Spiralis Antigen Research Articles

The involvement of the macrophage mannose receptor in the innate immune response to infection with parasite Trichinella spiralis

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. The MR can mediate endocytosis and phagocytosis, as well as activation of macrophages and antigen presentation. Since Trichinella spiralis antigens are rich in oligomannose residues, we investigated whether a mannose-recognizing receptor, such as the MR, participated in the host–parasite interaction. The results show that the MR (either on the surface of macrophages or in the purified form) recognizes and binds components of T. spiralis muscle larvae. The presence of parasites provoked activation of peritoneal macrophages, which was indicated by down-regulation of MR expression, and the stimulation of NO secretion. In vitro stimulation of macrophages with T. spiralis components resulted in increased NO and IL-6 production. However, while the MR was partially involved in stimulation of NO production, it did not mediate IL-6 secretion.

Immunization with Trichinella spiralis Korean Isolate Larval Excretory–Secretory Antigen Induces Protection and Lymphocyte Subset Changes in Rats

Protection and immune responses were studied in rats immunized with Trichinella spiralis muscle stage larval excretory–secretory antigen (ES Ag) without adjuvant. Protection was assessed by the degree of adult worm burden and the yield of muscle (diaphragmatic) larvae after challenge infection with live larvae. Lymphocyte subsets were identified by flow cytometry in the spleen and peripheral blood. Cytokine production and specific IgG, IgG1 and IgG2a antibody responses were measured. Immunization with ES Ag produced highly significant protection against adult stages (98.4%) and muscle larvae (82.9%). Th2 type cytokines (IL‐10, IL‐4) were predominant. Anti‐muscle stage larval ES Ag antibody was significantly elevated in the order IgG2a > IgG1 > IgG on the 2nd day after final immunization and on the 7th day after challenge infection. Expression of CD4+ and the CD4+/CD8+ ratio from spleen and blood were significantly increased compared to the control. These results demonstrate that immunization with T. spiralis antigen can elicit robust immune response, resulting in complete protection against infective larvae, and this protection can be achieved without the use of any adjuvant.

Imported human trichinellosis: sequential IgG4 antibody response to Trichinella spiralis.

Two out of five members of one household presented with clinical signs of trichinellosis after their return to the Netherlands. The family had consumed Trichinella-infected pork in Montenegro, formerly Yugoslavia. Serological tests were performed at 1, 2, 6, and 18 months after ingestion of the infected meat. Trichinella-specific IgM, IgG, and IgA antibodies measured in sera from symptomatic and asymptomatic patients remained positive up to 18 months after ingestion. The measured IgG4 antibody response directed to a 45 kDa Trichinella spiralis antigen also persisted 18 months after ingestion for three of the family members.

The effects of fexofenadine on eosinophilia and systemic anaphylaxis in mice infected with Trichinella spiralis

Background: The effects of the non-impairing, H 1-receptor antagonist fexofenadine were investigated in in vivo mouse models of eosinophilia and systemic anaphylaxis. Methods: Eosinophilia was investigated in C57BL/6 mice ( n=5 per group) infected with Trichinella spiralis, with and without administration of fexofenadine HCl (5, 10 and 20 mg/kg/day). Eosinophilia was also studied, with and without fexofenadine administration, in mice with a congenital mast-cell deficiency (W/W v) and controls (+/+). The effect of fexofenadine HCl (20 mg/kg/day) on IL-5 and eotaxin blood levels was also investigated in C57BL/6 mice. In a separate model, systemic anaphylaxis was induced in C57BL/6 mice using T. spiralis antigen. Fexofenadine HCl (5, 10 and 20 mg/kg) or vehicle was administered 20 min before antigen challenge ( n=5 per group). The effect of fexofenadine on systemic anaphylaxis caused by IgE and anti-IgE was also examined in CBF1 mice injected with serum from NC/Nga mice with high IgE levels. Rectal temperature was measured as an indicator of anaphylaxis. Results: In C57BL/6 mice, repetitive oral administration of fexofenadine HCl (5, 10 and 20 mg/kg/day) resulted in dose-dependent suppression of eosinophilia ( p<0.05–0.0001). No suppression was observed in mast-cell deficient W/W v mice. In addition, single oral administration of fexofenadine HCl (10 and 20 mg/kg) significantly suppressed the decrease in rectal temperature ( p<0.01), a marker for systemic anaphylaxis, in C57BL/6 mice. In CBF1 mice injected with serum from NC/Nga mice with high IgE levels, the decrease in rectal temperature was suppressed by single administration of fexofenadine HCl (10 and 20 mg/kg; p<0.01 and p<0.001, respectively). Fexofenadine had no effect on peripheral IL-5 and eotaxin levels. Conclusion: These results indicate that fexofenadine suppresses both eosinophilia and systemic anaphylaxis, both of which are fundamental reactions in allergic diseases.

A putative serine protease among the excretory–secretory glycoproteins of L1 Trichinella spiralis

Trichinella spiralis first-stage larvae infect susceptible hosts by invading epithelial cells that line the small intestine. During this process the larva disgorges several glycoproteins that bear an unusual, highly antigenic sugar moiety, tyvelose (3,6-dideoxy arabinohexose). Monoclonal antibodies specific for tyvelose protect the intestine against infection, implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment in the intestinal epithelium. In order to investigate these glycoproteins at the molecular level, we first prepared monoclonal anti-peptide antibodies. The antibodies bind a family of glycoproteins that are present in excretory–secretory products of first-stage larvae and are delivered to epithelial cells during invasion by T. spiralis. The major species present in an affinity purified fraction of crude T. spiralis antigens were subjected to tryptic peptide digestion. De novo amino acid sequencing of the peptides using Q-TOF tandem mass spectrometry, in combination with database searches and antibody screening of an L1 cDNA library, showed that the glycoproteins are variably glycosylated homologues of the serine protease family.

Detection of circulating and fecal Trichinella spiralis antigens during experimental infection using monoclonal antibodies against the new born larvae.

Different assays to detect antigens of Trichinella spiralis during current infection have been standardized, although sensitivity values have been the main limitation to use them as routine diagnostic test. We report the production and use of monoclonal antibodies (MAbs) against the new born larvae which recognize both somatic and metabolic antigens from adult and muscular larvae (ML). We used two IgG3 MAbs (4B1, 4B2) and two IgG2a (2D3, 2D4) to detect antigens during experimental infection. All MAbs detect fecal antigens starting the second to third week post infection (wpi), although it was less clear with MAbs 4B1 and 4B2, while circulating antigens were detected from third to fourth wpi. Thus, the recognition of shared antigens among T. spiralis developmental stages can be used for early diagnosis of trichinellosis.

Murine model study of the practical implication of trypanosome-induced immunosuppression in vaccine-based disease control programmes

The relevance of trypanosome-induced immunosuppression in relation to the efficacy of vaccine-induced immunity was studied in mice. Mice were immunised with crude Trichinella spiralis muscle larvae homogenate vaccine and infected with T. spiralis and/or Trypanosoma brucei. Vaccination significantly decreased adult worm burden ( p<0.05) and accelerated worm expulsion in mice infected with T. spiralis only. T. brucei superinfection resulted in monocytosis, suppressed eosinophilia, significant decrease in PCV ( p<0.001), higher numbers of adult worms ( p<0.001) and failure to expel all adult worms by Day 12 post infection (p.i.). Regardless, they produced anti- Trichinella IgG 1 responses similar to those of the vaccinated non- T. brucei-infected group. T. brucei also suppressed the proliferative responses of spleen cells to stimulation with Con A and T. spiralis antigen, and induced strong production of interferon-gamma (IFN-γ) in culture supernatants of antigen stimulated spleen and mesenteric lymph node cells. Interleukin-5 (IL-5) production was suppressed by T. brucei in supernatants of Con A- and antigen-stimulated spleen cells. It was concluded that trypanosome infections and the associated immunosuppression are of great practical significance in trypanosome endemic areas, especially with regards to disease control programmes involving vaccine-induced herd immunity.

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 IFN γ when stimulated in vitro, cells from animals infected with T. spiralis and with trypanosomes released large amounts of IFN γ but no IL-4. These observations are consistent with the known, IFN γ-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.

Host nuclear abnormalities and depletion of nuclear antigens induced in Trichinella spiralis-infected muscle cells by the anthelmintic mebendazole

Infection by the parasitic nematode Trichinella spiralis induces cell cycle repositioning (chronic suspension in apparent G 2/M) and genetic reprogramming in differentiated mammalian skeletal muscle cells. These changes occur in association with dramatic enlargement of infected host cell nuclei (as large as 17 μm in diameter) and nucleoli. Nuclear antigens (NA) that colocalize with host chromatin have been detected by antibodies to T. spiralis antigens, but the functions of these NA are unresolved. Mebendazole (MBZ) preferentially binds parasite versus host β-tubulins, is implicated in inhibiting secretion in nematodes and induces cytoplasmic changes in muscle cells infected with T. spiralis. These infected cell changes might be indirect via MBZ inhibition of parasite secretions. This effect would have implications for host/parasite interactions and was evaluated here. MBZ treatment of chronically infected mice caused: (1) a significant deformation of host nuclei and diminution of nucleoli by 4 and 6 days of treatment (dot), respectively; (2) a reduction of nuclear lamins A/C in infected cell nuclei that was concomitant with nuclear deformation; and (3) significant reductions in total RNA, general protein and acid phosphatase activity levels. These changes were associated with the depletion of NA from host nuclei detected by 4 dot. However, DNA content of infected cell nuclei was not detectably reduced and muscle gene expression was not reactivated. The cellular changes documented are likely to account for previously described cytoplasmic alterations induced by MBZ. Concomitant depletion of NA from infected cell nuclei suggests a role of these products in regulating nuclear functions of host cells.

Early induction and augmentation of parasitic antigen-specific antibody-producing B lymphocytes in the non-Peyer's patch region of the small intestine.

In this study, B lymphocytes from the small intestine of immunized rats were examined for their expression of specific antibodies against Trichinella spiralis (TS) antigen. The isotypes of the antigen-specific antibodies on B cells were examined via immunofluorescence microscopy. Monoclonal mouse anti-rat IgE, IgG1, IgG2a, IgG2b, IgG2c, IgA and IgM primary antibodies in conjunction with FITC-conjugated goat anti-mouse Ig secondary antibody and XRITC-conjugated 9D4 T. spiralis antigen were used to study the dynamics of the appearance of activated B lymphocytes in the small intestine, Peyer's patch, both the germinal center (PP-GC) and the non-germinal center (PP-NGC), the mesenteric lymph node (MLN), and the spleen. The results demonstrate that activated B cells are elicited by TS in the non-Peyer's patch region of the small intestine to express all isotypes of antibodies against TS antigen. IgG- and IgE-producing cells (Ab-PC) began proliferation only 1 and 2 days after infection, respectively. The strongest response was mounted by the IgE-PC in the lamina propria of the intestine. The response by IgA-PC generated was not only significantly delayed and also much weaker than that of the IgE- and IgG-PC. Peyer's patches failed to be a significant contributor in this immune response. Although this antigen-specific immune response was produced in the MLN and the spleen, it was weaker than that of the small intestine. The study indicates the potential ability of an immunized host to generate an early, yet effective, humoral immunity against T. spiralis in the non-Peyer's patch region of the small intestine."

Workshop on a detailed characterization of Trichinella spiralis antigens: a platform for future studies on antigens and antibodies to this parasite.

In order to characterize immunodominant components of T. spiralis a workshop was organized. In this the reactivity of monoclonal and polyclonal antibodies, provided by different research groups, towards total extracts from adult, new born larvae and muscle larvae as well as to excretory/secretory components of muscle larvae were tested by ELISA, Western blot and immunoprecipitation assays. As a result of this workshop T. spiralis ML antigens have been classified into eight groups (TSL-1-TSL-8) according to their recognition by monoclonal and polyclonal antibodies. Among them, TSL-1 antigens have been the most extensively characterized both biochemically and immunologically. These antigens are stage specific, originate in the muscle stichosome and are abundant in both E/S and on the larval cuticular surface. The TSL-1 antigens share an immunodominant carbohydrate epitope (tyvelose), which is unique for Trichinella and is not associated with phosphorylcholine. The data collected in this workshop has allowed both the unification of the nomenclature for T. spiralis antigens and their biochemical characterization. It also has provided a platform for further studies on the characterization of other T. spiralis antigens and indeed for other Trichinella species.

Effects of maternal ethanol consumption on the subsequent development of immunity to Trichinella spiralis in rat neonates.

The immune response of rat pups to the intestinal parasite Trichinella spiralis was studied to determine if maternal pre- and/or postnatal ethanol consumption affected neonatal immune responses. Female rats were fed ethanol-containing (36% of calories) or pair-fed control liquid diets and include groups that were maintained on ethanol as follows: group 1, from day 1 of pregnancy through weaning and whose pups were then placed on ethanol to sacrifice; group 2, from day 1 of pregnancy through lactation; group 3, from day 1 of pregnancy through pup delivery; and group 4, from day 1 of lactation through weaning. A parallel group of animals was pair-fed isocaloric control diet until sacrifice. The pups of all litters were immunized orally with 500 L1 (T. spiralis) larva 5 days after weaning. To examine the effects of maternal ethanol on primary immune responses, one-fourth of the pups from each litter were sacrificed on days 10 and 20 after immunization. To examine the effects on neonatal secondary immune responses, the remaining pups were challenged with 1,000 larva 30 days after the initial immunization and sacrificed either 3 or 8 days after challenge. At the time of sacrifice, blood samples were collected, the intestine removed to determine T. spiralis worm burdens, and suspensions of mesenteric lymph node (MLN) cells prepared. Intestinal worm counts and serum levels of anti-T. spiralis IgM and IgG antibodies, interleukin-2 (IL-2), and tumor necrosis factor (TNF) were determined. In vitro proliferation responses of MLN cells to T. spiralis antigen and to the mitogen concanavalin A (Con A) were also examined. Pups from groups 1 to 3 demonstrated significantly higher intestinal worm counts (decreased immunity) than the pair-fed controls at the day 20 primary immune response sacrifice, and pups from group 1 had significantly higher worm counts at day 3 after a secondary immune challenge. Pups of dams from groups 1, 3, and 4 had significantly lower IgM antibody titers at the day 20 primary immune response sacrifice. All experimental ethanol groups (1 to 4) demonstrated significantly lower IgG antibody titers than that observed in pair-fed control pups at the 20-day sacrifice. IgM antibody titers showed significant reductions for ethanol-treated groups at 3 and 8 days after T. spiralis secondary challenge. In addition, IgG antibody titers were also significantly reduced for all alcohol groups at 3 and 8 days during the secondary immune response. Serum IL-2 and TNF levels were significantly lower in all experimental ethanol groups (1 to 4) relative to pair-fed controls at day 20 during a primary immune response, and IL-2 levels at 3 days postchallenge were lower in groups 2 to 4 after a secondary immune challenge. MLN proliferation responses to antigen and Con A were significantly reduced in ethanol groups 1 to 3 and to Con A in group 4 at day 10 after a primary immune challenge. Ethanol group 3 pups also demonstrated a reduced response to antigen at day 20. For animals undergoing a secondary immune response, ethanol group 2 demonstrated a reduced response to antigen at day 3, whereas groups 2 and 4 showed increased reactivity to antigen at days 3 and 8 postchallenge. These results show that maternal ethanol consumption diminishes the capacity of neonates to respond to T. spiralis antigen and that the depressed immune response involves T- and B-cell-mediated reactions and also affects the production of certain cytokines. These results also suggest that the diminished immune responses are increased with longer periods of maternal and neonatal exposure to ethanol.

Cytokine profile of protective anti‐Trichinella spiralis CD4+ OX22− and non‐protective CD4+ OX22+ thoracic duct cells in rats: secretion of IL‐4 alone does not determine protective capacity

We analysed the cytokine profile of a T cell subset (CD4+ CD45 RC-) that confers protection against Trichinella spiralis infection in rats. These CD4+ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-gamma and functional cytokine secretion for IL-4, IL-5, IFN-gamma, TNF-alpha and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4+ CD45 RC+ or CD8+), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-gamma in the protective CD4+ CD45 RC- cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-gamma or TNF-alpha was secreted by the protective CD4+ CD45- RC- cells. Whereas the non-protective CD4+ CD45 RC+ cells secreted significant levels of IL-4, IFN-gamma, mast cell differentiating activity and TNF-alpha but little IL-5 activity. Non-protective CD8+ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-gamma secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.

Effect of maternal ethanol consumption on in vitro tumor necrosis factor, interleukin-6 and interleukin-2 production by rat milk and blood leukocytes.

It has been shown that the transfer of immunity via lactation plays an important role in providing early protection to the neonate. Maternal ethanol consumption also results in a reduced transfer of immunity to their neonates against a Trichinella spiralis infection. Because of the known presence of cytokines in milk and their important role in inflammation, we tested the effects of maternal ethanol consumption on cytokine production by milk and blood cells from T. spiralis-infected rats. With T. spiralis antigen, Concanavalin A (Con A) or lipopolysaccharide stimulation, milk cells from both ethanol-treated and pair-fed groups were capable of producing tumor necrosis factor (TNF), interleukin (IL)-6 and IL-2. There were no differences between groups for TNF or IL-6 production by milk cells. Milk cells from the ethanol group produced a significantly higher amount of IL-2 upon Con A stimulation, as compared with that from the pair-fed group (16 +/- 4 units/10(6) cells vs. 4 +/- 1 units/10(6) cells, p < 0.05). After stimulation with Con A, blood cells from the ethanol group produced significantly lower amounts of TNF (17 +/- 15 units/10(6) cells) than that from the pair-fed group (102 +/- 64 units/10(6) cells, p < 0.05). The amount of TNF and IL-6 produced by milk cells was significantly lower, as compared with that produced by blood cells. This study suggests that ethanol consumption has some modulatory effects on cytokine production by milk and blood cells.

Assessment of Host Resistance to Trichinella spiralis in Mice Following Preinfection Exposure to 2,3,7,8-TCDD

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been reported to decrease host resistance to a variety of infectious agents when exposure occurs prior to infection. Resistance to viral infection has been observed at doses as low as 0.1 μg TCDD/kg body wt, well below the thymolytic dose in mice. In the present study, female B6C3F1 mice were exposed to a single intraperitoneal injection of 0, 0.1, 1.0, 10.0, or 30.0 μg TCDD/kg 7 days prior to infection to determine the effects of TCDD exposure on resistance to the nematode parasite Trichinella spiralis. Exposure to 10 or 30 μg TCDD/kg delayed adult parasite elimination from the small intestine. Significantly more larvae were released by female parasites and greater numbers of encysted larvae were recovered from the muscle of mice exposed to TCDD. Proliferative responses of splenocytes and mesenteric lymph node cells stimulated with T. spiralis antigen were significantly suppressed at exposure levels of TCDD ≥ 1.0 μg/kg 7 days after infection and in splenocytes only at 14 days after infection, demonstrating the greater sensitivity of proliferative responses to TCDD exposure than actual host resistance to Ts infection. Suppressed proliferation was observed at doses which produced TCDD concentrations ≥ 0.2 pmol/g of lymphoid tissue on Day 7 of infection. In addition, it was determined that infected mice had higher TCDD levels than noninfected mice given the same dose. These results suggest an interaction between TCDD exposure and infection, i.e., that exposure to TCDD altered the host response to infection, while infection delayed elimination of TCDD from the host.

Mast cell-mediated colonic immune function and its inhibition by dietary aspirin in mice infected with Trichinella spiralis.

Because of the integrated nature of cellular elements in the gut wall, an understanding of the local mucosal immune system and its adaptive capacity should provide more insight into diseases of the colon, such as inflammatory bowel disease and colorectal cancer. To develop a method to quantify colonic mucosal immune function in situ, ion transport mediated by a type I hypersensitivity reaction was measured in the colon of mice infected with Trichinella spiralis. Segments of sensitized distal colon mounted in Ussing chambers and challenged with T. spiralis-derived antigen resulted in a rise in short-circuit current (delta Isc) that was antigen-specific and inhibited by furosemide. Colonic segments from infected, mast cell-deficient W/Wv mice were unresponsive to challenge with T. spiralis antigen. Inhibition of anaphylactic mediators with various pharmacological agents implicated prostaglandins and leukotrienes as the principal mediators of the antigen-induced delta Isc, with 5-HT also playing a role. Neural blockade with tetrodotoxin or blockade of histamine H1 receptors with diphenhydramine failed to inhibit the colonic immune response. Distal colon from immune mice fed an aspirin-containing diet (800 mg/kg powdered diet) ad libitum for 6 weeks had a decreased response to antigen. However, dietary aspirin had no effect on antigen-induced delta Isc in the jejunum or on Cl- secretagogue-stimulated delta Isc in the distal colon. These results suggest that products of arachidonic acid metabolism are important mediators of mast cell-dependent, antigen-stimulated Cl- secretion in the distal colon of mice immunized by infection with T. spiralis.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro TNF, IL-6 and IL-2 production by rat milk cells following Trichinella spiralis infection

Trichinella spiralis-specific immunity can be transferred from immune mothers to suckling neonates via lactation, suggesting that milk from immune dams contains specific factors to T. spiralis. TNF and IL-6 are important cytokines in inflammatory processes, and IL-2 is essential in lymphocyte activation. Using cell line bioassays, we examined the capacity of rat milk mononuclear cells from immune and non-immune control dams to produce these cytokines following in vitro stimulation with mitogens or T. spiralis antigen. Milk cells were capable of producing IL-2, IL-6 and TNF upon Con A stimulation, and TNF and IL-6 upon LPS stimulation. The amount of these cytokines produced by mitogen-stimulated milk cells from T. spiralis-infected rats was similar to that produced by non-infected controls, although lower than that of corresponding blood mononuclear cells. Upon stimulation with T. spiralis antigen, milk cells from infected rats produced a significantly higher amount of TNF (158 ± 39U/10 6 cells) compared to non-infected controls (16 ± 6 U/10 6 cells, P < 0.01), and also higher than that of the corresponding blood cells (60 ± 10 U/10 6 cells, P < 0.01). Only small amounts of IL-6 and no IL-2 was secreted by milk cells from control or infected groups after stimulation with antigen. This study shows that rat milk cells are capable of synthesizing cytokines, and TNF produced by immune mothers may play a role in augmenting the neonate resistance to T. spiralis infection.

Trichinella spiralis--characterization of circulating and immune-complex-associated antigens.

Recent studies on Trichinella spiralis have indicated that both surface components and secretions from T. spiralis muscle larvae play an important role in host-parasite relationships (Silberstein and Despommier 1984; Gamble 1985). Antigens released by nematodes may circulate within the host and become complexed by antibodies, In the present study, we investigated the molecular weight of circulating T. spiralis antigens and of antigens associated with immune complexes (ICs). We report on the identification of a T. spiralis antigen localized in the stichosome of muscle larvae. Outbred Wistar rats were infected with 1000 T. spiralis muscle larvae each in the course of this investigation. Anti-T. spiralis monoclonal antibody (mAb) TS 3G6 was obtained using standard methods (Tankov et al. 1987). An enzyme-linked immunosorbent assay (ELISA) was performed as described elsewhere (Kehayov et al. 1991). An indirect immunoperoxidase technique was applied as described by Barcley (1981). Pure ICs were isolated according to the method of Chia et al. (1979) from the sera of rats on the 21st day of infection with T. spiralis. Sera with a light-scattering index (%LSI) of > 50 as determined by direct laser nephelometry (Bartoloni et al. 1985) were used for the isolation of ICs. The antigens were electrophoresed in 0.1% polyacrylamide gel under reducing conditions as described by Laemmli (1970). The gel slabs were either used f o r blotting or stained with 0.25% Coomassie blue. Western blotting was done according to the method of Spandidos and Dimitrov (1985). When a total extract from Trichinella muscle larvae was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with mAb TS 3G6, three bands were stained specifically. These had mol. wt. of 76, 41, and 36 kDa, respectively (Fig. 1). Specific binding to the stichocytes was clearly seen in both longitudinal and cross sections of the larvae (Fig. 2). When mAb TS 3G6 was used in ELISA to demonstrate the presence of Trichinella antigen in ICs, the

Characteristics of T lymphocyte cell lines established from NIH minipigs challenge inoculated with Trichinella spiralis

MHC restricted, antigen specific, cytokine dependent T-cell lines were established from peripheral blood mononuclear cells (PBMC) of NIH minipigs that were challenge inoculated with Trichinella spiralis. Swine lymphocyte antigen (SLA) inbred NIH minipigs of the SLA a/a ( aa) haplotype can be divided into Responder (R) and Non-responder (NR) phenotypes on the basis of their ability to destroy encysted muscle larval (ML) forms of the parasite T. spiralis. When orally inoculated with 300 infectious ML and challenge inoculated with 10 000 ML, R pigs are able to mount an immune attack against encysted ML resident in host muscle cells since the primary inoculation. Because T-lymphocyte mediated mechanisms are likely to be operative in this response, a series of T-lymphocyte cell lines was established from PBMC of ten aa minipigs 10 days after the challenge infection with T. spiralis. These T-lymphocyte lines showed antigen specific, cytokine dependent proliferation in response to T. spiralis antigen stimulation. T-lymphocyte cell lines from R and NR pigs were compared and found to be similar in the magnitude of proliferative responses, in the duration of antigen specificity, and in the alterations in T-lymphocyte subsets and cell surface phenotype after antigen stimulation. In vitro antigen stimulation of T-lymphocyte cell lines and evaluation of small lymphocytes and lymphoblasts by density gradient centrifugation, immunofluorescent staining and flow cytometry, revealed that up to 55% of the cells were lymphoblasts at 4 days after stimulation, and that the majority of the lymphoblast population expressed CD4 and CD8, as well as the interleukin-2 receptor and the Class II alloantigen, SLA-DR (DR+). These results indicate that pig T-lymphocytes of the CD4+CD8+ phenotype may be involved in the anamnestic cell mediated immune response to T. spiralis.

Detection of circulating antigens in human trichinellosis

A ‘sandwich’ enzyme-linked immunosorbent assay was established to detect circulating antigens of Trichinella spiralis in human sera, its sensitivity and specificity was evaluated using 4 antigens ( Trichinella spiralis, Trichuris trichiura, Dirofilaria immitis and Ascaris suum), and it was found to be sensitive and specific for T. spiralis antigen. Samples of 347 individuals with suspected trichinellosis, who had eaten incompletely cooked bear meat containing larvae of T. spiralis, were examined. Among individuals showing clinical symptoms, circulating antigens were detected in 29·9%, and the prevalence of antibodies was 18·9%. Among individuals lacking clinical symptoms, antigens were detected in 21·4% and antibodies in 5·0%. It was concluded that detection of circulating antigens was more useful for making diagnoses than measurement of specific antibodies.