Schistosoma mansoni Infection in Mice Depleted of Thymus-Dependent Lymphocytes: I. Eosinophilia and Immunologic Responses to a Schistosomal Egg Preparation

Chronic murine schistosomiasis is characterized by a host cell-mediated immune response to schistosomal egg antigens. This response is manifested in vitro by lymphocyte blastogenic reactivity to a soluble schistosomal egg antigen preparation (SEA) and in vivo by delayed dermal hypersensitivity to SEA. In addition, mice develop reaginic, agglutinating, and early dermal reactive antibodies to SEA and a characteristic pattern of peripheral blood eosinophilia. In this study, depletion of thymus-dependent lymphocytes abolished the anti-SEA lymphocyte blastogenic response and delayed dermal reactivity, reaginic antibody response and the major peak of peripheral blood eosinophilia, which in normal mice occurs at the same time as SEA-induced blastogenesis. Antibodies mediating the early dermal reaction to SEA and agglutinins for SEA-coated particles were not inhibited by T-lymphocyte depletion. The experimental group did not develop the normally observed granulomatous response, but rather suffered focal hepatic and mucosal liquefactive necrosis, bacteremia and accelerated mortality.     

Allergens of Schistosoma mansoni. II. Fractionation and characterization of S. mansoni egg allergens

The interaction of Schistosoma mansoni crude soluble egg antigen (SEA) with IgE antibodies in sera from S. mansoni-infected mice, rats and humans has been studied by the radioallergosorbent test (RAST) and the Prausnitz-Küstner (PK) technique. IgE antibodies recognizing egg antigens were present as early as day 21 after the infection in the mouse sera and day 28 in rat sera. IgE in sera of infected humans reacted with antigenic components in the Mr range 70,000-150,000 and focusing as a broad peak in the pH range 4.5-6.5 as measured by RAST. SDS-PAGE followed by western blotting showed the presence of major components at molecular weights of 117,000 and 35,000-43,000. In the PK test, using mouse sera, components focusing in the alkaline pH range also gave a positive reaction. Most of the allergenic activity was bound by concanavalin A-Sepharose and by wheat germ agglutinin-Ultrogel. IgE in serum from an infected non-permissive host (the Fischer rat) apparently recognized egg-stage-specific allergen as indicated by differences in the time course of the IgE response to egg allergens compared to the adult material. When analyzed by SDS-PAGE and western blotting with day 45-infected rat serum, SEA showed some qualitative and quantitative differences to adult worm antigen. Molecules at molecular weights between 25,000 and 30,000 and at about 43,000 in SEA reacted with rat serum IgE and were absent from adult worm antigen. The allergenic similarities between egg and adult worm are discussed.     

Therapeutic Administration of the Direct Thrombin Inhibitor Argatroban Reduces Hepatic Inflammation in Mice with Established Fatty Liver Disease

Thrombin generation is increased in patients with nonalcoholic fatty liver disease (NAFLD) and in mouse models of diet-induced obesity. Deficiency in the thrombin receptor protease activated receptor-1 reduces hepatic inflammation and steatosis in mice fed a Western diet. However, it is currently unclear whether thrombin inhibitors can modify the pathogenesis of established NAFLD. We tested the hypothesis that thrombin inhibition could reverse hepatic steatosis and inflammation in mice with established diet-induced NAFLD. Low-density lipoprotein receptor–deficient LDLr^−/− mice were fed a control diet or a Western diet for 19 weeks. Mice were given the direct thrombin inhibitor argatroban ∼15 mg/kg/day or its vehicle via a miniosmotic pump for the final 4 weeks of the study. Argatroban administration significantly reduced hepatic proinflammatory cytokine expression and reduced macrophage and neutrophil accumulation in livers of mice fed a Western diet. Argatroban did not significantly impact hepatic steatosis, as indicated by histopathology, Oil Red O staining, and hepatic triglyceride levels. Argatroban reduced serum triglyceride and cholesterol levels in mice fed a Western diet. Argatroban reduced both α-smooth muscle actin expression and Type 1 collagen mRNA levels in livers of mice fed a Western diet, indicating reduced activation of hepatic stellate cells. This study indicates that therapeutic intervention with a thrombin inhibitor attenuates hepatic inflammation and several profibrogenic changes in mice fed a Western diet.More than 70% of patients with abdominal obesity develop concurrent nonalcoholic fatty liver disease (NAFLD).¹ NAFLD, the hepatic manifestation of metabolic syndrome, is characterized by excess accumulation of lipids in the liver (ie, hepatic steatosis)^2,3 and affects approximately 25% of the Western population.⁴ Steatosis accompanied by marked histological inflammation is termed nonalcoholic steatohepatitis (NASH), which is the most severe form of NAFLD and a major cause of liver fibrosis and cirrhosis.^5,6 Progression from simple steatosis to NASH is indicative of a poor clinical outcome and currently has no effective pharmacological treatment options. In addition, both obesity and NAFLD are associated with an increased risk of developing type 2 diabetes mellitus⁷ and cardiovascular disease.^8,9 Therefore, there is an immediate need to identify novel pharmacological approaches to treat NAFLD.A significant commonality among obesity-related diseases is inflammation. Obesity and hepatic steatosis are associated with increased expression of many inflammatory mediators in the liver.¹⁰ The expression of several of these mediators, particularly those involved in leukocyte recruitment, is further increased in patients with NASH.¹⁰ Several compelling studies have demonstrated that inflammatory chemokines such as monocyte chemoattractant protein-1 (MCP-1) and the subsequent recruitment and activation of hepatic macrophages (ie, Kupffer cells) are essential components of NAFLD pathogenesis.^11–14 A systemic proinflammatory state, driven in part by hepatic inflammation, is associated with an increased risk of type 2 diabetes^15,16 and adverse cardiovascular outcomes.¹⁷ In particular, systemic levels of high sensitivity C-reactive protein (hs-CRP), a biomarker of risk for acute cardiovascular events,¹⁸ are primarily dictated by the proinflammatory environment in the liver. Indeed, hs-CRP levels are independently associated with hepatic steatosis in patients with metabolic syndrome.⁸ These studies indicate that increased hepatic inflammation is a focal point of multiple diseases stemming from the metabolic syndrome. Of importance, the molecular triggers of hepatic inflammation in metabolic diseases such as obesity are not completely understood. To this end, understanding the cellular and molecular pathways coordinating hepatic inflammation in metabolic disease could lead to the development of clinical therapies that target inflammation as an underlying cause of multiple interrelated diseases.Because the liver is the primary site of coagulation factor synthesis, liver diseases are often accompanied by a rebalancing of the hemostatic profile.¹⁹ Indeed, abdominal obesity, metabolic syndrome, and NAFLD are each associated with activation of the blood coagulation cascade, including increased generation of the serine protease thrombin.^20–23 Moreover, thrombin generation is increased in mouse models of diet-induced obesity and hypercholesterolemia.^24,25 Previous studies have shown that the induction of tissue factor on monocytes is essential for thrombin generation in mice fed a Western diet.²⁶ Various hepatic manifestations of diet-induced obesity, including hepatic steatosis, are reduced in tissue factor–deficient mice.²⁴ Moreover, we found previously that mice lacking a thrombin receptor, protease activated receptor-1 (PAR-1), did not develop hepatic steatosis when fed a Western diet.²⁴ Although compelling, these genetic approaches do not directly address the question of whether intervention with pharmacological agents, perhaps anticoagulants, can reduce established liver disease. Indeed, it is currently unclear whether pharmacological inhibition of thrombin alters the course of established diet-induced fatty liver disease in mice.To this end, we tested the hypothesis that pharmacological inhibition of thrombin could therapeutically reverse diet-induced hepatic inflammation and steatosis in hypercholesterolemic low density lipoprotein receptor–deficient (LDLr^−/−) mice.     

Schistosoma mansoni: autoantibodies and polyclonal B cell activation in infected mice.

The appearance of autoantibodies was investigated during the course of Schistosoma mansoni infection in C57Bl/6 mice. Anti-liver autoantibodies or lymphocyte-reactive alloantibodies were detected respectively without cell-mediated immunity against liver antigen or lymphocytotoxic activity. Anti-liver, anti-DNA, anti-Ig and anti-lymphocyte antibodies were shown 6-7 weeks after the beginning of the infection concomitantly with the increase of immunoglobulin levels and circulating immune complexes. At this period, the antibody response to polyvinylpyrrolidone (PVP) was increased and the injection of spleen cells from day-45-infected mice to uninfected recipients increased the anti-PVP antibody response. Conversely, the injection of spleen cells from uninfected to infected mice did not modify the anti-PVP Ab response. After 6 weeks of infection, the basal thymidine incorporation of spleen cells was increased contrasting with the marked inhibition of spleen cell response to PHA. The present data are consistent with the induction of a polyclonal non-specific B cell activation by S. mansoni.     

Schistosoma mansoni Infection in Mice Depleted of Thymus-Dependent Lymphocytes: II. Pathology and Altered Pathogenesis

Murine schistosomiasis mansoni is a more rapidly fatal disease in hosts deprived of their cell-mediated immune capabilities. Analysis of the histopathology of the disease under these circumstances indicates that rather than the hepatic granulomata characteristic of the normal infection, the host develops zones of liquefactive necrosis in the liver and intestinal mucosa. These lesions are associated with severe parenchymal cell destruction. Such hepatic and mucosal damage, with subsequent toxemia and septicemia, is presumed central to the altered course of the disease.     

Cathepsin B-like activity predominates over cathepsin L-like activity in adult Schistosoma mansoni and S. japonicum

Both cathepsin B-like and cathepsin L-like endopeptidase activities have been described in schistosomes, but their relative contribution to proteinolysis remains controversial. In an attempt to clarify which type of activity predominates, the selective mammalian cathepsin B inhibitor CA-074 was tested under standardized assay conditions with different preparations from Schistosoma mansoni and S. japonicum. CA-074 (0.94 μM) inhibited at least 92% and 80% of proteinolytic activity, respectively, for these species: completely inhibited bovine-spleen cathepsin B activity; but showed only marginal inhibition (4%) of rat-liver cathepsin L activity. We discuss the results with respect to previous studies and conclude that schistosome cathepsin B-like, not L-like, activity predominates. Received: 13 November 1996 / Accepted: 13 January 1997     

A comparison of the susceptibility to praziquantel of Schistosoma haematobium,S. japonicum,S. mansoni,S. intercalatum and S. mattheei in hamsters

Summary Groups of six- to eight-week-old hamsters each experimentally infected with Sudanese and South African strains of S. haematobium, Puerto Rican and Liberian strains of S. mansoni, S. japonicum from mainland China, a Congo strain of S. intercalatum and a Nelspruit South African strain of S. mattheei, were treated with different dosage regimens of a new schistosomicide — praziquantel.The drug is more effective against S. haematobium when administered by the intramuscular route than per os, a complete parasitological cure being obtained following a single intramuscular injection of 200 mg/kg bwt. Per os the best results were obtained using the three highest regimens of 5×100 mg/kg, 5×50 mg/kg and 3×100 mg/kg bwt. It was observed that S. haematobium female worms are more susceptible to the compound than male worms.The results show that S. japonicum in the hamster is very susceptible to the compound, a dosage of 100 mg/kg administered orally for three days resulting in a complete parasitological cure. More than a 90% reduction in adult worms was obtained at all the dosage regimens used except 1×50 mg/kg (73.2%). Female worms were again found to be more susceptible to the drug than male worms. Hatching tests performed on ova in liver tissue of control and treated animals were positive up to four weeks after treatment, thus showing that praziquantel has no ovicidal properties.Immature S. japonicum worms were found to be markedly less susceptible to treatment than the mature schistosomes (5×100 mg/kg reduced mature adults by 99.7%, but immature forms were reduced by only 54.2%).A 100% cure rate was obtained in the hamster infected with the Liberian strain of S. mansoni following treatment with praziquantel at 3×100 mg/kg given on consecutive days, and at 3×50 mg/kg administered in one day. Treatment of the hamsters infected with the Puerto Rican strain of S. mansoni also resulted in substantial reductions of adult worms. Praziquantel was also found to be highly effective against S. intercalatum and S. mattheei.It is noted that the efficacy of the compound against S. haematobium, S. japonicum and S. mattheei in the hamster is significantly greater than that of metrifonate (Bilarcil). Following treatment with praziquantel most S. haematobium and S. japonicum worms undergo a classic hepatic shift and are trapped and killed in the liver tissue.It is considered that the present study shows that this new compound exhibits a high degree of activity against the three major schistosome species S. haematobium, S. japonicum and S. mansoni, against S. intercalatum, and against the cattle schistosome S. mattheei in the hamster, with no apparent significant differences in efficacy against the different geographical strains of the parasites used in the trials.

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Zusammenfassung Gruppen von 6–8 Wochen alten Goldhamstern wurden jeweils mit einem sudanesischen und südafrikanischen Stamm von S. haematobium, mit einem puertoricanischen und liberianischen Stamm von S. mansoni, mit S. japonicum aus Kontinentalchina, S. intercalatum vom Kongo und mit S. mattheei aus Nelspruit, Südafrika, infiziert und mit verschiedenen Dosierungen des neuen Schistosomenmittels Praziquantel behandelt.Praziquantel ist gegen S. haematobium bei intramuskulärer Gabe wirksamer als bei oraler Gabe. Eine parasitologische Heilung konnte durch eine einmalige intramuskuläre Injektion von 200 mg/kg erreicht werden. Bei oraler Gabe ergaben die drei höchsten Dosen 5×100, 5×50 und 3×100 mg/kg die besten Ergebnisse. Es wurde beobachtet, daß die Weibchen von S. haematobium empfindlicher gegen Praziquantel sind als die Männchen.Die Ergebnisse zeigen, daß S. japonicum im Goldhamster sehr empfindlich gegen Praziquantel ist. Eine parasitologische Heilung wird durch 3×100 mg/kg erreicht. Mit allen verabreichten Dosen wurde die Zahl der adulten Parasiten um mehr als 90% reduziert. Nur bei Gabe von 1×50 mg/kg betrug die Parasitenreduktion 73,2%. Wieder waren die Weibchen empfindlicher gegen Praziquantel als die Männchen. Der Miracidienschlüpfversuch wurde an Eiern aus der Leber behandelter und unbehandelter Kontrolltiere durchgeführt. Er war bis zu vier Wochen nach der Behandlung positiv; Praziquantel wirkt also nicht ovizid.Jugendliche S. japonicum waren gegen die Behandlung deutlich unempfindlicher als adulte Schistosomen (5×100 mg/kg bewirkten bei Adulten eine Reduktion um 99,7%, bei den Jugendlichen eine um 54,2%).Eine parasitologische Heilung wurde bei Goldhamstern, die mit einem liberianischen Stamm von S. mansoni infiziert waren, mit 100 mg/kg (verabreicht an drei aufeinanderfolgenden Tagen) und mit 3×50 mg/kg/die erreicht. Die Behandlung von Goldhamstern, die mit einem puertoricanischen S. mansoni-Stamm infiziert waren, ergab ebenfalls eine sehr starke Reduktion der Anzahl adulter Schistosomen. Gegen S. intercalatum und S. mattheei war Praziquantel ebenfalls sehr gut wirksam.Es wurde gefunden, daß die Wirksamkeit von Praziquantel gegen S. haematobium, S. japonicum und S. mattheei im Hamster deutlich besser ist als die von Metrifonat (Bilarcil). Nach Behandlung mit Praziquantel zeigen die meisten S. haematobium- und S. japonicum-Würmer eine klassische liver shift und werden im Lebergewebe festgehalten und abgetötet.Die Untersuchung zeigt, daß Praziquantel gegen alle drei wichtigen Schistosomenarten, S. haematobium, S. japonicum und S. mansoni, und auch gegen S. intercalatum sowie gegen den Rinderparasiten S. mattheei im Hamster hoch wirksam ist. Zwischen den in dieser Untersuchung verwendeten Parasitenstämmen unterschiedlicher geographischer Herkunft bestehen in der Wirksamkeit keine wesentlichen Unterschiede.

     

A method for the in vivo labelling of Schistosoma mansoni and S. intercalatum cercariae with radioselenium

Summary In order to develop a standard technique for in vivo labelling of Schistosoma mansoni and S. intercalatum cercariae by incubation of infected host snails with radioselenium, studies were conducted on the relationship between mode of administration, dose size (Ci), and time of administration of the radioisotope on the one hand and the production of cercariae and the amount of cercaria-bound radioactivity obtained on the other.The ability to take up radioactivity by B. glabrata snails was influenced by the following factors: living/heat-killed snails, size of snails, length of exposure period, volume of exposure medium, and the amount of radioactivity per snail in the exposure medium. A linear proportionality existed between the amount of radioactivity in the exposure medium and the subsequent snail-bound radioactivity. The rate of uptake and the final level of snail-bound radioactivity reached was increased by reducing the volume of the exposure medium to a minimum. Thus, at exposure to ⁷⁵Se-methionine in 1 ml exposure medium the maximum snail-bound radioactivity (70–85% of available amount) was obtained after 5 h. Therefore, the labelling procedure used comprised an individual exposure of snails in 1 ml for 20 h to radioselenium. Irrespective of the exposure level (Ci) used the labelling of infected B. glabrata prior to or 3 days after the start of shedding of cercariae resulted in a significant interference with the production of cercariae. The same interference was observed when snails were labelled 12 days after patency using exposure levels at or above 6.6 Ci/snail. However, a dose level of or below 5.5 Ci/snail administered 12 days after the start of shedding cercariae did not interfere with the host-parasite relationship, i.e. a normal and continuous production of labelled cercariae was obtained during a period of at least 54 days.The maximum cercaria-bound radioactivity obtained, using non-influential dose levels, was obtained 3 days after exposure of snails to the isotope. The maximum amount obtained was roughly comparable when exposure levels of 3.0–19.5 Ci per snail were used after the start of shedding of cercariae.The labelling procedure did not interfere with the biological characteristics of the labelled larvae. Thus their behavioural activity, longevity, and infectivity to mice were comparable with those of unlabelled cercariae.The possible application of labelled larval schistosomes to various problems in research on schistosomiasis is discussed.     

Schistosoma japonicum,S. mansoni,S. haematobium,S. intercalatum,and S. rodhaini: cysteine-class cathepsin activities in the vomitus of adult worms

 Vomitus from adults of five Schistosoma species was screened for biochemical homologues of the mammalian cysteine proteinases cathepsins B, H, and L. Bovine cathepsin B and rat cathepsin L served as references. Using the substrate Arg-NMec, a schistosome cathepsin H-like activity was never detected. All species degraded the cathepsin B substrate Z-Arg-Arg-NMec, but distinct species differences were observed with respect to pH optima and buffer preferences. The cathepsin B and L substrate Z-Phe-Arg-NMec was similarly degraded by all species, and activity was abolished by the cysteine proteinase inhibitor E-64. Preferences by vomitus proteinase activities for Z-Phe-Arg-NMec over Z-Arg-Arg-NMec were similar to or higher than those found for bovine cathepsin B but well below those observed for rat cathepsin L; also, the preferential cathepsin L inhibitor Z-Phe-PheCHN₂ only partially inhibited proteinolytic activity. The results suggest the possible presence in vomitus of a minor cathepsin L-like activity and demonstrate a major cathepsin B-like activity that is biochemically variable between schistosome species. Received: 1 April 1996 / Accepted: 19 June 1996     

Egg laying is delayed but worm fecundity is normal in SCID mice infected with Schistosoma japonicum and S. mansoni with or without recombinant tumor necrosis factor alpha treatment

Mice with severe combined immunodeficiency (SCID mice) lack functional B and T cells. Egg laying by Schistosoma mansoni and S. japonicum was delayed in SCID mice, but in a matter of weeks worm fecundity was equivalent to that in intact mice. SCID mice formed smaller hepatic granulomas and showed less fibrosis than did intact mice. The reduction in egg-associated pathology in SCID mice correlated with marked reductions in interleukin-4 (IL-4), IL-5, IL-13, and gamma interferon mRNA expression in the liver. S. mansoni infections were frequently lethal for SCID mice infected for more than 9 weeks, while S. japonicum-infected SCID mice died at the same rate as infected intact mice. We were unable to affect hepatic granuloma formation or egg laying by worms in SCID mice by administration of recombinant murine tumor necrosis factor alpha (TNF-alpha). In fact, SCID and BALB/c mice appeared to express nearly equivalent levels of TNF-alpha mRNA in their granulomatous tissues, suggesting that there is little or no deficit in TNF-alpha expression in infected SCID mice. The data indicate that TNF-alpha may be in large part derived from a non-T-cell source. Together, these findings provide little evidence that TNF-alpha alone can reconstitute early fecundity, granuloma formation, or hepatic fibrosis in schistosome-infected SCID mice.     

Susceptibility of neonate mice born to Schistosoma mansoni-infected and noninfected mothers to subsequent S. mansoni infection

The present study tested the hypothesis that prenatal exposure of neonate Outbred albino mice to Schistosoma mansoni antigens (Ags) or antibodies (Abs) modulates their immunity against postnatal responses to infection. Persistence of maternal S. mansoni Abs and/or Ags in mice born to S. mansoni-infected mothers (IF-IMs) and noninfected mothers (IF-NMs) for up to 8 weeks after delivery was investigated. A higher level of anti-S. mansoni IgG Ab was detected in sera of 1-week-old mice born to IF-IM compared to controls. Then, immunoglobulin (Ig)G gradually decreased to the eight week. No anti-S. mansoni IgM Ab was detected in sera of these offspring at any week after delivery. Schistosoma Ags were detected in liver and kidney tissues of mice born to infected mothers. However, Ags decreased markedly till the sixth week in the liver but increased significantly at the sixth week in the kidney. Eight-week-old mice born to infected and noninfected mothers were infected with 200 S. mansoni ceracriae. Their sera and livers were collected for testing IgG and granuloma formation 6 weeks postinfection. Worms were collected via portal perfusion and counted. Anti-S. mansoni IgG level, size and number of liver granuloma, and worm burden were significantly reduced in the offspring of infected mothers. These data suggest that in utero exposure of Outbred albino mice to S. mansoni may attenuate the pathogenesis of S. mansoni in subsequent challenge.     

Schistosoma haematobium,S. intercalatum,S. japonicum,S. mansoni,and S. rodhaini in mice: relationship between patterns of lung migration by schistosomula and perfusion recovery of adult worms

The development of five schistosome species was compared in mice by the recovery of schistosomula from chopped lung tissue and of adult worms by portal perfusion. Three developmental patterns appeared. (1) Schistosoma japonicum was unique in showing an early establishment of schistosomula in and a rapid departure from the lungs together with the highest worm recovery; (2) S. haematobium contrasted by establishing later and persisting in the lungs for at least 2 weeks while yielding the lowest adult worm recovery; and (3) S. intercalatum, S. mansoni, and S. rodhaini had an intermediate pattern – they resided in the lungs for several days, then disappeared and produced intermediate numbers of adults. Lung petechiae, known to accompany the migration of S. japonicum, were never detected after infection with the other species. We speculate that the three migration patterns of schistosomes are related to the size of the relative spectra of naturally infected definitive hosts. Received: 31 August 1997 / Accepted: 15 October 1997     

Enhancement of neutrophil- and eosinophil-mediated complement-dependent killing of schistosomula of Schistosoma mansoni in vitro by leukotriene B4

We have studied the ability of leukotrienes and other lipoxygenase products of arachidonic acid (AA) to influence complement-dependent killing of schistosomula of Schistosoma mansoni in vitro by human neutrophils or eosinophils. These lipid mediators, which included LTB4, LTC4, LTD4, 5-HETE and 5-HPETE, had no apparent effect, by themselves, on schistosomular motility or viability. However, in the presence of granulocytes and fresh serum (as a source of complement) LTB4 (but not LTC4, LTD4, 5-HETE or 5-HPETE) enhanced neutrophil- and (to a much lesser extent) eosinophil mediated, complement-dependent killing. These effects varied with the concentration of LTB4, the dilution of complement and time of incubation. The percentage of LTB4-induced enhancement obtained with neutrophils was greater than that observed with eosinophils (although the latter were obtained from patients with helminthic parasitic disease). The synthetic bacterial analogue f-Met-Leu-Phe, also known to amplify complement associated granulocyte events, was comparable to LTB4 in its ability to enhance neutrophil- and eosinophil-mediated, complement-dependent killing of schistosomula. These results indicate that LTB4, which is released in mast cell associated reactions and promotes cell locomotion and enhancement of complement receptors in vitro, increases neutrophil- and eosinophil-mediated, complement-dependent damage of schistosomula, possibly through enhancement of C3b receptors and that this may be an important amplification mechanism in IgE related immunity to migrating helminthic larvae.