Progress 10/01/00 to 09/30/05
Outputs
Sequestration in the microvessels of the deep tissues is a signal characteristic of the human malaria Plasmodium falciparum.The adhesion of P.falciparum-infected cells to the postcapillary endothelial cells in various tissues contributes to both the pathology of the disease (i.e. organ infarcts and coma) and parasite survival(i.e.the microaerophilic environment favors plasmodial growth while avoiding passage through and destruction in the spleen). We have identified a conformational change in a region of band 3 protein, called the DIDS-binding region that is involved in the enhanced adhesiveness of P.falciparum-infected erythrocytes to CD36 on the endothelial cell Further, anti-peptide antibodies generated against an amino acid sequence of the DIDS-binding region (YETFSKLIKIFQDH) recognized P. falciparum- infected erythrocytes. In addition, sera from individuals living in a malaria endemic area (and who are presumably immune) contained immunoglobulins specific for this
region of band 3. The anti-peptide antibodies reacted with the surface excrescences (knobs) on falciparum-infected erythrocytes. In uninfected erythrocytes the band 3 region was cryptic and its exposure on the falciparum-infected erythrocyte surface required clustering of band 3 protein. Thus, a parasite-induced modification of band 3 promotes adhesion and induces antigenic changes in the P. falciparum-infected erythrocyte. In an attempt to identify other surface antigens of falciparum-infected erythrocytes (iRBC), antibodies were eluted from iRBCthat had been treated with a pool of sera from malaria-infected individuals (IHS), and were used to screen a phage display library (PDL). After repeated panning of the PDL on immobilized antibodies, phage that selectively bound to IHS were accumulated. Of 23 randomly chosen clones that were sequenced, 13 individual sequences were detected at varying frequencies and 3 of the 13 sequences had homology with membrane proteins known to exist on
iRBC. The majority of phage clones (7 out of 8 clones) selected after the 4th panning bound selectively to IgG in IHS. Specific binding of the selected phage to IgG in IHS was also confirmed using 24 IHS and 11 sera from uninfected individuals. One phage clone was the most frequently found in the sequenced clones after the 4th panning, and the binding of this clone to IgG in all IHS was greater than in any serum from uninfected individuals. A rabbit antiserum against the peptide expressed on the clone specifically recognized the surface of iRBC and resulted in iRBC haemolysis.
Impacts
Malaria remains one of the world's greatest public health problems. At present, there are more than 300 million human infections and annually more than 3 million deaths, mostly children under 5 years of age. Because there is no protective vaccine against the most deadly of all the malarias, Plasmodium falciparum, identification of protective antigens remains a high priority. The surface antigens of the falciparum-infected erythrocyte, especially those involved in its increased adhesiveness, have been considered as prime Candidates because they are exposed to the immune system for more than 24 hours. This work provides the basis for novel vaccines and therapies for falciparum malaria.
Publications
- Winograd, E., S. Eda, and I. W. Sherman. 2004. Chemical modifications of band 3 protein affect the adhesion of Plasmodium falciparum-infected erythrocytes to CD36. Mol. Biochem. Parasitol. 136:243-248.
- Winograd, E. and I. W. Sherman. 2004. Malaria infection induces a conformational change in erythrocyte band 3 protein. Mol. Biochem. Parasitol. 138:83-87.
- Eda, S. and I. W. Sherman. 2005. Selection of peptides recognized by human antibodies against the surface of Plasmodium falciparum-infected erythrocytes. Parasitology 130:1-11.
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Progress 01/01/03 to 12/31/03
Outputs
Erythrocytes infected with the mature stages of PLASMODIUM FALCIPARUM sequester in the deep tissues by binding to the surface of endothelial cells (EC). Sequestered parasitized erythrocytes (PE) contribute to the pathology of falciparum malaria by mechanically blocking small blood vessels, leading to unarousable coma and death. The significance of this research is to use molecular approaches to discover the interactive regions of the PE and EC and then to develop anti-adhesive agents that can act to unplug microvessels in the deep tissues of humans infected with malaria. Surface changes in the malaria-infected erythrocyte membrane are involved in antigenic variation and also play a role in sequestration. We have used a phage display library in order to find specific surface antigens and to identify peptidic sequences recognized by immune sera from malaria patients. Some of the peptidic sequences show significant homology with proteins on the surface of infected
erythrocytesboth parasite-encoded and an intrinsic membrane protein of the red cell the band 3 protein. Antibody raised against one peptide specifically recognized the surface of malaria-infected erythrocytes and caused hemolysis of the infected cells through complement activation. In addition, another peptide was found to be able to specifically target infected red cells and to cause destruction of infected cells when coupled with a lytic protein. Based on our finding that treatment of red cells with DIDS (a reagent that specifically binds to band 3 protein) ablates the adhesion of infected red cells by greater than 50% we were able to synthesize a peptide (corresponding to the DIDS-binding region) that effectively inhibited the binding of PE to CD36. In addition, we found that patients from malaria endemic areas contain antibodies that specifically recognize this peptide. We were able to induce exposure of this peptide by treatment of red cells with acridine orange, an agent that
acts to cluster band 3 protein. These data strongly suggest that cytoadhesion and neoantigen exposure in malaria-infected erythrocytes result from parasite induced aggregation of the band 3 protein.
Impacts
Malaria infects 300 million people and kills 1-2 annually, mostly children under the age of 5. For the most part death is due to blood vessel blockage by infected red blood cells and organ infarcts. Determination of the molecules involved in the binding of malaria-infected red blood cells to the cells lining the blood vessels of the deep tissues, could provide the basis for the discovery of novel therapies that would unplug blood vessels, thus preventing host tissue destruction and death.
Publications
- Sherman, I.W., S. Eda, and E. Winograd. 2003. Cytoadherence and sequestration in PLASMODIUM FALCIPARUM: defining the ties that bind. Microbes and Infection 5:897-909.
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Progress 01/01/02 to 12/31/02
Outputs
Erythrocytes infected with the mature trophozoite and schizont stages of PLASMODIUM FALCIPARUM sequester in the deep tissues by binding to the surface of endothelial cells (EC). Sequestered parasitized erythrocytes (PE) contribute to the pathology of falciparum malaria by mechanically blocking small blood vessels, leading to unarousbale coma and death. The significance of this research is to use molecular approaches to discover the interactive regions of the PE and EC and then to develop "molecular teflons" that can be therapeutic to unplug the microvessels. Surface changes in the malaria-infected erythrocyte membrane are involved in antigenic variation and enable the parasites to evade the host immune attack. Some parasite proteins inserted in infected erythrocyte membrane have shown to serve as variant antigens as well as ligands for endothelial receptors; however, the identification of conserved and potent epitopes for vaccine development is still under
investigation. We have used a phage display library in order to find specific surface antigens and have identified peptidic sequences that are specifically recognized by immune sera from malaria patients. Some of the peptidic sequences show significant homology with proteins on the surface of infected erythrocytes. Antibody raised against one peptides specifically recognized the surface of malaria-infected erythrocytes and caused hemolysis of the infected cells through complement activation. This peptide will be tested with a variety of parasite strains to detrmine whether the epitope is conserved. We have found that treatment of red cells with DIDS (a reagent that specifically binds to band 3 protein) ablates the adhesion of infected red cells by greater than 50%. Furthermore, peptides derived from the DIDS binding region of band 3 can effectively inhibit the binding of PE to CD36. In addition, we have found that patients from malaria endemic areas contain antibodies that
specifically recognize the DIDS binding region. These data strongly suggests that cytoadhesion and neoantigen exposure in malaria infected erythrocytes can result from parasite induced modifications of host cell membrane proteins.
Impacts
Malaria results in 1-2 million death annually. In addition, falciparum malaria may result in unarousable coma (cerebral malaria) as well as organ infarct due to blood vessel blockage by infected red blood cells. Determination of the molecules involved in the attachment of malaria-infected red blood cells to the endothelial cells lining the blood vessels, could provide the basis for the discovery of novel therapies that would unplug the microscopic blood vessels, thus reversing coma in the tens of thousands of patients who suffer and die from cerebral malaria.
Publications
- Shigetoshi Eda, and Irwin W. Sherman (2002) Cytoadherence of malaria-infected red blood cells involves exposure of phosphatidylserine. Cellular Physiology and Biochemistry 12 (5-6), 373-38
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Progress 01/01/01 to 12/31/01
Outputs
Erythrocytes infected with the mature trophozoite and schizont stages of PLASMODIUM FALCIPARUM sequester in the deep tissues by binding to the surface of endothelial cells (EC). Sequestered parasitized erythrocytes (PE) contribute to the pathology of falciparum malaria by mechanically blocking small blood vessels, leading to unarousbale coma and death. The significamce of this research is to use molecular approaches to discover the interactive regions of the PE and EC and then to develop "molecular teflons" that can be therapeutic to unplug the microvessels. To assess the activity of anti-adhesive substances we have developed a rapid, high capacity in vitro assay using 96 well microplates and have tested the effects of cominatorial libraries of peptides on the binding of malaria-infected red cells; we found 11 active mixtures out of 120 mixtures. We have developed a flow system for studying the adherence of P. FALCIPARUM-infected red cells to EC and immobilized
proteins. Under physiological flow conditions the effect of recombinant human lactoferrin has been shown to be anti-adhersive. We have found that exposure of phosphatidyl-serine (PS) on the P. FALCIPARUM-infected red blood cell membrane is involved in the adhesion of PE to CD36 and thrombospondin, and found that small, soluble form of PS, glycerophosphorylserine could inhibit CD36- and thrombospondin-mediated infected cell binding, suggesting that this molecule may assist in the development of novel and effective anti-adhesive agents. We have identified changes in the molecular size of band 3 protein during the intra-erythrocytic growth of P. FALCIPARUM. Further, red cells treated with DIDS (a reagent that specifically binds to band 3 protein) ablates the adhesion of infected red cells by greater than 50%. This suggests that modifications in an intrinsic protein of the erythrocyte play a role in adhesiveness and antigenicity similar to the parasite-encode protein PLASMODIUM FALCIPARUM
erythrocyte membrane protein 1 (PfEMP1).
Impacts
Malaria results in 3 million deaths, mostly children under the age of 5. Specific information on the molecules involved in the attachment of malaria-infected red blood cells to the cells that line the blood vessels, and which blocks blood flow, should lead to the discovery of novel therapies that can unblock the blood vessels, thus reversing coma in the tens of thousands of patients who suffer and die from cerebral malaria.
Publications
- No publications reported this period
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Progress 01/02/00 to 12/31/00
Outputs
Erythrocytes infected with the human malaria PLASMODIUM FALCIPARUM sequester in the deep tissues by binding to the surface of microvessels (post-capillary venules). Sequestered parasitized erythrocytes, which do not circulate, may contribute to the pathophysiology of malignant tertian malaria by mechanically blocking microvessels, leading to local hypoxia and lactic acid acidosis. The goal of this research program is to use molecular approaches to design new anti-adhesive drugs for falciparum malaria. In collaboration with Pharming Inc. we have evaluated the anti-adhesive properties of recombinant human lactoferrin. We have found that during the intracellular growth of P. FALCIPARUM there is exofacial exposure of phosphatidylserine in the red blood cell membrane, and this is involved in the adhesion to CD36. We have found that infected red cells bind to the RGD (Arg-Gly-Asp) sequence of fibronectin found on the microvessel surface.
Impacts
Since none of the therapies for malaria specifically target the adhesive phenomena associated with sequestration our findings could lead to novel anti-adhesive therapeutics (=molecular teflon).
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
A simple, efficient, sensitive, reproducible and high throughput assay for measuring the cytoadherence of PLASMODIUM FALCIPARUM-infected red blood cells (human malaria) was developed. The assay format uses 96-well microplates, with the number of P. FALCIPARUM parasitized erythrocytes bound determined by measuring PLASMODIUM specific lactic dehydrogenase activity colorimetrically (absorbance at 655 nm) using the 3-acetylpyridine analog of nicotinamide adenine dinucleotide, nitro blue tetrazolium and diaphorase. The results of the microplate assay were found to be comparable to those using microscopic analysis but much less time consuming. Lectoferrin (LF), a human serum protein, strongly inhibited the adherence of PLASMODIUM FALCIPARUM-infected erythrocytes (PE) to immobilized chondroitin sulfate A (CSA)-conjugated albumin at a concentration of 100 microgram/mL and blocked the PE binding to CD36-expressing Chinese hamster ovary (CHO) cells, as well as immobilized CD36
at concentrations of 5 microgram/mL and 100 microgram/mL, respectively. Biotinylated LF bound to CD36 in a saturable manner, and such binding was inhibited by unlabeled LF and the anti-CD36 monoclonal antibody, 8A6, suggesting specificity of binding. Additionally, LF inhibited PE binding to immobilized thrombospondin (TSP) at a concentration of 100 microgram/mL, and specific binding of LF to TSP was confirmed using biotinylated LF. LF inhibited PE binding to C32 amelanotic melanoma cells in a dose-dependent manner. A peptide of LF, Arg-Asn-Met Arg-Lys-Val Arg-Gly-Pro-Pro-Val-Ser-Cys (amino acid residues 25-37 of LF), which has been suggested to contribute to LF binding to various materials, including CSA, inhibited PE binding to immobilized CSA-conjugated albumin, immobilized CD36, CD36-expressing CHO cells, immobilized TSP, and C32 amelanotic melanoma cells, as well as LF itself. These results suggest that LF peptide may provide the basis for developing agents that are able to
inhibit CSA-, CD36-, and TSP-mediated cytoadherence of PE.
Impacts
Falciparum malaria kills more than 3 million people annually; therefore, our work on the development of anti-adhesive therapeutics for malaria could improve the health and well-being of those who live in tropical (mostly African) agricultural nations.
Publications
- Eda, S., Eda, K., Prudhomme, J.G., and Sherman, I.W. 1999. Inhibitory activity of human lactoferrin and its peptide on chondroitin sulfate A-, CD36- and thrombospondin-mediated cytoadherence of PLASMODIUM FALCIPARUM-infected erythrocytes. Blood 94(1):326-332.
- Prudhomme, J.G. and Sherman, I.W.. 1999. A high capacity in vitro assay for measuring the cytoadherence of PLASMODIUM FALCIPARUM-infected erythrocytes. Journal of Immunological Methods 229:169-176.
- Eda, S., Lawler, J. and Sherman, I.W. 1999. PLASMODIUM FALCIPARUM-infected erythrocyte adhesion to the type 3 repeat domain of thrombospondin-1 is mediated by a modified band 3 protein. Molecular and Biochemical Parasitology 100:195-205.
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Progress 01/01/98 to 12/01/98
Outputs
Specific regions of the human erythrocyte anion transport protein, AE 1 or band 3, have been identified as being adhesive in PLASMODIUM FALCIPARUM-infected erythrocytes (iRBC). Previously, the binding site for the Plasmodium falciparum-infected erythrocyte (PE) was determined to be the C-terminal 120 or 140 kDa region but not the N-terminal 25 kDa domain of thrombospondin (TSP). In this work, we have localized the TSP binding site for PE more precisely. PE adhered to glutathione-S-transferase-fusion proteins containing the type 3 repeat (T3) of TSP, but not to other functional domains of TSP (i.e., N-terminal domain, procollagen domain, type 1 and 2 repeat, and C-terminal domain). Soluble T3 inhibited PE binding to immobilized TSP. PE binding to immobilized T3 was inhibited by soluble TSP, a monoclonal antibody directed against the T3, glycine-arginine-glycine-aspartic acid-serine-proline (GRGDSP) peptide, and *cysteine-GRGDSP-cysteine*, where *cysteine and cysteine*
form a disulfide linkage, suggesting involvement of an RGD-containing motif in the T3. In support of this, a fusion protein which excluded the RGD motif showed no PE binding activity. Earlier it was shown that the amino acid sequence of the band 3 protein, histidine-proline-leucine-glutamine-lysine-threonine-tyrosine (HPLQKTY), was exposed on PE and mediated PE binding to TSP. Monoclonal antibodies, which recognize HPLQKTY and inhibit PE binding to TSP, also inhibited P binding to the T3. The involvement of the sequence was confirmed by the fact that an octamer of HPLQKTY-containing peptide bound to the T3.
Impacts
(N/A)
Publications
- SHERMAN, I. W. (Editor). 1998. Maleria Parasite Biology, Pathogenesis and Protection. ASM Press. 700 pp.
- LUCAS, J. Z. and I. W. SHERMAN. 1998. Plasmodium-falciparum: Thrombospondin mediates parasitized erythrocyte band 3-related adhesin binding. Exper. Parasit. 89:78-85.
- PRUDHOMME, J. G., G. ALMEIDA-PORADA, J. L. ASCENSAO and I. W. SHERMAN. 1998. Plasmodium falciparum-infected erythrocytes adhere to immortalized human bone marrow endothelial cells. (Letter to the Editor) In Vitro Cell. Dev. Biol. 43:524.
- SHIGETOSHI EDAL, JACK LAWLER2 and IRWIN W. SHERMAN1.
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Progress 01/01/95 to 12/30/95
Outputs
Specific regions of the human erythrocyte anion transport protein, AE 1 or band 3, have been identified as being adhesive in PLASMODIUM FALCIPARUM-infected erythrocytes. In addition, synthetic peptides based on these sequences and murine monoclonal antibodies (Mabs) to these block cytoadherence/sequestration. These findings suggest the possibility that humans who are able to control P. FALCIPARUM infections may produce anti-adhesin (and thus anti-band 3) antibodies. To test this hypothesis, sera from individuals living in The Gambia and Southern California were assays for reactivity to decapeptides patterned on putative exofacial regions of the human anion transporter, band 3 protein. Sera from an area highly endemic for malaria, The Gambia, were found to have strong reactivity to well-defined regions of the band 3 protein (some of which contain the adhesin), whereas minimal reactivity was observed with sera from individuals living in a geographic area where malaria
transmission is rare (California). Sera from The Gambia reacted strongly with residue blocks 534-560, 638-660, and 808-842. Gambian sera that reacted strongly with peptides patterned on band 3 failed to react with native band 3 on an immunoblot, but did react with fixed P. FALCIPARUM-infected erythrocytes. Artificial malaria-infected red cells were produced using polystyrene microspheres with covalently bound peptides fashioned on band 3 sequences previously found to be adherent (residues 546-553 and 820-829 and called pfalhesin).
Impacts
(N/A)
Publications
- No publications reported this period.
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