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1.
BMC Biotechnol ; 12: 88, 2012 Nov 21.
Article in English | MEDLINE | ID: mdl-23171216

ABSTRACT

BACKGROUND: An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. RESULTS: By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. CONCLUSION: In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.


Subject(s)
Antibodies, Monoclonal/chemistry , Biomarkers, Tumor/immunology , DNA-Binding Proteins/immunology , Endonucleases/immunology , Protein Array Analysis/methods , Antibodies, Monoclonal/immunology , Antibody Specificity , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/chemistry , Carcinoma, Non-Small-Cell Lung/metabolism , Choline-Phosphate Cytidylyltransferase/immunology , Choline-Phosphate Cytidylyltransferase/metabolism , Cross Reactions , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , HEK293 Cells , Humans , Immunohistochemistry/methods , Lung Neoplasms/chemistry , Lung Neoplasms/metabolism
2.
Nature ; 435(7045): 1117-21, 2005 Jun 23.
Article in English | MEDLINE | ID: mdl-15973412

ABSTRACT

Haemoglobin C, which carries a glutamate-to-lysine mutation in the beta-globin chain, protects West African children against Plasmodium falciparum malaria. Mechanisms of protection are not established for the heterozygous (haemoglobin AC) or homozygous (haemoglobin CC) states. Here we report a marked effect of haemoglobin C on the cell-surface properties of P. falciparum-infected erythrocytes involved in pathogenesis. Relative to parasite-infected normal erythrocytes (haemoglobin AA), parasitized AC and CC erythrocytes show reduced adhesion to endothelial monolayers expressing CD36 and intercellular adhesion molecule-1 (ICAM-1). They also show impaired rosetting interactions with non-parasitized erythrocytes, and reduced agglutination in the presence of pooled sera from malaria-immune adults. Abnormal cell-surface display of the main variable cytoadherence ligand, PfEMP-1 (P. falciparum erythrocyte membrane protein-1), correlates with these findings. The abnormalities in PfEMP-1 display are associated with markers of erythrocyte senescence, and are greater in CC than in AC erythrocytes. Haemoglobin C might protect against malaria by reducing PfEMP-1-mediated adherence of parasitized erythrocytes, thereby mitigating the effects of their sequestration in the microvasculature.


Subject(s)
Erythrocytes/metabolism , Erythrocytes/parasitology , Hemoglobin C/metabolism , Malaria/blood , Malaria/prevention & control , Plasmodium falciparum/physiology , Protozoan Proteins/metabolism , Animals , Antibodies/immunology , CD36 Antigens/metabolism , Cell Adhesion , Erythrocyte Aggregation , Erythrocytes/pathology , Flow Cytometry , Hemeproteins/metabolism , Humans , Intercellular Adhesion Molecule-1/metabolism , Malaria/parasitology , Plasmodium falciparum/pathogenicity
3.
Mol Microbiol ; 53(2): 445-55, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15228526

ABSTRACT

The complications of malaria in pregnancy are caused by the massive sequestration of parasitized erythrocytes (PE) in the placenta. Placental isolates of Plasmodium falciparum are unusual in that they do not bind the primary microvasculature receptor CD36 but instead bind chondroitin sulphate A (CSA). Pregnant mothers develop antibodies that recognize placental variants worldwide, suggesting that a vaccine against malaria in pregnancy is possible. Some members of the Duffy binding-like gamma (DBL-gamma) domain of the large and diverse P. falciparum erythrocyte membrane protein-1 (PfEMP-1) family, when expressed on Chinese hamster ovary (CHO) cells, bind CSA. To characterize better the molecular requirements for DBL-gamma adhesion to CSA, we determined the binding of various DBL-gamma domains. Most DBL-gamma did not bind CSA, and no conserved region was identified that strictly differentiated binders from non-binders. Structure-function analysis of the FCR3-CSA DBL-gamma domain localized the minimal CSA binding region to a 67-residue fragment. This region was partially conserved among some binding sequences. Serum from a rabbit immunized with the minimal domain reacted with CSA-binding parasite lines, but not with non-CSA-adherent PE lines that adhered to CD36 and other receptors. The identification of a minimal binding region from a highly variable cytoadherent family may have application for a vaccine against malaria in pregnancy.


Subject(s)
Antibodies, Protozoan/immunology , Chondroitin Sulfates/metabolism , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Antigens, Protozoan/metabolism , CD36 Antigens/metabolism , Cell Adhesion , Cell Line , Conserved Sequence , Cricetinae , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/immunology , Membrane Proteins/metabolism , Phylogeny , Plasmodium falciparum/pathogenicity , Protein Binding , Protein Structure, Tertiary , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Rabbits , Sequence Homology , Structure-Activity Relationship
4.
Infect Immun ; 71(8): 4536-43, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12874333

ABSTRACT

The variant surface antigens of Plasmodium falciparum are an important component of naturally acquired immunity and an important vaccine target. However, these proteins appear to elicit primarily variant-specific antibodies. We tested if naked DNA immunization can elicit more cross-reactive antibody responses and allow simultaneous immunization with several variant constructs. Mice immunized with plasmid DNA expressing variant cysteine-rich interdomain region 1 (CIDR1) domains of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) developed antibodies that were reactive to the corresponding PfEMP1s as measured by an enzyme-linked immunosorbent assay, flow cytometry, and agglutination of parasitized erythrocytes (PEs). We observed some cross-reactive immune responses; for example, sera from mice immunized with one domain agglutinated PEs of various lines and recognized heterologous domains expressed on the surface of Chinese hamster ovary (CHO) cells. We found no significant antigenic competition when animals were immunized with a mixture of plasmids or immunized sequentially with individual constructs. Moreover, mixed or sequential immunizations resulted in greater cross-reactive agglutination responses than immunization with a single domain. Recombinant protein (Sc y179) immunization after priming with DNA (prime-boost regimen) increased antibody titers to the homologous domain substantially but seemed to diminish the cross-reactive responses somewhat. The titer of agglutinating antibodies was previously shown to correlate with protection. Surprisingly, the agglutination titers of sera from DNA immunization were high, similar to those of pooled human hyperimmune sera. These sera also appeared to give limited low-titer variant transcending agglutination. Thus, DNA immunization appears to be a very useful tool for developing variant antigen vaccines.


Subject(s)
Antibodies, Protozoan/biosynthesis , Antigens, Protozoan/genetics , Malaria Vaccines/pharmacology , Plasmodium falciparum/genetics , Plasmodium falciparum/immunology , Vaccines, DNA/pharmacology , Animals , Antigens, Protozoan/chemistry , CHO Cells , Cricetinae , Cross Reactions , Female , Hemagglutination , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Mice , Mice, Inbred BALB C , Plasmids/genetics , Vaccines, DNA/genetics , Vaccines, DNA/immunology
5.
Blood ; 101(7): 2850-7, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12517811

ABSTRACT

The pathogenicity of Plasmodium falciparum is due to the unique ability of infected erythrocytes (IRBCs) to adhere to vascular endothelium. We investigated whether adhesion of IRBCs to CD36, the major cytoadherence receptor on human dermal microvascular endothelial cells (HDMECs), induces intracellular signaling and regulates adhesion. A recombinant peptide corresponding to the minimal CD36-binding domain from P falciparum erythrocyte membrane protein 1 (PfEMP1), as well as an anti-CD36 monoclonal antibody (mAb) that inhibits IRBC binding, activated the mitogen-activated protein (MAP) kinase pathway that was dependent on Src-family kinase activity. Treatment of HDMECs with a Src-family kinase-selective inhibitor (PP1) inhibited adhesion of IRBCs in a flow-chamber assay by 72% (P <.001). More importantly, Src-family kinase activity was also required for cytoadherence to intact human microvessels in a human/severe combined immunodeficient (SCID) mouse model in vivo. The effect of PP1 could be mimicked by levamisole, a specific alkaline-phosphatase inhibitor. Firm adhesion to PP1-treated endothelium was restored by exogenous alkaline phosphatase. In contrast, inhibition of the extracellular signal-regulated kinase 1/2 (ERK 1/2) and p38 MAP kinase pathways had no immediate effect on IRBC adhesion. These results suggest a novel mechanism for the modulation of cytoadherence under flow conditions through a signaling pathway involving CD36, Src-family kinases, and an ectoalkaline phosphatase. Targeting endothelial ectoalkaline phosphatases and/or signaling molecules may constitute a novel therapeutic strategy against severe falciparum malaria.


Subject(s)
Endothelium, Vascular/cytology , Plasmodium falciparum/cytology , Signal Transduction , src-Family Kinases/physiology , Alkaline Phosphatase/pharmacology , Animals , Binding Sites , CD36 Antigens/chemistry , CD36 Antigens/metabolism , CD36 Antigens/physiology , Cell Adhesion/drug effects , Endothelium, Vascular/chemistry , Erythrocytes/parasitology , Erythrocytes/pathology , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Mice , Mice, SCID , Microcirculation , Peptide Fragments/pharmacology , Perfusion , Signal Transduction/drug effects , Signal Transduction/physiology
6.
Blood ; 101(1): 331-7, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12393525

ABSTRACT

The parasite ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and host endothelial receptors represent potential targets for antiadhesive therapy for cytoadherence. In the present study, the major host receptor CD36 was targeted in vitro and in vivo with a recombinant peptide, PpMC-179, corresponding to the minimal CD36-binding domain from the cysteine-rich interdomain region 1 (CIDR1) within the MCvar1 PfEMP1. The in vitro inhibitory effect of PpMC-179 on human dermal microvascular endothelial cells (HDMECs) expressing multiple relevant adhesion molecules was investigated using a parallel-plate flow chamber. Pretreatment of endothelial monolayers with PpMC-179 (2 microM) inhibited the adhesion of infected erythrocytes (IRBCs) from all clinical isolates tested by 84.4% on resting and 62.8% on tumor necrosis factor alpha (TNF-alpha)-stimulated monolayers. Adhesion to stimulated cells was further inhibited (90.4%) when PpMC-179 was administered with an inhibitory anti-intercellular adhesion molecule 1 (ICAM-1) monoclonal antibody 84H10 (5 microg/mL). To determine the in vivo effectiveness of PpMC-179, we used a human/severe combined immunodeficiency (SCID) mouse chimeric model that allowed direct visualization of cytoadherence on intact human microvasculature. In unstimulated skin grafts, PpMC-179 inhibited adhesion by 86.3% and by 84.6% in TNF-alpha-stimulated skin grafts. More importantly, PpMC-179 administration resulted in the detachment of already adherent IRBCs by 80.7% and 83.3% on resting and stimulated skin grafts, respectively. The antiadhesive effect of PpMC-179 was rapid and sustained in vivo for at least 30 minutes. Our data indicate that targeting cytoadhesion in vivo is feasible and may offer a rapid antimalarial therapy.


Subject(s)
Plasmodium falciparum/drug effects , Protozoan Proteins/pharmacology , Adhesiveness/drug effects , Animals , Endothelium, Vascular/metabolism , Humans , Mice , Mice, Transgenic , Microcirculation , Microscopy, Video , Plasmodium falciparum/pathogenicity , Protozoan Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology
7.
Article in English | MEDLINE | ID: mdl-12446418

ABSTRACT

Because of the breakdown of malaria control programs, the constant emergence of drug resistant parasites, and, possibly, climatic changes malaria poses a major problem for the developing countries. In addition, because of the speed of international travel it is being seen with increasing frequency as an imported disease in non-tropical countries. This update explores recent information about the pathophysiology of the disease, its protean hematological manifestations, and how carrier frequencies for the common hemoglobin disorders have been maintained by relative resistance to the malarial parasite. In Section I, Dr. Louis Miller and colleagues consider recent information about the pathophysiology of malarial infection, including new information about interactions between the malarial parasite and vascular endothelium. In Section II, Dr. David Roberts discusses what is known about the complex interactions between red cell production and destruction that characterize the anemia of malaria, one of the commonest causes of anemia in tropical countries. In Section III, Dr. David Weatherall reviews recent studies on how the high gene frequencies of the thalassemias and hemoglobin variants have been maintained by heterozygote advantage against malaria and how malaria has shaped the genetic structure of human populations.


Subject(s)
Erythrocytes/parasitology , Malaria , Adult , Animals , Blood Group Antigens/genetics , Child , Erythrocytes/pathology , Genetic Variation , HLA Antigens/genetics , Hemoglobins/genetics , Humans , Immunity/genetics , Malaria/complications , Malaria/etiology , Malaria/transmission , Plasmodium/physiology , Transfusion Reaction
8.
Proc Natl Acad Sci U S A ; 99(15): 10020-4, 2002 Jul 23.
Article in English | MEDLINE | ID: mdl-12096191

ABSTRACT

Plasmodium falciparum-infected erythrocytes adhere dichotomously to the host receptors CD36 and chondroitin sulfate A (CSA). This dichotomy is associated with parasite sequestration to microvasculature beds (CD36) or placenta (CSA), leading to site-specific pathogenesis. Both properties are mediated by members of the variant P. falciparum erythrocyte membrane protein 1 (PfEMP-1) family and reside on nonoverlapping domains of the molecule. To identify the molecular basis for the apparent dichotomy, we expressed various domains of PfEMP-1 individually or in combination and tested their binding properties. We found that the CD36-binding mode of the cysteine-rich interdomain region-1 (CIDR1) ablates the ability of the Duffy binding-like gamma domain to bind CSA. In contrast, neither a non-CD36-binding CIDR1 nor an intercellular adhesion molecule 1 binding domain had any affect on CSA binding. Our findings point out that interactions between different domains of PfEMP-1 can alter the adhesion phenotype of infected erythrocytes and provide a molecular basis for the apparent dichotomy in adhesion. We suggest that the basis for the dichotomy is structural and that mutually exclusive conformations of PfEMP-1 are involved in binding to CD36 or CSA. Furthermore, we propose a model explaining the requirement for structural dichotomy between placental and nonplacental isolates.


Subject(s)
CD36 Antigens/physiology , Chondroitin Sulfates/physiology , Erythrocytes/parasitology , Plasmodium falciparum/physiology , Protozoan Proteins/genetics , Animals , CHO Cells , Cell Line , Cricetinae , Erythrocyte Membrane/parasitology , Genetic Variation , Mammals , Mutagenesis , Plasmodium falciparum/genetics , Protozoan Proteins/chemistry , Protozoan Proteins/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
9.
Nature ; 418(6895): 320-3, 2002 Jul 18.
Article in English | MEDLINE | ID: mdl-12124623

ABSTRACT

Widespread use of antimalarial agents can profoundly influence the evolution of the human malaria parasite Plasmodium falciparum. Recent selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite, resembling effects attributed in current debates to a historic population bottleneck. Chloroquine-resistant (CQR) parasites were initially reported about 45 years ago from two foci in southeast Asia and South America, but the number of CQR founder mutations and the impact of chlorquine on parasite genomes worldwide have been difficult to evaluate. Using 342 highly polymorphic microsatellite markers from a genetic map, here we show that the level of genetic diversity varies substantially among different regions of the parasite genome, revealing extensive linkage disequilibrium surrounding the key CQR gene pfcrt and at least four CQR founder events. This disequilibrium and its decay rate in the pfcrt-flanking region are consistent with strong directional selective sweeps occurring over only approximately 20-80 sexual generations, especially a single resistant pfcrt haplotype spreading to very high frequencies throughout most of Asia and Africa. The presence of linkage disequilibrium provides a basis for mapping genes under drug selection in P. falciparum.


Subject(s)
Antimalarials/pharmacology , Chloroquine/pharmacology , Drug Resistance/genetics , Genetic Variation/genetics , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Selection, Genetic , Alleles , Animals , Evolution, Molecular , Founder Effect , Genome, Protozoan , Haplotypes/genetics , Humans , Linkage Disequilibrium/genetics , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Microsatellite Repeats/genetics , Phenotype , Polymorphism, Genetic/genetics
11.
Proc Natl Acad Sci U S A ; 99(6): 3860-5, 2002 Mar 19.
Article in English | MEDLINE | ID: mdl-11904437

ABSTRACT

Immunity to Plasmodium falciparum in African children has been correlated with antibodies to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) variant gene family expressed on the surface of infected red cells. We immunized Aotus monkeys with a subregion of the Malayan Camp variant antigen (MCvar1) that mediates adhesion to the host receptor CD36 on the endothelial surface and present data that PfEMP1 is an important target for vaccine development. The immunization induced a high level of protection against the homologous strain. Protection correlated with the titer of agglutinating antibodies and occurred despite the expression of variant copies of the gene during recurrent waves of parasitemia. A second challenge with a different P. falciparum strain, to which there was no agglutinating activity, showed no protection but boosted the immune response to this region during the infection. The level of protection and the evidence of boosting during infection encourage further exploration of this concept for malaria vaccine development.


Subject(s)
Antigens, Protozoan/chemistry , Antigens, Protozoan/immunology , Aotus trivirgatus/immunology , Aotus trivirgatus/parasitology , Genetic Variation/genetics , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Animals , Antibodies, Protozoan/immunology , Antigens, Protozoan/genetics , Malaria Vaccines/immunology , Malaria, Falciparum/parasitology , Malaria, Falciparum/prevention & control , Male , Mutation/genetics , Parasitemia/immunology , Parasitemia/parasitology , Parasitemia/prevention & control , Plasmodium falciparum/classification , Plasmodium falciparum/genetics , Protein Structure, Tertiary , Vaccination
12.
Nature ; 415(6872): 673-9, 2002 Feb 07.
Article in English | MEDLINE | ID: mdl-11832955

ABSTRACT

Malaria is today a disease of poverty and underdeveloped countries. In Africa, mortality remains high because there is limited access to treatment in the villages. We should follow in Pasteur's footsteps by using basic research to develop better tools for the control and cure of malaria. Insight into the complexity of malaria pathogenesis is vital for understanding the disease and will provide a major step towards controlling it. Those of us who work on pathogenesis must widen our approach and think in terms of new tools such as vaccines to reduce disease. The inability of many countries to fund expensive campaigns and antimalarial treatment requires these tools to be highly effective and affordable.


Subject(s)
Malaria, Falciparum/etiology , Animals , Endothelium, Vascular/parasitology , Erythrocytes/parasitology , Humans , Life Cycle Stages , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Placenta/parasitology , Plasmodium falciparum/growth & development , Plasmodium falciparum/physiology
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