Increased IL-2, IL-4 and interferon-gamma (IFN-gamma) in steroid-sensitive nephrotic syndrome.

We investigated the production of cytokines by peripheral blood mononuclear cells (PBMC) and serum cytokine concentrations in children with steroid-sensitive idiopathic nephrotic syndrome (SSNS). PBMC from patients off treatment were collected during remission and relapse and cultured in medium alone or stimulated with calcium ionophore plus phorbol myristate acetate. Control PBMC were taken from healthy age-matched children. IL-2 was measured by bioassay, IL-4 by immunoradiometric assay, and IL-8 and IFN-gamma by ELISA. After 24 h culture without stimulation, IL-2, IL-4 and IFN-gamma were not detectable in the supernatant in any of the children. After stimulation, the supernatant concentrations of IL-2 (median 172 U/ml at 24 h) and IL-4 (160 pg/ml at 24 h; 210 pg/ml at 72 h) were significantly increased in relapse compared with remission (IL-2 37 U/ml; IL-4 65 pg/ml and 60 pg/ml) and controls (IL-2 69 U/ml; IL-4 40 pg/ml and 40 pg/ml) (P < 0.05). The concentration of IFN-gamma was not significantly increased in relapse compared with remission and controls (600, 325, and 145 U/ml, respectively, at 72 h). IL-8 concentrations were similar in relapse, remission and controls with stimulation (median 32, 40 and 40 ng/ml, respectively) and without (30, 17 and 10 ng/ml). IL-2 was not detectable in serum, but IL-4, IL-8 and IFN-gamma were measurable in about half the patients, both in relapse and remission, though were virtually undetectable in controls. We conclude that relapse of SSNS in children is associated with T lymphocyte activation with release of IL-2, IL-4 and IFN-gamma.     

Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.     

IMMUNE RESPONSES IN AUTOIMMUNE HEPATITIS: EFFECT OF PREDNISONE AND AZATHIOPRINE TREATMENT: CASE REPORT

The role of the immune response in autoimmune hepatitis has not been studied before and after prednisone and azathioprine treatment. Distributions of blood lymphocytes (CD4+, CD8+, CD19+, CD23+, CD16/56+), levels of serum immunoglobulins (IgM, IgG, IgE, IgA) and cytokines (IFN-γ, IL-4, IL-12, TNFα ) were studied in a child (f/14 y/o) with autoimmune hepatitis before and after prednisone (20 mg/d) and azathioprine (50 mg/d) treatment (nephelometry, UniCAP Total IgE Fluoroenzymeimmunoassay, flow cytometry, cytokine ELISA). Patient was studied for 0-2.5 yrs; treatment was initiated 12 weeks post diagnosis. Numbers of CD4+ T cells increased (50%), while CD19+ and CD23+ cells decreased (>50%) post treatment; other lymphocyte subsets were unaffected by treatment. Serum IgG and IgE levels decreased (>50%) after treatment; serum IgM and IgA were within normal range and were not affected by treatment High levels of IFN-γ (5-23 pg/ml) were initially detected in serum, which decreased after treatment (<0.1 pg/ml). Furthermore, low levels of IL-4 (0.2 pg/mL) were detected before treatment, which were not detected after treatment (<0.1 pg/ml). In contrast, before treatment, IL-12 and TNFα were not detected in serum; however after treatment the levels of IL-12 and TNFα dramatically increased. Prednisone and azathioprine treatment decreased total serum IgG, IgE, IFN-γ and IL-4 levels, and blood CD19+ and CD23+ cells; however serum IL-12, TNFα and blood CD4+ T cells increased with treatment. Understanding immunomodulation in autoimmune hepatitis will provide better insight and mechanisms of this disease and may tailor more effective therapeutic intervention.     

IL-23R rs11209026 polymorphism modulates IL-17A expression in patients with rheumatoid arthritis

The interleukin (IL)-17/IL-23 axis is an important pro-inflammatory pathway in rheumatoid arthritis (RA). IL-23 maintains CD4(+) T-helper 17 (Th(17)) cells, whereas IL-12 negates IL-17A production by promoting Th(1)-cell differentiation. We sought evidence for any effect of polymorphisms within the interleukin-23 receptor (IL-23R), IL-12 or IL-21 genes on serum cytokine concentrations in 81 patients with RA. Serum cytokines were measured using bead-based multiplex assays. Targeted cytokines were detected in up to 66% of samples. A subgroup of 48 patients had detectable serum IL-17A. Within this subgroup, patients, homozygous for the IL-23R rs11209026 major allele had significantly higher serum IL-17A concentrations compared with patients with the minor allele (394.51 ± 529.72 pg ml(-1) vs 176.11 ± 277.32 pg ml(-1); P = 0.017). There was no significant difference in any of the cytokine concentrations examined in patients positive for the minor allele vs homozygosity for the major allele of IL-12B rs3213337, IL-12Bpro rs17860508 and IL-21 rs6822844. Our results suggest the IL-23R Arg381Gln substitution may influence serum IL-17A concentrations. In patients with the 381Gln allele higher IL-23 concentrations may be needed to produce similar IL-17A concentrations to those in patients with the 381Arg allele. This suggests altered IL-23R function in patients with the minor allele and warrants further functional studies.     

polyethylene glycol-conjugated adenosine deaminase (ADA) therapy provides temporary immune reconstitution to a child with delayed-onset ADA deficiency

We describe the effects of polyethylene glycol-conjugated adenosine deaminase (ADA) replacement therapy on lymphocyte counts, activation, apoptosis, proliferation, and cytokine secretion in a 14-month-old girl with "delayed-onset" ADA deficiency and marked immunodysregulation. Pretreatment lymphopenia affected T cells (CD4, 150/microl; CD8, 459/microl), B cells (16/microl), and NK cells (55/microl). T cells were uniformly activated and largely apoptotic (CD4, 59%; CD8, 82%); and T-cell-dependent cytokine levels in plasma were elevated, including the levels of interleukin 2 (IL-2; 26 pg/ml), IL-4 (81 pg/ml), IL-5 (46 pg/ml), gamma interferon (1,430 pg/ml), tumor necrosis factor alpha (210 pg/ml), and IL-10 (168 pg/ml). Mitogen-stimulated peripheral blood mononuclear cells show reduced IL-2 secretion and proliferation. During the first 5 months of therapy there was clinical improvement and partial immune reconstitution, with nearly normal lymphocyte subset numbers, reduced T-cell activation and CD4-cell apoptosis, and decreased plasma cytokine levels. In parallel, IL-2 secretion and the lymphocyte mitogenic response improved. Between 4 and 7 months, immunoglobulin G antibodies to bovine ADA developed and resulted in the complete reversal of immune recovery.     

卵巢癌患者外周血CD4+CD25hiCD127lo调节性T细胞格局变化及临床意义

目的:观察卵巢癌患者外周血中CD4+CD25hiCD127lo调节性T细胞格局变化及其相关免疫细胞因子TGF-β1、IL-10的变化及与临床病理特征之间的关系,探讨其临床意义.方法:采用流式细胞术(FCM)和酶联免疫吸附法(ELISA)检测70例卵巢癌患者、50例卵巢良性疾患及70例健康者外周血单个细胞中调节性T细胞的比率和血浆TGF-β1、IL-10的水平.结果:①卵巢癌患者外周血中CD4+CD25hiCD127loTreg调节性T细胞占CD4+细胞的比例为6.32%±1.46%(n=70),显著高于卵巢良性疾患4.03%±1.25%(n=50)和健康对照组3.21%±0.96%(n=70),均P＜0.01.术后患者CD4+CD25hiCD127loTreg比率与术前比较无明显差异.②卵巢癌患者血浆中TGF-β1、IL-10水平(256.68±56.34) pg /ml、(28.24±3.12) ng/ml,明显高于良性疾患(156.48±43.68) pg /ml、(20.58±2.39) ng/ml与健康对照组(130.24±35.60) pg/ml、(18.38±2.98) ng/ml,有统计学差异,分别P＜0.001,P＜0.01.③术前卵巢癌患者外周血CD4+CD25hiCD127loTreg比率、血浆中TGF-β1、IL-10水平与患者的临床分期、淋巴结转移以及远处转移有关,P＜0.05～P＜0.001.④相关分析显示,卵巢癌患者外周血 CD4+CD25hiCD127loTreg比率与血浆中TGF-β1水平、IL-10水平呈正相关,r=0.734,P＜0.01;r=0.665,P＜0.01.结论:①CD4+CD25hiCD127loTreg在卵巢癌患者外周血中表达显著增高,这可能是卵巢癌患者免疫功能下降的一个重要原因,并与临床病理特征存在显著相关性;②卵巢癌患者血浆中抑制性细胞因子TGF-β1、IL-10水平明显升高,并与临床病理特征存在显著相关性;③CD4+CD25hiCD127loTreg与TGF-β1、IL-10水平存在正相关,CD4+CD25hiCD127loTreg可能通过产生抑制性细胞因子TGF-β1、IL-10 对效应性T细胞发挥抑制作用.     

骨关节炎与免疫关联性初探

为了探讨免疫因素是否介导骨关节炎的发病 ,应用直接免疫荧光法检测了 6例晚期骨关节炎患者滑膜液中浸润淋巴细胞分化抗原的表达 ,同时用微量酶联免疫技术测定了这些患者滑膜液中IL 10、IL 12和sFasL的含量。结果显示关节滑膜液中的淋巴细胞以CD8+ 细胞为主 (6 7 0 %± 14 6 %) ,远远高于CD4+ 细胞 (15 0 %± 12 8%) ,两者相差显著 (P <0 0 1)。CD4+ /CD8+ 比值 <1。进一步分析表明其T细胞受体主要为αβ型TCR (6 4 0 %± 12 6 %)。滑膜液中IL 12含量 (2 0 3 84±49 2 )pg/ml高于IL 10 (37 8± 14 5 )pg/ml,有明显统计学差异 (P <0 0 1)。外周血中IL 10和IL 12的含量分别为 (4 3 37±14 2 )pg/ml和 (4 5 5 8± 10 6 )pg/ml。血清中sFasL含量较高 (15 3 90± 5 6 )pg/ml,而滑膜液中则很低 (<5pg/ml,P <0 0 1)。提示在骨关节炎的晚期CD8+ 淋巴细胞和IL 12增高可能参与了关节的免疫损伤。     

Independent regulation of interleukin 4 (IL-4)-induced expression of human B cell surface CD23 and IgM: functional evidence for two IL-4 receptors.

Activation of human B cells with interleukin 4 (IL-4) is known to result in increased expression of CD23 (the low-affinity receptor for IgE) and sIgM. However, whereas CD23 expression is increased by several B cell mitogens, including phorbol 12-myristate 13-acetate, Epstein-Barr virus, anti-immunoglobulin (Ig), and IL-4, surface IgM (sIgM) expression is increased only with IL-4, suggesting that expression of each surface antigen is regulated independently. This was confirmed in three different ways. First, in dose-response experiments, it was shown that 10 times the concentration of IL-4 was required for CD23 than for sIgM expression. Similar or even higher concentrations of IL-4 were required for proliferation. In fact, optimal sIgM expression was obtained in some experiments with concentrations of IL-4 (1-5 units/ml) which had little or no effect on either CD23 expression or B cell proliferation. Secondly, IL-4 is known to activate the phosphatidyl inositol pathway in human B cells followed 8-10 min later by an increase in cAMP. Pharmacologically mimicking this pathway by brief exposure of resting B cells to phorbol dibutyrate plus ionomycin followed 10 min later with dibutyryl cAMP resulted in an increase in expression of CD23 but not sIgM. Thirdly, CD19 monoclonal antibody, which inhibits B cell proliferation in response to IL-4 plus anti-Ig, was found to inhibit IL-4-induced CD23 but not sIgM expression. These results show that CD23 and sIgM expression are regulated independently and are consistent with the existence of two separate signal transduction pathways stimulated by IL-4, which may be coupled to distinct IL-4 receptors.     

Polyethylene Glycol-Conjugated Adenosine Deaminase (ADA) Therapy Provides Temporary Immune Reconstitution to a Child with Delayed-Onset ADA Deficiency

We describe the effects of polyethylene glycol-conjugated adenosine deaminase (ADA) replacement therapy on lymphocyte counts, activation, apoptosis, proliferation, and cytokine secretion in a 14-month-old girl with “delayed-onset” ADA deficiency and marked immunodysregulation. Pretreatment lymphopenia affected T cells (CD4, 150/μl; CD8, 459/μl), B cells (16/μl), and NK cells (55/μl). T cells were uniformly activated and largely apoptotic (CD4, 59%; CD8, 82%); and T-cell-dependent cytokine levels in plasma were elevated, including the levels of interleukin 2 (IL-2; 26 pg/ml), IL-4 (81 pg/ml), IL-5 (46 pg/ml), gamma interferon (1,430 pg/ml), tumor necrosis factor alpha (210 pg/ml), and IL-10 (168 pg/ml). Mitogen-stimulated peripheral blood mononuclear cells show reduced IL-2 secretion and proliferation. During the first 5 months of therapy there was clinical improvement and partial immune reconstitution, with nearly normal lymphocyte subset numbers, reduced T-cell activation and CD4-cell apoptosis, and decreased plasma cytokine levels. In parallel, IL-2 secretion and the lymphocyte mitogenic response improved. Between 4 and 7 months, immunoglobulin G antibodies to bovine ADA developed and resulted in the complete reversal of immune recovery.     

类风关滑膜浸润T细胞特性与致病机制研究

为研究类风湿关节炎时关节滑膜浸润性T细胞生物学特性与致病机制 ,对 10例RA患者滑膜液中淋巴细胞的免疫表型、细胞因子分泌格局与趋化因子受体表达进行了分析。用双色荧光标记法分别测定滑膜液中和外周血淋巴细胞表型与趋化因子受体表达。用ELISA方法检测滑膜液与外周血中IFN γ、IL 10、IL 4与IL 12的含量。结果是滑膜液中的CD4 + T淋巴细胞为 4 0 0 %± 11% ,CD8+ T细胞为 34 0 %± 6 % ,CD4 + 与CD8+ T细胞的比值为 1 2 ,显著低于外周血中CD4 /CD8的比值。滑膜液中CD3和CD2 5双阳性的活化T细胞占 16 %± 6 0 %。趋化因子受体CCR5表达较低 ,与外周血无明显差异。但CX CR3表达水平较高 ,为 16 %± 4 0 % ,远远高于外周血 (仅为 0 5 %± 0 3% )。IFN γ在滑膜液中含量很高 ,达 (36 6 7± 4 3 2 )pg/ml,而外周血中含量仅为 (2 0 1± 3 2 )pg/ml。IL 4含量未能测得 (<15pg/ml ) ,与外周血相似。IL 12含量为 (4 19 9±89 2 )pg/ml,远高于外周血中的含量 (6 5 32± 34 2 )pg/ml。IL 10含量为 (187 7± 34 5 )pg/ml,高于外周血中的含量 (85±12 7)pg/ml。在所测细胞因子中 ,关节滑膜液中IFN γ和IL 12的含量与外周血相比具有显著的统计学差异。表明RA关节滑膜液中有相当数量的T细胞浸润。这些T细胞     

Detection of Tumour Necrosis Factor from Lipopolysaccharide-Stimulated Human Mononuclear Cells by Enzyme-Linked Immunosorbent Assay and Cytotoxicity Bioassay

Tumour necrosis factor (TNF) or cachectin is an important mediator of endotoxic activity. To investigate the production of TNF from human mononuclear cells (MNC) in response to lipopolysaccharide (LPS), we developed a sensitive and specific enzyme immunoassay (ELISA) and a cytotoxicity bioassay for TNF. The ELISA utilizes the biotin/avidin system and includes four incubation steps. The detection limit was 25 pg recombinant TNF (rTNF)/100 microliter. There was no interference of medium, serum, plasma, spinal fluid, or urine and no cross-reaction with natural or recombinant IL-1-alpha, IL-1-beta, IL-2, IFN-gamma, or lymphotoxin (TNF-beta). Recovery of TNF added to the media was 85-123% (n = 22). The relative standard deviations within and between assays were 7% and 8%, respectively. TNF-induced cytotoxicity was measured on actinomycin-D-treated L-M mouse fibroblasts. The detection limit in this bioassay was 0.5 U/30 microliter or 12.5 pg/30 microliter of rTNF. IL-1-alpha and IL-1-beta slightly inhibited the cytotoxic activity of rTNF. In this bioassay, cytotoxic activity (50-300 U/ml) was detected only when MNC were stimulated with high concentrations of LPS (100-1000 ng/ml). In contrast, using 0.01-100 ng/ml of LPS, the ELISA detected TNF in a dose-dependent manner (0.25 ng/ml to 40 ng/ml). It is concluded that TNF is liberated from human blood MNC if stimulated with minute amounts of LPS. It is suggested that human TNF may be secreted in a relatively inactive form or that inhibitors of TNF are generated along with the monokine. Because of this, and because commonly used bioassays for TNF fail to distinguish between TNF and lymphotoxin, specific ELISA are recommended to supplement TNF bioassays.     

Circulating antiinflammatory cytokine IL-10 in patients with inflammatory bowel disease (IBD).

IBD is characterized by increased serum concentrations of different cytokines. IL-10 inhibits the production of proinflammatory cytokines such as IL-1, tumour necrosis factor-alpha (TNF-a), interferon-gamma (IFN-gamma) and IL-6 through inhibitory action on Th1 cells and macrophages, and it is thought to be a suppressor type cytokine. In the present study we determined serum concentrations of IL-10 in patients with ulcerative colitis (UC) and Crohn's disease (CD). We measured human IL-10 by our own newly established ELISA system using PharMingen antibodies. Serum antibodies were assessed in 44 patients with UC, 40 patients with CD, and in 30 healthy controls. Human IL-10 serum levels were significantly increased in patients with active UC (144 +/- 34 pg/ml (mean +/- s.e.m.), P < 0.001) and in active CD (132 +/- 32 pg/ml, P < 0.001) compared with healthy controls (44 +/- 9.5 pg/ml). Only patients with active CD and active UC presented with significantly increased IL-10 serum levels, while patients with inactive disease did not show any significant increase. There was no statistically significant difference between IL-10 serum levels in patients with CD or UC. Compared with clinical disease activity indices there was a significant correlation between IL-10 serum concentration and CDAI in patients with CD (r = 0.45, P < 0.01) and CAI in UC patients (r = 0.39, P < 0.05). Comparing IL-10 serum levels with serum concentrations of other proinflammatory cytokines there was a significant correlation to serum levels of sIL-2R (r = 0.417, P < 0.05) and IL-6 (r = 0.387, P < 0.05) in patients with CD. Serum cytokine levels in patients with UC did not show any significant correlation to IL-10 serum concentration. IL-10 is elevated in serum of patients with active CD and UC, suggesting that IL-10 acts as a naturally occurring damper in the acute inflammatory process of IBD.     

Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel

Whereas the involvement of Th1- and Th2-type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL-10 is less clear. We therefore investigated the impact of IL-10 on Ni-induced Th1- (IFN-gamma) and Th2-type (IL-4 and IL-13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni-PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine-producing cells and the levels of secreted cytokines were analysed by ELISpot (IL-4, IL-13 and IFN-gamma) and ELISA (IL-10, IL-13 and IFN-gamma), respectively. The Ni-induced response was further assessed in the presence of recombinant IL-10 (rIL-10) or neutralizing antibody to IL-10 and the phenotype of the Ni-specific cytokine-producing cells regulated by IL-10 was determined by cell depletion experiments. Ni induced IL-10 production in Ni-PBMC (mean, (range); 33.1 pg/ml (0-93.4 pg/ml)) but not control PBMC (2.2 pg/ml (0-14.9 pg/ml)) (P = 0.002). Ni also induced significant production of IL-4, IL-13 and IFN-gamma that correlated with the IL-10 response. Addition of rIL-10 down-regulated the Ni-induced production of all cytokines but with a more pronounced effect on IFN-gamma. However, neutralization of Ni-induced IL-10 enhanced the levels of IFN-gamma induced by Ni (P = 0.004) but did not affect the number of IFN-gamma-producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni-specific IFN-gamma (and Th2-type cytokine) producing cells were CD4(+) T cells. The impact of IL-10 on Ni-induced IFN-gamma responses by CD4(+) T cells suggests that an important role of IL-10 in vivo is to counteract the allergic reactions mediated by Th1-type cytokines.     

Ovarian follicular concentration of IL-12, IL-15, IL-18 and p40 subunit of IL-12 and IL-23

BACKGROUND: The aim of the study was to determine the presence of interleukin (IL)-12, IL-15, IL-18 and p40 subunit of IL-12/IL-23 in follicular fluid from spontaneous cycles and the relation between the concentration of selected cytokines and IVF-embryo transfer outcome. METHODS: IVF-embryo transfer and enzyme immunoassay (EIA) (R&D Systems, Minneapolis, MN, USA and MBL, Nagoya, Japan) were used. RESULTS: Follicular fluid of women included in the IVF-embryo transfer procedure contained common p40 subunit of IL-12/IL-23 (median 70.1 pg/ml), IL-15 (median 1.3 pg/ml) and IL-18 (median 38.2 pg/ml). There was a significant negative correlation between follicular fluid concentrations of IL-15 and IL-18 (R=-0.392, P=0.003). Significantly higher concentrations of common p40 subunit of IL-12/IL-23 (median 79.8 pg/ml) were found in the follicular fluid taken from follicles containing oocytes, when compared with those without an oocyte (median 44.5 pg/ml, P=0.006). Patients who achieved clinical pregnancy had significantly decreased concentration of IL-15 (median 0.8 pg/ml) compared with patients without successful IVF-embryo transfer outcome (median 1.4 pg/ml, P=0.047). CONCLUSION: Follicular fluid collected from spontaneous cycles contains detectable levels of p40 subunit of IL-12/IL-23, IL-15 and IL-18. Increased concentrations of p40 subunit of IL-12/IL-23 in follicles containing oocytes suggest an important role of this cytokine in reproduction. Possible negative value of IL-15 as a predictor of IVF-embryo transfer success remains to be determined.     

当归及其多糖对隐孢子虫感染小鼠的免疫调节作用

目的 探讨当归及当归多糖对隐孢子虫感染小鼠的免疫调节作用.方法 连续8 d灌服给小鼠醋酸地赛米松后,于第9 d经口接种1×10^6个隐孢子虫卵囊1次,建立隐孢子虫感染模型,再经当归水煎剂及当归多糖灌胃治疗10 d,计数粪便隐孢子虫卵囊,观察肠组织病理学改变,检测T细胞亚群和血清IL-2、IL-4、IFN-γ水平.并设正常对照、模型对照和西药对照组.结果 与模型组比较,西药组、当归水煎剂组和当归多糖组小鼠卵囊排除数量减少（第4天分别为300个、50个、238个和230个）,肠组织病理学改变较轻,免疫学检测发现CD4^＋T细胞[分别为（38.85±8.16）%、（47.98±11.90）%、（55.85±6.63）%、（51.43±8.42）%]CD4^＋/CD8^＋[分别为1.98±0.47、2.25±0.53、2.51±0.55、2.42±0.23）]及IL-2的水平[分别为（28.75 4±1.93）pg/ml、（69.02±10.47）ps/ml、（42.91±4.48）pg/ml、（40.90±0.79）pg/ml]IL-4的水平[分别为（42.00±6.79）pg/ml、（64.26±6.07）pg/ml、（58.31±9.13）ps/ml、（54.95±8.99）pg/ml]和IFN-γ的水平[分别为（28.73±8.71）ps/ml、（45.40±6.11）pg/ml、（84.40±7.63）pg/ml、（78.40±6.32）pg/ml]明显升高（F值分别为13.58、19.37、24.22、54.36和35.74 P值均小于0.05）.结论 当归水煎剂和当归多糖通过增强机体免疫功能促进隐孢子虫感染的免疫抑制小鼠的恢复.     

T helper type 2-like cells and therapeutic effects of interferon-γ in combined immunodeficiency with hypereosinophilia (Omenn's syndrome)

We characterized the defects of CD4+ cells in a 17-month-old girl suffering from combined immunodeficiency with hypereosinophilia (Omenn's syndrome). Because the vast majority of peripheral blood CD4⁺ cells expressed the CD45R0 isoform, we purified circulating CD4⁺ CD45R0⁺ cells from the patient and healthy individuals in order to compare their production of cytokines. The patient's CD4⁺ CD45R0⁺ cells spontaneously produced high levels of interleukin-5 (IL-5) in vitro (1600 pg/ml after 24 h of culture) and this was associated with the presence of IL-5 in serum (323 pg/ml). After stimulation with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187, they produced higher levels of IL-4 (306 vs. 55 ± 4 pg/ml) and IL-5 (2900 vs. 213 ± 72 pg/ml) and lower levels of IL-2 (17 vs. 63 ± 17 IU/ml) and interferon-γ (IFN-γ) (16 vs. 299 ± 70 IU/ml) than controls CD4⁺ CD45R0⁺ cells. This T helper type 2 (Th2) pattern was confirmed by the detection using reverse polymerase chain reaction of IL-4, IL-5 and IL-10 mRNA within peripheral blood mononuclear cells. During a therapeutic trial with human IFN-γ (40 μg/day) which ameliorated the clinical status of the patient, we observed a down-regulation of the in vivo expression of IL-5 and IL-10, a normalization of the eosinophil count and an improvement of the Tcell response to phytohemagglutinin. This observation indicates for the first time that Th2-like cells might be involved in certain forms of congenital immunodeficiency and that IFN-γ might down-regulate their activities in vivo.     

脐血来源间充质干细胞可抑制异体T细胞的反应

目的 研究脐血间充质干细胞(HUCB-MSCs)对异体T细胞的抑制作用.方法 体外培养HUCB-MSCs,流式细胞术测表面标记;取正常人外周血,免疫磁珠分离CＤ３+T细胞,将分离的CD3+T与HUCB-MSCs 1:1混合培养5d,PHA刺激或不刺激,采用3H-TdR掺入法观察T细胞增殖,ELISA方法检测细胞因子,流...     

A sensitive and versatile bioassay for ligands that signal through receptor clustering.

The induced expression of xanthine-guanine phosphoribosyl transferase (XGPRT) by low concentrations (-2 pg/ml) of interferon-alpha (IFN-alpha) or IFN-beta, in the 2fTPGH cell line caused a 50% cytotoxicity when these cells were grown in medium containing 6-thioguanine. We extended the application of this sensitive, reliable, and easy bioassay to other members of the cytokine family. To activate the IFN signaling pathway, we made receptor chimeras, consisting of the IFN type I receptor intracellular and transmembrane domains, fused to either the interleukin-5 (IL-5) receptors or erythropoietin (Epo) receptor extracellular domains as model systems. 2fTGH cells, stably transfected with these receptor chimeras, responded to very low concentrations of IL-5 or Epo (IC50 values of approximately 15 pg and 3 pg/ml, respectively) and thus can be used as a very sensitive bioassay for both ligands. Background activity of IL-5, Epo, tumor necrosis factor (TNF), IL-6, or leptin on cells that did not carry the receptor chimeras was very low. This methodology can in principle be extended to any ligand that acts via clustering of its receptors.     

Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma

Allergen-reactive T helper type-2 (Th2) cells and proinflammatory cytokines have been suggested to play an important role in the induction and maintenance of the inflammatory cascade in allergic asthma. We compared the plasma concentrations of novel proinflammatory cytokines IL-17 and IL-18, other proinflammatory cytokines IL-6 and IL-12, Th2 cytokines IL-10 and IL-13, and intracellular interferon-gamma (IFN-gamma) and IL-4 in Th cells of 41 allergic asthmatics and 30 sex- and age-matched health control subjects. Plasma cytokines were measured by enzyme-linked immunosorbent assay. Intracellular cytokines were quantified by flow cytometry. Plasma IL-18, IL-12, IL-10, IL-13 concentrations were significantly higher in allergic asthmatic patients than normal control subjects (IL-18: median 228.35 versus 138.72 pg/ml, P < 0.001; IL-12: 0.00 versus 0.00 pg/ml, P = 0.001; IL-10: 2.51 versus 0.05 pg/ml, P < 0.034; IL-13: 119.38 versus 17.89 pg/ml, P < 0.001). Allergic asthmatic patients showed higher plasma IL-17 and IL-6 concentrations than normal controls (22.40 versus 11.86 pg/ml and 3.42 versus 0.61 pg/ml, respectively), although the differences were not statistically significant (P = 0.077 and 0.053, respectively). The percentage of IFN-gamma-producing Th cells was significantly higher in normal control subjects than asthmatic patients (23.46 versus 5.72%, P < 0.001) but the percentage of IL-4 producing Th cells did not differ (0.72 versus 0.79%, P > 0.05). Consequently, the Th1/Th2 cell ratio was significantly higher in normal subjects than asthmatic patients (29.6 versus 8.38%, P < 0.001). We propose that allergic asthma is characterized by an elevation of both proinflammatory and Th2 cytokines. The significantly lower ratio of Th1/Th2 cells confirms a predominance of Th2 cells response in allergic asthma.