Japonicum-infected Mice Research Articles

Kinetic study of eosinophil chemotactic factor production with reference to eosinophilia and granuloma formation in mice infected with Schistosoma japonicum

Kinetic changes of eosinophil chemotactic factor (ECF) production from granulomas, splenic T-cells or mast cells were examined with reference to granuloma formation around newly deposited single eggs in Schistosoma japonicum-infected mice. The peri-ovular granulomas began to appear at around 5 weeks post-infection (p.i.). Their size reached a peak at 6 weeks and then decreased gradually. Up to 8 weeks p.i., eosinophils were the predominant cell type in the granulomas. ECF-release from isolated granulomas paralleled the size of granulomas. Circulating ECF-A, which was assumed to be derived from mast cells, was also detected 6 weeks afterwards in parallel with the level of specific IgE antibody level against egg antigens in the serum. The circulating ECF-A peaked at 8 weeks and decreased after 10 weeks. Spleen cells began to produce ECF specific to bone-marrow eosinophils began at 5 weeks p.i., reached a peak at 6 weeks and then decreased rapidly. On the other hand, the production of ECF specific to eosinophils obtained from the peritoneal cavity began at 6 weeks and decreased rapidly thereafter. These results suggest that various kinds of host-derived ECFs seem to contribute, in one way or an other, to the accumulation of eosinophils in and around granulomatons lesions. The possible role of these ECFs in eosinophil mobilization from the site of production to the inflamed site is discussed.

Serum levels of circulating anodic antigen and circulating cathodic antigen detected in mice infected with Schistosoma japonicum or S. mansoni.

Serum concentrations of circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were studied in mice infected with either Schistosoma japonicum or S. mansoni cercariae. Sera from uninfected mice were negative for both antigens. CAA was detectable in the S. japonicum-infected mice as early as at 2 weeks post-infection (p.i.), and levels were higher in these animals than in the S. mansoni-infected group during the full study period. At the moment of perfusion, 10 weeks p.i., a median of 9 and 29 worms, respectively, were recovered from the S. japonicum- and S. mansoni-infected mice, and the median CAA levels were 326 and 27 ng/ml, respectively. In contrast, CCA levels were much lower in the S. japonicum-infected group (27 ng/ml) as compared with the S. mansoni-infected mice (282 ng/ml). These results suggest an important difference between S. japonicum and S. mansoni infections in CAA and CCA production and/or clearance and indicate a significant role for CAA in the diagnosis of human schistosomiasis japonicum.

Anti-interleukin-4 treatment diminishes secretion of Th2 cytokines and inhibits hepatic fibrosis in murine schistosomiasis japonica.

Anti-interleukin-4 (IL-4) treatment of Schistosoma japonicum-infected mice markedly inhibited in vitro secretion of the Th2 cytokines IL-4 and IL-5 from antigen-stimulated spleen cells, but enhanced the secretion of the Th1 cytokine IFN-gamma. IL-2 secretion was unaffected. Hepatic fibrosis was markedly diminished in anti-IL-4-treated-mice at ten weeks of infection while granulomas around S. japonicum eggs in the livers were slightly-to-moderately increased in size. The number of eggs per worm pair in the tissues and feces did not differ significantly in treated and untreated mice. These findings suggest that Th2 cytokine responses are important in the genesis of schistosomal hepatic fibrosis.

Parasite infection and cancer: with special emphasis on Schistosoma japonicum infections (Trematoda). A review

This article contains a review of current knowledge on the association of parasite infections and cancer formation, especially that of Schistosoma japonicum (Trematoda) in man and experimental animals. The association of S. haematobium infection and bladder cancer is well known and documented. However, S. japonicum infection has also been reported to be associated with cancer, in this case hepatocellular carcinoma and/or colorectal cancer. Pathological records and analyses have shown a correlation between this infection and cancer, and pathohistological descriptions have been numerous, together with clinical case reports. Epidemiological analyses have been conducted in China and Japan and support a role of S. japonicum infection as one of the risk factors in cancer formation, along with others, such as hepatitis virus infection and alcoholic intake. Experimental results have also shown that cancer appears early and in larger numbers in experimentally infected animals given a known carcinogen. In spite of these positive end-point associations, the mechanism of schistosome-mediated enhancement of carcinogenesis is obscure. A suggestive observation is that in S. japonicum-infected mice carcinogen-metabolizing hepatic activity including P-450 was decreased so that an administered carcinogen persisted for a longer period than in uninfected mice. Further studies, both epidemiological and experimental, are needed to firmly establish the relationship between schistosome infection and cancer.

Immune attrition of adult schistosomes

Mouse infection models are described that demonstrate reduction in the rate of egg production in Schistosoma haematobium worms 6-10 weeks after the onset of oviposition and loss of Schistosoma bovis worms around 10 weeks after infection. Neither phenomenon has been shown in Schistosoma mansoni- or Schistosoma japonicum-infected mice. The immunological basis for these anti-adult responses was inferred by comparison with infections in T-cell deprived mice and by transference of the ability to reduce a S. bovis worm burden in immuno-compromised hosts with immune serum. Vaccination with irradiation-attenuated parasites of S. haematobium was also shown to have consequences for the adults of challenge infections of either S. haematobium or of S. bovis, but not of S. mansoni. Thus, prior vaccination resulted in an abrogation of the reduced egg production by S. haematobium and S. bovis worms and also of the adult worm elimination that occurred in non-vaccinated S. bovis-infected mice. These models are being used to define the targets and mechanisms involved in the attrition of adult worms of schistosomes with terminal spined eggs.

Schistosoma japonicum-infected mice show reduced hepatic fibrosis and eosinophilia and selective inhibition of interleukin-5 secretion by CD4+ cells after treatment with anti-interleukin-2 antibodies.

Schistosoma japonicum-infected mice were injected with antibodies to interleukin-2 (IL-2) and/or IL-2 receptor to clarify the role of IL-2 on the granulomatous reaction around schistosome eggs in the liver. Granulomas were of normal or slightly increased size in animals subjected to IL-2 blockade, but hepatic fibrosis was markedly decreased in treated animals 10 weeks after infection. Anti-IL-2 treatment significantly decreased the in vitro secretion of IL-5 by antigen-stimulated spleen cells, and peripheral eosinophilia and tissue eosinophilia were diminished. Secretion of IL-2, IL-4, and gamma interferon was unaffected. Our results indicate that IL-2 is not an essential determinant of granuloma size in S. japonicum-infected mice but that, as in Schistosoma mansoni infection, the development of hepatic fibrosis is critically dependent on IL-2 levels and granuloma size and hepatic fibrosis are differentially regulated.

Modified metabolism of a carcinogen, 3-amino-1-methy-5 H-pyrido[4,3- b]indole (Trp-P-2), by liver S9 from Schistosoma japonicum-infected mice

schitosoma japonicum infection has been associated with an increased incidence of liver and colorectal cancers in humans. To explore the mechanisms underlying this association, we investigated the carcinogen-metabolizing properties of liver S9 preparations from S. japonicum-infected mice and compared then with those of S9 from uninfected animals. When the carcinogen 3-amino-1-methyl-5 H-pyridol[4,3- b]indole (Trp-P-2) was incubated with these S9s and the products were analyzed by high-performance liquid chromatography, we observed that the S9 from infected mice had a lower ability to convert Trp-P-2 into 30-hydroxyamino-1-methyl-5 H-pyridol[4,3- b]indole (Trp-2(NHOH)), an activated form of promutagenic Trp-P-2, than the S9 from uninfected mice. We found that both of these S9 preparations have a high ability to reduce Trp-P-2(NHOH) into Trp-P-2; however, the infected-mouse S9 showed a significantly greater reducing power than the control S9. This difference appears to be responsible for the observed lower mutagen-activating potential of the infected mouse S9. These results suggest that hepatic enzyme activities of S. japonicum-infected mice are quantitatively different from those of normal mice.

Analysis of egg granuloma formation in Schistosoma japonicum-infected mice treated with antibodies to interleukin-5 and gamma interferon

Schistosoma japonicum-infected mice were treated with antibodies to interleukin-5 (IL-5) or gamma interferon (IFN-gamma) from week 3 or 4 to week 10 of infection. Neither antibody affected egg production by the parasite, and neither had a consistent effect on the secretion of IFN-gamma or IL-5 cell-related cytokines by spleen cells from infected mice. Mice treated with antibody to murine IL-5 had only rare eosinophils in hepatic circumoval granulomas. Granulomas around single eggs were reduced in volume by a third, but hepatic fibrosis was unaffected. Treatment with antibody to murine IFN-gamma also reduced the size of granulomas and also did not affect hepatic fibrosis, which was measured as hydroxyproline. Our results, taken together with the studies of others, indicate that a complex interaction of cytokines affects granuloma size and that the size and fibrosis of granulomas are to some extent regulated independently.

Uptake and Effect of Praziquantel and the Major Human Oxidative Metabolite, 4-Hydroxypraziquantel, by Schistosoma japonicum

After exposure to praziquantel in vitro at a concentration of 1 microgram/ml for 0.5-2 hr, amounts of praziquantel in Schistosoma japonicum varied from 2.1 +/- 1.2 to 3.7 +/- 1.6 ng/male worm and 1.3 +/- 1.2 to 2.2 +/- 1.5 ng/female worm during the time studied. At 30 micrograms/ml, praziquantel amounts were 11-33-fold higher. However, within 2 hr after removal from a medium containing 30 micrograms/ml praziquantel, 95% of the drug was released from the parasites. When S. japonicum worm pairs were incubated in vitro with 1, 10, and 30 micrograms/ml of 4-hydroxypraziquantel, the major human oxidative metabolite of praziquantel, 0.2 +/- 0.2, 3.8 +/- 1.3, and 7.4 +/- 1.3 ng/worm pair, respectively, were found after a 2-hr incubation. 15-30-fold lower than corresponding worm pair amounts of praziquantel. In vivo, when 4- or 5-wk S. japonicum-infected mice were treated orally with praziquantel (300 mg/kg), peak concentrations of praziquantel in plasma determined by high pressure liquid chromatography were 14.7 +/- 1.5 micrograms/ml (4-wk infection) and 16.7 +/- 2.8 micrograms/ml (5-wk infection) 15 min after treatment. Corresponding in vivo worm praziquantel amounts were 1.8 +/- 0.4 ng/male worm and 2.4 +/- 1.1 ng/female worm, respectively, in the 4-wk infection and 4.6 +/- 1.6 ng/male worm and 5.6 +/- 1.2 ng/female worm in the 5-wk infection. Peak plasma concentrations of 4-hydroxypraziquantel were similar but corresponding in vivo worm amounts were 1-20-fold lower, depending on the time after drug administration.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Praziquantel on the Circulating Antigen SJ 70 in Murine Schistosomiasis Japonica

A circulating schistosome 70 kDa antigen (SJ 70) has been detected in the sera of mice infected with Schistosoma japonicum. For the detection of this antigen, a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was employed. SJ 70 is first detected in the serum of S. japonicum-infected mice 4 weeks after infection. Antigen levels rise rapidly, plateau by 7 weeks after infection, and remain relatively unchanged for at least another 9 weeks. In mice infected with S. japonicum for 7 weeks and then treated with praziquantel (100 mg/kg body weight), there is a significant decrease in serum antigen levels within 2 weeks after treatment, and an almost complete disappearance of antigen from the serum 3-4 weeks after treatment.

Extramedullary eosinophilopoiesis in the liver ofSchistosoma japonicum-infected mice, with reference to hemopoietic stem cells

Extramedullary hemopoiesis recognized as hemopoietic foci increased in the liver of Schistosoma japonicum-infected mice in parallel with kinetic changes in periovular granuloma formation. At the peak of the response, about 65% of the hepatic hemopoietic foci were of eosinophil lineage. When S. japonicum-infected mice were irradiated, hepatic hemopoietic foci rapidly disappeared within 3 days, whereas inflammatory cells in the periovular granulomas slowly reduced in number. When the number of hemopoietic stem cells in the liver were examined by spleen-colony assay, kinetic changes in the number of hemopoietic stem cells in hepatic nonparenchymal cells paralleled those of hepatic hemopoietic foci. Hemopoietic stem cells were rare in the granuloma cells. These results indicate that in response to the increased demand for eosinophils and other inflammatory cells, the liver acts as an extramedullary hemopoietic organ in which inflammatory cells are generated from hemopoietic stem cells.

Reduced levels of mutagen processing potential in the Schistosoma japonicum-infected mouse liver

Epidemiological studies have shown the presence of a positive correlation between the infection of Schistosoma japonicum and colorectal and/or liver cancers in the humans. To explore the mechanism underlying this correlation, we have investigated the mutagen-activating potentials of the liver homogenate fraction (S9) from Schistosoma japonicum infected mice and those from control mice, by use of the Ames test with 2-acetylaminofluorene, aflatoxin B 1 and 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2) as test mutagens. Liver S9 prepared from the infected group at the 15th week after the infection showed a potential significantly lower than that from the control group. The hepatic cytochrome P-450 concentration in the infected mice was persistently low, about a half of that in the uninfected mice, during the period of 6–18 weeks after the infection. Thus, in mice bearing chronic schistosomiasis, mutagen-processing potentials are decreased.

Effect of praziquantel on Schistosoma mansoni eggs: leucine aminopeptidase (LAP) activity and anti-LAP antibodies.

High levels of leucine aminopeptidase (LAP) have been detected in Schistosoma mansoni eggs. In this report we demonstrate that this enzyme is immunogenic in mice infected with either S. mansoni or S. japonicum. The anti-schistosomal agent, praziquantel, causes rapid release of this enzyme from schistosome eggs in vitro. Praziquantel treatment of S. japonicum infected mice is associated with a significant decrease in anti-LAP antibodies. Anti-LAP IgM antibody levels decreased more than did anti-LAP IgG antibodies.

Granulocyte-macrophage colony-stimulating factor enhances the production of eosinophil chemotactic lymphokine by egg-associated granulomas of Schistosoma japonicum-infected mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by splenic lymphocytes obtained from Schistosoma japonicum-infected mice was partially purified by a combination of DEAE anion-exchange chromatography, concanavalin A-Sepharose affinity chromatography, and high-pressure liquid chromatography. When this partially purified GM-CSF was added to the culture of isolated intact granulomas, eosinophil chemotactic factor (ECF) lymphokine production by granulomas was significantly enhanced. The partially purified GM-CSF also enhanced ECF lymphokine production by granuloma T cells cocultured with syngeneic macrophages and specific antigen. The partially purified GM-CSF itself had neither ECF activity nor a synergistic effect with ECF lymphokine. When normal splenic macrophages were preincubated with the partially purified GM-CSF, they potentiated the ECF production by granuloma T cells under the presence of specific antigen. Augmentation of ECF lymphokine production by partially purified GM-CSF was further confirmed by using T-cell clones that were established from granuloma T cells. These results suggest that T-cell-derived GM-CSF primarily activate macrophages so that these activated macrophages can cooperate more effectively with T lymphocytes to produce ECF. Such potentiation of macrophage-T-cell interaction by GM-CSF may be important in the mechanisms of granuloma formation during an acute stage of schistosomiasis.

Isolation of two immunogenically different allergens from Schistosoma japonicum eggs.

Two allergenic components, termed J1 and J2, were isolated from a soluble egg antigen preparation (SEA) of Schistosoma japonicum by anion-exchange chromatography on DE52 and gel chromatography on Sephacryl S-200. The apparent molecular weights of J1 and J2 were 260,000 and 46,000, respectively, by gel chromatography on Sephadex G-150. By SDS-polyacrylamide gel electrophoresis, both J1 and J2 showed apparent homogenicity and their estimated molecular weights were 135,000 and 45,000, respectively. The isoelectric point of J1 (pI 4.9) was similar to that of J2 (pI 4.8). Both J1 and J2 bound to Con A-Sepharose 4B, indicating their glycoprotein nature. The amino acid compositions of J1 and J2 have some similarities. However, phenylalanine and leucine, which contain large hydrophobic groups, were dominant in J1, whereas serine and threonine, which contain a hydroxyl group, were dominant in J2. J1 was sensitive to heating or pronase treatment, whereas J2 was rather stable to these treatments. Both J1 and J2 were sensitive to 0.1 M periodate treatment. When mice were immunized with either J1 or J2 with A1(OH)3 as an adjuvant, anti-J1 or anti-J2 IgE antibody was highly specific to the respective antigens. Since S. japonicum-infected mouse serum has high PCA titer to J1 and J2, these two components are the major allergens of S. japonicum eggs.

Eosinophil Chemotactic Lymphokine Produced by Spleen Cells of <i>Schistosoma japonicu</i><i>m</i>-lnfected Mice

Eosinophil chemotactic factor (ECF) was detected in the cell-free supernatant of the culture of spleen cells obtained from Schistosoma japonicum-infected mice. ECF production by spleen cells was dependent on both the amount of soluble egg antigen (SEA) added and the number of cells in the culture. When sufficient amount of SEA was added to the culture, ECF production was detectable by 6 h after incubation, reached a plateau by 24 h and then rapidly decreased. ECF was produced by nonadherent, Thy 1.2-positive cells, indicating that it is a lymphokine. After Sephadex G-75 gel chromatography, estimated molecular weight of this ECF was 15,000-20,000 daltons. When eosinophils were preincubated with this ECF lymphokine, their chemotactic reactivity to S. japonicum egg-derived ECF was significantly enhanced, although this pretreatment caused neither the deactivation of chemotactic reactivity to homologous ECF nor the enhancement of the random migration of eosinophils. Thus, this ECF lymphokine seems to be important not only as a chemoattractant but also as an activator of eosinophilotaxis around deposited eggs in schistosomiasis japonica.

Eosinophil chemotactic lymphokine produced by egg-associated granulomas in murine schistosomiasis japonicum.

Eosinophil chemotactic factor (ECF) was detected in the culture supernatant of isolated intact egg granulomas from the livers of Schistosoma japonicum-infected mice. This factor had an apparent molecular weight of 15,000 by high-pressure liquid chromatography with an SW3000 column and bound to concanavalin A-Sepharose 4B. When cells obtained by enzymatic digestion of isolated granulomas were cultured under the presence of soluble egg antigen of S. japonicum or concanavalin A, ECF was also detected in the conditioned medium. The physicochemical nature of the ECF produced by concanavalin A-stimulated granuloma cells was similar to that produced by isolated intact granulomas. The ECF-producing activity of the cells was abolished by pretreatment with anti-Thy-1.2 or anti-Lyt-1.2 monoclonal antibody and complement but not by anti-Lyt-2.2 antibody. Furthermore, nylon wool-passed, T-enriched granuloma cells required collaboration of syngeneic macrophages to produce ECF. These results suggest that Lyt-1-positive T cells in the granuloma could, in collaboration with macrophages, produce ECF and thereby attract eosinophils to this lesion.

Detection of high molecular weight eosinophil chemotactic factor in murine schistosomiasis sera.

Eosinophil chemotactic activity in sera from mice undergoing an acute stage of schistosomiasis japonica and mansoni was examined. Eosinophilotactic activity in the serum was dependent on the dose and time of infection. Eosinophilotactic activity in sera from S. japonicum-infected mice was higher than that from S. mansoni-infected mice when they were compared at the comparable dose and time of infection. After gel chromatography on Sephadex G-200, eosinophilotactic activity in sera from mice infected with 30 cercariae of S. japonicum for 5 weeks was detected in the high molecular weight component. On the other hand, when sera from mice infected with 30 cercariae of S. japonicum for 8 weeks was chromatographed through Sephadex G-200 columns, eosinophilotactic activity was segregated into high (greater than 455,000) and low (less than 13,000) molecular weight components. High molecular weight ECF in sera from mice infected with 30 cercariae of S. japonicum for 8 weeks had high affinity to Con A, and was stable to heating or pronase digestion, but was sensitive to periodate oxidation, indicating its polysaccharide or glycoprotein nature. This high molecular weight ECF could be adsorbed by, and eluted from immunoaffinity beads coated with rabbit IgG anti-S. japonicum adult worm antibody. Thus, at least some part of circulating high molecular weight ECF would be derived from adult parasites.

Inhibition of immediate and Arthus responses to schistosome egg antigens by T cells from Schistosoma japonicum-infected mice.

Schistosoma japonicum-infected mice develop hepatic granulomas, immediate hypersensitivity (IH), and delayed hypersensitivity (DH) to soluble egg antigens (SEA) released by parasite eggs trapped in liver sinusoids. All of these responses spontaneously regress after 7 to 9 weeks of infection. This study aimed to develop an in vivo system for the further dissection of cellular and humoral immune responses to SEA. C57BL/6 mice immunized subcutaneously with SEA in complete Freund adjuvant developed IH, an Arthus reaction, and DH to this antigen 5 to 9 days later. IH and the Arthus reaction, but not DH, were markedly inhibited if, 1 day before injection of SEA in complete Freund adjuvant, the mice were injected intravenously with purified T cells from the spleens of mice infected for at least 9 weeks. This in vivo model system can be used to study various aspects of cellular and humoral immune responses to SEA and their modulation. These results raise questions about the role of antibodies in the pathogenesis of granulomatous inflammation and about the mechanisms of its cellular regulation in infections with S. japonicum.

Granulocyte-macrophage colony-stimulating factor produced by splenic T lymphocytes of mice infected with Schistosoma japonicum.

The production of granulocyte-macrophage (GM) colony-stimulating factor (CSF) by splenic lymphocytes was examined in murine schistosomiasis japonica. When splenic lymphocytes obtained at various weeks after infection were cultured with soluble egg antigen, GM-CSF activity in the conditioned medium became detectable at 3 weeks after infection, reached a peak at week 5, and persisted at least up to week 7. Not only soluble egg antigen but also concanavalin A was highly effective in stimulating splenic lymphocytes to produce GM-CSF. When splenic lymphocytes were treated with anti-Thy-1.2 antibody and complement, GM-CSF-producing activity was completely abolished. The molecular weight of this T-cell-derived GM-CSF was estimated to be 30,000 by gel filtration on Sephadex G-150. After isoelectric focusing, GM-CSF activity was detected as two major peaks at pH 3.7 and 5.5. The physicochemical nature of this T-cell-derived GM-CSF was compared with those of known lymphokine GM-CSFs or with that of a previously reported GM-CSF in the serum of S. japonicum-infected mice (M. Owhashi and Y. Nawa, Infect. Immun. 49: 533-537, 1985).