Comparison of Schistosoma mansoni migration patterns in normal and irradiated cercaria-immunized mice by means of autoradiographic analysis. Evidence that worm elimination occurs after the skin phase in immunized mice

Migration and elimination of radiolabeled Schistosoma mansoni were compared in naive and irradiated cercaria-immunized mice by autoradiography of compressed host tissues. The results indicated that 1) most of the normal elimination of schistosomula in unimmunized mice and the additional elimination in immunized mice occur at some point(s) after arrival of schistosomula in the lungs and before their development into adult worms, 2) migration of schistosomula from skin to lungs is delayed for several days but not reduced in immunized mice, 3) migration of schistosomula from lungs to liver is delayed for several days in immunized mice, and 4) schistosomula reach the liver in reduced numbers or are killed and cleared in the liver in greater numbers in immunized mice. The lung chop procedure was shown to recover schistosomula from control and irradiated cercaria-immunized mice with equal efficiency. Autoradiography of all tissues of the body demonstrated that, in both control and immunized mice, at least 20-25% of the schistosomula detectable 2 and 3 weeks after infection were present in tissues other than the skin, lungs and liver.     

Autoradiographic analysis of resistance to reinfection with Schistosoma mansoni in mice. Evidence that the liver is a major site of worm elimination

Migration and elimination of 75Se-labeled Schistosoma mansoni were studied in previously infected mice by means of autoradiography and worm recovery. As in control mice, there appeared to be little if any worm elimination in the skin of previously infected mice. In previously infected mice, there appeared to be some worm elimination in the lungs and in migration sites between the lungs and liver, though much less than would have been expected from previous studies. In two of three experiments most of the challenge worm elimination in previously infected mice, above the normal attrition level occurring in the controls, appeared to take place in the liver. In the third experiment, it appeared that all of the challenge worm elimination occurred after migration to the lungs, and at least one-third of it in the liver. It was also observed that the lung chop procedure recovered viable schistosomula much less efficiently from previously infected mice than from controls indicating that the reduction in lung schistosomulum recovery overestimates the amount of lung and prelung killing that occurs in reinfected mice.     

Schistosoma mansoni: age-dependent susceptibility to immune elimination of schistosomula artificially introduced into preinfected mice

Mice chronically infected with Schistosoma mansoni exhibited a significant resistance to a second infection with the same parasite, as demonstrated by their challenge worm burdens measured by portal perfusion. A decreased worm recovery was also exhibited by chronically infected mice when the challenge was administered intravenously using 3-h schistosomula obtained by the isolated skin technique or using 5-, 6-, 7- and 9-day-old schistosomula obtained from the lungs ofinfected donor mice. Variable results were obtained with 10- and II-day-old forms, while schistosomula which were 12days old or older, did not undergo significant rejection when introduced into the mesenteric veins of preinfected mice. Attempts to analyse these phenomena using the‘lung assay’were made complicated by the observation that the day of maximum recovery from the lungs was dependent upon the age of injected worms.     

Autoradiographic analysis of Schistosoma mansoni migration from skin to lungs in naive mice. Evidence that most attrition occurs after the skin phase

A study was performed to determine the extent of attrition of Schistosoma mansoni in naive mice (innate resistance) during the 1st week of infection. Each mouse was exposed to exactly 50 cercariae radiolabeled with [75Se] selenomethionine. On 1, 4, and 7 days postexposure, skin, lungs and liver were analyzed by compressed organ autoradiography for the presence of labeled larvae. Using this technique it was determined that no more than one-third of the 59% attrition that occurred between the cercarial and adult worm stages could be attributed to losses during the skin phase; most of the attrition in naive mice occurred after the migration of larvae to the lungs.     

Site requirements and kinetics of immune-dependent elimination of intravascularly administered lung stage schistosomula in mice immunized with highly irradiated cercariae of Schistosoma mansoni

Experiments were performed to compare the migration and survival of 75Se-labeled schistosomes, introduced by percutaneous cercarial exposure or by intravascular administration of 7-day-old lung stage schistosomula, in control and irradiated cercaria-immunized mice. Schistosomula were intravascularly introduced into the lungs, systemic organs and liver by injection via the femoral vein (FV), left ventricle (LV), and superior mesenteric vein (SMV), respectively. The fate of challenge larvae was examined by autoradiography of host tissues and by recovery of adult worms. It was found that both normal and immune elimination were site-dependent. In control mice 45%-60% of cercarial penetrants and lung schistosomula injected into the FV and LV were recoverable as adult worms, while a significantly greater number (70%-85%) were recoverable when lung schistosomula were injected into the SMV. In immunized mice, parasites introduced as either cercariae or FV-injected schistosomula were both highly sensitive to immune elimination. LV-injected schistosomula were also sensitive but to a slightly lesser degree. In contrast, schistosomula placed directly in the liver by SMV injection were totally insensitive to immune elimination. It was concluded that elimination of schistosomula in irradiated cercaria-immunized mice occurs in the lungs and/or in the systemic organs, but not in the liver. Also, it was concluded that immune elimination is not a rapid process, since more than 7 days were required after intravascular challenge for the development of demonstrable differences between control and immunized mice.     

Schistosoma mansoni: host cell adhesion to the different stages of the parasite, in vivo.

The peritoneal cavity of laboratory mice was used to study the phenomenon of host cell adhesion to different evolutive stages of the Schistosoma mansoni (cercaria, adult worm, developing and mature eggs, miracidium, young and mature daughter sporocysts). Material recovered from the peritoneal cavity 30 and 180 min after the inoculation of each evolutive form was examined with the help of a stereomicroscope. The free swimming larvae (cercaria and miracidium), and the evolutive forms producing such larvae (mature egg and mature daughter sporocyst) elicited the host cell adhesion phenomenon. In all forms but cercariae the adherent cells remained as so till 180 minutes after inoculation.     

Evidence that spontaneous mitotic recombination occurs at the two-strand stage.

Spontaneous reciprocal mitotic recombination in the yeast Saccharomyces cerevisiae, associated with heteroallelic recombination, occurs almost exclusively at the two-strand stage and involves recombination of unduplicated chromosomes (i.e., during G1) or the unduplicated regions of chromosomes during the S phase of mitosis. The associated heteroallelic recombination frequently reflects the formation of symmetric Holliday structures, is not strongly polarized with respect to conversion at the heteroallelic trp5 sites studied, occasionally results in simultaneous conversion of widely separated genetic markers, and is positively correlated with recombination of flanking markers.     

Isolation of cDNA clones for differentially expressed genes of the human parasite Schistosoma mansoni.

Little is known about the mechanisms that control transformations during the life cycle of Schistosoma mansoni. To enable isolation of DNA sequences encoding developmentally regulated antigens a cDNA expression library in the vector lambda gt11 amp3 was constructed from adult mRNA and immunologically screened with sera from infected individuals. We report here on the properties of three recombinant clones that derive from developmentally regulated genes. Clone 10-3 encoded a beta-galactosidase fusion protein present in high abundance in infected Escherichia coli. Clones 7-2 and 8-2 also produced immunologically recognized proteins; however, the peptides did not appear to be beta-galactosidase fusion proteins. The expression of mRNAs hybridizing to these cDNAs was examined in the different stages of the parasite life cycle. Messenger RNA corresponding to clone 10-3, approximately equal to 1000 bases in length, was present in higher abundance in male worms than in females but was not detected in schistosome eggs. A 900-base mRNA hybridizing to clone 7-2 was observed in adult worms and eggs. Both clone 10-3 and clone 7-2 hybridized to smaller mRNAs in cercariae and freshly transformed schistosomula than in adult worms. Clone 8-2 contained tandem cDNA inserts. One cDNA hybridized to a 1700-base mRNA present in all stages, while the second hybridized to an 800-base mRNA specific to adult female worms.     

The use of aldehydes to show a relationship between host and parasite antigens at the surface of adult male Schistosoma mansoni

An indirect radiolabelled antibody method has been developed to measure the effects of aldehydes on the amount of antigen detectable at the surface of adult male Schistosoma mansoni. Incubation of schistosomes in formaldehyde (0.01-10% wt/vol.) or glutaraldehyde (0.01-0.1% wt/vol.) was found to result in increased exposure of parasite antigens with a concomitant decrease in the amount of surface located host RBC antigens. Those concentrations of formaldehyde or glutaraldehyde which were most effective in removing or displacing host antigens were also most able to expose parasite antigens. These findings are consistent with the hypothesis that host antigens mask parasite antigens at the surface of the adult schistosome.     

Simple school questionnaires can map both Schistosoma mansoni and Schistosoma haematobium in the Democratic Republic of Congo

The use of self-administered questionnaires has been shown in different African countries to be inexpensive and reliable for the rapid identification of communities at highest risk of urinary schistosomiasis. For intestinal schistosomiasis due to Schistosoma mansoni there is a clear need for a similar approach. We report the results from a large-scale study undertaken in the western part of the Democratic Republic of Congo (DRC, formerly Zaire). Within 4 weeks questionnaires were correctly completed in 136 out of 160 schools (85%). In 57 of these schools children were screened for infections with schistosomes and geohelminths. The prevalence of 'schistosomiasis' as reported in the questionnaires showed the best correlation with the prevalence of S. mansoni infections (r = 0.77, P < 0.0001). Calculations of the diagnostic performance of reported 'schistosomiasis' to detect schools with a high risk of intestinal schistosomiasis gave positive predictive values of 87 and 62%, and negative predictive values of 74 and 87% for moderate and high infection thresholds, respectively. Reported 'blood in stool' was another useful indicator for intestinal schistosomiasis. Reported 'blood in urine' showed the best correlation with urinary schistosomiasis (r = 0.75, P < 0.001) and the positive predictive values were 81 and 50%, and the negative predictive values were 89 and 95% for moderate and high infection thresholds, respectively. We conclude that school children in DRC have a distinct perception of intestinal and urinary schistosomiasis and that questionnaires could be useful to identify high-risk schools for both parasites.     

Immunosuppression in the definitive and intermediate hosts of the human parasite Schistosoma mansoni by release of immunoactive neuropeptides.

Evidence supporting the concept that the parasitic trematode Schistosoma mansoni may escape immune reactions from its vertebrate (man) or invertebrate (the freshwater snail Biomphalaria glabrata) hosts by using signal molecules it has in common with these hosts was obtained by the following experiments. The presence of immunoactive proopiomelanocortin (POMC)-derived peptides [corticotropin (ACTH), beta-endorphin] in, and their release from, S. mansoni was demonstrated. Coincubation of adult worms with human polymorphonuclear leukocytes or B. glabrata immunocytes led to the appearance of alpha-melanotropin (MSH) in the medium. The conclusion that this alpha-MSH resulted from conversion of the parasite ACTH by neutral endopeptidase 24.11 (NEP) present on these cells was supported by the fact that the alpha-MSH level in the medium was markedly reduced by addition of the specific NEP inhibitor phosphoramidon. This interpretation is substantiated by the fact that no conversion was observed in comparable tests with human monocytes, which exhibit no NEP activity. alpha-MSH has the capacity to inactivate formerly active immunocytes not only from the definitive host (man, hamster) but also from the intermediate host (B. glabrata), as determined by microscopic computer-assisted examination of conformational changes. POMC-derived peptides have been detected in B. glabrata hemolymph 2, 10, and 24 days after infection by S. mansoni miracidia. Immunocytes from infected snails were found to be inactivated, and this inactivation was prevented by antibodies directed against ACTH and alpha-MSH. The immunoactive beta-endorphin released from S. mansoni does not appear to be subject to enzymatic conversion. Since it is active at lower concentrations, it may be used for distant signaling.     

A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni.

We present evidence that a subset of mRNAs in the human parasitic trematode Schistosoma mansoni contain an identical 36-nucleotide spliced leader (SL) sequence at their 5' termini. The SL is derived from a 90-nucleotide nonpolyadenylylated RNA (SL RNA), presumably by trans-splicing. Neither the SL nor the SL RNA share significant sequence identity with previously described trans-spliced leaders and SL RNAs in trypanosomatid protozoans or nematodes. However, several features, such as predicted secondary structure, trimethylguanosine cap, and potential Sm binding site, suggest similarities among SL RNAs in widely divergent organisms. Our evidence also indicates that the exon 3 acceptor site of the 3-hydroxy-3-methylglutaryl-CoA reductase gene can be spliced either to the SL by trans-splicing or to an upstream exon, 2, by cis-splicing. The presence of a SL sequence in S. mansoni, a member of the phylum Platyhelminthes, suggests that transplicing may be a common feature of other lower invertebrates.     

Evidence that the reduced surface antigenicity of developing Schistosoma mansoni schistosomula is due to antigen shedding rather than host molecule acquisition

Antibody and lectin binding characteristics of Schistosoma mansoni schistosomula maturing in vivo and in vitro were quantitatively assessed and compared in order to investigate the basis of the reduced surface antigenicity of host derived larval schistosomes. Quantitative indirect immunofluorescence assays showed that schistosomula recovered from mice at 24 h and 5-10 days post infection bound low or insignificant amounts of a variety of anti-schistosome antibodies including those from chronically infected and radiation attenuated cercariae-vaccinated mice, a vaccinated rabbit and rabbits hyper-immunized with non-living larval and adult schistosome antigen preparations. In contrast, parasites maturing in vitro continued to bind highly significant levels of each of these antibody preparations until at least 10 days post transformation. To investigate the basis of the decreased surface antigenicity of parasites maturing in vivo, 6-day-cultured parasites were injected intravenously into mice and recovered from the lungs at various times thereafter and examined for their ability to bind both anti-parasite and anti-host antibodies. After 30 min in vivo, cultured schistosomula exhibited a significantly decreased capacity to bind anti-parasite antibodies and concanavalin A (Con A), and by 16 h had lost their binding sites for fucose binding protein (FBP) as well. That this reduction in antigenicity was due to shedding of surface antigens was suggested by the observation that the reduced ability of these parasites to bind anti-parasite antibodies coincided closely with the loss of 125I-labelled surface proteins. Furthermore unlike 6 day schistosomula which had developed wholly in vivo, 6-day-cultured parasites recovered after 30 min in vivo failed to bind anti-host antibodies suggesting that in these organisms parasite antigens were not masked by host molecules. These data argue that surface antigen shedding may explain the reduced surface antigenicity of schistosomula developing in vivo. While this surface modulation apparently occurs independently of host antigen uptake, it is dependent upon an as yet unidentified host factor.     

Evidence that cellular immune responses to soluble and membrane associated antigens are independently regulated during human schistosomiasis mansoni

We have made a comparative analysis of human cellular and antibody responses to membrane associated adult worm antigens (Mb-A), soluble adult worm antigens (SWAP) and soluble egg antigens (SEA) derived from Schistosoma mansoni . Chronically infected patients with the intestinal (I) and hepatosplenic (HS) forms of the disease as well as non-infected putative immune 'endemic normals' (EN), were studied. We observed that the cellular responses, of individuals, to the two adult worm preparations, SWAP and Mb-A, may be distinct and can be related to the occurrence of resistance or pathology. The resistant group (EN) presented higher levels of both cellular proliferation, and IFN-γ production, in response to Mb-A as compared with SWAP whereas HS individuals presented higher levels of cellular proliferation to SWAP as compared with Mb-A. Individuals with intestinal disease had similar levels of proliferation to both antigens. The response to SEA by all groups was generally similar, and not predictive of any clinical form. The specific antibody response to the three antigens were in general higher among infected patients than in resistant EN individuals. These results support the hypothesis that the response to adult worm antigens may be pivotal in determining both the development of resistance and severity of disease.     

Synergistic action of praziquantel and host specific immune response against Schistosoma mansoni at different phases of infection

The interaction between specific immune response to Schistosoma mansoni and praziquantel (PZQ) was studied in mice. In mice harboring concomitant immunity, 6-day-old parasites treated with PZQ were more effectively removed than 24 h treated parasites despite both had a significant worm burden reduction when compared with respective treated controls. These results show that PZQ can be effective at the skin and lung stages of parasite's development mainly acting with a established specific immune response, and particularly at the lung phase.     

Molecular cloning and sequence analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from the human parasite Schistosoma mansoni.

cDNA clones encoding the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [(S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] from the human parasite Schistosoma mansoni have been isolated and characterized. The composite 3459 base pairs of cDNA sequence contains a 2844-base-pair open reading frame corresponding to a protein of 948 amino acids. The predicted S. mansoni HMG-CoA reductase protein contains a hydrophobic amino terminus consisting of seven potential transmembrane domains that are structurally conservative but are not identical in amino acid sequence with HMG-CoA reductases from other species. The hydrophilic carboxyl terminus of the S. mansoni HMG-CoA reductase protein, however, shares 48-52% sequence identity with the carboxyl termini of other HMG-CoA reductases in a region that contains the catalytic domain. When expressed as a fusion protein in Escherichia coli, the carboxyl-terminal domain of the schistosome protein exhibits HMG-CoA reductase enzyme activity.     

Evidence against the existence of specific Schistosoma mansoni subpopulations which are resistant to irradiated vaccine-induced immunity

When mice are immunized with irradiated Schistosoma mansoni cercariae a proportion of the subsequent cercarial challenge always escapes killing and matures to egg-laying adults. This report investigates the possibility that incomplete immunity in this system is governed by a genetically-determined insusceptibility of a particular schistosome subpopulation. To do this we tested whether more immunoresistant schistosomes would develop following successive passages of progeny of the resistant worms through immunized mice. Mice were immunized with 500 50 Krad-irradiated cercariae, and challenged with normal cercariae when immunity was at its peak. After five successive passages through snails and immune mice, progeny of those parasites which escaped immune killing were no more refractory to vaccine-induced resistance than the original stock maintained in nonimmune mice. Additionally, the "passaged" isolates did not differ from the original stock in their ability to induce protection following irradiation. Our results indicate that with this model of acquired resistance incomplete immunity is unlikely to be due to a subpopulation of the parasites possessing a genetically-determined insusceptibility to killing.     

Endothelial cells are activated by cytokine treatment to kill an intravascular parasite, Schistosoma mansoni, through the production of nitric oxide.

Like many pathogens that undergo an intravascular stage of development, larvae of the helminth parasite Schistosoma mansoni migrate through the blood vessels, where they are in close contact with endothelial cells. In vitro exposure of murine endothelial cells to various cytokines (interferon gamma, tumor necrosis factor alpha, and interleukin 1 alpha or 1 beta) resulted in their activation to kill schistosomula through an arginine-dependent mechanism involving production of nitric oxide (NO). Cytokine-treated endothelial cells showed increased expression of mRNA for the inducible form of the NO synthase, and both NO production and larval killing were suppressed by treatment with competitive inhibitors. The effector function of cytokine-treated endothelial cells was similar to that of activated inflammatory tissue macrophages, although activation appeared to be differentially regulated in these two cell types. Activated endothelial cells killed older (18-day) forms of the parasite, such as those currently thought to be a primary target of immune elimination in the lungs of mice previously vaccinated with radiation-attenuated cercariae, as well as newly transformed larvae. In C57BL/6 mice, which become resistant to S. mansoni infection as a result of vaccination with irradiated cercariae, endothelial cell morphology characteristic of activation was observed in the lung by 1-2 weeks after challenge infection. Similar endothelial cell changes were absent in P-strain mice, which do not become resistant as a result of vaccination. Together, these observations indicate that endothelial cells, not traditionally considered to be part of the immune system, may play an important role in immunity to S. mansoni and, by means of NO-dependent killing, could serve as effectors of resistance to other intravascular pathogens.