Interferon-gamma treatment in mice experimentally infected withTrichinella spiralis

Interferon- (IFN-) treatment ofTrichinella spiralis-infected BALB/c mice was investigated. The therapeutic regimen consisted of daily intraperitoneal injection of 10⁴ U murine IFN- for 7 days, starting at 2 weeks post-infection. Striated muscle samples (diaphragm, thigh) were collected at 4, 8 and 12 weeks after infection. The muscle larval burden, the degree of encystation and the digestion ofT. spiralis larvae were investigated. Furthermore, immunohistochemical studies of the inflammatory cell infiltrate around encysted larvae were performed. The results demonstrated an influence of IFN- treatment on the CD4⁺ and CD8⁺ subset distribution during the immune response but revealed no difference in the degree of encystation or digestion of encapsulated larvae as compared with control values.Abbreviations in figures A artrficial shrinking zone (due to preparation of TEM) - CO collagen - CS cross sectioned Trichinella larvae - CU cuticle - D defence cell of host - DG degenerating laryae - HN hypertrophied host cell nucleus - TC inner cyst wall - Il inner layer of inner cyst wall - LS longitudinally sectioned larvae - NC normal host cell nucleus - NU nucleus - OC outer cyst wall - OL outer layer of inner cyst wall - SF striated muscle fibre - Z zone of host defence cells     

Effect of Trichinella spiralis infection on passive cutaneous anaphylaxis in mice.

Infection of CFW mice with Trichinella spiralis induced a state of relative unresponsiveness to passive cutaneous anaphylaxis (PCA) induced with hen egg albumin and its corresponding antibodies. The unresponsiveness was to PCA produced either with immunoglobulin G1 (IgG1) or IgE type of antibodies, but was more pronounced with the latter. As few as 25 larvae given by stomach tube 20 days before induced this resistance, although 400 larvae induced a greater resistance. When 400 to 600 larvae were fed to mice, the refractoriness of these mice to PCA was noticed 15 days later. The sera of infected mice had the ability to inhibit mainly PCA induced by IgE. This inhibitory property of sera from infected mice was more pronounced 35 days after infection than 10 months later, when only weak inhibitory activity was detected. Purified rat IgE inhibited the PCA reactions induced in both mice and rats with mouse IgE-type antibody. At high concentrations, evidence of inhibition of the IgG1-induced PCA in mice was also obtained. We believe that the relative unresponsiveness of infected mice is due to an increase in production of IgE which competitively blocks the mast cell sites for other IgE molecules.     

小鼠感染流感病毒后可抵抗致死性流感病毒攻击的研究

目的 BALB/c小鼠初次感染流感病毒(A/California/7/2009(H1N1)(pCA07)和A/Guangzhou/333/99(H9N2)(GZ333))后,用流感病毒鼠肺适应株A/PR/8/34(H1N1)(PR8) 10倍致死剂量攻击,观察攻毒后小鼠的反应.方法 BALB/c小鼠150只,分成3组,空白对照组PBS滴鼻,实验组分别感染甲型H1N1流感病毒pCA07和禽源H9N2病毒GZ333;感染56 d后用10倍致死剂量的鼠肺适应株流感病毒PR8攻击两个实验组和对照组小鼠,比较PR8病毒攻击后,病毒载量和抗体水平,以及对小鼠存活率的影响.结果 感染过pCA07和GZ333的小鼠在致死病毒PR8攻击后全部存活,并分别在病毒攻击6d和9d后小鼠肺组织中检测不到攻击病毒.对初次感染病毒的抗体水平在病毒PR8攻击后迅速升高,在保持初次感染病毒抗体的同时能够产生针对PR8病毒的抗体.结论 不同亚型流感病毒感染小鼠后可以提供交叉保护.     

Efficacy of Lasia spinosa leaf extract in treating mice infected with Trichinella spiralis

Trichinellosis is a widespread zoonoses for which no effective drug treatment is available at this time. Though anthelmintics such as mebendazole and albendazole are commonly used to treat human trichinellosis, none of these drugs are fully effective against the encysted or new-born larvae of Trichinella spiralis. In recent years, there has been a growing interest in developing newer anthelminthics from medicinal plants, particularly the ones used in traditional medicines in many parts of the world, due to the increasing spread of anthelminthic resistance and/or decreasing activity against encapsulated larval stages of parasites. The aim of the present study was to investigate the efficacy of leaf extract of Lasia spinosa (Araceae) against different life cycle stages of T. spiralis, i.e. adult (days 3 and 4 post-infection), migrating larvae (days 8, 9 and 10 post-infection) and encysted muscle larvae (days 31–37 post-infection). The study showed that L. spinosa leaf extract is effective against all the three life cycle stages of parasite. Against the adult stage, an oral administration of plant extract at 800 mg/kg dose revealed a 75.30% reduction in the number of adult worms, as compared to untreated controls at day 10 post-infection. Whereas against migrating larvae, the same dose of plant extract given for 3 days, reduced the number of larvae recovered from musculature of treated animals by 72.23%. However, in comparison of preceding two stages, the extract showed comparatively less efficacy against the encysted larvae of parasite. In this case, the 800 mg/kg dose of extract given for 7 days (after 30 day of post-infection) revealed only 64.84% reduction in the number of encysted larvae, as was evident from larval count on day 49 post-infection. Therefore, the results of this study indicate that leaf extract of L. spinosa possesses significant anthelminthic efficacy against the adult stages and migrating larvae of T. spiralis. On the other hand, the encysted muscle larvae of parasite are comparatively less sensitive to L. spinosa leaf extract treatment.     

Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis

Background

The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children.

Objective

We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model.

Methods

The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified.

Results

Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis.

Conclusion

We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

     

Phospholipase B activity in congenitally athymic (nude) mice infected withTrichinella spiralis

The effects of an infection with 200Trichinella spiralis larvae on the intestinal phospholipase B activity and bone marrow eosinophilia of congenitally athymic (nude) mice (BALB/c; NU/NU) were studied. Nude mice were used since it had been shown that they do not undergo a typical worm expulsion and also they lack a thymus. The results showed that nude mice do not develop either an increased bone marrow eosinophilia or an elevation in intestinal phospholipase B activity. The findings thus support the hypothesis that phospholipase B is involved in the expulsion of parasitic worms and that elevated enzyme levels and expulsion are thymus cell dependent.     

Identification and partial characterization of a T-cell-derived antigen-binding factor from mice infected with the intestinal helminth Trichinella spiralis

Immunochemical and biological characterization was performed of an antigen-binding factor derived from culture supernatants of T cells from mice infected 4 days previously with the intestinal helminth Trichinella spiralis. Affinity chromatography with T. spiralis antigen resulted in the purification of a protein, provisionally designated Trichinella factor (Tric-F), that shared antigenic and other properties with a known T-cell-derived antigen-binding factor of different antigenic specificity, picryl chloride factor, which mediates an early 2-hour component of contact sensitivity. Tric-F lacked determinants of immunoglobulins and possessed determinants shared by other antigen-specific T cell factors, as determined by ELISA and antibody affinity chromatography. Biological activity of Tric-F was assayed in vivo and in vitro. Mice injected intravenously with Tric-F developed an antigen-specific early 2-hour ear swelling response following local challenge with T. spiralis antigen. These results corresponded to delayed-type hypersensitivity responses in the ears of T. spiralis-infected mice that comprised early 2-hour and late classical 24-hour responses. In vitro, Tric-F induced serotonin release by mast cells in the presence of T. spiralis antigen. Mast cells sensitized with Tric-F formed rosettes with antigen-coated sheep erythrocytes. It is suggested that Tric-F, an antigen-binding molecule that is T-cell-derived, mediates the early 2-hour component of delayed-type hypersensitivity and is involved in the initiation and regulation of T-cell-mediated intestinal inflammation during a T. spiralis infection in mice.     

旋毛虫对三硝基苯磺酸诱导的实验性小鼠肠炎模型的干预研究

目的 研究旋毛虫对三硝基苯磺酸（TNBS）诱导的实验性小鼠肠炎模型的影响及其免疫作用机制。方法观察感染和未感染旋毛虫小鼠于TNBS诱导肠炎后3d及7d不同指标的变化，包括小鼠生存率、疾病活动指数（DAI）、结肠大体损伤和病理损伤评分、炎症指标髓过氧化物酶（MPO）活性检测，结肠细胞因子IFN-γ和IL-4 mRNA的表达量分析。结果 预先感染旋毛虫后诱导TNBS模型组小鼠在造模后3d及7d与单纯模型组相比小鼠生存率升高（P〈0．05），DAI、结肠大体损伤和病理损伤评分及MPO活性下降（P〈0．05），结肠中IFN-γmRNA的表达量下调（P〈0．05），而IL-4 mRNA的表达量增加（P〈0．05）。结论 旋毛虫对TNBS诱导的实验性小鼠肠炎具有良好的干预作用，其免疫作用机制可能是通过下调炎症性肠病过度的．TH1型免疫反应、上调TH2型免疫反应而实现的。     

Injection of recombinant interleukin 3 hastens worm expulsion in mice infected with Trichinella spiralis

 The effects of interleukin 3 (IL-3) on worm expulsion were studied in mice infected with Trichinella spiralis. C3H/He mice were treated with a total of 10⁴ U IL-3 or saline by daily peritoneal injection from day-5 to day-1. Muscle larvae were given orally to both groups of mice on day 0. The muscle worm burden in infected mice was assessed on day 28. The worm burden in mice treated with recombinant IL-3 (rIL-3) was significantly suppressed as compared with that in control mice. A reduction in the worm burden was observed in mice treated with rIL-3 from day-5 to day-1 but not in those treated day 16 to day 20. This suggests that IL-3 could up-regulate the host defense response to intestinal worms but not to parenteral stage worms. When various doses of rIL-3 were given to mice and the intestinal worm burden was assessed on day 5, protection was observed only in mice treated with a total of 10⁴ U rIL-3 but not in those given either 3.5×10³ or 10³ U. A kinetics study on the recovery of intestinal adult worms showed that rIL-3 treatment hastened worm expulsion. The mucosal mast-cell response observed in the small intestine of rIL-3-treated mice was induced earlier and was greater than that seen in the control. The host defense response induced by rIL-3 could not be inhibited by treatment with anti-IL-4 or anti-IL-5 monoclonal antibody. Under such an experimental condition in this study, at least, the numbers of mast cells per villus crypt unit observed in mice treated with rIL-3 and anti-IL-4 antibody were slightly lower than those seen in mice treated with rIL-3, but the difference was not significantly different. These results suggest that IL-3 can induce the expulsion of T. spiralis worms without the cooperation of IL-4 or IL-5 in mice. Received: 24 March 1995 / Accepted: 20 May 1995     

Characterization and mast cell origin of a chymotrypsin-like proteinase isolated from intestines of mice infected with Trichinella spiralis.

A proteinase was purified by cation exchange and affinity chromatography from the small intestines of mice infected with Trichinella spiralis. The enzyme was highly soluble and was chymotrypsin-like in its substrate specificities and susceptibility to inhibitors. It had a MW of 26,000, as determined by SDS-PAGE electrophoresis. Antibodies raised against the proteinase were affinity purified and their specificity confirmed by Western blot analysis. When used to localize the enzyme immunohistochemically, they reacted with granules of mast cells in the epithelium and lamina propria of the parasitized small intestine. The antibodies also bound to mast cell granules in a number of other sites, including tracheal epithelium, gastric mucosa, skin and tongue. Affinity-purified antibodies raised against rat mast cell proteinase II (RMCPII) cross-reacted with the mouse mast cell proteinase on Western blots.     

Genetic factors controlling the intestinal mast cell response in mice infected with Trichinella spiralis.

Inbred strains of mice showed marked variation in their mast cell (MC) response to infection with Trichinella spiralis. Variation was under genetic control, the ability to respond to infection being inherited as a dominant trait. MHC-linked genes may influence the absolute level of response, but overall response kinetics appear to be controlled by genes which are not linked to the MHC. An enhanced MC response was transferred adoptively with immune mesenteric lymph node cells (IMLNC), but reciprocal adoptive transfers between H-2 compatible rapid (NIH) and slow (B10.G) responder strains showed that the degree of enhancement was determined by the response phenotype of the recipient, not that of the donor. Similarly, in bone marrow (BM) chimaeras, produced by reconstituting lethally irradiated F1 (B10.G x NIH) mice with parental BM, the MC response to T. spiralis was determined by the response phenotype of the BM donor, whether or not rapid responder IMLNC were transferred. The data are discussed in terms of a T lymphocyte regulated, bone marrow stem cell origin of mucosal MC and interpreted as showing that genetic regulation of the MC response is expressed at the level of stem cell or precursor response to T cell derived mastopoietic factors.     

Pure CD4+ T lymphocytes fail to protect perorally infected SCID mice from lethal microsporidiosis caused by Encephalitozoon cuniculi

The role of CD4+ and CD8+ T lymphocytes in the protection against intraperitoneally (i.p.) or perorally (p.o.) acquired Encephalitozoon cuniculi (Levaditi et al., C R Soc Biol Paris 89:984–986, 1923) infection was studied by means of reconstitution of severe combined immunodeficiency (SCID) mice with well-defined populations of naive CD8+ or CD4+ T lymphocytes. Adoptive transfer of pure CD8+ T lymphocyte subpopulation protects SCID mice against a lethal microsporidiosis caused by E. cuniculi. The protective effect of CD8+ T lymphocytes is manifested in both i.p. and p.o. infection. On the contrary, the host defense against peroral infection does not require CD8+ T cells. The protective role is not mediated by CD4+ T lymphocytes only. SCID mice reconstitution with pure CD4+ T cell subpopulation led to prolonged survival of perorally infected mice. However, these mice died due to lethal encephalitozoonosis caused by i.p. infection.     

The immunological response of mice infected with Trichinella spiralis: Biological and physico-chemical distinction of two homocytotropic antibodies

(1) Infection of mice with Trichinella spiralis induced the appearance in serum of two homocytotropic antibodies that could be distinguished by their biological and chromatographic behaviour.

(2) Biologically, the antibodies could be distinguished by their ability to persist in homologous skin after passive transfer. One antibody was able to induce PCA only when a short latent period was used, whereas the other was able to induce PCA even after 72 hours.

(3) They could also be separated when antiserum was passed through a DEAE-cellulose column. Antibody present in the first eluates was able to induce PCA only if a short latent period was used whereas antibody present in the subsequent eluates was able to induce PCA 72 hours after sensitization.

(4) Both antibodies appeared in the circulation 5 weeks after infection and reached their highest levels around the 9th week. Later, the 72-hour PCA antibody disappeared from the serum in some animals, whereas the 4-hour PCA antibody remained.

(5) Re-infection resulted in an increase in the levels of both antibodies.

(6) In animals subjected to repeated reinfections the reagin-like antibody either decreased or disappeared from the serum. On the other hand, the 4-hour PCA antibody increased.

(7) Immunization with `dead' T. spiralis antigen led to the appearance of both antibodies in the serum. A second dose of antigen resulted in increases in the levels of both antibodies, but further injections resulted in a high level of 4-hour PCA antibody and in the disappearance of the reagin-like antibody.

     

Adaptive changes in muscle fibers infected with Trichinella spiralis.

Ultrastructural changes which occurred in infected skeletal muscle fibers after infection with larvae of Trichinella spiralis were followed on a daily basis utilizing synchronous infections. No changes were observed in muscle fiber architecture during the first 2 days of intracellular infection. However, on Day 3, a space containing various sarcoplasmic elements developed between the plasma membrane and myofilaments. Widening near the regions of triads was also observed at this time. On Day 4 the space at the outer edge had increased, as did the ones at the triads. In addition, the myofilaments throughout the infected fiber were in a state of partial disarray. Finally, the nuclei were enlarged and had migrated to the central portion of the infected cytoplasm. On Day 5 day, sarcomeres were highly disorganized, and an increase in sarcoplasmic reticulum (SR) was noted. By Day 8, only the extreme periphery of the infected fiber contained Z bands with actin filaments attached. Proliferation of the t tuble system was also evident. At Day 10, myofilaments were completely replaced with SR. Further, the plasma membrane became hyperinvoluted and was associated with a 36-fold increase in the thickness of the glycocalyx. No further enlargement of nuclei occurred after Day 10. Finally, a host-derived double membrane completely surrounded the larva.     

Impaired protective immunity and T helper 2 responses in alymphoplasia (aly) mutant mice infected with Trichinella spiralis

The alymphoplasia (aly) mutation of mice prevents the development of systemic lymph nodes and Peyer's patches. The mutant homozygotes (aly/aly) are partially deficient in both humoral and cell-mediated immune functions. In the present study, we show that adult worm expulsion was slightly delayed and that T helper 2 (Th2)-type responses were partially defective in aly/aly mice after infection with Trichinella spiralis. Male aly/aly and aly/+ mice (8-weeks old) were infected with 400 muscle larvae. There was no difference in worm recovery between the two groups on day 5. However, worm recovery in aly/aly mice was significantly higher than that in aly/+ mice on day 14. Mucosal mast cells increased in number and peaked 14 days after infection in aly/+ mice. aly/aly mice were deficient in their mucosal mast cell response through out the primary infection. To examine the existence of mast cell precursors, aly/aly mice were treated with recombinant interleukin-3 (rIL-3) before infection. The mast cell response was poorly induced in aly/aly mice treated with rIL-3. An immunoglobulin E (IgE) response was not detected in aly/aly mice during the course of infection. Serum IgG1 levels in aly/aly mice were significantly lower than that of aly/+. The serum IgG2a levels increased in both strains of mice. However, IgG2a production in aly/aly mice on day 14 was half as much as that in aly/+mice. Stimulation of splenic T cells in vitro with anti-CD3 monoclonal antibody (mAb) showed that spleen cells from aly/+ mice on day 5 produced more IL-4 than spleen cells from aly/aly mice. IL-4 production from aly/aly mice on day 14 was half that from aly/+ mice. Interferon-gamma (IFN-gamma) was produced in both aly/aly and aly/+ mice on day 14. Proliferation assay showed that T cells of aly/aly mice responded poorly when cultured with antigen-presenting cells. These results suggest that aly gene is needed for the induction of protective immunity and Th2 responses in mice infected with T. spiralis.     

MCP-1 and MIP-2 response in Trichinella spiralis infected mice treated with 4-deoxypyridoxine (4-DPD)

Chemokines are involved in a number of pathophysiological conditions, such as inflammatory processes and are divided in two major subfamilies, C-X-C and C-C chemokines. The C-C chemokines are monocyte chemotactic protein 1-2-3-4-5, while C-X-C chemokines include MIP-2, IL-8, etc. We studied the levels of MCP-1 and MIP-2 in diaphragmatic and intercostal muscle tissue and serum in Trichinella spiralis infected mice treated and not treated with 4-deoxypyridoxine, a potent Vit. B6 antagonist which inhibits humoral and cellular immune response. MCP-1 and MIP-2 were measured in homogenized tissue and serum and determined by a specific ELISA. Here we found the levels of MCP-1 and MIP-2 in diaphragmatic and intercostal muscle tissue of T. spiralis infected mice were significantly increased after 10 days and peaked on day 20 post-infection; however, the levels of MIP-2 in mice treated with 4-DPD was lower than that of untreated mice at day 20. MCP-1 also peaked at days 20 and 40. Animals treated with 4-DPD also inhibited the production of MCP-1, compared with untreated animals. The maximum inhibition was at day 40. These inhibitory effects on MIP-2 and MCP-1 were also repeated in the serum determinations, but were not significant. This study demonstrates that MIP-2 and MCP-1 are stimulated in serum and tissue of T. spiralis infected mice and 4-DPD-treated animals significantly inhibited them.     

Interleukin‐4 protects mice against lethal influenza and Streptococcus pneumoniae co‐infected pneumonia

Streptococcus pneumoniae co‐infection post‐influenza is a major cause of mortality characterized by uncontrolled bacteria burden and excessive immune response during influenza pandemics. Interleukin (IL)‐4 is a canonical type II immune cytokine known for its wide range of biological activities on different cell types. It displays protective roles in numerous infectious diseases and immune‐related diseases, but its role in influenza and S. pneumoniae (influenza/S. pneumoniae) co‐infected pneumonia has not been reported. In our study, we used C57BL/6 wild‐type (WT) and IL‐4‐deficient (IL‐4^−/−) mice to establish co‐infection model with S. pneumoniae after influenza virus infection. Co‐infected IL‐4^−/− mice showed increased mortality and weight loss compared with WT mice. IL‐4 deficiency led to increased bacterial loads in lungs without altering influenza virus replication, suggesting a role of IL‐4 in decreasing post‐influenza susceptibility to S. pneumoniae co‐infection. Loss of IL‐4 also resulted in aggravated lung damage together with massive proinflammatory cytokine production and immune cell infiltration during co‐infection. Administration of recombinant IL‐4 rescued the survival and weight loss of IL‐4^−/− mice in lethal co‐infection. Additionally, IL‐4 deficiency led to more immune cell death in co‐infection. Gasdermin D (GSDMD) during co‐infection was induced in IL‐4^−/− mice that subsequently activated cell pyroptosis. Treatment of recombinant IL‐4 or inhibition of GSDMD activity by disulfiram decreased immune cell death and bacterial loads in lungs of IL‐4^−/− co‐infected mice. These results suggest that IL‐4 decreases post‐influenza susceptibility to S. pneumoniae co‐infection via suppressing GSDMD‐induced pyroptosis. Collectively, this study demonstrates the protective role of IL‐4 in influenza/S. pneumoniae co‐infected pneumonia.     

Gene delivery of SOCS3 protects mice from lethal endotoxic shock

Suppressor of cytokine signaling 3 (SOCS3) was reported as a feedback inhibitor of cytokine receptor signaling byinhibiting the JAK-STAT signal transduction pathway.We sought to test the anti-endotoxic septic shock effect ofliposome mediated gene delivery of SOCS3 in a lethal endotoxic shock mouse model.BALB/c mice were injectedintraperitoneally with 200 μg pcDNA3.1-SOCS3 cationic liposomes,while pcDNA3.1-IL-10 and empty vector aspositive and negative control respectively.Forty-eight hours after gene delivery,mice were challenged with 4 μg ofE.coli 0127:B8 LPS and 18 mg D-GaIN administered i.p.90 min later,serum TNF-α level was determined.Survivalover the next 48h was evaluated.Peritoneal macrophages from survival mice were stimulated in vitro with 1 μg/mlLPS for 18h,and the supernatants were harvested for determination of the amount of TNF-α.We found that genedelivery of SOCS3 significantly increase the mouse survival rate from 27.8±9.6% of control group to 61.1±9.6%(p<0.01).In comparison with control group (218±13pg/ml) and sham delivery group (219±22pg/ml),genedelivery of SOCS3 reduced the level of serum TNF-α(68±9pg/ml) significantly(p<0.01).Furthermore,genedelivery of SOCS3 displayed the capacity of prevention of tolerance of peritoneal macrophages to LPS.Thesefindings suggest that gene delivery of SOCS3 mediated by liposome is a promising approach for endotoxic septicshock treatment.Cellular & Molecular Immunology.2005;2(5):373-377.