川崎病患儿树突状细胞亚群变化及意义初探

目的?探讨急性期川崎病（KD）患儿外周血树突状细胞（DC）亚群的变化及意义。方法 回顾性分析2020年12月—2021年5月就诊的初发急性期KD患儿的临床资料。KD患儿分别于静脉注射用免疫球蛋白（IVIG）治疗前、后取血,采用流式细胞术检测外周血DC亚群浆细胞样树突状细胞（pDC）及CD1c⁺髓样树突状细胞（mDC）数量和比例及表面功能分子HLA-DR、CD86、CD40蛋白表达水平。选择同期健康体检儿童作为对照组。结果 KD组患儿32例,男19例、女13例,中位年龄37.0（22.3～58.5）月。对照组23例,男14例、女9例,中位年龄52.0（30.0～65.0）月。KD组患儿IVIG治疗前、治疗后的pDC、CD1c⁺mDC占外周血总DC比例均低于对照组,治疗后组CD1c⁺mDC占外周血总DC比例高于治疗前;治疗前组pDC、CD1c⁺mDC数量低于对照组和治疗后组,差异均有统计学意义（P<0.05）。治疗前组pDC表面CD86、CD40表达以及CD1c⁺mDC表面H...     

儿童免疫性血小板减少症树突状细胞与T淋巴细胞分化失衡的关系

目的探讨树突状细胞和T淋巴细胞分化失衡与儿童免疫性血小板减少症(ITP)的关系及临床意义。方法用流式细胞术分别检测ITP患儿和对照者外周血Th细胞、Ts细胞、Treg细胞及树突状细胞变化。结果在持续性和慢性ITP组中Th细胞、Treg细胞和Th/Ts细胞比值降低,而Ts细胞升高,与对照组相比差异有显著性(P<0.05),与新诊断ITP组相比差异亦有显著性(P<0.05);Treg细胞在新诊断ITP组降低,与对照组比较差异有显著性(P<0.05);在慢性ITP组中浆细胞样树突状细胞(pDC)绝对值降低,髓样树突状细胞(mDC)/pDC比值增高,与对照组和新诊断ITP组比较差异有显著性(P<0.05)。60例ITP患儿经过糖皮质激素治疗后有39例完全缓解,12例部分缓解,9例无效。在无效组中Th细胞降低,而Tc细胞增高,与对照组和完全缓解组比较差异有显著性(P<0.05);Treg细胞和pDC绝对值在无效组和部分缓解组中降低,与对照组比较差异有显著性(P<0.05),无效组与完全缓解组比较差异亦有显著性(P<0.05)。结论 T淋巴细胞亚群和DC亚群比例失衡与儿童持续性和慢性ITP的发病及儿童ITP的临床分期和预后有关。     

大剂量丙种球蛋白与环孢素A治疗儿童再生障碍性贫血的疗效分析

目的：阶段性总结大剂量丙种球蛋白（HDIG）联合环孢素A(CsA)、小剂量泼尼松（P）、雄激素等治疗儿童再生障碍性贫血（AA）的疗效,同时探讨初诊时血常规、外周血淋巴细胞亚群百分比、骨髓CD34+细胞等各项实验室指标与疗效的相关性。方法：对该院46例应用大剂量丙种球蛋白与环孢素A治疗的AA 患儿进行临床回顾分析及追踪随访。结果：HDIG＋CsA治疗46例AA患儿,总有效率67.4%（31/46）。经二分类logistic回归分析,中性粒细胞计数降低(B=4.703, P<0.05)是AA治疗效果不良的危险因素。外周血淋巴细胞亚群CD4+、CD8+分布百分比值以及CD4+/CD8+比值与HDIG＋CsA方案的疗效显著相关,CD4+、CD4+/CD8+比值正常患者治疗有效率显著高于比值降低患者（P<0.01）。而CD8+比值正常患者治疗有效率显著高于CD8+比值增高患者（P<0.05）。AA患儿CD34+细胞/有核细胞比值均低于正常水平,但其比值对疗效无显著影响（P>0.05）。结论：中性粒细胞计数、外周血淋巴细胞CD4+百分比及CD4+/CD8+比值是影响AA治疗效果的重要因素。HDIG+CsA+P联合治疗儿童AA有效率较高,安全经济,是目前治疗儿童AA的理想方案,值得临床推广。［中国当代儿科杂志,2009,11（1）：33-36］     

特发性血小板减少性紫癜患儿治疗前后外周血长寿命浆细胞的检测及其意义

目的探讨长寿命浆细胞在特发性血小板减少性紫癜(ITP)外周血治疗前后的变化。方法用流式细胞术检测21例急性特发性血小板减少性紫癜(aITP)患儿、20例慢性特发性血小板减少性紫癜(cITP)患儿治疗前后和41名正常对照外周血长寿命浆细胞表面分子的改变;采用SPSS10.0进行统计学分析。结果cITP患儿外周血长寿命浆细胞表面分子表达率治疗前高于正常对照组(P<0.05),aITP患儿长寿命浆细胞表达率治疗前与正常对照组无显著性差异(P>0.05),cITP患儿外周血长寿命浆细胞表面分子表达率高于aITP患者(P<0.05),aITP患儿长寿命浆细胞表达率治疗前后无显著性差异(P>0.05),aITP患儿长寿命浆细胞表达率治疗前后无显著性差异(P>0.05)。结论长寿命浆细胞的改变提示其可能参与cITP自身免疫的病理机制,并为慢性ITP患儿治疗提供理论指导。     

中性粒细胞CD64在儿童社区获得性肺炎诊断中的价值

目的：探讨外周血中性粒细胞CD64的表达在儿童社区获得性肺炎（CAP）诊断中的价值。方法：依据病原体不同将98例社区获得性肺炎患儿分为细菌感染组（48例）、病毒感染组（29例）以及支原体感染组（21例）;另设健康对照组（20例）。细菌感染组依据患儿的入院情况分为轻症感染组（36例）和重症感染组（12例）。采用流式细胞术检测外周血中性粒细胞CD64的表达,同时免疫比浊法检测外周血C反应蛋白（CRP）的水平。结果：治疗前细菌感染组CD64指数和CRP水平显著高于其他3组,差异有统计学意义（P<0.05）。重症组CD64指数和CRP水平较轻症组显著增高,差异有统计学意义（P<0.05）。细菌感染组经过有效的抗菌治疗后,CD64表达水平下降,和治疗前相比差异有统计学意义（P<0.05）。相关分析结果显示CD64指数与CRP呈正相关（r=0.545,P＜0.01）。 ROC曲线分析结果显示CD64、CRP最佳临界值分别为2.8和8 mg/L,CD64指数的特异性（90%）远高于CRP（74%）。结论：外周血中性粒细胞CD64测定有助于肺部细菌感染的早期诊断,并可以判断病情的严重程度及疗效。     

糖皮质激素对哮喘小鼠CD4+CD25+调节性T细胞的作用

目的：建立哮喘小鼠动物模型并给予吸入糖皮质激素治疗,通过分析CD4+CD25+调节性T细胞及相关细胞因子在哮喘发病中的变化情况,以及观察糖皮质激素治疗对该细胞的影响和干预作用,探讨哮喘发作的新的免疫机制。方法：分别制备哮喘组（OVA）,哮喘吸入激素治疗组（OVA/GC）及对照组（NS）动物模型。所有小鼠于末次激发后24 h,取右肺中叶标本作病理切片,观察炎症改变;同时收集外周抗凝血及肺组织细胞悬液,通过流式细胞仪及酶联免疫吸附试验（ELISA）测定CD4+CD25+调节性T细胞占CD4+ T细胞的百分比和血浆中IL-10和TGF-β1水平。结果：①哮喘小鼠外周血及肺组织细胞悬液中CD4+CD25+调节性T细胞百分率明显降低（P<0.01）,吸入糖皮质激素治疗后CD4+CD25+调节性T细胞百分率明显升高（P<0.01）。②IL-10在哮喘组较对照组明显降低（P<0.01）,糖皮质激素治疗组IL-10明显升高（P<0.01）。③TGF-β1在哮喘小鼠较对照组明显降低（P<0.01）,吸入糖皮质激素治疗后TGF-β1有所升高,但差异无显著性。结论： ①哮喘小鼠CD4+CD25+调节性T细胞和IL-10,TGF-β1数量和分泌水平降低,尤其是肺部CD4+CD25+调节性T细胞不能有效聚集,导致全身和局部对炎症反应的抑制作用减弱,可能是导致哮喘发生的原因之一。②糖皮质激素可通过增加CD4+CD25+调节性T细胞及IL-10数量和分泌水平,增强其免疫抑制功能而发挥治疗作用。③外周血和肺部CD4+CD25+调节性T细胞百分率变化具有一致性,监测外周血CD4+CD25+调节性T细胞变化情况可以了解肺部变化趋势。     

癫癎患儿外周血中调节性T细胞的变化及其临床意义

目的：研究CD4+ CD25+ Foxp3+ 调节性T细胞（Treg细胞）在癫癎患儿外周血的表达水平,探讨其在癫癎发病机制中的作用。方法：采用流式细胞术分别检测41例癫癎患儿和38例健康儿童外周血Treg细胞占CD4+ T细胞的比例,及CD4+ T细胞、CD8+ T细胞、自然杀伤细胞（NK）、B细胞分别占淋巴细胞的比例。结果：癫癎组患儿外周血Treg细胞比例、CD4+ T细胞比例、CD4+ /CD8+ 比值分别为（2.4±0.5）%、（35±5）%、1.32±0.24,较对照组的（6.1±1.2）%、（38±4）%、1.60±0.24显著降低（P<0.05）;而癫癎组CD8+ T细胞比例、NK细胞比例、B淋巴细胞比例分别为（27±3）%、（11.1±5.1）%、（24±9）%,较对照组的（24±3）%、（8.5±1.9）%、(16±5)%显著升高（P<0.05）。结论：癫癎患儿外周血中存在 Treg 细胞比例异常,提示免疫机制可能参与了癫癎的发生和发展。     

胚胎干细胞诱导发育为树突状细胞的研究

目的探讨胚胎干细胞（ESC）诱导分化为树突状细胞（DCs）的实验方法,实现体外大规模扩增高纯度DCs用于临床免疫治疗。方法E14小鼠胚胎干细胞系在GM-CSF与IL-3联合作用下经胚胎体（EB）诱导分化为DCs。不成熟DCs（im-DCs）流式细胞仪检测CD11c、CD80、CD86、MHCⅡ-DR表达。用磷酸酯多糖（LPS）促进DCs成熟,收获细胞后观察细胞形态及摄片并做扫描电镜检查,分析细胞表型并与im-DCs细胞表型做对比,异体淋巴细胞增殖实验行功能检测。结果ESC来源DCs呈典型DCs形态学表现。im-DCCD11c、CD80、CD86、MHC-Ⅱ表达低,m-DCCD11c、CD80、CD86、MHC-Ⅱ的表达均较前明显升高。功能检测发现,ESC来源DCs具有强烈激发同种异体淋巴细胞增殖的作用,证实ESC来源的DCs具备正常的免疫学功能。结论使用GM-CSF联合IL-3能成功诱导E14胚胎干细胞发育为DCs。故ESC可作为DCs来源一条新途径,对于体外大规模制备DCs用于免疫过继治疗提供一个较好的方法。     

哮喘患儿CD4+CD25+调节性T细胞及IL-10和TGF-β1的变化

目的：通过测定和分析哮喘急性发作期与缓解期儿童外周血中CD4+CD25+调节性T淋巴细胞及其相关细胞因子白细胞介素-10（IL-10）和转化生长因子-β（TGF-β1）在哮喘发病中的变化情况,探讨哮喘发作的新的免疫机制。方法：分别采集74例哮喘发作期和28例哮喘缓解期患儿及20例健康儿童外周静脉血,通过流式细胞仪分析及酶联免疫吸附试验（ELISA）测定CD4+CD25+调节性T细胞占CD4+T细胞的百分比和血浆中IL-10和TGF-β1水平。结果：①哮喘发作期患儿外周血中CD4+CD25+调节性T细胞百分率及IL-10水平较缓解期及对照组明显降低（P<0.01）,而哮喘缓解期明显升高接近正常水平; ②哮喘急性发作期患儿TGF-β1水平较缓解期及对照组明显升高（P<0.05）。哮喘缓解期患儿TGF-β1水平明显降低,仍高于对照组水平; ③哮喘发作期、缓解期及对照组各组之间CD4+CD25+T细胞百分率与IL-10、TGF-β1无相关性（P>0.05）。结论：哮喘发作期患儿CD4+CD25+调节性T细胞和IL-10数量和分泌水平降低、TGF-β1水平增高,导致了全身和局部对炎症反应的抑制作用减弱,可能是导致哮喘发作的原因之一。［中国当代儿科杂志,2009,11（10）：829-832］     

幼年类风湿关节炎患儿血清白介素-15变化与T_H细胞亚群表达的研究

目的：探讨幼年类风湿关节炎(JRA)患儿血清白介素15变化及其TH细胞亚群CD4+CD45RA+,CD4+CD45RO+的表达变化。方法：采用ELISA方法检测39例JRA患儿的血清IL15的水平,并同期选择26例年龄、性别无差异的健康儿童为对照。对其中24例JRA患儿采用免疫荧光标记技术和流式细胞仪检测外周血CD4+T淋巴细胞亚群CD4+CD45RA+,CD4+CD45RO+的表达。结果：JRA患儿组血清IL15水平显著高于正常对照组(P<0.05);JRA常见亚型中全身型患儿血清IL15水平明显升高,与对照组比较差异有显著性(P<0.01),而少关节型、多关节型患儿IL15水平与对照组比较差异无显著性(P>0.05);治疗后IL15水平较治疗前明显下降(P<0.01);JRA患儿血清IL15水平与外周血白细胞计数呈正相关(r=0.347,P<0.05),与血沉无相关(r=0.307,P>0.05),与C反应蛋白呈显著正相关(r=0.452,P<0.01);IL15高表达组患儿外周血CD4+CD45RO+T淋巴细胞数明显高于IL15低表达组患儿(P<0.05),CD4+CD45RA+T淋巴细胞数、CD4+CD45RA+T/CD4+CD45RO+T比值略低于IL15低表达组患儿,差异无显著性(P>0.05)。结论：JRA患儿血清IL15的水平显著升高;IL15升高使JRA患儿外周血CD4+T细胞表面CD45RA分子向CD45RO分子转换,促使T淋巴细胞大量激活,进而介导组织免疫病理损伤;在临床上可通过检测IL15以判断JRA的病情状况,为JRA治疗提供理论依据。     

Monozygous twins with a microdeletion syndrome involving BTK, DDP1, and two other genes; evidence of intact dendritic cell development and TLR responses

We report for the first time monozygous twins with a microdeletion syndrome involving genes coding for Bruton’s tyrosine kinase (Btk) and deafness-dystonia peptide 1 (DDP1), and two other genes. Apart from its essential role in B cell development, Btk is indicated to affect signaling mediated by toll like receptors (TLRs) and development of dendritic cells (DCs) but results are conflictive. The twins revealed normal numbers of plasmacytoid and myeloid DCs (pDCs and mDCs). Moreover, BTK null cells from these patients exhibited robust responses to TLR agonists, normal natural killer (NK) cell activity, and normal pDC functions. Conclusion: Our results do not indicate the essential role of Btk in TLR signaling and DC development.     

Peripheral blood lymphocyte subsets in children with frequent upper respiratory tract infections

It is a common and well-known fact that infants and preschool children undergo frequent episodes of upper respiratory tract infections. The majority of these children do not have a recognized immunodeficiency. The aim of the present study was to evaluate the effects of frequent upper respiratory tract infections on cellular immunity, using peripheral blood lymphocyte subsets and activation markers as defining parameters. The study group consisted of 16 children (aged 2-6 years) with frequent upper respiratory tract infections; 30 age-matched healthy children served as controls. Peripheral blood T, B, NK cells; T lymphocyte subsets; naive and memory cells; and activation markers were analyzed by using monoclonal antibodies and flow cytometry. White blood cell count (WBC) was found to be markedly increased in the study group compared to controls (p < 0.05). The absolute number of lymphocytes was also higher than that of the healthy children. The relative size of the CD3+CD8+ T lymphocytes and the relative and absolute numbers of CD3-CD16+56+ NK cells were found to be higher in patients than the controls. All the remaining percentages and numbers of the T cell subgroups including naive and memory cells and B lymphocytes did not show any difference, while CD3+CD25+ cell numbers were markedly increased (p < 0.05). In conclusion, the examination of peripheral blood lymphocyte subsets in children with frequent upper respiratory tract infections is important in evaluating cellular immune alterations due to antigenic stimulation; however, it is neither essential nor cost-effective in the management of the disease. This study has shown that both the percentage and absolute numbers of peripheral blood lymphocyte subsets maintain their normal status in children with frequent upper respiratory tract infections.     

支气管肺炎患儿免疫功能变化的临床分析

目的：通过检测普通和重症支气管肺炎患儿血清补体、免疫球蛋白、淋巴细胞亚群变化,探讨免疫功能检测在支气管肺炎中可能的作用。方法：纳入符合诊断标准的普通和重症支气管肺炎患儿各20例,同时选取健康儿童20例为对照组。采用免疫速率散射比浊法和六色流式细胞仪分析法检测各组儿童血清IgA、IgG、IgM、补体C3、C4及淋巴细胞亚群CD3+、CD4+、CD8+、CD16+CD56+及CD19+指标变化。结果：普通及重症肺炎组IgA水平均较对照组明显降低（P0.05）;重症肺炎组CD4+、CD3+水平均较对照组降低（P0.05）。结论：支气管肺炎患儿存在免疫功能的紊乱,在重症组更为明显,免疫水平的变化与肺炎病情的严重程度相关。支气管肺炎患儿免疫功能检测具有重要的临床意义。     

Peripheral blood lymphocyte subsets in healthy Turkish children

Immunophenotyping of peripheral blood lymphocyte subpopulations is essential for the diagnosis and follow-up of children with immunodeficiencies and other immune disorders. The relative size and absolute number distributions (median and 5-95%) of lymphocyte subsets, including cord blood (Coulter, EPICS-XL) were examined by flow cytometry in 190 healthy subjects from birth to 18 years of age with a view to obtaining normal reference values for Turkish children of the following age groups: cord blood (n:29), birth to 1 year (n:41), 1 to 2 years (n:30), 2 to 6 years (n:30), 6 to 10 years (n:30), and 10 to 18 years (n:30). The relative size of CD2+, CD3+CD16-56-, CD3+CD8+ T lymphocytes increased while the relative size and absolute counts of those together with CD3+CD4+ and CD19+, CD20+ B lymphocytes decreased with age. The percentage of CD3-CD16+56+ NK cells increased from 0-1 year to 10-18 years; however, absolute count of CD3-CD16+56+ NK cells remained stable and unchanged in all age groups. The relative size and absolute count of activation markers (CD3+CD25+ and HLADR+) decreased from 0-1 year through 10-18 years age group. This study has once more demonstrated that both the percentage and the absolute number of lymphocyte subsets in cord blood and peripheral blood of healthy infants and children changed with age. Therefore, comparison of results to those of age-matched healthy controls is of utmost importance in the reliable and accurate evaluation of lymphocyte subsets reflecting cellular immunity in children.     

脓毒性休克患儿外周血淋巴细胞亚群的动态变化及临床意义

目的：探讨脓毒性休克(septic shock,SS)患儿外周血淋巴细胞亚群(总淋巴细胞、CD3＋T、CD4＋T、CD8＋T、CD19＋B、CD56＋NK)计数的变化特点及对预后的影响。方法检测 SS 重度组25例、SS轻度组24例入院时的外周血淋巴细胞亚群,并与同期健康体检小儿(正常对照组)25例进行比较分析。SS 重度组、轻度组于入院后第3、8天动态监测以上指标,同时记录病情变化和预后转归,并根据 SS 重度组转归分为死亡组与存活组,比较上述指标。结果入院时 SS 重度组、轻度组的淋巴细胞各亚群计数与正常对照组比较均有明显降低,三组相比差异具有显著性(P ＜0.05)。入院第3天,SS 轻度组的 CD4＋T、CD3＋T、总淋巴细胞计数较入院时有显著升高(P ＜0.05);SS 重度组除了 NK 细胞外,其余淋巴细胞亚群均较入院时显著降低(P ＜0.05)。入院第8天,SS 轻度组的 CD4＋T 计数与正常对照组比较已无显著差异(P ＞0.05);SS 重度组的 CD4＋T、CD3＋T、总淋巴细胞计数较入院时有显著升高,但与正常对照组比较差异仍有统计学意义(P ＜0.05),其余淋巴细胞亚群计数有所上升。SS 患儿重度组5例死亡(死亡组)均发生在入院后2～5 d,与其余生存的20例(存活组)相比,两组入院时的淋巴细胞各亚群计数比较无显著差异,死亡组入院第3天的淋巴细胞各亚群计数值明显降低,与存活组同期存在显著差异(P ＜0.05)。结论SS 患儿外周血淋巴细胞各亚群计数值越低,病情越严重,持续低水平提示死亡风险大;经过治疗,病情有效控制者首先表现为 CD4＋T、CD3＋T、总淋巴细胞计数上升,尤其是 CD4＋T。因此,动态监测 SS 患儿的淋巴细胞亚群对判断病情的严重程度、疗效、预后具有较高临床价值。     

Reduction of peripheral blood macrophages/monocytes in Kawasaki disease by intravenous gammaglobulin

The effects of intravenous gammaglobulin (IVGG) on changes in the peripheral blood mononuclear cell subsets during acute Kawasaki disease (KD) were studied by a random selection trial of IVGG plus Aspirin (group G) compared to Aspirin alone (group A). Group G received IVGG with 200 mg/kg per day × 5 dose. The absolute counts of peripheral blood mononuclear cell subsets were assayed by a fluorescence-activated cell sorter using monoclonal antibodies of Leu series. Before therapy, patients in each treatment group had increased counts of CD14+ macrophage/monocytes compared to healthy childhood controls (P<0.01). After IVGG treatment, group G underwent a greater decrease in their CD14+ macrophage/monocyte counts (P<0.01) than group A. The changes of CD3+ T cells, Leu 7+ NK/K cells and CD19+ B cells in the peripheral blood mononuclear cell subsets with treatment in group G, were similar to those in group A. These results suggest the possibility that IVGG therapy is effective in KD by modulating macrophages/monocytes.     

The role of Helicobacter pylori in children with chronic idiopathic thrombocytopenic purpura

Recent reports have suggested that Helicobacter pylori infection may be a causative agent of adult chronic idiopathic thrombocytopenic purpura (cITP) and antimicrobial treatment may increase platelet counts. As there is limited experience in pediatric age, we investigated the prevalence of H. pylori infection and the effects of H. pylori eradication therapy in a series of children with cITP. Twenty-four children with cITP were investigated for H. pylori infection using the C-urea breath test or H. pylori fecal antigen. In cases of H. pylori infection, antimicrobial treatment was given with amoxicillin, clarithromycin, and proton pump inhibitors. Response was assessed at 6 months and defined as complete (platelet count >150x10/L) or partial (platelet count between 50 and 150x10/L). H. pylori infection was found in 8 patients (33%) and 3 of them showed a response after eradication therapy, but 2 of them relapsed later on. Two patients had a spontaneous increase in platelet count in the group of H. pylori-negative patients. Given that spontaneous improvements in platelet count can occur in children with cITP, we were unable to demonstrate that H. pylori plays a major role in cITP occurring in pediatric age.     

Age-dependent changes in peripheral blood dendritic cell subsets in normal children and children with specific polysaccharide antibody deficiency (SPAD)

Myeloid and plasmacytoid dendritic cells (MDC/PDC) play crucial roles in bridging adaptive and innate immunity by affecting development of both cellular and humoral immunity. The immune system evolves after birth as reflected in dynamic changes in numbers and functions of various immune cells with age. However, age-associated changes in DC subsets in children have not been elucidated despite the fact that such normative data are crucial for evaluating alternations of DC subsets in various pediatric diseases. This study addressed age-associated changes in DC subsets and CD40/86 expression on PDC (markers of maturation/activation) in 50 healthy children in comparison with 25 children with specific polysaccharide antibody deficiency (SPAD). Our results revealed age-dependent decrease of PDC numbers (p < 0.0001), although there was no age-associated changes in CD40/CD86 expression. MDC1/MDC2 numbers did not reveal such linear age-dependent changes and MDC1/PDC ratio reached around 2 as typically seen in young adults after 10 years of age. In contrast, SPAD patients did not reveal such age-associated changes and showed decreased fluorescence intensity of CD86 in PDC cells. These results indicate lineage specific, age-dependent changes in DC subsets in normal children and possible altered development of these cells in SPAD children, emphasizing the importance of age-appropriate controls.