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As we all know, invasion of host intestinal epithelium is very important for T. spiralis to complete successfully their life cycle. However, the mechanisms that the intestinal infective larvae (IIL) invade and migrate in the intestinal epithailial cells (IECs) remain unclear until now. The related researches have been hindered since a readily operable in vitro normal model. In our earlier study, an in vitro normal IEC invasion model was established for the first time, and the abilities of the normal IECs to initiate mucosal inflammatory responses to invasion by the IIL in vitro were evaluated in this study. When the IIL were overlaid on the normal mouse IEC monolayers, they quickly within seconds invaded the monolayers and move within the IECs, leaving trails of damaged cells. Then the larvae were found to have started their molting at 12 h, and the complete cuticle was found at 24 h. The percentage of the first molt in the larvae was about 62.3%, and the percentage of the 2nd-4th molt was about 38.2% at 36 h. Real-time PCR showed that the mRNA levels of interleukin-1β (IL-1β), IL-8, epithelial neutrophil-activating peptide 78 (ENA-78), inducible nitric oxide synthase (iNOS), and monocyte chemotactic protein 2 (MCP-2) were elevated in the IECs after 7 h of infection after invasion by the IIL, and their levels were enhanced with the increase of larvae number. No changes in tumor necrosis factor-α (TNF-α) mRNA were observed after the IIL invasion. Secretion increases of IL-1β and IL-8 from the IEC monolayers invaded by T. spiralis were also detected by ELISA. Secretion increases of proinflammatory cytokines and inflammatory mediators in normal IECs can launch the acute inflammatory in response to the IIL invasion. This study would be helpful in further investigating the relationship between the host and T. spiralis, and the immune escape mechanisms of the niche established by T. spiralis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the functions of Trichinella spiralis Nudix hydrolase (TsNd) during the larval invasion of intestinal epithelial cells (IECs), development and survival in host by RNAi. The TsNd-specific double-stranded RNA (dsRNA) was designed to silence the expression of TsNd in T. spiralis larvae. DsRNA were delivered to the larvae by soaking incubation or electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of larvae treated with dsRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. After being soaked with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 65.8% and 56.4%, respectively. After being electroporated with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 74.2% and 58.2%, respectively. Silencing TsNd expression by both soaking and electroporation inhibited significantly the larval invasion of IECs in a dose-dependent manner (r1 = -0.96798, r2 = -0.98707). Compared with the mice inoculated with untreated larvae, mice inoculated with larvae soaked with TsNd dsRNA displayed a 49.9% reduction in adult worms and 39.9% reduction in muscle larvae, while mice inoculated with larvae electroporated with TsNd dsRNA displayed a 83.4% reduction in adult worms and 69.5% reduction in muscle larvae, indicating that electroporation has a higher efficiency than soaking in inhibiting the larval development and survival in mice. Our results showed that silencing TsNd expression in T. spiralis inhibited significantly the larval invasion and survival in host., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Gastrointestinal helminth infection, including Trichinella spiralis, initiates a series of intestinal structural, cellular and physiological changes. Intestinal invasion is an important stage of trichinellosis because it determines the development and subsequent course of the disease and its consequences. Apoptosis mediated by endoplasmic reticulum stress (ERS) plays a key role in infectious diseases, but the effect of T. spiralis infection on inducing apoptosis in the small intestine has been neglected. We investigated apoptosis and changes in ERS-associated apoptosis molecules in the intestine of mice with T. spiralis infection. TUNEL staining and detection of the apoptotic marker cleaved caspase 3 revealed that apoptosis occurred in the mouse intestine at days 3 and 7 post-infection. The ER chaperone 78-kDa glucose-regulated protein (GRP78) was upregulated at days 3 and 7 post-infection. The ERS-associated apoptosis molecules C/EBP homologous protein, cleaved caspase 12 and c-Jun NH2-terminal kinase were upregulated at days 3 and 7, days 3, 7 and 10 and days 7 and 10 post-infection, respectively. Thus, apoptosis occurred in the intestine of mice with T. spiralis infection, and the ERS-mediated apoptosis pathway was activated by infection with this small intestine dwelling nematode., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Trichinella spiralis causes a significantly higher parasite burden in rat muscle than Trichinella nativa. To assess whether the difference in infectivity is due to the early intestinal response, we analyzed gene expression changes in the rat jejunum during Trichinella infection with a whole-genome microarray. The rats were euthanized on day five of infection, and their jejunal mucosa was sampled for microarray analysis. In addition, intestinal histology and hematology were examined. Against our expectations, the gene expression changes were similar in both T.nativa- and T. spiralis-infected groups. The two groups were hence pooled, and in the combined Trichinella-infected group, 551 genes were overexpressed and 427 underexpressed when compared to controls (false discovery rate ≤ 0.001 and fold change at least 2 in either direction). Pathway analysis identified seven pathways significantly associated with Trichinella infection (p < 0.05). The microarray data suggested nonspecific damage and an inflammatory response in the jejunal mucosa. Histological findings, including hyperemia, hemorrhage and a marked infiltration of inflammatory cells, supported the microarray data. Trichinella infection caused complex gene expression changes that indicate a host response to tissue damage in the mucosa of the jejunum, but the changes were not notably dependent on the studied species of Trichinella., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This study was carried out to determine the influence of short chain fatty acids (SCFA) on spleen and mesenteric lymph node lymphocyte proliferation, goblet cells and apoptosis in the mouse small intestine during invasion by Trichinella spiralis. BALB/c mice were infected with 250 larvae of T. spiralis. An SCFA water solution containing acetic, propionic and butyric acids (30:15:20 mM) was administered orally starting 5 days before infection and ending 20 days post infection (dpi). Fragments of the jejunum were collected by dissection 7 and 10 dpi, and were examined for apoptotic cells in the lamina propria of the intestinal mucosa, and for goblet cells. The proliferation index of the cultured spleen and mesenteric lymph node lymphocytes with MTT test was also determined. The orally administered SCFA solution decreased the proliferation of mesenteric lymph node lymphocytes in the mice infected with T. spiralis at both examination times, but did not influence the proliferative activity of the spleen cells. Seven dpi, both in the spleen and mesenteric lymph nodes, the highest proliferation index of concanavalin A (Con A)-stimulated lymphocytes was found in the group of uninfected animals receiving SCFA animals. This tendency could still be seen 10 dpi in the mesenteric lymph nodes but not in the spleen, where the proliferation index in this group had significantly decreased. In vitro studies revealed, that butyric and propionic acids added to the cell cultures suppressed the proliferation of Con A-stimulated mesenteric lymph nodes and spleen lymphocytes taken from uninfected and T. spiralis-infected mice. Acetic acid stimulated proliferation of splenocytes taken from uninfected mice but did not affect lymphocyte proliferation in mesenteric lymph nodes from uninfected or infected mice. Orally administered SCFA increased the number of goblet cells found in the epithelium of the jejunum 7 dpi, but this number had decreased 10 dpi. The number of apoptotic cells in the lamina propria of the intestinal mucosa of animals infected with the T. spiralis and receiving SCFA was also lower, particularly 10 dpi. The above results show that SCFA can participate in the immune response during the course of trichinellosis in mice., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The differential effect of fox and pig bile and its corresponding low molecular weight fraction (LMW) was investigated on the in vitro invasion of MDCK-AA7 epithelial cell monolayers by Trichinella spiralis muscle larvae. Seven invasion experiments were performed and a total of 274 cell monolayers were examined. Fox and pig raw bile at 1:10 and 1:20 dilution and their LMW fractions at 1:10 dilution activated T. spiralis larvae to invade the cell monolayers. In addition, fox raw bile caused significantly larger cell damage than pig raw bile at both dilutions. The area of cell damage was larger at 1:10 than at 1:20 dilution for both fox and pig raw bile (p<0.05). On the other hand, there was no significant difference between the areas of cell damage caused by the LMW fractions of fox and pig bile. It is concluded that differences between host bile actions may account for differences in host susceptibility to T. spiralis.

To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.

The ability of a 43 kDa stichocyte protein from Trichinella spiralis (Tsp43) to interfere with mammalian skeletal muscle gene expression was investigated. A MYC-tagged Tsp43 construct was expressed as a recombinant protein in C2C12 myoblasts. Transfection with low amounts of expression plasmid was required for successful expression of the protein. This construct had apparent toxic effects on transfected myoblasts and ectopic green fluorescent protein expression was suppressed in myoblasts co-transfected with the Tsp43 construct. These effects may result from similarities of Tsp43 to DNase II. Use of the general DNase inhibitor aurintricarboxylic acid (ATA) enhanced expression of MYC-Tsp43 in transfected muscle cells. Myoblasts transfected with Tsp43 did not fuse well when cultured under differentiation conditions without ATA. In contrast, transfected myoblasts transiently cultured with ATA underwent fusion and differentiation. Under short-term differentiation conditions without ATA, unfused myoblasts nevertheless expressed both MYC-Tsp43 and myosin heavy chain. Collectively, the results support that Tsp43 has a role in the T. spiralis life cycle that is distinct from repressing muscle gene expression during the muscle phase of infection. While the function of Tsp43 as a DNase is under debate, the effects of ATA on transfected muscle cells were consistent with this possibility.

Duodenal and jejunal responses to infection with Trichinella spiralis were compared in weaned piglets with a "normal dirty" vs. a "clean SPF" gut flora. Histochemical staining of neutral, acidic, sialylated, and sulphated residues was used to assess biosynthetic responses in mucin-secreting goblet cells. Peanut and Ulex lectins were also used to assess responses within the intestinal glycocalyx. Histomorphometric analysis was undertaken to evaluate the distribution and staining patterns of goblet cells in villi and crypts. Our analysis showed that stored mucin within goblet cells increased more in the infected conventional animals than in the infected SPF group. This was accompanied by changes in the pattern of sulphation and sialylation in the duodenum and jejunum. The thickness of the glycocalyx was increased in both duodenum and jejunum in both infected groups. However, this effect was greater for the infected SPF animals than the infected conventional animals. No significant differences were observed between uninfected conventional and uninfected SPF pigs.

The activity of the cerebral pyruvate recycling pathway and energy metabolism in mice infected with Trichinella spiralis were investigated using (13)C-NMR and in vivo (31)P-NMR spectroscopy, respectively. The (13)C-NMR analysis, using [1,2-(13)C(2)] acetate as a substrate, of whole-brain extracts demonstrated that activity of the pathway increased when T. spiralis infection induced hypoglycemia in the host. The in vivo (31)P-NMR observation showed that the cerebral ATP in normal level sustained throughout this experiment. These findings indicate that the pyruvate recycling pathway plays a role in the energy supply to the host in hypoglycemia induced by T. spiralis infection.

The portion of skeletal muscle fiber (Nurse cell) harboring Trichinella spiralis is surrounded by an acellular capsule susceptible to digestion with collagenase. Antibodies recognized type IV and type VI collagen in the capsule, while the periodic acid Schiff reagent stained the capsule differentially, revealing at least two distinct layers. RNA analysis showed that mRNA specific for type IV and type VI collagen was present in muscle tissue on Days 9 and 15, but not on Day 3, following intracellular infection. In situ hybridization showed that most of the mRNA for both types was within the Nurse cell, and all enlarged Nurse cell nuclei were transcriptionally active for those messages. Synthesis of type IV collagen mRNA was absent by Day 24. In contrast, type VI collagen mRNA was still present at 24 days and 8 months. These results support the hypothesis that T. spiralis, either directly or indirectly, influences the synthesis of these two collagen types throughout its own developmental cycle in the Nurse cell.

Weaned rats were passively immunized with rat IgG2c monoclonal antibodies previously shown to protect infant rats against challenge with Trichinella spiralis. Although the antibodies did not protect rats against infection, the course of larval establishment in the intestine was altered in a way that has not been described previously. Specifically, larvae that invaded the epithelium were inhibited from migrating from intestinal tissue into saline during the standard recovery procedure. The inhibition was reversed at a time coincident with the first moult. Whole serum from infected rats had a similar effect. These results show that specific antibodies influence T. spiralis larvae in the intestinal epithelium, yet fail to effect their expulsion from adult rats.

The effect of oral immunization and the adjuvancy of cholera toxin (CT) were examined in mice infected with Trichinella spiralis. The mean of total muscle larvae was reduced by 36% in mice infected with Trichinella larvae in combination with CT. In mice fed soluble, particulate, or soluble/particulate antigens in combination with CT on Days 0, 14, and 21, and challenged with Trichinella larvae on Day 28, there was a significant reduction in adult worm fecundity (50%), worm size (20-30%), and the mean of total muscle larvae (75%) but no apparent effect on the rate of expulsion on Day 6 postchallenge. Following antigen feeding, but prior to challenge with Trichinella (Day 28), the immunoglobulin response was positive in only a small proportion of mice. On Day 6 following challenge with Trichinella larvae, the biliary immunoglobulin response was enhanced approximately 10-fold (P < 0.05) in all antigen-feeding treatments which included CT, compared with nonimmunized controls or antigen-feeding treatments which did not include CT. Similarly, the serum IgG response was enhanced following challenge with Trichinella larvae in treatments fed soluble, particulate, or soluble/particulate antigens with CT. The response was also enhanced in the particulate, but not in soluble or soluble/particulate antigen treatments without CT.