Influence of free radicals on Trichinella spiralis infection in mice

The aim of the study was to examine the influence of free radicals: nitric oxide (NO), hydrogen peroxide (H202) and superoxide anion (O2-) on Trichinella spiralis infection in mice. The studies were performed on two strains of mice: C57BL/6 and BALB/c, which differ in immunological response to T. spiralis infection. Also the influence of AG--inhibitor of inducible nitric oxide synthase (iNOS) administered in the first days after T. spiralis infection (1-5 dpi) on the cytotoxic immune response and on the number of adult parasites as well as the influence of AG administered at the beginning of muscle phase of the T. spiralis infection (16-29 dpi) on the cytotoxic immune response and the number of muscle larvae was studied. Activation of macrophages can cause pathology. Contact of macrophages with antigens stimulates these cells to produce, among others, highly reactive inorganic compounds. There are free radicals: NO, H2O2 and O2-. NO, O2-, and their metabolites are highly toxic for most pathogens, including parasites. However, little is known about their role in the defense against T. spiralis infection. The performed studies have proved, that free radicals play role in the host immune response during both intestinal and muscle phase of T. spiralis infection in mice. In the intestinal phase of the T. spiralis infection cytotoxic immune response is activated in mice peritoneal cavity and in the muscle phase, the local immune response activated in the neighborhood of larvae in muscles appeared as the higher level of free radicals in blood and urine. Administration of AG between 1-5 dpi causes opposite reactions in two different strains of mice. In BALB/c mice AG causes fast expulsion of adult T. spiralis from the intestine but in C57BL/6 mice the expulsion of parasites is slower after AG. However, there are no differences between two strains of mice after treatment with AG between 16 and 29 dpi. AG causes diminution of larvae in muscles of both BALB/c and C57BL/6 mice. Inflammatory response in peritoneal cavity is observed later during the infection in "low responders" (C57BL/6) mice in comparison with "high responders" (BALB/c) mice. Thl like mice (C57BL/6) react stronger to AG treatment than Th2 like mice (BALB/c). It occurs as changes and fluctuations in free radicals levels and the number of peritoneal cells after AG treatment in C57BL/6 mice.Weak or no reaction on AG injections in BALB/c mice is responsible for more stabile and more sufficient defense response of the host to T. spiralis infection.     

旋毛虫肌幼虫期外泌体的分离和小RNA鉴定

目的 分离纯化旋毛虫肌幼虫分泌的外泌体,并进行鉴定,为研究旋毛虫免疫逃逸机制提供新的线索。方法 采用超速离心法提取旋毛虫肌幼虫期外泌体,通过透射电子显微镜、纳米颗粒跟踪分析、免疫印记和小RNA测序对其进行鉴定。结果 旋毛虫肌幼虫期外泌体为有膜的泡状物,直径约80～200 nm,表达外泌体特异性标记蛋白CD63和Enolase,含1 266个已知的miRNA。结论 经形态、粒径及标记蛋白分析证实成功分离旋毛虫肌幼虫期外泌体,同时构建旋毛虫肌幼虫期外泌体的小RNA文库,鉴定出多种功能性小RNA,为深入研究旋毛虫肌幼虫期外泌体内小RNA分子的应用提供数据。     

旋毛虫DNA疫苗诱导小鼠免疫应答的研究

目的　观察旋毛虫DNA疫苗在小鼠体内诱导的免疫应答。方法　将旋毛虫肌幼虫编码 31kDa抗原结构基因真核表达质粒pcDNA3-TspE1分别用肌肉注射和基因枪免疫BALB/c小鼠 ,免疫血清用旋毛虫肌幼虫冰冻切片及石蜡切片抗原进行IFAT检测 ,并用旋毛虫肌幼虫可溶性抗原进行Westernblot分析。结果　IFAT表明 pcDNA3-TspE1接种BALB/c小鼠后产生的免疫血清与旋毛虫肌幼虫可产生阳性反应 ,在肌幼虫冰冻及石蜡切片上均显示黄绿色荧光。Westernblot检测结果显示 ,pcDNA3-TspE1接种小鼠后产生的免疫血清只能识别旋毛虫肌幼虫可溶性抗原中的 31kDa抗原组分 ,而 pcDNA3质粒接种小鼠后的血清不能与旋毛虫肌幼虫可溶性抗原发生免疫反应。结论　旋毛虫DNA疫苗 pcDNA3-TspE1能够诱导小鼠产生特异性体液免疫应答。     

Trichinella spiralis infection enhances protein kinase C phosphorylation in guinea pig alveolar macrophages

To learn more about the signalling pathways involved in superoxide anion production in guinea pig alveolar macrophages, triggered by Trichinella spiralis infection, protein level and phosphorylation of mitogen activated protein (MAP) kinases and protein kinase C (PKC) were investigated. Infection with T. spiralis, the nematode having ‘lung phase’ during colonization of the host, enhances PKC phosphorylation in guinea pig alveolar macrophages. Isoenzymes β and δ of PKC have been found significantly phosphorylated, although their location was not changed as a consequence of T. spiralis infection. Neither in macrophages from T. spiralis‐infected guinea pig nor in platelet‐activating factor (PAF)‐stimulated macrophages from uninfected animals, participation of MAP kinases in respiratory burst activation was statistically significant. The parasite antigens seem to act through macrophage PAF receptors, transducing a signal for enhanced NADPH oxidase activity, as stimulating effect of newborn larvae homogenate on respiratory burst was abolished by specific PAF receptor antagonist CV 6209. A suppressive action of T. spiralis larvae on host alveolar macrophage innate immunological response was reflected by diminished protein level of ERK2 kinase and suppressed superoxide anion production, in spite of high level of PKC phosphorylation.     

旋毛虫成虫可溶性抗原和排泄分泌抗原对小鼠免疫保护作用的比较研究

目的　比较旋毛虫成虫可溶性抗原和排泄分泌抗原对小鼠的免疫保护作用。方法　收集人工感染大鼠小肠内的成虫, 经研磨和冻融制备成虫可溶性抗原。采用体外培养的方法从培养液中提取旋毛虫成虫排泄分泌抗原。分别用两种抗原免疫小鼠, 间隔１ 周共免疫３ 次, 末次免疫后１ 周, 每只小鼠攻击感染１００ 条旋毛虫感染性肌肉幼虫。感染后１ 周检查小鼠小肠内成虫数量和雌虫生殖力, 感染后５ 周检查肌肉幼虫负荷。结果　成虫可溶性抗原诱导的成虫减虫率、新生幼虫减虫率和肌肉幼虫减虫率分别为７９５５ ％ 、６２２５ ％ 和６５０ ％ 。成虫排泄分泌抗原诱导的成虫减虫率、新生幼虫减虫率和肌肉幼虫减虫率分别是９７２７ ％ 、８６６０ ％ 和９００ ％ 。结论　实验结果表明旋毛虫成虫可溶性抗原和成虫排泄分泌抗原均能够诱导宿主产生较强的抗攻击感染的免疫力, 但后者的免疫原性更强。     

The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice

The complement system is an important component of the innate immune response to bacterial pathogens, including Streptococcus pneumoniae. The classical complement pathway is activated by antibody-antigen complexes on the bacterial surface and has been considered predominately to be an effector of the adaptive immune response, whereas the alternative and mannose-binding lectin pathways are activated directly by bacterial cell surface components and are considered effectors of the innate immune response. Recently, a role has been suggested for the classical pathway during innate immunity that is activated by natural IgM or components of the acute-phase response bound to bacterial pathogens. However, the functional importance of the classical pathway for innate immunity to S. pneumoniae and other bacterial pathogens, and its relative contribution compared with the alternative and mannose-binding lectin pathways has not been defined. By using strains of mice with genetic deficiencies of complement components and secretory IgM we have investigated the role of each complement pathway and natural IgM for innate immunity to S. pneumoniae. Our results show that the proportion of a population of S. pneumoniae bound by C3 depends mainly on the classical pathway, whereas the intensity of C3 binding depends on the alternative pathway. Furthermore, the classical pathway, partially targeted by the binding of natural IgM to bacteria, is the dominant pathway for activation of the complement system during innate immunity to S. pneumoniae, loss of which results in rapidly progressing septicemia and impaired macrophage activation. These data demonstrate the vital role of the classical pathway for innate immunity to a bacterial pathogen.     

Growth hormone and mannan-binding lectin: emerging evidence for hormonal regulation of humoral innate immunity

An increasing number of studies in animals and humans indicate that growth hormone (GH) and insulin-like growth factor-I (IGF-I) modulate immune function. It was recently reported that, surprisingly, GH therapy increased the mortality in critically ill patients. The excessive mortality was almost entirely due to septic shock or multi-organ failure, which could suggest that a GH-induced modulation of immune function was involved. Mannan-binding lectin (MBL) is a plasma protein that plays an important role in innate immunity through activation of the complement cascade and inflammation following binding to carbohydrate structures. The serum concentration of MBL is subject to large between-subjects differences, which primarily are caused by genetic factors. However, mounting evidence supports a significant influence from growth hormone on MBL levels. In the present review, we focus on the function of MBL, on the influence of growth hormone on MBL levels, and on the possible clinical consequences of this new link between the endocrine and the immune system.     

Contributions to the study of Trichinella spiralis TSL-1 antigens in host immunity

The observation on different hosts infected with Trichinella spiralis that recognized similar muscle larvae (ML) antigens and the fact that different monoclonal antibodies (mAb) had a similar reactivity to ML components prompted a proposal to define a useful classification system for these antigens. For this purpose, an international workshop provided a platform for the classification of T. spiralis antigens. ML antigens were classified in eight groups -- Trichinella spiralis larvae groups, TSL-1 to TSL-8. TSL-1 antigens are highly immunogenic and a number of important studies have been performed to analyse the role of these antigens in the host-parasite interplay. In this context, we have focused on the analysis of the role of TSL-1 antigens in the induction of innate immune responses with particular emphasis on the activation of mast cells (MC) by an IgE-independent pathway. These studies provided evidence on the role of mediator release from TSL-1-activated MC in the development of Type 2 immune responses. The protective role of TSL-1 in T. spiralis-infected mice has been described. In addition, it has been demonstrated that the use of TSL-1 antigens allows for a more sensitive and specific diagnosis of human and animal trichinellosis.     

Changes in the immune response of BALB/c mice coinfected with Heligmosomoides polygyrus and Trichinella spiralis

The influence of Heligmosomoides polygyrus on infection with Trichinella spiralis was studied in BALB/c mice. Mice coinfected with T. spiralis and previously given H. polygyrus harboured both nematode species till day 34. The number of T. spiralis muscle larvae was greater in mice coinfected with H. polygyrus/T. spiralis or T. spiralis/H. polygyrus than after infection with T. spiralis alone. Infection with H. polygyrus did not enhance eosinophil and IL-5 levels induced by T. spiralis. Additionally, the production of IgG1 specific to L1 T. spiralis was inhibited by co-infection. Changes in the levels of IFN-gamma and IgG2a implicated a disturbance in Th2 cell activation during protective response and resulted in the greater number of T. spiralis muscle larvae in coinfected mice.     

旋毛虫成虫可溶性粗抗原对小鼠的免疫保护作用

本文应用人工感染的方法从大白鼠获得大量成虫。经冻融、超声粉碎、高速离心制备出成虫可溶性粗抗原。将不同剂量的抗原与等体积弗氏完全佐剂乳化后间隔1周共免疫小白鼠3次。最后一次免疫后间隔1周每鼠攻击感染130条感染性肌幼虫,攻击感染后1周剖检成虫、新生幼虫,攻击感染后35天剖检肌幼虫。试验结果表明,以每只小白鼠免疫200μg成虫可溶性粗抗原所诱导的免疫力最好,诱导成虫减虫率达94.65％,肌幼虫减虫率达95.60％。初步研究表明,旋毛虫成虫可溶性抗原是很好的免疫原。     

The dynamics of LPS recognition: complex orchestration of multiple receptors

The molecular mechanisms that have been designed to protect the host from invading pathogens are responsible for sepsis, an often fatal response of the immune system against microbial pathogens. In the past few years, intense research in the field of innate immunity has identified a plethora of pattern recognition receptors that are responsible for bacterial-induced activation. Recognition of bacterial lipopolysaccharide seems to involve a complex orchestration of protein-protein interactions that eventually leads to cellular activation. In this review, we attempt to unravel the dynamic interactions that occur among the different receptors involved and dictate the outcome of the innate immune response.     

Trichinella spiralis-secreted products modulate DC functionality and expand regulatory T cells in vitro

Helminths and their products can suppress the host immune response which may benefit parasite survival. Trichinella spiralis can establish chronic infections in a wide range of mammalian hosts including humans and mice. Here, we aim at studying the effect of T. spiralis muscle larvae excretory/secretory products (TspES) on the functionality of DC and T cell activation. We found that TspES suppress in vitro DC maturation induced by both S- and R-form lipopolysaccharide(LPS) from enterobacteria. Using different toll-like receptor (TLR) agonists, we show that the suppressive effect of TspES on DC maturation is restricted to TLR4. These helminth products also interfere with the expression of several genes related to the TLR-mediated signal transduction pathways. To investigate the effect of TspES on T cell activation, we used splenocytes derived from OVA-TCR transgenic D011.10 that were incubated with OVA and TspES-pulsed DC. Results indicate that the presence of TspES resulted in the expansion of CD4(+) CD25(+) Foxp3+ T cells. These regulatory T (Treg) cells were shown to have suppressive activity and to produce TGF-β. Together these results suggest that T. spiralis secretion products can suppress DC maturation and induce the expansion of functional Treg cells in vitro.     

Mitochondrial ATP-ASE as a measure of uncoupling of rat muscle mitochondria in experimental infection with Trichinella spiralis and Trichinella pseudospiralis

Changes in a bioenergetic state of Trichinella spiralis and Trichinella pseudospiralis infected rat and mouse muscle mitochondria were evaluated enzymatically, and in both infections 3-4-fold increase of mitochondrial, Mg++-stimulated ATP-ase (EC 3.6.1.3) was observed. Looking for the dynamics of those bioenergetic changes in T. pseudospiralis infected rat and mouse muscle mitochondria 1-2 weeks, pi, the 5-6-fold stimulation of mATP-ase activity, followed by a significant drop between the week 3-4th was found. In in vitro experiments cytoplasmic fractions isolated from T. spiralis and T. pseudospiralis larvae stimulated strongly mATP-ase activity of control rat liver mitochondria, the effect being much more pronounced in case of T. spiralis larvae. The factor(s) present in cytoplasmic fractions seem(s) to be heat-labile, of high molecular weight. Those experiments in vitro prove the causative role of the presence of T. spiralis and T. pseudospiralis larvae in the uncoupling of host muscle mitochondria. Since some relationship between the intensity of infection and the degree of uncoupling was observed, the measurements of the activity of this enzyme might serve not only as a biochemical method of differentiation between infected and normal muscles, but may be useful in crude evaluation of the intensity of these tissue infections.     

旋毛虫肌幼虫三种抗原的免疫印迹分析

应用酶联免疫印迹分析旋毛虫肌幼虫的表面抗原、S_3 抗原和虫体粗抗原,结果显示上述三种抗原的多肽组成存在一定的差异。与同源感染兔血清反应,显示表面抗原有4个免疫反应区带,分子量范围在 102～44KD之间。S_3 抗原和虫体粗抗原分别有 8和10个主要区带,分子量范围在 73～16 KD之间。与异源感染兔血清反应,显示上述抗原的低分子量多肽有较强的特异性。家兔感染旋毛虫后,血清抗体对表面抗原的识别,在不同感染时期具有相同的反应图谱,而对其他两种抗原的识别则具有阶段性的改变。     

Mannose-binding lectin regulates the inflammatory response of human professional phagocytes to Neisseria meningitidis serogroup B

The influence of the innate immune protein mannose-binding lectin (MBL) on the response of human phagocytes to Neisseria meningitidis was investigated. MBL increased the association of killed meningococci with neutrophils, monocytes, and macrophages by increasing the proportion of cells that recognized bacteria. MBL down-regulated the normal change in expression of the leukocyte adhesion molecules CD11b and CD62L. In an ex vivo model, the addition of MBL to the blood of MBL-deficient donors influenced the production of monocyte-derived inflammatory cytokines. The addition of high concentrations of MBL (>6 microg/mL) profoundly decreased the production of interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha by monocytes in response to meningococci, whereas lower concentrations enhanced the production of IL-6 and IL-1beta. These results suggest that MBL not only is involved in complement activation but also is a potent regulator of inflammatory pathways and, as such, may affect the severity of meningococcal disease.     

旋毛虫成虫抗原的免疫保护性研究

目的 比较旋毛虫成虫虫体抗原、排泄分泌抗原和表面抗原对小鼠产生的免疫保护作用。 方法 检查免疫鼠和对照鼠肠道成虫、肌幼虫和血液中的嗜酸性粒细胞数;用ELISA测血清中抗旋毛虫肌幼虫IgG抗体滴度。 结果旋毛虫成虫虫体抗原、排泄分泌抗原和表面抗原免疫组的成虫减虫率分别为84.48％、89.98％和85.16％;肌幼虫减虫率分别为69.82％、78.80％和73.94％。3种抗原免疫组小鼠血中的嗜酸性粒细胞(EOS)数明显增多,血清中IgG抗体滴度明显升高,IgG抗体的几何平均倒数滴度(GMRT)分别是未免疫组的6.96、7.99和6.06倍。 结论 旋毛虫成虫虫体抗原、排泄分泌抗原和表面抗原均能诱导宿主产生较强的抗攻击感染保护力,且可激发特异性体液免疫和细胞免疫。成虫的排泄分泌抗原显示出更强的免疫原性。     

Establishment of T-Helper-2 immune response based gerbil model of enteric infection

BACKGROUND: The reciprocal antagonism of T-helper-1 (Th-1) and Th-2 type immune responses suggests that helminth parasitic infection may ameliorate disease where a Th-1 type response dominates. The Mongolian gerbil has been useful in the investigation of the pathogenesis of gastric cancer, since long-term infection of gerbils with Helicobacter pylori induces adenocarcinoma. In this study the kinetics of worm expulsion and associated immune responses in gerbils infected with Trichinella spiralis were investigated in an attempt to establish an animal model of parasitic infection that could be helpful when investigating the effect of a Th-2 type response on Th-1-based intestinal disorders. METHODS: Gerbils were infected with various doses of infective T. spiralis larvae and were euthanized on different days after infection to investigate the intestinal worm recovery, goblet cell population, eosinophil response and serum IgG1 responses. RESULTS: The number of worms recovered from the intestine was dependent on the number of larvae used for the infection. Almost all worms were expelled spontaneously by day 26 post-infection, when the gerbils had been infected with 375 or 750 larvae. The number of intestinal goblet cells, eosinophils and the serum IgG1 level significantly increased following infection compared with the control. CONCLUSION: This is the first comprehensive report on the time-course of T. spiralis infection in gerbils. The data indicate that the T. spiralis-infected gerbil could be used as a model of the Th-2-based response to investigate the effect of a parasite-induced Th-2 response on various Th-1-mediated intestinal disorders such as H. pylori-induced gastritis and gastric carcinoma.     

Mannose-binding lectin and maladies of the bowel and liver

Mannose-binding lectin (MBL) is a pattern-recognition molecule that binds to characteristic carbohydrate motifs present on the surface of many different pathogens.MBL binding stimulates the immune system via the lectin pathway of complement activation. In certain clinical situations, often characterized by pre-existing immune compromise, MBL deficiency increases the risk of infectious and other disease-specific complications. Many of the key pathogenic processes inherent to common gastroenterological diseases, such as infection, immunological damage, and carcinogenesis, have been linked to MBL. This editorial reviews the biology of MBL, outlines key disease associations to document the breadth of influence of MBL, and finally, highlights the relevance of MBL to both gastroenterological health and disease.     

Mannose-binding lectin and maladies of the bowel and liver

INTRODUCTIONMannose-binding lectin(MBL)is an important componentof the innate immune system.MBL is primarily produced by the liver,circulates throughout the body,and is able to recognize a wide array of common pathogens through repeating carbohydrate sequ…