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R L Coppel

Publications and source records attributed to R L Coppel.

At least 19 recordsLinked to original sources
PubMed2004

Merozoite surface protein 4/5 provides protection against lethal challenge with a heterologous malaria parasite strain.

Immunization with merozoite surface protein 4/5 (MSP4/5), the murine malaria homologue of Plasmodium falciparum MSP4 and MSP5, has been shown to protect mice against challenge by parasites expressing the homologous form of the protein. The gene encoding MSP4/5 was sequenced from a number of Plasmodium yoelii isolates in order to assess the level of polymorphism in the protein. The gene was found to be highly conserved among the 13 P. yoelii isolates sequenced, even though many of the same isolates showed pronounced variability in their MSP1(19) sequences. Nonsynonymous mutations were detected only for the isolates Plasmodium yoelii nigeriensis N67 and Plasmodium yoelii killicki 193L and 194ZZ. Immunization and challenge of BALB/c mice showed that the heterologous MSP4/5 proteins were able to confer a level of protection against lethal Plasmodium yoelii yoelii YM challenge infection similar to that induced by immunization with the homologous MSP4/5 protein. To explore the limits of heterologous protection, mice were immunized with recombinant MSP4/5 protein from Plasmodium berghei ANKA and Plasmodium chabaudi adami DS and challenged with P. y. yoelii YM. Interestingly, significant protection was afforded by P. berghei ANKA MSP4/5, which shows 81% sequence identity with P. y. yoelii YM MSP4/5, but it was abolished upon reduction and alkylation. Significant protection was not observed for mice immunized with recombinant P. c. adami DS MSP4/5, which shows 55.7% sequence identity with P. y. yoelii YM MSP4/5. This study demonstrates the robustness of MSP4/5 in conferring protection against variant forms of the protein in a murine challenge system, in contrast to the situation found for other asexual-stage proteins, such as MSP1(19) and AMA1.

Amino Acid Sequence↗
PubMed2003

The protective efficacy of MSP4/5 against lethal Plasmodium chabaudi adami challenge is dependent on the type of DNA vaccine vector and vaccination protocol.

The enhancement of immunogenicity of malarial DNA vaccines is important if they are to have practical application in protecting against blood-stage malaria. Here we describe three different DNA vaccine vector types used in conjunction with the blood-stage merozoite surface protein 4/5 (MSP4/5), the murine homologue of Plasmodium falciparum MSP4 and MSP5, in an attempt to enhance survival against lethal Plasmodium chabaudi adami DS blood-stage challenge. MSP4/5 was inserted into VR1020 (secretory), monocyte-chemotactic protein-3 (MCP-3) (chemoattractant), and cytotoxic T-lymphocyte antigen 4 (CTLA4) (lymph node targeting) vectors. Mice were immunized intradermally via gene-gun, IM injection, or boosting with recombinant MSP4/5 protein. Antibody responses after boosting were predominantly of the IgG1 and IgE isotypes, with low avidity antibodies produced in DNA primed groups. Despite antibody responses comparable to recombinant protein immunization, boosting mice primed with antigens encoded by MCP-3 and CTLA4 vectors did not enhance survival compared to vector control groups. Gene-gun vaccination using VR1020/MSP4/5 followed by recombinant MSP4/5 boosting, or gene-gun DNA vaccination alone using MCP-3/MSP4/5, resulted in enhanced survival compared to empty vector control mice. The results suggest that the enhancement of survival against lethal blood-stage malaria challenge after utilizing MSP4/5 DNA vaccination is therefore highly dependent on the route and type of vaccine vector employed.

Animals↗
PubMed2001

Comparison of the protective efficacy of yeast-derived and Escherichia coli-derived recombinant merozoite surface protein 4/5 against lethal challenge by Plasmodium yoelii.

The gene encoding the Plasmodium yoelii homologue of P. falciparum merozoite surface proteins 4 (MSP4) and 5 (MSP5) has been expressed in Escherichia coli and Saccharomyces cerevisiae. The protein contains a single epidermal growth factor (EGF)-like domain and is expressed in a form lacking the predicted N-terminal signal and glycosyl phosphatidylinositol (GPI) attachment sequences. The recombinant protein derived from E. coli (EcMSP4/5) was highly effective at protecting mice against lethal challenge with 10(5) parasites of the P. yoelii YM strain. In contrast, the protective efficacy of yeast-derived MSP4/5 (yMSP4/5) was considerably less. The antibody titres in both groups were significantly different with mice immunised with yeast-derived protein showing significantly lower pre-challenge antibody responses. There was a significant inverse correlation between antibody levels as measured by ELISA and peak parasitaemia. Mice immunised with EcMSP4/5 produced anti-PyMSP4/5 antibodies predominantly of the IgG2a and IgG2b isotypes, whereas, mice immunised with yMSP4/5 mainly produced antibodies of the IgG1 isotype. The differences in antibody titres and subtype distribution may account for the observed differences in protective efficacy of these protein preparations. Levels of protective efficacy of MSP4/5 were compared with that obtained using P. yoelii MSP1 produced in S. cerevisiae. Levels of protection induced by E. coli derived MSP4/5 were superior to those induced by MSP1 which in turn were better than those induced by yeast-derived MSP4/5.

Animals↗
PubMed2001

Immunoreactivity of organic mimeotopes of the E2 component of pyruvate dehydrogenase: connecting xenobiotics with primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), the major autoepitope recognized by both T and B cells is the inner lipoyl domain of the E2 component of pyruvate dehydrogenase. To address the hypothesis that PBC is induced by xenobiotic exposure, we took advantage of ab initio quantum chemistry and synthesized the inner lipoyl domain of E2 component of pyruvate dehydrogenase, replacing the lipoic acid moiety with synthetic structures designed to mimic a xenobiotically modified lipoyl hapten, and we quantitated the reactivity of these structures with sera from PBC patients. Interestingly, antimitochondrial Abs from all seropositive patients with PBC, but no controls, reacted against 3 of the 18 organic modified autoepitopes significantly better than to the native domain. By structural analysis, the features that correlated with autoantibody binding included synthetic domain peptides with a halide or methyl halide in the meta or para position containing no strong hydrogen bond accepting groups on the phenyl ring of the lysine substituents, and synthetic domain peptides with a relatively low rotation barrier about the linkage bond. Many chemicals including pharmaceuticals and household detergents have the potential to form such halogenated derivatives as metabolites. These data reflect the first time that an organic compound has been shown to serve as a mimeotope for an autoantigen and further provide evidence for a potential mechanism by which environmental organic compounds may cause PBC.

Amino Acid Sequence↗
PubMed2001

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis.

The characterization of differentially expressed genes provides a powerful tool for identifying molecules that may be involved in the pathogenesis of disease. We have used two independent techniques to identify overexpressed transcripts in bile duct cells and in liver from patients with primary biliary cirrhosis (PBC). In the first method, we used suppressive subtractive hybridization to compare mRNA from isolated PBC bile duct epithelial cells (BECs) to normal BECs and identified 71 clones as transcribed at higher levels in PBC-BECs. Amongst these clones, 62/71 had matches in a non-redundant nucleotide database and 9/71 had matches in an EST database. Of the 62 clones, 51/62 include a complexity of genes involved in cell proliferation, signal transduction, transcription regulation, RNA processing, carbohydrate metabolism and hypothetical/unknown proteins; 4/62 were identified as interstitial collagenase and collagenase precursors, 4/62 as ribosomal proteins, 3/62 as mitochondrial DNA. The mitochondrial cDNA sequences included cytochrome c oxidase, Wnt-13, and the pHL gene, a c-myc oncogene containing coxIII sequence. In the second method, we constructed cDNA libraries from three different PBC livers and sequenced a total of 12,324 independent clones. These 12,324 clones underwent virtual subtraction with 2,814,148 independent clones from Incyte LifeSeq libraries. Twenty one sequences were identified as unique to PBC liver. Collectively, these approaches identified a number of genes involved in signalling, RNA processing, mitochondrial function, inflammation, and fibrosis. Interestingly, both Wnt-13 and Notch transcripts are overexpressed in PBC liver. Further studies are needed to focus on the significance of these genes during the natural history of disease.

Bile Ducts↗
PubMed2001

The malaria-infected red blood cell: structural and functional changes.

The asexual stage of malaria parasites of the genus Plasmodium invade red blood cells of various species including humans. After parasite invasion, red blood cells progressively acquire a new set of properties and are converted into more typical, although still simpler, eukaryotic cells by the appearance of new structures in the red blood cell cytoplasm, and new proteins at the red blood cell membrane skeleton. The red blood cell undergoes striking morphological alterations and its rheological properties are considerably altered, manifesting as red blood cells with increased membrane rigidity, reduced deformability and increased adhesiveness for a number of other cells including the vascular endothelium. Elucidation of the structural changes in the red blood cell induced by parasite invasion and maturation and an understanding of the accompanying functional alterations have the ability to considerably extend our knowledge of structure-function relationships in the normal red blood cell. Furthermore, interference with these interactions may lead to previously unsuspected means of reducing parasite virulence and may lead to the development of novel antimalarial therapeutics.

Animals↗
PubMed2001

Merozoite surface protein 8 of Plasmodium falciparum contains two epidermal growth factor-like domains.

By motif searching of the unfinished sequences in the Malaria Genome Sequencing Project databases we have identified a novel EGF-like domain-containing protein of Plasmodium falciparum. The sequence lies within a single open reading frame of 1791 bp and is predicted to encode a polypeptide of 597 amino acids. There are hydrophobic regions at the extreme N- and C-termini, which could represent secretory signal peptide and GPI attachment sites, respectively. Similar to MSP1, there are two EGF-like domains located near the C-terminus. RT-PCR analysis of the novel gene shows that it is transcribed in asexual stages of the malaria parasite. We have expressed portions of the protein as recombinant GST fusions in Escherichia coli and raised antisera in rabbits. Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, MSP4 or MSP5 expressed as GST fusion proteins. Antiserum raised to the most C-terminal region of the protein reacts with four bands of 98, 50, 25 and 19 kDa in P. falciparum parasite lysates whereas antisera to the N-terminal fusion proteins recognise the 98 and 50 kDa bands, suggesting that the novel protein may undergo processing in a similar way to MSP1. Immunoblot analysis of stage-specific parasite samples reveals that the protein is present throughout the parasite asexual life cycle and in isolated merozoites, with the smaller fragments present in ring stage parasites. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites, schizonts and free merozoites by indirect immunofluorescence. Antisera to the C-terminus stain the surface of rings, whereas antisera to the N-terminus do not, suggesting that a fragment of the protein is carried into the developing ring stage parasite. Based on the accepted nomenclature in the field we designate this protein MSP8. We have shown that the MSP8 fusion proteins are in a conformation that can be recognised by human immune sera and that there is very limited diversity in the MSP8 gene sequences from various P. falciparum laboratory isolates. MSP8 shows significant similarity to the recently reported sequence of the protective P. yoelii merozoite surface protein pypAg-2 [Burns JM, Belk CC, Dunn PD. Infect Immun 2000;68:6189-95.] suggesting that the two proteins are homologues. Taken together, these findings suggest that MSP8/pypAg-2 may play an important role in the process of red cell invasion and is a potential malaria vaccine candidate.

Amino Acid Sequence↗
PubMed2001

Bioinformatics and the malaria genome: facilitating access and exploitation of sequence information.

The torrent of sequence information unleashed by the various genome sequencing projects, including that of Plasmodium falciparum, will lead to an unprecedented increase in the data available for research purposes. The scientific community is struggling to develop ways to assimilate this information and ensure that it is fully analysed in a way that enables rapid development of new therapeutic and diagnostic advances. This is particularly so for the field of tropical medicine where many of the scientists have had limited training in the area of Bioinformatics and may be further hampered by poor access to the sequence data. A number of collections of malaria genome sequence are available, each with their own advantages and disadvantages, however further improvements in these information resources are needed. In particular, there would be great benefit in integrating genomic sequence and functional genomics results with the large amount of pre-existing knowledge related to parasite biology and immunological interactions with the host. Attempts to achieve this include the PlasmoDB database, and the lessons learned in this effort could be of great utility to other organism-specific databases.

Animals↗
PubMed2001

Properties of the Plasmodium falciparum homologue of a protective vaccine candidate of Plasmodium yoelii.

We describe an unusual tryptophan-rich protein of Plasmodium falciparum that contains threonine-rich repeats. The protein is encoded by a 2.5 kb gene with a two-exon structure including a short AT-rich intron that is spliced out of the mature message. The 5' end of the gene encodes a hydrophobic region, which is assumed to be a signal peptide. The peptide sequence is characterised by a tryptophan-rich region and a block of degenerate threonine repeats. The protein is synthesised throughout the asexual life cycle and has an apparent molecular weight of approximately 94 kDa. It has a variable molecular weight in different strains of P. falciparum. Length polymorphisms can be found in the intron region and the second exon. Four single nucleotide mutations are localised in the tryptophan-rich region and two were found in the threonine-repeat block. Homology searches based on gene structure and amino acid sequence revealed a relationship with a P. yoelii antigen that has been used successfully in vaccine studies. Thus, this P. falciparum antigen should be considered an additional candidate for assessment in vaccination against the asexual blood-stages of P. falciparum.

Amino Acid Sequence↗
PubMed2001

Discovering patterns in Plasmodium falciparum genomic DNA.

A method has been developed for discovering patterns in DNA sequences. Loosely based on the well-known Lempel Ziv model for text compression, the model detects repeated sequences in DNA. The repeats can be forward or inverted, and they need not be exact. The method is particularly useful for detecting distantly related sequences, and for finding patterns in sequences of biased nucleotide composition, where spurious patterns are often observed because the bias leads to coincidental nucleotide matches. We show here the utility of the method by applying it to genomic sequences of Plasmodium falciparum. A single scan of chromosomes 2 and 3 of P. falciparum, using our method and no other a priori information about the sequences, reveals regions of low complexity in both telomeric and central regions, long repeats in the subtelomeric regions, and shorter repeat areas in dense coding regions. Application of the method to a recently sequenced contig of chromosome 10 that has a particularly biased base composition detects a long internal repeat more readily than does the conventional dot matrix plot. Space requirements are linear, so the method can be used on large sequences. The observed repeat patterns may be related to large-scale chromosomal organization and control of gene expression. The method has general application in detecting patterns of potential interest in newly sequenced genomic material.

Algorithms↗
PubMed2001

Heterogeneous response of antimitochondrial autoantibodies and bile duct apical staining monoclonal antibodies to pyruvate dehydrogenase complex E2: the molecule versus the mimic.

The 2-oxo-acid dehydrogenase complexes and, in particular, the E2 component of the pyruvate dehydrogenase complex (PDC) are the target of antimitochondrial antibodies (AMA). More than 95% of primary biliary cirrhosis (PBC) patients have detectable levels of autoantibodies to PDC-E2 and in general these react with a region of the molecule that contains the prosthetic group lipoic acid (LA). LA is vital to the function of the enzyme, although there is conflicting evidence as to whether its presence is required for PDC-E2 recognition by AMA. Some, but not all, monoclonal antibodies (mAbs) to PDC-E2 produce an intense staining pattern at the apical surface of bile duct epithelial cells (BEC) in patients with PBC, and it has been argued that the molecule at the apical surface of PBC bile duct cells is a modified form of PDC-E2 or a cross-reactive molecule, acting as a molecular mimic. Herein, we characterize the epitopes recognized by 4 anti-PDC-E2 mAbs that give apical staining patterns (3 mouse and 1 human). In particular, by using a combination of recombinant antigens, competitive inhibition assays, and a unique peptide-on-bead assay, we determined that these apically staining mAbs recognize 3 or 4 distinct epitopes on PDC-E2. More importantly, this suggests that a portion spanning the entire inner lipoyl domain of PDC-E2 can be found at the BEC apical surface. In addition, competition assays with patient sera and a PDC-E2-specific mAb showed significant epitope overlap with only 1 of the 3 mouse mAbs and showed a differential response to the peptide bound to beads. These findings further highlight the heterogeneous response of patient autoantibodies to the inner lipoyl domain of PDC-E2.

Amino Acid Sequence↗
PubMed2001

Immunoglobulin gene usage and immunohistochemical characteristics of human monoclonal antibodies to the mitochondrial autoantigens of primary biliary cirrhosis induced in the XenoMouse.

The immunodominant antimitochondrial antibody (AMA) response in primary biliary cirrhosis (PBC) is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). The nature of the clonal selection process is unclear, and to address this issue, we took advantage of a transgenic technology, XenoMouse, that contains 80% of the human immunoglobulin (Ig) variable gene repertoire and can produce high-affinity human antibodies to virtually any immunogen without evidence of clonal bias. We immunized mice with PDC-E2 to obtain 13 HmAbs, including 4 IgG(2) and 9 IgM isotypes. Immunoglobulin gene analysis was unique and demonstrated a clonal bias; the immunoglobulin gene usage was considerably different from other antibody responses analyzed in XenoMouse systems. Four of the 13 mAbs recognized the inner lipoyl domain of PDC-E2, 2 of 13 recognized the entire PDC-E2 molecule, 4 of 13 recognized PDC-E2 and OGDC-E2, 1 of 13 recognized OGDC only, 1 recognized BCOADC-E2 only, and 1 recognized an unidentified 100-kd mitochondrial protein. Immunohistochemical staining using these HmAbs produced mitochondrial staining of septal bile ducts in both PBC and control livers. Ig gene analysis showed that 7 of 13 HmAbs used the V(H)3 and 4 of 13 used VH4 gene repertoire, respectively. Three of 7 V(H)3 antibodies used the same Ig VH3-21 gene family found in human AMA from patients with PBC. The CDRs of these autoantibodies were slightly mutated when compared with the sequences present within the Ig germline genes. In conclusion, the XenoMouse not only recapitulates the unique specificity and restriction of PBC patients, but indicates that the autoantibodies are derived from a restricted clonal selection process. Such data suggest that the original immunogen leads to somatic mutation without subsequent development of determinant spreading.

Amino Acid Sequence↗
PubMed2001

Antibody responses to infections with strains of Plasmodium falciparum expressing diverse forms of merozoite surface protein 2.

Individuals living in areas where Plasmodium falciparum is endemic experience numerous episodes of infection. These episodes may or may not be symptomatic, with the outcome depending on a combination of parasite and host factors, several of which are poorly understood. One factor is believed to be the particular alleles of several parasite proteins to which the host is capable of mounting protective immune responses. We report a study examining antibody responses to MSP2 in 15 semi-immune teenagers and adults living in the Khanh-Hoa area of southern-central Vietnam, where P. falciparum is highly endemic; subjects were serially infected with multiple strains of P. falciparum. The MSP2 alleles infecting these subjects were determined by nucleotide sequencing. A total of 62 MSP2 genes belonging to both dimorphic families were identified, of which 33 contained distinct alleles, with 61% of the alleles being detected once. Clear changes in the repertoire occurred between infections. Most infections contained a mixture of parasites expressing MSP2 alleles from both dimorphic families. Two examples of reinfection with a strain expressing a previously encountered allele were detected. Significant changes in antibody levels to various regions of MSP2 were detected over the course of the experiment. There was no clear relation between the infecting form of MSP2 and the ensuing antibody response. This study highlights the complexity of host-parasite relationship for this important human pathogen.

Adolescent↗
PubMed2001

Naturally acquired antibody responses to Plasmodium falciparum merozoite surface protein 4 in a population living in an area of endemicity in Vietnam.

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein that is being developed as a component of a subunit vaccine against malaria. We report here the measurement of naturally acquired antibodies to MSP4 in a population of individuals living in the Khanh-Hoa region of Vietnam, an area where malaria is highly endemic. Antibodies to MSP4 were detected in 94% of the study population at titers of 1:5,000 or greater. Two forms of recombinant MSP4 produced in either Escherichia coli or Saccharomyces cerevisiae were compared as substrates in the enzyme-linked immunosorbent assay. There was an excellent correlation between reactivity measured to either, although the yeast substrate was recognized by a higher percentage of sera. Four different regions of MSP4 were recognized by human antibodies, demonstrating that there are at least four distinct epitopes in this protein. In the carboxyl terminus, where the single epidermal growth factor-like domain is located, the reactive epitope(s) was shown to be conformation dependent, as disruption of the disulfide bonds almost completely abolished reactivity with human antibodies. The anti-MSP4 antibodies were mainly of the immunoglobulin G1 (IgG1) and IgG3 subclasses, suggesting that such antibodies may play a role in opsonization and complement-mediated lysis of free merozoites. Individuals in the study population were drug-cured and followed up for 6 months; no significant correlation was observed between the anti-MSP4 antibodies and the absence of parasitemia during the surveillance period. As a comparison, antibodies to MSP1(19), a leading vaccine candidate, were measured, and no correlation with protection was observed in these individuals. The anti-MSP1(19) antibodies were predominantly of the IgG1 isotype, in contrast to the IgG3 predominance noted for MSP4.

Adolescent↗
PubMed2001

Primary biliary cirrhosis: from induction to destruction.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease that predominantly affects middle-aged women; fatigue and pruritus are the most common symptoms at presentation. Liver function tests are consistent with cholestasis and reveal an elevation of serum alkaline phosphatase and gamma-glutamyl transpeptidase with or without elevation of aminotransferase levels. Histologically, PBC is characterized by the destruction of the intrahepatic small bile ducts and subsequently fibrosis. The serological hallmark of the disease is the presence of antimitochondrial antibodies, which are found in 95% of patients with PBC. The antimitochondrial antibodies are directed against the 2-oxo-acid dehydrogenase complexes located on the inner membrane of the mitochondria. PBC generally slowly progresses, even over decades, and may lead to liver failure. In symptomatic patients, advanced age, elevated serum bilirubin levels, decreased serum albumin levels, and cirrhosis each correlate with shortened survival. Immunosuppressive and anti-inflammatory drugs have been used in the treatment of PBC based on the presumed autoimmune pathogenesis, but satisfactory agents leading to complete reversal or cure of the disease are not available. At present ursodeoxycholic acid appears to be the only effective therapy in preventing or delaying the need for liver transplantation and improving survival. However, a number of patients receiving ursodeoxycholic acid still develop progressive disease and require transplantation; transplantation is the only effective therapy at the end stage of the disease.

Adult↗
PubMed2000

Differences in epitope recognition, isotype and titer of antisera to Plasmodium falciparum merozoite surface protein 4 raised by different modes of DNA or protein immunization.

Plasmodium falciparum merozoite surface protein 4 (MSP4) is being developed as a component of a subunit vaccine against asexual stages of malaria. Three DNA constructs were produced that induced expression of MSP4 either in the cytoplasm of transfected cells or secreted from cells under the control of the human tissue plasminogen activator (TPA) signal or the native P. falciparum MSP4 signal. Only the construct containing the TPA signal induced detectable antibodies in mice, although gene expression was demonstrated in all constructs and MSP4 was shown to be secreted using either signal by in vitro transient transfection of COS cells. Two recombinant MSP4 proteins that encoded the same sequence as the plasmid DNA were produced in E. coli (EcMSP4-His) and S. cerevisiae (yMSP4-His) and used to raise antibodies in mice. Comparison of the antibodies elicited by these various antigen formulations showed differences in titer, isotype and epitope recognition. The titer of antibodies induced by DNA vaccination was lower than that induced by yMSP4-His, which in turn was lower than that induced by EcMSP4-His. The isotype profiles of the antibodies were also different, the plasmid DNA induced predominantly IgG(2a) responses whereas the two proteins induced predominantly IgG(1) responses. The antibodies induced by DNA and yMSP4-His recognized predominantly the C-terminal epidermal growth factor (EGF)-like domain of the protein, whereas EcMSP4-His induced antibodies recognizing all domains of the protein equally. The antibodies induced by DNA vaccination were directed almost extensively to conformational epitopes so that reactivity with native MSP4 was abolished after disulfide bonds in the protein were disrupted. Antibodies induced by recombinant proteins recognized linear epitopes as well and reactivity to native MSP4 was preserved after reduction and alkylation of parasite proteins.

Animals↗