抗独特型抗体对马来丝虫感染沙鼠的保护性免疫效…

目的：研究抗丝虫抗独特型抗体（抗ｆｉｌ－抗Ｉｄ－Ａｂ）对丝虫感染沙鼠的保护性免疫作用。方法：从班氏丝虫病乳糜尿和鞘膜积液患者血清中分离含有高滴度的ＩｇＧ，免疫家兔，获得兔抗ｆｉｌ－抗Ｉｄ－ＩｇＧ，免疫沙鼠，再用马来丝虫感染期幼虫攻击，观察其免疫效果。结果：抗ｆｉｌ－抗Ｉｄ－ＩｇＧ一次脾内注射或多次皮下及腹腔内免疫后，５０％和８０％沙鼠产生保护性免疫效果，沙鼠体查不到微丝蚴和成虫，而从马来丝虫成虫可     

抗独特型抗体对马来丝虫感染沙鼠的保护性免疫效果观察

目的 :研究抗丝虫抗独特型抗体 (抗 fil-抗 Id- Ab)对丝虫感染沙鼠的保护性免疫作用。方法 :从班氏丝虫病乳糜尿和鞘膜积液患者血清中分离含有高滴度的 Ig G,免疫家兔 ,获得兔抗 fil-抗 Id- Ig G,免疫沙鼠 ,再用马来丝虫感染期幼虫攻击 ,观察其免疫效果。结果 :抗 fil-抗 Id- Ig G一次脾内注射或多次皮下及腹腔内免疫后 ,50 %和 80 %沙鼠产生保护性免疫效果 ,沙鼠体内查不到微丝蚴和成虫 ,而从马来丝虫成虫可溶性抗原或兔抗正常人 Ig G为对照组的沙鼠中 ,80 %沙鼠感染丝虫 ,与正常感染组相近似。结论 :用抗 fil-抗 Id- Ab对马来丝虫感染沙鼠 ,证实有保护性免疫作用。     

克隆丝虫早期优势抗原:马来丝虫肌球蛋白重链基因家族的一员

用放射性元素照射过的丝虫第3期幼虫(L_3)免疫动物可产生抗丝虫的保护性免疫,而以单剂量正常L_3感染动物却不能。用免疫和感染动物的血清筛选马来丝虫cDNA库,可筛出能表达保护性重组抗原的克隆。基此作如下研究。 以~(60)Co照射马来丝虫L_3皮下注射沙鼠,获得免疫的沙鼠血清(VJS);沙鼠皮下或腹     

淋巴丝虫感染中T淋巴细胞的免疫应答──白细胞介素2和γ干扰素的体外诱生分析

目的：探讨淋巴丝虫感染中Ｔ淋巴细胞的免疫应答机制。方法：检测安徽省班氏丝虫病流行区人群及感染马来丝虫的长爪沙鼠体外诱生的白细胞介素２（ＩＬ－２）和γ干扰素（ＩＦＮ－γ）。结果：对非特异性有丝分裂原ＰＨＡ及ＣｏｎＡ的刺激，各组人群外周血单核细胞（ＰＢＭＣ）产生ＩＬ－２及ＩＦＮ－γ水平无显著差异。对马来丝虫成虫抗原的刺激，微丝蚴血症者ＰＢＭＣ产生ＩＬ－２及ＩＦＮ－γ水平比流行区无症状、无微丝蚴血症者显著低下，长爪沙鼠实验结果与人群的相似。结论：本实验提示，班氏丝虫病流行区微丝蚴血症者及马来丝虫微丝蚴血症长爪沙鼠的Ｔ细胞对丝虫抗原处于一种低应答状态，且这种受抑制的Ｔ细胞主要是Ｔｈ１亚群细胞。     

日本血吸虫卵黄铁蛋白基因的体外扩增及逆转录病毒pLXSN-Fer1重组质粒的构建

目的　构建日本血吸虫ｐＬＸＳＮ－ Ｆｅｒ１ 真核表达重组逆转录病毒,为进一步在细胞中表达及ＤＮＡ免疫作准备。方法　用ＰＣＲ技术从日本血吸虫成虫ｃＤＮＡ文库中扩增编码卵黄铁蛋白（ｙｏｌｋ ｆｅｒｒｉｔｉｎ,Ｆｅｒｌ） 的基因片段,定向克隆入ｐＬＸＳＮ 逆转录病毒载体,然后经氨苄ＬＢ培养基筛选,酶切、ＰＣＲ鉴定。结果　从日本血吸虫成虫ｃＤＮＡ文库中扩增出特异的卵黄铁蛋白的基因片段,克隆成功ｐＬＸＳＮ－ Ｆｅｒ１ 重组逆转录病毒。结论　构建成功ｐＬＸＳＮ－ Ｆｅｒ１ 重组逆转录病毒。     

马来丝虫病特异性诊断抗原研究

采用ＳＤＳ－ＰＡＧＥ和ＥＬＩＢ技术分析马来丝虫成虫（ＭＡＡ）和微丝蚴（ＭＦＡ）可溶性抗原。马来丝虫成虫和微丝蚴采自感染沙鼠腹腔。分析结果表明，ＭＦＡ蛋白组分含有沙鼠腹腔液蛋白组分（６４－６７ｋＤａ和５６－５８ｋＤａ）。健康沙鼠血清与ＭＦＡ作ＥＬＩＢ，可见３条淡反应带（６０ｋＤａ、７４ｋＤａ和１００ｋＤａ），与ＭＡＡ无反应带可见。ＭＡＡ蛋白组分中的４２ｋＤａ和１４．５ｋＤａ可被感染６个月的阳性沙鼠血清识别，而不被阴性沙鼠血清识别。     

日本血吸虫卵黄铁蛋白基因的体外扩增及逆转录病毒pLXSN-Ferl重组质粒的构建

目的 构建日本血吸虫ｐＬＸＳＮ－Ｆｅｒ１真核表达重组逆转录病毒，为进一步在细胞中表达及ＤＮＡ免疫作准备，方法 用ＰＣ座日本血吸虫成虫ｃＤＮＡ文库听话 增编码卵黄铁蛋白的基因片段，定向克隆入ＰＬＸＳＮ逆转录病毒载体，然后经氨苄ＬＢ培养基筛选，酶切、ＰＣＲ鉴定。结果 从日本血吸虫成虫ｃＤＮＡ文库中扩增出特异的卵黄铁蛋白的基因片段，克隆成功ＰＬＸＳＮ－Ｆｅｒ１重组逆转录病毒。结论构成功ＰＬＸＳＮ＿Ｆｅｒ     

马来丝虫感染沙鼠治疗前后血清抗体水平的动态研究

应用马来丝虫和牛丝虫(Setaria cervi)成虫冰冻切片抗原作间接荧光抗体试验(IFAT)及免疫酶染色试验(IEST),均能显示马来丝虫感染沙鼠治疗前后血清IgG及IgM水平的消长情况。两者高峰分别在感染后12～14wk及2～6wk。IgG水平与感染时间长短有密切关系,但与感染度无显著相关。感染沙鼠治后6个月抗体下降。认为用同种和异种抗原作IFAT及IEST,对感染沙鼠均具有诊断和考核疗效的价值。     

长爪沙鼠抗马来丝虫抗体的动态变化

用马来丝虫成虫和微丝蚴可溶性粗抗原作ELISA,观察了感染马来丝虫的长爪沙鼠血清中特异抗体的动态变化。结果表明:感染沙鼠腹腔液中的微丝蚴阳性检出率为36.7％;抗成虫和微丝蚴抗体最早分别在感染后第2周和第3周可测得;两种抗体在第1次出现后,均持续上升,抗成虫抗体水平在感染后的第16周稍有下降,而抗微丝蚴抗体却持续在高水平上;显性感染沙鼠的抗微丝蚴抗体在腹腔出现微丝蚴后,明显高于隐性感染鼠.     

鼠伤寒杆菌核糖体制剂用作血吸虫抗原佐剂的探讨

目的：探讨鼠伤寒杆菌核糖体制剂对血吸虫抗原的佐剂作用。方法：小鼠分别用鼠伤寒杆菌核糖体制剂（ＳＴＲＰ）加日本血吸虫成虫抗原（ＳＷＡ）和日本血吸虫成虫抗原免疫后，其体液免疫水平用ＥＬＩＳＡ检测，保护性免疫力用减虫率表示。结果：用ＳＴＲＰ＋ＳＷＡ免疫的小鼠的抗体水平显著高于单用ＳＷＡ免疫的小鼠。尾蚴攻击感染后，ＳＴＲＰ＋ＳＷＡ免疫组小鼠和ＳＷＡ免疫组小组的减虫率，分别为４７％和１７％，前者高于后者。结论：ＳＴＲＰ可以增强小鼠对血吸虫抗原的体液免疫应答反应，并且可诱导小鼠产生较强的抗尾蚴攻击感染能力。     

Immunoprophylactic potential of a 120 kDa Brugia malayi adult antigen fraction, BmA-2, in lymphatic filariasis

A 120 kDa antigen containing SDS-PAGE fraction BmA-2 isolated from Brugia malayi adult parasite was highly reactive with normal sera from filarial endemic area. BmA-2 was analysed for its propylactic potential in in vitro and in vivo. Sera collected from BmA-2 immunized jirds induced a significant level (80 to 90%) of protection against infective larvae and microfilariae in in vitro ADCC assay as well as in in situ micropore chamber implantation studies. Mastomys natalensis immunized with BmA-2 showed a significant level of protective response against circulating microfilariae by clearing 90% of them from circulation by fifth day after challenge infection. Immunization of jirds with BmA-2 resulted in an enhanced level of antibody response against BmA-2 and 88% reduction in the development of the parasites to the adult stage. Passive transfer of immunesera from jirds immunized with BmA-2 to naive jirds resulted in 71% reduction in adult worm recovery as observed 90 days after challenge infection with B. malayi. On the other hand the passive transfer of nonadherent spleen cells from immune jirds did not show any significant effect on the development of parasite. Administration of jirds anti BmA-2 serum to microfilaraemic jirds showed a temporary decrease in micrqfilarial count which was increased to pretherapeutic level within 100 days and there was no effect on the adult worms. This implies that the immune protective effect of BmA-2 is mainly antibody dependent and active immunization with BmA-2 is effective against filarial infection.,     

Evaluation of recombinant chitinase antigen in serological diagnosis and surveillance of lymphatic filariasis

Apply recombinant chitinase fusion protein antigen, enzyme-linked immunosorbent assays examined anti-filarial antibodies and evaluated of useful value in serological diagnosis and surveillance of lymphatic filariasis. The test jirds were immunized and infected by chitinase and B. malayi third stage larvae respectively. Functional protein molecular of chitinase was analyzed by SDS-PAGE, Western blot. The result shown that jirds from microfilaremia (mf) and donors with Mf were directly to react with chitinase antigen that positive rate was 100%, but Mf-xt antigen was only 80%. Normal jirds and persons sera from unepidemic control donors all were negative. False positives of 5% and 20% reacted with chitinase and Mf-xt antigens respectively. The results indicate that recombinant chitinase antigen is suitable for detection of active occult or patent lymphatic filariasis with daytime blood samples in residents of endemic areas, is easy to be performed and inexpensive.     

Parasite antigenemia in untreated and treated lymphatic filarial infections

To evaluate the merit of antigen detection assays as a tool to monitor the efficacy of chemotherapy for lymphatic filariasis, we serially measured antigen levels in sera from jirds infected with Brugia malayi and from humans with bancroftian filariasis. Antigenemia was detected in all animals with parasitologically proven infection and was present in jirds with prepatent or occult filariasis. Antigen levels correlated with worm burdens, and progressively declined in drug-cured animals. Treatment with diethylcarbamazine (DEC) triggered a transient increase in serum levels of filarial antigens bearing the epitope recognized by the monoclonal antibody HC 11. All patients with bancroftian filariasis became amicrofilaremic within one week after DEC treatment. Antigenemia levels slowly declined over a period of several months in all but one treated individual. Forty-two months after treatment, progressively rising antigen levels are present in 10 patients. Six of these remain amicrofilaremic; in the other 4, elevated antigenemia levels preceded or were detected at the same time as recurrent parasitemia. Periodic monitoring of antigenemia levels after treatment of patients with lymphatic filariasis can be used to identify individuals who are likely to develop recurrent microfilaremia before the parasites become detectable in blood samples, thereby allowing timely retreatment.     

Identification of phosphorylcholine epitope-containing antigens in Brugia malayi and relation of serum epitope levels to infection status of jirds with brugian filariasis

The Gib 13 monoclonal antibody was raised against eggs of Onchocerca gibsoni and subsequently found to react with a phosphorylcholine epitope designated as the T15 idiotype. Since an immunoradiometric assay based on the Gib 13 monoclonal antibody holds promise for serodiagnosis of filariasis, the goals of the current study were to evaluate phosphorylcholine epitope production and release by various parasite stages and to assess changes in serum epitope levels during different phases of Brugia malayi infection in jirds. Extracts of B. malayi adult male worms, female worms, and microfilariae contained Gib 13 monoclonal antibody-reactive antigens of Mr 25-30,000, 57-90,000, and approximately equal to 200,000. Adult female worms secreted ten-fold more epitope than microfilariae on a weight basis. Phosphorylcholine-containing antigens were localized in female and male worms, respectively, in egg-bearing regions and the intestines. Assessment of the relationship between serum levels of Gib 13 antibody-binding epitope and parasitologic status of B. malayi-infected jirds showed that the immunoradiometric assay distinguishes patent infected from uninfected control animals, detects a significant rise in epitope level during the prepatent phase of infection, and is unaffected by diethylcarbamazine-induced reduction in the intensity of microfilaremia. There was a direct positive correlation between serum epitope level and female adult worm load. Quantification of serum phosphorylcholine epitope of the T15 idiotype may be useful as an indirect measure of parasite burden in humans with lymphatic filariasis that is independent of microfilaremia.     

Granulomatous inflammatory response to recombinant filarial proteins of Brugia species

The lymphatic inflammatory response in Brugia-infected jirds peaks early during primary infections and then decreases in severity as judged by the numbers of lymph thrombi present within these vessels. Antigen-specific hypersensitivity reactions in these animals was measured by a pulmonary granulomatous inflammatory response (PGRN) induced by somatic adult worm antigen (SAWA)-coated beads, and by cellular proliferative responses of renal lymph node cells. The kinetics of these responses temporally correspond to lymphatic lesion formation. The importance of any single antigen to the induction of this inflammatory response has not been elucidated. In this study, the PGRN was used to measure the cellular immune response to four recombinant filarial proteins during the course of a primary B. pahangi infection. These proteins were BpL4, glycoprotein (glutathione peroxidase) gp29, heat shock protein (hsp) 70, and filarial chitinase. All were fusion proteins of maltose-binding protein (MBP). Control beads included those coated with diethanolamine (DEA), SAWA, or MBP. The measurements of PRGN were made at 14, 28, 56, and > 150 days postinfection (PI) in infected jirds, in jirds sensitized with SAWA, and in uninfected jirds. The secretory homolog of glutathione peroxidase gp29 was the only recombinant protein tested that induced a significantly greater PGRN (P < 0.05) than controls. This was seen at 28 days PI. These observations indicate that gp29 may be part of the worm antigen complex that induces an early inflammatory response, a response similar to that observed with SAWA. These studies indicate that this approach is useful in investigating the functional ability of specific proteins in the induction and down-regulation of immune-mediated inflammatory responses elicited by filarial parasites. Absence of a granulomatous response to the other recombinant proteins used may be related to the nature and sensitivity of the assay used or the character of recombinant proteins tested.     

马来丝虫虫体抗原检测长爪沙鼠血清抗体

长爪沙鼠实验感染马来丝虫后,用感染期幼虫、微丝蚴的超声粉碎片段抗原和成虫的冰冻切片抗原,进行间接荧光抗体试验,观察血清抗体动态变化。抗感染期幼虫抗体多于感染后前3Wk内出现,抗成虫及微丝蚴抗体多于感染后前5、6wk内出现。在微丝蚴出现前,腹腔微丝蚴阳性和阴性沙鼠抗体水平差别无显著性,而在微丝蚴出现后,抗体水平具有显著性差别。     

Brugia malayi: vaccination of jirds with 60cobalt-attenuated infective stage larvae protects against homologous challenge

Vaccination of inbred jirds (Meriones unguiculatus) with 60cobalt radiation-attenuated Brugia malayi infective stage larvae (L3) protected against homologous challenge given either subcutaneously (sc) or by the intraperitoneal (ip) route. Groups of jirds vaccinated once sc with 75, 15 Krad L3 showed from 69% to 91% reduction in recovered worms after ip challenge infection compared to infection in non-vaccinated control jirds, while 75% reduction in mean worm burden was seen in jirds receiving sc challenge infection. A single sc vaccination with 75, 10 or 20 Krad L3 produced no protection (10 Krad) and 64% reduction in recovered worms (20 Krad). Therefore the 15 Krad dose appeared to be best. A marked increase in anti-B. malayi antibody in vaccinated jirds was seen (by ELISA) immediately after challenge infection and an immunofluorescence assay showed that L3 incubated in serum from vaccinated jirds were completely and uniformly covered with specific antibody. Eosinophil-rich granulomas containing dead and moribund L3 were recovered from vaccinated jirds. This model of protective immunity in a Brugia-susceptible small rodent may provide a useful system for identification of molecularly defined filarial-protective immunogens.     

Withania somnifera chemotypes NMITLI 101R, NMITLI 118R, NMITLI 128R and withaferin A protect Mastomys coucha from Brugia malayi infection

Withania somnifera is an ayurvedic Indian medicinal plant whose immunomodulatory activities have been widely used as a home remedy for several ailments. We recently observed immunostimulatory properties in the root extracts of chemotypes NMITLI-101, NMITLI-118, NMITLI-128 and pure withanolide, withaferin A. In the present study, we evaluated the potential immunoprophylactic efficacies of these extracts against an infective pathogen. Our results show that administration of aqueous ethanol extracts (10 mg/kg) and withaferin A (0·3 mg/kg), 7 days before and after challenge with human filarial parasite Brugia malayi, offers differential protection in Mastomys coucha with chemotype 101R offering best protection (53·57%) as compared to other chemotypes. Our findings also demonstrate that establishment of B. malayi larvae was adversely affected by pretreatment with withaferin A as evidenced by 63·6% reduction in adult worm establishment. Moreover, a large percentage of the established female worms (66·2%) also showed defective embryogenesis. While the filaria-specific immunological response induced by withaferin A and NMITLI-101 showed a mixed Th1/Th2 phenotype, 118R stimulated production of IFN-γ and 128R increased levels of IL-4. Taken together, our findings reveal potential immunoprophylactic properties of W. somnifera, and further studies are needed to ascertain the benefits of this plant against other pathogens as well.     

A comparison of host responses of the Mongolian jird to infections of Brugia malayi and B. pahangi

Host responses of jirds receiving a single subcutaneous inoculation of subperiodic Brugia malayi were compared with those of jirds similarly infected with B. pahangi. Parasite burdens, lymphatic lesion severity, granulomatous reactivity, antibody responses to parasite antigens, and complete blood cell counts were assessed at 60 and 150 days post-inoculation. At 60 days post-inoculation, percentages of adults recovered at necropsy and lymphatic lesion severity were greater in B. pahangi-infected jirds. At 150 days post-inoculation, lesion severity and percentages of worms recovered were similar in both infections. No significant differences were noted in either infection in reactivity to homologous or heterologous parasite antigens in any parameter measured. Similarities in the kinetics of the inflammatory reactivities of the 2 infections suggest that previous observations made in the jird-B. pahangi model could be utilized in designing studies using B. malayi. Further, the more marked lesion severity observed in B. pahangi-infected jirds and the relative ease of maintaining B. pahangi in the laboratory support the continued use of this system as a conceptual model for the study of lymphatic lesion pathogenesis.