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Ross L Coppel

Publications and source records attributed to Ross L Coppel.

At least 73 records · Page 4Linked to original sources
PubMed2003

Oral immunization with a recombinant malaria protein induces conformational antibodies and protects mice against lethal malaria.

The increasing death toll from malaria, due to the decreasing effectiveness of current prophylactic and therapeutic regimens, has sparked a search for alternative methods of control, such as vaccines. Although several single proteins have shown some promise as subunit vaccines against sexual blood stages in experimental systems, it is clear that multicomponent vaccines are required. Many logistic difficulties make such an approach prohibitively expensive. In an effort to try to overcome some of these issues, we examined the possibility of oral immunization as a route for inducing host protective immunity. We report here that oral feeding of a malaria protein induced serum antibody levels similar to those induced by intraperitoneal immunization with Freund's adjuvant. Further, responses to conformational epitopes were induced. In the rodent challenge system, significant levels of protection to lethal challenge with malaria were induced in mice. The protective efficacy was highly correlated with antibody levels, which depended on the antigen dosage and required cholera toxin subunit B as an oral adjuvant. These findings offer new approaches to the development of a malaria vaccine and provide justification for the investigation of transgenic plants as a means of vaccine delivery.

Administration, Oral↗
PubMed2002

The Plasmodium vivax homologues of merozoite surface proteins 4 and 5 from Plasmodium falciparum are expressed at different locations in the merozoite.

Merozoite surface proteins of Plasmodium falciparum are one major group of antigens currently being investigated and tested as malaria vaccine candidates. Two recently described P. falciparum merozoite surface antigens, MSP4 and MSP5, are GPI-anchored proteins that each contain a single EGF-like domain and appear to have arisen by an ancient gene duplication event. The genes are found in tandem on chromosome 2 of P. falciparum and the syntenic region of the genome was identified in the rodent malarias P. chabaudi, P. yoelii and P. berghei. In these species, there is only a single gene, designated MSP4/5 encoding a single EGF-like domain similar to the EGF-like domain in both PfMSP4 and PfMSP5. Immunization of mice with PyMSP4/5 provides mice with high levels of protection against lethal challenge with blood stage P. yoelii. In this study, we show that in P. vivax, which is quite phylogenetically distant from P. falciparum, both MSP4 and MSP5 homologues can be found with their relative arrangements with respect to the surrounding genes mostly preserved. However, the gene for MSP2, found between MSP5 and adenylosuccinate lyase (ASL) in P. falciparum, is absent from P. vivax. The PvMSP4 and PvMSP5 genes have a two-exon structure and encode proteins with potential signal and GPI anchor sequences and a single EGF-like domain near the carboxyl-terminus. Rabbit antisera raised against purified recombinant proteins show that each of the antisera react with distinct proteins of 62 kDa for PvMSP4 and 86 kDa for PvMSP5 in parasite lysates. Indirect immunofluorescence assays (IFA) localized PvMSP4 over the entire surface of P. vivax merozoites, as expected, whereas, the MSP5 homologue was found to be associated with an apical organellar location consistent with micronemes or over the polar prominence.

Adenylosuccinate Lyase↗
PubMed2002

Contribution of parasite proteins to altered mechanical properties of malaria-infected red blood cells.

Red blood cells (RBCs) parasitized by Plasmodium falciparum are rigid and poorly deformable and show abnormal circulatory behavior. During parasite development, knob-associated histidine-rich protein (KAHRP) and P falciparum erythrocyte membrane protein 3 (PfEMP3) are exported from the parasite and interact with the RBC membrane skeleton. Using micropipette aspiration, the membrane shear elastic modulus of RBCs infected with transgenic parasites (with kahrp or pfemp3 genes deleted) was measured to determine the contribution of these proteins to the increased rigidity of parasitized RBCs (PRBCs). In the absence of either protein, the level of membrane rigidification was significantly less than that caused by the normal parental parasite clone. KAHRP had a significantly greater effect on rigidification than PfEMP3, contributing approximately 51% of the overall increase that occurs in PRBCs compared to 15% for PfEMP3. This study provides the first quantitative information on the contribution of specific parasite proteins to altered mechanical properties of PRBCs.

Animals↗
PubMed2002

Identification of HLA-A2-restricted CD8(+) cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross-presented by dendritic cells.

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4(+) and CD8(+) T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2-restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159-167 of PDC-E2, induces specific MHC class I-restricted CD8(+) CTL lines from 10/12 HLA-A2(+) PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2-specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.

Antigen Presentation↗
PubMed2002

PlasmoDB: the Plasmodium genome resource. An integrated database providing tools for accessing, analyzing and mapping expression and sequence data (both finished and unfinished).

PlasmoDB (http://PlasmoDB.org) is the official database of the Plasmodium falciparum genome sequencing consortium. This resource incorporates finished and draft genome sequence data and annotation emerging from Plasmodium sequencing projects. PlasmoDB currently houses information from five parasite species and provides tools for cross-species comparisons. Sequence information is also integrated with other genomic-scale data emerging from the Plasmodium research community, including gene expression analysis from EST, SAGE and microarray projects. The relational schemas used to build PlasmoDB [Genomics Unified Schema (GUS) and RNA Abundance Database (RAD)] employ a highly structured format to accommodate the diverse data types generated by sequence and expression projects. A variety of tools allow researchers to formulate complex, biologically based queries of the database. A version of the database is also available on CD-ROM (Plasmodium GenePlot), facilitating access to the data in situations where Internet access is difficult (e.g. by malaria researchers working in the field). The goal of PlasmoDB is to enhance utilization of the vast quantities of data emerging from genome-scale projects by the global malaria research community.

Animals↗
PubMed2002

Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase.

Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes. Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis. The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment. Furthermore, it has been shown that ASL is not subject to positive natural selection. We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P. falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium. The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution. Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships. We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed. Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa.

Adenylosuccinate Lyase↗
PubMed2002

Assignment of functional roles to parasite proteins in malaria-infected red blood cells by competitive flow-based adhesion assay.

Adhesion of parasitized red blood cells (PRBCs) to endothelial cells and subsequent accumulation in the microvasculature are pivotal events in the pathogenesis of falciparum malaria. During intraerythrocytic development, numerous proteins exported from the parasite associate with the RBC membrane skeleton but the precise function of many of these proteins remain unknown. Their cellular location, however, suggests that some may play a role in adhesion. The adhesive properties of PRBCs are best studied under flow conditions in vitro; however, experimental variation in levels of cytoadherence in currently available assays make subtle alterations in adhesion difficult to quantify. Here, we describe a flow-based assay that can quantify small differences in adhesion and document the extent to which a number of parasite proteins influence adhesion using parasite lines that no longer express specific proteins. Loss of parasite proteins ring-infected erythrocyte surface antigen (RESA), knob-associated histidine-rich protein (KAHRP) or Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) had a significant effect on the ability of PRBCs to adhere, whereas loss of mature parasite-infected erythrocyte surface antigen (MESA) had no effect. Our studies indicate that a number of membrane skeleton-associated parasite proteins, although not exposed on the RBC surface, can collectively affect the adhesive properties of PRBCs and further our understanding of pathophysiologically relevant structure/function relationships in malaria-infected RBCs.

Animals↗
PubMed2002

Quantitation and phenotypic analysis of natural killer T cells in primary biliary cirrhosis using a human CD1d tetramer.

BACKGROUND & AIMS: Natural killer T (NKT) cells are a subset of lymphocytes incriminated in playing an important role in the modulation of the innate immune response and the development of autoimmunity. However, there have been only limited studies attempting to quantitate the number of NKT cells in autoimmune disease, particularly because of difficulties associated with definition of this subpopulation. METHODS: We used a human CD1d (hCD1d) tetramer produced by a baculovirus expressing recombinant CD1d protein complexed with alpha-galactosylceramide (alpha-GalCer) and quantitated hCD1d tetramer reactive cells in blood and liver from controls and patients with primary biliary cirrhosis (PBC). RESULTS: The majority of CD1d-alphaGalCer-restricted NKT cells were positive for TCR Valpha24 and Vbeta11. There was a distinct CD4- CD8+ population within the CD1d-alphaGalCer-restricted NKT cells in addition to the CD4- CD8- and CD4+ CD8- population. The frequency of CD1d-alphaGalCer-restricted NKT cells was similar between blood and liver in healthy individuals. In contrast, the frequency of CD1d-alphaGalCer-restricted NKT cells in the liver was significantly higher than in the blood of PBC patients. The frequency of CD1d-alpha-GalCer-restricted NKT cells in the liver was also significantly higher in PBC patients than in healthy individuals. CONCLUSIONS: The frequency and function of such cells should be studied not only in blood but also in the target organ of the autoimmune disease. Selective enrichment of CD1d-alphaGalCer-restricted NKT cells at the site of inflammation is observed in PBC, suggesting a role of these cells in the development of PBC.

Antigens, CD1↗
PubMed2002

Contribution to antimitochondrial antibody production: cleavage of pyruvate dehydrogenase complex-E2 by apoptosis-related proteases.

Patients with PBC produce a directed, specific response to a single immunodominant autoepitope of PDC-E2 within the inner lipoyl domain. In contrast, immunized animals react to multiple epitopes and rarely recognize the inner lipoyl domain. In other autoimmune diseases, apoptosis plays a critical role in antigen presentation; the caspases and granzyme B are the key proteases in the generation of autoepitopes. To determine the specific cleavage pattern of full-length recombinant PDC-E2, we performed in vitro digestion with caspases-3, -6, -8 and granzyme B. The resulting fragments were immunoblotted and probed with an extensive panel of monoclonal anti-PDC-E2 antibodies and sera from patients with PBC. Interestingly, on granzyme B digestion, PDC-E2 lost reactivity, suggesting the destruction of the immunodominant epitope. Because this site contains the major epitope for both B cells and T cells, it suggests that granzyme B is unlikely to be involved in generation of autoepitopes in primary biliary cirrhosis (PBC). In contrast, following treatment with the caspase enzymes, immunoreactive fragments were generated. Indeed, by confocal microscopy, activated caspase-3 is found in the marginal hepatocytes and bile ducts. Moreover, caspase-3 staining was strongest in the small intrahepatic bile ducts, the major site of tissue destruction in PBC. In conclusion, these data suggest that following apoptosis, the caspase family of proteolytic enzymes have the potential to generate immunogenic fragments that contribute to the autoantigen reservoir and the production of antimitochondrial antibodies. These findings are also consistent with the generation of an autoimmune response against an intracellular antigen that evades catabolism during apoptosis.

Adult↗
PubMed2002

Analysis of TCR antagonism and molecular mimicry of an HLA-A0201-restricted CTL epitope in primary biliary cirrhosis.

Although the etiology and mechanism of primary biliary cirrhosis (PBC) is unknown, growing evidence suggests a major role for T cells. We have recently identified the first CD8 T-cell epitope, amino acid 159-167 of the E2 component of pyruvate dehydrogenase complexes (PDC-E2). To seek for analogue peptide-antagonizing effector function of CTLs specific for this autoantigen, we examined the effector functions of the PDC-E2-specific CTLs against alanine substituted peptides. Furthermore, because molecular mimicry has been postulated as a possible cause of initiating PBC, we carried out studies aimed at identifying naturally occurring peptides for the 159-167 peptide of PDC-E2 that may serve as agonists. An alanine substitution at position 5 of this epitope significantly reduced peptide-specific effector functions of CTLs. Moreover, this analogue peptide inhibited effector functions of the CTLs to the prototype peptide, including cytotoxicity and IFN-gamma production. We also identified a peptide derived from Pseudomonas aeruginosa, which showed a higher binding affinity to the HLA-A*0201 than the prototype peptide. This homologous peptide was recognized by CTLs specific for the prototype epitope on PDC-E2. In conclusion, a modification of the immunodominant autoepitope can be utilized to manipulate the CD8 T-cell responses against the autoantigen PDC-E2. Our finding also supports the thesis that molecular mimicry may be implicated in the initiation of the autoreactive CD8 T-cell responses and has implications for the use of such peptides for immunotherapy.

Bacterial Proteins↗
PubMed2002

Differential expression of intestinal trefoil factor in biliary epithelial cells of primary biliary cirrhosis.

Intestinal trefoil factor (ITF) promotes epithelial cell migration and mucosal restitution during inflammation. We used real-time quantitative PCR, in situ nucleic acid hybridization, and immunohistochemistry to study the expression of the ITF gene and protein expression in the liver of primary biliary cirrhosis (PBC) and controls. There were significantly higher levels of ITF messenger RNA (mRNA) in PBC liver compared with primary sclerosing cholangitis (PSC) (P <.05) or normal controls (P <.001) and also higher in hepatitis C virus (HCV) liver (P <.05) and cryptogenic cirrhosis (P <.01) compared with normal controls. However, only in PBC was there a significant difference between small (interlobular and bile ductules) and large (intrahepatic and septal) bile ducts. Using in situ hybridization, the highest levels of ITF gene expression were localized to the large bile ducts in PBC. This differential expression of ITF was also noted at the protein level. Thus, in PBC, although 92% of large bile ducts expressed the ITF protein, only 2% of small bile ducts (P <.0001) expressed ITF. In contrast, in control livers, 34% of large bile ducts and 13% of small bile ducts expressed ITF. ITF protein is absent in small bile ducts in all stages of PBC. In conclusion, the expression of ITF may play an important role in bile duct damage. In small bile ducts, ITF production in response to damage is absent, making such cells vulnerable to damage and providing a thesis for the selective loss of small, but not large, bile ducts in PBC.

Biliary Tract↗
PubMed2002

Comprehensive mapping of HLA-A0201-restricted CD8 T-cell epitopes on PDC-E2 in primary biliary cirrhosis.

Growing evidence has implicated the involvement of autoreactive T lymphocytes in the pathogenesis of primary biliary cirrhosis (PBC). We have recently taken advantage of motif prediction analysis of HLA-A*0201 and identified the first major histocompatibility complex (MHC) class I restricted epitope, amino acids 159 to 167 on E2 components of pyruvate dehydrogenase complexes (PDC-E2), the major mitochondrial antigens in PBC. The mechanisms involved in the selection of epitope peptide(s) that comprise the PDC-E2-specific autoreactive cytotoxic T lymphocytes (CTLs) are unknown and likely involve other epitopes on PDC-E2 restricted by MHC class I molecules. To address this issue, a comprehensive mapping of the CTL epitope repertoire on the PDC-E2 molecule that binds HLA-A*0201 was performed to provide further clues regarding the role of CTLs. We used the T2 cell line to screen 79 overlapping 15mer peptides, spanning the entire PDC-E2 molecule. Six of the 79 peptides exhibited significantly higher binding activity to HLA-A*0201 than the other 15mer peptides. Two of these 6 peptides induced CTL lines from patients with PBC. Fine mapping with N-terminus or C-terminus truncated peptides identified 10mer peptide, PDC-E2 amino acids 165 to 174, which is a novel CD8 epitope restricted by HLA-A*0201. In conclusion, using a combination of the 15mer peptide library screening with the T2 binding assay and also the induction of CTL lines with candidate peptides, we have defined a novel HLA-A*0201-restricted epitope PDC-E2 165 to 174 in patients with PBC. These data will become important in the development of altered peptide ligands to modulate disease activity.

Amino Acid Sequence↗
PubMed2002

Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pyruvate dehydrogenase complex (PDC-E2)-specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti-PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the Calpha constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6% nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further support of the hypothesis that PDC-E2-specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2-specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC.

Animals↗
PubMed2002

Immunization with a combination of merozoite surface proteins 4/5 and 1 enhances protection against lethal challenge with Plasmodium yoelii.

It is widely believed that subunit vaccines composed of multiple components will offer greater protection against challenge by malaria, and yet there is little experimental evidence to support this view. We set out to test this proposition in the Plasmodium yoelii challenge system in rodents by comparing the degree of protection conferred by immunization with a mixture of merozoite surface proteins to that conferred by single proteins. We therefore examined a defined protein mixture made of the epidermal growth factor-like domains of P. yoelli merozoite surface protein 1 (MSP1) and MSP4/5, the homologue of P. falciparum MSP4 and MSP5. In the present study we demonstrate that this combination of recombinant proteins dramatically enhances protection against lethal malaria challenge compared to either protein administered alone. Many mice immunized with the MSP4/5 plus MSP1(19) combination did not develop detectable parasitemia after challenge. Combined immunization with MSP1(19) and yMSP4/5, a product characterized by lower protective efficacy, also greatly enhanced protection by reducing peak parasitemias and increasing the numbers of survivors. In some combination trials, levels of antibodies to MSP1(19) were elevated compared to the MSP1(19) alone group; however, improved protection occurred regardless of whether boosting of the anti-MSP1(19) response was observed. Boosting of anti-MSP1(19) did not appear to be due to contaminating endotoxin in the EcMSP4/5 material since enhanced protection was observed in C3H/HeJ mice, which are endotoxin insensitive. Collectively, these experiments show that multiantigen combinations offer enhanced levels of protection against asexual stage infection and suggest that combinations of MSP1, MSP4, and MSP5 should be evaluated further for use in humans.

Animals↗
PubMed2002

Quantitative and functional analysis of PDC-E2-specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis.

While the pathologic mechanisms responsible for organ-specific tissue damage in primary biliary cirrhosis (PBC) remain an enigma, it has been suggested that the pathology is mediated by autoreactive T cells infiltrating the intrahepatic bile ducts. Previously, we have documented that there is 100-fold enrichment in the frequency of CD4(+) autoreactive T cells in the liver that are specific for peptides encoded by the E2 components of the pyruvate dehydrogenase complexes (PDC-E2). We have also recently characterized the first MHC class I-restricted epitope for PDC-E2, namely amino acid 159-167, a region very similar to the epitope recognized by MHC class II-restricted CD4(+) cells and by autoantibodies. The effector functions of these PDC-E2(159-167)-specific CD8(+) cytotoxic T lymphocytes (CTLs) are not well understood. We have taken advantage of tetramer technology and report herein that there is tenfold increase in the frequency of PDC-E2(159-167)-specific CTLs in the liver as compared with the blood in PBC. In addition, the precursor frequency of the CTLs in blood was significantly higher in early-stage PBC. Of interest was the fact that, upon stimulation with the peptide, the response of PDC-E2(159-167) tetramer-positive cells is heterogeneous with respect to IFN-gamma synthesis. These data, we believe for the first time, document the enrichment of autoantigen-specific CD8(+) T cells in the PBC liver, suggesting that CD8(+) T cells play a significant role in the immunopathogenesis of PBC.

Autoantigens↗