Biomedical subjects

H Eisen

Publications and source records attributed to H Eisen.

At least 19 recordsLinked to original sources

PubMed1992

Polyreactive autoantibodies to negatively charged epitopes following Trypanosoma cruzi infection.

During the course of many human autoimmune diseases, antibodies which recognize negatively charged epitopes on self antigens are detected. Trypanosoma cruzi, an intracellular protozoan parasite capable of infecting a wide variety of vertebrates, is the cause of Chagas disease in humans. Infection with the parasite frequently results in autoimmune and inflammatory pathology. We report here on an affinity-purified population of antibodies that bind to a broad class of antigens that contain runs of acidic amino acids, including tubulin. Although these antibodies can be isolated from both uninfected and T. cruzi chronically infected C3H/He mice, the antibodies from the normal mice (the natural autoantibodies) bind to tubulin poorly at physiological pH, whereas the antibodies isolated from the infected animals bind well at physiological pH. We propose that similar processes may occur in humans following other infections accounting for the detection of antibodies to negatively charged epitopes in a variety of autoimmune diseases.

3T3 Cells↗

PubMed1992

Evaluation of recombinant trypomastigote surface antigens of Trypanosoma cruzi in screening sera from a population in rural northeastern Brazil endemic for Chagas' disease.

A perfect serologic test for infection with Trypanosoma cruzi does not exist. This study uses recombinant T. cruzi surface proteins in the antibody capture enzyme linked immunoabsorption assay (ELISA); and compares this approach to the more standard immunofluorescence assay (IFA). Three recombinant antigens are studied: F1-160 corresponding to the 160 kDa flagellar associated surface protein of trypomastigotes (the motile form of T. cruzi in mammalian infections); and SA 85-1.1 and 1.2 corresponding to different members of the 85 kDa family of surface proteins expressed by trypomastigotes and amastigotes (the replicative, non-motile form of T. cruzi in mammalian infections). Each recombinant antigen is found to be highly specific (range 86-94%) but relatively insensitive (range 36-52%) when used to screen for antibodies to T. cruzi. Defining seropositivity as reactivity to any of the three recombinant antigens markedly increases the sensitivity (72%) with only a minor reduction in specificity (82%). Thus, employing recombinant T. cruzi antigens to screen for T. cruzi infection has promise, but improvements in sensitivity must be made before widespread utilization is recommended.

Animals↗

PubMed1992

An interspecific linkage map of mouse chromosome 15 positioned with respect to the centromere.

We have used an interspecific backcross between C57BL/6J and Mus spretus to derive a molecular genetic linkage map of chromosome 15 that includes 25 molecular markers and spans 93% of the estimated length of chromosome 15. Using a second interspecific backcross that was analyzed with a centromere-specific marker, we were also able to position our map with respect to the chromosome 15 centromere. This map provides molecular access to many discrete regions on chromosome 15, thus providing a framework for establishing relationships between cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease.

Animals↗

PubMed1992

Sodium butyrate inhibits myogenesis by interfering with the transcriptional activation function of MyoD and myogenin.

Sodium butyrate reversibly inhibits muscle differentiation and blocks the expression of many muscle-specific genes in both proliferating myoblasts and differentiated myotubes. We investigated the role of the basic helix-loop-helix (bHLH) myogenic determinator proteins MyoD and myogenin in this inhibition. Our data suggest that both MyoD and myogenin are not able to function as transcriptional activators in the presence of butyrate, although both apparently retain the ability to bind DNA. Transcription of MyoD itself is extinguished in butyrate-treated myoblasts and myotubes, an effect that may be due to the inability of MyoD to autoactivate its own transcription. We present evidence that the HLH region of MyoD is essential for butyrate inhibition of MyoD. In contrast to MyoD and myogenin, butyrate does not inhibit the ubiquitous basic HLH protein E2-5 from functioning as a transcriptional activator.

Base Sequence↗

PubMed1991

The major 85-kDa surface antigen of the mammalian-stage forms of Trypanosoma cruzi is a family of sialidases.

Trypanosoma cruzi, an intracellular protozoan parasite infecting a wide variety of vertebrates, is the agent responsible for Chagas disease in humans. An estimated 15-20 million people in South and Central America are infected with the parasite. Chagas disease often results in severe autoimmune and inflammatory pathology and is the major cause of heart failure in endemic areas. Nevertheless, little is known about the host-parasite interactions that lead to this pathology. We have previously cloned several members of a large gene family (SA85-1) and shown that these genes encode 85-kDa T. cruzi, mammalian-stage-specific, surface antigens. Here we report that members of the SA85-1 family possess sialidase activity and are shed by the parasite. We suggest that the sialidases may contribute to the pathology during T. cruzi infection by cleaving sialic acid from cells of the immune system.

Amino Acid Sequence↗

PubMed1991

Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.

Amino Acid Sequence↗

PubMed1991

The trypanosome VSG expression site encodes adenylate cyclase and a leucine-rich putative regulatory gene.

African trypanosomes are protozoan parasites that evade the host immune system by varying their dense antigenic coat. The Variant Surface Glycoprotein (VSG) is expressed exclusively from telomere-linked expression sites that contain in addition to the VSG gene, a number of open reading frames termed Expression Site Associated Genes (ESAGs). Here we demonstrate by complementation of a yeast mutant deleted for adenylate cyclase (cyr-1), that an ESAG from Trypanosoma equiperdum encodes an adenylate cyclase. Furthermore, we report that adjacent to adenylate cyclase in the expression site, is a separate open reading frame that encodes a protein sequence motif similar to the leucine-rich repeat regulatory domain of Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylate cyclases. The finding of two adjacent open reading frames homologous to a single enzyme in yeast suggests that the two expression site encoded proteins may interact to regulate adenylate cyclase activity during the course of an infection.

Adenylyl Cyclases↗

PubMed1991

Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes.

Classic galactosemia, an inborn error of human galactose metabolism, is characterized by a deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). The current model for the pathophysiology of this disease ascribes most of its symptoms to the toxicity of intracellular galactose-1-phosphate (Gal-1-P), one of the substrates of GALT which accumulates in the untreated disease state. Recently, a reduction in the intracellular concentration of UDP-Gal (uridine diphosphogalactose), one of the products of GALT, has been described in treated galactosemic patients. We investigated whether galactosemic patients might also have reduced amounts of those macromolecules that depend on UDP-Gal for their biosynthesis. We report a reduction in glycolipids that contain either galactose or its derivative N-acetylgalactosamine and an accumulation of the precursors to these compounds in the brain of a neonate with galactosemia. We also found an imbalance in glycolipids in galactosemic lymphoblasts. This novel biochemical abnormality observed in galactosemic patients is not addressed by dietary galactose-restriction therapy and could explain some of the chronic neurologic and other complications of galactosemia.

Acetylgalactosamine↗

PubMed1991

Mimicry in Trypanosoma cruzi: fantasy and reality.

Chronic infection of mammals by Trypanosoma cruzi often results in severe autoimmune and inflammatory pathology. Extensive antigen cross-reactivity between the parasite and its mammalian hosts has also been reported. These findings have stimulated speculation that Trypanosoma cruzi uses antigenic mimicry as a mechanism for escaping the host immune system. This may not be the case and the observed antigen cross-reactivity may be a result of perturbations of the immune system such that common, normally tolerated antigens are recognized in infected animals. The parasite, however, does appear to use functional mimicry to survive in the immune competent host.

Acute-Phase Reaction↗

PubMed1991

A family of genes related to a new expression site-associated gene in Trypanosoma equiperdum.

Two genes, belonging to a new expression site-associated gene family of six to eight members in Trypanosoma equiperdum and Trypanosoma brucei, have been cloned from a T. equiperdum variant. One of them, called ESAG-9c, is contained in the 1.78-C expression site and is found just upstream of the 5' barren region. The other one, called ESAG-9u, is unique in the family, is not telomere linked, and apparently is not expression site related. A 2-kb poly(A)+ mRNA is detected with probes for this ESAG-9 family in all T. equiperdum variants examined. By using polymerase chain reaction and restriction fragment length polymorphism techniques, it has been possible to distinguish between ESAG-9c and ESAG-9u and to show that ESAG-9c is transcribed in an expression site-specific manner. However, ESAG-9u (or another gene in the family having identical characteristics) is transcribed in all variants, regardless of the expression site used by these variants. Thus, this ESAG-9 family contains at least one gene that is under expression site control but might have other genes that are not. The function of these ESAG-9 genes is unknown. Transcripts homologous to ESAG-9 were detected in T. brucei bloodstream forms but not in procyclics.

Amino Acid Sequence↗

PubMed1991

Value of peak exercise oxygen consumption for optimal timing of cardiac transplantation in ambulatory patients with heart failure.

BACKGROUND: Optimal timing of cardiac transplantation in ambulatory patients with severe left ventricular dysfunction is often difficult. To determine whether measurement of peak oxygen consumption (VO2) during maximal exercise testing can be used to identify patients in whom transplantation can be safely deferred, we prospectively performed exercise testing on all ambulatory patients referred for transplant between October 1986 and December 1989. METHODS AND RESULTS: Patients were assigned into one of three groups on the basis of exercise data: Group 1 (n = 35) comprised patients accepted for transplant (VO2 less than or equal to 14 ml/kg/min); group 2 (n = 52) comprised patients considered too well for transplant (VO2 greater than 14 ml/kg/min); and group 3 (n = 27) comprised patients with low VO2 rejected for transplant due to noncardiac problems. All three groups were comparable in New York Heart Association functional class, ejection fraction, and cardiac index (p = NS). Pulmonary capillary wedge pressure was significantly lower in group 2 than in either group 1 or 3 (p less than 0.05), although there was wide overlap. Patients with preserved exercise capacity (group 2) had cumulative 1- and 2-year survival rates of 94% and 84%, which are equal to survival levels after transplantation. In contrast, patients rejected for transplant (group 3) had survival rates of only 47% at 1 year and 32% at 2 years, whereas patients awaiting transplantation (group 1) had a survival rate of 70% at 1 year (both p less than 0.005 versus patients with VO2 greater than 14 ml/kg/min). All deaths in group 2 were sudden. By univariate and multivariate analyses, peak VO2 was the best predictor of survival, with only pulmonary capillary wedge pressure providing additional prognostic information. CONCLUSIONS: These data suggest that cardiac transplantation can be safely deferred in ambulatory patients with severe left ventricular dysfunction and peak exercise VO2 of more than 14 ml/min/kg.

Evaluation Studies as Topic↗

PubMed1991

An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits.

Emulsion adjuvants have been used for production of polyclonal antisera in rabbits (Oryctolagus cunniculi) for decades. Complete Freund's adjuvant has a reputation as a very effective immunoenhancer, but adverse physiological effects, including fever, inflammation and sterile abscess formation, have prompted a search for alternatives to complete Freund's. In this study, we quantitatively compared five adjuvant regimens: (a) a primary inoculation with complete Freund's followed by three boosts with incomplete Freund's; (b) four serial inoculations of incomplete Freund's adjuvant augmented with 6-bromoguanisine; (c) four serial inoculations with RIBI's MPL + TDM + CWS adjuvant emulsion; (d) four serial inoculations with Montanide ISA 50 emulsion; and (e) four serial inoculations with Montanide ISA 70 emulsion. We chose a small (12 amino acid) chain polypeptide coupled to bovine serum albumin as our test antigen. When compared, no system could be seen to be significantly better than a regimen of a primary immunization with complete Freund's adjuvant followed by serial reimmunization with incomplete Freund's adjuvant. The commercially available RIBI adjuvant produced significantly lower antibody levels, while other systems produced essentially equivalent levels. With all five adjuvants, antibody quantities plateaued after the second injection and further immunization did not increase titers significantly. Boost injections did yield greater intradermal tissue reaction than primary inoculations, and intramuscular inoculum volumes of 0.4 cc caused chronic lesions still detectable by the gross necropsy 2 weeks after the final injection.

Adjuvants, Immunologic↗

PubMed1990

The major 85-kD surface antigen of the mammalian form of Trypanosoma cruzi is encoded by a large heterogeneous family of simultaneously expressed genes.

Trypanosoma cruzi is an obligate intracellular protozoan parasite. The parasite mammalian stage surface antigens exhibit extensive antigenic diversity. We have characterized a family of T. cruzi genes that code for a polymorphic set of 85-kD surface antigens, the SA85-1 antigens. The family contains greater than 100 genes and pseudogenes, of which a minimum of nine are transcribed. The gene family is expressed in the mammalian stage only. A subset of the gene family is present in two telomere-linked copies in the genome. Telomere linkage of other expressed SA85-1 genes has not been demonstrated. We have shown that at least three members of the SA85-1 gene family encode antigens at the surface of the mammalian stage of the parasite. Interestingly, these three antigens are expressed on all the trypanosomes examined. This suggests that T. cruzi simultaneously expresses a large repertoire of similar, but diverse antigens at its surface. Thus, T. cruzi exhibits extensive antigenic diversity in a system unique from that of African trypanosomes, perhaps reflecting its intracellular niche.

Amino Acid Sequence↗

PubMed1990

Trypanosome variable surface glycoproteins: composite genes and order of expression.

Combinatorial processes increase the diversity of variable surface glycoproteins (VSGs) expressed by Trypanosoma equiperdum. We show here that a single telomeric pseudogene provides the 3' portion of three distinct T. equiperdum VSG genes by recombination with different 5' donor pseudogenes. Regions of sequence homology among the pseudogenes determine the sites of recombination in the formation of the expressed copies. This suggests that the recombination between any given basic copy (BC) and the expression-linked copy (ELC) depends on their sharing homology. We present evidence that this is the case and propose that such rules account for the order of expression of the VSGs. These results demonstrate how homologous recombination can generate an ordered sequence of gene expression.

Animals↗

PubMed1989

Fl-160. A surface antigen of Trypanosoma cruzi that mimics mammalian nervous tissue.

Chagas' disease, caused by Trypanosoma cruzi, is an excellent model for autoimmune disease induced by an infectious agent. Transfer of T cells, directed against crossreactive antigens of T. cruzi and nervous tissue, have been shown to reproduce pathology found in chronic Chagas' disease. We used recombinant DNA technology to characterize one of these crossreactive antigens (Fl-160). We have cloned DNA from T. cruzi, which expresses a protein corresponding to a 160-kD protein found on the surface of the trypanosome, overlying the flagellum. This clone hybridizes to a 4.5-kb poly(A)+ RNA that is distributed in a differentiation-specific manner, suggesting expression of this protein is transcriptionally controlled. Antibodies to this protein crossreact with a 48-kD mammalian nervous tissue protein found in sciatic nerve, brain, and myenteric plexi of gut. The myenteric plexi are destroyed by inflammatory infiltrates in Chagas' disease, leading to the characteristic megaesophagus and megacolon Chagas' disease pathology. Thus, this antigen is a candidate antigen for autoimmune mimicry leading to nervous tissue pathology.

Animals↗

PubMed1989

Active late-appearing variable surface antigen genes in Trypanosoma equiperdum are constructed entirely from pseudogenes.

The expression of genes coding for variable surface glycoproteins (VSGs) in Trypanosoma equiperdum is linked to duplicative transpositions of silent, basic copy sequences into telomere-linked expression sites. Examination of three independently derived late-appearing trypanosome clones expressing VSG-78 revealed that the expressed gene in all cases is composed of sequences derived from three or four individual silent genes. The 182 base pairs at the 3' end of the coding sequence are derived from one silent gene, the 3' donor. The remaining 5' segment is a mosaic structure containing variable-length segments derived from two, or perhaps three, related silent genes. All of the silent genes that participate in the construction of the VSG-78 expression-linked copy (ELC) genes contain multiple stop codons and are unable to code for VSGs. Individual silent pseudogenes complement one another in the mosaic structure of the 5' portions of the ELC genes and create functional VSG genes. The joining of the 3' and 5' portions of the composite genes occurs in short regions of homology and suggests a mechanism by which the ordered expression of the VSG genes is generated.

Animals↗

PubMed1989

Antigenic diversity by the recombination of pseudogenes.

During the course of an infection, the hemoflagellate Trypanosoma equiperdum sequentially expresses an extensive repertoire of surface glycoproteins. There is evidence that combinations of silent genes are involved in the generation of this repertoire, but the combination rules are not known. To gain insight into these rules, we determined the fine structure of a composite gene. The gene coding for the variant surface glycoprotein 20 of T. equiperdum is a late gene generated by the partial duplication of three silent pseudogenes. Two closely related but not identical '5' donors' form a mosaic coding for the antigenic portion of the protein. A telomeric '3' donor' provides the last 200 nucleotides of the expressed gene. The sequences of the 5' and 3' donors are not related except for a short segment in which the hybrid junction is formed. These results demonstrate that recombinational processes generate diversity by reassorting sequences and also allow the expression of pseudogenes. Furthermore, the use of a short sequence similarity for the formation of the 5'-3' donor hybrid suggests a mechanism that may act in ordering the expression of the variant surface glycoproteins.

Animals↗

PubMed1989

Susceptible mice present higher macrophage activation than resistant mice during infections with myotropic strains of Trypanosoma cruzi.

The kinetics of macrophage activation were compared among inbred strains of mice (C3H, BALB, B6 and B10.A) that are known to differ in their relative resistance to infections with the myotropic strains (Colombian and CL) of Trypanosoma cruzi. The parameters utilized to measure macrophage activation were rapid spreading on glass surfaces, hydrogen peroxide release and tumour necrosis factor/cachectin production. Macrophages obtained from C3H (susceptible), BALB (intermediate) and B6 or B10.A (resistant) mice infected with both strains of T. cruzi began to spread rapidly at the onset of parasitaemia. Surprisingly, the amount of hydrogen peroxide released by peritoneal cells obtained from the more susceptible mouse strain (C3H) was significantly higher than in the other mouse strains. Also, only in the serum of C3H mice was tumour necrosis factor/cachectin detected. These results suggest that resistance against infections with myotropic strains of T. cruzi does not correlate with enhanced macrophage activation. It is also shown that the acquired macrophage activation is largely dependent on T-lymphocytes bearing the phenotypic marker CD4 (helper/inducer), since all parameters of macrophage activation were significantly inhibited in athymic mice or in C3H mice treated in vivo with monoclonal antibody anti-CD4+ T-cells.

Animals↗