Publications by authors named "Abhai K Tripathi"

33 Publications

Pf77 and male development gene 1 as vaccine antigens that induce potent transmission-reducing antibodies.

Sci Transl Med 2021 Jun;13(597)

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.

Malaria vaccines that disrupt the life cycle in mosquitoes and reduce parasite transmission in endemic areas are termed transmission-blocking vaccines (TBVs). Despite decades of research, there are only a few antigens that indisputably and reproducibly demonstrate transmission-blocking immunity. So far, only two TBV candidates have advanced to phase 1/2 clinical testing with limited success. By applying an unbiased transcriptomics-based approach, we have identified Pf77 and male development gene 1 (PfMDV-1) as two TBV antigens that, upon immunization, induced antibodies that caused reductions in oocyst counts in mosquito midguts in a standard membrane feeding assay. In-depth studies were performed to characterize the genetic diversity of, stage-specific expression by, and natural immunity to these two molecules to evaluate their suitability as TBV candidates. Pf77 and PfMDV-1 display limited antigenic polymorphism, are pan-developmentally expressed within the parasite, and induce naturally occurring antibodies in Ghanaian adults, which raises the prospect of natural boosting of vaccine-induced immune response in endemic regions. Together, these biological properties suggest that Pf77 and PfMDV-1 may warrant further investigation as TBV candidates.
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http://dx.doi.org/10.1126/scitranslmed.abg2112DOI Listing
June 2021

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding.

J Vis Exp 2020 07 3(161). Epub 2020 Jul 3.

Johns Hopkins Malaria Research Institute, W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University.

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.
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http://dx.doi.org/10.3791/61426DOI Listing
July 2020

Transcriptome analysis based detection of Plasmodium falciparum development in Anopheles stephensi mosquitoes.

Sci Rep 2018 08 1;8(1):11568. Epub 2018 Aug 1.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.

The Plasmodium life cycle within the mosquito involves the gamete, zygote, motile ookinete, and the oocyst stage that supports sporogony and sporozoite formation. We mapped the P. falciparum transcriptome as the parasite progresses through the oocyst stage of development on days 2, 4, 6, and 8 post-P. falciparum infectious blood meal. Through these genomic studies, we identified 212 novel transmission stage biomarkers including genes that are developmentally expressed at a single time point and genes that are pan-developmentally expressed at all four time points in P. falciparum oocysts. Validation of a small subset of genes at the transcriptional and translational level resulted in identification of a signature of genes/proteins that can detect parasites within the mosquito as early as day 2 post-infectious blood meal and can be used to distinguish early versus late stage P. falciparum oocyst development in the mosquito. Currently, circumsporozoite protein (CSP), which is detectable only after day 7 post-infection, is the only marker used for detection of P. falciparum infection in mosquitoes. Our results open the prospect to develop a non-CSP based detection assay for assessment of P. falciparum infection in mosquitoes and evaluate the effect of intervention measures on malaria transmission in an endemic setting.
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http://dx.doi.org/10.1038/s41598-018-29969-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070505PMC
August 2018

A mosquito salivary gland protein partially inhibits Plasmodium sporozoite cell traversal and transmission.

Nat Commun 2018 07 25;9(1):2908. Epub 2018 Jul 25.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, 06520, USA.

The key step during the initiation of malaria is for motile Plasmodium parasites to exit the host dermis and infect the liver. During transmission, the parasites in the form of sporozoites, are injected together with mosquito saliva into the skin. However, the contribution of vector saliva to sporozoite activity during the establishment of the initial infection of the liver is poorly understood. Here we identify a vector protein by mass spectrometry, with similarity to the human gamma interferon inducible thiol reductase (GILT), that is associated with saliva sporozoites of infected Anopheles mosquitoes and has a negative impact on the speed and cell traversal activity of Plasmodium. This protein, referred to as mosquito GILT (mosGILT) represents an example of a protein found in mosquito saliva that may negatively influence sporozoite movement in the host and could lead to new approaches to prevent malaria.
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http://dx.doi.org/10.1038/s41467-018-05374-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060088PMC
July 2018

Long-acting injectable atovaquone nanomedicines for malaria prophylaxis.

Nat Commun 2018 01 22;9(1):315. Epub 2018 Jan 22.

Division of Clinical Pharmacology, Departments of Medicine and of Pharmacology and Molecular Sciences, The Johns Hopkins University, 725 North Wolfe Street, Baltimore, MD, 21205, USA.

Chemoprophylaxis is currently the best available prevention from malaria, but its efficacy is compromised by non-adherence to medication. Here we develop a long-acting injectable formulation of atovaquone solid drug nanoparticles that confers long-lived prophylaxis against Plasmodium berghei ANKA malaria in C57BL/6 mice. Protection is obtained at plasma concentrations above 200 ng ml and is causal, attributable to drug activity against liver stage parasites. Parasites that appear after subtherapeutic doses remain atovaquone-sensitive. Pharmacokinetic-pharmacodynamic analysis indicates protection can translate to humans at clinically achievable and safe drug concentrations, potentially offering protection for at least 1 month after a single administration. These findings support the use of long-acting injectable formulations as a new approach for malaria prophylaxis in travellers and for malaria control in the field.
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http://dx.doi.org/10.1038/s41467-017-02603-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778127PMC
January 2018

A Novel Gametocyte Biomarker for Superior Molecular Detection of the Plasmodium falciparum Infectious Reservoirs.

J Infect Dis 2017 12;216(10):1264-1272

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases.

Background: Complete malaria eradication and optimal use of transmission-reducing interventions require knowledge of submicroscopic infectious reservoirs among asymptomatic individuals. Even submicroscopic levels of Plasmodium falciparum gametocytes can infect mosquitoes and promote onward transmission. Most efforts to identify gametocyte carriers use polymerase chain reaction amplification of the gametocyte-specific transcript Pfs25.

Methods: To expand the repertoire of biomarkers available for superior gametocyte detection, we compared the gene expression profiles of gametocytes and asynchronous blood-stage P. falciparum parasites by microarray technology. This allowed the identification of 56 molecules abundantly expressed in the gametocyte stage of the parasite. The analytical sensitivity for gametocyte detection was evaluated for 25 genes with the highest expression levels.

Results: One candidate, Pfg17, exhibited superior analytical sensitivity against a panel of gametocyte-spiked whole blood, detecting 10 gametocytes/mL; in comparison, Pfs25 detected only 25.3 gametocytes/mL. Pfg17 also exhibited superior clinical sensitivity, identifying 19.1% more samples from blood-film microscopy-negative Ghanaian children and 40% more samples from asymptomatic adults as gametocyte positive.

Conclusions: Cumulatively, our results suggest Pfg17 is an excellent biomarker for detecting asymptomatic infectious reservoirs otherwise missed by the most sensitive molecular method available. Our study has also improved the repertoire of transmission-stage antigens available for evaluation as candidate vaccines.
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http://dx.doi.org/10.1093/infdis/jix442DOI Listing
December 2017

A no film slot blot for the detection of developing P. falciparum oocysts in mosquitoes.

PLoS One 2017 21;12(4):e0174229. Epub 2017 Apr 21.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.

Non-microscopy-based assays for sensitive and rapid detection of Plasmodium infection in mosquitoes are needed to allow rapid and high throughput measurement of transmission intensity and malaria control program effectiveness. Here, we report on a modified enhanced chemiluminescence-based slot blot assay for detection of Plasmodium falciparum (Pf) circumsporozite protein (PfCSP) expressed on parasite oocysts developing inside the mosquito midgut. This modified assay has several novel features that include eliminating the need for exposure to autoradiography (AR) film, as well as utilizing a novel high affinity anti-CSP antibody, and optimizing assay procedures resulting in significant reduction in the time required to perform the assay. The chemiluminescent signal for the detection of PfCSP in mosquito samples was captured digitally utilizing the C-Digit blot scanner that, allowed the detection of 0.01 pg of recombinant P. falciparum CSP and as few as 0.02 P. falciparum oocysts in a little over two hours. The earlier ECL-SB detected rCSP and oocysts and took approximately 5 h to perform. Whole mosquito lysates from both high and low prevalence-infected mosquito populations were prepared and evaluated for PfCSP detection on the ECL-SB by both AR film and digital data capture and analysis. There was a 100% agreement between the AR film and the C-Digit scanner methods for PfCSP detection in randomly sampled mosquitoes. This novel "No Film" Slot Blot assay obviates the need for AR film exposure and development and significantly reduces the assay time enabling widespread use in field settings.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400236PMC
September 2017

Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony.

Nat Microbiol 2017 Feb 17;2:17017. Epub 2017 Feb 17.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 651 Huntington Avenue, FXB, Room 202, Boston, Massachusetts 02115, USA.

Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection. DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly. However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.
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http://dx.doi.org/10.1038/nmicrobiol.2017.17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328244PMC
February 2017

Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites.

PLoS One 2016 2;11(12):e0166814. Epub 2016 Dec 2.

Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States.

Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166814PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135057PMC
June 2017

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

PLoS Pathog 2016 07 28;12(7):e1005763. Epub 2016 Jul 28.

Department of Biochemistry and Molecular Biology and Huck Center for Malaria Research, Pennsylvania State University, University Park, Pennsylvania, United States of America.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.
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http://dx.doi.org/10.1371/journal.ppat.1005763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965013PMC
July 2016

Antimalarial iron chelator FBS0701 blocks transmission by Plasmodium falciparum gametocyte activation inhibition.

Antimicrob Agents Chemother 2015 Mar 15;59(3):1418-26. Epub 2014 Dec 15.

The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA

Reducing the transmission of the malarial parasite by Anopheles mosquitoes using drugs or vaccines remains a main focus in the efforts to control malaria. Iron chelators have been studied as potential antimalarial drugs due to their activities against different stages of the parasite. The iron chelator FBS0701 affects the development of Plasmodium falciparum early gametocytes and lowers blood-stage parasitemia. Here, we tested the effect of FBS0701 on stage V gametocyte infectivity for mosquitoes. The incubation of stage V gametocytes for up to 3 days with increasing concentrations of FBS0701 resulted in a significant dose-related reduction in mosquito infectivity, as measured by the numbers of oocysts per mosquito. The reduction in mosquito infectivity was due to the inhibition of male and female gametocyte activation. The preincubation of FBS0701 with ferric chloride restored gametocyte infectivity, showing that the inhibitory effect of FBS0701 was quenched by iron. Deferoxamine, another iron chelator, also reduced gametocyte infectivity but to a lesser extent. Finally, the simultaneous administration of drug and gametocytes to mosquitoes without previous incubation did not significantly reduce the numbers of oocysts. These results show the importance of gametocyte iron metabolism as a potential target for new transmission-blocking strategies.
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http://dx.doi.org/10.1128/AAC.04642-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4325789PMC
March 2015

Antimalarial chemotherapy: orally curative artemisinin-derived trioxane dimer esters.

Bioorg Med Chem Lett 2015 Jan 27;25(2):245-8. Epub 2014 Nov 27.

Department of Chemistry, School of Arts and Sciences, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, United States; The Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, United States. Electronic address:

Eight new artemisinin-derived trioxane dimer esters 5 have been prepared and tested for antimalarial efficacy in malaria-infected mice. At a single oral dose of only 6mg/kg combined with 18mg/kg of mefloquine, each of the dimer esters 5 outperformed the antimalarial drug artemether (2). The most efficacious dimer, dichlorobenzoate ester 5h, prolonged mouse survival past day 30 of infection with three of the four mice in this group having no detectable parasitemia and appearing and acting healthy on day 30.
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http://dx.doi.org/10.1016/j.bmcl.2014.11.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277730PMC
January 2015

A sensitive enhanced chemiluminescent-ELISA for the detection of Plasmodium falciparum circumsporozoite antigen in midguts of Anopheles stephensi mosquitoes.

J Microbiol Methods 2015 Jan 22;108:19-24. Epub 2014 Oct 22.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD USA. Electronic address:

Efforts to develop a successful malaria vaccine are hampered due to lack of assays that are predictive of protective immunity without conducting large clinical studies. The effect of experimental vaccines and drugs on malaria transmission is yet more difficult to measure. Knowledge on the Plasmodium infection rate in mosquito populations will aid the measurement of effects from intervention measures for malaria control. Here, we report the development of a chemiluminescent sandwich ELISA (ECL-ELISA) that can detect Plasmodium falciparum circumsporozoite protein (Pf CSP) produced in recombinant form at concentrations of 4.4pg and in P. falciparum sporozoites (Pf SPZ) derived from mosquito salivary glands at levels corresponding to 5 Pf SPZ. Most importantly, we demonstrate reliable Pf CSP-based detection of 0.056day 8 P. falciparum oocysts developing inside mosquito midguts in whole mosquito lysates. Cumulatively, the ECL-ELISA is 47× more sensitive for the detection of Pf CSP than a colorimetric ELISA while greatly simplifying sample preparation, obviating the need for cumbersome midgut dissections and allowing high throughput screening of Plasmodium infection in mosquito populations. The ECL-ELISA may also have broader application in diagnosis of infectious diseases and the prognostic value in cancer and other diseases such as auto-immunity and genetic disorders based on antigen detection, or quality validation of biological vaccine components.
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http://dx.doi.org/10.1016/j.mimet.2014.10.006DOI Listing
January 2015

Isoprenoid precursor biosynthesis is the essential metabolic role of the apicoplast during gametocytogenesis in Plasmodium falciparum.

Eukaryot Cell 2015 Feb 1;14(2):128-39. Epub 2014 Dec 1.

Department of Biochemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia, USA

The malaria parasite harbors a relict plastid called the apicoplast and its discovery opened a new avenue for drug discovery and development due to its unusual, nonmammalian metabolism. The apicoplast is essential during the asexual intraerythrocytic and hepatic stages of the parasite, and there is strong evidence supporting its essential metabolic role during the mosquito stages of the parasite. Supply of the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is the essential metabolic function of the apicoplast during the asexual intraerythrocytic stages. However, the metabolic role of the apicoplast during gametocyte development, the malaria stages transmitted to the mosquito, remains unknown. In this study, we showed that production of IPP for isoprenoid biosynthesis is the essential metabolic function of the apicoplast during gametocytogenesis, by obtaining normal gametocytes lacking the apicoplast when supplemented with IPP. When IPP supplementation was removed early in gametocytogenesis, developmental defects were observed, supporting the essential role of isoprenoids for normal gametocytogenesis. Furthermore, mosquitoes infected with gametocytes lacking the apicoplast developed fewer and smaller oocysts that failed to produce sporozoites. This finding further supports the essential role of the apicoplast in establishing a successful infection in the mosquito vector. Our study supports isoprenoid biosynthesis as a valid drug target for development of malaria transmission-blocking inhibitors.
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http://dx.doi.org/10.1128/EC.00198-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311923PMC
February 2015

Gametocytocidal screen identifies novel chemical classes with Plasmodium falciparum transmission blocking activity.

PLoS One 2014 26;9(8):e105817. Epub 2014 Aug 26.

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.

Discovery of transmission blocking compounds is an important intervention strategy necessary to eliminate and eradicate malaria. To date only a small number of drugs that inhibit gametocyte development and thereby transmission from the mosquito to the human host exist. This limitation is largely due to a lack of screening assays easily adaptable to high throughput because of multiple incubation steps or the requirement for high gametocytemia. Here we report the discovery of new compounds with gametocytocidal activity using a simple and robust SYBR Green I- based DNA assay. Our assay utilizes the exflagellation step in male gametocytes and a background suppressor, which masks the staining of dead cells to achieve healthy signal to noise ratio by increasing signal of viable parasites and subtracting signal from dead parasites. By determining the contribution of exflagellation to fluorescent signal and using appropriate cutoff values, we were able to screen for gametocytocidal compounds. After assay validation and optimization, we screened an FDA approved drug library of approximately 1500 compounds, as well as the 400 compound MMV malaria box and identified 44 gametocytocidal compounds with sub to low micromolar IC50s. Major classes of compounds with gametocytocidal activity included quaternary ammonium compounds with structural similarity to choline, acridine-like compounds similar to quinacrine and pyronaridine, as well as antidepressant, antineoplastic, and anthelminthic compounds. Top drug candidates showed near complete transmission blocking in membrane feeding assays. This assay is simple, reproducible and demonstrated robust Z-factor values at low gametocytemia levels, making it amenable to HTS for identification of novel and potent gametocytocidal compounds.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105817PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144897PMC
December 2015

Antimalarial chemotherapy: artemisinin-derived dimer carbonates and thiocarbonates.

Bioorg Med Chem Lett 2014 Jun 16;24(11):2440-3. Epub 2014 Apr 16.

Department of Chemistry, School of Arts and Sciences, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, United States; The Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, United States. Electronic address:

Several 2-carbon-linked trioxane dimer secondary alcohol carbonates 14 and thiocarbonates 15, combined with mefloquine and administered in a low single oral dose, prolonged the survival times of malaria-infected mice much more effectively than the popular monomeric antimalarial drug artemether plus mefloquine. Three dimer carbonates 14 and one dimer thiocarbonate 15 partially cured malaria-infected mice.
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http://dx.doi.org/10.1016/j.bmcl.2014.04.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074917PMC
June 2014

The survival times of malaria-infected mice are prolonged more by several new two-carbon-linked artemisinin-derived dimer carbamates than by the trioxane antimalarial drug artemether.

Bioorg Med Chem Lett 2014 Mar 30;24(5):1285-9. Epub 2014 Jan 30.

Department of Chemistry, School of Arts and Sciences, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, United States; The Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, United States. Electronic address:

Sixteen new artemisinin-derived 2-carbon-linked trioxane dimers were prepared to study chemical structure/antimalarial activity relationships (SAR). Administering a very low single oral dose of only 5mg/kg of dimer secondary alcohol 6a or 6b plus 15 mg/kg of mefloquine hydrochloride prolonged the lives of Plasmodium berghei-infected mice to an average of 25 days after infection. This ACT chemotherapy result is of high medicinal significance because the antimalarial efficacy of the popular trioxane drug artemether (2) plus mefloquine under the same conditions was significantly lower (only 20 day average survival). NH-aryl carbamate derivatives 7e, 7i, and 7j of 2-carbon-linked dimer alcohol 6b also significantly outperformed artemether (2) in prolonging the survival times (25-27 days) of malaria-infected mice.
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http://dx.doi.org/10.1016/j.bmcl.2014.01.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943161PMC
March 2014

Model system to define pharmacokinetic requirements for antimalarial drug efficacy.

Sci Transl Med 2013 Oct;5(205):205ra135

Division of Clinical Pharmacology, Department of Medicine, Johns Hopkins University, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Malaria presents a tremendous public health burden, and new therapies are needed. Massive compound libraries screened against Plasmodium falciparum have yielded thousands of lead compounds, resulting in an acute need for rational criteria to select the best candidates for development. We reasoned that, akin to antibacterials, antimalarials might have an essential pharmacokinetic requirement for efficacy: action governed either by total exposure or peak concentration (AUC/CMAX), or by duration above a defined minimum concentration [time above minimum inhibitory concentration (TMIC)]. We devised an in vitro system for P. falciparum, capable of mimicking the dynamic fluctuations of a drug in vivo. Using this apparatus, we find that chloroquine is TMIC-dependent, whereas the efficacy of artemisinin is driven by CMAX. The latter was confirmed in a mouse model of malaria. These characteristics can explain the clinical success of two antimalarial drugs with widely different kinetics in humans. Chloroquine, which persists for weeks, is ideally suited for its TMIC mechanism, whereas great efficacy despite short exposure (t1/2 in blood 3 hours or less) is attained by CMAX-driven artemisinins. This validated preclinical model system can be used to select those antimalarial lead compounds whose CMAX or TMIC requirement for efficacy matches pharmacokinetics obtained in vivo. The apparatus can also be used to explore the kinetic dependence of other pharmacodynamic endpoints in parasites.
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http://dx.doi.org/10.1126/scitranslmed.3006684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3845824PMC
October 2013

Malaria-infected mice live until at least day 30 after a new artemisinin-derived thioacetal thiocarbonate combined with mefloquine are administered together in a single, low, oral dose.

J Med Chem 2012 Sep 27;55(17):7892-9. Epub 2012 Aug 27.

Department of Chemistry, School of Arts and Sciences, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.

In only three steps and in 21-67% overall yields from the natural trioxane artemisinin, a series of 21 new trioxane C-10 thioacetals was prepared. Upon receiving a single oral dose of only 6 mg/kg of the monomeric trioxane 12c combined with 18 mg/kg of mefloquine hydrochloride, Plasmodium berghei-infected mice survived on average 29.8 days after infection. Two of the four mice in this group had no parasites detectable in their blood on day 30 after infection, and they behaved normally and appeared healthy. One of the mice had 11% blood parasitemia on day 30, and one mouse in this group died on day 29. Of high medicinal importance, the efficacy of this ACT chemotherapy is much better than (almost double) the efficacy under the same conditions using as a positive control the popular trioxane drug artemether plus mefloquine hydrochloride (average survival time of only 16.5 days).
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http://dx.doi.org/10.1021/jm3009986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460521PMC
September 2012

Antimalarial iron chelator, FBS0701, shows asexual and gametocyte Plasmodium falciparum activity and single oral dose cure in a murine malaria model.

PLoS One 2012 21;7(5):e37171. Epub 2012 May 21.

The Malaria Research Institute, W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.

Iron chelators for the treatment of malaria have proven therapeutic activity in vitro and in vivo in both humans and mice, but their clinical use is limited by the unsuitable absorption and pharmacokinetic properties of the few available iron chelators. FBS0701, (S)3"-(HO)-desazadesferrithiocin-polyether [DADFT-PE], is an oral iron chelator currently in Phase 2 human studies for the treatment of transfusional iron overload. The drug has very favorable absorption and pharmacokinetic properties allowing for once-daily use to deplete circulating free iron with human plasma concentrations in the high µM range. Here we show that FBS0701 has inhibition concentration 50% (IC(50)) of 6 µM for Plasmodium falciparum in contrast to the IC(50) for deferiprone and deferoxamine at 15 and 30 µM respectively. In combination, FBS0701 interfered with artemisinin parasite inhibition and was additive with chloroquine or quinine parasite inhibition. FBS0701 killed early stage P. falciparum gametocytes. In the P. berghei Thompson suppression test, a single dose of 100 mg/kg reduced day three parasitemia and prolonged survival, but did not cure mice. Treatment with a single oral dose of 100 mg/kg one day after infection with 10 million lethal P. yoelii 17XL cured all the mice. Pretreatment of mice with a single oral dose of FBS0701 seven days or one day before resulted in the cure of some mice. Plasma exposures and other pharmacokinetics parameters in mice of the 100 mg/kg dose are similar to a 3 mg/kg dose in humans. In conclusion, FBS0701 demonstrates a single oral dose cure of the lethal P. yoelii model. Significantly, this effect persists after the chelator has cleared from plasma. FBS0701 was demonstrated to remove labile iron from erythrocytes as well as enter erythrocytes to chelate iron. FBS0701 may find clinically utility as monotherapy, a malarial prophylactic or, more likely, in combination with other antimalarials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037171PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357340PMC
December 2012

A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells.

Proc Natl Acad Sci U S A 2012 Jun 22;109(26):E1782-90. Epub 2012 May 22.

Seattle Biomedical Research Institute, Seattle, WA 98109-5219, USA.

Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1-binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM.
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http://dx.doi.org/10.1073/pnas.1120534109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387091PMC
June 2012

Chemical genetics of Plasmodium falciparum.

Nature 2010 May;465(7296):311-5

Department of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library-many of which showed potent in vitro activity against drug-resistant P. falciparum strains-and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.
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http://dx.doi.org/10.1038/nature09099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874979PMC
May 2010

Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket.

Biochimie 2009 Nov-Dec;91(11-12):1509-17. Epub 2009 Sep 20.

National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University MS 38677, USA.

The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate binding pocket of PfMDH, which may be important in design of selective PfMDH/PfLDH inhibitors as potential antimalarials.
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http://dx.doi.org/10.1016/j.biochi.2009.09.005DOI Listing
April 2010

Plasmodium falciparum-infected erythrocytes induce NF-kappaB regulated inflammatory pathways in human cerebral endothelium.

Blood 2009 Nov 27;114(19):4243-52. Epub 2009 Aug 27.

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.

Cerebral malaria is a severe multifactorial condition associated with the interaction of high numbers of infected erythrocytes to human brain endothelium without invasion into the brain. The result is coma and seizures with death in more than 20% of cases. Because the brain endothelium is at the interface of these processes, we investigated the global gene responses of human brain endothelium after the interaction with Plasmodium falciparum-infected erythrocytes with either high- or low-binding phenotypes. The most significantly up-regulated transcripts were found in gene ontology groups comprising the immune response, apoptosis and antiapoptosis, inflammatory response, cell-cell signaling, and signal transduction and nuclear factor kappaB (NF-kappaB) activation cascade. The proinflammatory NF-kappaB pathway was central to the regulation of the P falciparum-modulated endothelium transcriptome. The proinflammatory molecules, for example, CCL20, CXCL1, CXCL2, IL-6, and IL-8, were increased more than 100-fold, suggesting an important role of blood-brain barrier (BBB) endothelium in the innate defense during P falciparum-infected erythrocyte (Pf-IRBC) sequestration. However, some of these diffusible molecules could have reversible effects on brain tissue and thus on neurologic function. The inflammatory pathways were validated by direct measurement of proteins in brain endothelial supernatants. This study delineates the strong inflammatory component of human brain endothelium contributing to cerebral malaria.
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http://dx.doi.org/10.1182/blood-2009-06-226415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925626PMC
November 2009

Analysis of quaternary structure of a [LDH-like] malate dehydrogenase of Plasmodium falciparum with oligomeric mutants.

Mol Cell Biochem 2009 May 29;325(1-2):141-8. Epub 2009 Jan 29.

National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA.

L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L: -lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archae and other prokaryotes. Initial sequence analysis and identification of critical amino acid residues involved in inter-subunit salt-bridge interactions predict tetrameric structure for PfMDH. The catalytically active recombinant PfMDH was characterized as a tetramer. The enzyme is localized primarily in the parasites cytosol. To gain molecular insights into PfMDH/PfLDH relationships and to understand the quaternary structure of PfMDH, dimers were generated by mutation to the potential salt-bridge interacting sites. The R183A and R214G mutations, which snapped the salt bridges between the dimers and resulted in lower dimeric state, did not affect catalytic properties of the enzyme. The mutant dimers of PfMDH were active equally as the wild-type PfMDH. The studies reveal structure of PfMDH as a dimer of dimers. The tetrameric state of PfMDH was not essential for catalytic functions of the enzyme but may be an evolutionary adaptation for cytosolic localization to support its role in NAD/NADH coupling, an important metabolic function for survival of the malaria parasite.
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http://dx.doi.org/10.1007/s11010-009-0028-2DOI Listing
May 2009

Plasmodium falciparum-infected erythrocytes decrease the integrity of human blood-brain barrier endothelial cell monolayers.

J Infect Dis 2007 Apr 21;195(7):942-50. Epub 2007 Feb 21.

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.

Background: Central to the pathologic progression of human cerebral malaria (CM) is sequestration of Plasmodium falciparum-infected red blood cells (Pf-IRBCs) to the blood-brain barrier (BBB) endothelium. The molecular interactions between Pf-IRBCs and the BBB endothelium and their implications for barrier function are unclear.

Methods: The effects of Pf-IRBCs on the integrity of the BBB were assessed by electrical cell substrate sensing and by transendothelial electrical resistance measurements in an in vitro human BBB model. In addition, Pf-IRBCs were subfractionated and treated with trypsin, artemisinin, or brefeldin A.

Results: Pf-IRBCs, but not normal red blood cells, significantly decreased BBB resistance. Subfractionation showed that both membrane-associated and soluble Pf-IRBC factors mediate the decrease in BBB resistance. Trypsin treatment significantly reduced Pf-IRBC binding but not their ability to decrease electrical resistance. Likewise, P. falciparum isolates with increased binding to human brain microvascular endothelial cells did not alter the electrical resistance response. Soluble factors from Pf-IRBC culture supernatant decreased resistance by 50%-70% and precipitated with 40% ammonium sulfate saturation. Brefeldin-A partially blocked the ability of Pf-IRBCs to reduce resistance.

Conclusion: The results suggest that, in CM, trypsin-resistant membrane components and soluble factors of Pf-IRBCs contribute to the impedance of BBB integrity in a multistep and multifactorial process.
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http://dx.doi.org/10.1086/512083DOI Listing
April 2007

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization.

Biochem J 2007 Feb;402(1):197-204

Malaria Research Institute, W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205, USA.

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.
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http://dx.doi.org/10.1042/BJ20060986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783988PMC
February 2007

Plasmodium falciparum-infected erythrocytes increase intercellular adhesion molecule 1 expression on brain endothelium through NF-kappaB.

Infect Immun 2006 Jun;74(6):3262-70

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 720 Rutland Avenue, Ross 1170, Baltimore, MD 21205, USA.

Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-IRBC) in postcapillary brain endothelium is a hallmark of cerebral malaria (CM) pathogenesis. There is a correlation between adherent Pf-IRBC and increased expression of intercellular cell adhesion molecule 1 (ICAM-1), which is also a receptor for Pf-IRBC on human brain microvascular endothelial cells (HBMEC). The underlying mechanism for the increased ICAM-1 expression has not been clearly defined. Therefore, we investigated the mechanisms of ICAM-1 expression on isolated HBMEC after exposure to Pf-IRBC. Ultrastructural characterization of the model confirmed that there was attachment through both Pf-IRBC knobs and HBMEC microvillus protrusions. Pf-IRBC induced a dose- and time-dependent increase in ICAM-1 expression on HBMEC that was specific for human brain endothelium and was not observed with human umbilical vein endothelium. Involvement of both membrane-associated Pf-IRBC proteins and parasite-derived soluble factors with the increase in ICAM-1 expression was demonstrated by surface trypsinization and fractionation. Pf-IRBC exposure induced nuclear translocation of NF-kappaB in HBMEC, which was linked to ICAM-1 expression, as shown by use of specific inhibitors of the transcription factor NF-kappaB and immunocytochemistry. In addition, inhibition of reactive oxygen species decreased Pf-IRBC-induced ICAM-1 expression on HBMEC. Parasite-induced ICAM-1 expression explains the localization of this molecule on brain endothelium in postmortem CM brain samples. By increasing ICAM-1 expression, Pf-IRBC may increase their sequestration, thereby perpetuating CM.
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http://dx.doi.org/10.1128/IAI.01625-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1479273PMC
June 2006