A Schistosoma mansoni 62-kDa band is identified as an irradiated vaccine T-cell antigen and characterized as calreticulin.

The Schistosoma mansoni soluble adult worm antigen (SAWA) bands of 62/60 kDa were found to contain immunodominant T-cell immunogen(s) in irradiated cercariae-immunized Swiss and C57BL/6 mice. In the present study, spleen T cells of BALB/c mice immunized twice with ultraviolet light-irradiated cercariae proliferated and produced interleukin 4 in response to the 62/60-kDa SAWA bands in T-cell western assays. To characterize the 62/60-kDa bands, an adult S. mansoni worm cDNA expression library constructed in lambdagt11 was immunoscreened with serum of mice immunized with the 62/60-kDa antigens, and the immunoreactive cDNA inserts were sequenced. Purified 62- and 60-kDa proteins were used for amino acid microsequencing and for immunization studies in Swiss, C57BL/6, and BALB/c mice and rabbits. Taken together, the data indicated that the 60-kDa molecules are poorly immunogenic in mice and rabbits, whereas the 62-kDa species identified as S. mansoni calreticulin, is a good T- and B-cell antigen and represents a potential vaccine candidate.

In vitro metabolism of N-(5-chloro-2-methylphenyl)-N'-(2-methylpropyl)thiourea: species comparison and identification of a novel thiocarbamide-glutathione adduct.

The in vitro metabolism of SDZ HDL 376, a thiocarbamide developed for the treatment of atherosclerosis, was investigated in rat, dog, monkey, and human liver microsomes, as well as in rat and human liver slices. [14C]SDZ HDL 376 was extensively metabolized in all the species except human. In rat liver microsomes an S-oxide was the major metabolite. In human and monkey microsomes, carbon hydroxylation was favored. The NADPH-dependent oxidation of SDZ HDL 376 resulted in covalent binding to microsomal protein. Addition of GSH to the incubations decreased protein binding in a concentration-dependent manner and resulted in a novel SDZ HDL 376-GSH adduct. Adduct formation required NADPH and was mediated predominantly by cytochrome P450. Inhibition of cytochrome P450 by 1-aminobenzotriazole resulted in a 95% decrease in adduct formation, while heat inactivation of flavin-containing monooxygenases resulted in a 10% decrease. Unlike other thiocarbamides which form disulfide adducts with GSH, the SDZ HDL 376 adduct contained a thioether linkage as characterized by LC/MS/MS and reference to a synthetic standard. Reactions performed with [35S]GSH resulted in a [35S]SDZ HDL 376-GSH adduct, demonstrating the sulfur was derived from GSH. Adduct formation was faster in rat microsomal reactions compared to human microsomes. Other structurally unrelated thiocarbamides (phenylthiourea, methimazole, 2-mercaptobenzimidazole, 2-mercaptoquinazoline, and 2-propyl-6-thiouracil) did not form similar adducts in rat liver microsomes supplemented with GSH. Therefore, the GSH adduct of SDZ HDL 376 is unique for this type of thiocarbamide. These results suggest that the bioactivation and detoxification of SDZ HDL 376 differ significantly from other thiocarbamides. Furthermore, the in vitro formation of S-oxides and GSH adducts in rat hepatic tissue, and ring hydroxylation and glucuronidation in human hepatic tissue, suggests rats may be more susceptible to the toxicity of SDZ HDL 376 compared to humans.

Homologous and heterologous protective immunity to Egyptian strains of Schistosoma mansoni and S. haematobium induced by ultraviolet-irradiated cercariae.

C57BL/6 and Balb/c mice were immunized with ultraviolet-irradiated cercariae of Egyptian strains of Schistosoma mansoni and S. haematobium, challenged with nonirradiated cercariae of the homologous or heterologous species, and assayed for protection against challenge infection by comparing the adult worm burdens of immunized and non-immunized mice. Homologous protection (per cent reduction in worm recovery) ranged from 56% to 69% for S. mansoni and 88% to 99% for S. haematobium. Significant heterologous protection was consistently induced against S. haematobium by immunization with S. mansoni, but not against S. mansoni by immunization with S. haematobium. These results are discussed in relation to those of previous studies and in terms of implications for vaccine development.

Induction of the adipose differentiation-related protein in liver of etomoxir-treated rats.

The effects of etomoxir, an irreversible carnitine palmitoyltransferase I inhibitor, on the liver protein pattern and on liver morphology were examined by two-dimensional gel electrophoresis in female Sprague-Dawley rats treated with 125 mg/kg/day etomoxir for 28 days. In livers of treated animals a protein spot was found which was not present in controls. The spot was identified by internal amino acid sequence analysis as the adipose differentiation-related protein (ADRP). The expression of ADRP in liver is a novel finding as the protein has been described previously as adipocyte-specific. Additionally we found histopathologic evidence of lipid accumulation in the livers of etomoxir rats. The data show that for each treated rat there was a good correlation between ADRP levels and degree of lipid droplet formation. This observation may suggest a potential relationship between drug-induced expression of ADRP in liver and lipid accumulation.

Comparison of two strains of Schistosoma mansoni with respect to the sites and kinetics of immune elimination in irradiated cercaria-immunized mice.

A comparison was made of the sites of elimination of NMRI and Mill Hill strains of Schistosoma mansoni in C57BL/6J mice previously immunized with 50 krad gamma-irradiated cercariae of the homologous strain. In the first experiment, the fate of percutaneous challenge infections with 75Se-labeled cercariae was evaluated by autoradiography of tissue squashes and hepatic portal perfusion. For both strains of parasite, migration from skin to lungs was delayed but not reduced in immunized mice relative to controls, with immune elimination taking place at some point after migration to the lungs. In a second experiment, resistance to the Mill Hill strain of S. mansoni was compared in mice challenged by percutaneous infection with cercariae and by intravenous injection with lung schistosomula. Both types of challenge were shown to be vulnerable to immune elimination. We conclude that under the conditions employed in this study, there is no significant difference between the NMRI and Mill Hill strains of S. mansoni in the patterns of migration and elimination, with most or all elimination in both control and immunized mice taking place after migration from the skin.

The role of IgG antibodies from irradiated cercaria-immunized rabbits in the passive transfer of immunity to Schistosoma mansoni-infected mice.

Antibodies of the IgG subclass isolated from the sera of rabbits immunized with cercariae subjected to 50 kilorads of gamma irradiation passively provided partial immunity against Schistosoma mansoni challenge in C57B1/6J mice. These mice exhibited reductions in adult worm burdens of 43-61% compared with recipients of normal rabbit antibodies. Passively transferred IgG antibodies were most effective when given 4-7-days postchallenge; they were less effective when given just before challenge, and were totally ineffective when given 15 days postchallenge. It was also shown that the Fc portion of the IgG molecule was important for passive transfer of immunity. Finally, we observed that although some antibodies from irradiated cercaria-immunized rabbits recognized keyhole limpet hemocyanin (KLH), these KLH cross-reacting antibodies were not necessary for successful passive transfer of immunity. Antibodies from a KLH-immunized rabbit also failed to passively protect mice.

Combined microautoradiographic and histopathologic analysis of the fate of challenge Schistosoma mansoni schistosomula in mice immunized with irradiated cercariae.

Combined microautoradiographic and histopathologic methods were used to locate and examine schistosomula of Schistosoma mansoni in the lungs of irradiated cercaria-immunized mice 21 days after percutaneous challenge infection with 75Se-labeled cercariae. Of 75 schistosomula examined in serial sections, 53% were located in the pulmonary microvasculature, 23% in alveolar spaces, 3% with one end in a vessel and the other in an alveolar space, and the locations of 21% were not identified. Inflammatory reactions of variable intensity were observed around schistosomula in both vascular and alveolar sites, although the most intense category of reactions was associated almost entirely with alveolar larvae. All autoradiographic foci contained recognizable schistosomula. Although the concentration of reduced silver grains precluded cyto-structural analysis, observations on schistosomular contour and shape provided no evidence of larval damage. Our findings suggest that immune elimination of schistosomula in mice immunized with irradiated cercariae is partly or largely effected by a process of alveolar extrusion of viable parasites during their lung migration.

Evidence that both normal and immune elimination of Schistosoma mansoni take place at the lung stage of migration prior to parasite death.

The number and distribution of autoradiographic foci observed in this and previous studies following percutaneous infection with 75Se-labeled Schistosoma mansoni cercariae indicate that the lungs are the principal site of worm elimination in both normal mice and mice immunized with irradiated cercariae. It was observed in the present study, however, that the intensities of the autoradiographic foci produced in the lungs during both the normal (early) and immune (late) phases of elimination were identical to those of foci produced in the livers of the same mice by larvae shown to be alive. In contrast, foci produced in the lungs by heat-killed, intravenously injected, lung schistosomula became smaller and fainter with time, disappearing completely between seven and 10 days after injection in normal mice and between four and six days in immunized mice. These results indicate that although the targets of both normal and immune elimination do not proceed beyond the lung stage of migration, they do not die in the lungs. A possible explanation for this paradoxical situation, for which there is some experimental evidence, is that unsuccessful migrators leave the blood stream, enter alveoli, pass up the trachea, and are eventually digested in the gastrointestinal tract or eliminated from the body intact.

Self-catalyzed irreversible inactivation of rat hepatic aryl sulfotransferase IV by N-hydroxy-2-acetylaminofluorene.

Rat hepatic aryl sulfotransferase IV catalyzes the sulfonation of the hepatocarcinogen, N-hydroxy-2-acetylaminofluorene. The resulting reactive N-O-sulfate ester is believed to be the ultimate carcinogenic species responsible for the induction of hepatic neoplasia. Previous studies have shown that dietary administration of either 2-acetylaminofluorene or N-hydroxy-2-acetylaminofluorene to rats is accompanied by a rapid decline in hepatic aryl sulfotransferase activity in vivo. In the present study, preincubation of purified rat hepatic aryl sulfotransferase IV with N-hydroxy-2-acetylaminofluorene resulted in rapid, time-dependent enzyme inactivation. This in vitro inactivation was not reversed by dialysis or gel filtration. Inclusion of excess nucleophile, methionine, resulted in considerable but not complete protection from inactivation. The inactivation was PAPS dependent and blocked by the sulfotransferase inhibitor, pentachlorophenol. The above observations and the apparent pseudo first-order kinetics observed suggest that the inactivation was in part mechanism based. Mechanism-based inactivation of the aryl sulfotransferases has not been previously reported. Furthermore, the results of the present study indicate that the previously reported in vivo decline in rat hepatic aryl sulfotransferase activity may be attributable in part to enzyme inactivation by its own reactive product.

Rat liver aryl sulfotransferase-catalyzed sulfation and rearrangement of 9-fluorenone oxime.

The role of hepatic cytosolic aryl sulfotransferase (3'-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1) in the enzymic rearrangement of 9-fluorenone oxime to phenanthridone was investigated. 9-Fluorenone oxime was found to be an excellent substrate for a partially purified rat liver aryl sulfotransferase preparation. This compound was in fact superior to 2-naphthol, the standard assay substrate. This is the first reported observation of aryl oxime sulfation by the aryl sulfotransferases. 9-Fluorenone oxime sulfation exhibited pronounced substrate inhibition at high substrate concentrations. However, despite virtually complete conversion of 9-fluorenone oxime to the corresponding N-O-sulfate conjugate in enzyme incubation mixtures, only small amounts of rearrangement product were detected after long-term incubations. In addition, 9-fluorenone oxime-O-sulfonic acid was chemically synthesized and tested for stability. The results showed that rearrangement was pH-dependent and occurred slowly over several hours. It is therefore concluded that aryl sulfotransferase-catalyzed sulfation likely plays an important role in the in vitro and in vivo disposition of 9-fluorenone oxime. Moreover, sulfation facilitates the Beckmann-like conversion of 9-fluorenone oxime to phenanthridone. Sulfation alone, however, appears insufficient to account for all of the previously reported in vitro and in vivo rearrangement.

Migration of Schistosoma mansoni in normal and passively immunized laboratory rats.

Normal and passively immunized Fischer rats were infected with 75Se-selenomethionine-labeled cercariae of Schistosoma mansoni. Migration of the parasites from skin to lungs to liver was monitored by autoradiographic analyses of these sites. Labeled parasites migrated from skin to lungs with high efficiency in normal and immune rats; disappearance of labeled parasites from the lungs was slower in immune rats. Labeled parasites accumulated in the liver, reaching maximal values by 11 days post-infection in both groups and remaining constant through day 21. Half the number of labeled parasites were detected in the liver of immune rats. The total number of labeled parasites detected in the skin, lungs, and liver was constant through day 5, then declined to about 60% of this value by day 11 in both groups. Over the next 10 days, the rate of decline decreased significantly in normal rats but did not change in immune rats. By day 21 post-infection, nearly 50% fewer labeled parasites were detectable in immune rats. We conclude that a subpopulation of parasites in the lungs is the target of protective antibody in the serum used for passive immunization. Target parasites, retained longer in the lungs, were probably prevented from migrating successfully to the liver. Another parasite subpopulation migrated to the liver with normal kinetics. Lung schistosomula isolated from normal and passively immunized rats were transferred by intravenous injection into recipient rats and their continued migration from lungs to liver compared. No differences in portal perfusion worm yields were detected in normal recipients; equally reduced yields were detected in passively immunized recipients. We conclude that the effects of antibodies during week 1 post-infection were insignificant or reversible.

Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae.

The humoral immune responses of mice patently infected with Schistosoma mansoni and of mice vaccinated with radiation-attenuated cercariae were compared by radioimmunoassays and one- and two-dimensional polyacrylamide gel analyses of radioimmunoprecipitates. The binding observed with antibodies of mice vaccinated twice with radiation-attenuated cercariae over a period of 7 to 11 wk was less than 50% of the binding observed with antibodies of mice patently infected for 20 wk, but three to four times greater than that obtained with antibodies of mice infected for 6 wk, irrespective of whether the test antigen extracts were derived from schistosomula or adult worms. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with [35S] methionine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by sera of infected mice. In contrast, sera of vaccinated mice uniquely precipitated a 38,000 m.w. glycoprotein of schistosomula cultured for 5 days and a 94,000 m.w. glycoprotein of adult male worms. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. In particular, an egg glycoprotein of 85,000 to 70,000 and isoelectric point of 4.8 showed an enhanced reactivity with sera of vaccinated mice in comparison with infected mice. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.

Passive transfer with serum and IgG antibodies of irradiated cercaria-induced resistance against Schistosoma mansoni in mice.

The role of humoral immunity to Schistosoma mansoni infection in C57BL/6J mice was examined by employing a passive transfer system. Sera from highly resistant mice that had been exposed to two or three immunizations with 50-kilorad-gamma-irradiated cercariae were tested for their ability to transfer protection against S. mansoni challenge. All five batches of serum tested were observed to have protective activity. Immune serum recipients exhibited statistically significant reductions in challenge worm burdens of 20 to 50% compared with recipients of normal serum or no serum. The most consistent level of resistance was obtained when immune serum was administered several days post-challenge, i.e., at a time coincident with schistosomulum residence in the lungs. Furthermore, it was shown that the protective activity in immune serum was associated with factors that bind to staphylococcal protein A and that are precipitated by 50% ammonium sulfate; thus it appears that the protective factors in immune serum are IgG antibodies.

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.

The migration and survival of gamma-irradiated Schistosoma mansoni larvae and the duration of host-parasite contact in relation to the induction of resistance in mice.

The migration in mice of 20, 50 and 90 krad. 60Co-irradiated Schistosoma mansoni larvae, biosynthetically radio-isotope labelled with [75Se]-selenomethionine, was evaluated by autoradiography of compressed tissues and compared to the migration of non-irradiated 75Se-labelled larvae. The migration of 20 krad.-irradiated schistosomula between skin and lungs was slightly delayed but otherwise paralleled the migration of normal, non-irradiated schistosomula during the first 8 days following exposure. By day 8 over 90% of both non-irradiated and 20 krad.-irradiated organisms were located in the lungs. In contrast to non-irradiated organisms, however, only a small proportion of 20 krad. organisms migrated to the liver. The delay in migration between skin and lungs was more pronounced with 50 krad.-irradiated schistosomula. Nevertheless, 45-93% of 50 krad.-irradiated organisms migrated to the lungs by 8 days post-exposure. Over 90% of the 50 krad. larvae detected in the mouse on day 21 were in the lungs; no more than an occasional 50 krad.-irradiated organism was ever detected in the liver. In three experiments, over 85% of the 90 krad.-irradiated organisms were retained in the skin; in a fourth experiment about half of the 90 krad.-irradiated organisms migrated as far as the lungs. As with 50 krad. organisms, only an occasional 90 krad. organism was ever detected in the liver. Removal of the skin exposure site within the first 4 days of immunization with either 50 or 90 krad.-irradiated cercariae completely blocked the induction of resistance. Removal between the 4th and 6th days gave variable results. Mice had to be in contact with the irradiated larvae for a minimum of 8-11 days to stimulate a level of resistance comparable to that of mice whose immunization site was not removed.

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.

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.

Immunization of mice with ultraviolet-irradiated Schistosoma mansoni cercariae: a re-evaluation.

Mice immunized by percutaneous exposure to ultraviolet-irradiated Schistosoma mansoni cercariae developed levels of resistance to subsequent S. mansoni infection comparable to those induced by gamma-irradiated cercariae (50-70% reduction in adult worm burden). Cercariae treated with ultraviolet doses ranging from one to three times the minimum dose required to prevent long-term survival induced the highest levels of resistance.