A novel method for the serum-free plating of stage-24 chick limb bud cells

Summary A modification of the standard plating of Stage 24 chick limb bud cells in serum-containing media (SCM) is described in which the cells are incubated in SCM for 3 hours before direct plating in a serum-free chemically defined media (SFM). This results in equal plating efficiencies to those obtained with plating in SCM and may allow for more defined culture conditions when testing mitogenic growth factors found in serum.     

CHO细胞无血清培养研究

目的:用无血清培养基培养中华仓鼠卵巢(Chinese hamster ovary,CHO)细胞。方法:应用无血清培养基采用小方瓶培养CHO细胞,观察细胞维持时间、培养过程的形态变化。结果:应用无血清培养基培养CHO细胞可维持细胞正常生长。结论:无血清培养基可以完全取代含血清培养基用于培养CHO细胞。     

Pluronic F-127水凝胶复合骨髓间充质干细胞构建组织工程化脂肪的体外研究

本实验应用Pluronic F-127水凝胶三维培养大鼠骨髓间充质干细胞(BMSCs),观察其生物相容性,并研究其对BMSCs诱导分化成脂肪细胞的影响。原代培养SD大鼠BMSCs,经诱导分化成脂、成骨和成神经鉴定后,用MTT法检测Pluronic F-127水凝胶对BMSCs的细胞毒性;将P4BMSCs均匀混合于水凝胶中,复合BMSCs的水凝胶经成胶后分别用成脂诱导液和完全培养基培养,观察并记录脂滴、脂肪细胞及脂肪组织形成情况,培养2周后油红O染色鉴定。结果表明BMSCs在Pluronic F-127水凝胶中能较好的黏附、生长及增殖。成脂诱导的水凝胶中BMSCs可分化为脂肪细胞,其中少部分聚集成脂肪细胞团,形成脂肪样组织,并经油红O染色鉴定证实。而仅加入完全培养基的水凝胶内BMSCs则未发生分化。提示BMSCs可在Pluronic F-127水凝胶三维支架中培养并被成功诱导分化为脂肪细胞,利用水凝胶负载BMSCs向脂肪细胞诱导分化可望为组织工程化脂肪提供新的思路,Plu-ronic F-127水凝胶可作为BMSCs治疗体外培养的良好支架。     

人血小板裂解液替代胎牛血清大规模扩增人脐带间充质干细胞

背景：目前国内外大多数实验仍采用经典的培养基和胎牛血清进行脐带间充质干细胞培养,血清培养潜在的不安全因素限制了未来临床应用的可行性。 目的：以人血小板裂解液替代胎牛血清培养、鉴定人间充质干细胞,以期进一步应用于临床低密度扩增人间充质干细胞。 方法：人血小板裂解液通过反复冻融、离心、过滤、浓缩等方法制备。以IMDM为基础培养基,添加5%浓缩血小板裂解液作为实验组培养基;以添加体积分数10%胎牛血清为对照组培养基。采用酶消化法分离培养人脐带间充质干细胞,以3 000/cm²的浓度进行传代培养,待细胞扩增至P5代后,进行细胞形态与直径、免疫表型、成骨成脂分化、克隆形成率等细胞生物学特性检测,并比较两者之间的差别。 结果与结论：人血小板裂解液扩增的脐带间充质干细胞形态细长,有更高的细胞累积群倍数;克隆形成检测结果显示两者形成的克隆效率差异无显著性意义,流式细胞术检测结果显示两者有相似的细胞表型,体外诱导分化显示两者都具有成骨、成脂分化能力,但人血小板裂解液扩增的间充质干细胞成骨分化能力更强。提示人血小板裂解液可以取代胎牛血清用于临床低密度扩增人间充质干细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

A-NK细胞培养和活化方法的改进

目的:比较A-NK细胞在完全培养基(CM)和无血清培养基(AIMV)中体外扩增及杀伤肿瘤细胞作用,同时探讨IL-12对于A-NK/IL-2治疗的辅助作用及其杀伤的形态学观察。方法:用MTT法比较不同培养条件下的A-NK细胞体外增殖能力,并测定细胞体外杀伤肿瘤细胞的活性。通过做扫描和透射电镜,对A-NK细胞杀伤的肿瘤细胞进行形态学观察。结果:不同培养条件下的A-NK细胞均可在短期内大量扩增(P<0.05),且均有很强的杀伤肿瘤的活性(P<0.05)。被A-NK细胞杀伤的肿瘤细胞的死亡形式是溶解坏死(necrosis)和凋亡(apoptosis)。结论:AIMV可替代CM用于A-NK细胞的培养。联合应用IL-12和IL-2激活A-NK细胞优于单独使用IL-2,可减轻高剂量IL-2带来的副作用。此研究为A-NK细胞的实验室研究和将来临床应用推广奠定了新的实验基础。     

无血清培养基SYL-SF对间充质干细胞的培养和扩增作用

目的探讨新型无血清培养基SYL-SF对脐带间充质干细胞的体外培养和扩增作用。方法机械分离方法获得脐带间充质干细胞,从P1代开始将含小牛血清的原代培养基SYL-NBCS更换为实验组SYL-SF和对照组MSCM-SF两种无血清培养基对细胞进行培养、传代、扩增和维持。利用Alexa Fluor 488 annexin V检测细胞凋亡率,CCK-8法比较传代细胞的增殖效率,并对细胞进行形态、表型、染色体核型和分化能力等方面的检测和评价。结果分离得到的单个脐带间充质干细胞在原代培养基SYL-NBCS中培养8~15d可获得原代(P0)细胞,更换为无血清培养基后,SYL-SF组细胞凋亡率明显低于MSCM-SF组;在细胞传代过程中SYL-SF组细胞增殖能力较强且维持MSCs特异性表面标志和向脂肪、骨、软骨等多向分化的潜能,并且在传代过程中维持正常染色体核型。结论 SYL-SF无血清培养基可以对UC-MSCs进行良好地体外扩增和维持。     

两种可注射Pluronic F-127水凝胶复合物修复兔软骨缺损的效果差异

文题释义：Pluronic F-127：是一种聚氧化乙烯-聚氧化丙烯-聚氧化乙烯三嵌段共聚物,具有在低温下为液态、常温下为固态的特性,为温固化水凝胶,具有良好的生物相容性与生物可降解性。作为组织工程中的细胞支架,Pluronic F-127已被广泛应用于软骨与皮肤等的构建。 SOX9基因：在性别决定与分化过程中具有重要的作用,在胚胎发育过程中参与骨的形成,同时其也参与神经系统与胰腺的发育及肿瘤的发生。在骨骼形成过程中,SOX9通过与 DNA 特定区域结合促进间充质细胞的聚集：首先是在软骨前体细胞中,随后是在分化中的或成熟的前体细胞中表达,维持软骨细胞增殖,抑制其向肥大软骨细胞分化,因此SOX9在软骨形成过程中起着十分重要的作用。 背景：预实验显示,SOX9基因转染的骨髓间充质干细胞可在Pluronic F-127水凝胶内良好的生长与增殖,促进细胞外基质的分泌,增加软骨基质的表达。 目的：利用慢病毒基因诱导方式将SOX9基因转导至骨髓间充质干细胞中,将其与可注射Pluronic F-127水凝胶复合,观察Pluronic F-127水凝胶复合物修复软骨缺损的效果。 方法：利用慢病毒基因诱导方式将SOX9基因转导至骨髓间充质干细胞中,转染48 h后与Pluronic F-127水凝胶复合。取60只新西兰大白兔(武汉科技大学实验动物中心提供),建立右侧膝关节股骨髁软骨缺损模型,随机分3组处理：模型组缺损部位未植入任何材料,对照组植入未转染的骨髓间充质干细胞与Pluronic F-127水凝胶复合物,实验组植入SOX9基因转染的骨髓间充质干细胞与Pluronic F-127水凝胶复合物。术后4,12周取缺损部位组织,分别进行Micro-CT三维重建、苏木精-伊红染色、番红O染色、Ⅱ型胶原免疫组织化学染色与Wakitani软组织损伤修复组织学评分。实验获得武汉科技大学伦理委员会批准。 结果与结论：①术后12周Micro-CT三维重建显示,模型组缺损区域未见明显的修复,中央仍有较大的凹陷;对照组可见明显的修复,中央凹陷区域明显减小,可见较多的再生骨小梁结构;实验组缺损部位基本完成修复;②术后12周苏木精-伊红染色显示,模型组缺损区仍未见骨小梁结构,细胞分布紊乱,未见软骨陷窝;对照组可见较多的骨组织重建,缺损区域主要由软骨样组织与纤维组织填充;实验组骨组织重建较充分,缺损区域主要由软骨样细胞与软骨样细胞外基质填充,细胞呈柱状排列,与周围软骨相似;③术后12周番红O染色与Ⅱ型胶原免疫组织化学染色显示,模型组可见少量糖胺多糖表达,未见Ⅱ型胶原表达;对照组可见较多的糖胺多糖与Ⅱ型胶原表达,实验组糖胺多糖与Ⅱ型胶原表达最多;④实验组Wakitani软组织损伤修复组织学评分高于对照组与模型组(P < 0.05);⑤结果表明,负载SOX9基因转染骨髓间充质干细胞的Pluronic F-127水凝胶复合物可促进软骨缺损的修复。 ORCID: 0000-0002-9648-5297(樊薰勤) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

缺氧和无血清培养对骨髓间充质干细胞内源性硫化氢生成的影响

背景：大鼠骨髓间充干细胞移植后的低存活率多与缺血微环境相关,而硫化氢可以对抗多种细胞与组织的凋亡和损伤模型。 目的：检测大鼠骨髓间充质干细胞在不同缺氧和无血清培养时间后的细胞凋亡、细胞活力、硫化氢含量及其合成酶体系的变化情况。 方法：取第3代大鼠骨髓间充质干细胞,设立5个(0,3,6,12和24 h)不同的缺氧和无血清培养时间点。用SubG1法检测细胞的凋亡率,用CCK-8法检测细胞的活力,并检测细胞培养基中硫化氢的含量以及细胞中硫化氢合成酶体系的表达变化情况。 结果与结论：与正常培养组相比,缺氧和无血清培养不同时间后,细胞的凋亡率显著增高,细胞活力明显下降。缺氧和无血清时间越长,细胞凋亡越多,活力越低,并且细胞培养基中硫化氢含量和细胞中硫化氢合成酶体系的表达也越低,差异有显著性意义。表明缺氧和无血清培养可以抑制大鼠骨髓间充质干细胞中硫化氢及其合成酶体系的生成和表达。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Elimination of allogeneic multipotent stromal cells by host macrophages in different models of regeneration

Allogeneic multipotent stromal cells were previously thought to be poorly recognized by host immune system; the prolonged survival in host environments was explained by their immune privileged status. As long as the concept is currently reconsidered, the routes of elimination of allogeneic multipotent stromal cells by host immunity must be taken into account. This is necessary for correct comprehension of their therapeutic action. The study was focused upon survival of umbilical cord-derived allogeneic multipotent stromal cells in different rat models of tissue regeneration induced by partial hepatectomy or by critical limb ischemia. The observations were carried out by means of vital labeling of the cells with PKH26 prior to injection, in combination with differential immunostaining of host macrophages with anti-CD68 antibody. According to the results, allogeneic multipotent stromal cells are specifically eliminated by host immune system; the efficacy can reach 100%. Massive clearance of transplanted cells by host macrophages is accompanied by appropriation of the label by the latter, and this is a pronounced case of misleading presentation of exogenous label by host cells. The study emphasizes the role of macrophages in host response and also the need of additional criteria for correct data interpretation.     

An improved procedure for the development of human mast cells from dispersed fetal liver cells in serum-free culture medium

The in vitro development of human mast cells from fetal liver cells with recombinant human stem cell factor in serum-containing RPMI was compared to that in AIM-V media with and without serum. Compared to serum-containing media, AIM-V medium caused mast cells to develop earlier and in greater numbers. By 2 weeks, about 60% of cells in serum-free AIM-V medium were phenotypic mast cells, 2 times the percentages in serum-containing media. By 6 weeks the percentages of mast cells were ≥80% under all conditions, but the number of mast cells was 3–4-fold greater in serum-free AIM-V medium than in serum-supplemented media. Mast cells obtained in serum-free AIM-V medium exhibited rounded nuclei, like tissue-derived mast cells; mast cells obtained in serum-supplemented media had segmented nuclei. By 10–12 weeks of culture about 40% of the AIM-V-derived cells showed strong chymase immunocytochemical staining, a pattern observed for only 14% of the cells in serum-containing media. AIM-V medium is a suitable medium for the development of human mast cells in vitro, and permits an earlier, more selective and greater expansion of mast cells than serum-containing media.     

Comparative intermolecular cross-relaxation studies of human hemoglobin in red blood cells and bovine serum albumin in solution

Intermolecular cross‐relaxation rate (CR) spectra [1/T_IS(HDO) or 1/T_IS(H₂O) vs f₂(ppm) profiles] for bovine serum albumin [BSA; molecular weight (MW), 66 kDa] solution, partially hydrolyzed BSA gel (BSA*gel) and packed human red blood cells (RBCs) with normal or unstable hemoglobin (Hb; MW, 65 kDa) were studied using f₂ irradiation ranging from – 100 to 100 ppm at γH₂/2π of 250 Hz. The CR spectra for BSA*gel (pD 4.01, 0.10 M NaCl, 4.83 and 14.39%) exhibited different features in the off‐resonance region (below – 2.00 and above 12.0 ppm) relative to that for BSA solution (pD 7.14, 0.10 M NaCl, 14.39%), indicating the association of BSA* molecules in the gel state. The CR spectrum for packed RBCs was compared with those for BSA*gel and BSA solution (14.39%) by correcting for differences in protein concentration. The corrected CR spectrum for packed normal RBCs in the off‐resonance region was similar to that for BSA solution, indicating that the physical characteristics of Hb in normal RBCs may be in a solution‐like state. Our results on normal RBCs were approximately consistent with the previously reported thermodynamic and hydrodynamic findings that Hb in RBCs and/or in concentrated solution seems to be in a suspension of hard scaled particles. Copyright © 2011 John Wiley & Sons, Ltd.     

A functional drug delivery platform for targeting and imaging cancer cells based on Pluronic F127

Functional polymeric micelles play an important role in the efficient delivery of therapeutic drugs into tumours. In this study, a functional drug delivery platform with ligands for targeting and fluorescent imaging was designed based on Pluronic F127 (PF127). Using folic acid (FA) and fluorescein isothiocyanate (FITC) to chemically conjugate with PF127, two functional polymers, Pluronic F127-FA (PF127-FA) and Pluronic F127-FITC (PF127-FITC), were synthesized. Solasodine-loaded micelles were then prepared via the thin-film hydration method. By employing A549 and HeLa cells, the results of in vitro cell assays performed using confocal laser scanning microscopy and flow cytometry suggested that the proposed micelles could provide the desired specific targeting and fluorescent imaging functions. In addition, the results of in vitro cytotoxicity experiments showed that the growth inhibition rates of A549 and HeLa cells treated with solasodine-loaded micelles were remarkably higher than those of cells treated with free solasodine. Solasodine-loaded micelles exhibited a more distinct inhibitory effect against HeLa cells than against A549 cells. Thus, an effective drug delivery system for targeting and imaging cancer cells was developed.     

Medium-associated luteinization expressed as progesterone release in granulosa-luteal cells isolated from patients undergoing in-vitro fertilization.

The present study describes the effect of culture medium components on progesterone release from human granulosa-luteal cells isolated from patients undergoing in-vitro fertilization (IVF). Progesterone release was selectively measured as a central parameter of in-vitro luteinization, a process believed to decrease the success rate of IVF treatments. Ten different media of relevance to embryo culture were investigated for their effect on progesterone release in unstimulated granulosa cell cultures and in cultures stimulated with human chorionic gonadotrophin (HCG) (1 IU/ml) during 4 days in vitro. Culture media supplemented with human serum yielded the greatest secretion of progesterone. Supplementation with fetal calf serum caused an intermediate pattern of progesterone release. Substitution of serum with a synthetic replacement (Medi-CultR SSR 1 and 2), lacking hormones, cholesterol and growth factors, led to a minimal output of progesterone from granulosa-luteal cells. Complex media (RPMI 1640 and Ham's F10) generally caused a greater progesterone release than simple salt solution (EBSS). No effect of insulin was detected when added to serum-free media.     

Dendritic cells as accessory cells in antigen-specific murine T lymphocyte proliferation in vitro

The role of dendritic cells in antigen-induced murine T lymphocyte activation was studied by addition of purified dendritic cells to purified lymph node T lymphocytes from ovalbumin-primed mice. In the presence of the priming antigen T cells generated an antigen-specific response. The response was at least 3-fold higher with the use of a modified IMDM culture medium. The complete requirement for accessory cells was demonstrated only when nylon wool-purified T lymphocytes were thoroughly depleted of Ia antigen-expressing cells. Dendritic cells as well as peritoneal exudate macrophages were equally effective as antigen-presenting cells.     

Cytoskeletal role in differential adhesion patterns of normal fibroblasts and breast cancer cells inside silicon microenvironments

In this paper we studied differential adhesion of normal human fibroblast cells and human breast cancer cells to three dimensional (3-D) isotropic silicon microstructures and investigated whether cell cytoskeleton in healthy and diseased state results in differential adhesion. The 3-D silicon microstructures were formed by a single-mask single-isotropic-etch process. The interaction of these two cell lines with the presented microstructures was studied under static cell culture conditions. The results show that there is not a significant elongation of both cell types attached inside etched microstructures compared to flat surfaces. With respect to adhesion, the cancer cells adopt the curved shape of 3-D microenvironments while fibroblasts stretch to avoid the curved sidewalls. Treatment of fibroblast cells with cytochalasin D changed their adhesion, spreading and morphology and caused them act similar to cancer cells inside the 3-D microstructures. Statistical analysis confirmed that there is a significant alteration (P < 0.001) in fibroblast cell morphology and adhesion property after adding cytochalasin D. Adding cytochalasin D to cancer cells made these cells more rounded while there was not a significant alteration in their adhesion properties. The distinct geometry-dependent cell–surface interactions of fibroblasts and breast cancer cells are attributed to their different cytoskeletal structure; fibroblasts have an organized cytoskeletal structure and less deformable while cancer cells deform easily due to their impaired cytoskeleton. These 3-D silicon microstructures can be used as a tool to investigate cellular activities in a 3-D architecture and compare cytoskeletal properties of various cell lines.

Dendritic cells as vectors for vaccination against infectious diseases.

Antigen presentation by dendritic cells (DCs) is critical for the induction of a specific immune response. The immunotherapeutic potential of antigen-pulsed DCs for the treatment of cancer has been confirmed in a number of experimental tumor models and in several preclinical trials. Recent advances in our understanding of the interaction of microbial pathogens with DCs have provided the basis to explore DCs as vaccine carriers for the induction of protective immune responses to infections. Support for this strategy comes from animal studies demonstrating that DCs, after ex vivo loading with microbial antigens, confer protection against microbial challenges in vivo. This may have important implications for the development of novel strategies for prophylactic or therapeutic immunizations against various microbial pathogens.     

脐带间充质干细胞在不同培养体系条件下的生物学特性

背景：体外培养的脐带间充质干细胞在不同培养体系下生长状态差异显著,因此选取一种更适合的培养基相当必要。 目的：对比观察人脐带间充质干细胞在3种培养基中的生长增殖情况,并检测细胞免疫表型以及间充质干细胞的诱导分化能力。 方法：在无菌条件下用贴块法收获人脐带间充质干细胞,用T75培养瓶培养传代后,取第3代脐带间充质干细胞分别种入到含体积分数为5%胎牛血清的DMEM/F12培养基、含体积分数为10%胎牛血清的DMEM/F12培养基和Mesen PRO RSTM培养基中,培养第1,3,5,7天进行细胞计数,绘制细胞生长曲线。采用流式细胞仪分析第3代脐带间充质干细胞免疫表型,并检测其成骨及成脂肪诱导分化能力。 结果与结论：培养出的第3代人脐带间充质干细胞高表达CD44、CD73、CD90、CD105,不表达CD29、CD31、CD34、HLA-DR。经成脂诱导后,油红O染色可见胞浆中有大量红色小脂滴;成骨诱导后,茜素红染色后镜下可见成骨样细胞团,说明脐带间充质干细胞在体外具有多向分化的潜能。在倒置显微镜下观察可见含体积分数为10%胎牛血清的DMEM/F12培养基中的细胞集落密集,形态均匀,而其他2种培养基中的细胞集落密集程度和形态都不如前者好。在培养传代细胞时,可优先选择含体积分数为10%胎牛血清的DMEM/F12培养基。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

A gamma-herpesvirus deficient in replication establishes chronic infection in vivo and is impervious to restriction by adaptive immune cells

Chronic gamma-herpesvirus infection is a dynamic process involving latent infection, reactivation from latency, and low level persistent replication. The gamma-herpesviruses maintain latent infection in restricted subsets of hematopoietic cells as a result of an intricate balance between host factors that suppress infection and viral factors that facilitate evasion of the immune response. Immune effectors limit reactivation and subsequent replication events, and the adaptive immune response ultimately restricts infection to a level compatible with life-long infection. However, it has not been possible to determine whether the immune system constrains chronic infection by directly targeting latently infected cells in vivo due to the complex nature of chronic infection. To begin to address this issue, we generated a murine gamma-herpesvirus 68 (gammaHV68) deficient in its ability to replicate or undergo reactivation from latency via a mutation in the single-stranded DNA binding protein encoded by ORF6. Even in the absence of lytic replication, this virus established long-term infection in peritoneal cells of wild-type mice at levels identical to that of wild-type gammaHV68, and generated an immune response that was sufficient to protect against secondary challenge with wild-type gammaHV68. Nevertheless, the number of latently infected cells was not significantly altered in mice deficient in T cells or both T cells and B cells, demonstrating that the adaptive immune system is incapable of altering infection with a virus lacking the capacity for lytic replication and reactivation from latency. Thus, these data support the conclusion that latency is immunologically silent.     

转基因兔骨髓间充质干细胞对异体跟腱重建前交叉韧带生物力学特性的影响

目的分别观察转PDGF-BB、VEGF165和TGFβ1基因的兔骨髓间充质干细胞(mesen chymal stem cells,MSCs)施加于异体跟腱重建兔前交叉韧带(anterior cruciate ligament,ACL)的影响,探讨改善和加强异体移植物重建ACL的途径。方法40只成年新西兰大白兔随机分为5组,每组8只。选用经Gamma射线照射,-80℃储存的同种异体跟腱进行前交叉韧带的重建。1组为对照组,2组为细胞组(MSCs),3组为经逆转录病毒转染稳定表达PDGF-BB的MSCs(PDGF-BB组),4组为经腺病毒转染稳定表达VEGF165的MSCs(VEGF165组),5组为经腺病毒转染稳定表达TGFβ1的MSCs(TGFβ1组)。各组细胞混合l00μL纤维蛋白胶粘附于重建韧带表面,术后12w取材。结果组织学观察发现,VEGF165组新生血管和成纤维细胞浸润高于其他各组。生物力学测试证明,PDGF-BB组和TGFβ1组移植物的最大载荷刚度、吸收能和最大拉张强度明显较对照组增强(P<0.05),细胞组和VEGF165组最大载荷、刚度和吸收能虽高于对照组,但无显著差异(P>0.05),VEGF165组拉断位移大于对照组(P<0.05)。所有实验组移植物、正常ACL和跟腱都在体部断裂。结论转VEGF165基因的MSCs可增加重建韧带的血管,但对生物力学功能无改善;转PDGF-BB基因和TGFβ1基因的MSCs有加速重建韧带成熟的作用。     

Lymphoid and non-lymphoid cells in the adenoid of children with otitis media with effusion: a comparative study

We characterized on immuno- and enzymecytochemical level the lymphoid and non-lymphoid cells in the adenoid of children with upper respiratory tract infections (URI) and otitis media with effusion (OME) and compared these with the adenoid of children with URI without OME and with the adenoid of 'healthy' children and adults. Besides macrophages and dendritic cells we also showed the presence of MHC class II positive, ciliated, epithelial cells. These non-lymphoid cells were present in all adenoids. However, their number was less than 1% of all cells. We found no difference in lymphocyte subsets from children with URI + OME compared with those from children with URI alone. These two groups showed a significant decrease of CD8-positive (suppressor/cytotoxic) cells and a slight increase in CD22-positive B cells in comparison to 'healthy' children. No difference was found in percentages of CD4-positive (helper/inducer) cells. The localization of the lymphoid subsets in adenoids of children with URI and/or OME did not differ from those of 'healthy' children and adults.