治疗和未治疗小鼠日本血吸虫病免疫应答的动态观察

本文报道定期检测治疗和未治疗日本血吸虫病小鼠循环抗原(CSA)、特异性抗体、循环免疫复合物(CIC)以及嗜酸性粒细胞(EOS)的结果。实验表明 CSA 于感染后2周的阳性检出率(ELISA 双抗体法)为40%,4周时全部呈现阳性反应。抗虫卵抗体在感染后3周阳性率为10%,6周时全部检出。结果表明检测 CSA 有早期诊断之价值。CIC 峰见于感染后7～10周,此时 EOS绝对计数亦最高。上述指标在感染后13周明显下降,但在未治疗鼠直至感染后12～30天 CSA逐渐转阴,而特异性抗体仍持续呈现阳性反应,用 ELISA 检测 CSA 在疗效考核上较用 IHA 检测抗虫卵抗体的方法为优。     

Role of IgE in primary murine Schistosomiasis mansoni

Schistosoma mansoni infection proceeds in normal mice in the absence of detectable levels of polyclonal or specific immunoglobulin (Ig)E until worms mature and deposit eggs. Hence, the course of a primary S. mansoni infection is not expected to vary appreciably in mice with defects in the IgE production. Experimental increase of IgE production early after infection may, however, influence worm development. In the first approach towards this goal, BALB/c mice were injected with interleukin(IL)4 to raise the level of endogenously synthesized IgE. A significant increase in serum polyclonal IgE and antischistosome IgG1 during the prepatent period was not associated with significant changes in worm and egg burden or liver pathology. During the second approach, mice were injected with IgE which was affinity purified from serum of BALB/c mice infected for 16 weeks with S. mansoni. The purified IgE bound to carbohydrate-independent epitopes of soluble antigens from 3 h larvae, adult worms and eggs and recognized the schistosomular surface membrane. No differences in worm and egg load or granuloma number and size were noted between untreated and exogenous IgE-injected mice. Together, the data demonstrate that by itself IgE does not influence the outcome of infection in primary murine S. mansoni.     

Kinetics of interleukin-6 production after experimental infection of mice with Schistosoma mansoni.

It has been reported that interleukin-6 (IL-6) is expressed in cells of acute inflammatory granulomas experimentally induced in mice by eggs of Schistosoma mansoni. Moreover, in vitro IL-6 was shown to enhance the cytotoxic activity of human platelets against larvae of S. mansoni. To elucidate further a proposed biological significance of this cytokine during the course of schistosomiasis, we studied the kinetics of IL-6 production and concomitantly performed a histopathological analysis of the livers in BALB/c mice subcutaneously infected with S. mansoni cercariae. Over a period of 24 weeks postinfection (p.i.) we monitored serum IL-6 levels, IL-6 production in vitro by pokeweed mitogen (PWM)-stimulated spleen cells as well as IL-6 mRNA expression in livers, spleens and kidneys. We found significantly elevated IL-6 levels in PWM-stimulated spleen cell-conditioned media (SCM) at weeks 6 to 20 p.i., peaking at week 10 p.i. In contrast, serum IL-6 concentrations started to rise not before week 8 but remained significantly elevated above normal control values until week 24 p.i. The time pattern of enhanced IL-6 mRNA expression detected in spleens and livers, but not in kidneys, as well as the rises of IL-6 in SCM and with a delay of 2 weeks in serum samples correlated with the onset of the egg-induced inflammatory reactions as well as the incidence and the number of the granulomas observed histopathologically in the livers of infected mice. Our data emphasize both a local and a systemic role of IL-6 in the host immune response following infection of mice with S. mansoni.     

Circulating antigens and immune complexes in Schistosoma mansoni-infected rats. Characterization by radioimmunoprecipitation-PEG assay (RIPEGA)

Circulating schistosome antigens (CSA) and circulating immune complexes (CLC) were investigated in rats infected with Schistosoma mansoni. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA), with 125I-labelled anti-S. mansoni anti-serum, detected CSA during two distinct periods of the infection; the first between the 11th and the 14th week of infection and the second between the 11th and 14th week after infection. The CH50 deviation test revealed the presence of CIC in sera from infected rats, approximately at the two periods when CSA were detected. At 6 weeks of infection, the levels of CIC in infected rats were not different from those in control rats. However, a more sensitive method characterized IgG2a in C1q-binding C1C from infected rats. At weeks 5 and 6, IgE immune complexes were also detected in the serum from infected rats. In fact, the use of RIPEGA on the material eluted from infected rat serum after passage through an anti-IgE immunosorbent showed the presence of schistosome antigen at week 4, and at higher levels at week 6. Levels of 50% haemolytic complement in infected rat serum were lowered between the 2nd and the 4th week, the 5th and the 8th week and after the 12th week of infection. The possible role played by CIC in the protective mechanisms to a S. mansoni challenge infection in rats is discussed.     

Modification of immune responsiveness in murine Schistosomiasis mansoni. I. Time course after cercarial exposure.

The immune responsiveness of mice infected with 90 cercariae of Schistosoma mansoni has been investigated. Post-infection kinetics of antibody responses, delayed hypersensitivity and mitogen responsiveness show that there are profound disturbances of these immunological parameters starting 2 weeks after the infection. Although the final outcome is immunodepression the S. mansoni infection can produce immunostimulation. We have observed differences in the kinetics of the depression of humoral antibody responses in two strains of mice. It seems that the worms and not the eggs are of major importance in determining the observed alterations of immune responsiveness.     

Influence of age of mice on the susceptibility to murine schistosomiasis infection

Intensity of human schistosomiasis infection increases with age, a peak being attained at early puberty. Hormones could be involved in the age-related changes in susceptibility to schistosomiasis. Male BALB / c mice were infected with Schistosoma mansoni either before or after puberty and worm numbers, cellular immune responses, hormonal levels and pathology analysed. Pre-puberty infected mice had a significantly higher number of adult worms (p < 0.05), more severe granulomas, higher mortality rate and higher proliferative responses as compared to post-puberty infected mice. Levels of the hormones were lower in the pre-puberty infected mice as compared to the post-puberty group early in the infection. Plasma levels of testosterone and luteinizing hormones decreased significantly (p < 0.05) in infected mice when compared to controls. Susceptibility to S. mansoni in male BALB / c mice seems to be influenced by levels of testosterone and luteinizing hormone at infection. Albeit, an infection with S. mansoni seems to lower the hormonal levels.     

Studies on the host-parasite relationship in Schistosoma mansoni-infected mice: the immunological dependence of parasite egg excretion.

CBA mice deprived of their T cells by means of thymectomy and anti-thymocyte serum and subsequently infected with Schistosoma mansoni were found to have substantially fewer parasite eggs in their faeces than similarly infected immunologically-intact control animals. The number of parasite eggs deposited in the tissues of T-cell deprived mice was by comparison only marginally lower than in control mice. Administration of serum obtained from normal mice with chronic S. mansoni infections partially restored the egg excretion rate in infected deprived mice, and also resulted in an increased number of eggs being deposited in the liver and intestine of these animals.     

Schistosoma japonicum soluble egg antigens: Separation by con a chromatography and immunoaffinity purification

A crude soluble egg antigen (SEA) preparation from Schistosoma japonicum eggs was used for immunoabsorbent fractionation of anti-SEA antibody. Anti-SEA antibodies were isolated from serum pooled from mice infected with S. japonicum for 7 weeks and from mice infected for 12 weeks. These anti-SEA immunoglobulins were then used for immunoabsorbent fractionation of specific SEA antigens. Crude SEA was also partially purified by Con A chromatography. Crude SEA, the Con A fraction, an antigen isolated with 7 week infection serum, and antigens isolated with 12 week infection serum were analysed by immunoprecipitation and SDS polyacrylamide gel electrophoresis.     

Altered immunoglobulin isotype profile and anti-immature worm surface immunoglobulins in mice harboring a praziquantel-resistant Schistosoma mansoni isolate

After placement in mice of PZQ-sensitive and -insensitive S. mansoni isolates obtained from villagers responding and not responding to PZQ, parasitological criteria reflecting their biological development and also the host anti-immature worm immunoglobulin isotypes were examined 8 and 10 weeks post infection. Hepatic granuloma diameter, hepatic histopathological changes and immunolocalization of IgG and IgM on the surface of PZQ-sensitive and -resistant worms were also examined 10 weeks post infection. Data showed that parasitological criteria were not significantly different between mice infected with the PZQ-sensitive and -insensitive S. mansoni isolates. As regards serum immunoglobulins, in mice infected with the PZQ-insensitive S. mansoni isolate, IgG and IgG1 were significantly (p<0.05) lower 8 and 10 weeks post infection, respectively (1.41+/-0.07 and 1.08+/-0.10 and 1.35+/-0.06 and 1.09+/-0.07) than in mice infected with the PZQ-sensitive S. mansoni isolate (1.73+/-0.15 and 1.38+/-0.10 and 1.73+/-0.17 and 1.54+0.21) after the same observation periods. IgM level was nearly the same while IgE was lower than that recorded in mice infected with the PZQ-sensitive S. mansoni isolate. IgG immunofluorescence was also lower (60%+/-6.78) on the surface of resistant worms than that of sensitive worms (66.6%+/-5.27); meanwhile, hepatic granuloma diameter was significantly larger (296.5+/-3.0 vs 283.6+/-4.0) in mice infected with the PZQ-insensitive S. mansoni isolate with higher percentage of intact eggs. Differences in the immunogenic make up of PZQ-sensitive and -insensitive S. mansoni isolates qualitatively and/or quantitatively favoring a certain Th cell subpopulation response could be the underlying reason for such differences recorded in the host immunoglobulin isotype response and also the egg-induced hepatic histopathological changes.     

Detection of circulating and urinary antigens in Mastomys natalensis experimentally infected with Brugia malayi,Brugia pahangi,or Litomosoides carinii

The time-course of the detection of circulating and urinary filarial antigens was followed with a 2S-IRMA assay, using a mouse monoclonal antibody raised against Brugia malayi larvae, in Mastomys natalensis experimentally infected with Brugia malayi, Brugia pahangi, or Litomosoides carinii. In the prepatent phase of the infections, filarial antigen was detected 4–7 weeks before microfilariae appeared in the peripheral blood. Moreover, the sensitivity of the test was greater with urine than with serum. During the patent phase of infection, the level of circulating antigens detected varied considerably. However, there was a positive correlation (P<0.05) between antigenemia and microfilaremia. In L. carinii infection, filarial antigen could be easily detected in spite of the disappearance of microfilariae in peripheral blood, 49 weeks post infection. If these results are extrapolated to man, the 2S-IRMA should be useful for epidemiological surveys in endemic areas where transmission has been eliminated.Abbreviations CA Circulating antigen - 2S-IRMA two-site immunoradiometric assay - Mab monoclonal antibody - p.i. post infection     

Detection and quantification of circulating antigen in schistosomiasis by monoclonal antibody. II. The quantification of circulating antigens in human schistosomiasis mansoni and haematobium: relationship to intensity of infection and disease status.

Circulating cathodic and circulating anodic antigens were quantified in sera of patients infected with S. mansoni, S. haematobium or both parasites. A monoclonal antibody and a polyclonal antiserum were applied in precipitation and solid phase immunosorbent techniques using radio- and enzyme-labelled antibody as a tracer to detect the cathodic and anodic antigen respectively. The results show that circulating cathodic antigen can frequently be detected in an immunoprecipitation or an immunoradiometric assay in serum of infected patients. The serum concentration of this antigen was found to be significantly correlated to the number of S. mansoni worms and to be higher in patients with the hepatosplenic form of the disease than in those without such complications. Examining paired serum samples before and after specific treatment the determination of this antigen by monoclonal antibody reliably indicated efficacy of chemotherapy in patients having received different forms of treatment.     

Elevated type 1, diminished type 2 cytokines and impaired antibody response are associated with hepatotoxicity and mortalities during Schistosoma mansoni infection of CD4-depleted mice

During murine Schistosoma mansoni infections parasite eggs evoke a type 2 cytokine-dependent and CD4(+) T cell-mediated granulomatous response in the liver. In this study CD4(+) T cell-depleted CBA / Ca mice developed hepatic steatosis and had high mortalities during early acute schistosome infection. CD4-depleted mice had smaller liver granulomas and reduced hepatic fibrosis. The hepatocytotoxicity was characterized by microvesicular steatosis and neutrophil infiltration. The livers of depleted mice had similar levels of apoptosis as control infected mice but had a marked increase in lipid peroxidation indicative of their livers being under oxidative stress. CD4-depleted mice had impaired egg excretion and exacerbated intestinal pathology. A type 1 cytokine-dominated response was present in infected CD4-depleted mice and relatively reduced production of type 2 cytokines. Antibody responses to parasite antigens were also substantially reduced. Transfer of immune serum or IgG significantly delayed mortalities in depleted mice and prevented hepatocyte damage. Although biasing the cytokine dichotomy to a type 1-dominated response during murine schistosome infection is desirable with respect to certain pathological processes, i. e. it will reduce the granulomatous inflammation and hepatic fibrosis, these effects contribute to fatal pathology if there is reduced protective type 2 cytokines and a defect in antibody responses.     

Schistosoma mansoni: unisexual infections sensitize mice for granuloma formation around intravenously injected eggs

 Mice carrying unisexual infection with male or female Schistosoma mansoni for 9 weeks developed accelerated and augmented reactions to S. mansoni eggs injected intravenously. The size of circumoval granulomas observed in the lungs of unisexually infected mice did not differ significantly from the reactions seen in bisexually infected mice. Tissue eosinophilia in the granulomas was also augmented similarly over that in naive mice by unisexual or bisexual infection. The cross-reactivity between worm and egg antigens is relevant to the development of acute toxemic schistosomiasis mansoni and, perhaps, to the consideration of antigens to be used for vaccination against S. mansoni infection. Received: 20 February 1996 / Accepted: 2 August 1996     

Diagnosis of canine Echinococcus multilocularis infections by copro-DNA tests: comparison of DNA extraction techniques and evaluation of diagnostic deworming

The use of copro-DNA detection methods for the diagnosis of canine Echinococcus multilocularis infection was evaluated with a focus on DNA extraction techniques: two commercial kits and a modified alkaline-sodium dodecyl sulfate (SDS) technique. Dog feces (0.2 g) mixed with a protoscolex or with 1 or 10 eggs of E. multilocularis were subjected to DNA detection following extraction by these methods. DNA was extracted from all protoscolex samples by all methods, but success for samples with eggs depended on extraction technique with the modified technique showing success on all samples. Following experimental infection of dogs, copro-DNA was successfully extracted from fecal samples (0.2 g) of dogs in the patent period by all methods. In the prepatent period, PCR testing of feces subsamples (0.2 g) extracted by each technique was positive at a rate of 79.6–94.4%. Extraction by the modified technique with fecal samples of over 1 g showed detection of copro-DNA in all samples in both the patent and prepatent periods, and it produced reproducible detection in the addition recovery test using feces from 72 different domestic dogs. As copro-DNA was detected for at least 1 day following deworming with administration of anthelmintic drugs in experimentally infected dogs, diagnostic deworming might be useful for clinical examination. Using the present detection method can provide quick and accurate diagnosis of canine E. multilocularis infection, which with prompt management and treatment of infected dogs can prevent pet owners from becoming infected and prevent echinococcosis from spreading into non-endemic areas.

     

Immune responses during human schistosomiasis mansoni. I. In vitro lymphocyte blastogenic responses to heterogeneous antigenic preparations from schistosome eggs, worms and cercariae.

In vitro lymphocyte blastogenesis assays were performed using peripheral blood lymphocytes obtained from patients with schistosomiasis mansoni. Their infection was well characterized both clinically and in regard to the duration and intensity of Schistosoma mansoni infection. Subjects who were either not infected with any helminths or who were negative for S. mansoni but infected with other helminths provided two control groups. Cultures were exposed to various concentrations of heterogeneous soluble antigens prepared either from S. mansoni eggs, worms, or cercariae. In this series there was no statistical correlation between the intensity of infection (as determined by eggs/ml of feces) and the degree of cell-mediated reactivity observed by lymphocyte blastogenesis to any of the antigenic preparations. Individual patient lymphocyte responsiveness varied considerably. However, analysis of the data by groups, based upon the longevity of their S. mansoni infection, demonstrated different patterns of reactivity in regard to the antigen preparations. Responses to the egg antigens were only present early in infection. In contrast, reactivities stimulated by either the worm or cercarial preparations increased in a direct relationship to the duration of S. mansoni infection.     

A mimotope peptide-based vaccine against Schistosoma mansoni: synthesis and characterization

A panel of four mimotopes of the epitope recognized by the highly protective monoclonal antibody against Schistosoma mansoni (152-66-9B) was obtained by screening a solid-phase 8mer random peptide library. Three of the four mimotopes (p28, p29 and p30) were efficiently recognized in an in vitro radioimmunoassay by the monoclonal antibody and by sera from infected mice and one (p30) induced in vitro proliferation of primed lymphocytes. When the mimotopes were conjugated to bovine serum albumin (BSA) and the conjugates used to immunize C57BL/6J mice, only the p30-BSA-induced antibodies which were effective at complement-mediated killing of schistosomula. The level of complement-mediated killing obtained with the anti-p30 antibodies was comparable to that seen with serum from mice immunized with the protective 9B-antigen. Furthermore, following challenge infection of mimotope-BSA-immunized mice, a greater than 40% reduction in worm burden was observed in p30-BSA-immunized mice, a level comparable to that seen following immunization with the intact 9B-antigen. These results show that a simple synthetic peptide immunogen comprising an eight-amino acid mimotope of a conformational epitope on the 9B-antigen can induce protective immune responses against S. mansoni that are comparable to those obtained following immunization with the far more complex intact antigen. This mimotope may well represent a potential component of a synthetic peptide vaccine against S. mansoni. The inclusion of other B-cell- and T-cell-stimulating synthetic epitopes in such a vaccine, together with a more appropriate carrier, adjuvant and delivery systems may well result in a level of protection even greater than that seen with the single mimotope.     

Hepatic granulomatous response to Schistosoma mansoni eggs in BALB/c mice and olive baboons (Papio cynocephalus anubis)

Hepatic granulomatous inflammation is one of the key pathological lesions of a patent Schistosoma mansoni infection. This study was concerned with the sequential induction, formation and eventual modulation of the schistosome egg granuloma in the mouse, which develops schistosome-induced hepatic fibrosis, and the olive baboon, which usually does not. Six baboons were each infected with 1500 S. mansoni cercariae and liver biopsies were collected at weeks 6, 9, 13 and 17 post-infection (p.i.). The mice (n=25) were each infected with 100 cercariae and killed in groups of five at weeks 6, 9, 12, 18 and 21 p.i. Peak granuloma size was observed at week 6 p.i. in baboons (mean 355 +/- 65.6 microm) but at week 12 p.i. in mice (299 +/- 40.5 microm). Eosinophils were more abundant in the baboon (60.6 +/- 8.9%) than in the mouse (41.2 +/- 8.4%) at the time of maximal granuloma size (P < 0.01). Neutrophils formed 21.1 +/- 6.3% of peak mouse granulomata but were virtually absent in baboon granulomata. A feature of the modulating baboon granulomata was the emergence of multinucleated giant cells (MGCs); modulating mouse granulomata, on the other hand, were characterized by infiltration of fibroblasts and collagen deposition. Thus, by week 21 p.i. mouse granulomata were 92 +/- 16.0 microm in diameter and well delineated by concentric layers of fibrous tissue. Granulomata, however, were present in only two of the baboons at week 17 p.i. (44 +/- 61.2 microm in diameter). The other four had peri-portal cellular infiltration without granuloma formation, implying that baboon granulomata resolve spontaneously. These data suggest that high tissue eosinophilia and MGC formation are particularly efficient in bringing about the destruction of schistosome eggs and subsequent resolution of the egg granuloma without fibrosis. In conclusion, the baboon model more closely mimics the pathogenesis observed in man than does the mouse model.     

Cloned Schistosoma mansoni proteinase (hemoglobinase) as a putative serodiagnostic reagent.

Expressed cDNA encoding a proteolytic enzyme from Schistosoma mansoni has been cloned recently. Circulating antibodies reacting with the recombinant protein have been detected in the blood of mice and humans infected with S. mansoni, S. japonicum, or S. haematobium. S. mansoni and S. haematobium infection can be distinguished by antibody titer.     

Schistosoma mansoni antigens modulate experimental allergic asthma in a murine model: a major role for CD4+ CD25+ Foxp3+ T cells independent of interleukin-10

In areas where schistosomiasis is endemic, a negative correlation is observed between atopy and helminth infection, associated with a low prevalence of asthma. We investigated whether Schistosoma mansoni infection or injection of parasite eggs can modulate airway allergic inflammation in mice, examining the mechanisms of such regulation. We infected BALB/c mice with 30 S. mansoni cercariae or intraperitoneally injected 2,500 schistosome eggs, and experimental asthma was induced by ovalbumin (OVA). The number of eosinophils in bronchoalveolar lavage fluid was higher in the asthmatic group than in asthmatic mice infected with S. mansoni or treated with parasite eggs. Reduced Th2 cytokine production, characterized by lower levels of interleukin-4 (IL-4), IL-5, and immunoglobulin E, was observed in both S. mansoni-treated groups compared to the asthmatic group. There was a reduction in the number of inflammatory cells in lungs of S. mansoni-infected and egg-treated mice, demonstrating that both S. mansoni infection and the egg treatment modulated the lung inflammatory response to OVA. Only allergic animals that were treated with parasite eggs had increased numbers of CD4(+) CD25(+) Foxp3(+) T cells and increased levels of IL-10 and decreased production of CCL2, CCL3, and CCL5 in the lungs compared to the asthmatic group. Neutralization of IL-10 receptor or depletion of CD25(+) T cells in vivo confirmed the critical role of CD4(+) CD25(+) Foxp3(+) regulatory T cells in experimental asthma modulation independent of IL-10.     

Serum protein concentrations during Schistosoma mansoni infection in intact and T-cell deprived mice. II. Immunoglobulin G and antibodies specific for heterologous erythrocytes.

Mice infected with from twenty to fifty Schistosoma mansoni cercariae were found to have elevated serum IgG concentrations following patency of the infection, and the parasite-induced increase in concentration of IgG was dependent on the possession by the host of an intact T-cell lymphocyte pool. Following passive transfer of hyperimmune anti-sheep erythrocyte (SRBC) antibody, there was a more rapid decrease of haemolytic and haemagglutinating antibody titres in S. mansoni-infected, immunologically intact recipients than in uninfected intact mice, and infected or uninfected T-cell deprived mice. However, primary and secondary active immunization with SRBC resulted in similar patterns of serum antibody titre increase and decay in infected and uninfected intact mice over a time course of 140 days. The number of direct and indirect haemolytic plaque-forming (PFC) cells per million spleen cells was similar in S. mansoni-infected and uninfected immunologically intact mice 6 days after either primary or secondary challenge with SRBC. It is concluded that S. mansoni-infected immunologically intact mice challenged with heterologous erythrocytes synthesize anti-erythrocyte antibody at a greater rate than similarly challenged, uninfected mice.