日本血吸虫慢性感染致CD+4T细胞凋亡的分子基础

目的　立足于全基因水平考察日本血吸虫慢性感染小鼠CD 4T细胞的凋亡相关基因的变化。方法　采用三色流式细胞术结合高密度寡核苷酸芯片 (Affemetrix芯片 ) ,对日本血吸虫感染 13周的小鼠脾脏中的CD 4T细胞进行凋亡观察及基因转录分析 ,获得凋亡相关基因的表达图谱。结果　日本血吸虫慢性感染小鼠CD 4T细胞的凋亡率均明显高于正常小鼠 (P <0 0 1) ;感染小鼠的CD 4T细胞中有 2 5个凋亡相关基因与正常小鼠相比有显著性差异 ,包括凋亡促进基因—生长抑制和DNA损伤 -GADD家族和肿瘤坏死因子α诱导蛋白 3,凋亡抑制基因—IAP等。结论　日本血吸虫慢性感染小鼠CD 4T细胞的凋亡可能是导致宿主免疫下调的原因之一。凋亡通路中存在复杂的凋亡促进基因和抑制基因的相互作用 ,其中肿瘤坏死因子α诱导蛋白 3可能是日本血吸虫感染诱导CD 4T细胞凋亡的特征性因子。     

NO介导Th1/Th2免疫偏移与日本血吸虫感染小鼠肝肉芽肿病变的作用

目的　观察日本血吸虫感染小鼠肝肉芽肿病变与一氧化氮 (NO)、Th1/Th2细胞因子水平的动态变化 ,探讨NO介导Th1/Th2免疫偏移在血吸虫卵肉芽肿病变中的作用。方法　采用石蜡切片 ,HE染色后观察感染小鼠肉芽肿病变。用硝酸还原酶法和ELISA夹心法分别检测日本血吸虫感染小鼠 0～ 12周血清及脾CD+ 4 T淋巴细胞培养上清NO、IFNγ和IL - 4表达水平 ,并进行相关分析。结果　感染小鼠 0～ 12周血清和脾CD+ 4 T淋巴细胞培养上清NO表达水平与小鼠肝肉芽肿病变动态相一致 ,并呈显著正相关 ,与IFNγ表达水平呈显著正相关 ,而与IL - 4在小鼠感染慢性期 (12周 )呈显著负相关。结论　NO在日本血吸虫感染小鼠肝肉芽肿病变过程中可能是一种与Th1/Th2细胞因子具有同样重要作用的调节因子 ,并可能是通过调节Th1/Th2细胞因子而发挥作用的 ,通过调控NO合成介导Th1/Th2免疫偏移可能是控制血吸虫卵肉芽肿病变的新途径     

ICOS信号与日本血吸虫感染小鼠Th9细胞极化的关系

目的 检测可诱导共刺激分子（ICOS）在日本血吸虫感染小鼠脾脏Th9细胞中的表达水平,探讨ICOS信号与Th9细胞极化之间的关系。方法 以日本血吸虫尾蚴感染小鼠,分别在感染后0、4、7、9周和12周,应用流式细胞术检测IL?9+细胞在CD4+ T细胞中的比例,以及ICOS+细胞在Th9细胞中的比例。结果 与0周比较,感染后4、7、9、12周,小鼠CD4+ T细胞中Th9细胞的比例显著增高（P < 0.05）,并且Th9细胞ICOS的表达也明显上调（P < 0.05）。结论 在日本血吸虫感染中,ICOS信号对Th9细胞的极化可能具有调控作用。     

日本血吸虫不同免疫原对小鼠Th1/Th2免疫偏移的影响

目的　观察单、双性日本血吸虫感染小鼠后Th1/Th2细胞因子水平的动态变化 ,探讨日本血吸虫不同免疫原对Th1/Th2免疫偏移的影响及与虫卵肉芽肿形成的相关性。方法　用ELISA夹心法检测单、双性日本血吸虫感染小鼠及单性感染 8w双性再感染 0～ 12w ,小鼠脾淋巴细胞培养上清Th1细胞因子IL - 2、IFN -γ和Th2细胞因子IL - 4表达水平。结果　单性感染小鼠 6～ 12w在脾淋巴细胞培养上清中可测出一定量的IL - 2和IFN -γ ,但无明显动态变化 ,而IL - 4未能测出 ;双性感染小鼠IL - 2和IFN -γ在感染后 4～ 6w开始上升 ,8w达高峰 ,随后下降 ,IL - 4则在感染后 8w时迅速上升且随着感染时间延长而明显升高 ,单性感染 8w双性再感染 4w时 ,小鼠脾淋巴细胞培养上清IL - 2、IFN -γ和IL - 4表达水平即迅速升高且在双性再感染 8～ 12w逐渐增强。结论　日本血吸虫不同免疫原对Th1/Th2免疫偏移的影响作用不同 ,Th1优势应答可能主要由虫体抗原诱导 ,Th2优势应答可能主要由虫卵抗原 (SEA)所诱导 ,后者是诱导宿主产生免疫病理反应的主要因素     

NO介导Th1／Th2免疫偏移与日本血吸虫感染小鼠肝肉芽肝病变的作用

目的：观察日本血吸虫感染小鼠肝肉芽肿病变为一氧化氮（NO）、Th1/Th2细胞因子水平的动态变化，探讨NO介导Th1/Th2免疫偏移在血吸虫卵肉芽肿病变中的作用。方法：采用石蜡切片，HE染色后观察感染小鼠肉芽肿病变。用硝酸还原酶法和ELISA夹心法分别检测日本血吸虫感染小鼠0－12周血清及脾CD4^ T淋巴细胞培养上清NO、IFNγ和IL－4表达水平，并进行相关分析。结果：感染小鼠0－12周血清和脾CD4^ T淋巴细胞培养上清NO表达水平与小鼠肝肉芽肿病变动态相一致，并呈显著正相关，与IFNγ表达水平呈显著正相关，而与IL－4在小鼠感染慢性期（12周）呈显著负相关。结论：NO在日本血吸虫感染小鼠肝肉芽肿病变过程中可能是一种与Th1/Th2细胞因子具有同样重要作用的调节因子，并可能是通过调节Th1/Th2细胞因子而发挥作用的，通过调控NO合成介导Th1/Th2免疫偏移可能是控制血吸虫卵肉芽肿病变的新途径。     

日本血吸虫感染过程中抑制性因子的表达特征

目的立足于基因组水平考察日本血吸虫慢性感染小鼠CD_4~+ T应答中抑制性因素的参与及可能的调控作用。方法采用高密度寡核苷酸芯片(Affymetrix芯片)对日本血吸虫感染0、3、6、13周小鼠脾脏中的CD_4~+ T细胞进行全基因组分析,获得相关抑制性因子的基因表达谱。结果基因芯片结果显示日本血吸虫感染从急性期至慢性期过程中抑制性因子的表达基因表达逐步上调,呈现基本一致的变化,包括抑制性细胞因子、阻断抗体、细胞凋亡、负调控分子的高表达等。结论从胞内信号转导通路至细胞凋亡的整体水平,多层次因素参与了日本血吸虫感染宿主免疫应答的负调控机制。     

TIGIT信号在日本血吸虫感染小鼠Th1/Th2 反应平衡中的作用研究

目的 研究TIGIT信号在日本血吸虫感染过程中Th1/Th2反应平衡中的作用。方法 C57BL/6小鼠感染日本血吸虫尾蚴,并以未感染小鼠作为正常对照。感染0、3、5、8、13周时取小鼠脾脏,采用流式细胞术检测小鼠脾脏淋巴细胞中TIGIT阳性细胞比例,以及TIGIT阴性和TIGIT阳性细胞上Ki67、IFN?γ、IL?4表达水平。结果 日本血吸虫感染组小鼠脾脏中TIGIT+细胞占CD4+ T细胞的比例显著高于正常对照组（29.30%±0.70% vs. 3.09%±0.50%;t = 29.09,P < 0.01）,但感染组和对照组小鼠脾脏中TIGIT+细胞占CD8+ T细胞的比例差异无统计学意义（3.61%±0.26% vs. 3.58%±0.16%;t = 0.108,P > 0.05）,且感染组和对照组小鼠脾脏中TIGIT+细胞占非T细胞的比例差异亦无统计学意义（1.86%±0.19% vs. 1.37%±0.17%;t = 1.931,P > 0.05）。进一步分析发现,Ki67在TIGIT+细胞上的表达比例（17.03%±0.64%）较其在TIGIT?细胞上表达的比例（6.59%±0.37%）显著升高（t = 14.09,P < 0.01）,而CD4+ TIGIT+细胞中Th2/Th1比值显著高于CD4+ TIGIT?细胞（39.28%±3.75% vs. 11.79%±1.64%;t = 6.721,P < 0.01）。结论 感染日本血吸虫后,CD4+ T细胞上TIGIT表达增加,且可能通过诱导Th2细胞增殖进而促进Th2细胞比例增加。     

日本血吸虫感染小鼠肠系膜淋巴结Th17细胞的免疫应答

目的观察C57BL/6小鼠感染日本血吸虫后肠系膜淋巴结Th17细胞的免疫应答。方法 20只C57BL/6小鼠随机分为感染组和对照组,每组10只,感染组小鼠经腹部皮肤感染日本血吸虫尾蚴,每鼠(40±5)条。感染后5~6周分离小鼠肠系膜淋巴结的淋巴细胞,分别用抗小鼠CD3单克隆抗体(anti-CD3,1μg/ml)和抗小鼠CD28单克隆抗体(anti-CD28,1μg/ml)刺激,培养4 h后收集细胞,RT-PCR检测小鼠肠系膜淋巴结淋巴细胞中白细胞介素17(IL-17)和维甲酸相关孤独受体(ROR-γt)mRNA的转录水平;培养72 h后,ELISA检测细胞培养上清液IL-17和γ干扰素(IFN-γ)的含量。同时用佛波酯(PMA,10 ng/ml)和离子霉素(1μg/ml)刺激淋巴细胞5 h后,胞内细胞因子染色,流式细胞术检测Th17细胞的含量和其他细胞因子的产生。结果 ELISA检测结果显示,感染小鼠肠系膜淋巴结培养物上清液中IFN-γ[(214.3±62.6)pg/ml]和IL-17[(176.8±62.1)pg/ml]的含量明显高于健康小鼠[(46.7±13.9)和0 pg/ml](P<0.05)。RT-PCR检测结果显示,IL-17和ROR-γt mRNA转录水平也明显高于健康小鼠。感染小鼠肠系膜淋巴细胞CD4+T细胞中,Th17细胞的比例为(0.55±0.03)%,明显高于健康小鼠[(0.16±0.01)%](P<0.05)。在CD4+T细胞中,IL-17+IL-4+细胞占0.06%,IL-17+IFN-γ+和IL-17+IL-5+细胞各占0.02%,IL-17+IL-9+细胞占0.01%,未检测到IL-17+IL-10+和IL-17+Foxp3+细胞。结论日本血吸虫感染C57BL/6小鼠的肠系膜淋巴结能诱导Th17细胞产生。Th17细胞能分泌IL-4,及少量的IFN-γ、IL-5和IL-9,不分泌IL-10,也不表达Foxp3。     

趋化因子在日本血吸虫感染模型中的表达特征

目的 研究日本血吸虫感染模型中趋化因子(chemokine)在CD4^ T细胞中的表达概貌，并探讨其与Th细胞极化和免疫病理的相关性。方法 采用高密度寡核苷酸芯片(Affymetrix芯片)技术，从基因水平获得日本血吸虫不同感染时期CD4^ T细胞表达的趋化因子概貌，并用反转录PCR(RT—PCR)凝胶成像半定量技术验证嗜酸性粒细胞趋化因子(ECF—L)的表达水平。同时观察趋化因子表达与病理的的关联。结果 日本血吸虫虫卵沉积前，MIG、RANTES明显增强；感染6周IP-10、MIP-2、MIP-1α、MIP-1β、ECF—L升高，持续到感染13周；GRO、Lymphotactin、MCP-1、RANTES在感染13周显著增强。RT-PCR半定量验证ECF-L表达模式与基因芯片结论一致。结论 日本血吸虫人工感染小鼠的CD4^ T细胞表达大量趋化因子，它们共同参与Th1／Th2免疫应答，影响免疫病理状态；改变趋化因子格局可能影响疾病的自然转归。     

日本血吸虫感染小鼠CD_4~+ T淋巴细胞协同刺激分子表达谱及其在介导Th1/Th2极化中的作用

目的观察日本血吸虫感染小鼠不同病期体内及体外培养后脾CD4+T淋巴细胞表面的协同刺激分子表达谱,探讨协同刺激分子介导Th1/Th2极化的作用。方法收集血吸虫感染后4w、7w、12w、16w和20w小鼠及正常小鼠脾CD4+T淋巴细胞,以及上述不同病期经SEA诱导72h后脾淋巴细胞培养上清,用流式细胞术检测细胞表面协同刺激分子CD80、CD86、ICOS、CD40、OX40、4-1BB及PD1表达水平,用ELISA法检测培养上清中的Th1细胞因子IFN-γ,Th2细胞因子IL-4、IL-5、IL-10、IL-13。结果血吸虫感染后小鼠脾CD4+T淋巴细胞表面CD86、ICOS、CD40和OX40的表达均随感染时间延长而上调,其中ICOS和CD86表达上调最为显著;经SEA诱导培养72h后CD86、ICOS亦显著上调表达;培养上清检测发现IFN-γ在4w时呈高表达,7w开始随感染时间延长呈下调表达;IL-4、IL-5、IL-10和IL-13的表达水平则从7w开始明显升高,IL-5的峰值出现在12w,其余均16w达高峰,致20w仍维持在较高水平。结论日本血吸虫感染小鼠CD4+T淋巴细胞协同刺激分子表达水平与Th1/Th2免疫偏移密切相关,其中ICOS、CD86在介导Th2极化中可能具有重要作用。     

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.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

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.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.