Mast cell-mediated toxicity to schistosomula of Schistosoma mansoni: potentiation by exogenous peroxidase

Mast cells, when incubated in vitro with hydrogen peroxide (H2O2) and iodide, are cytotoxic to schistosomula of Schistosoma mansoni, as determined morphologically by dye exclusion, motility, and refractility and by transmission and scanning electron microscopy. When intact mast cells were incubated with schistosomula, mast cell degranulation with extracellular release of mast cell granules (MCG) was only observed in the presence of added H2O2 (10(-4) M). The secreted MCG, which contain small amounts of endogenous peroxidase activity, adhered to the surface of schistosomula. By 15 to 30 min, the mast cell-H2O2 system in the presence of iodide (10(-4) M) produced marked disruption of the tegumental and internal structures of the schistosomula. No helminthic damage was noted if any component of the incubation mixture (mast cells, H2O2 or iodide) was omitted. MCG could substitute for intact mast cells in the H2O2 and iodide-dependent cytotoxic system; MCG-mediated killing of schistosomula was inhibited by the hemeprotein inhibitor azide, suggesting that the cytotoxic reaction required endogenous peroxidase. The cytotoxicity was increased by eosinophil peroxidase bound to the MCG surface. These findings suggest a mechanism by which mast cells may contribute to the host cytotoxic response to helminths. H2O2 formed by nearby inflammatory cells may induce mast cell secretion, and the released MCG, through their endogenous peroxidase content (or bound eosinophil or neutrophil peroxidase), may react with H2O2 and a halide to form a system toxic to the adjacent helminth.     

Trichinella spiralis: an intracellular parasite in the intestinal phase.

Trichinella spiralis has been examined by electron microscopy after fixation in situ in the intestine of mice. The worms lie within the cytoplasm of cells of the intestinal mucosa and may occupy both absorptive and goblet cells. They cause little damage to host cells. A few worms have been seen protruding from tissue by SEM techniques. These unusual observations suggest that the nematodes may be capable of exit and reentry into the epithelium.     

Trichinella spiralis: behavior, structure, and biochemistry of larvae following exposure to components of the host enteric environment

Four layers are present on the surface of infective larvae of Trichinella spiralis isolated from host muscle in pepsin-HCl. Trypsin treatment of pepsin-HCl isolated worms caused partial degradation and removal of large patches of the two outer surface layers. Following exposure to bile, only traces of the outer layers remained on the worms surface. These changes in the worm surface were accompanied by a shift from Type I behavior, typical of pepsin-HCl isolated larvae, to Type II behavior, (snakelike) following exposure to either trypsin or bile. Worm behavior was also temperature dependent. Type I behavior was typical of worms maintained at room temperature regardless of treatment, while Type II behavior displayed by worms held at 37 C was treatment dependent. The absorption of in vitro glucose or beta-methyl-D-glucoside was lowest in pepsin-HCl isolated first stage infective larvae, significantly higher in trypsin treated worms and greatest in worms following exposure to bile. Sugar uptake by worms isolated from the host small intestine after 1 hr of enteral infection was similar to that seen in worms isolated from host muscle in pepsin-HCl. Sugar uptake in vitro in worms 2 hr following enteral infection was similar to worms following exposure to bile. The highest levels of sugar absorption in vitro occurred in worms which had resided in the small intestine for 3 hr. The lowest rates of incorporation of label into worm tissues was seen in 1 hr enteral and pepsin-HCl isolated worms. Infective larvae treated with trypsin or bile incorporated significantly greater amounts of label than the two former groups. The highest levels of incorporation of label into worm tissues was seen in 3 hr enteral worms. These findings support the view that trypsin, bile, and temperature serve as environmental cues which lead to alteration of the parasite's behavioral and nutritional status.     

Intraintestinal migration to the epithelium of protective, dividing, anti-Trichinella spiralis CD4+ OX22- cells requires MHC class II compatibility

We have measured the intraintestinal migration of activated Th cells belonging to the OX8- OX22- and OX8- OX22+ subsets derived from thoracic duct lymph of rats infected with Trichinella spiralis. Cells in S-phase were labeled with 125I-UdR or 3H-TdR in vitro and transfused i.v. Identical proportions of both helper cell subsets localized in the small intestine but three to four times as many OX22- cells as OX22+ cells migrated to the intestinal epithelium. Experiments using MHC class I and II recombinant rats indicated that localization of OX8- OX22- cells in the lower lamina propria (LP) and muscularis was reduced by 50% in MHC class II incompatible rats as assessed by total gamma-counts but by a factor of three when labeled cells were counted. Movement of labeled OX8- OX22- cells to the epithelium was reduced by a factor of five to seven when assessed by the same methods. Quantitative cellular localization in the gut and further movement to the epithelium were normal in class I-mismatched rats. The movement from the lower-mid LP to the epithelium was undertaken principally by dividing cells as indicated by a progressive loss of grain counts in labeled cells and the appearance of doublet-cells in mitosis. In allogeneic combinations, extravasating cells remained localized close to their primary site of exit in the muscularis and lower-mid LP. The results suggest that the requirement for MHC class II compatibility for adoptive transfer of immunity in rats to T. spiralis is functionally related to localization of the protective OX22- cell subset in the epithelium.     

Glutathione peroxidase in yeast. Presence of the enzyme and induction by oxidative conditions

The presence of glutathione peroxidase activity is reported for the first time for a wild type strain of Saccharomyces cerevisiae. Both forms of enzyme, i.e. that specifically active toward H2O2 alone and that decomposing also organic peroxides, were found to be present. The H2O2 specific form disappeared when cells were grown in the absence of oxygen, while the other form was much increased under the same conditions. Addition of copper to the culture greatly increased both forms. The results show that glutathione peroxidase is to be included, as an important component that is also highly responsive to oxidative environments, in the enzyme defense system of yeast against oxidative damage.     

Trichinella spiralis: role of non-H-2 genes in resistance to primary infection in mice

Two strains of mice which share identical H-2 genes but differ in their genetic backgrounds were compared for their ability to resist infection with Trichinella spiralis. The two strains of mice, C3HeB/FeJ and AKR/J, share the H-2k haplotype which is associated with susceptibility to primary infection with T. spiralis in H-2 congenic strains of mice. AKR/J mice, infected with 150 infective muscle larvae, harbored significantly fewer muscle larvae 30 days postinfection than did mice of the strain C3HeB/FeJ. Approximately equal numbers of worms establish in the small intestine of AKR and C3H mice, but the AKR mice expelled adult worms from the gut more rapidly than did mice of the C3H strain. By Day 9 postinfection, 50% of the worms had been expelled by the AKR mice whereas expulsion of worms from C3H mice was delayed beyond Day 9 and occurred primarily between Days 10 and 12. Over this same experimental period (Days 6-12), fecundity of female worms from AKR mice, measured as the mean newborn larvae/female/hour, was approximately one-half that of worms taken from C3H mice. These results support the conclusion that genes outside of the mouse H-2 complex regulate expulsion of adult worms from the gut. These background genes also markedly influence the fecundity of female worms.     

Rat intestinal peroxidase: inhibition by endogenous xanthine and xanthine oxidase

The high-speed supernatant from homogenates of rat small intestine contains a heat-stable, dialyzable factor which showed a time-dependent inhibition of peroxidase activity in salt extracts of the tissue. The inhibitor was purified by chromatography on Dowex 50W-X8 and identified as xanthine. The inhibition of peroxidase by xanthine was prevented by allopurinol, an inhibitor of xanthine oxidase, and hypoxanthine was also found to be inhibitory. H2O2, produced in the reaction catalyzed by xanthine oxidase, was shown to be directly responsible for the observed inhibition. The time-dependent loss of peroxidase activity in the presence of xanthine or hypoxanthine occurred more rapidly in NH4Cl than in CaCl2 extracts of small intestine and was due to the difference in the initial concentration of H2O2 in these two extracts. The possible relationship between peroxidase and xanthine oxidase in the rat small intestine is discussed.     

Peroxidase Activity in the Abscission Zone of Bean Leaves during Abscission

Peroxidase activity and localization in the abscission zone of bean leaves were studied histochemically and by gel electrophoresis. Deblading of bean leaves resulted in an increase in peroxidase activity in the abscission zone 2 to 4 days after deblading with highest activity just prior to separation. In debladed plants, the cell division in six to eight layers of cells preceded separation. An ethylene treatment (8 microliters per liter) induced separation of debladed petioles in approximately 24 hours and of intact plants in 36 to 48 hours. Ethylene treatment produced similar results in both debladed and intact plants. In ethylene-treated plants, whether debladed or not, enzyme localization was restricted to only two to three layers of cells with no cell division apparent prior to separation. Infrequent cell divisions were observed after treatment with 2-chloroethylphosphonic acid (1000 micrograms per liter) (Ethephon); however, other changes were similar to those observed with ethylene. Deblading and ethylene treatment resulted in changes in the six peroxidase isozymes observed in the abscission zone. Only four were observed in samples collected 2 centimeters below the abscission zone. Peroxidase bands IV and V increased significantly in debladed and ethylene-treated plants and peroxidase VI decreased only in debladed plants. The changes in peroxidase activity were invariably observed prior to separation in all treatments.     

Trypanosome-induced suppression of responses to Trichinella spiralis in vaccinated mice.

Mice vaccinated against the gastro-intestinal (GI) nematode Trichinella spiralis by injection of muscle larval homogenate antigen express a strong immunity to subsequent infection, reflected in earlier expulsion of adult worms from the intestine and reduced female worm fecundity. Infection with Trypanosoma brucei at the time of vaccination, or at the time of infection with T. spiralis, significantly reduced the level of immunity expressed, the effect being greatest when vaccination and T. brucei infection were given together. Trypanosome infection reduced T. spiralis-specific antibody responses in vaccinated mice, the effect being most apparent against IgM, IgG1 and IgG2b, and ablated the eosinophil response to T. spiralis. In vaccinated mice infected with both trypanosomes and T. spiralis, the proliferative responses of lymphocytes to the mitogen Con A or to T. spiralis antigen were much lower than in vaccinated mice infected only with the nematode. Whereas cells from mice infected only with T. spiralis produced the cytokine IL-4 and little or no IFNgamma when stimulated in vitro, cells from animals infected with T. spiralis and with trypanosomes released large amounts of IFNgamma but no IL-4. These observations are consistent with the known, IFNgamma-dependent, nitric-oxide-mediated suppressive effects of trypanosomes on lymphocyte function and the Th1 bias associated with these infections, both of which reduce the effectiveness of the Th2-mediated responses involved in immunity against GI nematode infections. The data are discussed in the context of the possible use of vaccines against GI nematodes in ruminants in countries where concurrent trypanosome-GI nematode infections are widespread.     

The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury

Superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins form an antioxidant network protecting cells against reactive oxygen species (ROS). Catalase is a potent H2O2-detoxifying enzyme, which is unexpectedly absent in some members of the Kinetoplastida and Apicomplexa, but present in Toxoplasma gondii. In T. gondii, catalase appears to be cytosolic. In addition, T. gondii also possesses genes coding for other types of peroxidases, including glutathione/thioredoxin-like peroxidases and peroxiredoxins. This study presents a detailed analysis of the role of catalase in the parasite and reports the existence of antioxidant enzymes localized in the cytosol and the mitochondrion of T. gondii. The catalase gene was disrupted and, in addition, T. gondii cell lines overexpressing either catalase or a cytosolic 1-cys peroxiredoxin, TgPrx2, under the control of a strong promoter were created. Analysis of these mutants confirmed that the catalase activity is cytosolic and is encoded by a unique gene in T. gondii. Furthermore, the catalase confers protection against H2O2 exposure and contributes to virulence in mice. The overexpression of Prx2 also increases protection against H2O2 treatment, suggesting that catalase and other peroxidases function as a defence mechanism against endogenously produced reactive oxygen intermediates and the oxidative stress imposed by the host.     

Hydroperoxide metabolism in cyanobacteria

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.     

Transplantation of adult Trichinella spiralis between hosts: worm survival and immunological characteristics of the host--parasite relationship.

A technique for the transplantation of Trichinella spiralis worms directly into the host intestine is described. Infections established by the direct transfer of adult worms were essentially normal both in terms of their survival and reproduction and in their stimulation of, and susceptibility to, host immune responses. Worms transplanted from NIH mouse donors at intervals after infection had an equal ability to survive in the recipient, even when taken from the donor shortly before or during the process of worm expulsion, showing that expulsion does not require worms to be irreversibly damaged. It was noted, however, that after 7 days in the donor the ability of the worm to reproduce in the recipient was temporarily impaired.     

Live and dead GFP-tagged bacteria showed indistinguishable fluorescence in Caenorhabditis elegans gut

Caenorhabditis elegans has been used for studying host-pathogen interactions since long, and many virulence genes of pathogens have been successfully identified. In several studies, fluorescent pathogens were fed to C. elegans and fluorescence observed in the gut was considered an indicator for bacterial colonization. However, the grinder in the pharynx of these nematodes supposedly crushes the bacterial cells, and the ground material is delivered to the intestine for nutrient absorption. Therefore, it remains unclear whether intact bacteria pass through the grinder and colonize in the intestine. Here we investigated whether the appearance of fluorescence is indicative of intact bacteria in the gut using both fluorescence microscopy and transmission electron microscopy. In wild-type N2 C. elegans, Escherichia coli DH5α, and Vibrio vulnificus 93U204, both of which express the green fluorescence protein, were found intact only proximal to the grinder, while crushed bacterial debris was found in the post-pharyngeal lumen. Nevertheless, the fluorescence was evident throughout the lumen of worm intestines irrespective of whether the bacteria were intact or not. We further investigated the interaction of the bacteria with C. elegans phm-2 mutant, which has a dysfunctional grinder. Both strains of bacteria were found to be intact and accumulated in the pharynx and intestine owing to the defective grinder. The fluorescence intensity of intact bacteria in phm-2 worms was indistinguishable from that of crushed bacterial debris in N2 worms. Therefore, appearance of fluorescence in the C. elegans intestine should not be directly interpreted as successful bacterial colonization in the intestine.     

Sensitized photooxygenation and peroxidase-catalyzed inactivation of xanthine oxidase--evidence of cysteine damage by singlet oxygen

Xanthine oxidase (XO) has been investigated for its decreased activity in several cancerous tissues and constitutive generation of reactive oxygen species (ROS) in vivo seems to contribute significantly to its inactivation. Singlet oxygen (1O2) production has been suggested to be relevant when considering folic acid metabolism by cancer cells. Thus, the susceptibility of XO to inactivation by 1O2 generated either by the bioenergized systems folic acid/peroxidase/GSH/Mn2+/O2 and malonaldehyde/peroxidase/Mn2+/O2 or by methylene blue (MB) or eosin-sensitized photooxygenation was studied. Our results showed that other ROS were also responsible for XO inactivation when MB was used. In contrast, eosin produced almost exclusively 1O2. Kinetic studies of XO oxidation in the malonaldehyde/peroxidase system showed that histidine (His) is a competitive inhibitor with respect to XO. A similar result was observed in the eosin-photosensitized process, suggesting the involvement of 1O2 in both processes. In addition, an efficient quenching of XO oxidation by guanosine in the folic acid/peroxidase system was observed. Amino acid analysis revealed that cysteine (Cys) is more affected than other XO amino acids also prone to oxidation such as tyrosine (Tyr), methionine (Met) and His. These results indicate that 1O2 may cause oxidative damage to the Cys residues of XO, with loss of enzyme activity. Alteration of the flavin prosthetic site is hypothesized.     

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.     

Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals.

Glutathione peroxidase is a key enzyme in the antioxidant system of the cells. This enzyme has been shown to be irreversibly inactivated by H2O2, tert-butyl hydroperoxide (tert-BHP) and hydroxyl radicals when incubated without GSH. We observed that in our experimental conditions glutathione peroxidase was not degraded by trypsin or chymotrypsin while degraded by pronase, papa?n, pepsin, and lysosomal proteases. Hydroxyl radicals and superoxide anions but not H2O2 or tert-BHP could also fragment the enzyme on their own. A former incubation with H2O2, tert-BHP, or hydroxyl radicals also increased the proteolytic susceptibility of glutathione peroxidase. Like superoxide dismutase (SOD) and other oxidatively denatured proteins, glutathione peroxidase inactivated by peroxides or free radicals seems to be degraded preferentially by proteases. As hydroxyl radicals can fragment the enzyme by themselves, the increased proteolytic susceptibility afterwards is easily understood while the increased susceptibility induced by H2O2 and tert-BHP seems to be more specific.     

Mechanism of H2O2 production in porcine thyroid cells: evidence for intermediary formation of superoxide anion by NADPH-dependent H2O2-generating machinery.

Hydrogen peroxide (H2O2), which is required for thyroid hormone synthesis, has been believed to be produced at the apical cell surface of thyroid follicular cells. However, we recently found that plasma membrane from porcine thyroid exclusively generated superoxide anion (O2-) by employing a novel method for simultaneous determination of H2O2 and O2- with diacetyldeuterioheme-substituted horseradish peroxidase (diacetyl-HRP) as the trapping reagent [Nakamura, Y., Ohtaki, S., Makino, R., Tanaka, T., & Ishimura, Y. (1989) J. Biol. Chem. 264, 4759-4761]. The present study describes the mechanism of H2O2 production as analyzed by this new method. Incubation of cultured porcine follicular cells with ionomycin, a Ca-ionophore, caused an increase in oxygen uptake of about 80%. During enhanced respiration, the cells released H2O2 in an amount equivalent to the amount of oxygen consumed as judged by the formation of compound II of diacetyl-HRP, and H2O2 adduct of the peroxidase. No formation of compound III of the peroxidase, an O2- adduct, was detected during burst respiration. Thus, the intact cells exclusively released H2O2 to the outside of the cells. On the other hand, when the cell fragments from follicular cells were incubated with NADPH or NADH in the presence of Ca2+, the production of O2- was observed only during NADPH-dependent burst respiration, supporting our previous results that the plasma membrane exhibited NADPH-dependent O2(-)-generating activity. O2- production by the plasma membrane was further confirmed by analyses of the effects of superoxide dismutase (SOD) and catalase on the reaction. These results suggested that H2O2 is secondarily produced through the dismutation of O2-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peroxidase-mediated toxicity to schistosomula of Schistosoma mansoni

Guinea pig eosinophil peroxidase (EPO) was capable of killing schistosomula of Schistosoma mansoni in vitro when combined with hydrogen peroxide and a halide. Killing was measured by 51Cr release, by microscopic evaluation of viability, and by reinfection experiments in mice. Parasite killing was dependent on each component of the EPO-H2O2-halide system, was completely inhibited by catalase and azide, and was partially inhibited by cyanide. The EPO-mediated system required 10(-4) M H2O2 and 10(-4) M iodide at pH 7.0, and the schistosomula were killed with exposure to this system of less than 30 min at 37 degrees C. At pH 6.0, the EPO-mediated system showed significant cidal activity with 10(-6) M iodide. Canine neutrophil peroxidase (myeloperoxidase [MPO]) was also able to kill schistosomula in vitro in the presence of 10(-4) M H2O2 and 10(-4) iodide at pH 7.0 and pH 6.0. Physiologic concentrations of chloride (0.1 M) could substitute for iodide at pH 7.0 and pH 6.0 as the halide cofactor; however, at pH 7.0, a higher concentration of enzyme was required. These findings with isolated enzyme systems are compatible with a role for peroxidase in the host defense against schistosomula.     

Antigen production by encysted muscle larvae of Trichinella spiralis

The production of excretory-secretory antigens by encysted muscle larvae of Trichinella spiralis has been investigated immuno-histochemically using an antiserum raised by infection in rabbits and purified both before and after conjugation by ion-exchange chromatography. The specificity of the antibody for excretory-secretory products was demonstrated by the pattern of staining of live worms in vitro and the failure of the labelled antibody to stain dead, non-metabolizing worms. Using this labelled antibody, and unlabelled antibody in the immunoperoxidase system, the presence of parasite antigen-bearing cells in close proximity to encysted muscle larvae has been demonstrated. This is believed to be the first demonstration of antigen production by encysted muscle larvae in vivo. The implications of this observation to current concepts of immunity to Trichinella spiralis are discussed.     

Trichinella spiralis: peroxidase activity in isolated cells from the rat intestine

An understanding of physiologic events underlying resistance to parasitic worms depends on a knowledge of metabolic interactions between parasites and specific cells at the host-parasite interface. In the case of invasive intestinal parasites this interaction involves contact with epithelial cells and cells of the lamina propria. This investigation deals with the collection of epithelial cells and lamina propria cells from the small intestine of control rats and rats infected with the nematode, Trichinella spiralis, and measurement of peroxidase activity in these cells. Lamina propria cells were isolated by collagenase digestion of everted gut segments previously denuded of epithelium by treatment with hyaluronidase. Mean peroxidase activity in homogenates of lamina propria cells was equivalent to 40 nmoles H₂O₂ decomposed/min/mg of cell protein from control rats compared to 413 nmoles from infected animals. Epithelial cell peroxidase activity in homogenates of epithelial cells from both control and infected rats was less than 2 nmoles H₂O₂ decomposed/min/mg cell protein. The degree of contamination of lamina propria cells with epithelial cells was determined by measuring disaccharidase activity in both cell populations. The specific activity of maltase, sucrase, and trehalase in lamina propria cells was between 10 and 17% of that in epithelial cells. This work is a requisite for a study in which the role of intestinal cell peroxidase in resistance to Trichinella will be evaluated.