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.

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.

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.

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.

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.

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.

Persistent epithelial barrier alterations in a rat model of postinfectious gut dysfunction.

BACKGROUND: Mucosal mast cells (MMCs), epithelial barrier function (EBF) and the enteric nervous system (ENS) are interactive factors in the pathophysiology of functional gastrointestinal disorders. We characterized postinfectious EBF alterations in the Trichinella spiralis infection model of MMC-dependent intestinal dysfunction in rats. METHODS: Sprague-Dawley rats were infected with T. spiralis. 30 ± 2 days postinfection, jejunal EBF (electrophysiological parameters, fluorescein isothiocyanate-dextran fluxes and responses to secretagogues and MMC degranulators) was evaluated (Ussing chamber). In some experiments, participation of secretomotor neurons was examined by tetrodotoxin (TTX) pretreatment. Jejunal histology and MMC count and activity were also assessed. KEY RESULTS: 30 ± 2 days postinfection, when only a low grade inflammation was observed, increased MMC number and activity were associated with altered EBF. EBF alterations were characterized by increased mucosal permeability and ion secretion. In T. spiralis-infected animals, secretory responses to serotonin (5-HT) and immunoglobulin E (IgE)-dependent activation of MMCs were reduced. In contrast, responses to substance P (SP) and capsaicin were similar in infected and noninfected animals. Neuronal blockade with TTX altered secretory responses to SP and capsaicin only in infected rats. CONCLUSIONS & INFERENCES: Trichinella spiralis infection in rats, at late stages, results in persistent postinfectious intestinal barrier dysfunctions and mucosal mastocytosis, with other signs suggestive of a low grade inflammation. The altered permeability and the TTX-independent hyporesponsiveness to 5-HT and IgE indicate epithelial alterations. Changes in responses to SP and capsaicin after neuronal blockade suggest an ENS remodeling during this phase. Similar long-lasting neuro-epithelial alterations might contribute to the pathophysiology of functional and postinfectious gastrointestinal disorders.

The impact of Trichinella spiralis excretory-secretory products on dendritic cells.

Parasitic nematode Trichinella spiralis exert immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from the encysted muscle larvae. Rat bone-marrow derived dendritic cells (DCs) stimulated with ES L1 antigens acquire semi-matured status and induce Th2 and regulatory responses in vitro and in vivo. Priming naïve T cells in vitro with ES L1 pulsed DCs caused strong Th2 polarization, accompanied by elevated production of regulatory cytokines IL-10 and TGF-ß and no increase in the proportion of CD4+CD25+Foxp3+ among the effector T cell population. In vivo T cell priming resulted in mixed Th1/Th2 cytokine response, with the dominance of the Th2 type and elevated levels of regulatory cytokines. Significant increase in the proportion of CD4+CD25+Foxp3+ cells was found among recipient's spleen cells. We have achieved to create immune status characteristic for the live infection by in vivo application of DCs educated with ES L1 antigens.

Protein change of intestinal epithelial cells induced in vitro by Trichinella spiralis infective larvae.

The aim of this study was to observe the protein changes of intestinal epithelial cells induced in vitro by Trichinella spiralis infective larvae and their excretory-secretory (ES) or surface antigens and identity the proteins related with invasion. HCT-8 cells were incubated for 2 h in the culture medium contained ES or surface antigens of infective larvae, and observed by Immunofluorescent test (IFT). The infective larvae were inoculated into culture of HCT-8 cells to incubate for 18 h, and the lysates of HCT-8 cells were analyzed by SDS-PAGE and Western blot. IFA showed that normal HCT-8 cells had positively reactions with sera of the infected mice and mice immunized with ES or surface antigens. However, after incubating with ES or surface antigens, HCT-8 cells had stronger positively reaction with the above sera. On Western blot, after cultured with infective larvae, additional seven protein bands (66, 61, 57, 45, 34, 21, and 17 kDa) of HCT-8 cells were recognized by sera of the infected or immunized mice, but three protein bands (48, 43, and 23 kDa) of HCT-8 cells were not recognized by the above sera, compared with normal HCT-8 cells. Our results showed that after cultured with infective larvae the protein components of HCT-8 cell changed, suggesting that additional seven proteins recognized by sera of the infected or immunized mice may be related with invasion of intestinal epithelial cells by infective larvae, these proteins might mediate or facilitate entry into the cells, while the three proteins not recognized by the above sera may be the specific mediators released from the cells which permit invasion.

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages.

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.

[In vitro effect of immune sera on the invasion of Trichinella spiralis infective larvae into intestinal epithelial cells and their development].

OBJECTIVE: To observe the effect of sera from mice immunized with excretory-secretory (ES) antigen or surface antigen of Trichinella spiralis larvae on the invasion of intestinal epithelial cells in vitro by the larvae and on their development. METHODS: HCT-8 cells grown to confluence were overlaid with the larvae suspended in semisolid medium (RPMI 1640 medium +1.75% agarose), and the larvae were then observed by using an inverted microscope after being incubated at 37 degrees C under 5% CO2 for 12, 24, 36, 72, 96 h. The larval development and its invasion into intestinal epithelial cells were observed under inverted microscope after 15 min when HCT-8 cells were overlaid with the larvae suspended in semisolid medium supplemented with immune sera. Finally, the 1st stage and 2nd-4th stage larvae were observed and enumerated by indirect fluorescent antibody test (IFAT) after 36 h incubation. RESULTS: When the larvae were cultured in semisolid medium, they invaded the HCT-8 cell monolayer and molted 1-2 times at 36 h to 72 h of culture. Early adult was observed at 96 h of culture. Cephalic caps on larvae were found at 15 min of culture when the larvae were cultured with medium containing immune sera, but the caps were not observed on those cultured with sera of normal mice or without sera. And the larvae with cephalic caps did not invade the cell monolayer. When the larvae were cultured with immune sera for 36 h, the percentage of 2nd-4th stage larvae (2.25%, 2.20%) were significantly lower than that of those cultured in normal sera (24.7%) (P < 0.05). CONCLUSION: The sera from mice immunized with excretory-secretory antigen or surface antigen of T. spiralis larvae prevent the invasion of the larvae into intestinal epithelial cells in vitro and impede the development (ecdysis) of the larvae.

Parasite challenge as host resistance models for immunotoxicity testing.

Identification of potentially immunosuppressive compounds typically involves assessing a combination of observational endpoints as surrogates for functional endpoints and functional endpoints as surrogates for resistance to infectious or neoplastic disease. Host resistance assays are considered to be the "gold standard" against which suppression of immune function at the molecular or cellular level can be judged, because resistance to infection, regardless of the actual pathogen, involves multiple pathways of effector function to neutralize or eliminate pathogens. Resistance to infection with the parasitic nematode Trichinella spiralis has been used to assess immune function following exposure to a variety of immunotoxicants at the whole animal level. The various immunological mechanisms that are responsible for resistance to different phases of the life cycle are well documented, as are the effects of immunosuppression on the outcome of infection. This chapter describes methods to assess elimination of adult parasites from the small intestine, body burdens of larvae, as well as antibody responses and lymphocyte responses to parasite antigens.

Hypoglycaemia induced by Trichinella infection is due to the increase of glucose uptake in infected muscle cells.

The present study investigates how Trichinella infection induces host hypoglycaemia and explores a potential relationship between infection and the insulin signalling pathway. The results showed that mice infected with Trichinella spiralis or Trichinella pseudospiralis exhibited a temporary decrease in blood glucose level between 8 and 28 days p.i. and the kinetics of the glucose levels corresponded to the process of muscle larval growth and development. Histochemical results showed that glycogen accumulation increased in infected muscle cells during the period of hypoglycaemia. Analysis of gene expression profiles with quantitative PCR demonstrated that insulin signalling pathway-related genes, such as insulin receptor (IR), insulin receptor substance 1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and V-akt murine thymoma viral oncogene homologue 2 (Akt2) were up-regulated in infected muscle cells during infection and these expression changes correlated with the kinetics of blood glucose level, glycogen accumulation and the process of larval growth and development in infected muscle cells. Western blot analysis clarified that the expression of IR and Akt2 proteins increased in muscle tissues infected with both species of Trichinella. This study suggests that hypoglycaemia induced by Trichinella infection is the result of an increase in glucose uptake by infected muscle cells via up-regulation of insulin signalling pathway factors.

Current trends in tissue-affecting helminths.

Some helminths have by their evolution learnt to systemically invade a host organism, and to select specific organs or host cell types as predilection site to reside, maturate or even proliferate. These parasites needed to develop complex and unique strategies to escape host immune reactions. The present work sheds some light into the strategy developed by three different helminths (Echinococcus multilocularis, Trichinella spiralis and Toxocara conis) to survive in the host organ or host cell, respectively. The crucial role of periparasitic host reactions that may help the host to control the parasite, but which may also be responsible for immunopathological events harmful to the host himself, are elucidated as well. Finally, for these three parasites selected, the murine host appears an acceptable model for carrying out experimental studies, as for these parasites, rodents as well as humans become infected in the parasites natural life cycle. Therefore, conclusions drawn from murine experiments may provide much more reliable data in view of their relevance for the human infection, a fact that frequently lacks when using mice as experimental model for other helminths.

Candidate genes responsible for common and different pathology of infected muscle tissues between Trichinella spiralis and T. pseudospiralis infection.

The gene expression profiles were compared between Trichinella spiralis- and T. pseudospiralis-infected muscle tissues by means of a cDNA microarray. Out of 30,000 genes, the expressions of 55 genes were up-regulated in both T. spiralis and T. pseudospiralis infections, 24 genes were down-regulated in both Trichinella infections, 30 genes were up-regulated only in T. spiralis infection, 23 genes were down-regulated only in T. spiralis infection, 25 genes were up-regulated only in T. pseudospiralis infection, and 21 genes were down-regulated only in T. pseudospiralis infection. Many of these differentially expressed genes were associated with satellite cell activation and proliferation (paired box gene 7, Pax7; Pax3; desmin; M-cadherin), myogenesis and muscle development (eyes absent 2 homolog, Eya2; myocyte enhancer factor 2C, MEF2C; pre B-cell leukemia transcription factor 1, Pbx1; chordin-like 2, Chrdl2), cell differentiation (galectin 1; insulin like growth factors, IGFs; c-ski; msh-like 1, Msx1; Numb), cell proliferation and cycle regulation (retinoblastoma 1, Rb1; granulin; p21, CDK4, cyclin A2), and apoptosis (tumor necrosis factor receptor 1, TNF-R1; programmed cell death protein 11, Pdcd11; Pdcd1; nuclear protein 1, Nuprl; clusterin, CLU). The differential expression of 17 genes was validated by quantitative real time PCR and 15 genes showed identical results with the microarray analysis. The present study listed the candidate genes that were commonly and differentially expressed between T. spiralis and/or T. pseudospiralis infection, thus suggesting that these genes need to be further investigated to reveal the mechanism of the common and/or different pathological changes induced by the two species Trichinella.

Efecto de la temperatura en la viabilidad e infectividad de Trichinella spiralis / Effect of the temperature in the viability and infectividad of Trichinella spiralis

Trichinella spiralis es el parásito causante de la Trichinellosis una zoonosis endémica en nuestro estado la cual afecta a mamíferos entre ellos el humano. La principal causa de infección para este es el consumo de carne mal cocida de cerdo infectada con la larva infectante (LI) de T. spiralis. El propósito de este trabajo es determinar el efecto que tiene la temperatura sobre este parásito para poder proporcionar a la población medidas profilácticas las cuales se puedan poner en práctica de manera sencilla y económica contra esta enfermedad. Material y Métodos: En este estudio se utilizaron 2 modelos experimentales: A) 2 cerdos raza York de 6 meses de edad (control y problema) el control sin infección y un problema infectado con T. spiralis para evaluar el efecto de la temperatura (cocción, frita, horno de microondas, olla de presión, refrigeración y congelación) en la viabilidad e Infectividad por medio de digestión artificial para obtener paquete larvario y observación directa al microscopio por compresión para observar sus características físicas y B) 3 ratas Long Evans se inocularon con muestras de carne (de cada procesamiento de temperatura) infectada obtenida del modelo de cerdo en cuánto a los resultados de los procedimientos empleados a temperaturas altas (cocción y frita) por compresión en la cocción a 96ºC la viabilidad dió positiva hasta los 30 min. Después de este tiempo sus características no fueron viables (sus características físicas se observaron alteradas), en cuánto a la Infectividad (capacidad de completar su ciclo vital), se observó positiva hasta el minuto 30, después de este tiempo la Infectividad fue negativa. Al observar los resultados obtenidos podemos concluir que T. spiralis es un parásito con gran capacidad de resistencia a condiciones extremas de temperatura, siendo efectivo el cocimiento de la carne en la olla de presión a los 30 minutos la infectividad y viabilidad fue negativa, en caso de no contar con la olla de presión.

Trichinella pseudospiralis infection is characterized by more continuous and diffuse myopathy than T. spiralis infection.

A time course study was performed to reveal the sequence of histopathology after Trichinella spiralis or T. pseudospiralis infection in mice. A cyst was formed in the former case by about 18 days post infection and prominent myopathy was restricted within the cyst. In the latter case, however, no typical cyst was formed, and myopathy spread diffusely over the infected muscle tissues occupying half the area of muscle sections. An electron microscope observation revealed that the disintegration of muscle cells was delayed in T. pseudospiralis infection than in T. spiralis infection. Quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that apoptosis-related genes were expressed for a longer term in muscles infected with T. pseudospiralis than in those with T. spiralis, although the same spectrum of genes are mobilized. Examined apoptosis-related genes included tumor suppressor genes p53, p53; mouse double minute 2, MDM2; cyclin-dependent kinase inhibitor p21 (WAF1), p21(waf) ; Bcl-2 associated protein X, BAX; apoptotic protease activating factor 1, Apaf-1; Caspase 9 and serine/ threonine protein kinase, PKB. Micro-dissection of the infected muscle tissue and subsequent RT-PCR confirmed that the expressions of these genes are restricted to tissue with myopathy. Thus, the expression of the apoptosis-related genes correlated with continuous and diffuse myopathy caused by T. pseudospiralis infection.

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

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