OBJECTIVE: To evaluate the protective effect of excretory-secretory proteins from Trichinella spiralis muscle larvae (Ts-MES) on sepsis-induced myocardial injury in mice. METHODS: Eighty male BALB/C mice were randomized equally into sham-operated group, myocardial injury group, Ts-MES treatment group and dexamethasone treatment group. In the latter 3 groups, sepsis-induced myocardial injury models were established by cecal ligation and perforation; the sham operation was performed by exposure of the cecum without ligation or perforation. Forty minutes after the operation, the mice were given intraperitoneal injections 150 μL PBS, 20 μg TS-MES or 0.3 mg/kg dexamethasone as indicated. At 12 h after the operation, 6 mice were randomly selected from each group for echocardiography, and 8 mice were used for observing the survival rate within 72 h. The remaining 6 mice were examined for myocardial pathologies with HE staining and serum levels of NTPro-BNP and cTnI with ELISA; the expressions of TNF-α, IL-6, IL-10 and TGF-β in the serum and myocardial tissue were detected using ELISA and qRT-PCR. RESULTS: Compared with the sham-operated mice, the septic mice showed significantly decreased cardiac function indexes (LVEF, LVFS, and E/A) with lowered survival rate within 72 h (P < 0.001) and significantly higher myocardial injury scores and serum levels of NTPro-BNP and cTnI (P < 0.01). Treatment with TS-MES significantly improved the cardiac function and 72-h survival rate (P < 0.05) and lowered the myocardial injury scores and serum levels of NTPro-BNP and cTnI (P < 0.05) in the septic mice. Compared with the sham-operated mice, the septic mice had obviously increased TNF-α and IL-6 levels in the serum and myocardial tissue (P < 0.001), which were significantly lowered by treatment with TS-MES (P < 0.05). TS-MES and dexamethasone both increased the levels of IL-10 and TGF-β in the septic mice, but the changes were significant only in TS-MES-treated mice (P < 0.05). CONCLUSION Ts-MES are capable of protecting against myocardial injury in septic mice by reducing the production of pro-inflammatory cytokines and enhancing the levels of regulatory cytokines.
Animals ; Cytokines ; Dexamethasone ; Heart Injuries ; Interleukin-10 ; Interleukin-6 ; Larva ; Male ; Mice ; Mice, Inbred BALB C ; Myocardium ; Sepsis ; Transforming Growth Factor beta ; Trichinella spiralis ; Tumor Necrosis Factor-alpha

The immune response against Trichinella spiralis at the intestinal level depends on the CD4⁺ T cells, which can both suppress or promote the inflammatory response through the synthesis of diverse cytokines. During the intestinal phase, the immune response is mixed (Th1/Th2) with the initial predominance of the Th1 response and the subsequent domination of Th2 response, which favor the development of intestinal pathology. In this context, the glucocorticoids (GC) are the pharmacotherapy for the intestinal inflammatory response in trichinellosis. However, its therapeutic use is limited, since studies have shown that treatment with GC suppresses the host immune system, favoring T. spiralis infection. In the search for novel pharmacological strategies that inhibit the Th1 immune response (proinflammatory) and assist the host against T. spiralis infection, recent studies showed that resiniferatoxin (RTX) had anti-inflammatory activity, which decreased the serum levels of IL-12, INF-γ, IL-1β, TNF-α, NO, and PGE₂, as well the number of eosinophils in the blood, associated with decreased intestinal pathology and muscle parasite burden. These researches demonstrate that RTX is capable to inhibit the production of Th1 cytokines, contributing to the defense against T. spiralis infection, which places it as a new potential drug modulator of the immune response.
Cytokines ; Drug Therapy ; Eosinophils ; Glucocorticoids ; Immune System ; Inflammation* ; Interleukin-12 ; Parasites ; Pathology ; T-Lymphocytes ; Therapeutic Uses* ; Trichinella spiralis ; Trichinellosis*

BACKGROUND/AIMS: Currently, there exists no biomarker for visceral hypersensitivity in irritable bowel syndrome (IBS). Piezo proteins have been proven to play an important role in the mechanical stimulation to induce visceral pain in other tissues and may also be a biomarker candidate. The aim of this study was to test the expressions of Piezo1 and Piezo2 proteins in the intestinal epithelial cells from different intestinal segments and to explore the correlation between Piezo proteins expression and visceral pain threshold. METHODS: Post-infectious IBS was induced in mice via a Trichinella spiralis infection. Visceral sensitivity was measured with abdominal withdrawal reflex to colorectal distention. Inflammation in the small intestine and colon was scored with H&E staining. Expression location of Piezo proteins was confirmed by immunohistochemistry. Abundance of Piezo proteins were measured with real-time reverse transcriptase polymerase chain reaction. RESULTS: Piezo1 and Piezo2 proteins were expressed in the intestinal epithelial cells. The expression levels of Piezo1 and Piezo2 were abundant in the colon than the small intestine (P < 0.001 for Piezo1, P = 0.003 for Piezo2). Expression of Piezo2 in the colon significantly correlated to the visceral sensitivity (r = −0.718, P = 0.001) rather than the mucosal inflammation. CONCLUSION: Piezo2 is a candidate biomarker for visceral hypersensitivity in IBS.
Animals ; Colon ; Epithelial Cells ; Humans ; Hyperalgesia ; Hypersensitivity* ; Immunohistochemistry ; Inflammation ; Intestine, Small ; Ion Channels ; Irritable Bowel Syndrome* ; Mice ; Reflex ; Reverse Transcriptase Polymerase Chain Reaction ; Trichinella spiralis ; Visceral Pain

OBJECTIVETo observe the effect of Trichinella spiralis and its worm-derived proteins on cecal ligation and puncture (CLP)-induced sepsis in mice.

METHODSEighty male BALB/c mice were randomly divided into sham-operated group, CLP group, Trichinella spiralis muscle larvae (ML) pre-infection group (ML+CLP group), soluble muscle larvae proteins (SMP) treatment group (SMP+CLP group) and excretory-secretory proteins (MES) treatment group (MES+CLP group). In ML+CLP group, the mice were orally infected with 300 Trichinella spiralis muscle larvae at 28 days before CLP and those in the other groups were intraperitioneally injected with PBS or SMP (25 µg/mice) or MES (25µg/mice) 30 min after CLP. The general condition and 72-h survival after CLP of the mice were observed. The levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (Cr), TNF-α, IL-6, IL-1β, IL-10 and TGF-β were measured at 12 h after the operation, and the pathological changes of the liver and kidney were observed.

RESULTSs Compared with the sham-operated mice, the mice in CLP group showed decreased 72-h survival, obviously increased ALT, AST, BUN, Cr, TNF-α, IL-6, IL-1β, IL-10, and TGF-β with hepatic cords disorder, hepatocytes swelling, glomerulus shrinkage, and renal tubular cell edema. Compared with CLP group, the mice in ML+CLP group showed lowered levels of ALT, AST, Cr, TNF-α and IL-1β and increased levels of IL-10 and TGF-β; in SMP+CLP group, the levels of ALT, AST, Cr, TNF-α and IL-1β were decreased and TGF-β increased. In MES+CLP group, the mice showed obviously increased 72-h survival with lowered levels of ALT, AST, BUN, Cr, TNF-α, IL-6 and IL-1β, increased levels of IL-10 and TGF-β, and alleviated liver and kidney damages.

CONCLUSIONTrichinella spiralis and its worm-derived proteins can decrease the levels of pro-inflammatory cytokines and increase immunomodulatory cytokines, and MES has more potent effect to reduce structural and functional damages of the liver and kidney.

Alanine Transaminase ; blood ; Animals ; Antigens, Helminth ; pharmacology ; Aspartate Aminotransferases ; blood ; Blood Urea Nitrogen ; Cecum ; Creatinine ; blood ; Cytokines ; blood ; Helminth Proteins ; pharmacology ; Kidney ; physiopathology ; Kidney Diseases ; therapy ; Ligation ; Liver ; physiopathology ; Liver Diseases ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Sepsis ; therapy ; Trichinella spiralis

The immune correlate of host resistance induced by reinfection of Trichinella spiralis remains unclear. In this study, we investigated immune correlates between the resistance and serum IgG antibody level, CD23⁺ IgM⁺ B cells, and eosinophil responses induced by T. spiralis reinfection. Mice were primarily infected with 10 or 100 T. spiralis larvae (10 TS, 100 TS), respectively, and after 4 weeks, they were challenge infected with 100 T. spiralis larvae (10–100 TS, 100-100 TS). Upon challenge infections, 10–100 TS mice induced significantly higher levels of T. spiralis-specific total IgG antibody responses in sera and antibody secreting cell responses in spleens compared to 100-100 TS mice, resulting in significantly reduced worm burdens in 10–100 TS mice (60% and 70% reductions for adult and larvae, respectively). Higher levels of eosinophils were found in mice primarily infected with 10 TS compared to those of 100 TS at week 8 upon challenge. CD23+ IgM+ B cells were found to be increased significantly in mice primarily infected with 10 TS. These results indicate that primary infection of 10 larvae of T. spiralis, rather than 100 larvae, induces significant resistance against reinfection which closely correlated with T. spiralis-specific IgG, eosinophil, and CD23+ IgM+ B cell responses.
Adult ; Animals ; Antibody Formation ; B-Lymphocytes ; Eosinophils ; Humans ; Immunoglobulin G ; Larva ; Mice* ; Spleen ; Trichinella spiralis* ; Trichinella*

As most infections by the helminth parasite elicit the recruitment of CD4+CD25+Foxp3+ T (T(reg)) cells, many scientists have suggested that these cells could be used for the treatment of immune-mediated inflammation and associated diseases. In order to investigate the distribution and alteration of activated T(reg) cells, we compared the expression levels of T(reg) cell activation markers in the ileum and gastrocnemius tissues 1, 2, and 4 weeks after infection. The number of T(reg) cells was monitored using GFP-coded Foxp3 transgenic mice. In mice at 1 week after Trichinella spiralis infection, the number of activated T(reg) cells was higher than in the control group. In mice at 2 weeks after infection, there was a significant increase in the number of cells expressing Foxp3 and CTLA-4 when compared to the control group and mice at 1 week after infection. At 4 weeks after infection, T. spiralis was easily identifiable in nurse cells in mouse muscles. In the intestine, the expression of Gzmb and Klrg1 decreased over time and that of Capg remained unchanged for the first and second week, then decreased in the 4th week. However, in the muscles, the expression of most chemokine genes was increased due to T. spiralis infection, in particular the expression levels of Gzmb, OX40, and CTLA-4 increased until week 4. In addition, increased gene expression of all chemokine receptors in muscle, CXCR3, CCR4, CCR5, CCR9, and CCR10, was observed up until the 4th week. In conclusion, various chemokine receptors showed increased expressions combined with recruitment of T(reg) cells in the muscle tissue.
Animals ; Gene Expression ; Helminths ; Ileum ; Inflammation ; Intestines ; Mice* ; Mice, Transgenic ; Muscles ; Parasites ; Receptors, Chemokine ; T-Lymphocytes, Regulatory* ; Trichinella spiralis ; Trichinella*

Eosinophils are multifunctional leukocytes implicated in protection against helminth infections. Although eosinophils comprise between 1~5% of peripheral blood leukocytes, they primarily reside in the gastrointestinal tract under homeostatic conditions, and rapidly proliferate upon parasitic infection. Intestinal infection with Trichinella spiralis (T. spiralis) induces eosinophilia when the parasite enters the larval stages and larvae finally migrate to the skeletal muscle. Eosinophils are known to mediate parasite death through antibody-dependent cellular cytotoxicity. In this study, we aimed to address the functional significance of eosinophils in the intestinal phase of T. spiralis infection by analysis of immune responses in the Peyer's patch (PP) of infected BALB/c and eosinophil-ablated ΔdblGATA mice. Trafficking of eosinophils to the PP was significantly increased, with upregulation of interleukin-5 at 14 days post infection. Eosinophil deficiency led to a significant augmentation of serum immunoglobulin (Ig) M and IgG1 antibody levels. In accordance with this, IgG1+ B cells in the PP were substantially increased in ΔdblGATA mice compared to that in BALB/c mice. Transforming growth factor-β expression in the PP of infected ΔdblGATA mice was significantly decreased compared to that in BALB/c mice, whereas the number of T. spiralis larvae in the diaphragm was increased. Taken together, these findings indicate that eosinophils contribute to the regulation of Th2 immune responses, and protect the host from T. spiralis attempting to establish larvae in the skeletal muscle.
Animals ; B-Lymphocytes ; Diaphragm ; Eosinophilia ; Eosinophils* ; Gastrointestinal Tract ; Helminths ; Immunoglobulin G ; Immunoglobulins ; Interleukin-5 ; Larva ; Leukocytes ; Mice ; Muscle, Skeletal ; Parasites ; Trichinella spiralis* ; Trichinella* ; Up-Regulation

BACKGROUND/AIMS: Mesenteric afferent nerves (MANs) play a pivotal role in the visceral-nociceptive perception. Inappropriate activation of MANs may be involved in the pathogenesis of post-infectious irritable bowel syndrome (PI-IBS). However, the underlying mechanisms remain unclear. We assessed the effects of mucosal mediators from different bowel segments of guinea pigs with PI-IBS on MAN firing and the role of mast cells. METHODS: PI-IBS was induced in guinea pigs by Trichinella spiralis infection. Inflammation in terminal ileum, proximal and distal colon was scored with hematoxylin-eosin staining, and mast cell infiltration was assessed with immunofluorescence. We determined the effects of supernatant extracted from the mucosa of different bowel segments of PI-IBS on MANs activity, and assessed the role of mast cells in this process. RESULTS: Eight weeks after infection, intestinal inflammation resolved, whereas mast cell numbers increased significantly in terminal ileum and proximal colon (P < 0.05) compared with findings in controls. Mucosal supernatant from different bowel segments of PI-IBS models, but not from controls, significantly enhanced the frequency of MAN firing (terminal ileum 41.01 +/- 7.60 Hz vs. 26.55 +/- 0.67 Hz, P = 0.001; proximal colon 45.90 +/- 11.20 Hz vs. 30.88 +/- 6.92 Hz, P = 0.002; distal colon 48.25 +/- 9.70 Hz vs. 29.47 +/- 6.13 Hz, P < 0.001). In addition, the excitatory effects were inhibited by mast cell stabilizer Nasmil (terminal ileum, 32.71 +/- 2.52 Hz, P = 0.030; proximal colon, 30.94 +/- 4.44 Hz, P = 0.002; distal colon, 27.15 +/- 5.83 Hz, P < 0.001). CONCLUSIONS: Supernatant from the intestinal mucosa of different bowel segments of PI-IBS models markedly enhanced the MAN firing in a mast cell-dependent manner, indicating that mast cell-mediated MAN activation plays an important role in the pathogenesis of PI-IBS.
Animals ; Colon ; Fires ; Fluorescent Antibody Technique ; Guinea Pigs* ; Hyperalgesia ; Ileum ; Inflammation ; Intestinal Mucosa ; Irritable Bowel Syndrome* ; Mast Cells ; Mucous Membrane ; Trichinella spiralis ; Visceral Afferents

In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (T(reg)) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and T(reg) cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP-/- MEF cells, and quite substantially decreased in TRIF-/- MEF cells. In contrast, IL-10 and TGF-beta expression levels were not elevated in MyD88/TIRAP-/- MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and T(reg) cell mediated immune responses, although additional data are needed to convincingly prove this observation.
Animals ; Gene Expression ; Humans ; Interleukin-10/genetics ; Mice ; Mice, Knockout ; Th2 Cells/metabolism ; Toll-Like Receptors/*genetics/metabolism ; Trichinella spiralis/genetics/*physiology ; Trichinellosis/genetics/metabolism/*parasitology

OBJECTIVETo study the effect of Trichinella spiralis (T.spiralis) infection on the expression and distribution of colonic epithelial E-cadherin in mice and its mechanism.

METHODSBALB/c mice and STAT6-/- mice were infected with T.spiralis, and mice without infection were used as control. Seven days later, the horseradish peroxidase (HRP) was infused by rectal enema. Serum HRP was detected in the subsequent 0, 60 and 120 minutes. Then the mice were sacrificed and colon was taken out. The distribution of E-cadherin in colon was detected by immunofluorescence staining, and the expression of E-cadherin was detected by Western blot. The expression of interleukin-4 (IL-4) in mesenteric lymph nodes was detected by ELISA.

RESULTSSerum HRP level in infected BALB/c mice was significantly higher than that in control mice (P<0.05), while it was not significantly different between infected STAT6-/- mice and controls (P>0.05). In infected BALB/c mice, E-cadherin located in cytoplasm of colonic epithelial cells, while in controls, it located in cellular membrane. E-cadherin expression down-regulated significantly in infected BALB/c mice as compared to controls. E-cadherin expression and distribution did not change obviously in infected STAT6-/- and control mice. IL-4 level in mesenteric lymph nodes of infected BALB/c mice [(193.0±12.5) μg/L] was significantly higher as compared to control BALB/c and infected STAT6-/- mice [(21.0±2.3) μg/L and (15.0±3.1) μg/L, all P<0.05].

CONCLUSIONT.spiralis infection can increase colonic epithelial permeability of mice, which may be associated with induction of Th2 cytokine secretion.

Animals ; Cadherins ; metabolism ; Colon ; metabolism ; Disease Models, Animal ; Female ; Interleukin-4 ; metabolism ; Intestinal Diseases, Parasitic ; metabolism ; Intestinal Mucosa ; metabolism ; Lymph Nodes ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Knockout ; Trichinella spiralis ; Trichinellosis ; metabolism