Imaging trematode and nematode parasites.

Recent advances in molecular genetics and in imaging mean that it is now increasingly feasible to image biological processes within helminth parasites and to visualize interactions between worms and their hosts. Moreover, other innovative imaging approaches that are not dependent on transgenic parasites have been applied to, and or developed for, the study of helminth parasites and have provided novel and important insights into the biology of these important pathogens.

Schistosoma mansoni egg-induced early IL-4 production is dependent upon IL-5 and eosinophils.

The initial immune response to Schistosoma mansoni eggs presumably results in IL-4 production, as schistosome eggs are strong Th2-inducing antigens and the differentiation of antigen-specific Th2 cells is largely dependent on the presence of IL-4 during priming of naive Th cells. Consistent with this concept, intraperitoneal injection of mice with schistosome eggs results in an upregulation of IL-4 production by peritoneal exudate cells (PECs) within 12 h. Egg-induced IL-4 is rapidly bound by its receptor, suggesting that this cytokine is utilized by a cell type present at the site of antigen deposition or is complexed to soluble receptor. The peak of early IL-4 production is accompanied by a local eosinophilia and the apparent disappearance of mast cells. Studies utilizing either IL-4, IL-5, or mast cell-deficient mice indicate that the eosinophilia is dependent on mast cells and IL-5 and independent of IL-4. Strikingly, egg-induced IL-4 production is absent in animals lacking the early peritoneal eosinophilia. Immunocytochemical analysis of PEC following egg injection indicates that the eosinophils themselves make IL-4. These data strongly suggest that egg-induced IL-5 plays an essential role in recruiting eosinophils to the site of antigen deposition and that it is these eosinophils that then directly produce early IL-4.

Downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni.

In the mouse, infection with Schistosoma mansoni results in an egg-producing infection and associated disease, whereas vaccination with attenuated larval stages produces a substantial and specific immunity in the absence of egg-induced pathology. Preliminary data showing enhanced interleukin-5 (IL-5) production by T cells from infected mice and interferon gamma (IFN-gamma) synthesis by cells from vaccinated animals (7), suggested differential CD4+ subset stimulation by the different parasite stimuli. To confirm this hypothesis, lymphocytes from vaccinated or infected animals were compared for their ability to produce IFN-gamma and IL-2 (secreted by Th1 cells) as compared with IL-4 and IL-5 (characteristic Th2 cytokines). After stimulation with specific antigen or mitogen, T cells from vaccinated mice or prepatently infected animals responded primarily with Th1 lymphokines, whereas lymphocytes from patently infected mice instead produced Th2 cytokines. The Th2 response in infected animals was shown to be induced by schistosome eggs and directed largely against egg antigens, whereas the Th1 reactivity in vaccinated mice was triggered primarily by larval antigens. Interestingly, Th1 responses in mice carrying egg-producing infections were found to be profoundly downregulated. Moreover, the injection of eggs into vaccinated mice resulted in a reduction of antigen and mitogen-stimulated Th1 function accompanied by a coincident expression of Th2 responses. Together, the data suggest that coincident with the induction of Th2 responses, murine schistosome infection results in an inhibition of potentially protective Th1 function. This previously unrecognized downregulation of Th1 cytokine production may be an important immunological consequence of helminth infection related to host adaptation.

Identification of paramyosin as schistosome antigen recognized by intradermally vaccinated mice.

Mice immunized intradermally with extracts of Schistosoma mansoni in combination with the adjuvant BCG are significantly protected against subsequent infection with living larval forms of the parasite. Remarkably, these vaccinated animals produce antibodies predominantly against a single parasite protein of molecular weight 97 kilodaltons (Sm-97). A complementary DNA that encodes about half of the Sm-97 molecule has now been cloned and sequenced. Analysis of the deduced amino acid sequence reveals a protein containing periodic repeats of hydrophobic amino acids characteristic of an alpha-helical coiled-coil structure. The deduced amino acid composition of the cloned gene and several properties of the native protein are similar to that of paramyosin, an alpha-helical protein that forms the core for myosin filaments in invertebrate muscle. Paramyosin was isolated from Schistosoma mansoni adult worms and antibodies to Sm-97 were shown to react with this molecule as well as with a known paramyosin from molluscan muscle.

IL-4(-/-) mice with lethal Mesocestoides corti infections--reduced Th2 cytokines and alternatively activated macrophages.

Protection against Mesocestoides corti, a cestode that invades vital organs, is dependent on the production of IL-4, as IL-4(-/-) mice were found to have higher parasite burdens when compared with wild-type mice. The goal of this study was to investigate the role of IL-4 in immunity to M. corti, focusing on the immunological profile and on potential mediators of pathology. IL-4(-/-) mice infected with M. corti showed 100% mortality by 32 days, whereas wild-type mice survived for approximately 1 year. Parasite burdens were significantly increased in the liver, peritoneal, and thoracic cavities of IL-4(-/-) mice, associated with impaired recruitment of inflammatory cells and a reduction in monocytes and macrophages. IL-5 production by splenocytes and expression in liver tissue was decreased in infected IL-4(-/-) mice compared with wild-type mice. In contrast, IL-4(-/-) mice produced increased amounts of IFNgamma and TNFalpha. Alternatively activated macrophages were a major feature of liver granulomas in wild-type mice evidenced by Arginase I expression, while livers from infected IL-4(-/-) mice showed impaired alternative macrophage activation without increased classical macrophage activation. Thus, lethality during M. corti infection of IL-4(-/-) mice is associated with decreased Th2 cytokines, increased Th1 cytokines and impairment of alternatively activated macrophages.

Reverse genetics and the study of the immune response to schistosomes.

The sequencing of the schistosome genome and the establishment of techniques for RNAi and transient transfection in these parasites have opened the door for a reverse genetics approach to studying schistosomes. One of the most intriguing aspects of schistosome biology is the interaction of these parasites with the immune system. The immune response underlies the ability of the host to survive while infected and to eventually develop resistance to further infection. However, it is also instrumental in the development of disease due to its role orchestrating granuloma formation around tissue-trapped parasite eggs. While schistosomes have clearly evolved mechanisms for evading host immune responses, their normal development is, paradoxically, also dependent upon the presence of a normal immune system. This article will review recent advances in the development of tools for studying gene function in schistosomes, and discuss how these new tools may be exploited to investigate issues of key importance in the interaction of schistosomes with the host immune system.

Induction of Th2 responses in infectious diseases.

Recent work on T-helper (Th) cell subset maturation has focused on defining the cellular source of early IL-4, which promotes precursor (naive) CD4+ Th cells to differentiate into Th type 2 (Th2) cells, and also on the roles of counter-regulatory cytokines, costimulatory signals, and antigen in the induction of Th2 responses. Results suggest that not all Th2 cells are equivalent in their ontogeny.

Profound effect of the absence of IL-4 on T cell responses during infection with Schistosoma mansoni.

T cell responses of interleukin (IL)-4(-/-) and wild-type (WT) mice infected with the helper T cell 2 (Th2) response-inducing pathogen Schistosoma mansoni were compared. As expected, given the important role of IL-4 in Th2 response induction, the absence of IL-4 resulted in diminished Th2 responses, apparent as reduced production of IL-4, -5, and -10 by CD4(+) cells isolated from the spleens of infected IL-4(-/-) mice. Surprisingly, these cells produced significantly less interferon (IFN)-gamma and proliferated less than did those from infected WT mice after T cell receptor ligation. CD8(+) cells isolated from infected IL-4(-/-) mice also produced less IFN-gamma than WT CD8 cells, although there was no difference in the proliferative responses of these cell populations. After infection, spleens of infected IL-4(-/-) mice did not enlarge to the same extent as those of WT mice, and attrition of the CD8(+) cell population within this lymphoid organ was noted. Taken together, the data indicate that in addition to inhibiting Th2 response development, the lack of IL-4 during schistosomiasis significantly affects additional aspects of T cell responses.

Conserved role for 14-3-3epsilon downstream of type I TGFbeta receptors.

Schistosoma mansoni receptor kinase-1 (SmRK1) is a divergent type I transforming growth factor beta (TGFbeta) receptor on the surface of adult parasites. Using the intracellular domain of SmRK1 as bait in a yeast two-hybrid screen we identified an interaction with S. mansoni 14-3-3epsilon. The interaction which is phosphorylation-dependent is not specific to schistosomes since 14-3-3epsilon also binds to TbetaRI, the human type I TGFbeta receptor. 14-3-3epsilon enhances TGFbeta-mediated signaling by TbetaRI and is the first TbetaRI-interacting non-Smad protein identified that positively regulates this receptor. The interaction of 14-3-3epsilon with schistosome and human TbetaRI suggests a conserved, but previously unappreciated, role for this protein in TGFbeta signaling pathways.

Surface-associated serine-threonine kinase in Schistosoma mansoni.

Existing evidence suggests that parasites of the genus Schistosoma are responsive to external stimuli derived from the host and from parasites of the opposite sex. We hypothesize that these interactions are mediated by receptors at the parasite surface. To begin to address this issue, we have employed surface labelling by biotinylation to identify and isolate the surface molecules of adult S. mansoni. Isolated surface molecules were subsequently analyzed for the presence of protein kinases, since protein kinase activity is frequently associated with signal-transducing receptors. Our results demonstrate that serine-threonine kinase activity is associated with the parasite surface and that surface proteins of 145, 125, 95 and 57 kDa became phosphorylated on serine and threonine residues under in vitro conditions. No significant tyrosine phosphorylation of surface molecules was detected, despite the presence of many tyrosine-phosphorylated proteins in tegumental extracts. An additional unexpected finding of these studies was that adult schistosomes express considerably more surface molecules than previously indicated by radioiodination studies, and that the majority of these molecules are of parasite rather than host origin.

Atypical post-translational modification and targeting of a Schistosoma mansoni surface receptor, a member of the transforming growth factor beta receptor family of cell surface receptors.

The surface membrane of the intravascular parasite Schistosoma mansoni is composed of not one but two closely apposed lipid bilayers which overlie a syncytial cellular layer, known as the tegument or neodermis. To gain insights into how membrane proteins are transported to and displayed on this unusual surface structure, we have investigated the post-translational modification and targeting of SmRK-1, a receptor and type I membrane protein expressed on the parasite surface, using heterologous expression systems. While SmRK-1 enters the secretory pathway in these systems, our data indicate that the SmRK-1 N-terminal signal peptide is either not cleaved by signal peptidase or is only eleven amino acids long or less. Retention of the signal peptide is accompanied by N-linked glycosylation of an asparagine residue within the predicted signal peptide. The SmRK-1 signal peptide is not capable of directing another cytoplasmic protein to the secretory pathway, suggesting that the signal for insertion of the SmRK-1 extracellular domain into the endoplasmic reticulum resides elsewhere in the protein. Further, SmRK-1 is inefficiently transported to the cell surface in mammalian cells, suggesting that the schistosome neodermis possesses specialized systems for receptor targeting and localization.

Functional conservation of Schistosoma mansoni Smads in TGF-beta signaling.

To begin to understand the molecular basis of communication between the parasite Schistosoma mansoni and its mammalian host, we are studying the signaling pathway downstream of S. mansoni receptor kinase-1 (SmRK1), a divergent type I transforming growth factor-beta (TGF-beta) receptor found on the tegumental surface of the parasite. In this study, we have used a homology based PCR approach to clone two S. mansoni Smad (SmSmad) genes; Smads play a pivotal role in the most well understood signaling pathways initiated by the TGF-beta family of ligands in other organisms. Comparison of the amino acid sequences with those of other Smads reveals that the conserved MH1 and MH2 domains of SmSmads show a high degree of identity to homologues in Drosophila. Transcripts for both SmSmads are detected in the same developmental stages as SmRK1, and both are capable of interacting with the intracellular domain of the receptor in vitro. Functional characterization using the human type I TGF-beta receptor further confirms the highly conserved nature of these proteins, as both SmSmads show TGF-beta dependent enhancement of luciferase activity and nuclear translocation in mammalian cells. These data are the first to show a TGF-beta-like receptor/Smad signaling pathway in parasitic helminths and by analogy with other systems, is likely important in regulating schistosome development.

Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice.

Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from cDNA lambda gt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni beta-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blots with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within beta-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain lambda gt11-fusion protein clones.

Kinetic correlation of the acquisition of resistance to immune attack in schistosomula of Schistosoma mansoni with a developmental change in membrane potential.

When Schistosoma mansoni cercariae penetrate the skin of the mammalian host they rapidly pass from fresh water to a high salt physiologic environment and transform into schistosomula. Following this transition, the parasites migrate from the skin to the lungs during which time they change from being highly susceptible to immune attack to being refractory, as measured by in vitro cytotoxicity assays. In this study, in vivo or in vitro schistosomula of different ages were examined for developmentally linked changes in membrane function which might correlate with the attainment of the resistant state. In particular, alterations in the distribution of tetraphenylphosphonium (TPP+), a synthetic lipophilic cation which shows a potential dependent partition across membranes, were followed. Three-hour-old schistosomula, which are greater than 75% susceptible to antibody-dependent complement-mediated attack or lymphokine-activated macrophage-mediated cytotoxicity, acquired TPP+ at a similar rate and steady state level to 5-day-old lung worms, which were completely resistant to both these effector mechanisms. The addition of ouabain, a Na+/K+-ATPase inhibitor, caused a 50% decrease in both the rate and steady state of TPP+ uptake by 3 h parasites but had little effect on these parameters in lung worms. Valinomycin, a K+-ionophore, completely inhibited TPP+ influx in both stages. The characteristics of TPP+ efflux from 3-h and 5-day-old parasites preloaded with the cation were found to be dissimilar. Whereas 30% of acquired TPP+ was lost from lung worms within 2 h, only 10% of acquired cation was released from 3-h schistosomula during the same period.(ABSTRACT TRUNCATED AT 250 WORDS)

Caveolae-like structures in the surface membrane of Schistosoma mansoni.

Specialized regions of cellular membranes termed detergent-insoluble glycosphingolipid-enriched membrane domains (DIG) have been identified in mammalian cells and shown to contain signalling molecules, cholesterol, sphingolipids and caveolae. Here we report that the unusual double surface membrane of the tegument of the trematode parasite Schistosoma mansoni possesses biochemically distinct domains analogous to DIG. When subjected to sucrose density gradient centrifugation, a detergent-extracted tegument from adult parasites yielded a low-density fraction consisting of detergent-insoluble complexes (DIC). Several tegument proteins were concentrated in DIC and a subset of these were labelled when adult schistosomes were biotinylated using a membrane-impermeant reactive biotin prior to extraction. The GPI-linked proteins alkaline phosphatase (SmAP), Sm200, the membrane-bound protein Sm23, and a protein recognized by an antibody against human caveolin, co-purified with DIC whereas soluble proteins, such as paramyosin and aldolase, were found at the bottom of the gradient. Antibodies against DIC immunoprecipitated a subset of worm surface proteins and immunolabeled the dorsal tegument of adult worms. Transmission electron microscopy of DIC revealed caveolae-like structures in the double bilayer surface structure. These results suggest that the tegument of adult S. mansoni possesses specialized membrane domains that are resistant to detergent-extraction, contain a subset of total tegument membrane proteins, and bear caveola-like invaginations, and thus are analogous to DIG.

Antigens from the surface and excretions/secretions of the filariform larva of Strongyloides stercoralis.

The surface and excretory/secretory (ES) antigens of the infective, filariform larva (L3) of Strongyloides stercoralis were identified. These studies provide a basis for the purification of these proteins as diagnostic allergens for human strongyloidiasis. The Mr values of the surface and ES molecules were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, fluorography, or silver staining following the recovery of these molecules after the radiolabelling of living parasites. At least 10 surface proteins were radioiodinated extrinsically using chloroglycoluril as the catalyst for iodination, and then extracted with detergents and/or beta-mercaptoethanol. Several surface molecules of the L3 were immunogenic in humans, as determined by immunoprecipitation with sera (IHS) from infected patients. About 30 proteins were present in the ES preparation. Many ES antigens were labelled biosynthetically during the culture of larvae in media supplemented with either [35S]methionine or [14C]glucose. Furthermore, several of the surface proteins of the L3 were found with the ES antigens recoverable by culturing larvae in vitro. About 10 of the ES proteins were immunogenic as determined by immunoaffinity chromatography using IHS; and two of these antigens with Mr 50,000 and 90,000 incorporated [35S]methionine during culture of larvae. Moreover, some ES proteins were allergenic when tested in an in vitro assay of histamine release from basophils from infected humans or monkeys. The isotype of the homocytophilic antibodies involved in this immediate hypersensitivity assay, which is the basis of a diagnostic skin test for human strongyloidiasis, appears to be IgE.

The human immune response to defined immunogens of Schistosoma mansoni: elevated antibody levels to paramyosin in stool-negative individuals from two endemic areas in Brazil.

Sera from individuals living in 2 areas endemic for Schistosoma mansoni in Minas Gerais, Brazil were assayed for the presence of antibodies against paramyosin and glutathione-S-transferase (GST), molecules previously implicated as vaccine immunogens from studies in laboratory hosts. A group was identified consisting of subjects who were stool-negative and had no record of previous infection but who were seropositive by enzyme-linked immunosorbent assay against crude adult worm antigen (SWAP). These individuals had anti-paramyosin antibody levels which were dramatically elevated with respect to those measured in infected (stool-positive) individuals living in the same endemic area. In contrast, the same 2 groups of stool-positive and stool-negative subjects could not be distinguished on the basis of their seroreactivity to either GST or SWAP. After chemotherapy, anti-paramyosin antibodies rose above pre-treatment levels and remained elevated in those individuals who became stool-negative. In contrast, anti-paramyosin antibodies decreased to pretreatment values in drug-treated individuals who failed to show complete parasitological cure. These results suggest that the immune response of humans to paramyosin may play a role in natural resistance to schistosome infection, and that an elevated antibody level against this antigen may be a useful correlate of drug-induced cure.

The use of human faeces for fertilizer is associated with increased intensity of hookworm infection in Vietnamese women.

To investigate different factors associated with hookworm infections we conducted 2 studies in a commune in northern Viet Nam. The first was part of a larger study on anaemia and covered 213 women (15-49 years of age) and their 92 children (6 months to 5 years of age) in one commune; 90% of the families reported using human faeces for fertilizer. Women who reported using fresh human faeces as fertilizer had significantly higher hookworm egg counts than women who either used treated human faeces or who did not use human faeces as fertilizer. The second study examined how human faeces were used for fertilizer in 30 selected families. Women participated in preparation and application of human faeces to crops in 81% of the families using human faeces for fertilizer. Two methods of preparing the faeces were described: 48% of the families mixed the faeces with ash before applying them to the field; 18% mixed the faeces with water; 33% used both methods.

Interferon-gamma production by peripheral blood mononuclear cells from residents of an area endemic for Schistosoma mansoni.

During human schistosomiasis host responses to antigens of various parasite life-cycle stages may contribute to whether the severe, hepatosplenic state develops or the patient remains relatively asymptomatic throughout infection, and may play a role in resistance. This study evaluated production of interferon gamma (IFN-gamma) in vitro by schistosome antigen-stimulated peripheral blood mononuclear cells (PBMCs) from asymptomatic patients, and by PBMCs from apparently uninfected, untreated persons living in areas endemic for Schistosoma mansoni ('endemic normals'). IFN-gamma production parallels PBMC proliferation in that schistosomal egg antigens stimulate patent patients' cells poorly, but strongly stimulate PBMCs from 'endemic normals'. This is proportionally true for antigens from adult worms and cercariae. Although asymptomatic patent patients' cells produced little or no IFN-gamma in response to the 3 schistosomal antigenic extracts, their PBMCs, and PBMCs from 'endemic normals', produced expected amounts of IFN-gamma when exposed to phytohaemagglutinin. This implies that persons with patent infections have schistosome antigen-specific defects in their ability to respond to IFN-gamma production that are not exhibited by putatively resistant 'endemic normals'.

Detecting a history of childhood sexual experiences among women substance abusers.

This article examines a means of detecting a history of childhood sexual experience (CSE) to improve substance abuse treatment outcomes. The symptom profile of women with a history of CSE is discussed in relation to the difficulties it presents in detection of a history of CSE, especially in women who also have a past or current history of substance abuse. A symptom checklist is described, and its validity and reliability are reported as satisfactory. The use of the checklist in client assessment and treatment planning is discussed.