A novel trypsin of Trichinella spiralis mediates larval invasion of gut epithelium via binding to PAR2 and activating ERK1/2 pathway.

BACKGROUND: Proteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved. METHODOLOGY/PRINCIPAL FINDING: TsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo. CONCLUSIONS: TsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

Characterization of a novel dipeptidyl peptidase 1 of Trichinella spiralis and its participation in larval invasion.

The research aimed to describe a new Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1) and investigate its functions in the larval invasion of intestinal epithelial cells (IECs). The gene TsDPP1 was successfully replicated and produced in Escherichia coli BL21 (DE3), showing a strong immune response. TsDPP1 was detected in diverse stages of T. spiralis and showed significant expression in the intestine infective larvae (IIL) and adult worms at 6 days post infection, as confirmed by qPCR and Western blot analysis. The primary localization of TsDPP1 in this parasite was observed in cuticles, stichosomes, and embryos by using the indirect immunofluorescence assay (IIFA). rTsDPP1 exhibited the enzymatic function of natural dipeptidyl peptidase and showed specific binding to IECs, and the binding site was found to be localized on cell membrane. Following transfection with dsRNA-TsDPP1, the expression of TsDPP1 mRNA and protein in muscle larvae (ML) were decreased by approximately 63.52 % and 58.68 %, correspondingly. The activity of TsDPP1 in the ML and IIL treated with dsRNA-TsDPP1 was reduced by 42.98 % and 45.07 %, respectively. The acceleration of larval invasion of IECs was observed with rTsDPP1, while the invasion was suppressed by anti-rTsDPP1 serum. The ability of the larvae treated with dsRNA-TsDPP1 to invade IECs was hindered by 31.23 %. In mice infected with dsRNA-treated ML, the intestinal IIL, and adults experienced a significant decrease in worm burdens and a noticeable reduction in adult female length and fecundity compared to the PBS group. These findings indicated that TsDPP1 significantly impedes the invasion, growth, and reproductive capacity of T. spiralis in intestines, suggesting its potential as a target for anti-Trichinella vaccines.

A Bead-Based Assay for the Detection of Antibodies against Trichinella spp. Infection in Humans.

Human trichinellosis can be diagnosed by a combination of medical history, clinical presentation, and laboratory findings, and through detection of anti-Trichinella IgG in the patient's sera. ELISA using excretory-secretory (E/S) antigens of Trichinella spiralis larvae is currently the most used assay to detect Trichinella spp. antibodies. Bead-based assay can detect antibodies to multiple antigens concurrently; the ability to detect antibody to T. spiralis using a bead assay could be useful for diagnosis and surveillance. We developed and evaluated a bead assay to detect and quantify total IgG or IgG4 Trichinella spp. antibodies in human serum using T. spiralis E/S antigens. The sensitivity and specificity of the assay were determined using serum from 110 subjects with a confirmed diagnosis of trichinellosis, 140 subjects with confirmed infections with other tissue-dwelling parasites, 98 human serum samples from residents of the United States with no known history of parasitic infection, and nine human serum samples from residents of Egypt with negative microscopy for intestinal parasites. Sensitivity and specificity were 93.6% and 94.3% for total IgG and 89.2% and 99.2% for IgG4, respectively. Twelve percent of sera from patients with confirmed schistosomiasis reacted with the IgG Trichinella bead assay, as did 11% of sera from patients with neurocysticercosis. The Trichinella spp. bead assay to detect IgG total antibody responses has a similar performance as the Trichinella E/S ELISA. The Trichinella spp. bead assay shows promise as a method to detect trichinellosis with a possibility to be used in multiplex applications.

Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella.

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Trichinella spiralis co-infection exacerbates Plasmodium berghei malaria-induced hepatopathy.

BACKGROUND: Although Plasmodium parasites and intestinal helminths share common endemic areas, the mechanisms of these co-infections on the host immune response remain not fully understood. Liver involvement in severe Plasmodium falciparum infections is a significant cause of morbidity and mortality. However, the effect of pre-existing Trichinella spiralis infection on the immune response and liver immune-pathogenesis in P. berghei ANKA (PbANKA)-infected mice needs to be elucidated. METHODS: Outbred Kunming mice were infected with T. spiralis and 9 days later were challenged with P. berghei ANKA (PbANKA), and the investigation occurred at 13 days after co-infection. RESULTS: Compared with PbANKA-mono-infected mice, T. spiralis + PbANKA-co-infected mice had similar survival rate but lower PbANKA parasitaemia; however, there were more severe hepatosplenomegaly, increased liver and spleen indexes, and increased liver pathology observed by hematoxylin and eosin staining; higher expression levels of galectin (Gal)-1, Gal-3, CD68+ macrophages, and elastase-positive neutrophils measured by immunohistochemical staining; upregulated mRNA expression levels of Gal-1, Gal-3, cytokines (interferon-gamma (IFNγ) and interleukin (IL)-6), and M1 macrophage polarization marker (inducible nitric oxide synthase (iNOS)) in the liver, and increased expression levels of Gal-1, IFNγ, IL-6, eosinophil cationic protein, eosinophil protein X, and M1 (IL-1ß and iNOS) and M2 (Ym1) macrophage polarization markers in the spleen of co-infected mice detected by using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). In vitro study showed that compared with PbANKA-mono-infected mice, there were significantly increased expression levels of Gal-1, Gal-3, IL-6, IL-1ß, and iNOS in the peritoneal macrophage isolated from co-infected mice detected by using qRT-PCR. Correlation analysis revealed significant positive correlations between Gal-3 and IL-1ß in the peritoneal macrophages isolated from PbANKA-mono-infected mice, between Gal-3 and IFNγ in the spleen of co-infected mice, and between Gal-1 and Ym1 in the peritoneal macrophages isolated from co-infected mice. CONCLUSIONS: Our data indicate that pre-existing infection of T. spiralis may suppress P. berghei parasitaemia and aggravate malaria-induced liver pathology through stimulating Gal-1 and Gal-3 expression, activating macrophages, neutrophils, and eosinophils, and promoting mediator release and cytokine production.

The deficiency of myelin in the mutant taiep rat induces a differential immune response related to protection from the human parasite Trichinella spiralis.

Taiep rat is a myelin mutant with a progressive motor syndrome characterized by tremor, ataxia, immobility episodes, epilepsy and paralysis of the hindlimbs. Taiep had an initial hypomyelination followed by a progressive demyelination associated with an increased expression of some interleukins and their receptors. The pathology correlated with an increase in nitric oxide activity and lipoperoxidation. In base of the above evidences taiep rat is an appropriate model to study neuroimmune interactions. The aim of this study was to analyze the immune responses in male taiep rats after acute infection with Trichinella spiralis. Our results show that there is an important decrease in the number of intestinal larvae in the taiep rat with respect to Sprague-Dawley control rats. We also found differences in the percentage of innate and adaptive immune cell profile in the mesenteric lymphatic nodes and the spleen that correlated with the demyelination process that took place on taiep subjects. Finally, a clear pro-inflammatory cytokine pattern was seen on infected taiep rats, that could be responsible of the decrement in the number of larvae number. These results sustain the theory that neuroimmune interaction is a fundamental process capable of modulating the immune response, particularly against the parasite Trichinella spiralis in an animal model of progressive demyelination due to tubulinopathy, that could be an important mechanism for the clinical course of autoimmune diseases associated with parasite infection.

The effect of Trichinella spiralis on muscular activity of experimentally infected mice.

Nematodes of genus Trichinella are wide-spread zoonotic parasites, able to infect a large variety of vertebrates. Animal hosts are usually regarded as asymptomatic carriers. However, there is little data regarding the functional consequences that T. spiralis infection renders on muscle cells. The aim of the present study was to assess the effect of T. spiralis on the effort capacity of experimentally infected mice. Overall, 60 mice, divided into three groups were used: M (uninfected), L200 and L1000, infected with 200 or 1000 larvae/mouse respectively. The mice were periodically weighed and their effort capacity was evaluated (days 0, 7, 15, 35 and 60). From each group, two randomly selected mice were euthanized after evaluation carcasses were artificially digested in order to establish the number of larvae per gram (LPG). On day 0, there were no significant differences among groups. Starting with day 7, the effort capacity of infected groups decreased, with significant differences between group M and the infected groups. From day 15, the differences between the infected groups also became significant. The LPG gradually increased and the differences between groups were always significant. A strong correlation between the LPG and decreased effort capacity was noted. The present study demonstrates the reduction of muscular capacity in mice experimentally infected with Trichinella spiralis, in correlation with the infective dose, providing new insights in this parasite's transmission strategy.

EphrinB2/ephB2-mediated myenteric synaptic plasticity: mechanisms underlying the persistent muscle hypercontractility and pain in postinfectious IBS.

Patients with irritable bowel syndrome (IBS) show pain hypersensitivity and smooth muscle hypercontractility in response to colorectal distension (CRD). Synaptic plasticity, a key process of memory formation, in the enteric nervous system may be a novel explanation. This study aimed to explore the regulatory role of ephrinB2/ephB2 in enteric synaptic plasticity and colonic hyperreactive motility in IBS. Postinfectious (PI)-IBS was induced by Trichinella spiralis infection in rats. Isometric contractions of colonic circular muscle strips, particularly neural-mediated contractions, were recorded ex vivo. Meanwhile, ephrinB2/ephB2-mediated enteric structural and functional synaptic plasticity were assessed in the colonic muscularis, indicating that ephrinB2 and ephB2 were located on enteric nerves and up-regulated in the colonic muscularis of PI-IBS rats. Colonic hypersensitivity to CRD and neural-mediated colonic hypercontractility were present in PI-IBS rats, which were correlated with increased levels of cellular homologous fos protein (c-fos) and activity-regulated cystoskeleton-associated protein (arc), the synaptic plasticity-related immediate early genes, and were ameliorated by ephB2Fc (an ephB2 receptor blocker) or MK801 (an NMDA receptor inhibitor) exposure. EphrinB2/ephB2 facilitated synaptic sprouting and NMDA receptor-mediated synaptic potentiation in the colonic muscularis of PI-IBS rats and in the longitudinal muscle-myenteric plexus cultures, involving the Erk-MAPK and PI3K-protein kinase B pathways. In conclusion, ephrinB2/ephB2 promoted the synaptic sprouting and potentiation of myenteric nerves involved in persistent muscle hypercontractility and pain in PI-IBS. Hence, ephrinB2/ephB2 may be an emerging target for the treatment of IBS.-Zhang, L., Wang, R., Bai, T., Xiang, X., Qian, W., Song, J., Hou, X. EphrinB2/ephB2-mediated myenteric synaptic plasticity: mechanisms underlying the persistent muscle hypercontractility and pain in postinfectious IBS.

In vitro silencing of a serine protease inhibitor suppresses Trichinella spiralis invasion, development, and fecundity.

In a previous study, immunoproteomics was used to identify a serine protease inhibitor (TsSPI) of T. spiralis excretory/secretory (ES) proteins that exhibited an inhibitory effect on trypsin enzymatic activity, but the precise role of TsSPI on worm infection and development in its host is not well understood. The objective of the present study was to use RNA interference to ascertain the function of TsSPI in larval invasion and growth. TsSPI-specific small interference RNAs (siRNAs) were delivered to muscle larvae (ML) to silence TsSPI expression by electroporation. Four days after electroporation, the ML transfected with 2 µM siRNA-653 exhibited a 75.75% decrease in TsSPI transcription and a 69.23% decrease in TsSPI expression compared with control ML. Although the silencing of TsSPI expression did not decrease worm viability, it significantly suppressed the larval invasion of intestinal epithelium cells (IEC) (P < 0.01), and the suppression was siRNA dose-dependent (r = 0.981). The infection of mice with siRNA-653-treated ML produced a 63.71% reduction of adult worms and a 72.38% reduction of muscle larvae. In addition, the length of the adults, newborn larvae, and ML and the fecundity of female T. spiralis from mice infected with siRNA-treated ML were obviously reduced relative to those in the control siRNA or PBS groups. These results indicated that the silencing of TsSPI by RNAi suppressed larval invasion and development and decreased female fecundity, further confirming that TsSPI plays a crucial role during the T. spiralis lifecycle and is a promising molecular target for anti-Trichinella vaccines.

Isolation of a Novel Flavanonol and an Alkylresorcinol with Highly Potent Anti-Trypanosomal Activity from Libyan propolis.

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans, showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4'-methyl ether (4), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans, while the purified compounds had low activity against P. falciparum. Anti-trypanosomal activity (EC50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol (10) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction.

Protease Activated Receptor-2 Induces Immune Activation and Visceral Hypersensitivity in Post-infectious Irritable Bowel Syndrome Mice.

BACKGROUND: The role of protease activated receptor-2 (PAR-2) in the pathogenesis of abdominal pain in irritable bowel syndrome (IBS) is not well defined. AIMS: To investigate the role of PAR-2-mediated visceral hypersensitivity in a post-infectious IBS (PI-IBS) mouse model. METHODS: T. spiralis-infected PI-IBS mouse model was used. Fecal serine protease activity and intestinal mast cells were evaluated. Intestinal permeability was assessed by urine lactulose/mannitol ratio, and colonic expressions of PAR-2 and tight junction (TJ) proteins were examined by Western blot. Intestinal immune profile was assessed by measuring Th (T helper) 1/Th2 cytokine expression. Visceral sensitivity was evaluated by abdominal withdrawal reflex in response to colorectal distention. RESULTS: Colonic PAR-2 expression as well as fecal serine protease activity and intestinal mast cell counts were elevated in PI-IBS compared to the control mice. Decreased colonic TJ proteins expression, increased lactulose/mannitol ratio, elevated colonic Th1/Th2 cytokine ratio, and visceral hypersensitivity were observed in PI-IBS compared to the control mice. Administration of PAR-2 agonist in control mice demonstrated similar changes observed in PI-IBS mice, while PAR-2 antagonist normalized the increased intestinal permeability and reduced visceral hypersensitivity observed in PI-IBS mice. CONCLUSIONS: PAR-2 activation increases intestinal permeability leading to immune activation and visceral hypersensitivity in PI-IBS mouse model.

Effect of Muscle Strength by Trichinella spiralis Infection during Chronic Phase.

Introduction:Trichinella spiralis establishes a chronic infection in skeletal muscle by developing nurse cells within muscle fibers. During symbiosis in host, changes in the muscle fibers and inflammation may affect muscle function. Methods: We investigated muscle strength and inflammation in T. spiralis-infected mice during 1 to 48 weeks after infection. Results: Muscle strength decreased compared to that in uninfected control mice during the late infection stage. Additionally, inflammatory related cytokines increased significantly during early stage of infection and then rapidly decreased. In pathological study, nuclear infiltration maintained from the early infection stage to chronic infection stage. Moreover, vacuoles and eosinophil infiltration were observed in infected muscle in chronic stage. Conclusion: These results suggest that infection by T. spiralis significantly affects muscle function was continuously being weakness because vacuoles formation and maintained nucleus and eosinophil infiltration during chronic phase of T. spiralis infection.

Desialización eritrocitaria por larvas recién nacidas de Trichinella spiralis incubadas in vitro / Erythrocyte desialylation by Trichinella spiralis newborn larvae incubated in vitro / Dessialização eritrocitária por larvas recém-nascidas da Trichinella spiralis incubadas in vitro

Trichinella spiralis es la especie que causa la mayoría de los casos de infección humana en todo el mundo. Se comunicó que el contacto de los eritrocitos con concentrados de larvas recién nacidas (LRN) no viables provoca la disminución de ácido siálico globular. El objetivo del trabajo fue estudiar la desialización eritrocitaria producida por LRN mantenidas en cultivo. Se realizaron 2 experiencias en las que se incubaron 80 larvas con 100 μL de eritrocitos en 1 mL de medio RPMI suplementado durante 1, 2, 3, 4 y 24 horas a 37 ºC. Se aplicó el Método de Titulación de la Agregación por Polibrene y se calculó Título, Score Total y CexpCASP en los eritrocitos control e incubados con LRN. Los resultados mostraron que en la primera y segunda hora la captación de ácido siálico fue moderada. A las 3 horas el título disminuyó significativamente en relación al del control y el CexpCASP (0,14±0,014) indicó la pérdida casi total de ácido siálico globular. Ambos valores se mantuvieron a las 4 y 24 horas. Al comparar con estudios similares realizados con larvas infectantes, se sugiere que, in vivo, las LRN captarían más rápidamente el ácido siálico que las larvas musculares.

Trichinella spiralis is the species that causes most human cases of infection in the world. It was reported that contact of erythrocytes with concentrates of non-viable newborn larvae (NL) causes the decrease in erythrocyte sialic acid. The objective was to study erythrocyte desialylation produced by NL maintained in culture. Two experiments were conducted, in which 80 larvae were incubated with 100 μL of erythrocytes in 1 mL of supplemented RPMI medium for 1, 2, 3, 4 and 24 hours at 37 ºC. Titration of Aggregation by Polybrene Method was used and Title, Total Score and CexpCASP were calculated in Control erythrocytes and erythrocytes incubated with NL. The results showed that the sialic acid capture was moderated in the first and second hour. At three hours of incubation, the Title decreased significantly in relation to Control and CexpCASP (0.14±0.014) indicated almost total loss of erythrocyte sialic acid. Both values were maintained at 4 and 24 hours. When compared to similar studies conducted with infective larvae, it is suggested that, in vivo, NL would capture sialic acid faster than muscle larvae.

Trichinella spiralis é a espécie que causa a maioria dos casos de infecção humana em todo o mundo. Comunicou-se que o contato dos eritrócitos com concentrados de larvas recém-nascidas (LRN), não viáveis, provoca a diminuição de ácido siálico globular. O objetivo do trabalho foi estudar a dessialização eritrocitária produzida por LRN mantidas em cultivo. Foram realizadas duas experiências nas quais se incubaram 80 larvas com 100 μL de eritrócitos em 1 mL de meio RPMI suplementado durante 1, 2, 3, 4 e 24 horas a 37 °C. Foi aplicado o Método de Titulação da Agregação por Polibreno e se calculou Título, Pontuação Total (ST) e CexpCASP nos eritrócitos. Os resultados mostraram que na primeira e segunda hora a captação de ácido siálico foi moderada. Às 3 horas o título diminuiu significativamente em relação ao do controle e o CexpCASP (0,14±0,014) indicou a perda quase total de ácido siálico globular. Ambos os valores se mantiveram às 4 e 24 horas. Ao comparar com estudos similares realizados com larvas infetantes, sugere-se que, in vivo, as LRN captariam mais rapidamente o ácido siálico que as larvas musculares.

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.

Partially Protective Immunity Induced by a 20 kDa Protein Secreted by Trichinella spiralis Stichocytes.

BACKGROUND: Trichinella spiralis infection induces protective immunity against re-infection in animal models. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Trichinella infection and immunodiagnosis. METHODS AND FINDINGS: The T. spiralis adult cDNA library was immunoscreened with sera from pigs experimentally infected with 20,000 infective T. spiralis larvae. Total 43 positive clones encoding for 28 proteins were identified; one of the immunodominant proteins was 20 kDa Ts-ES-1 secreted by Trichinella stichocytes and existing in the excretory/secretory (ES) products of T. spiralis adult and muscle larval worms. Ts-ES-1 contains 172 amino acids with a typical signal peptide in the first 20 amino acids. The expression of Ts-ES-1 was detected in both the adult and muscle larval stages at the mRNA and protein expression levels. Mice immunized with recombinant Ts-ES-1 (rTs-ES-1) formulated with ISA50v2 adjuvant exhibited a significant worm reduction in both the adult worm (27%) and muscle larvae burden (42.1%) after a challenge with T. spiralis compared to the adjuvant control group (p<0.01). The rTs-ES-1-induced protection was associated with a high level of specific anti-Ts-ES-1 IgG antibodies and a Th1/Th2 mixed immune response. CONCLUSION: The newly identified rTs-ES-1 is an immunodominant protein secreted by Trichinella stichocytes during natural infection and enables to the induction of partial protective immunity in vaccinated mice against Trichinella infection. Therefore, rTs-ES-1 is a potential candidate for vaccine development against trichinellosis.

Secretory Products of Trichinella spiralis Muscle Larvae and Immunomodulation: Implication for Autoimmune Diseases, Allergies, and Malignancies.

Trichinella spiralis has the unique ability to make itself "at home" by creating and hiding in a new type of cell in the host body that is the nurse cell. From this immunologically privileged place, the parasite orchestrates a long-lasting molecular cross talk with the host through muscle larvae excretory-secretory products (ES L1). Those products can successfully modulate parasite-specific immune responses as well as responses to unrelated antigens (either self or nonself in origin), providing an anti-inflammatory milieu and maintaining homeostasis. It is clear, based on the findings from animal model studies, that T. spiralis and its products induce an immunomodulatory network (which encompasses Th2- and Treg-type responses) that may allow the host to deal with various hyperimmune-associated disorders as well as tumor growth, although the latter still remains unclear. This review focuses on studies of the molecules released by T. spiralis, their interaction with pattern recognition receptors on antigen presenting cells, and subsequently provoked responses. This paper also addresses the immunomodulatory properties of ES L1 molecules and how the induced immunomodulation influences the course of different experimental inflammatory and malignant diseases.

Increased VGLUT3 involved in visceral hyperalgesia in a rat model of irritable bowel syndrome.

AIM: To investigate the activity of vesicular glutamate transporter-3 (VGLUT3) in a visceral hyperalgesia rat model of irritable bowel syndrome, and the role of mast cells (MCs). METHODS: Transient intestinal infection was induced by oral administration of Trichinella spiralis larvae in rats. On the 100(th) day post-infection (PI), the rats were divided into an acute cold restraint stress (ACRS) group and a non-stressed group. Age-matched untreated rats served as controls. The abdominal withdrawal reflex was used to measure the visceromotor response to colorectal distension (CRD). The expression levels of VGLUT3 in peripheral and central neurons were analyzed by immunofluorescence and western blotting. RESULTS: VGLUT3 expression in the L6S1 dorsal root ganglion cells was significantly higher in the PI group than in the control group (0.32 ± 0.009 vs 0.22 ± 0.008, P < 0.01), and there was no significant difference in the expression of VGLUT3 between MC-deficient rats and their normal wild-type littermates. Immunofluorescence showed that the expression levels of VGLUT3 in PI + ACRS rats were enhanced in the prefrontal cortex of the brain compared with the control group. CONCLUSION: VGLUT3 is involved in the pathogenesis of visceral hyperalgesia. Coexpression of c-fos, 5-hydroxytryptamine and VGLUT3 after CRD was observed in associated neuronal pathways. Increased VGLUT3 induced by transient intestinal infection was found in peripheral nerves, and was independent of MCs. Moreover, the expression of VGLUT3 was enhanced in the prefrontal cortex in rats with induced infection and stress.

Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage.

BACKGROUND: The intestinal phase is the early invasion stage of Trichinella spiralis (T. spiralis), in which muscle larvae invade intestine epithelial cells and then develop into adult worms to breed newborn larvae. Thus, intestinal infective larvae are first exposed to the immune system of the host, and antigens from the worms may be the earliest marker in the diagnosis of trichinellosis and may contribute to vaccine development to prevent Trichinella infections in pigs. METHODS: A cDNA library of intestinal infective larvae of T. spiralis at 6 hours post infection (p.i.) was constructed and immunoscreened using serum collected from pigs that were infected with T. spiralis at 26 days p.i. T. spiralis cystatin-like protein (Ts-CLP) gene encoding a 45.9 kDa protein was cloned and expressed in Escherichia coli. The rabbit antisera were generated and used to determine the location of Ts-CLP in the parasite. Transcription levels of Ts-CLP in different developmental stages of T. spiralis were observed by RT-PCR. The potential application of recombinant Ts-CLP in diagnosis against T. spiralis infection was tested by ELISA. The immune protection of recombinant Ts-CLP protein against T. spiralis infection was evaluated in mice. RESULTS: Thirty-three positive clones were selected from cDNA library, among which 20 clones encoded the same novel cystatin-like protein (Ts-CLP). Immunolocalisation and real-time quantitative PCR revealed that native Ts-CLP was localised primarily to ß-stichocytes and that the Ts-clp gene was transcribed and expressed in all developmental stages of T. spiralis. The recombinant protein rTs-CLP was recognised by pig antiserum as early as 15 days p.i., and could induce protective immunity in mice, with a 61.21% reduction in the number of muscle larvae. CONCLUSIONS: These data preliminarily suggested that Ts-CLP may play an important role in the early infection of T. spiralis and that recombinant Ts-CLP protein is a candidate antigen for diagnosis and vaccine development in Trichinella infections.

Eosinophils mediate protective immunity against secondary nematode infection.

Eosinophils are versatile cells that regulate innate and adaptive immunity, influence metabolism and tissue repair, and contribute to allergic lung disease. Within the context of immunity to parasitic worm infections, eosinophils are prominent yet highly varied in function. We have shown previously that when mice undergo primary infection with the parasitic nematode Trichinella spiralis, eosinophils play an important immune regulatory role that promotes larval growth and survival in skeletal muscle. In this study, we aimed to address the function of eosinophils in secondary infection with T. spiralis. By infecting eosinophil-ablated mice, we found that eosinophils are dispensable for immunity that clears adult worms or controls fecundity in secondary infection. In contrast, eosinophil ablation had a pronounced effect on secondary infection of skeletal muscle by migratory newborn larvae. Restoring eosinophils to previously infected, ablated mice caused them to limit muscle larvae burdens. Passive immunization of naive, ablated mice with sera or Ig from infected donors, together with transfer of eosinophils, served to limit the number of newborn larvae that migrated in tissue and colonized skeletal muscle. Results from these in vivo studies are consistent with earlier findings that eosinophils bind to larvae in the presence of Abs in vitro. Although our previous findings showed that eosinophils protect the parasite in primary infection, these new data show that eosinophils protect the host in secondary infection.

[THERAPEUTIC ACTIVITY OF MICRONIZED MEBENDAZOLE IN THE MUSCULAR PHASE OF EXPERIMENTAL TRICHINELLA SPIRALIS INVASION IN ALBINO MICE].

The incidence of trichinosis in Russia was 0.07 per 100,000 population in 2014, which was 2.9-fold higher than that in 2013. Two WHO recommended medications mebendazole and albendazole are now used to treat humari trichinosis. The drugs are active against only mature helminths and non-encysted muscle larvae. The original oil suspension of micronized mebendazole was.found to have 100% efficacy against trichinosis in albino mice in the late muscular phase (encysted larvae) of hyperinvasion after intensive therapy under lifetime diagnostic guidance during and after a treatment cycle. The lifetime diagnostic method used to evaluate the larvicidal activity of anti-trichinosis agents in animals with experimental trichinosis revealed the signs of viaility, established a trend for deatih of Trichinella larvae, and determined their destructive changes.