Ubiquitin-Dependent Modification of Skeletal Muscle by the Parasitic Nematode, Trichinella spiralis.

Trichinella spiralis is a muscle-specific parasitic worm that is uniquely intracellular. T. spiralis reprograms terminally differentiated skeletal muscle cells causing them to de-differentiate and re-enter the cell cycle, a process that cannot occur naturally in mammalian skeletal muscle cells, but one that holds great therapeutic potential. Although the host ubiquitin pathway is a common target for viruses and bacteria during infection, its role in parasite pathogenesis has been largely overlooked. Here we demonstrate that the secreted proteins of T. spiralis contain E2 Ub-conjugating and E3 Ub-ligase activity. The E2 activity is attributed to TsUBE2L3, a novel and conserved T. spiralis enzyme located in the secretory organ of the parasite during the muscle stages of infection. TsUBE2L3 cannot function with any T.spiralis secreted E3, but specifically binds to a panel of human RING E3 ligases, including the RBR E3 ARIH2 with which it interacts with a higher affinity than the mammalian ortholog UbcH7/UBE2L3. Expression of TsUBE2L3 in skeletal muscle cells causes a global downregulation in protein ubiquitination, most predominantly affecting motor, sarcomeric and extracellular matrix proteins, thus mediating their stabilization with regards to proteasomal degradation. This effect is not observed in the presence of the mammalian ortholog, suggesting functional divergence in the evolution of the parasite protein. These findings demonstrate the first example of host-parasite interactions via a parasite-derived Ub conjugating enzyme; an E2 that demonstrates a novel muscle protein stabilization function.

Eosinophils preserve parasitic nematode larvae by regulating local immunity.

Eosinophils play important roles in regulation of cellular responses under conditions of homeostasis or infection. Intestinal infection with the parasitic nematode, Trichinella spiralis, induces a pronounced eosinophilia that coincides with establishment of larval stages in skeletal muscle. We have shown previously that in mouse strains in which the eosinophil lineage is ablated, large numbers of T. spiralis larvae are killed by NO, implicating the eosinophil as an immune regulator. In this report, we show that parasite death in eosinophil-ablated mice correlates with reduced recruitment of IL-4(+) T cells and enhanced recruitment of inducible NO synthase (iNOS)-producing neutrophils to infected muscle, as well as increased iNOS in local F4/80(+)CD11b(+)Ly6C(+) macrophages. Actively growing T. spiralis larvae were susceptible to killing by NO in vitro, whereas mature larvae were highly resistant. Growth of larvae was impaired in eosinophil-ablated mice, potentially extending the period of susceptibility to the effects of NO and enhancing parasite clearance. Transfer of eosinophils into eosinophil-ablated ΔdblGATA mice restored larval growth and survival. Regulation of immunity was not dependent upon eosinophil peroxidase or major basic protein 1 and did not correlate with activity of the IDO pathway. Our results suggest that eosinophils support parasite growth and survival by promoting accumulation of Th2 cells and preventing induction of iNOS in macrophages and neutrophils. These findings begin to define the cellular interactions that occur at an extraintestinal site of nematode infection in which the eosinophil functions as a pivotal regulator of immunity.

Trichinella spiralis: infection changes serum paraoxonase-1 levels, lipid profile, and oxidative status in rats.

Paraoxonase-1 (PON1) is an HDL-associated enzyme with anti-atherogenic properties. Reduced PON1 activity has previously been observed in Nippostrongylus brasiliensis-infected rats. However, the effect of chronic zoonotic nematode infections on serum PON1 activity has not yet been studied. Therefore, we evaluated the effect of Trichinella spiralis infection on serum PON1 activity, the lipid profile, and oxidative stress in rats. There were significant reductions in serum PON1 activities (Day 2-Week 7 post-infection) in rats infected with T. spiralis, and these reductions were associated with significant increases in the serum levels of triglyceride and LDL/VLDL, as well as a significant reduction in the level of HDL. Moreover, T. spiralis infection was associated with a status of oxidative stress indicated by increased concentrations of superoxide dismutase and malondialdehyde. Given the zoonotic prevalence of T. spiralis and the cardioprotective role of PON1, further mechanistic research in this area is warranted.

Expulsion of secondary Trichinella spiralis infection in rats occurs independently of mucosal mast cell release of mast cell protease II.

Our aim was to elucidate the contribution of mucosal mast cells to the effector phase of a secondary immune response to Trichinella spiralis. During secondary infection, rats expel 90-99% of T. spiralis first-stage larvae from the intestine in a matter of hours. This phenomenon appears to be unique to rats and has been called rapid expulsion. Primary intestinal infection by T. spiralis induces mastocytosis, and mast cell degranulation occurs when challenged rats exhibit rapid expulsion. These observations have engendered the view that mast cells mediate rapid expulsion. In this study, we report that immunization of adult Albino Oxford rats by an infection limited to the muscle phase did not induce intestinal mastocytosis, yet such rats exhibited rapid expulsion when challenged orally. Although mastocytosis was absent, the protease unique to mucosal mast cells, rat mast cell protease II (RMCPII), was detected in sera at the time of expulsion. We further evaluated mast cell activity in neonatal rats that display rapid expulsion. Pups born to infected dams displayed rapid expulsion, and RMCPII was detected in their sera. By feeding pups parasite-specific mAbs or polyclonal Abs before challenge infection, it was possible to dissociate mast cell degranulation from parasite expulsion. These results indicate that rapid expulsion can occur in the absence of either intestinal mastocytosis or RMCPII release. Furthermore, release of RMCPII is not sufficient to cause expulsion. The data argue against a role for mast cells in the mechanism underlying the effector phase of protective immunity against T. spiralis in rats.

Trichinella spiralis induces de novo expression of group IVC phospholipase A2 in the intestinal epithelium.

Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes.

[Effect in vitro of albendazole on the kinetics of cytosolic glutathione transferase from the rat liver]. / Badania in vitro wplywu albendazolu na kinetyke reakcji ka- talizowanej przez cytozolowa transferaze glutationowa (GST) z watroby szczura.

INTRODUCTION: Since the idea of multifunctional mode of action of anthelmintics is considered and in experimental trichinellosis in vivo albendazole seems to act as an allosteric activator of cytosolic GST from mice muscles, in this study a termosensitivity after in vitro incubation with albendazole of purified commercial cytosolic glutathione transferase (GST) from the rat liver was investigated. METHODS: Two extremal temperatures: -80 degrees C and +30 degrees C were used to destroy the dimer in quaternary structure of this enzyme. RESULTS: In control preparations both extremal temperatures destroy this structure, so the Michaelis-Menten kinetic curves of substrate saturation show the typical hyperbolic shape. After a long (15 h) freezing at -80 degrees C or heating (up to 14 h at +30 degrees C) the kinetics of substrate saturation of GST after incubation with albendazole show the sigmoidal or "double sigmoidal" shape, pointing out the quaternary GST structure as a complex of "frozen subunits". Drug inhibits about 6-times the total activity of GST after incubation at +30 degrees C. We conclude that albendazole in vitro influences the structure of cytosolic GST from the rat liver and inhibits its activity, but, in opposite to in vivo study in mouse muscles infected with Trichinella spiralis larvae, does not act as an activator of this enzyme.

Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain.

Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation.

Effect of aminoguanidine and albendazole on inducible nitric oxide synthase (iNOS) activity in T. spiralis-infected mice muscles.

The aim of this study was to provide evidence for the expression of iNOS in the cells of inflammatory infiltrates around larvae in skeletal muscles of T. spiralis infected mice. The BALB/c mice (n = 8) divided into subgroups, received either aminoguanidine (AMG)--a specific iNOS inhibitor or albendazole (ALB)--an antiparasitic drug of choice in trichinellosis treatment. Control animals (n = 2 in each subgroup) were either uninfected and treated or uninfected and untreated. Frozen sections of hind leg muscles from mice sacrificed at various time intervals after infection were cut and subjected to immunohistochemistry, using monoclonal anti-iNOS antibody. The ALB-treated mice revealed stronger iNOS staining in the infiltrating cells around larvae than the infected and untreated animals. On the contrary, in the AMG-treated animals, the infiltrating cells did not show any specific iNOS reaction. These data confirm the specificity of iNOS staining in the cellular infiltrates around T. spiralis larvae and shed some light on the role of nitric oxide during ALB treatment in experimental trichinellosis.

Enhanced lysophospholipase activity in sensitized mice challenged with Trichinella spiralis: a role for cell-cooperation.

The relation between lysophospholipase activity and eosinophils was studied using in vivo and in vitro systems. The results support a role for cell cooperation in the eosinophils production of lysophospholipase during a parasitic infection. In vivo experiments demonstrated that the enzyme activity and numbers of tissue eosinophils in immune mice challenged with 200 Trichinella spiralis larvae were not temporally related as previously hypothesized. The data showed that the peak tissue enzyme response occurred well before that of the eosinophil, suggesting increased tissue lysophospholipase activity to be due to more than an increase in numbers of eosinophils infiltrating parasitized tissue. In vitro experiments demonstrated that the eosinophils production of lysophospholipase was influenced by lymphocytes and macrophages after antigen stimulation. We hypothesize that a significant amount of lysophospholipase in parasitized tissue results from each eosinophil synthesizing more of the enzyme after interaction with these cells of the specific immune system.

Up-regulation of the 31 kDa dehydroascorbate reductase in the modified skeletal muscle cell (nurse cell) during Trichinella spp. infection.

Ascorbic acid (AA) is an important factor of defence against oxidative stress. AA is maintained in the reduced functional form by glutathione (GSH)-dependent dehydroascorbate (DHA) reducing enzymes, including the cytosolic glutaredoxin, the microsomal protein disulphide isomerase, and a DHA reductase of 31 kDa, hereafter referred to as DHAR, purified from rat liver cytosol and human red cells. As these mechanisms have rarely been studied in parasites, we looked for the possible presence of this 31 kDa protein in Trichinella spiralis L(1) larvae. Biochemical data, immunoblot analysis and immunohistochemical studies suggested the absence of this protein within parasites at this stage. However, they possess a low DHA reducing ability, which is probably due to the presence of glutaredoxin. On the other hand, immunohistochemical studies performed in histological sections of muscle tissue from Trichinella-infected animals showed an increase in DHAR in the nurse cell (NC) of T. spiralis- and Trichinella britovi-infected animals, compared with the surrounding muscle fibres. This result was confirmed by immunoblot analysis, whereas no such increase was observed in Trichinella pseudospiralis-infected animals. In the modified skeletal muscle cell also haeme oxygenase 1 increased, as well as lipoperoxidised proteins. Both findings suggest an oxidative stress of the NC, which might be related to the intense inflammatory reaction which surrounds the NC-parasite complex. Another possibility to explain the increase in DHAR could be that the NC needs to recycle a substantial amount of AA to synthesise the collagen capsule.

Enteral and parenteral feeding to evaluate malabsorption in intestinal parasitism.

General evidence of malnutrition such as loss in body weight associated with intestinal parasitism has been attributed to decreased food intake, to intestinal malabsorption, and to change in host basal metabolism. To establish the relative importance of these factors in this regard, rats with trichinosis were studied. The weights of infected and uninfected animals were followed after being placed on one of three feeding regimens for 1 week--stock diet ad libitum, intraduodenal nutrition, and intravenous nutrition. Infected rats on a stock diet lost weight whereas those on the other two regimens maintained the same weight pattern as uninfected counterparts. The maintainance of body weight occurred despite alterations at the level of the intestinal brush border as indicated by a depression of intestinal disaccharidase activities (sucrase and lactase) and by reduction of monosaccharide absorption (measured as accumulation of beta-methyl glucoside) in the proximal, heavily infected region of the small intestine. There was no compensatory increase in enzyme activity nor in the absorptive capacity in the distal gut. Results support the conclusion that inadequate oral food intake rather than changes in basal metabolism or intestinal pathophysiology accounts for weight loss during the intestinal phase of infection.

Phospholipase B levels in fecal pellets from mice infected with Trichinella spiralis, Hymenolepis nana, and Schistosoma mansoni.

Fecal pellets of mice infected with Trichinella spiralis, Hymenolepis nana, and Schistosoma mansoni have been found to contain high levels of phospholipase B activity. The rise, time course and decline of the enzymatic content of the pellets correlate with the known patterns of intestinal injury and reaction due to the parasites or their eggs. Treatment with drugs (thiabendazole, niclosamide, niridazole) which are effective in suppressing the infection also prevents the rise, or causes an early decline, in the titers of phospholipase B appearing in the excreta. These findings complement the previous reports of a close correlation between accumulation of this enzyme in the intestine and infection of mice with T. spiralis and H. nana. Determination of fecal phospholipase B activity constitutes a relatively simple, quantitative, and blood-free method of following the course of infection and its response to treatment, which might be of particular advantage in long term studies and preliminary therapeutic screening.

Response of ADA and its isoenzymes in mice infected by Trichinella spiralis and treated with mimosine.

Infections caused by the nematode Trichinella spiralis (T. spiralis) are characterized by an inflammatory response in the host. The aim of this study was to identify and evaluate markers for monitoring mice infected with T. spiralis and treated with or without mimosine. The markers that have been used were total and differential white blood cell counts, subpopulations of lymphocytes, serum tADA and its isoenzymes ADA1 and ADA2 activity. The study included 3 groups of BALB/c mice. Group A consisted of 16 healthy mice, Group B of 16 mice infected with T. spiralis and treated with saline, and Group C of 16 mice infected with T. spiralis and treated with mimosine. The measurements were made once per week for the first six weeks continuously following the infection. According to our results, leukocytosis, lymphocytosis and increased percentages of adhesion molecules and CD4 lymphocytes were present in groups B and C one week post-infection. Total ADA activity as well as ADA1 and ADA2 was higher in groups B and C versus group A from the first week post-infection. The levels of tADA activity, ADA1 and ADA2 were higher in group B compared to those of group C and the difference was statistically significant (p<0.05) during the 4th week post-infection. The majority of tADA activity, essential for an efficient immune response, was derived from ADA1 which may have been produced by infected tissues. The elevated activities of tADA and ADA1 may be sensitive markers for infection of T. spiralis and for monitoring the course of the infection.

mRNA expression and immunohistochemical localization of inducible nitric oxide synthase (NOS-2) in the muscular niche of Trichinella spiralis.

The aim of this study was to demonstrate iNOS mRNA expression in muscular phase of experimental trichinellosis and to localize iNOS protein in T. spiralis-infected muscles using specific anti-iNOS monoclonal antibodies. The expression of iNOS mRNA in skeletal muscles from Trichinella spiralis-infected mice was examined using the reverse transcription PCR assay. Fragments of skeletal muscles were also subjected to the immunohistochemical reaction using specific anti-iNOS monoclonal antibodies followed by Dako-Ark test. mRNA for iNOS measured on day 21 after infection was expressed in the muscular phase of trichinellosis. Positive immunostaining for iNOS occurred in infiltrating mononuclear cells around the encapsulated larvae. iNOS-positive cells could be traced from the 21st day post infection (dpi); on 42 dpi and 90 dpi most cells expressed iNOS. By assessing expression of protein and its mRNA it can be concluded that iNOS is active in the pathology of skeletal muscle tissue in experimental trichinellosis.

Effect of anti-eosinophil serum on phospholipase B activity in mice infected with Trichinella spiralis.

Anti-eosinophil serum (AES) was administered to mice infected with 400 Trichinella spiralis larvae and its effect on peripheral eosinophilia and intestinal phospholipase B activity was assayed. The AES caused a significant, temporally related, suppression in both peripheral eosinophilia and phospholipase B activity when compared to responses found in infected mice given normal rabbit serum. The findings support the hypothesis that a parasite-induced eosinophilia is the source of elevated phospholipase B activity present in parasitized tissues.

Muscle fiber selectivity of Trichinella spiralis and Trichinella pseudospiralis (1983).

The biceps, semimembranosus, biceps femoris, and soleus muscles of female Rockland Wistar mice infected with either 1,000 Trichinella spiralis or 1,000 Trichinella pseudospiralis larvae were removed on days 12, 14, 16, and 18 post-infection (PI), sectioned and stained histochemically for their myosin ATPase activity. Light microscopic examination of the sections revealed that larvae of T. spiralis invade only the slow twitch muscle fibers, and those of T. pseudospiralis invade both the fast twitch and the slow twitch fibers. In sections obtained from mice infected with either parasite and killed on days 16 and 18 PI, identification of the majority of the infected fibers as fast twitch or slow twitch was not possible due to pathological modification of infected fibers.

The response of the intestinal epithelium in B10.A mice to infection with Trichinella spiralis.

Previous studies on intestinal trichinosis have dealt mainly with areas other than the intestinal epithelium. Since the epithelium is now known to be the parasite's habitat, its response to infection is important. Infection with Trichinella spiralis in immunologically slow-responding B10.A mice was associated with crypt hyperplasia and villus atrophy. With similar infection levels in both primary and challenge infections, there was no difference in the maximal degree of atrophy or hyperplasia between the 2 groups. However, challenged mice underwent these mucosal changes in about half the time. Expulsion of worms always occurred during regeneration of the intestinal epithelium suggesting that the host's defense mechanism of altering the kinetics of the epithelium was not the prime factor causing expulsion. Pulse labelling of enterocytes with [3H] thymidine showed that there was no significant increase in the relative size of the proliferation zone. This indicates that the crypt cell output was not altered by this parasite. Atrophy of the villus was analysed with respect to its 3-dimensional shape. There was a decrease in both height and width of the villus but not thickness. Thus, there is a real decrease in the size of the enterocyte population per villus. Histochemical staining of the enterocyte brush border by an alkaline phosphatase method showed that (1) hyperplastic crypts have an enlarged maturation zone and (2) the villus epithelium is composed entirely of mature cells. The distribution of the nematode population was compared to these changes in the intestine. Trichinella spiralis showed a marked anteriad (distal to proximal) migration prior to expulsion. Thus, utilizing a novel approach to study intestinal trichinosis, the response of the mucosal epithelium has been characterized.

Immunological characteristics and localization of the Trichinella spiralis glutathione S-transferase.

Trichinella spiralis glutathione S-transferase (TsGST) was isolated from crude extracts of L1 larvae by glutathione-affinity chromatography. Two closely migrating polypeptides with molecular masses of 28.5 and 28 kDa were identified by electrophoresis. Three isoforms of pI 5.6, 5.8, and 6.0 were detected by isoelectric focusing. Purified TsGST showed a low transferase activity as measured with 1-chloro-2,4-dinitrobenzene; glutathione peroxidase activity was also demonstrated using cumene hydroperoxide. A rabbit antiserum against TsGST reacted by western blot with crude extracts of Trichinella britovi and Trichinella nativa but not with extracts of Trichinella pseudospiralis, Fasciola hepatica, Schistosoma bovis, Schistosoma mansoni, Dirofilaria immitis, Toxocara canis, or Anisakis sp. TsGST was detected by western blot in extracts of T. spiralis adults, but not in newborn larvae or L1 excretory-secretory products; yet, an antiserum against T. spiralis excretory-secretory products reacted with TsGST. By immunoelectron microscopy, TsGST was found in the granules of the alpha- and beta-stichocytes of L1 larvae, as well as in some granules of the stichocytes of 72-hr adults. Rabbits experimentally infected with T. spiralis developed substantial levels of anti-TsGST antibodies. Moreover, circulating TsGST was detected in serum by a sandwich-enzyme-linked immunosorbent assay, isolated from serum by glutathione-affinity chromatography, and characterized as TsGST by western blot.

Intestinal phospholipase B activity in swine inoculated with Trichinella spiralis.

Trichinella spiralis was studied in outbred swine to determine whether infection would cause an increase in intestinal phospholipase B (EC 3.1.1.5) activity and in number of peripheral eosinophils. Intestinal phospholipase B activities increased and were accompanied by eosinophilia. The response was similar to that found in rodents infected with helminth parasites, thus demonstrating that phospholipase B is not unique to rodent models and is probably part of the complex immune response of the host in defense against parasitic infections.

Antioxidant enzyme levels in the erythrocytes of riboflavin-deficient and Trichinella spiralis-infected rats.

The erythrocyte antioxidant enzyme levels of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of riboflavin-deficient and Trichinella spiralis-infected rats were investigated. The rats were deprived of riboflavin at the 8th week of the experiment. At that time, the erythrocyte glutathione reductase activity coefficient (EGR AC), as an indicator of riboflavin status, was > or = 1.30 in rats fed a riboflavin-deficient diet and T. spiralis-infected rats fed a riboflavin-deficient diet showed no biochemical sign of riboflavin deficiency. At the 12th week of the experiment, the levels of catalase, SOD and GSH-Px were significantly lower in the riboflavin-deficient, T. spiralis-infected, and combined riboflavin-deficient and T. spiralis-infected, rats, compared to the control group. This may have been due to an increase in free oxygen radicals caused by riboflavin deficiency and parasitic infection.