Human serum antibodies against shared antigens of different stages of<Emphasis Type="Italic"> Trichinella spiralis</Emphasis>: relevance of glycan and protein epitopes

Taking into account that antibodies against the surface antigens of newborn larvae (anti-NBL Abs) present in sera from individuals with chronic trichinellosis recognize antigenic determinants of the excretory-secretory muscle larva products (ML-ESP), and that these products are mainly glycoproteins, the aim of this work was to assess the frequency of anti-NBL Abs in sera from individuals with acute and chronic trichinellosis, to analyse the relevance of glycan and protein epitopes of the ML-ESP in the cross-reactivity phenomenon, and its correlation with the host's serum response towards these products. Anti-NBL surface Abs were determined in sera by indirect immunofluorescence. The degree of recognition by serum and purified anti-NBL Abs was evaluated comparatively before and after chemical deglycosylation of ML-ESP by immunoelectrotransfer blot assay. Results showed that 64% of the sera from individuals with acute trichinellosis and 35% of those belonging to the chronic phase had anti-NBL Abs, and also that the protein epitopes are the major ones responsible for the cross-reactivity phenomenon involving the ML-ESP and the NBL surface during both phases of the infection, while glycan epitopes are immunodominant in the stimulation of the host's immune system. A modulatory phenomenon in the immune response generated towards Trichinella spiralis NBL driven by the ML-ESP is postulated.     

Identification of diagnostic antigens fromTrichinella spiralis

The Western blotting technique was used to determine the antigens ofTrichinella spiralis muscle larvae that were recognized by antibodies in sera from humans and pigs displayingT. spiralis infections. This resulted in the identification of several antigens that were recognized by all sera. Some of these antigens, notably those that were recognized during the early stage of infection, cross-reacted with antibodies to other parasites. This cross-reactivity was caused by the presence of phosphorylcholine on these antigens. A large portion of the antigens that were recognized by antibodies from infected humans and pigs were found to share a singleTrichinella-specific determinant. TheTrichinella-specific antigen population could be isolated from phosphorylcholine-containing antigens by a simple two-step affinity chromatography procedure using monoclonal antibodies to both determinants. The resulting preparation consisted primarily of a single antigen showing an apparent molecular weight of 45 kDa that corresponded to a mamor constituent of excretory-secretory (ES) products of muscle larvae. When tested in an enzyme-linked immunosorbent assay (ELISA), this antigen displayed diagnostic specificity that was comparable with the ES fraction and diagnostic sensitivity comparable with the crude muscle-larvae extract.     

Trichinella spiralis Secreted Enzymes Regulate Nucleotide-Induced Mast Cell Activation and Release of Mouse Mast Cell Protease 1

Extracellular nucleotides are important triggers of innate immunity, acting on a wide variety of cells via signaling through purinergic receptors. Mucosal mast cells contribute to expulsion of a number of gastrointestinal nematode parasites, and mouse mast cell protease 1 has been shown to have a critical role in clearance of Trichinella spiralis from the intestinal tract. We show here that adenosine, ADP, ATP, UDP, and UTP all stimulate calcium mobilization in bone marrow-derived mast cells with a mucosal phenotype. Secreted proteins from T. spiralis infective larvae inhibit nucleotide-induced mast cell activation, and that induced by ADP and UDP is specifically blocked by parasite secretory 5′-nucleotidase. Release of mouse mast cell protease 1 is stimulated by ADP and ATP. Both parasite secreted products and the 5′-nucleotidase inhibit ADP-induced release of mast cell protease, whereas that stimulated by ATP is partially inhibited by secreted products alone. This indicates that the 5′-nucleotidase contributes to but is not solely responsible for inhibition of nucleotide-mediated effects on mast cell function. Secretion of nucleotide-metabolizing enzymes by parasitic nematodes most likely evolved as a strategy for suppression of innate immune responses and is discussed in this context.     

Calcification of Trichinella spiralis larval capsule

Trichinella spiralis larvae were examined in TEM to identify calcareous corpuscles in the outer part of parasite capsule. The microroentgenographic analysis of calcareous corpuscles mainly demonstrated the presence of phosphorus and calcium. The physiological importance of calcareous corpuscles, as well as their significance in decay of T. spiralis larvae were discussed.     

Antigenic differences betweenTrichinella spiralis andT. pseudospiralis detected by monoclonal antibodies

Antigenic differences betweenTrichinella spiralis andT. pseudospiralis were established using two monoclonal antibodies (mAbs) that show different specificities to muscle larvae of the two variants. Enzymelinked immunosorbent assay (ELISA) revealed that mAb 3G6 reacts positively againstT. spiralis, T. nelsoni, T. nativa andT. pseudospiralis, whereas mAb 3E10 does not react withT. pseudospiralis under the same experimental conditions. These antigenic differences were confirmed after preabsorption of the antibodies with serial dilutions of extracts ofT. spiralis orT. pseudospiralis muscle larvae. The indirect immunofluorescence technique showed that the antigen corresponding to mAb 3G6 is located in the stichosomes and the cuticle surface of bothT. spiralis andT. pseudospiralis. In contrast, mAb 3E10 positively stained cryostat sections ofT. spiralis, forming a dense reaction product on the surface of the whole larvae and the surrounding capsule. This antibody can be quite useful as a specific probe for distinguishingT. spiralis fromT. pseudospiralis in taxonomic studies. Using an avidin-biotin system, we could prove that mAb 3G6 recognizes an excretory/secretory-type antigen.     

A mouse monoclonal antibody that binds to an α-stichocyte of Trichinella spiralis

Monoclonal antibodies were generated for the isolation of specific antigens from Trichinella spiralis. A monoclonal antibody (TS32D12) of the Igg₁ class was selected according to its reactivity and specificity by enzyme-linked immunosorbent assay and immunofluorescent technique. The TS32D12 antibody was purified from ascites by fast protein liquid chromatography. The purified antibody showed a sensitive reaction to the T. spiralis antigen, but not to any other heterologous parasite antigens so far examined. Western blot analysis showed that the monoclonal antibody bound to epitopes present on the 160-kDa molecule. The antigen molecule was fragmented into 56-kDa molecules by heat treatment. The epitopes seemed to be destroyed since the antibody could not bind to the 56-kDa molecule. Staining with the periodic acid-Schiff (PAS) reagent suggested that the two molecules of 160 kDa and 56 kDa were glycoproteins. The 160-kDa molecule was detected only in the -stichocyte of T. spiralis muscle larvae.     

Preotection againstTrichinella spiralis induced by purified stage-specific surface antigens of infective larvae

The stage-specific surface antigens of the infective larvae ofTrichinella spiralis, isolated using an affinity column of monoclonal antibody (Mab) NIM-M1, consisted of four components with molecular weights of 72, 65, 52, and 47 kDa, respectively. These four components may have unique as well as shared structural features and appear to be products of the stichosome. When injected i.p. as an emulsion in complete Freund's adjuvant, the purified antigens induced protection against infection of BALB/c mice withT. spiralis, as assessed by reductions in both the muscle larvae load and the number of adult intestinal worms.     

Isolation of specific antigens fromTrichinella spiralis by the rotating horizontal ampholine column method

By preparative isoelectric focusing in a rotating ampholine column, specific antigens with molecular weights of 45, 47 and 53 kDa were successfully isolated from crude somatic extracts ofTrichinella spiralis muscle larvae. These antigens, with pI 5.5, 4.3 and 4.4, were found to possess the same epitope found in specific antigens in the excretory/secretory products that were recovered by in vitro culture of muscle larvae. When the antigens obtained by the two methods were compared using enzyme-linked immunosorbent assays, they showed similar specificity and sensitivity.     

Partial characterization of serine proteinases secreted by adult <Emphasis Type="Italic">Trichinella spiralis</Emphasis>

Serine proteinases secreted by adult Trichinella spiralis were isolated from excretory/secretory products (ES) of in-vitro-cultured parasites by affinity chromatography with p-benzamidine-celite. The purified enzymes had molecular weights of approximately 18, 40, and 50 kDa and displayed enzyme activity against a range of low-molecular-weight substrates, gelatin, and azocasein. The antigenicity of these parasite proteinases was demonstrated by the inhibition of enzymatic activity with IgG purified from infected hosts. The inactivation of major secreted proteinases of adult T. spiralis by immune antibody could presumably contribute to impairment of the survival of the parasite in sensitized hosts. Received: 29 January 2000 / Accepted: 8 February 2000     

Cross-resistance betweenTrichinella spiralis andAngiostrongylus cantonensis in laboratory rats

Summary Rats were sensitized with one dose of 20, 30, or 100, or three doses of 30Trichinella spiralis larvae and then given a challenge infection with 100 third-stage larvae ofAngiostrongylus cantonensis during different periods of development ofT. spiralis. A significant reduction in the mean number of adultA. cantonensis was found in all the experimental groups. The mean number of lung worms found in the groups varied from 56 to 68, whereas a mean of 82 was found in the control. This reduction is probably caused by the non-specific inflammatory reaction and cell-mediated response induced byT. spiralis in the intestine and other organs of the host. A weaker and slower haemagglutinating antibody response againstA. cantonensis was observed in animals infected with both species of nematodes. This may be attributed to a suppressive action elicited byT. spiralis against unrelated antigens. Changes in differential and total white blood cell counts were followed in some infected animals. An increase in neutrophils 3–4 weeks after infection with either or both species of parasites was observed. Eosinophilia occurred primarily in the lung phase of the metastrongyloid infection.     

Cloning and characterization of the mitochondrial heat-shock protein 60 gene of<Emphasis Type="Italic"> Trichinella spiralis</Emphasis>

The full-length cDNA of mitochondrial heat-shock protein (hsp) 60 of the infective-stage larva of Trichinella spiralis was cloned by degenerative PCR and rapid amplification of cDNA end reactions. The 1,945 bp full-length cDNA sequence contained an open reading frame of 576 amino acids. A mitochondrial signal peptide was located at the N-terminal and a GGM motif at the C-terminal. The gene contained ten exons and nine introns. RT-PCR analysis indicated that thermal, cold, acidic and oxidative treatment did not elicit significant changes in the expression of mitochondrial hsp 60 in the larvae. Cluster analysis showed that the sequence of the hsp 60 gene of T. spiralis is closely related to that of Drosophila melanogaster.     

Correlates of Immune Response in Trichinella spiralis Infection

Trichinella spiralis infection induces Trichinella-specific IgG antibody and high level of blood eosinophil. However, the kinetics induced by different parasite burdens during infectious periods remains unclear. In this study, rats were infected with 100, 1000, or 3000 larvae of T. spiralis (100 TS, 1000 TS, or 3000 TS). Correlates of eosinophils, antibody responses, and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) with worm burdens were evaluated at 1 week, 2 weeks, 1 month, and 2 months postinfection. Heavy infections (1000 TS and 3000 TS) showed significantly higher levels of eosinophil, IgG, and IgG1 antibody responses at 2 weeks postinfection compared to light infection (100 TS). The highest RANTES mRNA expression was also found in the heavy infection group (3000 TS). The results indicate, at early stage of infection (week 2), heavy infection induced higher levels of IgG, IgG1, eosinophil, and RANTES responses. However, at late stage of infection (month 2), there were no correlates of immunity with parasite burdens. Higher levels of IgG and IgG1 antibody responses are critical in heavy T. spiralis infection. These results provide important information in evaluating immune responses by T. spiralis infective stage during the T. spiralis infection.     

Differentiation ofTrichinella isolates by polymerase chain reaction

Oligonucleotide primers were synthesized for the polymerase-chain-reaction amplification of target DNA from two sequences ofTrichinella spiralis. Six strains belonging toT. spiralis, T. nativa, T. britovi, T. pseudospiralis, andT. nelsoni were tested. Amplification products were obtained withT. spiralis, T. britovi, andT. nelsoni DNA from a 53-kDa antigen cDNA sequence and withT. spiralis andT. nelsoni DNA from a 1.6-kb repetitive DNA sequence. Differences in the length of the amplification products obtained from the repetitive sequence would enable a differentiation betweenT. spiralis andT. nelsoni, suggesting that the 1.6 kb repetitive DNA sequence would not be specific forT. spiralis. No amplification was detected forT. nativa orT. pseudospiralis DNA from the two sequences and forT. britovi DNA from the 1.6-kb repetitive DNA sequence.     

Development of cellular immune response of mice to infection with low doses of Trichinella spiralis, Trichinella britovi and Trichinella pseudospiralis larvae

The murine cellular immune response to the infection with ten larvae of encapsulating (Trichinella spiralis, Trichinella britovi) and non-encapsulating species (Trichinella pseudospiralis) was studied. Both T. spiralis and T. britovi stimulated the proliferation of splenic T and B lymphocytes during the intestinal phase of infection, but T. spiralis activated the proliferative response also at the muscle phase, particularly in B cells. Non-encapsulating T. pseudospiralis stimulated the proliferation of T and B cells only on day 10 post-infection (p.i.) and later at the muscle phase. The numbers of splenic CD4 and CD8 T cells of T. spiralis infected mice were significantly increased till day 10 p.i., i.e., at the intestinal phase, and then at the late muscle phase, on day 60 p.i. T. britovi infection increased the CD4 and CD8 T cell numbers only on day 30 p.i. Decreased numbers of CD4 and CD8 T cells after T. pseudospiralis infection suggest a suppression of cellular immunity. Both encapsulating Trichinella species induced the Th2 response (cytokines interleukin-5 (IL-5) and interleukin-10) at the intestinal phase and the Th2 dominant response at the advanced muscle phase. Interferon-γ (IFN-γ) production (Th1 type) started to increase with migrating newborn larvae from day 15 p.i. till the end of the experiment. IL-5 production was suppressed during the intestinal phase of T. pseudospiralis infection. The immune response to T. pseudospiralis was directed more to the Th1 response at the muscle phase, the high IFN-γ production was found on day 10 p.i. and it peaked on days 45 and 60 p.i.     

The involvement of the cysteine proteases of<Emphasis Type="Italic"> Clonorchis sinensis</Emphasis> metacercariae in excystment

The effects of trypsin, bile, trypsin-bile, pepsin, dithiothreitol (DTT) and metacercarial excretory-secretory product (ESP) on the in vitro excystment of Clonorchis sinensis metacercariae were investigated. The majority of metacercariae excysted immediately in trypsin-bile in PBS solution, a process which was complete after 30 min of incubation. When incubated in metacercarial ESP in PBS, excystment was potentiated in the presence of 5 mM DTT, but was inhibited dose-dependently by a cysteine protease inhibitor, iodoacetic acid. Two active protease bands of 28 and 40 kDa were identified in the ESP of metacercariae by gelatin substrate SDS-PAGE. Scanning electron microscopy demonstrated that the larvae in solutions of DTT and ESP migrated through a small hole on the metacercarial wall, whereas larvae were liberated by entire wall disruption in trypsin solution. These results suggest that trypsin is a major extrinsic factor of the rapid excystment of C. sinensis metacercariae, and that endogenous cysteine proteases are also involved in metacercarial excystment.     

A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis

Newborn larvae of Trichinella spiralis were collected for 30 min from female worms in culture, incubated in vitro for various times up to 18 h, and surface-labelled with iodine. The detergent-solubilised products were examined by SDS-polyacrylamide gel electrophoresis. At time periods up to 6 h these larvae expressed only one M_r 64 000 iodine-labelled surface protein. Some time between 6 h and 18 h a further three components (apparent M_r 58 000, 34 000 and 32 000) became accessible to surface labelling. All four of these components are antigenic in that they can be immunoprecipitated with T. spiralis immune sera. Tryptic peptide analysis revealed that the 32 and 34 kDa antigens were structurally very similar, but the 58 and 64 kDa proteins differed from each other and the 32–34 kDa pair. Thus T. spiralis not only undergoes a total change in surface antigens between moults, but also major changes in surface antigen expression within one stage.     

Immunomodulation by Trichinella spiralis: primary versus secondary response to phosphorylcholine-containing antigens

The present work was designed to determine in detail the capacity of the nematode Trichinella to modulate the plaque-forming cell (PFC) response of BCF1 mice to the parasite's own antigens. To this end, we studied the PFC responses shown by infected and non-infected BCF1 mice using as the target antigen phosphorylcholine, an epitope which is found in the parasite. From the results presented here, the following conclusions can be drawn: i) Trichinella spiralis is capable of modulating the immunoresponse to thymus-dependent (TD), but not to thymus-independent (TI), parasite antigens; ii) Trichinella spiralis suppresses the PFC response to the parasite-derived TD antigen FCp1 (a particulate antigen containing PC) during the muscle stage of its life cycle, but does not affect the responses to other parasite-derived PC-bearing antigens; this seems to indicate that the suppressive activity exerted by Trichinella is highly specific; iii) anti-PC PFC production in the secondary response was also suppressed by the parasite. Finally, the inability of the FCp1 antigen to induce detectable anti-PC PFC, other than IgM, is discussed.     

Participation of Parasite Surface Glycoproteins in Antibody-Mediated Protection of Epithelial Cells against Trichinella spiralis

The L1 stage of the parasitic nematode Trichinella spiralis displays on its surface glycoproteins that are immunologically cross-reactive with several larval excretory-secretory (ES) products. The basis for the cross-reactivity is tyvelose, the terminal residue on the complex glycans shared by these surface and ES glycoproteins. In neonatal rats, tyvelose-specific monoclonal antibodies mediate the expulsion of larvae from the intestine. The aim of the studies described in this report was to determine how antibody binding to larval surfaces contributes to expulsion. In these experiments, which involve an in vitro assay of epithelial cell invasion, surface proteins on living larvae were biotinylated to distinguish them from ES products. Biotinylated and nonbiotinylated larvae were cocultured with avidin, biotin-specific antibodies, or anti-tyvelose monoclonal antibodies. Biotinylated larvae cultured with avidin or biotin-specific antibodies invaded Madin-Darby canine kidney (MDCK) cells equally as well as biotinylated larvae cultured with medium alone. Anti-tyvelose monoclonal antibodies were highly protective in this assay; however, biotinylation of larval surfaces hindered the ability of anti-tyvelose monoclonal antibodies to prevent larval invasion of epithelial cells. This correlated with a reduction in the binding of anti-tyvelose antibody to biotinylated larval surfaces. Our results indicate that antibody binding to surface glycoproteins contributes to protection against T. spiralis invasion but that surface binding alone is not sufficient for protection. Our findings support the notion that protection is effected by cross-linking of ES products to surface antigens.     

Analysis of differentially expressed genes of Trichinella spiralis larvae activated by bile and cultured with intestinal epithelial cells using real-time PCR

The activation of Trichinella spiralis muscle larvae (ML) by exposure to intestinal contents or bile and the intestinal epithelial cells (IECs) themselves are two pivotal requirements for the in vitro larval invasion of IECs. However, it is yet unknown which genes are involved in the process of larval invasion. The purpose of the present study was to analyze the differentially expressed genes of T. spiralis larvae activated by bile and cultured with IECs by using real-time polymerase chain reaction. Ten T. spiralis genes encoded the proteins produced by the larvae after co-culture with IECs were selected. Compared with untreated ML, four genes were up-regulated in both bile-activated and cell-cultured larvae, including calcium-dependent secretion activator (Csa; 2.55- and 16.04-fold, respectively), multi cystatin-like domain protein precursor (Mcd; 4.36 and 52), serine protease (Sp; 2.03 and 20.02), and intermediate filament protein ifa-1 (Ifa 1; 2 and 3.31). The expression of two genes, enolase (Eno; 1.51) and ribosomal protein S6 kinase beta-1 (Rsk; 1.49), was up-regulated only in cell-cultured larvae, not in bile-activated larvae. The expression of secreted 5′-nucleotidase (5 N; 1.42) and putative serine protease (Psp; 1.41) was up-regulated in bile-activated larvae, but was not changed or down-regulated after cultured with IECs. ATP synthase F1, beta subunit (ATPase; 0.58 and 0.51) and serine protease precursor (Spp; 0.42 and 0.65) were down-regulated in both bile-activated and cell-cultured larvae. This study provide some differentially expressed genes among the untreated (normal), bile-activated and cell-cultured larvae of T. spiralis. The up-regulated genes might be related with the larval invasion of IECs, but their exact biological functions need to be further investigated. This study will be helpful to further elucidate the molecular mechanism of the invasion of IECs by T. spiralis larvae and to better understand the interaction between parasite and host enterocytes.     

Antibodies in sera from multiple sclerosis patients recognize Trichinella spiralis muscle larvae excretory–secretory antigens

Helminths, as complex pathogens, possess a large number of different epitopes, some of which may be similar to the epitopes of the host. Besides being the cause for the activation of self-reactive immune cells, molecular mimicry may also be the cause for the expansion of regulatory T cells, crucial for the host tolerance of self-antigens. Amelioration of experimental autoimmune encephalomyelitis (EAE), animal model of multiple sclerosis (MS), caused by Trichinella spiralis infection or application of its muscle larvae excretory-secretory products (ES L1), was achieved through activation of Th2 and regulatory responses. The present study aimed to reveal whether the cause of observed immunomodulation could be the existence of shared epitopes between ES L1 antigens and auto-antigens. Serum samples from 92 MS patients were tested in Western blot for the reactivity toward components of ES L1. Immunoglobulins from the sera of MS patients recognized several ES L1 components, but 45, 49 and 58 kDa proteins dominated others by the frequency of interaction. According to the logistic regression analysis, these interactions were statistically significantly associated with MS, regardless of the disease phenotype or severity. Selected molecules might share homology with self-antigens and as such are worthy of further investigation in terms of potential immunomodulatory capacity and involvement in the parasite’s provoked amelioration of EAE.