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S L Stanley

Publications and source records attributed to S L Stanley.

At least 55 records · Page 3Linked to original sources
PubMed1995

Interaction of laminin with Entamoeba histolytica cysteine proteinases and its effect on amebic pathogenesis.

The Entamoeba histolytica 27-kDa cysteine proteinases exhibit striking binding specificities for immobilized laminin over other components of the extracellular matrix, such as collagen and fibronectin. Inactivation of the proteinase with the active-site inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane abolishes laminin binding by the enzyme, and conversely, laminin inhibits cleavage of a fluorogenic dipeptide substrate of the amebic cysteine proteinase, suggesting that the substrate binding pocket of the enzyme is involved in the binding of laminin. The addition of laminin but not fibronectin or collagen to E. histolytica trophozoites significantly reduces amebic liver abscess formation in severe combined immunodeficient mice, further supporting the hypothesis that E. histolytica cysteine proteinases play an important role in amebic pathogenesis. The specific interaction of amebic proteinases with laminin may be exploited in designing new inhibitors of these enzymes.

Amino Acid Sequence↗
PubMed1995

Oral immunization with the dodecapeptide repeat of the serine-rich Entamoeba histolytica protein (SREHP) fused to the cholera toxin B subunit induces a mucosal and systemic anti-SREHP antibody response.

The intestinal protozoan parasite Entamoeba histolytica causes amebic dysentery, a major cause of morbidity worldwide. The induction of a mucosal antibody response capable of blocking amebic adhesion to intestinal cells could represent an approach to preventing E. histolytica infection and disease. Here we describe the expression of a chimeric protein containing an immunogenic dodecapeptide derived from the serine-rich E. histolytica protein (SREHP), fused to the cholera toxin B subunit (CtxB). The CtxB-SREHP-12 chimeric protein was purified from Escherichia coli lysates and retained the critical GM1 ganglioside-binding activity of the CtxB moiety. Mice fed the CtxB-SREHP-12 fusion protein along with a subclinical dose of cholera toxin developed mucosal immunoglobulin A and immunoglobulin G and systemic antibody responses that recognized recombinant and native SREHP. Our study confirms the feasibility of inducing mucosal immune responses to immunogenic peptides by their genetic fusion to the CtxB subunit and identifies the CtxB-SREHP-12 chimeric protein as a candidate oral vaccine to prevent E. histolytica infection.

Administration, Oral↗
PubMed1995

Role of the Entamoeba histolytica cysteine proteinase in amebic liver abscess formation in severe combined immunodeficient mice.

Evidence from in vitro studies suggest that the Entamoeba histolytica cysteine proteinase plays a role in the tissue lysis and cytopathic effects seen in invasive amebiasis. We used affinity-purified antibodies against a recombinant E. histolytica cysteine proteinase to demonstrate that the proteinase is present extracellularly in amebic liver abscesses in mice with severe combined immunodeficiency (SCID mice). Treatment of E. histolytica trophozoites with specific cysteine proteinase inhibitor E-64 blocked or greatly reduced liver abscess formation at 48 h in SCID mice. Our study suggests an important role for a functional cysteine proteinase in amebic liver abscess formation.

Animals↗
PubMed1994

Entamoeba histolytica has an alcohol dehydrogenase homologous to the multifunctional adhE gene product of Escherichia coli.

Entamoeba histolytica ferments glucose to ethanol under the anaerobic conditions of the human colon. There is special interest in this metabolic pathway because it provides an opportunity for parasite-specific chemotherapy. Peptide sequences from a 97-kDa E. histolytica protein, which was originally isolated because of extracellular matrix binding properties, were used to clone and sequence a gene that was found to encode an E. histolytica alcohol dehydrogenase and acetaldehyde dehydrogenase (EhADH2). The EhADH2 cDNA clone had an open reading frame encoding 870 amino acids with a predicted molecular weight of 95,758. The EhADH2 cDNA clone was identical in 48% of its amino acids to the multifunctional enzyme (alcohol dehydrogenase, acetyl-CoA reductase, and pyruvate-formate-lyase-deactivase) encoded by the Escherichia coli adhE gene. The isolation of the EhADH2 protein helps define a new family of ADH enzymes that may be specific to anaerobic and facultatively anaerobic organisms.

Alcohol Dehydrogenase↗
PubMed1994

Antibodies to the serine rich Entamoeba histolytica protein (SREHP) prevent amoebic liver abscess in severe combined immunodeficient (SCID) mice.

Amoebic liver abscess caused by Entamoeba histolytica is a major cause of morbidity and mortality worldwide. We used mice with severe combined immunodeficiency (SCID mice) to study the role of antibody in protection from amoebic liver abscess, and to identify protective antigens of E. histolytica. Antisera to recombinant versions of two major surface antigens of E. histolytica, the serine rich E. histolytica protein (SREHP) and the 170 kDa adhesin were used in this study. We found that 100% of SCID mice passively immunized with antiserum to the recombinant SREHP molecule were protected from developing amoebic liver abscess after intrahepatic challenge with virulent E. histolytica trophozoites. In contrast, preimmune serum, antiserum to a portion of the 170 kDa adhesin, and antiserum to the trpE fusion partner of SREHP did not protect SCID mice from amoebic liver abscess. Our study demonstrates that antibodies to a recombinant version of the amoebic SREHP molecule can protect against amoebic liver abscess, and suggest the recombinant SREHP molecule should be considered as a possible vaccine candidate to prevent amoebic liver abscess.

Animals↗
PubMed1994

Entamoeba histolytica interactions with polarized human intestinal Caco-2 epithelial cells.

To model the initial pathogenic effects of Entamoeba histolytica trophozoites on intestinal epithelial cells, the interactions of E. histolytica HM1-IMSS trophozoites with polarized human intestinal Caco-2 cell monolayers grown on permeabilized filters were examined. Trophozoites, when incubated with the apical surface of the monolayers at 37 degrees C, induced a rapid decrease in transepithelial resistance over 15 to 60 min. The transmonolayer resistance response was not associated with changes in short-circuit current but was associated with an increase in mannitol flux, suggesting that the drop in resistance reflected a nonselective increase in epithelial permeability rather than stimulation of electrogenic ion transport. This response preceded the earliest detection of morphologic disruption of monolayer integrity by light or electron microscopy. Apical injury to the monolayer was detected by ultrastructural studies which revealed a loss of brush border in regions of contact between epithelial cells and amebas and by chromium release assays where a small increase in the apical release of 51Cr from the monolayer (6% over background) was observed. The transmonolayer resistance response was inhibited when the temperature was reduced to 4 degrees C and by addition of cytochalasin D (1 microgram/ml) to the medium at concentrations that did not directly affect transmonolayer resistance. Application of amebic lysates or medium conditioned by coincubation of amebas with Caco-2 monolayers failed to lower transmonolayer resistance, suggesting that this effect was not mediated by soluble amebic cytotoxins. Polarized Caco-2 monolayers grown on permeable filters provide a useful model for studying the initial interactions of E. histolytica trophozoites with intestinal epithelial cells.

Animals↗
PubMed1994

Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen.

Amebiasis, infection by the intestinal protozoan parasite Entamoeba histolytica, is a leading parasitic cause of death. As a step in the development of a recombinant antigen vaccine to prevent E. histolytica infection, we looked at the ability of a recombinant version of the serine-rich E. histolytica protein (SREHP) to elicit a protective immune response against invasive amebic disease. Gerbils, a standard model for amebic liver abscess, were immunized with either a recombinant SREHP/maltose-binding protein (MBP) fusion, recombinant MBP alone, or phosphate-buffered saline (PBS), all combined with complete Freund's adjuvant. In the first trial (group 1), gerbils received a primary and two booster immunizations intraperitoneally; in the second trial (group 2), gerbils were immunized by a single intradermal injection. SREHP/MBP-immunized gerbils in both groups produced antibody to native SHEHP and developed delayed-type hypersensitivity responses to recombinant SREHP. All gerbils were challenged by an intrahepatic injection with 5 x 10(4) virulent E. histolytica HM1-IMSS trophozoites. Complete protection from amebic liver abscess was seen in 64% of the SHEHP/MBP-immunized gerbils in group 1 and in 100% of the SREHP/MBP-immunized gerbils in group 2. There was no protection observed in MBP- or PBS-immunized gerbils in either group. Our results indicate that the SREHP molecule has potential as a vaccine to prevent amebic infection and demonstrate that successful vaccination of animals with recombinant E. histolytica antigen vaccines is possible.

Animals↗
PubMed1994

Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites.

Animals↗
PubMed1993

SCID mice as models for parasitic infections.

Mice with severe combined immunodeficiency (SCID mice) have become a favored model system for the study of many parasitic diseases. In this review, Samuel Stanley Jr and Herbert Virgin IV provide a brief overview of the biology of the SCID mouse, and review some examples of how the SCID mouse model has been applied to the study of the immunology of a number of different parasitic diseases.

Journal Article↗
PubMed1993

Expression of a recombinant Entamoeba histolytica antigen in a Salmonella typhimurium vaccine strain.

The expression of a major surface antigen of the intestinal protozoal parasite Entamoeba histolytica in an attenuated Salmonella typhimurium vaccine strain is described. A polymerase chain reaction fragment derived from cDNA encoding the serine-rich Entamoeba histolytica protein, SREHP, was introduced into S. typhimurium chi 3987 (delta cya delta crp delta asd) using a plasmid expression vector (pYA292) containing the aspartate semialdehyde (asd) gene. S. typhimurium expressing recombinant SREHP as a SREHP/maltose binding protein fusion protein was administered orally to mice and gerbils (an important animal model for E. histolytica infection) and was recovered from splenic tissue in both species. Our study demonstrates the feasibility of expressing recombinant amoebic proteins in attenuated S. typhimurium strains, and shows that vaccine strains of S. typhimurium can successfully infect the gerbil, a widely used model for amoebic liver abscess and intestinal amoebiasis.

Animals↗
PubMed1993

Cysteine-dependent zinc binding by membrane proteins of Giardia lamblia.

The abundant, highly variable surface proteins (VSPs) which cover the surface of Giardia lamblia trophozoites compose a group of extremely cysteine (C)-rich proteins in which more than half of the cysteines are in the motif CXXC. Because of the constancy of these features among the known VSPs and the prominence of cysteine and particularly CXXC in proteins that bind zinc and other metals, we asked whether G. lamblia VSPs bind zinc in vitro. VSPs are the major protein component of Triton X-114 detergent-phase extracts of G. lamblia trophozoites and can be readily identified by surface iodination of intact cells. The partitioning of 65Zn binding into the Triton X-114 detergent phase and the correspondence between surface iodination and zinc binding patterns of four G. lamblia strains or sublines with different VSPs support the idea that VSPs bind zinc. The requirement for renaturation of blots with a reducing agent indicates that Zn2+ is coordinated by cysteine residues, rather than by other amino acids. Binding did not appear to be specific to zinc since it was inhibited by competition with other divalent metal ions. The abundance of the VSPs and the prevalence of metal binding motifs among all known variants suggest that they may play an important role in trophozoite survival and colonization in the host.

Animals↗
PubMed1992

A severe combined immunodeficient (SCID) mouse model for infection with Entamoeba histolytica.

We used severe combined immunodeficient (SCID) mice to study resistance to invasive infection with Entamoeba histolytica. Seven of seven SCID mice developed liver abscesses when challenged intrahepatically with virulent HM1:IMSS strain E. histolytica trophozoites. Only one of seven similarly challenged immunocompetent congenic C.B-17 mice developed an abscess. Adoptive transfer of polyclonal rabbit anti-E. histolytica antiserum, but not preimmune rabbit serum, completely protected 7 of 12 SCID mice from intrahepatic challenge with ameba. These results demonstrate that lymphocyte-based immunity is important in protection against amebic liver abscess, and that anti-E. histolytica antibody can protect against amebic infection in this system. The SCID mouse may provide a powerful model for studying the components of protective immunity to invasive amebiasis.

Animals↗
PubMed1992

Molecular analysis of carbohydrate antigen-induced monoclonal IgM anti-IgG antibodies (rheumatoid factors).

Light and heavy chain variable regions of 11 monoclonal rheumatoid factors (MRF) produced after carbohydrate antigen immunization, and one MRF produced after protein immunization have been sequenced. Most carbohydrate antigen induced MRF utilized light chains that were homologous to light chains of MRF obtained from protein immune or LPS stimulated mice, and MRF derived from the autoimmune MRL/lpr mouse strain. VH gene usage was diverse for carbohydrate antigen induced MRF that bound all four isotypes of IgG, or that bound only the IgG3 isotype. In contrast VH gene use among our panel of MRF that bound the IgG1 isotype appeared restricted. Four of the five IgG1 binders used VH genes that were highly homologous to the VH nucleotide sequence of a gene encoding an NP binding monoclonal antibody. Our study confirms the use of a particular group of light chain genes among murine MRF, confirms that there is diversity in the heavy chain genes utilized among MRF, and suggests that a gene(s) homologous to the VH NP 23 J558 gene may be preferentially associated with murine MRF specificity for the IgG1 isotype.

Amino Acid Sequence↗
PubMed1992

Isolation and partial characterization of a surface glycoconjugate of Entamoeba histolytica.

To study surface molecules of Entamoeba histolytica we produced monoclonal antibodies from mice immunized with lysates from the pathogenic amebic strain HM1:IMSS, and screened them for the ability to inhibit E. histolytica adhesion. One monoclonal antibody, CC 8.6, was a potent inhibitor of amebic adhesion to a Chinese hamster ovary cell line, and was capable of inhibiting HM1:IMSS mediated cytotoxicity by 50%. We found that monoclonal antibody CC 8.6 bound to an amebic glycoconjugate. The glycoconjugate is present only in E. histolytica and not in other Entamoeba sp. It migrates as a polydisperse band on SDS-PAGE, and can be metabolically radiolabeled with [14C]glucose, [32P]phosphate, and [3H]palmitate. The glycoconjugate can be purified by hydrophobic interaction chromatography on octyl-Sepharose; enzymatic hydrolysis with phosphatidylinositol-specific phospholipase C alters the hydrophobic properties of the molecule. HPLC analysis of [14C]glucose-labeled glycoconjugate saccharides revealed that approximately 82% of the incorporated label was in glucose and 12% in galactose. Our studies demonstrate that one of the immunogenic surface molecules of E. histolytica is a phosphorylated, lipid-containing, glycoconjugate, and that antibodies to this antigen may have the potential to protect against E. histolytica adhesion and cytotoxicity.

Animals↗
PubMed1992

Use of a recombinant 170-kilodalton surface antigen of Entamoeba histolytica for serodiagnosis of amebiasis and identification of immunodominant domains of the native molecule.

We expressed the gene that encodes one of the major surface antigens of Entamoeba histolytica, the 170-kDa protein (1,270 amino acids), as a glutathione S-transferase fusion protein containing amino acids 1 to 1202 (lacking the putative transmembrane and cytoplasmic regions) and as separate fusion proteins containing each of three major domains of the 170-kDa molecule. Lysates from bacteria induced to express one of these proteins were used as the target antigens in a Western blot (immunoblot) analysis to determine whether a recombinant 170-kDa antigen could serve as the basis for a serologic test used to detect invasive amebiasis and whether there are differences in humoral immunogenicity among the three major domains of the 170-kDa antigen. Among patients with invasive amebiasis from three major areas where the disease is endemic and two sites in the United States, 54 (90%) of 60 had antibodies to the recombinant 170-kDa protein. Among 37 patients from regions where the disease is endemic and 20 patients from the United States without amebic disease, 1 (2%) of 57 had antibodies to the recombinant 170-kDa protein. We found significant differences in seroreactivity to each of three major domains of the molecule among patients seropositive for the complete construct, ranging from 100% seroreactivity with the fusion protein containing the domain designated cysteine rich and 89% seropositivity with the fusion protein incorporating a portion of the region designated cysteine poor to only 9% seropositivity for the fusion protein containing the pseudorepeat domain. Our study indicates that a serologic test based on the recombinant 170-kDA antigen could serve as a highly sensitive and specific test for acute invasive amebiasis.

Animals↗