ,Host-Parasite Interactions ,Antimalarials ,Liver ,Sporozoites ,parasitic diseases ,Aminoquinolines ,Hepatocytes ,Malaria, Vivax ,Animals ,Humans ,Other ,Plasmodium vivax ,Transcriptome ,Research Advance ,Biomarkers ,hypnozoite ,Plasmodium cynomolgi - Abstract
Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.
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- 2018
6. Characterization of the type I IFN response to liver stage infection with genetically attenuated malaria parasite vaccines
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Nana K Minkah, Brandon K Sack, Jessica L Miller, Ashley Vaughan, and Stefan H Kappe
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Immunology ,Immunology and Allergy - Abstract
Malaria, a disease caused by Plasmodium parasites, kills nearly 600,000 people annually. Inoculated by an infectious mosquito bite, Plasmodium sporozoites travel to the liver and infect hepatocytes. Here, they develop into merozoites that are released into the blood to cause malaria associated mortality and morbidity. No fully protective malaria vaccine exists, but immunizations with genetically attenuated parasites (GAPs) that arrest in the liver confer sterile protection from challenge in mice. Moreover, using a novel super-infection assay, we observed that GAP immunization induces a potent type I IFN response to control liver stage infection. However, the factors required for the induction and propagation of the IFN response, and the influence of this response on adaptive immunity had not been elucidated. To identify the upstream inducers of IFN signaling, we immunized mice with either mosquito debris or GAPs and measured gene expression in hepatocytes by RNA-Seq. 800 genes were significantly upregulated upon GAP immunization, including transcripts for cytosolic RNA and DNA sensors. To examine the influence of the IFN response on adaptive immunity, WT, IRF3−/− and IFNAR−/− mice were immunized with a suboptimal dose of GAP, and challenged 4 weeks later with WT parasite. The immunization regimen elicited sterile protection in 20% of the WT mice, while 100% protection was achieved in IRF3−/− or IFNAR−/− mice. Taken together, the data suggest that hepatocytes utilize RNA and DNA sensors to induce a type I IFN response that is necessary for early control of liver stage infection, yet detrimental to adaptive immunity. Current studies are examining the mechanisms behind the IFN-mediated suppression of adaptive immunity.
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- 2016
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7. Contrasting CTL responses in protective and non-protective models of malaria (45.3)
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Megha Gupta, Akihide Takagi, Thomas L Richie, Stefan H Kappe, and Ruobing Wang
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Immunology ,Immunology and Allergy - Abstract
Protracted sterile protection conferred by a P. yoelii genetically attenuated parasite (PyGAP) vaccine was found to be completely dependent on CD8+ T lymphocytes. Immunization studies with perforin and IFN-γ knock out mice indicated that the protection was largely dependent on perforin as compared to IFN-γ. Both liver and spleen CD8+ T cells from PyGAP immunized mice induced massive apoptosis of liver stage (LS)-infected hepatocytes in vitro without the release of detectable IFN-γ and TNF-a, which directly correlated with GAP vaccine efficacy in vivo. Most importantly, we demonstrated that CD8+ T cells isolated from naïve mice that had survived wild-type P. yoelii sporozoite infection targeted mainly sporozoite-traversed and uninfected hepatocytes, revealing an immune evasion strategy that might have been used by wild-type parasites to subvert host immune responses. Differential processing of the parasite antigens in liver of immunized or naturally infected mice may be responsible for contrasting outcomes of their CTL responses. A controversial role of circumsporozoite protein (CSP) in host immune protection versus immune evasion against malaria is also highlighted by our findings. This research was supported by SBRI innovation grant.
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- 2009
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8. Safety and comparability of controlled human Plasmodium falciparum infection by mosquito bite in malaria-naïve subjects at a new facility for sporozoite challenge.
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Angela K Talley, Sara A Healy, Olivia C Finney, Sean C Murphy, James Kublin, Carola J Salas, Susan Lundebjerg, Peter Gilbert, Wesley C Van Voorhis, John Whisler, Ruobing Wang, Chris F Ockenhouse, D Gray Heppner, Stefan H Kappe, and Patrick E Duffy
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Medicine ,Science - Abstract
Controlled human malaria infection (CHMI) studies which recapitulate mosquito-borne infection are a critical tool to identify protective vaccine and drug candidates for advancement to field trials. In partnership with the Walter Reed Army Institute of Research, the CHMI model was established at the Seattle Biomedical Research Institute's Malaria Clinical Trials Center (MCTC). Activities and reagents at both centers were aligned to ensure comparability and continued safety of the model. To demonstrate successful implementation, CHMI was performed in six healthy malaria-naïve volunteers.All volunteers received NF54 strain Plasmodium falciparum by the bite of five infected Anopheles stephensi mosquitoes under controlled conditions and were monitored for signs and symptoms of malaria and for parasitemia by peripheral blood smear. Subjects were treated upon diagnosis with chloroquine by directly observed therapy. Immunological (T cell and antibody) and molecular diagnostic (real-time quantitative reverse transcriptase polymerase chain reaction [qRT-PCR]) assessments were also performed.All six volunteers developed patent parasitemia and clinical malaria. No serious adverse events occurred during the study period or for six months post-infection. The mean prepatent period was 11.2 days (range 9-14 days), and geometric mean parasitemia upon diagnosis was 10.8 parasites/µL (range 2-69) by microscopy. qRT-PCR detected parasites an average of 3.7 days (range 2-4 days) earlier than blood smears. All volunteers developed antibodies to the blood-stage antigen merozoite surface protein 1 (MSP-1), which persisted up to six months. Humoral and cellular responses to pre-erythrocytic antigens circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) were limited.The CHMI model was safe, well tolerated and characterized by consistent prepatent periods, pre-symptomatic diagnosis in 3/6 subjects and adverse event profiles as reported at established centers. The MCTC can now evaluate candidates in the increasingly diverse vaccine and drug pipeline using the CHMI model.ClinicalTrials.gov NCT01058226.
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- 2014
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