儿童难治性自身免疫性疾病自体外周血干细胞动员采集和分选的临床研究

目的 探讨儿童难治性自身免疫性疾病进行自体外周血干细胞动员采集的安全性和CD34+细胞分选纯化的可行性及其临床意义.方法 8例儿童难治性自身免疫性疾病,包括4例系统性红斑狼疮、2例皮肌炎,1例幼年型类风湿关节炎和1例多发性硬化,予行CD34+细胞纯化的自体外周血干细胞移植.首先采用环磷酰胺(CTX)联合粒细胞集落刺激因子(G-CSF)方案动员外周血干细胞,然后采用CS-3000血细胞分离机采集外周血,通过CliniMACS细胞分选仪分选自体外周血CD34+细胞,将其用保养液配置冻存于-80℃冰箱.采用非清髓内去除T的预处理方案,即卡氮芥+足叶乙苷+阿糖胞苷+马法兰+抗胸腺球蛋白(ATG)或CTX+ATG或CTX+马法兰+ATG,于第0天回输自体外周血CD34+细胞.结果 儿童能够耐受自体外周血干细胞动员采集过程,无动员相关死亡,动员后获得的单个核细胞数和CD34+细胞数的平均值分别为8.35×108/kg和7.92×106/kg,纯化后的白体外周血CD34+和CD3+细胞数的平均值分别为6.28×106/kg和0.71×105/kg.回输后中性粒细胞和血小板的植入中位时间分别为+11d和+15 d.结论 经CTX联合G-CSF方案可动员出足量的外周血干细胞,经CS-3000血细胞分离机采集可获得足够的单个核细胞,在动员过程中原发病无明显进展恶化,患儿能耐受动员方案,采集过程顺利安全;经CliniMACS细胞分选仪分选的自体外周血CD34+细胞纯度高,移植后造血恢复;采用CD34+细胞纯化的自体外周血移植治疗是常规治疗无效的儿童难治性自身免疫性疾病的可选择治疗措施之一.     

大剂量化疗并自体外周血干细胞移植治疗重症原发性干燥综合征

目的探讨大剂量化疗（HDC）并自体外周血干细胞移植（PBSCT）对于常规治疗反应不佳的重症原发性干燥综合征（pSS）的可行性、疗效及安全性。方法选择1999年起3例长期激素和免疫抑制剂治疗病情不能缓解的重症pSS患者作为治疗对象，给予大剂量免疫抑制剂治疗及自体外周血干细胞移植。自体干细胞动员采用环磷酰胺（CTX）2—3g／m^2和粒细胞集落刺激因子（G—CSF），并行CD34^＋细胞分选。干细胞回输前用CTX200mg／kg、抗胸腺细胞免疫球蛋白（ATG）90mg／kg或CTX200mg／kg、全身照射（TBI）4gy进行预处理。结果1例患者完成2次CTX2g／m^2动员，尚未行干预处理和细胞回输，2例重症pSS顺利完成治疗全过程，3例患者随诊时间分别达48、60和18个月。治疗后3例患者的B淋巴细胞功能亢进均得以抑制，2例患者抗SSB抗体转阴，1例患者重复唇腺活检示灶性淋巴细胞浸润消失：2例患者的肺功能改善，间质性肺炎得以逆转。结论自体外周血干细胞移植可控制重症pSS患者的病情，具有可行性和安全性，疗效较为肯定。     

不同来源的CD34^＋细胞归巢相关分子的表达

目的 研究不同来源CD34^＋细胞归巢相关分子（HRM）的表达情况。方法采用免疫磁珠法（MACS）分选不同来源的CD34^＋细胞，免疫荧光标记流式细胞仪测定HRM的表达。结果骨髓（BM）、动员后的外周血（mPB）及脐血（UCB）来源的CD34^＋细胞均高表达细胞黏附分子CD49d、CD49e、CD54、CD11a、CD62L、CD44、CD31。UCB来源的CD34^＋细胞表面表达的细胞黏附分子中，CD49e表达显著低于BM和mPB来源的CD34^＋细胞（P〈0．05），CD54表达显著低于mPB来源者（P〈0．05），CXCR4表达显著低于BM来源者（P〈0．05）。结论UCB来源的造血干／祖细胞归巢能力低可能是UCB移植造血重建延迟的原因之一。     

粒细胞集落刺激因子动员对CD34^＋细胞黏附分子表达的影响

目的探讨粒细胞集落刺激因子（G—CSF）动员对CD34^＋细胞黏附分子表达的影响。方法应用流式细胞仪检测健康供者稳态及G—CSF动员过程中骨髓、外周血CD34^＋细胞的黏附分子表达变化，并应用结晶紫染色测定CD34^＋细胞的黏附功能。结果G-CSF动员后CD34^＋CD49d^＋细胞比例无显著下降，外周血CD34^＋CD621．^＋、CD34^＋CD54^＋和CD34^＋CDlla^＋细胞比例增高。动员后CD34^＋细胞表面CD49d的平均荧光强度显著减弱，但CD49e、CD62L、CDlla、CD54的平均荧光强度虽呈减弱趋势，却无统计学差异。动员后CD34^＋细胞黏附纤连蛋白能力下降。结论G—CSF通过降低造血干细胞与骨髓基质的黏附而产生动员效应。     

自体外周血造血干细胞移植治疗系统性红斑狼疮

目的：探讨自体外周血造血干细胞（APBSCT）治疗系统性红斑狼疮（SLE）的临床效果。方法：采集经用环磷酰胺（CTX）2000mg/m^2静脉注射和粒细胞集落刺激因子（G－CSF）250ug/d皮下注射动员的外周血造血干细胞，预处理CTX48mg.kg^-1.d^-1，连续静脉滴注3d，回输干细胞后用抗胸腺细胞球蛋白（ATG）13．5mg/kg，体内去细胞，分3d静脉滴注，用G－CSF刺激升高白细胞，观察APBSCT前后免疫指标与临床表现，结果：APBSCT后患者的临床表现基本消失，免疫指标正常，抗体转阴，结论：APBSCT治疗SLE有较好的疗效，远期疗效还需长期随访观察。     

不同免疫抑制诱导治疗对肝移植受者CD4+CD25+high细胞的影响

目的CD4^＋CD25^＋Treg细胞介导的免疫调节对诱导和维持移植物免疫耐受有非常重要的作用，本研究探讨抗胸腺淋巴细胞球蛋白（ATG）和噻呢哌这两种不同作用途径的免疫抑制诱导药物对CD4^＋CD25^＋high／CD4^＋T细胞及其表面标志性因子CTLA-4、Foxp3的影响，为临床合理选择应用免疫诱导药物提供依据。方法选择15例在我院行同种异体肝移植患者，随机分为ATG组（8例）和噻呢哌组（7例），两组除免疫抑制诱导药物不同外，均接受相同治疗。分别于术前、术后3周、6周、8周取血，应用流式细胞仪检测CD4^＋CD25^＋high/CD4^＋T细胞的数量及其表面因子CTLA-4和Foxp3表达状况，比较不同诱导药物对其影响。结果这两种免疫抑制诱导药物对CD4^＋CD25^＋Treg细胞短期内有一定影响，但可逐渐恢复，两者比较，使用ATG后各指标恢复较噻呢哌快，尤其是Foxp3更加明显。结论作为诱导治疗药物，ATG更有利于CD4^＋CD25^＋Treg细胞功能恢复，从而有助于更好形成移植免疫耐受。     

粒细胞集落刺激因子对供者外周血干细胞动员的影响

目的：探讨外周血造血干细胞移植（PBSCT）供者应用粒细胞集落刺激因子（G-CSF）后细胞成分的变化和药物对供者身体状况的近期影响。方法：对18例健康PBSCT供者应用一定剂量G-CSF,4-5d后采集外周血单个核细胞进行检测及观察用药后出现的不良反应。结果：外周血白细胞在动员后4-5 d达峰值,动员后比动员前高7-14倍。供者单个核细胞（MNC）和CD34^＋细胞值之间差异无统计学意义。结论：对PBSCT供者应用G-CSF 5-10μg/（kg.d）,4-5 d可有效动力员MNC和CD34^＋细胞。     

外周血造血干细胞与白细胞在脑外伤后的动态变化及意义

脑外伤患者38例,采用流式细胞仪、全自动血细胞分析仪动态检测第1、3、5、7、14天外周血CD34^＋的绝对值和白细胞的变化。结果显示,脑外伤后,轻症组、重症组均出现CD34^＋细胞逐步上升,重症组上升幅度较大;而白细胞值在伤后迅速升高并逐步下降,与CD34^＋细胞变化呈负相关。认为脑外伤后CD34^＋细胞与白细胞计数可作为重要观察指标,脑外伤后机体存在CD34^＋细胞的动员,参与脑外伤的修复。     

大鼠部分肝移植后自体骨髓干细胞动员的研究

目的 探讨大鼠部分肝移植后骨髓干细胞动员的情况。方法 建立大鼠性别交叉部分肝移植模型，分为部分肝移植组、全肝移植组和假手术组，分别与术后1、3、5、7d取标本。用流式细胞法检测骨髓中干细胞标记的细胞群体的数量变化，荧光原位杂交检测移植肝脏内Y染色体特异的Sry基因的表达，免疫组织化学法检测移植肝脏内干细胞标志CD34，c—kit和Thy-1．1的表达。结果 与全肝移植组相比，部分肝移植组术后第1天，骨髓细胞中，β2微球蛋白（β2m）／Thy-1．1^＋，CD45^＋／CD34^＋有不同程度的升高，然后呈递减趋势。免疫组织化学检测汇管区和炎性灶周围可见CD34、c—kit和Thy-1．1单个核细胞表达，并于第5至7天后减少。同时免疫组织化学双染可检出CD34^＋／CD45^＋细胞。全肝移植组汇管区CD34、c—kit和Thy-1．1表达较少，假手术组CD34、c-kit和Thy-1．1偶见表达。在部分肝移植物可检出Sry^＋细胞，但Srg^＋／CD34^＋，Sry^＋／Thy-1．1^＋细胞较少。全肝移植组Sry^＋偶见。结论 部分肝移植中，骨髓源性的干细胞发生动员；同时肝内有干细胞被激活，其中外源性干细胞较少。     

免疫清除性化疗结合自体外周血造血干细胞移植治疗重症系统性红斑狼疮

目的 观察免疫清除性化疗结合自体外周血造血干细胞移植（移植）治疗重症系统性红斑狼疮（SLE）的疗效及安全性。方法 重症SLE4例，分别合并狼疮性肾炎，狼疮脑，狼疮心或股骨头坏死，干细胞的动员采用环磷酰胺加重组人粒细胞集落因子（G－CSF）；预处理为回输前3天每天应用环磷酰胺50mg/kg 回输后3天每天应用抗胸腺细胞球蛋白（ATG）5mg/kg。观察移植前后临床症状，体征，狼疮相关抗体等指标的改变，并动态观察移植后免疫功能的重建。结果 移植后患者的临床症状完全缓解，狼疮相关抗体全部转阴，移植后患者的免疫功能均明显降低，约半年后恢复正常，但不伴有临床症状的复发。结论 免疫清除性化疗结合自体外周血造血干细胞移对难治性SLE有明显的疗效，尤其适用对各种药物治疗无效者，治疗是安全的，远期疗效还需长期随访。     

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.     

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.     

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.