经口灌胃感染弓形虫诱导的BALB/c小鼠肠道粘膜免疫应答

目的 研究BALB/c小鼠经口感染(灌胃)弓形虫速殖子后肠液IgA抗体分泌的动态变化及肠道粘膜组织诱导与效应部位T淋巴细胞亚群的变化,探讨肠道粘膜免疫应答机制.方法 将6～8周龄BALB/c小鼠100只随机分为对照组和感染组.感染组经口感染(灌胃)弓形虫RH株速殖子5×104个/只,对照组给予等量PBS.于感染后第2、4、6、8、10、13、16、19、22、25 d处死小鼠,每次5只.收集各时间点肠道冲洗液,用ELISA法测定肠液IgA水平;分离第2、4、6、8、10 d Peyer's Patches(PP)、肠系膜淋巴结(MLN)及小肠上皮内淋巴细胞(IEL),制备悬液并涂片,用免疫细胞化学方法测定CD4+、CD8+T细胞亚群水平.结果 在感染后第4、6、8、13 d实验组的肠液IgA抗体分泌显著高于对照组(P＜0.01).实验组PP结CD4+T细胞水平在6、8、10 d显著高于对照组(P＜0.05);CD8+T细胞水平与对照组无显著性差异,CD4+/CD8+比值在6、8、10 d显著升高(P＜0.05).肠系膜淋巴结的CD4+、CD8+及CD4+/CD8+比值各时间点均无显著变化.感染后第6、8、10 d,效应部位小肠上皮内淋巴细胞CD8+T细胞增高显著(P＜0.01)、CD4+/CD8+比值倒置(P＜0.05).结论 经口感染弓形虫BALB/c小鼠的肠道产生高水平的IgA抗体,作为局部首道屏障,发挥着重要的抗虫作用.诱导部位PP CD4+T细胞水平明显增高,诱导对弓形虫抗原的处理提呈作用;效应部位IEL CD8+T细胞亚群增殖明显,在清除虫体的过程中起主导作用.     

致敏IEL过继转移诱导抗弓形虫感染的机制

目的分离供体鼠感染弓形虫后不同时间点的小肠上皮内淋巴细胞(intraepithelial lymphocyte,IEL)并过继转移,研究其诱导受体鼠抗弓形虫感染作用及其机制。方法BALB/c小鼠经口感染弓形虫速殖子5×104个/只,对照组未感染,作为供体提供IEL。同品系小鼠36只为受体鼠,分为6组,每组6只,经尾静脉分别接受分离自供体鼠感染弓形虫后第7(IEL7组)、9(IEL9组)、11(IEL11组)、13(IEL13组)、15d(IEL15组)的致敏IEL或未致敏(IEL0组,即对照组)的IEL3×105/0.2ml·只。各组小鼠过继转移后第4d,用弓形虫速殖子灌胃攻击,攻击后第13d分离纯化脑、肺、脾组织内速殖子并计数,测定小肠Peyer′s Patch(PP)CD4+、CD8+T细胞亚群水平。结果致敏IEL过继免疫可使受体鼠脑、肺、脾组织内速殖子数显著减少,接受致敏后第13d IEL的小鼠组织内速殖子数减少最为显著(Ρ<0.01),与IEL0组相比,脑、肺和脾组织内速殖子数分别减少81.13%、58.43%和70.97%;同时,小肠PP结CD4+T细胞增多,CD4+/CD8+比值增高(Ρ<0.01)。结论过继转移致敏IEL能显著提高受体鼠的抗弓形虫感染能力,且保护作用同致敏时间关系密切,以感染后13d分离的致敏IEL保护性最强。     

经口感染弓形虫诱导小鼠黏膜免疫动物模型的建立

目的建立经口感染弓形虫速殖子诱导黏膜免疫的动物模型。方法BALB/c小鼠分别灌胃接种5×103、5×104、5×105、5×106个RH株速殖子,观察小鼠的体况、病理变化;检测肠道分泌型IgA(SIgA)和Peyer’spatches(PP)淋巴细胞及小肠上皮内淋巴细胞(IEL)T细胞亚群的变化。结果经口接种5×104个弓形虫RH株速殖子可使小鼠出现临床症状和病理改变;SIgA水平升高;黏膜诱导部位的CD4 T亚群及效应部位的CD8 T亚群水平升高。结论5×104个弓形虫RH株速殖子灌胃接种小鼠可以诱导机体黏膜免疫应答。     

霍乱毒素联合弓形虫排泄-分泌抗原鼻内免疫小鼠诱导的GALT和NALT黏膜免疫应答

目的观察霍乱毒素(CT)联合弓形虫排泄-分泌抗原(ESA)鼻内免疫小鼠诱导的肠相关淋巴组织(GALT)和鼻相关淋巴组织(NALT)黏膜部位的免疫应答。方法将48只5~6周龄BABL/c小鼠随机均分为3组,分别用PBS、ESA和CT+ESA滴鼻免疫小鼠2次,间隔2周。末次免疫后14 d,处死小鼠,ELISA测定小鼠粪便和鼻咽冲洗液sIgA抗体水平;计数派伊尔淋巴集结(PP)、肠上皮淋巴细胞(IEL)、NALT和鼻通道(NC)淋巴细胞数,免疫细胞化学法检测CD4+、CD8+T细胞亚群水平。结果免疫后14 d,CT+ESA组小鼠鼻咽冲洗液和粪便sIgA水平显著高于ESA组和PBS组(P<0.01);CT+ESA和ESA组小鼠GALT部位的PP淋巴细胞显著增生,主要以CD4+T细胞为主;而IEL以CD8+T细胞增生为主,CD4+/CD8+比值降低(P<0.05)。CT+ESA NALT内淋巴细胞明显增生。CT+ESA组NC淋巴细胞显著升高(P<0.01),以CD4+T细胞增生为主。结论 CT佐剂联合ESA滴鼻免疫BALB/c小鼠可诱导GALT和NALT黏膜部位免疫应答。     

弓形虫复合粘膜疫苗滴鼻免疫小鼠诱导的Peyer''''s patches持续性细胞免疫应答

目的以可溶性速殖子抗原（soluble tachyzoite antigen，STAg）和霍乱毒素（choleratoxin，CT）佐剂制备的弓形虫复合粘膜疫苗滴鼻免疫小鼠，观察肠粘膜诱导部位Peyer’s patches（PP）的细胞免疫应答及持续时间，探讨其免疫机制。方法BALB／c小鼠96只，随机分为实验组和对照组，实验组以STAg（20μg／只）为抗原，CT（1／μg／只）为佐剂滴鼻免疫，对照组PBS滴鼻。滴鼻2次（间隔2周）后，每组6只小鼠分别于第1、2、3、4、6、8、10、12周处死。计数PP个数，制备PP淋巴细胞悬液，计数并涂片；免疫细胞化学法检测CD4^＋、CD8^＋T细胞亚群。结果实验期间两组小鼠PP数目均无明显变化；实验组免疫后PP淋巴细胞数量明显增生，第2周达高峰，第1、2、3周显著高于对照组（P〈0.05），其中以CD4^＋T细胞增生为主，第1周～第8周高于对照组（P〈0.01），CD8^＋T细胞第1周～第4周显著增高（P〈0.01），CD4^＋／CD8^＋比值无显著变化（P〉0．05）。结论弓形虫复合粘膜疫苗滴鼻免疫BALB／c小鼠可有效诱导肠PP部位持续性的免疫应答，从而激活肠粘膜效应部位淋巴细胞的抗弓形虫感染作用。     

弓形虫复合粘膜疫苗滴鼻免疫小鼠诱导的Peyer’s patches持续性细胞免疫应答

目的以可溶性速殖子抗原（soluble tachyzoite antigen，STAg）和霍乱毒素（choleratoxin，CT）佐剂制备的弓形虫复合粘膜疫苗滴鼻免疫小鼠，观察肠粘膜诱导部位Peyer’s patches（PP）的细胞免疫应答及持续时间，探讨其免疫机制。方法BALB／c小鼠96只，随机分为实验组和对照组，实验组以STAg（20μg／只）为抗原，CT（1／μg／只）为佐剂滴鼻免疫，对照组PBS滴鼻。滴鼻2次（间隔2周）后，每组6只小鼠分别于第1、2、3、4、6、8、10、12周处死。计数PP个数，制备PP淋巴细胞悬液，计数并涂片；免疫细胞化学法检测CD4^＋、CD8^＋T细胞亚群。结果实验期间两组小鼠PP数目均无明显变化；实验组免疫后PP淋巴细胞数量明显增生，第2周达高峰，第1、2、3周显著高于对照组（P〈0.05），其中以CD4^＋T细胞增生为主，第1周～第8周高于对照组（P〈0.01），CD8^＋T细胞第1周～第4周显著增高（P〈0.01），CD4^＋／CD8^＋比值无显著变化（P〉0．05）。结论弓形虫复合粘膜疫苗滴鼻免疫BALB／c小鼠可有效诱导肠PP部位持续性的免疫应答，从而激活肠粘膜效应部位淋巴细胞的抗弓形虫感染作用。     

可溶性弓形虫速殖子抗原联合蜂胶和IFN-γ鼻内免疫小鼠诱导的细胞免疫应答

目的比较蜂胶、IFN-γ佐剂以及两种佐剂混合鼻内免疫辅助STAg增强机体细胞免疫应答的水平,探讨两种佐剂联合应用的免疫效果。方法将5~6周龄雌性BALB/c小鼠60只随机分为4组:20μg STAg组,20μg STAg+40μg蜂胶组,20μg STAg+1 000 U IFN-γ组和20μg STAg+40μg蜂胶+1 000 U IFN-γ组。将抗原和佐剂溶于20μl PBS中,双侧鼻孔(10μl/鼻孔)滴鼻免疫。免疫2次,间隔14 d,末次免疫后第10 d用RH株弓形虫速殖子4×104个/只灌胃攻击。逐日观察小鼠存活情况。攻击后第43 d处死全部存活小鼠,免疫细胞化学(ICC)法检测小鼠PP结、IEL和脾T淋巴细胞亚群水平。结果与STAg组相比,各佐剂组小鼠T淋巴细胞亚群比例有升高的趋势,其中IFN-γ、蜂胶+IFN-γ佐剂组小鼠PP结CD4+、CD8+T淋巴细胞,IEL、脾CD8+T淋巴细胞数显著高于STAg组,CD4+/CD8+T淋巴细胞比值倒置。结论在抗弓形虫感染中,IFN-γ的佐剂作用优于蜂胶,有效激发机体细胞免疫应答,可用作鼻内免疫抗弓形虫感染的粘膜佐剂。IFN-γ+蜂胶佐剂鼻内免疫效果优于单独蜂胶、IFN-γ佐剂,应用联合佐剂是抗弓形虫感染免疫的一个新思路。     

弓形虫感染大鼠外周血T细胞亚群动态变化

目的观察弓形虫感染大鼠外周血T淋巴细胞亚群的动态变化。方法将58只清洁级SD成年大鼠随机分成9组,其中8组为实验组,1组为对照组,实验组每鼠腹腔注射2×105/LCF-11纤维素纯化的活弓形虫速殖子悬液2ml,对照组腹腔注射2ml生理盐水,并留4只为正常对照鼠。分别于弓形虫感染后1、3、7、14、28、35、42、60d时取大鼠外周全血,用流式细胞术检测外周血T细胞亚群CD3+、CD4+、CD8+百分比。结果弓形虫感染后3d、7d时,CD3+水平降至最低,但与正常鼠和对照鼠无显著性差异(P>0.05),至感染14d基本恢复至正常水平,此后再无明显变化。CD8+亚群水平在感染后3d、7d、14d时均显著低于正常水平(P<0.05),至感染后28d恢复正常水平。而CD4+亚群水平在整个感染过程中无明显变化(P>0.05)。CD4+/CD8+比例在感染后3d、7d、14d时显著高于正常鼠和对照鼠(P<0.05)。结论弓形虫感染影响大鼠外周血T淋巴细胞亚群比例,但可自行恢复正常。     

重组小鼠干扰素-γ对感染弓形虫孕鼠T细胞亚群自然杀伤细胞的影响

目的 探讨重组小鼠γ-干扰素（rmu-IFN-γ）对妊娠期感染弓形虫的小鼠脾脏T细胞亚群和自然杀伤细胞（NK细胞）水平的影响。 方法 20只BALB/c孕鼠随机分为空白对照、感染对照及rmu-IFN-γ治疗组。妊娠第7～9 天孕鼠急性感染弓形虫,rmu-IFN-γ分为2个剂量组（按小鼠体重1 U/g和10 U/g）腹腔注射给药,3 d后每组随机处死5只,取脾细胞,流式细胞术检测T细胞亚群和NK细胞水平。 结果 两种剂量rmu-IFN-γ治疗组CD4+ T细胞水平在妊娠第10、12、14 天高于感染对照组,CD8+ T细胞水平在妊娠第10、14天低于感染对照组,CD4+/CD8+ T细胞比值在妊娠第10、12、14 天高于感染对照组（P<0.05）,孕鼠存活时间长于感染对照组。 结论 注射适量rmu-IFN-γ可改善CD4+/CD8+ T细胞比值,促进NK细胞增殖,提高宿主免疫水平。     

香菇多糖预处理对急性弓形虫感染小鼠中枢神经系统损伤的保护性作用研究北大核心CSCD

目的探究香菇多糖预处理对急性弓形虫感染小鼠脑中枢神经系统的保护性作用。方法将C57BL/6小鼠随机分为对照组(NC)、模型组(TDI)和香菇多糖预处理组(pre-10dLNT TDI)。弓形虫感染前10d,pre-10dLNT组小鼠连续10d腹腔注射LNT(1mg/kg体重,1次/d),弓形虫感染后无任何处理;TDI组小鼠腹腔注射弓形虫悬液0.5 mL/只鼠;NC组小鼠感染前10d开始腹腔注射同等体积的生理盐水(1次/d)。通过观察小鼠的死亡时间及数量计算各组小鼠的存活率,使用流式细胞仪检测感染后第8d小鼠脑组织中CD8+IFN-γ+T细胞百分比,ELISA法检测感染后第4d和第8d小鼠外周血中IFN-γ、GABA和DA水平,应用SoftMax Pro 4.3.1LS软件绘制标准曲线计算各项指标的含量(pg/ml)。采用Grapad 8.0软件进行处理和分析。结果TDI组小鼠第8d存活率为0%,pre-10dLNT TDI组小鼠第8d存活率为60%;与NC组相比,TDI组和pre-10dLNT TDI组CD8+IFN-γ+T细胞亚群水平均明显增高;与TDI组小鼠相比,pre-10dLNT预处理明显降低了弓形虫感染的C57BL/6J小鼠脑组织中CD8+IFN-γ+细胞数量(P<0.01)、明显降低了小鼠第4d和8d的外周血中IFN-γ产生水平(P<0.05;P<0.01)、第8d小鼠外周血中DA和GABA的产生水平显著增加(P<0.01)。结论LNT预处理可以显著提高急性弓形虫感染小鼠的存活率,通过减少CD8+T细胞通过血脑屏障进入CNS,降低神经系统的广泛炎症反应,通过降低脑组织中CD8+IFN-γ+T细胞数量,调节神经递质的分泌水平,从而改善小鼠脑部的炎症反应,对急性弓形虫小鼠中枢神经系统的损伤具有明显的保护作用。     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

N-acetyl-L-cysteine combined with mesalamine in the treatment of ulcerative colitis： Randomized,placebo-controlled pilot study

AIM： To evaluate the effectiveness and safety of oral N-acetyl-L-cysteine （NAC） co-administration with mesalamine in ulcerative colitis （UC） patients.
METHODS： Thirty seven patients with mild to moderate UC were randomized to receive a four-wk course of oral mesalamine （2.4 g/d） plus N-acetyl-L-cysteine （0.8 g/d） （group A） or mesalamine plus placebo （group B）. Patients were monitored using the Modified Truelove-Witts Severity Index （MTWSI）. The primary endpoint was clinical remission （MTWSI ≤ 2） at 4 wk. Secondary endpoints were clinical response （defined as a reduction from baseline in the MTWSI of ≥ 2 points） and drug safety. The serum TNF-α, interleukin-6, interleukin-8 and MCP-1 were evaluated at baseline and at 4 wk of treatment. RESULTS： Analysis per-protocol criteria showed clinical remission rates of 63% and 50% after 4 wk treatment with mesalamine plus N-acetyl-L-cysteine （group A） and mesalamine plus placebo （group B） respectively （OR = 1.71; 95% CI： 0.46 to 6.36; P = 0.19; NNT = 7.7）. Analysis of variance （ANOVA） of data indicated a significant reduction of MTWSI in group A （P = 0.046） with respect to basal condition without significant changes in the group B （P = 0.735） during treatment. Clinical responses were 66% （group A） vs 44% （group B） after 4 wk of treatment （OR = 2.5; 95% CI： 0.64 to 9.65; P = 0.11; NNT = 4.5）. Clinical improvement in group A correlated with a decrease of IL-8 and MCP-1. Rates of adverse events did not differ significantly between both groups.
CONCLUSION： In group A （oral NAC combined with mesalamine） contrarily to group B （mesalamine alone）, the clinical improvement correlates with a decrease of chemokines such as MCP-1 and IL-8. NAC addition not produced any side effects.     

Delorme's transrectal excision for internal rectal prolapse

Surgical therapy of functional outlet obstruction in patients with internal rectal intussusception may include abdominal, perineal, or transrectal procedures. Because abdominal procedures often result in significant physiologic impact but unrelieved constipation, the authors have elected Delorme's transrectal excision for management of these patients. Since a short-term placebo effect attends many therapies, this report describes results of transrectal excision only after a threeyear postoperative period. Delorme's transrectal excision of internal intussusception accomplished sustained symptomatic relief in over 70 percent of otherwise refractory constipated patients. The association of internal intussusception with other abnormalities underscores the importance of defining both anatomic and functional components when selecting patients whose constipation may require surgical therapy. Critical technical elements, surgical pitfalls, and potential complications of the procedure are discussed.Poster presentation at the meeting of The American Society of Colon and Rectal Surgeons, Toronto, Canada, June 11 to 16, 1989.     

The oral glucose tolerance test: A comparison of the time points on the basis of limit values,normal dispersion,and reproducibility

Summary Time points in the glucose tolerance test (GTT) are compared on the basis of limit values, dispersion within a reference population, and reproducibility. We suggest using the distance between a limit value and the median reference value as a measure of the magnitude of abnormality. The distance between 140 mg/100 ml and the median fasting plasma glucose value is chosen as a standard distance and limits for other points in the GTT are calculated to equal this standard distance of abnormality. We suggest that the probability of correctly interpreting an inividual result is directly related to the reproducibility of the test and inversely related to the percentage of the total range of values which is dispersed among the normal population. The ratio of reproducibility to percentage normal dispersion is proposed as an index of the probability of correctly interpreting an individual result. According to this index, the probability of correct interpretation varies in order: fasting plasma glucose concentration>3-h>2-h>0.5-h>1-h plasma glucose concentration.