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R L Coppel

Publications and source records attributed to R L Coppel.

At least 55 records · Page 3Linked to original sources
PubMed1999

Are infectious agents involved in primary biliary cirrhosis? A PCR approach.

BACKGROUND/AIMS: A variety of data suggest that microbial infections and, in particular, atypical mycobacteria infections, may either initiate and/or be associated with the pathogenesis of primary biliary cirrhosis. METHODS: To address this hypothesis, use was made of polymerase chain reaction techniques and primers specific for the 16s rRNA gene of Eubacteria, Archaeabacteria, Mycobacteria and Helicobacter to determine if such sequences were detectable in liver tissue specimens from 29 patients with primary biliary cirrhosis. Similar liver tissues from patients with primary sclerosing cholangitis, chronic hepatitis, alcoholic liver disease and otherwise normal donors were analyzed in parallel. Genomic DNA was extracted from each of these liver tissue specimens using sterile techniques to avoid possible laboratory contamination. The DNA was subjected to polymerase chain reaction amplification using bacterial genus specific primers and the amplified products cloned and sequenced. Sequence data were analyzed by searching for homology to existing genes. RESULTS: Sequences from primary biliary cirrhosis and control livers corresponded to those found in a variety of bacteria, but no consensus sequence was found in primary biliary cirrhosis specimens. Neither Archaeabacteria nor Mycobacteria products were detected in liver specimens of patients with primary biliary cirrhosis, and Helicobacter pylori DNA was detected in only one primary biliary cirrhosis patient. CONCLUSIONS: Although bacterial infection, particularly with intracellular organisms, has been suggested to play a role in the initiation of primary biliary cirrhosis, there is no evidence from this study to suggest an ongoing chronic infectious process.

Archaea↗
PubMed1999

Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis.

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild-type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O-methylated rhamnose and O-acylated 6-deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild-type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.

Alleles↗
PubMed1999

Is there a serological difference between men and women with primary biliary cirrhosis?

OBJECTIVE: Primary biliary cirrhosis (PBC) is an autoimmune disease affecting small intrahepatic bile ducts of the liver, causing destruction of the epithelium that results in eventual fibrosis and scarring. We still lack a complete epidemiological description of this disease, although interesting geographic differences in prevalence have been described. One consistent feature has been the relative scarcity of men with PBC. In fact, published ratios of women to men range from 3:1 to as high as 22:1. Thus far, the only clinical difference reported between men and women with PBC is a putative higher risk of hepatocarcinoma in men. Previous serological studies have shown that about 95% of all patients possess antimitochondrial antibodies to members of the highly conserved 2-oxo-acid dehydrogenase family of proteins, namely pyruvate dehydrogenase complex E2 (PDC-E2), branched-chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2), and 2-oxo glutarate dehydrogenase complex E2 (OGDC-E2). However, there has been no information as to whether there is a difference in serological response between men and women. Using the serological hallmark of antimitochondrial antibodies (AMAs) and taking advantage of the availability of recombinant mitochondrial autoantigens, investigations were performed to determine if there were any serological differences between men and women with PBC. METHODS: Sera were collected from 88 patients with PBC, of whom 46 were men and 42 were women. Using a combination of immunoblotting and enzyme-linked immunoabsorbent assay (ELISA) against beef heart mitochondria (BHM), recombinant PDC-E2, BCOADC-E2, and OGDC-E2, we determined the relative autoantibody reactivities of our study population. RESULTS: Both men and women with PBC produced high titer antimitochondrial antibodies. The frequency of reactivity was similar in both groups and included, in descending order, PDC-E2, E3BP (Protein X), BCOADC-E2, and finally OGDC-E2. More importantly, antigenic specificity was nearly identical regardless of gender. CONCLUSIONS: AMAs are the serological hallmark of PBC in both men and women, and there is no significant difference in reactivity between the two groups of patients.

Adult↗
PubMed1999

Characterization of a Mycobacterium smegmatis mutant lacking penicillin binding protein 1.

The ponA gene of Mycobacterium smegmatis encodes a 95-kDa penicillin binding protein, PBP1, that is similar to PBP1s of Mycobacterium tuberculosis and Mycobacterium leprae. Transposon disruption of ponA in M. smegmatis resulted in a PBP1-deficient mutant that was sensitive to beta-lactam antibiotics, was more permeable to glycine, and grew slowly in liquid culture.

Bacterial Proteins↗
PubMed1999

Identification of the Plasmodium chabaudi homologue of merozoite surface proteins 4 and 5 of Plasmodium falciparum.

Previous studies of Plasmodium falciparum have identified a region of chromosome 2 in which are clustered three genes for glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins, MSP2, MSP5, and MSP4, arranged in tandem. MSP4 and MSP5 both encode proteins 272 residues long that contain hydrophobic signal sequences, GPI attachment signals, and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. The locations and similar structural features of these genes suggest that they have arisen from a gene duplication event. Here we describe the identification of the syntenic region of the genome in the murine malaria parasite, Plasmodium chabaudi adami DS. Only one open reading frame is present in this region, and it encodes a protein with structural features reminiscent of both MSP4 and MSP5, including a single EGF-like domain. Accordingly, the gene has been designated PcMSP4/5. The homologue of the P. falciparum MSP2 gene could not be found in P. chabaudi; however, the amino terminus of the PcMSP4/5 protein shows similarity to that of MSP2. The PcMSP4/5 gene encodes a protein with an apparent molecular mass of 36 kDa, and this protein is detected in mature stages of the parasite. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites and developing and free merozoites. The PcMSP4/5 gene is transcribed in both ring and trophozoite stages but appears to be spliced in a stage-specific manner such that the central intron is spliced from the mRNA in the parasitic stage in which the protein is expressed.

Amino Acid Sequence↗
PubMed1999

Structural and antigenic properties of merozoite surface protein 4 of Plasmodium falciparum.

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carboxyl terminus. We have expressed both full-length MSP4 and a number of fragments in Escherichia coli and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific antibodies that reacted with parasite-derived MSP4 by immunoblotting. Antibody reactivity was highly dependent on the protein conformation. For example, reduction and alkylation of MSP4 almost completely abolished the reactivity of several antibody preparations, including specificities directed to regions of the protein that do not contain cysteine residues and are far removed from the cysteine-containing EGF-like domain. This indicated the presence of conformation-dependent epitopes in MSP4 and demonstrated that proper folding of the EGF-like domain influenced the antigenicity of the entire molecule. The recombinant proteins were used to map epitopes recognized by individuals living in areas where malaria is endemic, and at least four distinct regions are naturally antigenic during infection. Binding of human antibodies to the EGF-like domain was essentially abrogated after reduction of the recombinant protein, indicating the recognition of conformational epitopes by the human immune responses. This observation led us to examine the importance of conformation dependence in responses to other integral membrane proteins of asexual stages. We analyzed the natural immune responses to a subset of these antigens and demonstrated that there is diminished reactivity to several antigens after reduction. These studies demonstrate the importance of reduction-sensitive structures in the maintenance of the antigenicity of several asexual-stage antigens and in particular the importance of the EGF-like domain in the antigenicity of MSP4.

Animals↗
PubMed1998

Identification and precursor frequency analysis of a common T cell epitope motif in mitochondrial autoantigens in primary biliary cirrhosis.

The immunodominant antimitochondrial antibody response in patients with primary biliary cirrhosis (PBC) is directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Based on our earlier observations regarding peripheral blood mononuclear cell (PBMC) T cell epitopes, we reasoned that a comparative analysis of the precursor frequencies of PDC-E2 163-176-specific T cells isolated from PBMC, regional hepatic lymph nodes, and from the liver of PBC patients would provide insight regarding the role of T cells in PBC. Results showed a disease-specific 100-150-fold increase in the precursor frequency of PDC-E2 163-176-specific T cells in the hilar lymph nodes and liver when compared with PBMC from PBC patients. Interestingly, autoreactive T cells and autoantibodies from PBC patients both recognize the same dominant epitope. In addition, we demonstrated cross-reactivity of PDC-E2 peptide 163-176-specific T cell clones with PDC-E2 peptide 36-49 and OGDC-E2 peptide 100-113 thereby identifying a common T cell epitope "motif" ExETDK. The peptide 163-176-specific T cell clones also reacted with purified native PDC-E2, suggesting that this epitope is not a cryptic determinant. These data provide evidence for a major role for PDC-E2 peptide 163-176 and/or peptides bearing a similar motif in the pathogenesis of PBC.

Adult↗
PubMed1998

Monoclonal antibodies to mitochondrial E2 components define autoepitopes in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial Abs (AMA). The autoantigens recognized by AMA are the E2 components of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxoacid dehydrogenase complex E (BCOADC-E2), and the 2-oxoglutarate dehydrogenase complex E (OGDC-E2). Previous studies using murine monoclonal and human combinatorial Abs to PDC-E2 have demonstrated an intense linear staining pattern in the apical region of biliary epithelial cells (BEC) in PBC but not control liver. We therefore examined whether mAbs to the other mitochondrial autoantigens BCOADC-E2 and OGDC-E2 demonstrated disease-specific patterns of reactivity. Using an expressed recombinant "trihybrid" protein containing the lipoyl domains of PDC-E2, OGDC-E2, and BCOADC-E2, we immunized BALB/c mice to produce 35 mAbs specific for one or more of the above mitochondrial autoantigens. Seven of these mAbs uniquely stained the apical region of BEC in PBC. Of these seven, one was reactive to PDC-E2, two recognized BCOADC-E2, three were reactive to OGDC-E2, and one recognized all three Ags. Our current data demonstrate that, similar to our previous studies regarding PDC-E2, mAbs to BCOADC-E2 and OGDC-E2, or a molecule that cross-reacts with the inner lipoyl domain of all three enzymes, also show a uniquely intense staining pattern in the apical region of BEC in patients with PBC when compared with diseased controls. The abundance of such disease-specific determinants in the target cells of PBC raises interesting possibilities regarding the role of these autoantigens in the pathogenesis of this disease.

Animals↗
PubMed1998

Close linkage of three merozoite surface protein genes on chromosome 2 of Plasmodium falciparum.

We have analysed a 10.5 kb region of chromosome 2 in Plasmodium falciparum that encompasses the coding region of four genes. Three genes are arranged in a head-to-tail orientation and encode the merozoite surface proteins MSP2 and MSP4 as well as a previously unreported sequence that encodes a polypeptide with the characteristics of a merozoite surface protein, now designated MSP5. The fourth gene, asl, is arranged in a tail-to-tail orientation with msp2 and has homology with prokaryotic and eukaryotic genes encoding adenylosuccinate lyase (ASL), an enzyme involved in purine biosynthesis and salvage. The genes, arranged in the order msp4, msp5, msp2 and asl, are separated by intergenic distances of 1021, 1017 and 722 bp, respectively. msp4 and msp5 are clearly related genes, each being composed of 2 exons and encoding proteins of identical length. Both msp4 and msp5 encode proteins that contain hydrophobic signal sequences, apparent glycosylphosphatidylinositol (GPI) attachment signals and a single epidermal growth factor-like (EGF-like) domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. It appears that one of these genes arose as a result of a relatively ancient gene duplication event and both genes have subsequently diverged considerably. This study shows that msp5 is transcribed in asexual stages and its encoded product is a 40 kDa protein that appears to be located on the merozoite surface as determined by immunofluorescence assays.

Amino Acid Sequence↗
PubMed1998

Characterization of antimitochondrial antibodies in health adults.

The detection of antimitochondrial antibodies (AMAs) is an important criterion for the diagnosis of primary biliary cirrhosis (PBC). During the last decade, the mitochondrial autoantigens have been cloned, sequenced, and identified as members of the 2-oxo-acid dehydrogenase pathway, including the E2 subunits of pyruvate dehydrogenase (PDC-E2), branched-chain 2-oxo-acid dehydrogenase (BCOADC-E2), and 2-oxo-glutarate dehydrogenase (OGDC-E2). We have developed a rapid and sensitive diagnostic test for use in PBC based on a triple hybrid recombinant molecule (r-MIT3) that contains the autoepitopes of PDC-E2, BCOADC-E2, and OGDC-E2. To help understand the frequency and antigen specificity of AMAs in an asymptomatic population and to identify patients with early disease, we investigated the prevalence of AMA, by enzyme-linked immunosorbent assay (ELISA), in a cohort of 1,530 people from northern Italy. Positive sera were further analyzed for immunoglobulin (Ig) isotypes, subclasses, and epitopes of AMA by a combination of ELISA and immunoblotting. In this cohort of 1,530 people, 9 (0.5%) reacted to r-MIT3 by ELISA. Of the 9 reactive sera, 2 recognized PDC-E2, 2 of 9 recognized BCOADC-E2, 1 of 9 recognized OGDC-E2, 2 of 9 recognized both PDC-E2 and BCOADC-E2, and 1 of 9 recognized PDC-E2 and OGDC-E2. AMA reactivity was primarily IgM and IgA. Epitope mapping revealed an AMA pattern of reactivity to PDC-E2 that differed from that found in patients with histologically proven PBC in most of the sera. However, 1 sera of a 72-year-old female with a normal alkaline phosphatase had an AMA profile identical to typical PBC. After a variable follow-up period (8-14 months), sera from 8 of 9 of these people were re-obtained for AMA and relative epitope mapping. Interestingly, the reactivity had a wider AMA pattern than before.

Adult↗
PubMed1998

Induction and persistence of immune-mediated cholangiohepatitis in neonatally thymectomized mice.

The availability of recombinant autoantigens allows the experimental study of the relationships between primary biliary cirrhosis (PBC) and mitochondrial antigens. We took advantage of these recombinant autoantigens and attempted to induce autoimmune cholangitis by immunizing neonatally thymectomized (NTx) lipopolysaccharide (LPS)-treated A/J mice, known to be prone to organ-specific autoimmune diseases. We employed a recombinant protein containing a dual-headed molecule that coexpresses the immunodominant epitope of the E2 subunits of the pyruvate dehydrogenase complex and the branched-chain keto-acid dehydrogenase complex. We report herein that an immune-mediated cholangiohepatitis was induced by such immunization and the concurrent injection of LPS into NTx mice. The incidence of cholangitis was 79% in the NTx, immunized, LPS group compared to 14% in the NTx, nonimmunized, LPS group. The histopathology ranged from mild to severe and included bile duct damage, focal hepatic necrosis, and endotheliitis, but no granulomas. Moreover, almost all such lesions persisted for 12 weeks after the discontinuation of immunization and LPS injections in the NTx mice. Interestingly, we were successful (89%) in transferring the cholangiohepatitis by injection of liver infiltrating mononuclear cells from the NTx, immunized, LPS mice into congenic nonimmunized NTx mice; such lesions could not be transferred with spleen cells. Although the pathology is not typical of PBC, this model offers a unique venue for the study of immune-mediated hepatobiliary injury.

Adoptive Transfer↗
PubMed1998

Plasmodium falciparum: influence of malarial and host erythrocyte skeletal protein interactions on phosphorylation in infected erythrocytes.

Phosphorylation of components of the erythrocyte membrane skeleton has major effects on the physical properties of the membrane. Infection of red cells by the protozoan parasite Plasmodium falciparum leads to a marked increase in the level of phosphorylation of red cell protein 4.1 and the insertion into the red cell skeleton of parasite-encoded phosphoproteins, including the mature-parasite-infected erythrocyte surface antigen (MESA). Because of the tight association of MESA with protein 4.1, we set out to determine the importance of this interaction and that of other parasite-encoded skeletal-associated proteins to phosphorylation of the infected red cell membrane. Our results show that neither MESA nor protein 4.1 is required for phosphorylation of its binding partner. Further, phosphorylation of MESA and protein 4.1 occurs independently of the presence of knobs, the expression of PfHRP1, or cytoadherence phenotype. In contrast to previous studies, we were unable to detect a change in the molecular weight of protein 4.1 in erythrocytes infected with cytoadherent parasite lines. In red cells infected with parasites expressing PfHRP1 (K+), MESA and protein 4.1 are substrates for a kinase with the inhibitor profile of a casein kinase. Surprisingly, however, when we examined phosphorylation of MESA and protein 4.1 in K(-)-infected erythrocytes, we found that casein kinase I and II inhibitors had no, or greatly reduced, effectiveness, and in fact, phosphorylation of these two proteins was enhanced in some instances.

Animals↗
PubMed1998

Penetration and co-localization in MDCK cell mitochondria of IgA derived from patients with primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease of unknown etiology characterized by high-titer anti-mitochondrial antibodies. The major autoantigen has been identified as the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). The fact that PDC-E2 is present in all nucleated cells, but autoimmune damage is confined to biliary epithelial cells, prompted us to investigate the possibility that mucosally-derived IgA may be pathogenic for biliary epithelial cells. Serum IgA was purified from six patients with PBC and its localization and ability to penetrate cells was studied using Madine-Darby canine kidney (MDCK) cells transfected with the human IgA receptor (MDCK-pIgR). The potential of IgA to be transported through the cells was studied by a combination of immunohistochemistry and dual color fluorescent microscopy. Interestingly, IgA from all PBC patients co-localized with PDC-E2 (the major autoantigen of PBC) inside the cells; this was demonstrated by dual staining with anti-human IgA and a mouse monoclonal antibody directed to PDC-E2. In contrast, no co-localization was observed for IgA controls. Furthermore, dual staining of liver sections from PBC patients demonstrated co-localization of IgA and PDC-E2, both cytoplasmically and at the apical surface. We postulate that there may be a direct effect of these autoantibodies on the mitochondrial function of biliary epithelial cells.

Animals↗
PubMed1998

Adhesive proteins of the malaria parasite.

Malaria infection of the host cells requires host-parasite recognition events mediated by adhesion and signaling molecules. Recent development of systems for stable transformation and targeted integration of exogenous DNA in malaria parasites provides a powerful tool to study the structure and function of Plasmodium attachment motifs, and their role in infection and disease.

Amino Acid Sequence↗
PubMed1998

A recombinant peptide based on PfEMP-1 blocks and reverses adhesion of malaria-infected red blood cells to CD36 under flow.

During falciparum malaria infection, severe complications ensue because parasitized red blood cells (PRBCs) adhere to endothelial cells and accumulate in the microvasculature. At the molecular level, adhesion is mediated by interaction of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1) on the PRBC surface with receptors on the surface of endothelial cells, including CD36. We have shown that a recombinant 179-residue subfragment of PfEMP-1 (rC1-2[1-179]), which encompasses the CD36-binding region, inhibits and reverses adhesion of PRBCs to CD36 under physiologically relevant flow conditions. rC1-2[1-179] inhibited adhesion in a concentration-dependent manner over the range 100 pM to 2 microM, with up to 99% of adhesion blocked at the highest concentration tested. The antiadhesive activity of rC1-2[1-179] was not strain specific and almost totally ablated adhesion of four different parasite lines. Furthermore, rC1-2[1-179] showed remarkable ability to progressively reverse adhesion when flowed over adherent PRBCs for 2h. The effect of rC1-2[1-179] was, however, specific for CD36-mediated adhesion and had no effect on adhesion mediated by CSA. Interference with binding of PRBCs to the vascular endothelium using rC1-2[1-179] or smaller organic mimetics may be a useful therapeutic approach to ameliorate severe complications of falciparum malaria.

Animals↗
PubMed1998

Malaria and the erythrocyte.

In terms of global health, the most important disease involving human erythrocytes is infection by protozoan parasites of the genus Plasmodium, particularly Plasmodium falciparum. Our understanding of the complex processes of erythrocyte invasion, remodeling, and cytoadherence has advanced considerably over the past few years. Considerable advances have been made in identifying the players in each of these phenomena, although identification of the exact functional roles for many molecules is still missing. The cloning of the parasite adhesin, the development of a transfection system, and a series of new imaging and cell biology assays are recent achievements that promise to further our understanding not only of the pathogenesis of malaria, but also the functioning of erythrocytes.

Animals↗
PubMed1998

Temporal variation of the merozoite surface protein-2 gene of Plasmodium falciparum.

Extensive polymorphism of key parasite antigens is likely to hamper the effectiveness of subunit vaccines against Plasmodium falciparum infection. However, little is known about the extent of the antigenic repertoire of naturally circulating strains in different areas where malaria is endemic. To address this question, we conducted a study in which blood samples were collected from parasitemic individuals living within a small hamlet in Western Irian Jaya and subjected to PCR amplification using primers that would allow amplification of the gene encoding merozoite surface protein-2 (MSP2). We determined the nucleotide sequence of the amplified product and compared the deduced amino acid sequences to sequences obtained from samples collected in the same hamlet 29 months previously. MSP2 genes belonging to both major allelic families were observed at both time points. In the case of the FC27 MSP2 family, we observed that the majority of individuals were infected by parasites expressing the same form of MSP2. Infections with parasites expressing 3D7 MSP2 family alleles were more heterogeneous. No MSP2 alleles observed at the earlier time point were detectable at the later time point, either for the population as a whole or for individuals who were assayed at both time points. We examined a subset of the infected patients by using blood samples taken between the two major surveys. In no patients could we detect reinfection by a parasite expressing a previously encountered form of MSP2. Our results are consistent with the possibility that infection induces a form of strain-specific immune response against the MSP2 antigen that biases against reinfection by parasites bearing identical forms of MSP2.

Alleles↗