Proteomic analysis of excretory/secretory products released by Teladorsagia circumcincta larvae early post-infection

Teladorsagia circumcincta is an important parasitic nematode of domestic small ruminants. Drug resistance in this species is common so alternative methods of control are required. As animals develop immunity to T. circumcincta, vaccination is a valid option. Little is known about the antigens that play a role in stimulating immunity at this host/parasite interface. As responses generated between 1 and 5 dpi are known to affect development of these nematodes in their gastric niche, we focused on proteins released during the early stages of infection. To identify molecules potentially involved in immunity, we undertook a proteomics analysis of proteins released from larvae harvested at 1-, 3- and 5-days post-infection (dpi). This analysis produced peptide sequence data that was used to search information available in T. circumcincta expressed sequence tag (EST) databases and enabled identification of a number of excretory/secretory (ES) proteins. Immunoblots were performed to assess the relative molecular weight of ES antigens that were targets of local IgA responses in mucus from sheep rendered immune to infection. ELISA was performed to assess antigen-specific mucus IgA levels in individual sheep. These experiments provided preliminary evidence that the proteins identified in the larval secretome were subject to these antibody responses.     

Hookworm excretory/secretory products modulate immune responses to heterologous and species‐specific antigens

Approximately one billion people are currently infected with hookworm. Despite its high prevalence and the concomitant immune suppression seen in infected individuals, little research has been performed on the mechanism of immunosuppression by hookworm. Our study focused on characterizing mechanisms utilized by hookworm to suppress the host immune response. Splenocytes and draining lymph node cells from mice injected with hookworm excretory/secretory (ES) proteins showed decreased proliferation in response to both heterologous and species‐specific antigens while also having increased nitric oxide secretion. Analysis by fluorescence‐activated cell sorting revealed that mice injected with ES had reduced percentages of CD4⁺ T cells indicating potential effects of ES proteins on lymphocyte homeostasis. Antibody and cytokine response analyses demonstrated that immunization with ES proteins decreased IgG and IgG1 levels, also decreased interleukin (IL‐)‐4 and increased IL‐12 and interferon‐gamma (IFN‐γ) cytokine production suggesting impairment of B‐cell activation and a shift towards a nonhealing IL‐12 directed T helper‐1 immune response. Together, these data demonstrate for the first time that host immunosuppression by hookworms is orchestrated by ES proteins and provide mechanisms underlying the shift towards a nonhealing Th‐1 profile as seen in humans suffering from hookworm infection.     

Excretory/secretory antigen of Toxocara canis: recognition profiles of polyclonal and larvicidal monoclonal antibodies

Summary Five monoclonal antibodies against the excretory/secretory (E/S) antigen of Toxocara canis were obtained and characterized. Immunoprecipitating activity was demonstrated in an in-vitro micropre-cipitating assay using live T. canis larvae. Their capacity to kill larvae was also shown in an in-vitro assay. Two zones of reactivity were observed in 7.5 and 12.5% SDS-PAGE (177-77 kD, 43-15 kD) of immunoprecipitates of human and mouse positive polyclonal anlisera. The murine monoclonal antibodies showed a common pattern of reactivity with the proteins in the 177-77 kD range.     

Genetic control of the antibody repertoire against excretory/secretory products and acetylcholinesterases of Dictyocaulus viviparus

Outbred Dunkin-Hartley and inbred strain 2 and strain 13 guinea pigs were immunized with Dictyocaulus viviparus adult ES products prior to challenge with third stage larvae. Antibody responses of the three strains to adult ES products and the acetylcholinesterase (AChE) isoforms which they contain were examined. Using immunoprecipitation and ELISA, it was observed that responses in the three strains to adult ES products were distinct: considerable heterogeneity in the antibody repertoire was observed between outbred Dunkin-Hartley animals, with only slight variation occurring amongst the inbred individuals. Responses to the AChE isoforms were heterogeneous amongst individual outbred guinea pigs but were more consistent in inbred strain 2 and 13 animals in which strain-specific patterns of recognition were observed. Previous studies with nematode infections have indicated a role for the major histocompatibility complex in determining the nature and level of the immune response. As the inbred strains bear different alleles at the Class II region but are identical at the Class I region, the differences observed are likely to be due to genes mapping to the Class II locus. This is therefore the first report of genetic restriction of the antibody repertoire to secreted AChEs of a parasitic nematode.     

Potent immunosuppression by secretory/excretory products of larvae from the sheep blowfly Lucilia cuprina

Secretory/excretory products (sec/ex) of parasitic larvae of the sheep blowfly Lucilia cuprina potently inhibited proliferation of peripheral blood leucocytes stimulated by mitogens in vitro. Suppression of proliferation was not due to irreversible damage because cells cultured for 24 h in high concentrations of sec/ex appeared viable (assessed by Trypan blue exclusion) and did not show impaired proliferation after washing. Furthermore, suppression induced by sec/ex could be overcome by increasing concentrations of mitogen. The inhibitory activity could be demonstrated in cultures where sec/ex was added at different times during the culture period. Inhibitory activities in sec/ex were heat-labile and sensitive to treatment with trypsin. In addition to effects in vitro, sec/ex was strongly immunosuppressive in vivo. Sheep given combined injections of myoglobin and sec/ex had markedly lower anti-myoglobin antibody levels in sera than sheep that received injections of myoglobin alone. There was no significant antibody response to sec/ex itself. Immunosuppressive moieties in sec/ex produced by blowfly larvae may promote parasite survival by inhibiting the immune response of host sheep.     

Production and characterization of genus and species specific monoclonal antibodies against surface tegumental antigens of Schistosoma mekongi

A panel of monoclonal antibodies (MoAbs) against freeze thaw surface tegumental antigens of Schistosoma mekongi were produced from naturally infected BALB/c mice. In this study, we have characterized two MoAbs which have different antigenic specificity for S. mekongi, S. japonicum and S. mansoni. The target epitopes of these two hybridoma antibodies are contained in the Mr 38 kDa (designated Sme 38) and Mr 97 kDa (designated Sme 97) proteins of adult worms as analysed by immunoblotting. The Sme 38 epitope was genus-specific, since it is also detectable in S. japonicum and S. mansoni. The Sme 97 was not detected in S. japonicum and S. mansoni. therefore it is considered as species-specific epitope. Immunocytochemical analysis revealed the presence of Sme 38 epitope in the surface tegument, the tegumental cells lying underneath the muscle layer and gut surface. The Sme 97 epitope was detectable only in the surface tegumental area.     

Secreted antigens of filarial nematodes: a survey and characterization of in vitro excreted/secreted products of adult Brugia malayi

We report here a broad analysis of the excretory/secretory (E/S) products of adult Brugia malayi, collected by in-vitro cultivation of the parasite. Culture media and conditions were optimized, and non-essential amino acids were found to be crucial for efficient protein synthesis under cell- and serum-free culture conditions. A close correlation was found between total protein secretion, phosphorylcholine-bearing antigen release and lactate production on each day of culture, indicating that E/S molecules are actively secreted. Parasites cultured in vitro take 2-3 days to adjust to the new environment, and show peak levels of secretion at days 3 and 4. The active secretion of phosphorylcholine by the parasite therefore justifies the measurement of this molecule as an indication of active infection, possibly reflecting total worm burdens. By comparing metabolically labelled E/S from male and female worms, several molecules of low mol. wt, namely 10,000, 13,000, 14,000 and 22,000, together with high mol. wt components of above 12,000 were found to be female specific. Tracing the origin of the E/S products, several molecules were also found to be associated with the surface. Among these, there are at least two glycoproteins, 29,000 and 51,000 of which the 29,000 molecule is a major surface protein. The immunogenicity of the E/S was examined and antigenic cross-reactivity was found with sera from most filarial infections but not with non-filarial nematodiases such as hookworm or Trichinella. However, two molecules of low mol. wt, 15,000 and 19,000, were not recognized by anti-Onchocerca sera and appeared to be potential Brugia-specific diagnostic molecules. Possible functional roles of the adult E/S products were examined but we could find no evidence of protease activity in the E/S or glutathione S-transferase activity in either the E/S or in whole somatic extract.     

旋毛虫成虫排泄分泌蛋白抗CLP诱导的小鼠脓毒症的观察

目的 目的 探讨旋毛虫成虫排泄分泌蛋白 （AES） 对盲肠结扎穿孔术 （CLP） 诱导的小鼠脓毒症的抑制作用。方法 方法 将48只BALB/c小鼠随机分为3组, 即假手术对照组 （PBS + sham组, A组）、 脓毒症组 （PBS + CLP组, B组）、 AES蛋白治疗 组 （AES + CLP组, C组）。A组和B组于CLP术后经腹腔注射200 μl PBS, C组术后经腹腔注射含25 μg AES蛋白的等量 PBS。每组随机抽取8只, 建模后观察各组小鼠的一般情况及96 h生存率; 每组其余8只小鼠于建模后12 h取肝、 肺、 肾, HE染色观察病理改变; 以酶联免疫吸附试验 （ELISA） 检测小鼠血清中TNF?α、 IL?1β、 IL?6、 IL?10及TGF?β细胞因子水平 的变化。结果 结果 3组小鼠96 h生存率差异有统计学意义 （χ2 = 21.16, P < 0.05）; 与A组 （100%） 相比, B组小鼠生存率 （0） 降低 （P < 0.05）, 且肺、 肝及肾脏损伤明显加重; 经AES治疗后, C组存活率 （75%） 明显增加 （P < 0.05）, 小鼠肺脏、 肝脏及 肾脏病理损伤亦显著缓解。3组小鼠血清中的促炎因子TNF?α （F = 27.11, P < 0.05）、 IL?1β （F = 18.75, P < 0.05） 及IL?6 （F = 100.93, P < 0.05） 水平差异均有统计学意义; B组小鼠的促炎因子水平均高于A组 （P均< 0.05）; 经AES治疗后, C组 小鼠上述3种促炎因子水平较B组均显著下降 （P均< 0.05）。3组小鼠血清中的调节性细胞因子IL?10 （F = 10.88, P < 0.05） 及TGF?β （F = 11.37, P < 0.05） 水平差异亦有统计学意义; 经AES治疗后, C组小鼠IL?10及TGF?β分泌水平较B组亦 明显增加 （P < 0.05）。结论 结论 旋毛虫AES对CLP诱导的BALB/c小鼠脓毒症有显著的缓解作用。     

The recognition of antigens on the surface of adult and L4 Necator americanus by human and hamster post-infection sera

The surface antigens of adult Necator americanus were recognized by post-infection hamster sera and resolved at molecular weight 93,000, 67,000, 46,000, 43,000, 32,000 and 25,000. L4 larvae in contrast had one major surface antigen, resolving at 93,000. These antigens were also recognized by a range of human sera, although on a differential basis. This suggests that the human sera tion. However, the results do indicate that the hamster model might be of immunological relevance to the human disease state, in that infected hamster recognized the full cuticular antigen spectrum of adult Necator. This, at least, gives the experimenter a convenient reference point from which to conduct further experiments incorporating parameters such as re-infection, anthelmintic treatment and genetic variability to study the effect of these modifications on the serological response.     

Neurocysticercosis immunodiagnosis using Taenia solium cysticerci crude soluble extract, excretory secretory and lower molecular mass antigens in serum and urine samples of Indian children

Neurocysticercosis (NCC), the most common neurological disorder of parasite etiology, results from lodgment of Taenia solium cysticerci in the central nervous system and is now increasingly being recognized in children. The confirmed diagnosis is based collectively on radiological findings and serodiagnostic techniques. The serodiagnostic techniques have variable sensitivity and specificity depending upon the technique, antigens used, location and number of cysts. Crude soluble extract (CSE), excretory secretory (ES) and lower molecular mass (LMM) (10-30 kDa) antigenic fraction of T. solium cysticerci were evaluated for antibody detection in serum and urine samples by ELISA. Serum and urine samples were collected each from 125 clinically suspected and radiologically proven NCC (111 with single Computed Tomography (CT) lesions and 14 with multiple CT lesions) and 125 control subjects (60 with neurological disorders other than NCC, 40 with other parasitic diseases and 25 apparently healthy subjects). The sensitivity of the ELISA with the use of CSE, ES and LMM antigenic fractions was 38.4%, 63.2% and 30.4% with serum (cut off dilution 400), 46.4%, 44% and 47.2% with neat urine and the specificity was 88%, 76.8% and 85.6% with serum (cut off dilution 400), 66.4%, 65.2% and 58.4% with neat urine samples, respectively. The study suggests that detection of antibody to ES antigen in serum samples may serve useful purpose for the serodiagnosis of human NCC.     

Excretory/secretory products from plerocercoids of Spirometra erinacei reduce iNOS and chemokine mRNA levels in peritoneal macrophages stimulated with cytokines and/or LPS

During infection with plerocercoids of Spirometra erinacei , organisms in the peritoneal cavity of infected animals have many bound inflammatory leukocytes yet survive apparently unharmed. Coculture of IFNγ and LPS stimulated mouse peritoneal macrophages with live plerocercoids suppressed the mRNA expression of the inducible isoform of nitric oxide synthase (iNOS) and JE, the murine homologue of monocyte chemotactic protein-1 (MCP-1). Excretory/secretory (ES) products from plerocercoids also suppressed the induced iNOS and JE mRNA and reduced nitrite production of macrophages in a dose dependent manner. The suppression of inducible mRNA levels in macrophages cultured for 24 h with ES products varied with the nature of the stimuli; IFNγ/LPS-induced iNOS mRNA levels were effected less than were iNOS mRNA levels induced by IFNγ/IL-2 or IFNγ/TNFα. Similar findings were obtained when nitrite production was measured. Thus modulation of LPS and cytokine inducible mRNA levels appear to be the primary target of ES products. We speculate that a major physiological role for this inhibitory activity in ES products might be the down regulation of pro-inflammatory gene expression.     

Immunochemical localization of major hydatid fluid antigens in protoscoleces and cysts of Echinococcus granulosus from human origin

Monospecific rabbit antisera obtained through experimental immunization with previously purified proteins were used in the structural localization of two hydatid fluid antigens, antigen 5 and antigen B, in cyst membranes and protoscoleces of E. granulosus from human origin. The antigen-antibody reaction was revealed by an avidin-biotin-peroxidase technique. Antigen 5 was not evident in the laminated membrane of the cyst wall, but it was associated with the germinal membrane of the cyst wall and brood capsules. The parenchyma of invaginated and evaginated protoscoleces was heavily labelled. The tegument, the calcareous corpuscles, the suckers and the hooks did not contain antigen 5. Degenerated protoscoleces were also labelled. Antigen B localization was essentially identical to antigen 5, but degenerated protoscoleces were not recognized by anti-antigen B antiserum. Technical aspects and differences with previously published work are discussed.     

Detection of circulating E/S antigens in the sera of patients with fascioliasis by IELISA: a tool of serodiagnosis and assessment of cure

An IELISA was developed to evaluate the performance of Fasciola E/S antigens in diagnosis and cure assessment of human Fasciola infection. Twenty patients with acute (prepatent) fascioliasis and another 20 with patent infection were enrolled in the study. Patients were treated with TCZ and followed at 1, 3 and 6 months after therapy. At inspection, the sensitivity of the test to diagnose prepatent cases was 100% compared to 70% for patent infections. There was a gradual decrease of antigenaemia over the follow-up period in acute cases. In chronic cases antigen disappeared from 13 cases (65%) at 1 month; this proportion did not change at 3 or 6 months.     

''Arc 5'' antibodies in sera of sheep infected with Echinococcus granulosus, Taenia hydatigena and Taenia ovis

The 'arc 5' precipitin band, formed when test human serum is reacted against immunoelectophoresed hydatid cyst fluid antigen, has provided a positive diagnosis of Echinococcus granulosus infection. These antibodies to 'arc 5' antigen have now been found in the sera of sheep. They appear 2 weeks after infection with Taenia ovis, after 3 weeks with T. hydatigena and after 16 weeks with E. granulosus. An antigen similar to the 'arc 5' antigen of E. granulosus cyst fluid was also demonstrated in cyst fluid from T. hydatigena, but it would not be positively identified in T. ovis cyst fluid. The presence of 'arc 5' in immunoelectrophoresis tests is not suitable for specific immunodiagnosis of E. granulosus infections in sheep in New Zealand. 'Arc 5' antibodies were only associated with living E. granulosus cysts and were not present if cysts were dead. The location of the cysts in either liver or lungs and the onset of brood capsule production did not influence the presence of 'arc 5' antibodies.     

日本血吸虫不同病期血清抗体对虫源性及卵源性组分抗原的识别及鉴定

目的分析不同病期感染兔血清所识别的日本血吸虫成虫抗原(adult worm antigen,AWA)和可溶性虫卵抗原(soluble egg antigen,SEA)的组分蛋白,探索可能与Th1/Th2极化相关的主要功能性抗原。方法常规方法制备成虫和虫卵抗原,用SDS-PAGE电泳和Western blotting技术,分析鉴定感染动物血清抗体识别的虫源性及卵源性组分抗原。结果不同病期血清抗体识别的AWA、SEA组分蛋白谱不同。其中AWA中的11kDa、37kDa、57kDa、79kDa、95kDa a组分分子能被各病期(2~20w)感染血清抗体所识别;SEA中的10kDa、18kDa、41kDa、47kDa、74kDa则可被急性期及以后各病期(6~20w)血清抗体所识别,25kDa、32kDa仅为急性期(6~12 w)血清抗体所识别,提示这些组分抗原可能为血吸虫免疫应答的主要功能性抗原,并且同感染血清的反应性明显与病期相关。结论以上AWA和SEA的组分蛋白和不同病期感染血清的反应与血吸虫感染后的Th1/Th2免疫偏移相关,其中能被急性期及以后各病期血清抗体共同识别的SEA主要功能性组分抗原中可能存在诱导Th2极化反应的重要抗原分子。     

HnRNP A2/B1及p21WAF1蛋白在非小细胞肺癌中表达及意义

目的探讨核内不均一核糖核蛋白A2/B1（hnRNP A2/B1）和p21WAF1蛋白在人非小细胞肺癌（NSCLC）组织中的表达,并进行相关性分析。方法采用免疫组化S-P法检测53例NSCLC组织、40例癌旁组织、7例肺良性病变组织中hnRNPA2/B1和p21WAF1表达。结果 NSCLC组织中hnRNP A2/B1和p21WAF12种蛋白的阳性表达率分别为66.0%和45.2%,显著高于癌旁肺组织和肺良性病变肺组织,差异有统计学意义（P〈0.05）。它们均与分化程度、淋巴结转移有关（P〈0.05）,与组织类型、临床分期无关（P〉0.05）。两种蛋白在NSCLC表达呈显著负相关（P=0.001）。结论 hnRNP A2/B1可能通过下调p21WAF1的表达而缩短细胞周期,促进NSCLC的癌细胞增殖。     

Establishment of an in vitro bioassay and radio receptor assay for LH/CG in human sera using immortalized granulosa cells transfected with LH/CG receptor

Levels of gonadotropic hormones in human sera or urine are routinely measured by radioimmunoassay or by enzyme-linked immunoassay (ELISA), which determine the immunoactivity of the hormone, but not its biological activity. We have utilized immortalized stable steroidogenic granulosa cells, which express 5–10 times more of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptors than the primary cells, to develop a biological assay and radioreceptor assay for this hormone. We found that stimulation of cells expressing LH/CG receptor with increasing doses of human LH or human CG resulted in a dose-dependent increase of cAMP and progesterone with an ED₅₀ of 30 and 57 mlU/mL, respectively. These dose-response data served as calibration curves for measuring the gonadotropin bioactivity in human serum samples at concentrations as low as 1–5 mlU/mL. We found a close correlation between LH levels measured by enzyme immunoassay (EIA) and the in vitro bioassay in normal cycling and menopausal women, as well as in normal adult men. Also, a close correlation was found between the EIA and the in vitro biological assay of hCG in pregnant women. In addition, we have developed a radioreceptor assay (RRA) for this hormone using enriched cell membranes of the appropriate cell line, which corresponds well to both the EIA and the bioassay in human sera. Deglycosylated hCG was fully active in RRA, but failed to activated cAMP response in these cells, demonstrating the importance of the bioassay in the biologically inactive form of gonadotropins. We believe this novel in vitro bioassay of gonadotropic hormones will serve as a useful tool for a more comprehensive set of assays that will determine not only the amount, but also the possible modulation in bioactivity of the gonadotropin associated with gonadal failure and miscarriage.     

Surface-displayed glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis enhance the activation of the fibrinolytic system of the host

Cardiopulmonary dirofilariosis is a cosmopolitan disease caused by Dirofilaria immitis, a filaroid parasite whose adult worms live for years in the vascular system of its host. Previous studies have shown that D. immitis can use their excretory/secretory (ES) and surface antigens to enhance fibrinolysis, which could limit the formation of clots in its surrounding environment. Moreover, several isoforms of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL) were identified in both antigenic extracts as plasminogen-binding proteins. The aim of this work is to study the interaction of the GAPDH and GAL of D. immitis with the fibrinolytic system of the host. This study includes the cloning, sequencing and expression of the recombinant forms of the GAPDH and GAL of D. immitis (rDiGAPDH and rDiGAL) and the analysis of their capacity as plasminogen-binding proteins. The results indicate that rDiGAPDH and rDiGAL are able to bind plasminogen and stimulate plasmin generation by tissue plasminogen activator (tPA). This interaction needs the involvement of lysine residues, many of which are located externally in both proteins as have been shown by the molecular modeling of their secondary structures. In addition, we show that rDiGAPDH and rDiGAL enhance the expression of the urokinase-type plasminogen activator (uPA) on canine endothelial cells in culture and that both proteins are expressed on the surface of D. immitis in close contact with the blood of the host. These data suggest that D. immitis could use the associated surface GAPDH and GAL as physiological plasminogen receptors to shift the fibrinolytic balance towards the generation of plasmin, which might constitute a survival mechanism to avoid the clot formation in its intravascular habitat.