日本血吸虫卵对TNBS诱导的小鼠结肠炎的保护作用及其机制研究
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摘要
克隆病(Crohn’s disease)是一种发生于肠道的自身免疫性疾病。近五十年来,其发病率在发达国家呈上升趋势,而在卫生条件不足、欠发达的第三世界国家则较低,由此有学者提出“卫生假说(hygiene hypothesis)”,即儿童早期对一些病原体如某些蠕虫、病毒或细菌等的接触有助于自身耐受的形成和维持免疫平衡,从而减少过敏性和自身免疫性疾病的发生。此机制可能与蠕虫感染可以调节Th1或Th2免疫反应有关。
蠕虫的慢性感染可以引起以IL-4升高为主的Th2类反应的发生,而IL-4可抑制Th1类细胞的分化,因此IL-4可以对宿主的免疫进行调节,并且在自身免疫性疾病的发生发展中起重要的作用,它可以抑制Th1类反应相关的自身免疫性疾病。2,4,6三硝基苯磺酸(trinitrobenzesulfonic acid, TNBS)诱导的结肠炎具有与克隆病类似的发病特点,是以Th1型介导为主的免疫反应,产生高水平的前炎症因子IFN-γ,从而在结肠引发严重的炎症。血吸虫感染后幼虫和成虫引起Th1为主的免疫反应,产卵后虫卵诱发以Th2为主的免疫反应。而调节性T淋巴细胞(regulatory T cells,Tregs),这群具有免疫抑制作用的细胞亚群可以识别自身抗原肽,分泌抑制性细胞因子如IL-10,从而抑制自身反应性T细胞和辅助T细胞的活化,抑制过度的Th1/Th2极化,防止自身免疫的发生并控制各种形式的免疫反应。在血吸虫的感染中,调节性T淋巴细胞也参与其免疫调节,但有关血吸虫诱导产生的Tregs在CD中的作用目前还未见报道。
本文探讨了日本血吸虫卵对TNBS诱导的小鼠结肠炎的影响及调节性T淋巴细胞在日本血吸虫卵对小鼠溃疡性结肠炎的免疫调节中的作用。本论文包括日本血吸虫卵对TNBS诱导的小鼠结肠炎保护作用的观察和日本血吸虫卵对TNBS诱导的小鼠结肠炎保护作用机制的研究两部分:
一、日本血吸虫卵对TNBS诱导的小鼠结肠炎保护作用的观察
目的观察日本血吸虫卵对TNBS诱导的小鼠结肠炎的保护作用。方法50只6～8周龄雌性BALB/c小鼠,随机分组,10只/组,分别为正常对照组、乙醇对照组、血吸虫卵免疫对照组、TNBS诱导的结肠炎组和虫卵免疫后TNBS诱导的结肠炎组。虫卵悬于100μl pH 7.4 PBS,以10 000个虫卵/鼠,分别于第0、7、14和21天经腹腔注射免疫小鼠,正常对照组小鼠仅给予相同剂量PBS。末次免疫后第5天,小鼠乙醚轻度麻醉后,用直径2 mm的灌肠器从肛门插入结肠约4 cm处,实验组小鼠注入2% TNBS (50%乙醇配制) 0.1ml,正常对照组和乙醇对照组分别注入等体积的生理盐水、50%乙醇,使灌注液能够到达盲肠,并保持小鼠垂直状态约30 s。诱导结肠炎后第6天,剖杀小鼠,剪取肛门至盲肠肠管,沿肠系膜纵轴剪开,用冰生理盐水冲洗后,常规10%甲醛固定24 h,石蜡包埋切片,苏木精-伊红染色后,显微镜下进行组织学观察,并对炎症进行分级。同时无菌取脾,制备淋巴细胞悬液,调整细胞数至3×106/ ml。将小鼠脾淋巴细胞悬液加至24孔培养板中,1 ml/孔,每孔分别加Con A 5μg/ml,双抗(青霉素和链霉素) 100 U/ml,置37℃, 5 % CO2培养箱,72 h后离心取上清液,用ELISA双抗体夹心法检测小鼠脾淋巴细胞培养上清中的IFN-γ和IL- 4水平。观察各组小鼠体重、粪便情况、结肠病理改变及细胞因子水平的变化。结果生理盐水、50%乙醇处理和单纯虫卵免疫组小鼠未见稀便、粘液便或血便,其体重随着时间的延长呈逐渐增长趋势;未给予虫卵免疫的TNBS诱导结肠炎组小鼠在诱导后第1天即出现不同程度的稀便或粘液血便;给予虫卵的结肠炎小鼠则多为稀便,粘液血便少见,但两者在TNBS诱导后体重均明显下降,与其它组比较有显著性差异(P < 0.01)。给予虫卵免疫的结肠炎小鼠体重从第3天开始逐渐恢复,至第5天接近未诱导结肠炎小鼠水平。TNBS诱导结肠炎后第6天,剖杀各实验组小鼠, 50%乙醇灌肠小鼠结肠未见充血水肿;TNBS模型组小鼠结肠明显充血水肿,有散在小溃疡形成;虫卵免疫结肠炎组小鼠则无充血水肿,结肠表面光滑。镜下,TNBS模型组小鼠肠壁明显增厚,组织上皮破损,炎性渗出,大量白细胞浸润并伴明显组织坏死,腺体结构部分消失;虫卵免疫的结肠炎小鼠则炎症较轻,黏膜结构完整。给予虫卵和未给予虫卵的结肠炎小鼠组织学分级分别为3.6±0.49和1.6±0.70 (P<0.0005)。小鼠经TNBS诱导后,Th1类细胞因子IFN-γ水平为3.47±0.87 ng/ml, Th2类细胞因子IL-4水平为146.06±45.76 pg/ml。预先经虫卵免疫的结肠炎小鼠则炎症得到明显控制,症状较轻,体重恢复较快,且IL-4水平显著升高至598.50±135.90 pg/ml而IFN-γ表达被明显抑制其水平为1.53±0.51 ng/ml。结论本研究首次证实日本血吸虫卵对TNBS诱导的小鼠结肠炎具有保护作用。
二、日本血吸虫卵对TNBS诱导的小鼠结肠炎保护作用机制的研究
目的探讨日本血吸虫卵对小鼠溃疡性结肠炎保护作用的免疫调节机制。方法40只6～8周龄雌性BALB/c小鼠,随机分组,10只/组,分为PBS对照组、血吸虫卵免疫对照组、TNBS诱导的结肠炎组和虫卵免疫后TNBS诱导的结肠炎组。将虫卵悬于100μl pH 7.4 PBS中,以10 000个虫卵/鼠分别于第0、7、14和21 d经腹腔注射免疫小鼠,对照组小鼠给予等体积PBS。末次免疫后第5 d,小鼠禁食24 h,自由饮水。乙醚轻度麻醉后,用直径2 mm的灌肠器从肛门插入结肠约4 cm处,实验组小鼠注入2% TNBS 0.1 ml,对照组注入等体积的生理盐水,使灌注液能够到达盲肠,并保持小鼠垂直状态约30 s。诱导结肠炎后第6 d剖杀小鼠,无菌取脾,制备淋巴细胞悬液,调整细胞数至3×106/ ml。将小鼠脾淋巴细胞悬液加至24孔培养板中,1 ml/孔,每孔分别加Con A 5μg/ml,双抗(青霉素和链霉素) 100 U/ml,置37℃, 5 % CO2培养箱,72 h后离心取上清液,用ELISA双抗体夹心法检测小鼠脾淋巴细胞培养上清中IFN-γ、IL- 4、IL- 5和IL- 10水平。取上述各组1×106脾细胞,用流式细胞术检测脾细胞中CD4~+ CD25~+ Foxp3~+ T细胞亚群水平。结果未经虫卵免疫的小鼠经TNBS诱导后结肠炎症状明显,细胞因子IFN-γ水平高达3.47±0.87 ng/ml,IL-4、IL-5和IL-10水平则较低分别为146.06±45.76 pg/ml, 140.32±39.01 pg/ml, 385.91±134.89 pg/ml;经虫卵免疫的结肠炎小鼠的炎症得到明显控制,IFN-γ表达被明显抑制其水平为1.53±0.51 ng/ml,IL-4、IL-5和IL-10水平显著升高分别为598.50±135.90 pg/ml, 217.86±77.82 pg/ml, 799.651±197.93 pg/ml。经虫卵免疫的结肠炎小鼠脾脏CD4~+ CD25~+ Foxp3~+ T细胞亚群比例为3.55±0.81%显著高于未免疫组结肠炎小鼠的2.31±0.39%。结论:1.首次确定了日本血吸虫卵对TNBS诱导的结肠炎小鼠有保护作用,在TNBS诱导小鼠结肠炎之前给予日本血吸虫卵,可以升高Th2类细胞因子IL- 4、IL- 5以及IL- 10的水平,抑制过度的Th1反应及引起的炎症反应;2.首次证实日本血吸虫卵对TNBS诱导的结肠炎小鼠的保护作用与CD4~+ CD25~+ Foxp3~+ T细胞有关; 3.提示诱生抗原特异性调节性T细胞可能成为治疗自身免疫性疾病的一条新途径,为变害为利,进一步开发利用寄生虫资源奠定了基础。
Crohn's disease (CD) is an idiopathic chronic disease in colon. It occurs with increasing frequency in developed and industrial countries, but remains sporadically in tropical third world countries with poor sanitation. Hygiene hypothesis may explain this phenomenon. It stated that pathogen infections may protect the host from immunological disease. Although the cause of CD is complicated, it is proved that CD is not only the consequence of inappropriate stimulation of effector responses but also due to a dysregulation of the normal immunosuppressive mechanisms. The development of CD is related to a polarized type-1 immune response and TNBS-induced colitis shares features with CD. Trinitrobenzene sulfonic acid (TNBS)-induced colitis is a murine model of human CD. In TNBS-induced murine model, ethanol was used to break the mucosa barrier of intestine, TNBS served as a hapten reagent and combined with the protein on the damaged intestinal mucosa to form the complete antigen. The similar immune lesion to CD happens in the intestine of murine. It is known that helminth infections might regulate a polarized type-1 or type-2 immune response and the immune regulatory process is associated with Tregs (CD4~+ CD25~+ regulatory T cells). Shistosome infection switches immunologically from an early Th1 response to a Th2-dominated response after the onset of parasite egg-laying. During schistosome infection,Tregs are also involved in its immunomodulation in its host.
Tregs are important components of the homeostasis of the immune system, they prevent the development of autoimmunity and control virtually all forms of immune response, including inflammation. Foxp3 (forkhead box P3), a member of the forkhead/winged-helix family of transcriptional regulators, is a master regulatory gene for the development of Tregs and specifically expressed in CD4~+CD25~+ Tregs. In mice, the lack of foxp3-expressing Tregs is sufficient to break down self-tolerance and induce autoimmune disease. CD4~+ CD25~+ Foxp3~+ T cells can suppress and modulate the excessive immune response. The role of CD4~+ CD25~+ Foxp3~+ regulatory T cells in murine colitis with schistosome eggs treatment has not been reported.
Elliott showed that exposure to Schistosoma mansoni eggs protects mice from TNBS-induced colitis. In the present paper, we demonstrate that exposure to Schistosoma japonicum eggs protects mice from TNBS-induced colitis and Tregs may be involved in the protection.
Specific pathogen-free 6-8 week-old female BALB/c mice were used. Eggs of S. japonicum were collected from the livers of schistosome-infected rabbits, which had been infected with 1,500 cercariae 46 days previously. The eggs were collected and washed in phosphate buffered saline (PBS). Mice were intraperitoneally injected with 10,000 eggs in 100μl PBS at day 0. At days 7, 14 and 21, the animals were boosted in the same way and with the same dosage. Control mice received sterile PBS with the same volume and timing. In each group 5 mice were used and all experiments were duplicated. Mice were lightly anesthetized with metofane (methoxyflurane) at day 6 after the last immunization. A 3.5F catheter, fitted onto a l-ml syringe, was carefully inserted into the colon such that the tip was 4 cm proximal to the anus and 0.5 mg of TNBS in 50% ethanol was slowly administered into the lumen of the colon. In control experiments, mice were given 50% ethanol alone using the same technique described above. The total injection volume was 100μl for each mouse allowing TNBS or ethanol to reach the entire colon, including the caecum and appendix. Animals were then kept in a vertical position for 30 s and returned to their cages. Colons of mice were removed on day 6 after the induction of colitis and embedded in paraffin. Sections were stained with hematoxylin and eosin. The degree of inflammation of the colon was graded semiquantitatively from 0 to 4 (0, no evidence of inflammation; 1, low level of lymphocyte infiltration with infiltration seen in10% high-power field (hpf), no structural changes observed; 2, moderate lymphocyte infiltration with infiltration seen in 10%–25% hpf, crypt elongation, bowel wall thickening which does not extend beyond the mucosal layer, no evidence of ulceration; 3, high level of lymphocyte infiltration with infiltration seen in 25%– 50% hpf, high vascular density, thickening of bowel wall which extends beyond mucosal layer; and 4, marked degree of lymphocyte infiltration with infiltration seen in 50% hpf, high vascular density, crypt elongation with distortion, transmural bowel wall thickening with ulceration). Grading the inflammation in colons of mice was done in a blinded fashion by the same pathologist. Spleen cells were isolated from the experimental mice on day 6 after TNBS treatment. Cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM HEPES buffer, 10μg/ml gentamicin, 10% FCS, 100 U/ml penicillin and 100μg/ml streptomycin. Cells were cultured for 72 h at 3×106 cells/well in 1 ml microtiter wells and stimulated with 5μg/ml Con A. The concentrations of IFN-γ, IL-4, IL-5 and IL-10 in cell culture supernatants were determined by ELISA Kits. In brief, mouse IFN-γ, IL-4, IL-5 and IL-10 were detected by biotinylated monoclonal antibodies, which were evidenced by avidin-conjugated horseradish peroxidase followed by incubation with TMB substrate. The reactions were measured at 450 nm in an ELISA reader. Single-cell suspensions of spleen cells were stained and analyzed on FACS Calibur using CellQuest software. For determination of the phenotype of lymphocyte populations,mouse regulatory T cell staining Kit was used. The following conjugated antibodies were incubated with lymphocyte populations: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4, allophycocyanin (APC)-conjugated anti-mouse CD25 and phycoerythrin (PE)-conjugated anti-mouse Foxp3 FJK-16s (a master regulatory gene for the development of Tregs). Before incubation with PE anti-mouse Foxp3 FJK-16s,cells were fixed with permeabilization buffer and Fc receptors were blocked with anti-CD16/32 antibody. PE -conjugated rat IgG2a served as the isotype control.
The colons of mice were removed on day 6 after administration of TNBS or ethanol. TNBS-treated mice showed ulcerations of the colons surrounded by hyperemia and inflammation, whereas the colons of control mice treated with ethanol alone or S. japonicum eggs followed by TNBS showed neither macroscopic signs of inflammation nor obvious hyperemia and edema. Histologically, transmural inflammation affecting the entire colon was observed in TNBS-treated mice, lymphocyte infiltrates were associated with thickening of the colon wall, ulcerations, loss of goblet cells, and the presence of granulomas in TNBS-treated mice. The inflammatory responses were significantly milder in the colitis mice exposed to egg prior to TNBS-treatment. Histological score from egg-unexposed and -exposed mice was 3.6±0.49 and 1.6±0.70, respectively (P< 0.0005), thus confirming the prevention of TNBS-induced inflammation by previous injection of eggs. Spleen cells from TNBS-treated mice generated in vitro large amounts of IFN-γ, which were about twice the level of IFN-γafter administration of schistosome eggs prior to TNBS treatment (P<0.01). The secretion of IL-4, IL-5 and IL-10 in vitro was significantly (P<0.01) higher in colitis mice previously exposured to schistosome eggs compared to the animals treated with TNBS alone. The percentage of CD4~+ CD25~+ Foxp3~+ T cells in spleens of egg-treated mice was not significantly different from untreated control mice. These cells increased, however significantly (P<0.05) after TNBS-treatment, egg-exposed mice, but not after TNBS treatment alone.
In the research, the results indicated that CD4~+ CD25~+ Foxp3~+ regulatory T cells may be involved in the modulation during TNBS-induced colitis with schistosome eggs exposure and the excessive Th1 response was converted. As a result, the lesion of TNBS-induced colitis in mice was attenuated.
蠕虫的慢性感染可以引起以IL-4升高为主的Th2类反应的发生,而IL-4可抑制Th1类细胞的分化,因此IL-4可以对宿主的免疫进行调节,并且在自身免疫性疾病的发生发展中起重要的作用,它可以抑制Th1类反应相关的自身免疫性疾病。2,4,6三硝基苯磺酸(trinitrobenzesulfonic acid, TNBS)诱导的结肠炎具有与克隆病类似的发病特点,是以Th1型介导为主的免疫反应,产生高水平的前炎症因子IFN-γ,从而在结肠引发严重的炎症。血吸虫感染后幼虫和成虫引起Th1为主的免疫反应,产卵后虫卵诱发以Th2为主的免疫反应。而调节性T淋巴细胞(regulatory T cells,Tregs),这群具有免疫抑制作用的细胞亚群可以识别自身抗原肽,分泌抑制性细胞因子如IL-10,从而抑制自身反应性T细胞和辅助T细胞的活化,抑制过度的Th1/Th2极化,防止自身免疫的发生并控制各种形式的免疫反应。在血吸虫的感染中,调节性T淋巴细胞也参与其免疫调节,但有关血吸虫诱导产生的Tregs在CD中的作用目前还未见报道。
本文探讨了日本血吸虫卵对TNBS诱导的小鼠结肠炎的影响及调节性T淋巴细胞在日本血吸虫卵对小鼠溃疡性结肠炎的免疫调节中的作用。本论文包括日本血吸虫卵对TNBS诱导的小鼠结肠炎保护作用的观察和日本血吸虫卵对TNBS诱导的小鼠结肠炎保护作用机制的研究两部分:
一、日本血吸虫卵对TNBS诱导的小鼠结肠炎保护作用的观察
目的观察日本血吸虫卵对TNBS诱导的小鼠结肠炎的保护作用。方法50只6～8周龄雌性BALB/c小鼠,随机分组,10只/组,分别为正常对照组、乙醇对照组、血吸虫卵免疫对照组、TNBS诱导的结肠炎组和虫卵免疫后TNBS诱导的结肠炎组。虫卵悬于100μl pH 7.4 PBS,以10 000个虫卵/鼠,分别于第0、7、14和21天经腹腔注射免疫小鼠,正常对照组小鼠仅给予相同剂量PBS。末次免疫后第5天,小鼠乙醚轻度麻醉后,用直径2 mm的灌肠器从肛门插入结肠约4 cm处,实验组小鼠注入2% TNBS (50%乙醇配制) 0.1ml,正常对照组和乙醇对照组分别注入等体积的生理盐水、50%乙醇,使灌注液能够到达盲肠,并保持小鼠垂直状态约30 s。诱导结肠炎后第6天,剖杀小鼠,剪取肛门至盲肠肠管,沿肠系膜纵轴剪开,用冰生理盐水冲洗后,常规10%甲醛固定24 h,石蜡包埋切片,苏木精-伊红染色后,显微镜下进行组织学观察,并对炎症进行分级。同时无菌取脾,制备淋巴细胞悬液,调整细胞数至3×106/ ml。将小鼠脾淋巴细胞悬液加至24孔培养板中,1 ml/孔,每孔分别加Con A 5μg/ml,双抗(青霉素和链霉素) 100 U/ml,置37℃, 5 % CO2培养箱,72 h后离心取上清液,用ELISA双抗体夹心法检测小鼠脾淋巴细胞培养上清中的IFN-γ和IL- 4水平。观察各组小鼠体重、粪便情况、结肠病理改变及细胞因子水平的变化。结果生理盐水、50%乙醇处理和单纯虫卵免疫组小鼠未见稀便、粘液便或血便,其体重随着时间的延长呈逐渐增长趋势;未给予虫卵免疫的TNBS诱导结肠炎组小鼠在诱导后第1天即出现不同程度的稀便或粘液血便;给予虫卵的结肠炎小鼠则多为稀便,粘液血便少见,但两者在TNBS诱导后体重均明显下降,与其它组比较有显著性差异(P < 0.01)。给予虫卵免疫的结肠炎小鼠体重从第3天开始逐渐恢复,至第5天接近未诱导结肠炎小鼠水平。TNBS诱导结肠炎后第6天,剖杀各实验组小鼠, 50%乙醇灌肠小鼠结肠未见充血水肿;TNBS模型组小鼠结肠明显充血水肿,有散在小溃疡形成;虫卵免疫结肠炎组小鼠则无充血水肿,结肠表面光滑。镜下,TNBS模型组小鼠肠壁明显增厚,组织上皮破损,炎性渗出,大量白细胞浸润并伴明显组织坏死,腺体结构部分消失;虫卵免疫的结肠炎小鼠则炎症较轻,黏膜结构完整。给予虫卵和未给予虫卵的结肠炎小鼠组织学分级分别为3.6±0.49和1.6±0.70 (P<0.0005)。小鼠经TNBS诱导后,Th1类细胞因子IFN-γ水平为3.47±0.87 ng/ml, Th2类细胞因子IL-4水平为146.06±45.76 pg/ml。预先经虫卵免疫的结肠炎小鼠则炎症得到明显控制,症状较轻,体重恢复较快,且IL-4水平显著升高至598.50±135.90 pg/ml而IFN-γ表达被明显抑制其水平为1.53±0.51 ng/ml。结论本研究首次证实日本血吸虫卵对TNBS诱导的小鼠结肠炎具有保护作用。
二、日本血吸虫卵对TNBS诱导的小鼠结肠炎保护作用机制的研究
目的探讨日本血吸虫卵对小鼠溃疡性结肠炎保护作用的免疫调节机制。方法40只6～8周龄雌性BALB/c小鼠,随机分组,10只/组,分为PBS对照组、血吸虫卵免疫对照组、TNBS诱导的结肠炎组和虫卵免疫后TNBS诱导的结肠炎组。将虫卵悬于100μl pH 7.4 PBS中,以10 000个虫卵/鼠分别于第0、7、14和21 d经腹腔注射免疫小鼠,对照组小鼠给予等体积PBS。末次免疫后第5 d,小鼠禁食24 h,自由饮水。乙醚轻度麻醉后,用直径2 mm的灌肠器从肛门插入结肠约4 cm处,实验组小鼠注入2% TNBS 0.1 ml,对照组注入等体积的生理盐水,使灌注液能够到达盲肠,并保持小鼠垂直状态约30 s。诱导结肠炎后第6 d剖杀小鼠,无菌取脾,制备淋巴细胞悬液,调整细胞数至3×106/ ml。将小鼠脾淋巴细胞悬液加至24孔培养板中,1 ml/孔,每孔分别加Con A 5μg/ml,双抗(青霉素和链霉素) 100 U/ml,置37℃, 5 % CO2培养箱,72 h后离心取上清液,用ELISA双抗体夹心法检测小鼠脾淋巴细胞培养上清中IFN-γ、IL- 4、IL- 5和IL- 10水平。取上述各组1×106脾细胞,用流式细胞术检测脾细胞中CD4~+ CD25~+ Foxp3~+ T细胞亚群水平。结果未经虫卵免疫的小鼠经TNBS诱导后结肠炎症状明显,细胞因子IFN-γ水平高达3.47±0.87 ng/ml,IL-4、IL-5和IL-10水平则较低分别为146.06±45.76 pg/ml, 140.32±39.01 pg/ml, 385.91±134.89 pg/ml;经虫卵免疫的结肠炎小鼠的炎症得到明显控制,IFN-γ表达被明显抑制其水平为1.53±0.51 ng/ml,IL-4、IL-5和IL-10水平显著升高分别为598.50±135.90 pg/ml, 217.86±77.82 pg/ml, 799.651±197.93 pg/ml。经虫卵免疫的结肠炎小鼠脾脏CD4~+ CD25~+ Foxp3~+ T细胞亚群比例为3.55±0.81%显著高于未免疫组结肠炎小鼠的2.31±0.39%。结论:1.首次确定了日本血吸虫卵对TNBS诱导的结肠炎小鼠有保护作用,在TNBS诱导小鼠结肠炎之前给予日本血吸虫卵,可以升高Th2类细胞因子IL- 4、IL- 5以及IL- 10的水平,抑制过度的Th1反应及引起的炎症反应;2.首次证实日本血吸虫卵对TNBS诱导的结肠炎小鼠的保护作用与CD4~+ CD25~+ Foxp3~+ T细胞有关; 3.提示诱生抗原特异性调节性T细胞可能成为治疗自身免疫性疾病的一条新途径,为变害为利,进一步开发利用寄生虫资源奠定了基础。
Crohn's disease (CD) is an idiopathic chronic disease in colon. It occurs with increasing frequency in developed and industrial countries, but remains sporadically in tropical third world countries with poor sanitation. Hygiene hypothesis may explain this phenomenon. It stated that pathogen infections may protect the host from immunological disease. Although the cause of CD is complicated, it is proved that CD is not only the consequence of inappropriate stimulation of effector responses but also due to a dysregulation of the normal immunosuppressive mechanisms. The development of CD is related to a polarized type-1 immune response and TNBS-induced colitis shares features with CD. Trinitrobenzene sulfonic acid (TNBS)-induced colitis is a murine model of human CD. In TNBS-induced murine model, ethanol was used to break the mucosa barrier of intestine, TNBS served as a hapten reagent and combined with the protein on the damaged intestinal mucosa to form the complete antigen. The similar immune lesion to CD happens in the intestine of murine. It is known that helminth infections might regulate a polarized type-1 or type-2 immune response and the immune regulatory process is associated with Tregs (CD4~+ CD25~+ regulatory T cells). Shistosome infection switches immunologically from an early Th1 response to a Th2-dominated response after the onset of parasite egg-laying. During schistosome infection,Tregs are also involved in its immunomodulation in its host.
Tregs are important components of the homeostasis of the immune system, they prevent the development of autoimmunity and control virtually all forms of immune response, including inflammation. Foxp3 (forkhead box P3), a member of the forkhead/winged-helix family of transcriptional regulators, is a master regulatory gene for the development of Tregs and specifically expressed in CD4~+CD25~+ Tregs. In mice, the lack of foxp3-expressing Tregs is sufficient to break down self-tolerance and induce autoimmune disease. CD4~+ CD25~+ Foxp3~+ T cells can suppress and modulate the excessive immune response. The role of CD4~+ CD25~+ Foxp3~+ regulatory T cells in murine colitis with schistosome eggs treatment has not been reported.
Elliott showed that exposure to Schistosoma mansoni eggs protects mice from TNBS-induced colitis. In the present paper, we demonstrate that exposure to Schistosoma japonicum eggs protects mice from TNBS-induced colitis and Tregs may be involved in the protection.
Specific pathogen-free 6-8 week-old female BALB/c mice were used. Eggs of S. japonicum were collected from the livers of schistosome-infected rabbits, which had been infected with 1,500 cercariae 46 days previously. The eggs were collected and washed in phosphate buffered saline (PBS). Mice were intraperitoneally injected with 10,000 eggs in 100μl PBS at day 0. At days 7, 14 and 21, the animals were boosted in the same way and with the same dosage. Control mice received sterile PBS with the same volume and timing. In each group 5 mice were used and all experiments were duplicated. Mice were lightly anesthetized with metofane (methoxyflurane) at day 6 after the last immunization. A 3.5F catheter, fitted onto a l-ml syringe, was carefully inserted into the colon such that the tip was 4 cm proximal to the anus and 0.5 mg of TNBS in 50% ethanol was slowly administered into the lumen of the colon. In control experiments, mice were given 50% ethanol alone using the same technique described above. The total injection volume was 100μl for each mouse allowing TNBS or ethanol to reach the entire colon, including the caecum and appendix. Animals were then kept in a vertical position for 30 s and returned to their cages. Colons of mice were removed on day 6 after the induction of colitis and embedded in paraffin. Sections were stained with hematoxylin and eosin. The degree of inflammation of the colon was graded semiquantitatively from 0 to 4 (0, no evidence of inflammation; 1, low level of lymphocyte infiltration with infiltration seen in10% high-power field (hpf), no structural changes observed; 2, moderate lymphocyte infiltration with infiltration seen in 10%–25% hpf, crypt elongation, bowel wall thickening which does not extend beyond the mucosal layer, no evidence of ulceration; 3, high level of lymphocyte infiltration with infiltration seen in 25%– 50% hpf, high vascular density, thickening of bowel wall which extends beyond mucosal layer; and 4, marked degree of lymphocyte infiltration with infiltration seen in 50% hpf, high vascular density, crypt elongation with distortion, transmural bowel wall thickening with ulceration). Grading the inflammation in colons of mice was done in a blinded fashion by the same pathologist. Spleen cells were isolated from the experimental mice on day 6 after TNBS treatment. Cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM HEPES buffer, 10μg/ml gentamicin, 10% FCS, 100 U/ml penicillin and 100μg/ml streptomycin. Cells were cultured for 72 h at 3×106 cells/well in 1 ml microtiter wells and stimulated with 5μg/ml Con A. The concentrations of IFN-γ, IL-4, IL-5 and IL-10 in cell culture supernatants were determined by ELISA Kits. In brief, mouse IFN-γ, IL-4, IL-5 and IL-10 were detected by biotinylated monoclonal antibodies, which were evidenced by avidin-conjugated horseradish peroxidase followed by incubation with TMB substrate. The reactions were measured at 450 nm in an ELISA reader. Single-cell suspensions of spleen cells were stained and analyzed on FACS Calibur using CellQuest software. For determination of the phenotype of lymphocyte populations,mouse regulatory T cell staining Kit was used. The following conjugated antibodies were incubated with lymphocyte populations: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4, allophycocyanin (APC)-conjugated anti-mouse CD25 and phycoerythrin (PE)-conjugated anti-mouse Foxp3 FJK-16s (a master regulatory gene for the development of Tregs). Before incubation with PE anti-mouse Foxp3 FJK-16s,cells were fixed with permeabilization buffer and Fc receptors were blocked with anti-CD16/32 antibody. PE -conjugated rat IgG2a served as the isotype control.
The colons of mice were removed on day 6 after administration of TNBS or ethanol. TNBS-treated mice showed ulcerations of the colons surrounded by hyperemia and inflammation, whereas the colons of control mice treated with ethanol alone or S. japonicum eggs followed by TNBS showed neither macroscopic signs of inflammation nor obvious hyperemia and edema. Histologically, transmural inflammation affecting the entire colon was observed in TNBS-treated mice, lymphocyte infiltrates were associated with thickening of the colon wall, ulcerations, loss of goblet cells, and the presence of granulomas in TNBS-treated mice. The inflammatory responses were significantly milder in the colitis mice exposed to egg prior to TNBS-treatment. Histological score from egg-unexposed and -exposed mice was 3.6±0.49 and 1.6±0.70, respectively (P< 0.0005), thus confirming the prevention of TNBS-induced inflammation by previous injection of eggs. Spleen cells from TNBS-treated mice generated in vitro large amounts of IFN-γ, which were about twice the level of IFN-γafter administration of schistosome eggs prior to TNBS treatment (P<0.01). The secretion of IL-4, IL-5 and IL-10 in vitro was significantly (P<0.01) higher in colitis mice previously exposured to schistosome eggs compared to the animals treated with TNBS alone. The percentage of CD4~+ CD25~+ Foxp3~+ T cells in spleens of egg-treated mice was not significantly different from untreated control mice. These cells increased, however significantly (P<0.05) after TNBS-treatment, egg-exposed mice, but not after TNBS treatment alone.
In the research, the results indicated that CD4~+ CD25~+ Foxp3~+ regulatory T cells may be involved in the modulation during TNBS-induced colitis with schistosome eggs exposure and the excessive Th1 response was converted. As a result, the lesion of TNBS-induced colitis in mice was attenuated.
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13. La Flamme AC, Ruddenklau K, Backstrom. BT. Schistosomiasis decreases central nervous system inflammation and alters the progression of experimental autoimmune encephalomyelitis. Infect Immun, 2003, 71: 4996-5004.
14. Sanchez JL, Aguirre C, Arcos-Burgos OM, et al. Prevalence of multiple sclerosis in Colombia. Rev Neurol, 2000, 31: 1101-1103.
15. Callegaro D, Goldbaum M, Morais L, et al. The prevalence of multiple sclerosis in the city of S?o Paulo, Brazil, 1997. Acta Neurol Scand, 2001, 104: 208-213.
16. Poser S, Stickel B, Krtsch U, et al. Increasing incidence of multiple sclerosis in South Lower Saxony, Germany. Neuroepidemiology, 1989, 8: 207-213.
17. Itoh T, Aizawa H, Hashimoto K, et al. Prevalence of multiple sclerosis in Asahikawa, a city in northern Japan. J Neurol Sci, 2003, 214: 7-9.
18. Elliott DE, Urban JJ, Argo CK, et al. Does the failure to acquire helminthic parasites predispose to Crohn’s disease? Faseb J, 2000, 14: 1848-1855.
19. Farrokhyar F, Swarbrick ET, Irvine E J. A critical review of epidemiological studies in inflammatory bowel disease. Scand J Gastroenterol, 2001, 36: 2-15.
20. Bodansky HJ, Staines A, Stephenson C, et al. Evidence for an environmental effect in the aetiology of insulin dependent diabetes in a transmigratory population. Bmj, 1992,304: 1020-1022.
21. Staines A, Hanif S, Ahmed S, et al. Incidence of insulin dependent diabetes mellitus in Karachi, Pakistan. Arch Dis Child, 1997b, 76: 121-123.
22. Bach JF. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med, 2002, 347: 911-920.
23. Aberg N, Hesselmar B, Aberg B, et al. Increase of asthma, allergic rhinitis and eczema in Swedish schoolchildren between 1979 and 1991. Clin Exp Allergy, 1995, 25: 815-819.
24. Demeure CE, Rihet P, Abel L, et al. Resistance to Schistosoma mansoni in humans: influence of the IgE/IgG4 balance and IgG2 in immunity to reinfection after chemotherapy. J Infect Dis , 1993, 168: 1000-1008.
25. Comoy EE, Pestel J, Duez C, et al. The house dust mite allergen, Dermatophagoides pteronyssinus, promotes type 2 responses by modulating the balance between IL-4 and IFN-gamma. J Immunol,1998, 160: 2456-2462.
26. Else KJ, Finkelman FD. Intestinal nematode parasites, cytokines and effector mechanisms. Int J Parasitol, 1998, 28: 1145-1158.
27. Keane-Myers AM, Gause WC, Finkelman FD, et al. Development of murine allergic asthma is dependent upon B7-2 costimulation. J Immunol, 1998, 160: 1036-1043.
28. Urban Jr JF, Noben-Trauth N, Donaldson DD, et al. IL-13, IL-4Ralpha, and Stat6 are required for the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis. Immunity, 1998, 8: 255-264.
29. Grzych JM, Pearce E, Cheever A, et al. Egg deposition is the major stimulus for the production of Th2 cytokines in murine schistosomiasis mansoni. J Immunol, 1991,146: 1322-1327.
30. Pearce EJ, Caspar P, Grzych JM, et al. Downregulation of Th1 cytokine productionaccompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J Exp Med, 1991, 173: 159-166.
31. de Jesus AR, Silva A, Santana LB, et al. Clinical and immunologic evaluation of 31 patients with acute schistosomiasis mansoni. J Infect Dis, 2002, 185: 98-105.
32. Araujo MI, Bacellar O, Ribeiro-de-Jesus A, et al. The absence of gamma-interferon production of S. mansoni antigens in patients with schistosomiasis. Braz J Med Biol Res, 1994, 27: 1619-1625.
33. Malaquias LC, Falcao PL, Silveira AM, et al. Cytokine regulation of human immune response to Schistosoma mansoni: analysis of the role of IL-4, IL-5 and IL-10 on peripheral blood mononuclear cell responses. Scand J Immunol, 1997, 46: 393-398.
34. Joseph S, Jones FM, Kimani G, et al. Cytokine production in whole blood cultures from a fishing community in an area of high endemicity for Schistosoma mansoni in Uganda: the differential effect of parasite worm and egg antigens. Infect Immun, 2004, 72: 728-734.
35. Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med, 1989, 170: 2081-2095.
36. Del Prete G, De Carli M, Almerigogna F, et al. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J Immunol, 1993, 150: 353-360.
37. Royer B, Varadaradjalou S, Saas P, et al. Inhibition of IgE-induced activation of human mast cells by IL-10. Clin Exp Allergy, 2001, 31: 694-704.
38. Araujo MI, de Jesus AR, Bacellar O, et al. Evidence of a T helper type 2 activation in human schistosomiasis. Eur J Immunol, 1996, 26: 1399-1403.
39. Sabin EA, Araujo MI, Carvalho EM, et al. Impairment of tetanus toxoid-specificTh1-like immune responses in humans infected with Schistosoma mansoni. J Infect Dis, 1996, 173: 269-272.
40. Zaccone P, Fehervari Z, Blanchard L, et al. Autoimmune thyroid disease induced by thyroglobulin and lipopolysaccharide is inhibited by soluble TNF receptor type I. Eur J Immunol, 2002, 32: 1021-1028.
41. Sewell D, Qing Z, Reinke E, et al. Immunomodulation of experimental autoimmune encephalomyelitis by helminth ova immunization. Int Immunol, 2003,15: 59-69.
42. Panitch HS, Hirsch RL, Haley AS, et al. Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet, 1987,1: 893-895.
43. Panitch HS. Interferons in multiple sclerosis. A review of the evidence. Drugs, 1992, 44: 946-962.
44. Chitnis T, Najafian N, Benou C, et al. Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis. J Clin Invest,2001, 108: 739-747.
45. Hou J, Schindler U, Henzel WJ, et al. An interleukin-4-induced transcription factor: IL-4 Stat. Science, 1994, 265: 1701-1706.
46. Schindler C, Kashleva H, Pernis A, et al. STF-IL-4: a novel IL-4-induced signal transducing factor. Embo J,1994, 13: 1350-1356.
47. Neurath MF, Fuss I, Kelsall BL, et al. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J Exp Med, 1995, 182: 1281-1290.
48. Moreels TG, Nieuwendijk RJ, De Man JG, et al. Concurrent infection with Schistosoma mansoni attenuates inflammation induced changes in colonic morphology, cytokine levels, and smooth muscle contractility of trinitrobenzene sulphonic acid induced colitis in rats. Gut, 2004, 53: 99-107.
49. Elliott DE, Li J, Blum A, Metwali A, et al. Exposure to schistosome eggs protects micefrom TNBS-induced colitis. Am J Physiol Gastrointest Liver Physiol,2002, 284: G385-391.
50. Summers RW, Elliott DE, Qadir K, et al. Trichuris suis seems to be safe and possibly effective in the treatment of inflammatory bowel disease. Am J Gastroenterol, .2003, 98: 2034-2041.
51. Lynch NR, Hagel IA, Palenque ME, et al. Relationship between helminthic infection and IgE response in atopic and nonatopic children in a tropical environment. J Allergy Clin Immunol, 1998, 101: 217-221.
52. Velupillai P, dos Reis EA, dos Reis MG, et al. Lewis(x)-containing oligosaccharide attenuates schistosome egg antigen-induced immune depression in human schistosomiasis. Hum Immunol, 2000, 61: 225-232.
53. van den Biggelaar AH, van Ree R, Rodrigues LC, et al. Decreased atopy in children infected with Schistosoma haematobium: a role for parasite-induced interleukin-10. Lancet, 2000, 356: 1723-1727.
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1. Herz U, Lacy P, Renz H, et al. The influence of infections on the development and severity of allergic disorders. Curr Opin Immunol, 2000, 12: 632-640.
2. Weinstock JV, Summers RW, Elliott DE, et al. The possible link between de-worming and the emergence of immunological disease. J Lab Clin Med, 2002, 139: 334-338.
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4. von Mutius E, Martinez FD, Fritzsch C, et al. Prevalence of asthma and atopy in two areas of West and East Germany. Am J Respir Crit Care Med, 1994, 149: 358-364.
5. Staines A, Bodansky HJ, McKinney PA, et al. Small area variation in the incidence of childhood insulin-dependent diabetes mellitus in Yorkshire, UK: links with overcrowding and population density. Int J Epidemiol, 1997a, 26: 1307-1313.
6. Blanchard JF, Bernstein CN, Wajda A, et al. Small-area variations and sociodemographic correlates for the incidence of Crohn’s disease and ulcerative colitis. Am J Epidemiol, 2001, 154: 328-335.
7. Lynch NR, Hagel I, Perez M, et al. Effect of anthelmintic treatment on the allergic reactivity of children in a tropical slum. J Allergy Clin Immunol, 1993, 92: 404-411.
8. Araujo MI, Lopes AA, Medeiros M, et al. Inverse association between skin response to aeroallergens and Schistosoma mansoni infection. Int Arch Allergy Immunol , 2000, 123: 145-148.
9. Cooper PJ, Chico ME, Bland M, et al. Allergic symptoms, atopy, and geohelminth infections in a rural area of Ecuador. Am J Respir Crit Care Med, 2003, 168: 313-317.
10. Medeiros Jr M, Figueiredo JP, Almeida MC, et al. Schistosoma mansoni infection is associated with a reduced course of asthma. J Allergy Clin Immunol, 2003, 111: 947-951.
11. Cooke A, Tonks P, Jones FM, et al. Infection with Schistosoma mansoni prevents insulin dependent diabetes mellitus in non-obese diabetic mice. Parasite Immunol, 1999, 21: 169-176.
12. Kurtzke JF. Multiple sclerosis in time and space—geographic clues to cause. J Neurovirol, 2000,6 (Suppl. 2): S134-140.
13. La Flamme AC, Ruddenklau K, Backstrom. BT. Schistosomiasis decreases central nervous system inflammation and alters the progression of experimental autoimmune encephalomyelitis. Infect Immun, 2003, 71: 4996-5004.
14. Sanchez JL, Aguirre C, Arcos-Burgos OM, et al. Prevalence of multiple sclerosis in Colombia. Rev Neurol, 2000, 31: 1101-1103.
15. Callegaro D, Goldbaum M, Morais L, et al. The prevalence of multiple sclerosis in the city of S?o Paulo, Brazil, 1997. Acta Neurol Scand, 2001, 104: 208-213.
16. Poser S, Stickel B, Krtsch U, et al. Increasing incidence of multiple sclerosis in South Lower Saxony, Germany. Neuroepidemiology, 1989, 8: 207-213.
17. Itoh T, Aizawa H, Hashimoto K, et al. Prevalence of multiple sclerosis in Asahikawa, a city in northern Japan. J Neurol Sci, 2003, 214: 7-9.
18. Elliott DE, Urban JJ, Argo CK, et al. Does the failure to acquire helminthic parasites predispose to Crohn’s disease? Faseb J, 2000, 14: 1848-1855.
19. Farrokhyar F, Swarbrick ET, Irvine E J. A critical review of epidemiological studies in inflammatory bowel disease. Scand J Gastroenterol, 2001, 36: 2-15.
20. Bodansky HJ, Staines A, Stephenson C, et al. Evidence for an environmental effect in the aetiology of insulin dependent diabetes in a transmigratory population. Bmj, 1992,304: 1020-1022.
21. Staines A, Hanif S, Ahmed S, et al. Incidence of insulin dependent diabetes mellitus in Karachi, Pakistan. Arch Dis Child, 1997b, 76: 121-123.
22. Bach JF. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med, 2002, 347: 911-920.
23. Aberg N, Hesselmar B, Aberg B, et al. Increase of asthma, allergic rhinitis and eczema in Swedish schoolchildren between 1979 and 1991. Clin Exp Allergy, 1995, 25: 815-819.
24. Demeure CE, Rihet P, Abel L, et al. Resistance to Schistosoma mansoni in humans: influence of the IgE/IgG4 balance and IgG2 in immunity to reinfection after chemotherapy. J Infect Dis , 1993, 168: 1000-1008.
25. Comoy EE, Pestel J, Duez C, et al. The house dust mite allergen, Dermatophagoides pteronyssinus, promotes type 2 responses by modulating the balance between IL-4 and IFN-gamma. J Immunol,1998, 160: 2456-2462.
26. Else KJ, Finkelman FD. Intestinal nematode parasites, cytokines and effector mechanisms. Int J Parasitol, 1998, 28: 1145-1158.
27. Keane-Myers AM, Gause WC, Finkelman FD, et al. Development of murine allergic asthma is dependent upon B7-2 costimulation. J Immunol, 1998, 160: 1036-1043.
28. Urban Jr JF, Noben-Trauth N, Donaldson DD, et al. IL-13, IL-4Ralpha, and Stat6 are required for the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis. Immunity, 1998, 8: 255-264.
29. Grzych JM, Pearce E, Cheever A, et al. Egg deposition is the major stimulus for the production of Th2 cytokines in murine schistosomiasis mansoni. J Immunol, 1991,146: 1322-1327.
30. Pearce EJ, Caspar P, Grzych JM, et al. Downregulation of Th1 cytokine productionaccompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J Exp Med, 1991, 173: 159-166.
31. de Jesus AR, Silva A, Santana LB, et al. Clinical and immunologic evaluation of 31 patients with acute schistosomiasis mansoni. J Infect Dis, 2002, 185: 98-105.
32. Araujo MI, Bacellar O, Ribeiro-de-Jesus A, et al. The absence of gamma-interferon production of S. mansoni antigens in patients with schistosomiasis. Braz J Med Biol Res, 1994, 27: 1619-1625.
33. Malaquias LC, Falcao PL, Silveira AM, et al. Cytokine regulation of human immune response to Schistosoma mansoni: analysis of the role of IL-4, IL-5 and IL-10 on peripheral blood mononuclear cell responses. Scand J Immunol, 1997, 46: 393-398.
34. Joseph S, Jones FM, Kimani G, et al. Cytokine production in whole blood cultures from a fishing community in an area of high endemicity for Schistosoma mansoni in Uganda: the differential effect of parasite worm and egg antigens. Infect Immun, 2004, 72: 728-734.
35. Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med, 1989, 170: 2081-2095.
36. Del Prete G, De Carli M, Almerigogna F, et al. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J Immunol, 1993, 150: 353-360.
37. Royer B, Varadaradjalou S, Saas P, et al. Inhibition of IgE-induced activation of human mast cells by IL-10. Clin Exp Allergy, 2001, 31: 694-704.
38. Araujo MI, de Jesus AR, Bacellar O, et al. Evidence of a T helper type 2 activation in human schistosomiasis. Eur J Immunol, 1996, 26: 1399-1403.
39. Sabin EA, Araujo MI, Carvalho EM, et al. Impairment of tetanus toxoid-specificTh1-like immune responses in humans infected with Schistosoma mansoni. J Infect Dis, 1996, 173: 269-272.
40. Zaccone P, Fehervari Z, Blanchard L, et al. Autoimmune thyroid disease induced by thyroglobulin and lipopolysaccharide is inhibited by soluble TNF receptor type I. Eur J Immunol, 2002, 32: 1021-1028.
41. Sewell D, Qing Z, Reinke E, et al. Immunomodulation of experimental autoimmune encephalomyelitis by helminth ova immunization. Int Immunol, 2003,15: 59-69.
42. Panitch HS, Hirsch RL, Haley AS, et al. Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet, 1987,1: 893-895.
43. Panitch HS. Interferons in multiple sclerosis. A review of the evidence. Drugs, 1992, 44: 946-962.
44. Chitnis T, Najafian N, Benou C, et al. Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis. J Clin Invest,2001, 108: 739-747.
45. Hou J, Schindler U, Henzel WJ, et al. An interleukin-4-induced transcription factor: IL-4 Stat. Science, 1994, 265: 1701-1706.
46. Schindler C, Kashleva H, Pernis A, et al. STF-IL-4: a novel IL-4-induced signal transducing factor. Embo J,1994, 13: 1350-1356.
47. Neurath MF, Fuss I, Kelsall BL, et al. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J Exp Med, 1995, 182: 1281-1290.
48. Moreels TG, Nieuwendijk RJ, De Man JG, et al. Concurrent infection with Schistosoma mansoni attenuates inflammation induced changes in colonic morphology, cytokine levels, and smooth muscle contractility of trinitrobenzene sulphonic acid induced colitis in rats. Gut, 2004, 53: 99-107.
49. Elliott DE, Li J, Blum A, Metwali A, et al. Exposure to schistosome eggs protects micefrom TNBS-induced colitis. Am J Physiol Gastrointest Liver Physiol,2002, 284: G385-391.
50. Summers RW, Elliott DE, Qadir K, et al. Trichuris suis seems to be safe and possibly effective in the treatment of inflammatory bowel disease. Am J Gastroenterol, .2003, 98: 2034-2041.
51. Lynch NR, Hagel IA, Palenque ME, et al. Relationship between helminthic infection and IgE response in atopic and nonatopic children in a tropical environment. J Allergy Clin Immunol, 1998, 101: 217-221.
52. Velupillai P, dos Reis EA, dos Reis MG, et al. Lewis(x)-containing oligosaccharide attenuates schistosome egg antigen-induced immune depression in human schistosomiasis. Hum Immunol, 2000, 61: 225-232.
53. van den Biggelaar AH, van Ree R, Rodrigues LC, et al. Decreased atopy in children infected with Schistosoma haematobium: a role for parasite-induced interleukin-10. Lancet, 2000, 356: 1723-1727.
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