狼疮肾炎患者外周血单个核细胞CD134表达的动态变化及意义

目的 探讨CD134共刺激分子的动态变化特点及其存很狼疮肾炎（LN）发病机制中的可能作用。方法 采用流式细胞仪检测活动期和非活动期LN患者外周血单个核细胞（PBMC），在加入抗CD3单抗、白细胞介素（IL）-2刺激培养前、后的不同时段CD134^＋细胞数的动态改变。结果 经抗CD3ε单抗／rIL-2刺激前，活动期LN患者PBMC的CD134^＋细胞百分比已明显增加，主要表现在CD4^＋T细胞亚群上，CD8^＋T细胞则不明显。在刺激活化后，相比于正常对照组，活动期和非活动期LN患者PBMC的CD134^＋细胞百分比12h已明显增加，24h达到高峰，持续到48h仍处于高水平，72～96h后开始下调；至96hi活动期组仍高于正常对照组。结论 不论是活动期或非活动期LN患者，当T细胞受刺激活化后，主要是CD4^＋T细胞表面的CD134分子处于高表达状态和持续时间延长，CD8^＋T细胞只少量且呈一过性地表达：LN患者，特别是活动期LN患者的T细胞处于异常活化状态。     

人GL50转基因细胞的构建及其生物学功能研究

目的 构建稳定表达人GL50基因的小鼠成纤维母细胞L929细胞株,探讨GL50 信号通路对T细胞的共刺激效应.方法 Trizol一步法抽提人扁桃体细胞RNA,RT-PCR法扩增出GL50基因,双酶切装入逆转录病毒载体pEGZ-Term,与辅助病毒载体脂质体法共转染包装细胞293T,用其培养上清感染L929细胞72 h,经Zeocin 筛选出稳定表达GL50分子的L929细胞株;采用3H-TdR掺入实验观察GL50信号对T 细胞体外培养的共刺激作用.结果 成功构建含GL50基因的重组逆转录病毒载体,并获得含有GL50基因的重组病毒,筛选获得能稳定高表达人GL50分子的L929转基因细胞;该转基因细胞对体外培养的T细胞具有促增殖和活化作用;同时,GL50/ICOS信号与CD28/B7 信号具有协同作用.结论 GL50/ICOS信号在机体免疫应答过程中起重要作用;GL50转基因细胞的成功构建为进一步研究GL50分子功能奠定了良好基础.     

PI3K在CD_3mAb活化T细胞信号转导中的作用

目的探讨磷脂酰肌酶-3激酶（PI3K）在CD3mAb活化T细胞信号转导途径中的作用。方法分离获取健康人外周血单个核细胞（PBMC）,用不同浓度的PI3K特异性抑制剂LY294002处理后再用CD3mAb活化T细胞。0、6、12、24、48、72h后检测总T细胞CD69分子的表达及IL-2表达情况,培养10d后计数总T细胞的增殖情况。结果LY294002呈浓度依赖性地抑制总T细胞CD69的表达、IL-2的产生和T细胞增殖。结论PI3K参与CD3mAb诱导T淋巴细胞活化的信号转导途径,对T细胞的充分活化必不可少。     

CD69与支气管哮喘

CD69是一种细胞早期活化标记物，以磷酸化的形式在细胞表面表达。CD69基因属于NK细胞信号传导基因复合体家族。哮喘患者气道中T细胞有CD69的表达，它在T细胞激活中起着共刺激作用，可促进T细胞分泌细胞因子而进一步活化与增殖；在细胞激活后，CD69通过对TGF-β生成与分泌的调节来激发T细胞的凋亡。嗜酸粒细胞（EOS）向气道迁移过程中在各种细胞及细胞因子作用下表达CD69，而应用抗CD69单抗进行抗体交联可诱导EOS的凋亡。CD69的诱导活化细胞凋亡功能为哮喘的治疗提供了新的思路。     

CD137和CD28分子对衰老T细胞活化的调节作用

目的观察两种共刺激分子CD137及CD28对D-半乳糖致亚急性衰老模型小鼠及自然衰老小鼠T细胞活化后的细胞增殖、白介素2(IL-2)分泌及细胞存活的影响。方法7周龄BALB／c雄性小鼠随机分为D-半乳糖致亚急性衰老模型组(衰老模型组)、对照组和青年组,衰老模型组小鼠每日背部皮下注射D-半乳糖溶液(120 mg／kg,溶于0．1 ml蒸馏水中),连续注射5个月,建立亚急性衰老小鼠模型;对照组小鼠每日背部皮下注射0．1 ml蒸馏水,连续注射5个月;青年组小鼠未行处理。自然衰老组为16月龄BALB／c雄性小鼠。分离各组小鼠的脾脏T细胞,分别用ConA+ IgG、ConA+CD137单抗、ConA+CD28单抗体外活化,测定T细胞的增殖水平、培养上清液中IL-2的浓度及细胞凋亡率。结果(1)衰老模型组T细胞经ConA+IgG活化后,细胞增殖、IL-2的分泌水平(A值)及细胞存活率分别为0．422±0．057、0．632±0．066及68．0％±2．4％,与自然衰老组T细胞经ConA+IgG活化后的相应指标的差异无统计学意义,但两组衰老T细胞的活化指标均显著低于ConA+IgG活化的青年组及对照组;(2)衰老模型组T细胞应用ConA+CD137单抗活化后,细胞增殖、IL-2的分泌水平分别为0．639±0．053、1．119±0．035,显著高于ConA+IgG活化组,细胞存活率为53．3％±2．4％,显著低于ConA+IgG活化组,自然衰老组也显示同样的结果;并且发现CD137单抗对衰老T细胞功能的调节作用弱于CD28单抗。结论(1)衰老模型组与自然衰老组的T细胞功能均降低,呈现相似的增龄性变化;(2)CD137及CD28分子对两组衰老T细胞有近似的调节作用,均能促进衰老T细胞的活化及存活,CD28分子的调节作用强于CD137分子。     

急性冠状动脉综合征患者外周血CD4~+及CD4~+ CD28~(null) T细胞活化后IKCa1通道的表达及意义

目的:研究急性冠状动脉综合征(ACS)患者外周血中CD4+T细胞及CD4+CD28null亚型活化前后IKCa1钾通道数目的变化以及IKCa1钾通道阻滞剂对活化CD4+T细胞效应分子表达的影响,探讨IKCa1钾通道对不稳定斑块的意义.方法:用免疫磁珠法分离出24例ACS患者外周血中的CD4+T细胞,其中12例进一步分出亚型CD4+CD28nullT细胞,采用全细胞膜片钳技术记录细胞活化前及活化3 d后的IKCa1钾电流.CD4+T细胞活化3 d后分别加入终浓度为0 2、1、5 μmol/L特异性IKCa1钾通道阻滞剂TRAM-34,再继续活化3 d,用反转录-PCR法检测干扰素-γ及颗粒酶B mRNA的表达.结果:活化后CD4+、CD4+CD28nullT细胞的IKCa1通道数目分别增加了9倍和8倍[活化前后2种细胞的通道数分别为(45±3)∶(439±33), (56±4)∶(497±45),均P<0 01].2种细胞的通道密度也分别增加了约3倍(P<0 01).活化前及活化后2种细胞的通道数目及通道密度均无差别.不同浓度的TRAM-34均下调CD4+T细胞活化后干扰素-γ、颗粒酶B mRNA的表达,各浓度组间干扰素-γ、颗粒酶B mRNA的表达差异均有统计学意义(P<0 01),浓度越高,各mRNA表达越低.结论:ACS患者外周血CD4+及CD4+CD28nullT细胞活化后IKCa1的通道数目及通道密度均明显增加,特异性IKCa1通道阻滞剂TRAM-34呈浓度依赖性地抑制CD4+T细胞活化时干扰素-γ及颗粒酶B mRNA的表达,提示CD4+T细胞的IKCa1钾通道可作为预防动脉粥样斑块不稳定的潜在治疗靶点.     

Graves病甲状腺共刺激分子GL50-ICOS的表达及其免疫病理意义

目的 研究共刺激分子GL50-ICOS在Graves病(GD)甲状腺组织中的表达及其免疫病理意义.方法运用细胞培养、流式细胞术、RT-PCR、Western印迹和免疫组化技术,检测GD甲状腺组织和原代培养的甲状腺滤泡细胞(TFC)共刺激分子GL50和ICOS的表达.结果 (1)与正常同龄对照相比,CD4+CD28-T细胞群体在GD患者外周血中显著升高,其表面ICOS表达上调.(2)RT-PCR显示,GD患者甲状腺组织中有ICOS mRNA表达,与对照非毒性甲状腺肿(NTG)组相比具有统计学差异(P＜0.01).(3)Western印迹显示,GL50蛋白在10例GD患者组织中全部表达,较对照组差异有统计学意义(P＜0.01).(4)与对照甲状腺腺瘤组相比,GL50在20例GD患者组织切片中全部检出,而对照组无阳性表达(P＜0.01).(5)炎性细胞因子刺激体外原代培养的甲状腺滤泡细胞表面上调表达GL50(P＜0.05).结论共刺激分子GL50-ICOS在Graves病甲状腺组织异常表达.     

急性冠状动脉综合征患者外周血CD4+T细胞及CD28null/CD28+亚型活化后Kv1.3钾通道的表达和意义

目的 研究急性冠状动脉综合征(ACS)患者外周血中CD4+T细胞及CD28null/CD28+亚型活化前后Kv1.3钾通道数目的 变化以及Kv1.3钾通道阻滞剂对CD4+T细胞活化表达的影响,探讨Kv1.3钾通道在不稳定斑块中的意义.方法 用免疫磁珠法分离出27例ACS患者外周血中的CD4+T细胞,其中12例进一步分出亚型CD4+CD28null和CD4+CD28+T细胞,采用全细胞膜片钳技术记录细胞活化前及经CD3抗体活化72 h后的Kv1.3钾电流.CD4+T细胞活化时分别加入终浓度为0.1、1、10 nmol/L特异性Kv1.3钾通道阻滞剂rMargatoxin(rMgTX),共同培养72 h后用反转录-PER法检测干扰素-γ、肿瘤坏死因子(TNF)-α及颗粒酶B mRNA的表达.结果 活化后CD4+、CD4+CD28null、CD4+CD28+T细胞的Kv1.3钾通道的峰电流均明显增加,细胞平均通道数分别增加约90%、60%、80%[活化前后每个细胞的通道数分别为(402±88)个比(752±275)个、(553±328)个比(874±400)个、(392±133)个比(716±251)个,均P<0.05].活化前CD4+CD28nullT细胞Kv1.3钾通道的平均数目比CD4+CD28+T细胞多约40%(P<0.05),活化后两者差异无统计学意义(P=0.102).不同浓度的rMgTX均下调CD4+T细胞活化后干扰素-γ、TNF-α、颗粒酶B mRNA的表达,各浓度组间干扰素-γ、TNF-α、颗粒酶B mRNA的表达差异均有统计学意义(均P<0.01),浓度越高,各mRNA表达越低.结论 ACS患者外周血CD4+T细胞及CD28null/CD28+亚型活化后Kv1.3钾通道表达增加,特异性Kv1.3通道阻滞剂rMgTX呈浓度依赖性地抑制CD4+T细胞活化时干扰素-γ、TNF-α及颗粒酶B mRNA的表达,提示CD4+T细胞特别是CD4+CD28nullT细胞的Kv1.3钾通道可作为预防动脉粥样斑块不稳定的潜在治疗靶点.     

4-1BB/4-1BBL共刺激信号通路在T淋巴细胞活化和自身免疫性疾病中的作用

T淋巴细胞在参与机体免疫调节及免疫效应的过程中发挥了重要的作用。T细胞参与这些活动的前提是必须首先处于活化状态．而活化程度的过高或过低均可导致免疫功能紊乱。T淋巴细胞活化需要双信号，即T细胞受体(TCR)特异性识别靶细胞膜上主要组织相容性复合体(MHC)-抗原肽复合物，并通过CD3分子传递该识别信号，此为第一活化信号；T细胞表面的多种辅助分子CD2、白细胞功能相关性抗原(LFA)-1、CD28和靶细胞表面相应配体如LFA-3、细胞间黏附分子(ICAM)-1、B7分子等结合，     

共刺激分子CD28 CD152在类风湿关节炎患者T细胞亚群上表达异常的研究

目的观察共刺激分子CD28,CD152在类风湿关节炎(RA)患者的T细胞亚群上的表达异常情况,探讨RA的发病机制及治疗手段.方法用流式细胞仪采用直接免疫荧光法测定39例RA患者和20名健康对照人外周血T细胞表面标志CD3,CD4,CD8的表达情况及CD28,CD152在CD4+T和CD8+T细胞上的表达.结果 RA患者CD3+CD4+细胞较正常对照组显著增高(P＜0.01),CD3+CD8+细胞较正常对照组显著降低(P＜0.05),CD4+T细胞上CD28的表达较对照组显著降低(P＜0.05),而CD8+T细胞上CD28的表达与对照组差异无显著性(P＞0.05);CD4+T和CD8+T细胞上CD152的表达都较对照组显著增高(P＜0.01).结论在RA患者的细胞免疫活化过程中首先表现为B7/CD28信号途径占优势,T细胞被激活,激活的T细胞大量分泌CD152,它与CD28竞争结合B7分子,CD152/B7途径转而占优势,下调或终止T细胞反应.同时CD28+细胞数目的减少或功能缺陷造成RA患者外周血单个核细胞凋亡加速,是诱发RA患者的局部病理损害的原因.阻断CD152和B7的相互作用可增强特异性T细胞应答,为RA的免疫学治疗提供理论依据.     

Comparison of CD3 and CD2 activation pathways in T cells from young and elderly adults.

The ability of purified T cells to be activated by immobilized anti-CD3 and soluble anti-CD2 monoclonal antibodies (mAbs) was compared using cells from young and old donors. Purified T cells from elderly humans activated with immobilized anti-CD3 mAb incorporated less [3H]thymidine (58,780 vs 92,258 cpm; p < 0.02) into cellular DNA, and secreted less IL-2 into the culture supernatants than did T cells from young donors. In contrast, T cells activated with anti-CD2 mAbs displayed no age-related differences in proliferation or IL-2 production. Anti-CD2 stimulation resulted in equal IL-2 synthesis by cells from young and old donors that was comparable to the amount produced by cells from elderly donors stimulated with immobilized anti-CD3. Northern blot analysis of early cell cycle gene expression by anti-CD2 activated T cells demonstrated no age differences in the expression of p55 IL-2R or c-myc specific mRNA, although T cells from elderly individuals activated with immobilized anti-CD3 showed statistically significant decreases in both mRNAs. T cell receptor beta chain mRNA levels did not differ between cells from young or old donors after activation by either anti-CD3 or anti-CD2. The discordance in proliferative ability, IL-2 secretion, and specific mRNA expression between T cells from elderly donors activated through the CD3-TCR complex or by soluble anti-CD2 mAbs provides additional evidence for a multifactorial causation of age-related T cell proliferative defects, and may indicate that the difference in proliferative ability is, in part, attributable to responsiveness to secreted IL-2.     

Estrogen increases CD40 ligand expression in T cells from women with systemic lupus erythematosus.

OBJECTIVE: To examine the in vitro effects of estrogen on CD40 ligand (CD40L) expression in peripheral blood T cells isolated from patients with systemic lupus erythematosus (SLE) and normal controls. METHODS: T cells from female patients with SLE and controls were cultured in serum-free medium without and with 2-fluoroestradiol. Some T cells were activated by further culture on anti-CD3 coated plates. Calcineurin was activated in some T cells by culture in ionomycin. Cell surface CD40L was quantitated by FACS analysis. mRNA expression was measured using semiquantitative PCR. RESULTS: Lupus T cells cultured in medium containing 2-fluoroestradiol showed a significant (p = 0.04) increase in the amount of CD40L on the cell surface, but not in the number of positive cells, compared to the same T cells cultured without estradiol. Estradiol did not significantly change CD40L expression on the surface of T cells from normal women. In addition, the difference in cell surface CD40L between T cells cultured without and with estradiol was significantly greater (p = 0.048) on SLE than on normal T cells. Culture of SLE T cells in medium containing 2-fluoroestradiol followed by T cell receptor (TCR) activation for 2 h using anti-CD3 resulted in a significant (p = 0.04) estrogen dependent increase in CD40L mRNA. The estrogen dependent increases in SLE T cell CD40L mRNA and cell surface protein were blocked by the estrogen receptor antagonist ICI 182,780. SLE and normal T cells pretreated with estradiol and cultured with ionomycin for 2 h to activate calcineurin showed no significant differences in CD40L mRNA. CONCLUSION: These results suggest that estradiol, working through the estrogen receptor, stimulates the expression of CD40L in unstimulated and activated SLE T cells. Estradiol effects may be exerted on multiple regulatory steps that control CD40L expression. The estrogen dependent increase in CD40L expression could hyperstimulate SLE T cells and thereby contribute to the pathogenesis of SLE.     

Mechanisms of CD23 hyperexpression on B cells from patients with rheumatoid arthritis.

OBJECTIVE: To analyze the mechanisms involved in the characteristic hyperexpression of CD23 on peripheral blood B cells from patients with rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from patients with active disease and activated during 18 h with an anti-CD3 monoclonal antibody in the presence or absence of blocking antibodies to CD154 or CD40. PBMC were further purified by rosetting and CD23 expression was assessed on B cells by flow cytometry after double staining (CD19/CD23). Lymphocytes were also isolated from synovial fluid (SF). CD154 expression was analyzed on PB or SF CD4+ T cells after double staining (CD4/CD154) by flow cytometry at basal conditions and after different stimuli [anti-CD3 or phorbol myristic acetate (PMA) plus ionomycin]. Co-culture experiments between SF and PB cells were performed to analyze the involvement of the CD40-CD154 interaction on CD23 expression. CD154 and CD23 expression was also analyzed on synovial membrane by immunohistochemical techniques. RESULTS: A high proportion of activated CD23 B cells was detected in patients with RA. Blocking experiments with both anti-CD40 and anti-CD154 Mab showed a significant reduction in the proportion of PB B cells expressing CD23. Following activation with anti-CD3 Mab or PMA plus ionomycin, CD154 expression was mainly induced on PB CD4+ T cells. In co-culture experiments, SF T cells were more efficient than PB T cells in inducing CD40 dependent CD23 expression on PB B cells. In addition, CD4+ T cells from synovial membrane clearly expressed CD154. CONCLUSION: Our results establish a link between CD154-CD40 pathway and CD23 expression on PB B cells from patients with RA. T cells from the synovial microenvironment were active participants in this CD23 expression, presumably in the context of cell recirculation.     

Effect of 17β-oestradiol on expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar cells

Oestradiol (E(2)) alters lymphocyte functionin vitro including T cell DNA synthesis and B cell immunoglobulin production in human tonsillar, splenic and peripheral blood cells. We have investigated whether one mechanism for this effect is that E(2) modifies the expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar mononuclear cells. Without E(2), addition of PHA (1 μg ml(-1)) for 10 h increased the expression of IL-2 and IL-6 mRNA but had no significant effect on IFN-γ mRNA. In separated T cells after 24 h incubation, E(2) (7×10(-8) M: ) increased only the IFN-γ mRNA levels. However, when E(2) was present in PHA-stimulated T cell cultures, mRNA levels from all cytokines were suppressed. E(2) decreased IL-2 mRNA levels in the T cell preparation after 24 h culture. For IL-6, E(2) decreased mRNA both in mononuclear cells and T cells after 10 h incubation. For IFN-γ, E(2) decreased mRNA levels in the mononuclear cell preparation after 24 h culture. Stimulation of the T cell preparation with PHA after 24 h incubation with E(2) decreased the IFN-γ mRNA levels compared to the cultures incubated with E(2) only. One part of the action of E(2) may be through a block in the up-regulation of the mRNA of IL-2, IL-6 and IFN-γ in activated cells.     

Age-related impairment of T cell proliferative responses related to the decline of CD28+ T cell subsets

The impairment of phytohaemagglutinin-triggered lymphocyte proliferation represents a prominent immunologic abnormality in elderly individuals. To assess whether the reduced function is related to a CD28/B7 signalling deficiency, purified T lymphocytes and antigen presenting cells (APCs) were analyzed for their phenotypic profile and/or functional capacities. T cell responses to immobilized OKT3 monoclonal antibodies (mAb) or a combination of anti-CD2 mAb and phorbol esters were unaffected in old subjects when compared to the younger counterpart. In contrast, CD28 costimulation in the presence of OKT3 or anti-CD2 mAb, gave rise to significantly diminished T cell proliferative responses. These findings correlated with a marked decline of CD28(+) T cell frequency, which mainly involved the CD4(-)CD45RO(-) cell subset. The defect in CD28 expression could not be reversed by T cell stimulation, as a comparable increase in CD28 levels occurred in both ;aged' and ;young' T cells after in vitro activation. Moreover, the elderly group did not exhibit a reduction of interleukin (IL)-2 synthesis, as assessed at 24 h of culture, regardless of the stimulant used. Finally, B7.2 (CD86) expression by ;aged' CD14(+) APCs was unaffected in both resting and interferon-gamma activated cells. These results suggest that an intrinsic defect in CD28 expression might in part account for the age-related decline of T cell proliferative responses.     

Preferential expression of B7.2 (CD86), but not B7.1 (CD80), on B cells induced by CD40/CD40L interaction is essential for anti-DNA autoantibody production in patients with systemic lupus erythematosus

OBJECTIVE: B7 (CD80/CD86) molecules are over-expressed in patients with SLE. However, it is not clear whether CD80/CD86 molecules are involved in the pathogenic autoantibody production specifically or in the polyclonal antibody production in human SLE. The present study was carried out to characterize B7 molecules on B cells in autoantibody production. METHODS: Expression of costimulatory molecules was analyzed by RT-PCR and two-color immunofluorescence staining. Purified B cells were co-cultured with T cells in the presence of anti-costimulatory molecule antibody. RESULTS: Excessive expression of CD86 and CD80 molecules was evident on freshly isolated B cells in patients with SLE. Normal B cells did not express CD86 molecules spontaneously and expressed it after co-culture with activated T cells. CD86 expression on normal and SLE B cells induced by the activated T cells was inhibited by the addition of anti-CD40L into the cell culture. Furthermore, CD40L expression on T cells upon activation was enhanced in SLE patients. Anti-DNA antibody production by SLE B cells in the presence of activated T cells was markedly inhibited by anti-CD86, but not anti-CD80. Anti-CD86 treatment inhibited polyclonal Ig and anti-SS-A antibody production of SLE B cells, suggesting the preferential involvement of CD86 in polyclonal antibody production. CONCLUSION: SLET T cells express CD40L excessively, and the CD40/CD40L pathway is involved in the CD86 over-expression of SLE B cells; thus T cell abnormality is at least partially involved in B cell hyperactivity. Enhanced CD86 expression of B cells by CD40L is essential for polyclonal antibody production.     

Regulation of proopiomelanocortin messenger ribonucleic acid levels in the ovine fetal anterior pituitary in vitro

Parturition in sheep is dependent upon maturation of the fetal hypothalamo-pituitary-adrenocortical (HPA) axis. Anterior pituitary expression of the ACTH precursor, proopiomelanocortin (POMC), increases during the final days of gestation in spite of exponentially increasing fetal plasma cortisol levels. Lesion of the hypothalamic paraventricular nucleus prevents the late gestation increase in POMC mRNA. The purpose of this study was to examine glucocorticoid, corticotropin releasing factor (CRF) and arginine vasopressin (AVP) regulation of POMC mRNA levels in fetal anterior pituitary corticotropes in vitro and to address potential interactions between glucocorticoids and neuropeptides in regulating POMC. Anterior pituitaries from fetal sheep at two gestational ages (dGA; 118–125 dGA, n=9; 140–144 dGA, n=7) were enzymatically dispersed. POMC mRNA levels were determined at 24, 48 and 72 h post-dispersion. CRF, AVP and dexamethasone (DEX) regulation of POMC mRNA were determined at 24 and 72 h post-dispersion. The capacity of CRF and AVP to modulate DEX suppression of POMC mRNA levels was also examined. POMC mRNA was elevated at 24 h (P<0.01) and 48 h (P<0.05) post-dispersion compared to 0 h (immediately post-dispersion) in 140–144 dGA but not 118–125 dGA corticotropes. DEX suppressed POMC mRNA in a dose-dependent manner (when administered at 24 h post-dispersion) in the 140–144 dGA anterior pituitary cells but not 118–125 dGA anterior pituitary cells. Administration of DEX (10 nM) at 0 h prevented the increase in POMC mRNA levels observed at 24 h post dispersion in the 140–144 dGA group. Neither CRF nor AVP (administered at either 24 or 72 h post-dispersion) altered POMC mRNA levels in either 118–125 or 140–144 dGA anterior pituitary cells. Continuous exposure of anterior pituitary cells with either CRF or AVP (50 pM) through 96 h increased (P<0.05) POMC mRNA. No synergistic or additive effects were observed with CRF and AVP. Four hour pretreatment with CRF but not AVP (100 nM at 24 h post-dispersion) attenuated (P<0.05) DEX suppression of POMC mRNA levels in 140–144 dGA corticotropes. In conclusion, our results indicate that direct glucocorticoid suppression of POMC expression in fetal sheep initiates between 120 and 140 dGA, coincident with the period of gestation when fetal plasma cortisol is exponentially rising. Further, while short duration exposure of fetal corticotropes to either CRF or AVP had no effect on POMC mRNA, CRF appears capable of interfering with glucocorticoid suppression of POMC mRNA. The latter observation provides a potential mechanism via which the fetal PVN may counter rising fetal plasma cortisol concentrations resulting in the previously observed late gestation increase in anterior pituitary POMC mRNA.     

Age-related impairment of T cell proliferative responses related to the decline of CD28+ T cell subsets

The impairment of phytohaemagglutinin-triggered lymphocyte proliferation represents a prominent immunologic abnormality in elderly individuals. To assess whether the reduced function is related to a CD28/B7 signalling deficiency, purified T lymphocytes and antigen presenting cells (APCs) were analyzed for their phenotypic profile and/or functional capacities. T cell responses to immobilized OKT3 monoclonal antibodies (mAb) or a combination of anti-CD2 mAb and phorbol esters were unaffected in old subjects when compared to the younger counterpart. In contrast, CD28 costimulation in the presence of OKT3 or anti-CD2 mAb, gave rise to significantly diminished T cell proliferative responses. These findings correlated with a marked decline of CD28⁺ T cell frequency, which mainly involved the CD4⁻CD45RO⁻ cell subset. The defect in CD28 expression could not be reversed by T cell stimulation, as a comparable increase in CD28 levels occurred in both `aged' and `young' T cells after in vitro activation. Moreover, the elderly group did not exhibit a reduction of interleukin (IL)-2 synthesis, as assessed at 24 h of culture, regardless of the stimulant used. Finally, B7.2 (CD86) expression by `aged' CD14⁺ APCs was unaffected in both resting and interferon-γ activated cells. These results suggest that an intrinsic defect in CD28 expression might in part account for the age-related decline of T cell proliferative responses.