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R L Coppel

Publications and source records attributed to R L Coppel.

At least 73 records · Page 4Linked to original sources
PubMed1998

X-ray microscopic visualization of specific labeling of adhesive molecule CD36 and cytoadherence by Plasmodium falciparum infected erythrocytes.

We investigated the cytoadherence of Plasmodium falciparum infected erythrocytes to target cells that express CD36 by soft x-ray microscopy. Using immunogold beads enhanced with silver, we localized CD36 on the surface of intact melanoma cells and throughout Triton extracted melanoma cells. We examined the orientation of parasites within erythrocytes that bound to target cells, and the interactions between the red cell membrane and the target cell, and we confirmed that fibrillar structures on the surface of melanoma and endothelial cells can be involved in the association between infected erythrocytes and melanoma cells or endothelial cells.

Animals↗
PubMed1997

Defining the minimal domain of the Plasmodium falciparum protein MESA involved in the interaction with the red cell membrane skeletal protein 4.1.

During part of its life cycle, the protozoan parasite Plasmodium falciparum lives within the human red blood cell and modifies both the structural and functional properties of the red cell. It does this by synthesizing a number of polypeptides that it transports into the red cell cytoplasm and to the red cell membrane. One of these transported proteins, MESA (mature parasite-infected erythrocyte surface antigen), is anchored to the red cell membrane by noncovalent interaction with erythrocyte protein 4.1. We have utilized a combination of in vitro transcription and translation and a membrane binding assay to identify the protein sequence involved in anchoring MESA to the membrane. Labeled fragments of different regions of the MESA protein were evaluated for their ability to bind to inside-out vesicle membrane preparations of human red cells. Binding was dependent on the presence of red cell membrane proteins and was abolished either by trypsin treatment or by selective depletion of membrane proteins. Binding was specific and could be inhibited by the addition of competing protein, with an IC50 of (6.3 +/- 1.2) x 10(-7) M, indicative of a moderate affinity interaction. Fractionation studies demonstrated that binding fragments interacted most efficiently with membrane protein fractions that had been enriched in protein 4.1. Binding inhibition experiments using synthetic peptides identified the binding domain of MESA for protein 4.1 as a 19-residue sequence near the amino terminus of MESA, a region capable of forming an amphipathic helix.

Animals↗
PubMed1997

Intracellular structures of normal and aberrant Plasmodium falciparum malaria parasites imaged by soft x-ray microscopy.

Soft x-ray microscopy is a novel approach for investigation of intracellular organisms and subcellular structures with high spatial resolution. We used x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes and in infected erythrocytes treated with cysteine protease inhibitors. Investigations in normal red blood cells enabled us to recognize anomalies in parasite structures resulting from growth under unfavorable conditions. X-ray microscopy facilitated detection of newly elaborated structures in the cytosol of fixed, unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. In cysteine protease inhibitor-treated, infected erythrocytes, high concentrations of material were detected in abnormal digestive vacuoles and aggregated at the parasite plasma membrane. We have demonstrated that an abnormal host erythrocyte skeleton affects structural development of parasites and that this aberrant development can be detected in the following generation when parasites from protein 4.1-deficient red blood cells infect normal erythrocytes. This work extends our current understanding of the relationship between the host erythrocyte membrane and the intraerythrocytic malaria parasite by demonstrating for the first time that constituents of the erythrocyte membrane play a role in normal parasite structural development.

Animals↗
PubMed1997

Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress.

Knobs at the surface of erythrocytes infected with Plasmodium falciparum have been proposed to be important in adherence of these cells to the vascular endothelium. This structure contains the knob-associated histidine-rich protein (KAHRP) and the adhesion receptor P. falciparum erythrocyte membrane protein 1. We have disrupted the gene encoding KAHRP and show that it is essential for knob formation. Knob-transfectants adhere to CD36 in static assays; when tested under flow conditions that mimic those of postcapillary venules, however, the binding to CD36 was dramatically reduced. These data suggest that knobs on P. falciparum-infected erythrocytes exert an important influence on adherence of parasitized-erythrocytes to microvascular endothelium, an important process in the pathogenesis of P. falciparum infections.

Animals↗
PubMed1997

In situ nucleic acid hybridization of pyruvate dehydrogenase complex-E2 in primary biliary cirrhosis: pyruvate dehydrogenase complex-E2 messenger RNA is expressed in hepatocytes but not in biliary epithelium.

Pyruvate dehydrogenase-E2, or a cross-reactive molecule, has been shown by a variety of immunohistochemical methods to be present in increased amounts in biliary epithelial cells (BEC) in primary biliary cirrhosis (PBC). In this study, to further understand the nature of the immunoreactive molecule in BEC, we examined the expression of pyruvate dehydrogenase complex-E2 (PDC-E2) messenger RNA (mRNA) and PDC-E2 protein in sections of livers from patients and controls to help identify the molecule found in BEC. We performed in situ hybridization using an antisense probe against the major epitope of PDC-E2. The data were very striking and suggested that there was no increased production of PDC-E2 in BEC. For example, in livers from patients with PBC, PDC-E2 mRNA was found in periportal hepatocytes in 16 of 17 cases (94%). In contrast, interlobular bile ducts and septal bile ducts had detectable levels of PDC-E2 mRNA in only 1 of 17 (6%) and 3 of 8 (38%) cases, respectively. Interestingly, proliferating bile ductules contained detectable levels of mRNA in 12 of 15 cases (80%). In control liver, periportal hepatocytes were positive in 15 of 17 cases (88%). Interlobular bile ducts, septal bile ducts, and proliferating bile ductules expressed mRNA signals in 4 of 17 (24%), 2 of 10 (20%), and 14 of 16 (88%), respectively. When formalin-fixed, paraffin-embedded sections were examined by immunohistochemical staining with anti-PDC-E2 monoclonal antibody (mAb) C355.1, the interlobular bile ducts showed typical aberrant apical staining in all 10 PBC cases, but 0 of 9 liver controls. Periportal hepatocytes, proliferating bile ductules and infiltrating mononuclear cells stained with C355.1 but in a characteristic mitochondrial staining pattern. The presence of a PDC-E2-like molecule recognized by C355.1 is not reflected by the expression levels of PDC-E2 mRNA in the BEC of patients with PBC.

Bile Ducts↗
PubMed1997

In situ nucleic acid hybridization of cytokines in primary biliary cirrhosis: predominance of the Th1 subset.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by destruction of the intrahepatic bile ducts. It is generally believed that cellular immune mechanisms, particularly involving T cells, result in this bile duct damage. The relative strength of Th1 and Th2 responses has recently been proposed to be an important factor in the pathophysiology of various autoimmune diseases. In this study, we have attempted to identify the Th subset balance in PBC, by detection of cytokines specific to the two T-cell subsets, i.e., interferon gamma (IFN-gamma) for Th1 cells and interleukin-4 (IL-4) for Th2 cells. We analyzed IFN-gamma and IL-4 messenger RNA (mRNA) positive cells in liver sections from 18 patients with PBC and 35 disease controls including chronic active hepatitis C, extrahepatic biliary obstruction (EBO), and normal liver, using nonisotopic in situ hybridization and immunohistochemistry. Mononuclear cells expressing IFN-gamma and IL-4 mRNA were aggregated in inflamed portal tracts in PBC livers, but were rarely present in extrahepatic biliary obstruction, alcoholic fibrosis, or normal liver sections. The IFN-gamma and IL-4 mRNA positive cells in PBC livers were detected in significantly higher numbers than in control livers (P < .01). Moreover, IFN-gamma mRNA expression was more commonly detected than IL-4 expression in PBC livers, and the levels of IFN-gamma mRNA expression were highly correlated with the degree of portal inflammatory activity. IFN-gamma mRNA-positive cells were detected primarily around damaged bile ducts that were surrounded by lymphoid aggregates. The data indicate that Th1 cells are the more prominent T-cell subset in the lymphoid infiltrates in PBC.

Actins↗
PubMed1997

Comparative studies of antimitochondrial autoantibodies in sera and bile in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by destruction of intrahepatic bile ducts. Although the pathogenesis of this disease is still unknown, high titers of antimitochondrial autoantibodies (AMA) have long been recognized in patient sera. However, little is known about the presence of AMA in bile. In this study, we investigated bile and sera from patients with PBC and healthy controls for the presence of AMA and mitochondrial autoantigens. AMA were detected in the bile of 17 of 19 patients (89.4%) with PBC; they were specifically directed against the pyruvate dehydrogenase complex (PDC-E2) in 15 of 19 patients (78.9%), to the branched-chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2) in 6 of 19 patients (31.6%), and to the 2-oxoglutarate dehydrogenase complex E2 (OGDC-E2) in 1 of 19 patients (5.3%). In a comparative study of sera from the same patients, anti-PDC-E2 antibodies were found in 19 of 19 patients (100%), anti-BCOADC in 9 of 19 patients (47.3%), and anti-OGDC-E2 in 4 of 19 patients (21.1%) patients. AMA in bile were always found together with antibodies of corresponding specificities in the serum from the same patient. Immunoglobulin (Ig)A AMA were found in the bile of 9 of 19 patients (47.7%) with PBC; they were specifically directed against PDC-E2 in 8 of 19 patients (42.1%) and to BCOADC in 2 of 19 patients (10.5%). Epitope mapping of IgA anti-PDC-E2 antibodies indicated that, like serum autoantibodies, the immunodominant epitope is directed against the inner lipoyl domain of PDC-E2. The prevalence and antigen reactivity of IgA AMA in sera correlated completely with IgA AMA in bile. Autoantibodies against nuclear envelope pore proteins (gp210) were found in 1 of 8 (12.5%) sera of patients with PBC, but not in bile. Furthermore, and of particular interest, we detected the autoantigens, PDC-E2, OGDC-E2, and BCOADC-E2, in the bile of 12 of 19 patients (63.2%), 9 of 19 patients (47.4%), and 9 of 19 patients (47.4%), respectively; PDC-E2 was found in only 1 of 17 (5.9%) disease controls. Although the presence of AMA in bile may merely reflect the presence of these antibodies in sera, the simultaneous detection of mitochondrial autoantigens in bile suggests an increase of mitochondrial autoantigens at inflammatory sites. Such autoantigens, coupled with AMA, may augment the local immune response and disease progression.

Autoantibodies↗
PubMed1997

A putative Plasmodium falciparum exported serine/threonine protein kinase.

An 8kb gene coding for a putative serine/threonine protein kinase from Plasmodium falciparum has been cloned and sequenced. It is arranged in two exons: exon I is 2 kb and exon II is 5.6 kb. The gene codes for a large protein of 2510 amino acids. Antibodies raised against a fusion protein were used to localize the putative kinase. By immunofluorescence microscopy, it was found in the cytoplasm of infected red cells. By immunoelectron microscopy it was associated with membranous structures in the red cell and with the red cell membrane, particularly at parasite-induced knobs. This is the first putative protein kinase of P. falciparum to be exported from the parasite into its host cell.

Amino Acid Sequence↗
PubMed1997

Antimitochondrial antibodies in primary biliary cirrhosis.

In the last decade, the cloning and biochemical identification of mitochondrial autoantigens in primary biliary cirrhosis (PBC) as members of the 2-oxoacid dehydrogenase complex has greatly advanced the detection of antimitochondrial antibodies (AMA) and the understanding of the immunobiology of the disease. Here, we discuss the methods of detecting AMA and its isotypes, methods of epitope mapping, and using these methods in PBC liver immunohistochemistry and Ig gene usage. Increasing evidence, including the specific association of AMA with PBC, the unique similar but noncross-reactive conformational epitope of the lipoyl domains of the mitochondrial autoantigens, the specific binding of anti-PDC-E2 monoclonal antibodies and human combinatorial antibodies derived from PBC patients to the apical area of bile duct epithelial cells in PBC livers, and Ig gene usage of AMA, suggests that AMA is not an epiphenomenon of the disease but plays a significant role in the pathogenesis of PBC.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1997

A second merozoite surface protein (MSP-4) of Plasmodium falciparum that contains an epidermal growth factor-like domain.

Merozoite surface proteins of Plasmodium falciparum play a critical role in the invasion of human erythrocytes by the malaria parasite. Here we describe the identification of a novel protein with a molecular mass of 40 kDa that is found on the merozoite surface of P. falciparum. We call this protein merozoite surface protein 4 (MSP-4). Evidence for the surface location of MSP-4 includes (i) a staining pattern that is consistent with merozoite surface location in indirect immunofluorescent studies of cultured parasites, (ii) localization of MSP-4 in the detergent phase in Triton X-114 partitioning studies, and (iii) nucleotide sequencing studies which predict the presence of an N-terminal signal sequence and a hydrophobic C-terminal sequence in the protein. Immunoprecipitation studies of biosynthetically labelled parasites with [3H] myristic acid indicated that MSP-4 is anchored on the merozoite surface by a glycosylphosphatidylinositol moiety. Of considerable interest is the presence of a single epidermal growth factor-like domain at the C terminus of the MSP-4 protein, making it the second protein with such a structure to be found on the merozoite surface.

Amino Acid Sequence↗
PubMed1997

Primary biliary cirrhosis an epithelitis: evidence of abnormal salivary gland immunohistochemistry.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease of unknown etiology. Nearly 93% of patients with PBC exhibit evidence of focal sialoadenitis. In an earlier study, we reported evidence of aberrant expression of PDC-E2, or a mimeotope, in the salivary glands of patients with PBC that had Sjogren's syndrome. At the time of the previous study, data was not yet available regarding patients with PBC without sicca complaints. Therefore, to investigate the extent of salivary gland involvement in PBC, we collected lip biopsy sections from 9 PBC patients diagnosed as PBC by liver biopsy, without clinical or histologic features of Sjogren's syndrome and 9 PBC patients with established Sjogren's syndrome. Using immunohistochemical staining with both a murine monoclonal antibody. C355.1, and a human combinatorial antibody, SP4, we examined the ducts of these salivary glands for the presence of the characteristic aberrant staining pattern found in patients with PBC. We report that 6/9 PBC patients fulfilling established Sjogren's syndrome criteria and 6/9 PBC patients lacking features of Sjogren's syndrome showed intense staining of the ductal epithelial cells of the salivary gland. These data suggest that the PBC-specific antigen recognized by C355.1 and SP4 in bile duct epithelial cells is expressed aberrantly in the salivary gland in 66% of patients with PBC, independent of Sjogren's syndrome. This finding suggests a common disease process in these two tissues. Further, expression of this molecule may be an early marker of salivary gland involvement in patients with PBC.

Animals↗
PubMed1997

A study of antimitochondrial antibodies in a random population in Estonia.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the spontaneous destruction of the small intrahepatic bile ducts. The hallmark serologic feature of PBC is the presence of high-titer antimitochondrial antibodies (AMA). Both the incidence and prevalence of PBC varies geographically; epidemiological data may provide valuable insight regarding the pathogenic mechanisms and etiology of disease. Thus far, the majority of studies on the occurrence of PBC and AMAs have been derived from autopsy, mortality figures, or hospital admission records. The numbers reported reflect only those patients with clinical disease. To address this issue, an adult population sample representing all age groups in the village of Karksi-Nuia in southern Estonia was selected for a study of AMA incidence. This village has unique features that make it ideal for such a study. First, the village is remote and a substantial number of families have lived in the area for generations. There is also a limited influx of new families into the village, therefore providing a limited genetic repertoire. In this unselected adult population, we examined AMA incidence by both immunoblot and ELISA, using native and recombinant antigens. Of the 1461 people studied, 13 (0.89%) were AMA positive. A similar frequency (0.96%) was found among 104 persons from a neighboring village, who subsequently joined the study. Our study suggests that the presence of AMA in Estonia is in agreement with the reported incidence of less than 1% AMA in a mixed hospital population.

Adolescent↗
PubMed1996

Cryptic antigenic determinants on the extracellular pyruvate dehydrogenase complex/mimeotope found in primary biliary cirrhosis. A probe by affinity mass spectrometry.

Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). Two different antibodies to PDC-E2, the immunodominant mitochondrial autoantigen in patients with PBC, were used. AMS was performed directly on frozen liver sections and purified bile duct epithelial cells. Mass spectrometric signals associated with the molecular recognition of PBC-specific antigenic determinants were enhanced by an in situ enzyme-linked signal amplification process. Samples from patients with PBC gave strong positive signals for the antigen(s) recognized by the monoclonal antibody C355.1. Conversely, tissues from normal and disease controls showed only a minimal signal. AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. PDC components bound to C355.1 were mapped and identified by mass before dissociation from the E2 component. A similar approach was used to identify unknown antigenic determinants associated with PBC. We believe AMS may be an important new approach with wide application to the identification of molecules associated with a number of disease states.

Antibodies, Monoclonal↗
PubMed1996

Adhesion of malaria-infected red blood cells to chondroitin sulfate A under flow conditions.

Adhesion of parasitized red blood cells (PRBCs) to microvascular endothelial cells (ECs) is a distinctive feature of Plasmodium falciparum malaria and is a central event in the development of life-threatening complications such as cerebral malaria. PRBCs adhere to several EC-expressed molecules in vitro, but the relative importance of these interactions in vivo remains unclear. Chondroitin sulfate A (CSA) is the most recent EC surface-associated molecule to be implicated in the adhesive process. Accordingly, we have studied adhesion of PRBCs to CSA in vitro using a parallel-plate flow chamber. Under controlled flow conditions, PRBCs adhered to CSA in a concentration-dependent manner at wall-shear stresses up to 0.2 Pa, a value that is within the physiological range for venules. Once adhered, PRBCs remained stationary (rather than rolling) and continued to remain stationary even when the wall-shear stress was raised to supravenular levels. The adhesive interaction was strong and a proportion of adherent PRBCs could withstand detachment at stresses up to 2.5 Pa. Soluble CSA at pharmacological concentrations prevented adhesion of flowing PRBCs in a concentration-dependent manner but failed to reverse established adhesion. Adhesion of PRBCs to CSA could contribute to the pathogenesis of malaria, and soluble CSA may have a useful therapeutic effect.

Cell Adhesion↗
PubMed1996

Analysis of isoniazid-resistant transposon mutants of Mycobacterium smegmatis.

The emergence of multidrug-resistant tuberculosis has renewed interest in the study of drug resistance in mycobacteria with the objective of improved chemotherapy. The genetic basis of isoniazid resistance in a model mycobacterium was studied. Eleven isoniazid-resistant mutants of Mycobacterium smegmatis were created using transposon mutagenesis. Genetic and enzymatic characterisation of the mutants showed that katG, encoding T-catalase, was inactivated. The nucleotide sequence of M. smegmatis katG was determined and the mutation sites mapped demonstrating that both the amino and carboxyl halves of T-catalase are important for enzymatic activity.

Bacterial Proteins↗
PubMed1996

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.

Amino Acid Sequence↗
PubMed1996

Epitope mapping and reactivity of autoantibodies to the E2 component of 2-oxoglutarate dehydrogenase complex in primary biliary cirrhosis using recombinant 2-oxoglutarate dehydrogenase complex.

Five different target mitochondrial autoantigens recognized by sera from patients with primary biliary cirrhosis (PBC) have been identified as subunits of the following 2-oxo acid dehydrogenase complexes: the pyruvate dehydrogenase complex (PDC), the branched chain 2-oxo acid dehydrogenase complex (BCOADC), and the 2-oxoglutarate dehydrogenase complex (OGDC). Unlike the E2 subunits of PDC (PDC-E2) and BCOADC (BCOADC-E2), the E2 subunits of OGDC (OGDC-E2) reactivity of PBC sera and the reactive epitope of OGDC-D2 have not hitherto been studied in detail. In this report, we took advantage of a recombinant fusion protein for OGDC-E2 to address these issues. Eighty of 268 (29.9%) PBC patient sera but none of the 45 controls reacted with recombinant OGDC-E2. The recombinant OGDC-E2 was judged to express the immunodominant epitope, because when sera from patients with PBC were preabsorbed with the recombinant fusion protein, such sera were depleted of reactivity against 48 kD OGDC-E2 when probed on beef heart mitochondria (BHM) but retained reactivity toward PDC-E2 and/or BCOADC-E2. Furthermore, affinity-purified PBC sera against recombinant OGDC-E2 reacted only with native OGDC-E2 and not with any other enzyme components of the 2-oxo acid dehydrogenase complex. Antimitochondrial autoantibodies (AMA) against OGDC-E2 included immunoglobulin (Ig)G2, IgG3 and IgM and the relative titers were as follows: IgG2 > IgG3 > IgM. Finally, using overlapping recombinant polypeptides, it was determined that a minimum of 81 amino acids (residues 67-147) corresponding to the lipoyl domain of OGDC-E2 are necessary for reactivity, suggesting that a conformational autoepitope is recognized by AMA. These data suggest that each of the 2-oxo acid dehydrogenase enzymes has distinct antigenicity despite their similarities in structure and function. The availability of recombinant OGDC-E2 autoantigen will allow the design of additional studies to further our understanding of the role of mitochondrial autoantigens in the pathogenesis of PBC.

Animals↗