In Vitro Generation of Functional Dendritic Cells from Human Umbilical Cord Blood CD34<Superscript>+</Superscript> Cells by a 2-Step Culture Method

Dendritic cells (DCs) are the most potent antigen-presenting cells in terms of initiating primary T-cell-dependent immune responses. We devised a 2-step culture method for obtaining sufficient numbers of functional DCs from umbilical cord blood (CB) CD34+ cells. In the first step, CB CD34+ cells were expanded by stimulation with early-acting cytokines such as stem cell factor (SCF), flt3 ligand (FL), and thrombopoietin (TPO) to amplify the hematopoietic progenitor cells. In the second step, granulocyte-macrophage colony-stimulating factor and interleukin 4 were added, and incubation was continued for another 5 days to induce differentiation of the expanded cells into DCs. During the first step of culturing with TPO, SCF, and FL, the total numbers of nucleated cells gradually increased, peaking at 4 weeks (245.3-fold). During the second step, expression of CD1a, CD83, and CD86 increased. Electron microscopic findings showed that these cells had cytosolic expansion to form dendrites and major histocompatibility complex class II compartments, which are characteristic of DCs. Functional analyses revealed that these cells had phagocytic activity and were capable of stimulating allogeneic T-cells in vitro.     

慢性心力衰竭患者CD4+CD25+调节性T细胞检测及意义

目的:探讨慢性心力衰竭(CHF)患者外周血CD4 CD25 调节性T细胞(Treg)水平及意义。方法:采用流式细胞分析法,检测42例CHF患者(CHF组)和16例正常对照者(对照组)外周血CD4 CD25 Treg/CD4 T细胞比例。结果:CHF患者外周血CD4 CD25 Treg/CD4 T细胞比例[(12.2±3.8)%]显著低于对照组[(16.3±5.2)%]。CD4 CD25 Treg/CD4 T比例在缺血性心脏病者[(12.6±4.1)%]与非缺血性心脏病者(12.0±3.7%)间差异无统计学意义,但心功能NYHA分级Ⅲ~Ⅳ级者[(10.4±3.2)%]比例明显低于Ⅰ~Ⅱ级者[(13.3±3·8)%]。结论:CHF患者外周血Treg比例减少,且与心功能有一定关系。CD4 CD25 Treg比例降低可能打破了外周免疫耐受,参与了心力衰竭的发生发展。     

Pleural tuberculosis in Harare, Zimbabwe: the relationship between human immunodeficiency virus, CD4 lymphocyte count, granuloma formation and disseminated disease

objective   To elucidate the relationship between HIV, CD4¹ count and pleural TB. method   In a prospective study, 94 patients presenting at two large Harare hospitals with clinically suspected pleural TB were enrolled over a 10-month period. All underwent standardized evaluation, closed pleural aspiration and biopsy. Patients receiving directly observed anti-TB therapy were followed-up. results   Pleural TB was diagnosed in 90 individuals (median age 33 years; range 18-65; 64 males); the seroprevalence of HIV was 85%. HIV-positive patients were older than HIV-negative individuals (median age 33 vs 23 years, P = 0.013) and had a significantly lower median CD4¹ count (191 vs 1106 × 10⁶/l respectively, P = 0.004). A CD4¹ count of <200 × 10⁶/l was associated with a length of illness >30 days (65% vs 37%; P = 0.05), a positive pleural fluid smear (37% vs 0%; P = 0.0006) and a positive pleural biopsy Ziehl-Neelsen stain (35% vs 7%; P = 0.021). However, a relationship between CD4¹ count and either pleural granuloma formation or radiological evidence of disseminated disease was not observed. conclusion   In sub-Saharan Africa, TB pleural effusions have become associated with older age, a chronic onset, and an increased mycobacterial load. These data emphasize the complex relationship between pleural TB, HIV infection and a low CD4¹ count.     

High cyclin-dependent kinase inhibitors in Bcl-2 and Bcl-xL-expressing CD34+-proliferating haematopoietic progenitors

We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE(bright) (primitive) and CFDA-SE(dim) (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA-SE(bright)/CD34+/slow-proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA-SE(bright) (bright) cells, whereas they increased in CFDA-SE(dim) (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C-myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl-xL, which followed the trend we have previously described for Bcl-2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin-dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.     

系统性红斑狼疮患者CD5+B细胞的测定和意义

目的探讨外周血中CD5 B细胞在系统性红斑狼疮(SLE)活动中的作用及相关性。方法利用流式细胞分析法对57例SLE患者和35名正常人群外周血CD5~ B细胞进行检测,并且同时检测抗dsDNA抗体、抗核抗体(ANA)、抗心磷脂抗体(ACL)、补体C3、C4。结果SLE患者CD5~ B细胞水平[(2．1 0．4)%]与正常人[(1．5±0．4)％]比较差异有统计学意义(P＜0．05),活动期SLE患者CD5~ B细胞水平(2．5±0．5)％显著高于稳定期(1．4±0．5)％；CD5 B细胞与dsDNA、ANA、抗心磷脂抗体(ACL)升高呈正相关,与补体c3呈负相关。结论系统性红斑狼疮患者外周血CD5~ B细胞明显升高,与SLE疾病活动有一定关系。     

IL-27 impact on NK cells activity: Implication for a robust anti-tumor response in chronic lymphocytic leukemia

Interleukin 27 (IL-27) belongs to IL-12 cytokine family, has shown anti-tumor potential in several solid tumors, as well as hematologic malignancies. IL-27 can inhibit tumor growth and progression through direct and indirect mechanisms, such as inhibition of proliferation, angiogenesis, induction of apoptosis in tumor cells, and anti-tumor immune response. B-CLL is characterized by remarkable immune perturbation, which leads to disease complications and reduced effectiveness of the treatment. Natural killer cells (NK) are considered as an important arm for the elimination of transformed cells. However, NK cells have shown significant impairment in patients with CLL. Here we analyzed the activity of recombinant human (rh) IL-27-stimulated NK cells in bone marrow (BM) and peripheral blood (PB) of CLL patients using cell surface flow cytometry assessment, and cytotoxicity assay. We showed that rhIL-27 can increase CD69 on NK cells both in BM and PB. Interestingly, BM-NK cells treated with rhIL-27 exhibited a significant increase in degranulation and NK cell-mediated cytotoxicity as compared with untreated NK cells, whereas it did not improve NK cell activity of PB. These observations added further explanation to the anti-tumor activity of IL-27 and also could pave the way to adoption immunostimulatory adjuvant for therapies in CLL.     

Elevated antigen-specific Th2 type response is associated with the poor prognosis of hand,foot and mouth disease

The immunopathogenesis of severe hand, foot and mouth disease (HFMD) remains elusive. This study revealed that enterovirus 71 (EV71) epitope-specific CD4+ T cell responses of HFMD patients were skewed toward a Th2 cytokine profile. Patients that demonstrated higher levels of IL-4 expression in their CD4 T cells following antigen stimulation in vitro tended to have a more prolonged period of high fevers and a longer duration of illness. Thus, an increase of EV71 epitope-specific Th2 type response may portend the poor prognosis for some HFMD patients.