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S L Stanley

Publications and source records attributed to S L Stanley.

At least 19 recordsLinked to original sources
PubMed2001

Protective immunity to amebiasis: new insights and new challenges.

Amebiasis, infection with the protozoan parasite Entamoeba histolytica, is the third leading cause of death from parasitic diseases. Despite its importance, we know little about protective immunity to amebiasis. Now, studies from a cohort of children in Bangladesh have provided some critical insights into immunity to intestinal amebiasis. Children with mucosal IgA antibodies to the amebic adherence lectin were found to be resistant to reinfection with E. histolytica. However, immunity was short-lived, and approximately 20% of children in the cohort had a second episode of E. histolytica infection during the study period. Theses findings indicate that immunity to amebiasis can develop in some children after intestinal infection, but protective immunity may be transient, and its importance in preventing disease remains to be established.

Animals↗
PubMed2001

The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) protein is necessary for amebic growth and survival and requires an intact C-terminal domain for both alcohol dahydrogenase and acetaldehyde dehydrogenase activity.

The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N- and C-terminal halves of the molecule.

Alcohol Dehydrogenase↗
PubMed2001

Pathophysiology of amoebiasis.

Few organisms are more aptly named than Entamoeba histolytica, an intestinal protozoan parasite that can lyse and destroy human tissue. Within the past four years, new models of E. histolytica infection have begun to illuminate how amoebic trophozoites cause intestinal disease and liver abscess, and have expanded our understanding of the remarkable killing ability of this parasite. Here, I summarize recent work on the interactions between E. histolytica and human intestine, and between E. histolytica and hepatocytes, and discuss what these studies tell us about the role of inflammation and programmed cell death in the pathogenesis of amoebiasis.

Animals↗
PubMed2001

Blockade of caspases inhibits amebic liver abscess formation in a mouse model of disease.

We looked at the effect of inhibiting caspases on amebic liver abscess in the mouse model of infection. A dose of the pan-caspase inhibitor benzyloxycarbonyl-V-A-D-O-methyl fluoromethyl ketone (Z-VAD-FMK; R & D Systems) given to SCID mice 2 h prior to direct hepatic inoculation with Entamoeba histolytica trophozoites, and 12 h after amebic inoculation, reduced the mean liver abscess size by 70% at 24 h compared to a control group. These data indicate that apoptosis plays a significant but not an exclusive role in amebic liver abscess formation in the mouse model.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Shigella infection in a SCID mouse-human intestinal xenograft model: role for neutrophils in containing bacterial dissemination in human intestine.

Shigellae infect human intestine and cause intense inflammation and destruction of colonic and rectal mucosa. To model the interactions of shigella with human intestine in vivo, we have studied shigella infection in human intestinal xenografts in severe combined immunodeficient mice (SCID-HU-INT mice). Inoculation of shigella into human intestinal xenografts caused severe inflammation and mucosal damage, which was apparent as soon as 4 h following infection. Shigella infection was associated with human intestinal production of interleukin-1B (IL-1B) and IL-8 and a marked neutrophil influx into the graft. Depletion of neutrophils from SCID-HU-INT mice reduced inflammation in the human intestinal xenograft in response to shigella infection but failed to significantly alter tissue damage. However, the number of intracellular bacteria was more than 20-fold higher in the human intestinal xenografts from neutrophil-depleted SCID-HU-INT mice. Infection of human intestinal xenografts with an attenuated vaccine strain of shigella (CVD1203) induced lower levels of IL-1B and IL-8 than wild-type shigella and caused only moderate damage to the intestinal permeability barrier. Our studies establish the SCID-HU-INT mouse as a viable model for studying the interactions between shigella and human intestine and indicate that neutrophils are important for controlling the invasion of human intestine by shigella.

Animals↗
PubMed2001

Amebic infection in the human colon induces cyclooxygenase-2.

We sought to determine if infection of the colon with Entamoeba histolytica induces the expression of cyclooxygenase-2 and, if it does, to determine the contribution of prostaglandins produced through cyclooxygenase-2 to the host response to amebic infection. Human fetal intestinal xenografts were implanted subcutaneously in mice with severe combined immunodeficiency and allowed to grow; the xenografts were then infected with E. histolytica trophozoites. Infection with E. histolytica resulted in the expression of cyclooxygenase-2 in epithelial cells and lamina propria macrophages. Infection with E. histolytica increased prostaglandin E(2) (PGE2) levels 10-fold in the xenografts and resulted in neutrophil infiltration, as manifested by an 18-fold increase in myeloperoxidase activity. Amebic infection also induced an 18-fold increase in interleukin 8 (IL-8) production and a >100-fold increase in epithelial permeability. Treatment of the host mouse with indomethacin, an inhibitor of cyclooxygenase-1 and cyclooxygenase-2, or with NS-398, a selective inhibitor of cyclooxygenase-2, resulted in (i) decreased PGE(2) levels, (ii) a decrease in neutrophil infiltration, (iii) a decrease in IL-8 production, and (iv) a decrease in the enhanced epithelial permeability seen with amebic infection. These results indicate that amebic infection in the colon induces the expression of cyclooxygenase-2 in epithelial cells and macrophages. Moreover, prostaglandins produced through cyclooxygenase-2 participate in the mediation of the neutrophil response to infection and enhance epithelial permeability.

Animals↗
PubMed2001

Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VI. Entamoeba histolytica: parasite-host interactions.

The protozoan intestinal parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. E. histolytica causes two major clinical syndromes, amebic colitis and amebic liver abscess. Recent advances in the development of in vitro and in vivo models of disease, new genetic approaches, the identification of key E. histolytica virulence factors, and the recognition of crucial elements of the host response to infection have led to significant insights into the pathogenesis of amebic infection. E. histolytica virulence factors include 1) a surface galactose binding lectin that mediates E. histolytica binding to host cells and may contribute to amebic resistance to complement, 2) amebapores, small peptides capable of lysing cells, which may play a role in killing intestinal epithelial cells, hepatocytes, and host defense cells, and 3) a family of secreted cysteine proteinases that play a key role in E. histolytica tissue invasion, evasion of host defenses, and parasite induction of gut inflammation. Amebae can both lyse host cells and induce their suicide through programmed cell death. The host response is also an important factor in the outcome of infection, and neutrophils may play a key role in contributing to the tissue damage seen in amebiasis and in controlling amebic infection.

Animals↗
PubMed2000

Entamoeba histolytica cysteine proteinases with interleukin-1 beta converting enzyme (ICE) activity cause intestinal inflammation and tissue damage in amoebiasis.

The protozoan parasite Entamoeba histolytica causes intestinal inflammation and ulceration. Amoebic trophozoites activate the transcription factor NF-kappa B in human intestinal epithelial cells, initiating an inflammatory response programme with resultant damage to the intestinal tissue. Amoebic cysteine proteinases have been proposed as important virulence factors for amoebiasis. To test the role of amoebic cysteine proteinases in the pathogenesis of amoebic colitis, human intestinal xenografts in SCID mice were infected with E. histolytica trophozoites expressing an antisense message to ehcp5. The cysteine proteinase-deficient amoeba failed to induce intestinal epithelial cell production of the inflammatory cytokines interleukin (IL)-1B and IL-8, and caused significantly less gut inflammation and damage to the intestinal permeability barrier. The critical role of amoebic cysteine proteinases in human gut inflammation and tissue damage may be explained by our discovery that amoebic cysteine proteinases possess IL-1B converting enzyme (ICE) activity. This ICE activity could contribute to intestinal inflammation by activating human pIL-1B released by damaged intestinal cells. These results demonstrate for the first time that amoebic cysteine proteinases are a key virulence factor in amoebic colitis, and provide a novel mechanism for their activity.

Amebiasis↗
PubMed2000

Innate immunity to amebic liver abscess is dependent on gamma interferon and nitric oxide in a murine model of disease.

Evidence from in vitro studies suggests that gamma interferon (IFN-gamma) and nitric oxide (NO) are important in host defense against the protozoan parasite Entamoeba histolytica. We used SCID mice with targeted disruption of the IFN-gamma receptor gene and mice with targeted disruption of the gene encoding inducible NO synthase to show that IFN-gamma plays a role in the innate immunity to amebic liver abscess seen in SCID mice while NO is required for control of amebic liver abscess in immunocompetent mice.

Animals↗
PubMed2000

Protection of gerbils from amebic liver abscess by vaccination with a 25-mer peptide derived from the cysteine-rich region of Entamoeba histolytica galactose-specific adherence lectin.

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality through intestinal infection and amebic liver abscess. Here we show that immunization of gerbils with a single keyhole limpet hemocyanin-coupled 25-mer peptide derived from the 170-kDa subunit of the E. histolytica galactose-binding adhesin is sufficient to confer substantial protection against experimentally induced amebic liver abscesses. Vaccination provided total protection in 5 of 15 immunized gerbils, and abscesses were significantly smaller (P < 0.01) in the remaining vaccinated animals. The degree of protection correlated with the titer of antibodies to the peptide, and results of passive transfer experiments performed with SCID mice were consistent with a role for antibodies in protection. In addition, parenteral or oral vaccination of gerbils with 13-amino-acid subfragments of the peptide N-terminally fused to the B subunit of cholera toxin also significantly inhibited liver abscess formation (P < 0.05). These data indicate that small peptides derived from the galactose-binding adhesin administered by the parenteral or oral route can provide protection against amebic liver abscess and should be considered as components of a subunit vaccine against invasive amoebiasis.

Adjuvants, Immunologic↗
PubMed1999

DNA vaccination with the serine rich Entamoeba histolytica protein (SREHP) prevents amebic liver abscess in rodent models of disease.

Amebiasis remains one of the leading parasitic causes of death worldwide. A vaccine that prevented amebic liver abscess would significantly reduce mortality from this disease. To test the feasibility of a DNA vaccine to prevent amebic liver abscess, we immunized both mice and gerbils with plasmid DNA encoding the serine rich Entamoeba histolytica protein (SREHP). Animals receiving the SREHP DNA vaccine developed both antibody and cell mediated immune responses that recognized amebic trophozoites. A single dose of the SREHP DNA vaccine protected 80% of vaccinated mice and 60% of vaccinated gerbils from developing amebic liver abscess after direct hepatic inoculation of amebic trophozoites. Our study indicates that DNA vaccination with SREHP can provide high levels of protection against amebic liver abscess in animal models of disease.

Animals↗
PubMed1999

Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases.

Pure populations of early and late endosomes of Entamoeba histolytica were isolated by magnetic fractionation and characterized. It was shown that these vesicles were enriched in acid phosphatase and cysteine protease activities. An important virulence factor, a 27-kDa cysteine protease, was also enriched in early and late endosomes of E. histolytica. These data suggest that E. histolytica hydrolases reside in compartments that are part of or communicate with the endosomal pathway. To begin to identify the role of Rab GTPases in E. histolytica, an oligonucleotide approach was employed to screen an E. histolytica cDNA library for genes encoding Rab-like proteins. cDNAs encoding a Rab11-like protein (EhRab11) and a novel Rab protein (EhRabA) were isolated and characterized. The EhRab11 cDNA predicts a polypeptide of at least 206 amino acids with a molecular mass of at least 23.2 kDa. Phylogenetic analysis and alignment of EhRab11 with other Rab proteins demonstrated that EhRab11 shared significant homology at the amino acid level with Rab11-like proteins from a number of other eukaryotes, suggesting that EhRab11 is a Rab11 homolog for E. histolytica. The EhRabA clone predicts a polypeptide of 219 amino acids with a molecular mass of at least 24.5 kDa. EhRabA shared only limited homology at the amino acid level with other Rab proteins, suggesting that it is a novel member of this family of GTP-binding proteins. Finally, Western blot analysis demonstrated that EhRab11 and a previously described Rab7-like GTPase from E. histolytica was enriched in magnetically purified endosomal compartments of this organism.

Acid Phosphatase↗
PubMed1998

Epithelial cell-initiated inflammation plays a crucial role in early tissue damage in amebic infection of human intestine.

BACKGROUND & AIMS: Entamoeba histolytica infection of the intestine can induce severe gut inflammation. The aims of this study were to assess the role of the host inflammatory response in the tissue damage observed with amebiasis and the role of the intestinal epithelial cell in initiating that response. METHODS: E. histolytica infection was established in human intestinal xenografts in severe combined immunodeficient (SCID-HU-INT) mice. Human intestinal epithelial cell inflammatory responses to amebic infection were inhibited by the intraluminal administration of an antisense oligonucleotide to the human p65 subunit of nuclear factor kappaB, and the role of neutrophils in tissue damage observed with amebiasis was studied by depleting neutrophils from SCID-HU-INT mice. RESULTS: Administration of the antisense oligonucleotide blocked the production of human interleukin 1beta and interleukin 8 by intestinal epithelial cells and inhibited neutrophil influx into the E. histolytica-infected intestinal xenografts. Inhibition of the gut inflammatory response by the antisense oligonucleotide or the depletion of neutrophils from SCID-HU-INT mice blocked the increase in intestinal permeability observed with amebic infection. CONCLUSIONS: Intestinal epithelial cells initiate an inflammatory response with resulting neutrophil-mediated tissue damage in response to E. histolytica infection; this inflammatory cascade can be blocked by inhibiting the transcription of genes regulated by nuclear factor kappaB.

Amebiasis↗
PubMed1998

Mucosal immunogenicity of a holotoxin-like molecule containing the serine-rich Entamoeba histolytica protein (SREHP) fused to the A2 domain of cholera toxin.

One strategy for the induction of mucosal immune responses by oral immunization is to administer the antigen in conjunction with cholera toxin. Cholera toxin consists of one A polypeptide (CTA) which is noncovalently linked to five B subunits (CTB) via the A2 portion of the A subunit (CTA2). Coupling of antigens to the nontoxic B subunit of cholera toxin may improve the immunogenicity of antigens by targeting them to GM1 ganglioside on M cells and intestinal epithelial cells. Here, we describe the construction of a translational fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective amebic antigen, fused to a maltose binding protein (MBP) and to CTA2. When coexpressed in Escherichia coli with the CTB gene, these proteins assembled into a holotoxin-like chimera containing MBP-SREHP-CTA2 and CTB. This holotoxin-like chimera (SREHP-H) inhibited the binding of cholera toxin to GM1 ganglioside. Oral vaccination of mice with SREHP-H induced mucosal immunoglobulin A (IgA) and serum IgG antiamebic antibodies and low levels of mucosal anti-CTB antibodies. Our studies confirm that the genetic coupling of antigens to CTA2 and their coexpression in E. coli can produce holotoxin-like molecules that are mucosally immunogenic without the requirement for supplemental cholera toxin, and they establish the SREHP-H protein as a candidate for evaluation as a vaccine to prevent amebiasis.

Animals↗
PubMed1998

Cryptosporidium parvum infection of human intestinal xenografts in SCID mice induces production of human tumor necrosis factor alpha and interleukin-8.

The protozoan parasite Cryptosporidium parvum invades intestinal epithelial cells and can cause life-threatening diarrhea in immunocompromised individuals. Despite the clinical importance of this organism, much remains to be learned about the pathogenesis of C. parvum-induced diarrhea. To explore the role of the intestinal inflammatory response in C. parvum disease, using C. parvum oocysts we infected human intestinal xenografts in severe combined immunodeficient (SCID) mice. Seven days after infection, we found levels of human tumor necrosis factor alpha and interleukin-8 in C. parvum-infected human intestinal xenografts that were significantly higher than those seen in uninfected control xenografts. These results demonstrate that human intestinal cells produce proinflammatory cytokines in response to C. parvum infection and establish SCID-HU-INT mice as a model system to study the interactions of C. parvum with the human intestine.

Animals↗
PubMed1998

Entamoeba histolytica induces host cell death in amebic liver abscess by a non-Fas-dependent, non-tumor necrosis factor alpha-dependent pathway of apoptosis.

Amebic liver abscess is characterized by extensive areas of dead hepatocytes that form cavities surrounded by a thin rim of inflammatory cells and few Entamoeba histolytica trophozoites. E. histolytica produces pore-forming proteins and proteinases, but how trophozoites actually kill host cells has been unclear. Here, we report that E. histolytica induces apoptosis in both inflammatory cells and hepatocytes in a severe combined immunodeficient (SCID) mouse model of amebic liver abscess. By studying infection in C57/BL6.lpr and C57/BL6.gld mice, we found that E. histolytica-induced apoptosis does not require the Fas/Fas ligand pathway of apoptosis, and by using mice with a targeted deletion of the tumor necrosis factor receptor I gene, we have shown that E. histolytica-induced apoptosis is not mediated by tumor necrosis factor alpha. Our data indicate that apoptosis plays a prominent role in the host cell death seen in amebic liver abscess in a mouse model of disease and suggest that E. histolytica induces cell death without using two common pathways for apoptosis.

Animals↗
PubMed1998

Longitudinal study of the antibody response to recombinant Entamoeba histolytica antigens in patients with amebic liver abscess.

Serology is a critical component in the diagnosis of amebic liver abscess. However, in areas endemic for amebiasis there is a high background level of seropositivity for amebiasis (owing to previous infection with Entamoeba histolytica), which may complicate the interpretation of a positive serologic test result. Recently, we reported that serologic tests based on recombinant E. histolytica antigens might offer improved diagnosis of current invasive amebiasis because they apparently differentiated active infection from past exposure to the parasite. To confirm this finding, we have performed a longitudinal study on 20 patients with amebic liver abscess by examining their seroreactivity over time with recombinant versions of two major E. histolytica proteins, the serine rich E. histolytica protein (SREHP), and the 170-kD subunit of the galactose-specific adhesin. We found that more than 50% of the patients examined had become seronegative by one or both recombinant tests within 180 days of their diagnosis of amebic liver abscess. In the case of the recombinant SREHP-based tests, 12 patients had become seronegative 90 days after presentation. In contrast, all patients remained seropositive by a standard conventional test, an indirect hemagglutination test, at more than six months after presentation. Our study shows that patients lose seroreactivity with the recombinant SREHP or 170-kD antigen-based tests more rapidly than with a conventional serologic test; this may make them useful for the serologic diagnosis of amebiasis in endemic areas.

Animals↗