Proliferation of T lymphocytes from atopic dermatitis skin is enhanced upon anti-CD3, reduced upon mitogen and superantigen, and negligible upon tuberculin stimulation

Knowledge about the nature of lymphocytes infiltrating atopic dermatitis skin is restricted to allergen-specific T cells. We investigated the proliferative capacities of T lymphocytes cultured in an antigen-independent way from biopsies of atopic dermatitis skin. When compared with peripheral blood mononuclear cells (PBMC) from healthy donors or atopic dermatitis patients, the skin-homing lymphocytes proliferated more vigorously in response to stimulation with anti-CD3 antibodies (1 microglml), reflecting their high response capacity. When stimulated with phytohemagglutinin (10 microg/ml) or staphylococcal enterotoxin A (0.1 microg/ml) the skin-homing lymphocytes achieved significantly lower proliferation levels than PBMC. In contrast to normal and atopic PBMC the skin-homing lymphocytes did not respond to tuberculin purified protein derivative (10 microg/ml). In the mixed lymphocyte reaction the skin-homing lymphocytes did not stimulate autologous PBMC to proliferate. We conclude that skin-homing lymphocytes have more pronounced immune deviations than PBMC in patients with atopic dermatitis. They represent a valuable approach for further investigating the pathogenesis of the disease.     

Atopic dermatitis in adults: evaluation of peripheral blood mononuclear cells proliferation response to Staphylococcus aureus enterotoxins A and B and analysis of interleukin-18 secretion

Background:  Atopic dermatitis (AD) is a chronic, inflammatory skin disease with a high prevalence and complex pathogenesis. The skin of AD patients is usually colonized by Staphylococcus aureus ( S. aureus ); its exotoxins may trigger or enhance the cutaneous inflammation. Several mediators are related to the AD immune imbalance and interleukin-18 (IL-18), an inflammatory cytokine, may play a role in the atopic skin inflammation.
Aims:  To evaluate peripheral blood mononuclear cells (PBMC) proliferation response to staphylococcal enterotoxins A (SEA) and B (SEB) and the levels of IL-18 in adults with AD.
Methods:  Thirty-eight adult patients with AD and 33 healthy controls were analysed. PBMC were stimulated with SEA and SEB, phytohemaglutinin (PHA), pokeweed (PWM), tetanus toxoid (TT) and Candida albicans (CMA). IL-18 secretion from PBMC culture supernatants and sera were measured by ELISA.
Results:  A significant inhibition of the PBMC proliferation response to SEA, PHA, TT and CMA of AD patients was detected ( P  ≤ 0.05). Furthermore, increased levels of IL-18 were detected both in sera and non-stimulated PBMC culture supernatants from AD patients ( P  ≤ 0.05).
Conclusions:  A decreased PBMC proliferation response to distinct antigens and mitogens (TT, CMA, SEA and PHA) in adults with AD suggest a compromised immune profile. IL-18 secretion from AD upon stimulation was similar from controls, which may indicate a diverse mechanism of skin inflammation maintained by Staphylococcus aureus. On the other hand, augmented IL-18 secretion from AD sera and non-stimulated cell culture may enhance the immune dysfunction observed in AD, leading to constant skin inflammation.     

The importance of CD54 and CD86 costimulation in T cells stimulated with Candida albicans and Dermatophagoides farinae antigens in patients with atopic dermatitis

Abstract A number of studies have demonstrated an increased frequency of allergen-specific T cells producing increased amounts of interleukin-4 (IL-4) and IL-5, but little interferon-γ in both the peripheral blood and skin lesions of patients with atopic dermatitis (AD). In this study, to further clarify the characteristics of T cells obtained from AD patients, we examined the dependency of the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) from AD patients on costimulatory molecules. The antigens used were Candida albicans and Dermatophagoides farinae, for which AD patients show increased levels of IgE antibodies. PBMC from control healthy donors stimulated with these antigens incorporated [³H]-thymidine much more than PBMC from AD patients. The addition of anti-CD54, -CD40, -CD80 and -CD86 monoclonal antibodies to the cultures showed that the PBMC required only CD54 and CD86 for stimulation with C. albicans, but required CD54, CD80 and CD86 for stimulation with D. farinae. Among these monoclonal antibodies, the anti-CD54 antibody suppressed the proliferative responses of most PBMC, most effectively followed by the anti-CD86 antibody. However, there were no significant differences in the requirement for costimulatory molecules of PBMC proliferation stimulated with C. albicans or D. farinae between AD patients and healthy donors. Since many studies have suggested that T-helper type 1 and T-helper type 2 immune responses are different in their dependency on CD80 or CD86 costimulation, our present results suggest that the allergen-specific T cells of AD patients are not completely shifted to a T-helper type 2 subset. Received: 10 February 1998 / Received after revision: 2 July 1998 / Accepted: 2 July 1998     

特应性皮炎患者外周血T淋巴细胞Rho激酶活性变化及其意义

目的 观察特应性皮炎（AD）患者外周血T淋巴细胞中Rho激酶活化情况,分析其临床意义。方法 收集60例AD患者和60例健康儿童外周肝素抗凝血8 ml,分离提取T细胞和血清。分别用AD血清和健康对照血清培养AD患者T细胞或健康对照T细胞,分为患者T细胞 + 自身血清组、患者T细胞 + 健康对照血清组、健康对照T细胞 + 自身血清组、健康对照T细胞 + AD血清组。此外,分别用Rho激酶特异性抑制剂Y27632（Y27632组）、CD3/CD28单抗（CD3/CD28单抗组）、Y27632 + CD3/CD28单抗（Y27632 + CD3/CD28单抗组）处理AD患者T细胞,自身血清培养AD患者T细胞为患者T细胞组。采用Western印迹法检测各组Rho激酶活性,四甲基偶氮唑蓝比色法（MTT）检测T细胞增殖活性,ELISA检测白细胞介素（IL）6、IL-10水平。 结果 新鲜分离的AD患者外周血T细胞Rho激酶活性（2.47% ± 0.89%）显著高于健康对照组（0.65% ± 0.35%,t = 2.729,P < 0.05）。AD患者T细胞在体外经10%胎牛血清培养24 h后Rho激酶活性（0.70% ± 0.38%）较培养前显著降低（t = 2.658,P < 0.05）,但与培养24 h后健康对照T细胞（0.63% ± 0.32%）相比,差异无统计学意义（t = 1.010,P > 0.05）。与健康对照血清培养T细胞相比,AD血清培养T细胞会增加Rho激酶活性（F = 8.22,P < 0.001）,患者T细胞 + 自身血清培养24 h后Rho激酶活性（2.41% ± 0.87%）明显高于患者T细胞 + 健康对照血清组（0.76% ± 0.41%）,健康对照T细胞 + AD血清组（2.17% ± 0.85%）显著高于健康对照T细胞 + 自身血清组（0.64% ± 0.33%）,差异均有统计学意义（P < 0.05）。Y27632可显著抑制AD患者T细胞的增殖和IL-6的分泌（F = 18.68、22.95,P < 0.001）,Y27632组T细胞增殖率、IL-6的表达均显著低于患者T细胞组（均P < 0.05）,且Y27632 + CD3/CD28单抗组也显著低于CD3/CD28单抗组（均P < 0.05）,而Y27632对AD患者T细胞IL-10分泌无显著影响。 结论 AD患者T淋巴细胞存在Rho激酶信号活化异常,提示Rho激酶信号异常在AD发病过程中可能发挥着一定的作用。     

银屑病患者外周血单个核细胞端粒酶活性及与疾病严重程度的关系

目的探讨银屑病患者外周血单个核细胞(PBMC)端粒酶活性及与疾病严重程度和病程的关系。方法分离银屑病患者和正常人外周血单个核细胞,采用聚合酶链反应-酶联免疫吸附(PCR-ELISA)法检测寻常型银屑病患者和正常人外周血单个核细胞的端粒酶活性水平;用银屑病皮损严重程度指数(PASI)评价银屑病患者病情严重程度。结果银屑病组PBMC的端粒酶活性(0.9±0.18)较正常组(0.49±0.11)显著升高(t=8.096,P<0.01),进行期(0.97±0.19)高于消退期或静止期(0.90±0.17),端粒酶活性与疾病严重程度成正相关(r=0.889,P<0.001),而与病程无明显线性关系。结论银屑病患者外周血单个核细胞端粒酶活性水平升高,且与疾病严重程度成正相关,而与病程无关,可作为银屑病发病及病情判定的一个指标。     

Impaired responses of peripheral blood mononuclear cells to nickel in patients with nickel-allergic contact dermatitis and concomitant atopic dermatitis

BACKGROUND: Allergic contact dermatitis (ACD) is pathogenetically dependent on cell-mediated immune responses mediated by type 1 T lymphocytes. Atopic dermatitis (AD), in contrast, occurs as a result of sustained activation of type 2 subsets of T cells. Although atopic patients may become sensitized to various contact allergens, little is known about the influence of atopy on delayed-type hypersensitivity. OBJECTIVES: To investigate the in vitro responses of peripheral blood mononuclear cells (PBMC) to nickel stimulation in groups of atopic and nonatopic patients with patch test-verified nickel ACD. METHODS: Ten nonatopic patients with nickel ACD, 10 patients with nickel ACD and concomitant AD, 10 patients with AD but with no contact allergy, and 10 healthy persons participated in the study. PBMC were cultured in the presence or absence of nickel sulphate, phytohaemagglutinin (PHA) or tetanus toxoid (TT). [(3)H]thymidine incorporation was used to measure the rate of antigen-induced DNA synthesis and enzyme-linked immunosorbent assay was used to measure the production of interleukin (IL)-2 (type 1 cytokine) and IL-5 (type 2 cytokine). RESULTS: Nickel-stimulated PBMC of nickel-allergic patients with AD proliferated significantly less and secreted significantly lower amounts of IL-2 than cells of nonatopic nickel-allergic patients. IL-5 production was also lower in the former group, although the difference was nonsignificant. Moreover, neither the nickel-specific DNA synthesis nor the cytokine production by PBMC of atopic nickel-allergic patients differed significantly from those of healthy control persons and AD patients without contact allergy. Proliferative and secretory responses of PBMC to PHA or TT stimulation differed nonsignificantly between the groups. Nickel-induced IL-2 production correlated well with IL-5 production in nickel-allergic patients regardless of their atopic status. CONCLUSIONS: Our results indicate that PBMC of nickel-allergic patients with concomitant AD are characterized by impaired in vitro proliferative and secretory responses to the contact allergen nickel but not to the mitogen PHA or the recall antigen TT. The type 2 cytokine IL-5 may play a role in the development of ACD.     

T cell epitope-specific defects in the immune response to cat allergen in patients with atopic dermatitis

Atopic dermatitis (AD) is often associated with high titer IgE antibodies (ab) to allergens, and IL-10-mediated regulation of IFN-gamma has been proposed to contribute to this IgE ab production. However, the relevance of IL-10 and IFN-gamma to IgE associated with AD has not been examined in the context of an allergen-specific system. Analysis of PBMC responses in vitro showed deficient T cell proliferation to overlapping IL-10- (peptide (P) 2:1) and IFN-gamma- (P2:2) inducing chain 2 major epitopes of cat allergen (Fel d 1) in cultures from sensitized AD patients (mean IgE to cat=20.9 IU/ml). Diminished IFN-gamma induction by Fel d 1 and P2:2, along with elevated peptide-induced IL-10 (except for P2:1) was observed in PBMC cultures from AD subjects compared with non-AD (sensitized and non-sensitized) subjects. Neither T cell proliferation nor IFN-gamma production to chain 2 epitopes could be restored by anti-IL-10 mAb in cultures from sensitized AD subjects. Moreover, allergen avoidance was associated with a paradoxical decrease in both IL-10 and IFN-gamma in peptide-stimulated PBMC from these subjects. Control of IFN-gamma production to chain 2 epitopes by IL-10 may be relevant to sensitization status. Development of high titer IgE ab in AD could reflect a failure of this mechanism.     

Augmented telomerase activity and reduced telomere length in parthenium‐induced contact dermatitis

Background Parthenium dermatitis is a common chronic inflammatory disease with activated T lymphocytes that recognize the antigens, which leads to proliferation and differentiation. Telomeres and telomerase play an important role in the regulation of life span of the cell. Telomere length maintained by telomerase, are specialized repeats present at the end of chromosomes which protect it from degradation, end‐to‐end fusion and are important for integrity of chromosomes. Objectives The aim of this study was to measure telomerase activity and telomere length in Peripheral blood mononuclear cell (PBMC), CD4⁺ and CD8⁺ T lymphocytes from parthenium dermatitis patients. Methods The study includes 50 patients of parthenium dermatitis confirmed by patch testing and 50 healthy controls. Telomerase activity was measured using the telomere repeat amplification protocol using PCR–ELISA kit. Telomere length was measured by using Telo TAGGG Telomere Length Assay Kit. Results Significantly elevated levels of telomerase activity was observed in PBMC, CD4⁺ and CD8⁺ T cells of parthenium dermatitis patients as compared with healthy controls. However, significantly reduced telomere length in PBMC, CD4⁺ and CD8⁺ T cells have been found in patients than healthy subjects, but there was no difference between CD4⁺ and CD8⁺ T cells in patients. Conclusion This study might have provided insight into the role of telomerase in parthenium dermatitis that is characterized by the recruitment of T lymphocytes, which play an important role in this inflammatory disease. The augmented telomerase activity and reduced terminal restriction fragment length might be explored as a potential diagnostic/prognostic marker for parthenium dermatitis in future.     

Interleukin-4 promotes the expansion of skin-infiltrating lymphocytes from atopic dermatitis in vitro

Functional studies of lymphocytes in atopic dermatitis (AD) have so far focused on peripheral blood mononuclear cells (PBMC), whereas cells at the involved site, the skin, have not been examined. Accordingly, we have developed methods to generate lymphocyte cultures from biopsies of inflammatory skin areas. Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of 6 patients with severe AD and expanded in vitro in the presence of interleukin-2 (IL-2) without additional antigens. After 6-10 d in culture, outgrowth of mononuclear cells from biopsy tissue was observed in all cases. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T-helper/inducer phenotype in SIL populations. Parallel cultures of SIL and PBMC showed an increase and expansion of CD8+ T cells in cultured PBMC, whereas the CD4+ phenotype was predominant in SIL cultures. As indicated by their expression of HLA-DR and CD25 antigens, most of the SIL were activated and the cells mainly expressed T-cell receptors (TCR) composed of alpha and beta chains. Different strategies for expansion of SIL in vitro were examined. High levels of IL-4 (1,000 U/ml) in combination with IL-2 (50 U/ml or 1,000 U/ml) preferentially promoted growth of SIL derived from AD and were much more effective than IL-2 alone. No cells expanded in cultures with IL-4 alone. SIL grown with high concentrations of IL-4 contained a significant proportion of double-positive CD4+8+ cells. No other marked differences were observed in the distribution of T cell subsets in cultures propagated under different conditions for 21 d. Our results demonstrate the feasibility of growing infiltrating T lymphocytes from inflammatory skin of AD patients. The use of high concentrations of IL-2 in combination with high levels of IL-4 allows a large expansion of these cells and thus represents a useful strategy to expand cells for further functional and molecular biologic studies.     

蕈样肉芽肿的端粒酶活性与细胞增殖指数相关性的研究

目的：检测蕈样肉芽肿（MF）的端粒酶活性和细胞增殖情况，探讨它们在MF肿瘤发生机制中的作用及关系。方法：使用免疫组化法检测增殖细胞核抗原（PCNA）的表达情况；聚合酶链反应－酶联免疫吸附测定法检测端粒酶活性。结果：肿瘤期MF、斑片期MF、扁平苔藓和正常皮肤的PCNA细胞增殖指数分别为22．8、12．1、8．8、1．7和0；端粒酶活性的A值分别为0．737、0．313、0．240、0．045和0．044。端粒酶阳性的MF的PCNA细胞增殖指数高于端粒酶阴性的MF。结论：端粒酶活性存在于大部分MF标本中，端粒酶活性与MF的恶性程度及分期有关。端粒酶活性与细胞增殖指数有关，提示端粒酶的的活化可能为肿瘤细胞的无限增殖提供了先决条件。     

Enhanced IL-4 production and IL-4 receptor expression in atopic dermatitis and their modulation by interferon-gamma.

The in vivo and in vitro immunomodulatory effects of interferon gamma (IFN-gamma) treatment on peripheral blood mononuclear cells (PBMC) from patients with atopic dermatitis (AD) and elevated IgE levels were studied. As part of a double-blind placebo-controlled clinical trial, 14 AD patients were treated with IFN-gamma (n = 7) or saline (n = 7) for 12 weeks. To assess the in vivo effects of IFN-gamma treatment on interleukin (IL)-4-dependent lymphocyte function, we assessed the proliferation of AD PBMC in response to IL-4. Prior to IFN-gamma treatment, AD PBMC had proportionately decreased proliferative responses to IL-4 when compared to IL-2. After 12 weeks of in vivo treatment with IFN-gamma, there was an increase of IL-4- but not IL-2-induced lymphocyte proliferation in seven of eight AD patients. To further study the immunologic basis of these observations, we studied the expression of IL-4 receptor (IL-4R) mRNA and the production of IL-4 by PBMC from AD patients. PBMC from AD patients expressed higher levels of IL-4R mRNA and produced significantly higher amounts of IL-4 than normal controls (p less than 0.05). More importantly, the in vitro addition of IFN-gamma caused significant reduction in both IL-4R and mRNA expression and IL-4 production of PBMC from AD and non-atopic controls. These data indicate that AD is characterized by an in vivo overstimulation of the IL-4-IL-4R pathway. The poor proliferative responses of untreated AD PBMC to exogenous IL-4 may be due to increased levels of endogenous IL-4 production with constant occupancy on the IL-4R. Furthermore, in vivo and in vitro treatment with IFN-gamma down-regulates this pathway.     

Cytokine-mediated effects of peripheral blood mononuclear cells from patients with atopic eczema on keratinocytes (HaCaT) in a new coculture system

Summary Interactions between keratinocytes and mononuclear cells via cytokines and adhesion molecules are thought to play a crucial part in inflammatory skin diseases. The cytokine-mediated effects of peripheral blood mononuclear cells (PBMC) from patients with atopic eczema (AE) and healthy individuals on keratinocytes (HaCaT) were investigated in vitro. A new coculture model (Transwell system) which consists of a lower and an upper compartment, which are separated by a polycarbonate-treated membrane, was established. ³[H]thymidine incorporation of keratinocytes and lymphocytes, as well as IL-6. IL-8 and IFN-γ synthesis, were measured. Keratinocyte proliferation was significantly enhanced in the presence of PBMC from patients with AE. In contrast, PBMC from normal donors did not enhance HaCaT cell proliferation when they were cocultttred. Lymphocytes from patients with AH showed a significantly enhanced proliferation after coculture with keratinocytes. However, PBMC from normal donors did not proliferate in the presence of HaCaT cells. Keratinocyte supernalants incubated with PBMC from either atopic or normal volunteers induced a suppression of lymphocyte ³[H]thymidine incorporation. In supernatants from cocultures of PBMC from patients with AE and keratinocytes. significantly enhanced amounts of IL-6 and IL-8. compared with normal donor's lymphocytes and HaCaT cells, were measured. No differences in TFN-γ production were observed. When PBMC were cultured without HaCaT cells, supernatants contained equal levels of IL-6, IL-8 and IFN-γ in normal donors and in patients with AE. Interestingly. HaCaT cells spontaneously secrete measurable amounts of IL-6, IL-8 and IFN-γ. Blocking experiments with neutralizing antibodies against these interleukins showed a complete inhibition of keratinocyte proliferation when PBMC from normal donors were used whereas the proliferative potency of PBMC supernalants from patients with AE on keratinocytes remained. Our data indicate that (i) PBMC from patients with AE stimulate keratinocyte proliferation via soluble factor(s) that are different from IL-6. IL-8 and IEN-γ (ii) probably, HaCaT cells spontaneously produce lymphocyte/monocyle inhibitory soluble factors and IL-6, IL-8 as well as IFN-γ and (iii) secretion and/or activity of keratinocytederived inhibitory mediators is regulated via cytokines of PBMC infiltrating inflammatory skin.     

特应性皮炎患者外周血CD4+CD25+调节性T细胞的检测

目的 探讨CD4+CD25+调节性T细胞（CD4+CD25+ Treg）在特应性皮炎（AD）发病中的作用机制及临床意义。方法 流式细胞仪分析AD患者外周血中CD4+CD25+ Treg细胞数量,实时荧光定量PCR检测外周血单核细胞（PBMC）中Foxp3 mRNA水平,ELISA检测血清中IL-2、IL-4、IL-10、IFN-γ水平。结果 AD患者外周血中CD4+CD25+ Treg细胞占CD3+ T细胞及CD4+ T细胞的比例均明显低于正常人对照组（t′ = 3.775、4.533,P值均 ＜ 0.01）;外周血中CD4+CD25+ Treg细胞占CD3+ T细胞比例在AD患者急性期明显低于慢性期（t = 2.217,P < 0.05）,而在急性期与亚急性期、亚急性期与慢性期之间差异均无统计学意义（t = 1.558、0.49,P值均 ＞ 0.05）。AD患者PBMC中Foxp3 mRNA的水平低于正常人对照组（z = -2.368,P ＜ 0.05）;其外周血中CD4+CD25+ Treg细胞与血清中IL-2和IL-10成正相关（r = 0.512、0.494,P值均 ＜ 0.05）,与IL-4和IFN-γ的相关性无统计学意义（r = -0.110、-0.237,P值均 ＞ 0.05）。结论 在AD患者中,外周血中CD4+CD25+ Treg细胞数量及Foxp3 mRNA水平均下降,从而可能减少对Th2细胞增生及其细胞因子分泌的抑制,使Th2占优势,参与AD的发病。     

Lidocaine inhibits staphylococcal enterotoxin-stimulated activation of peripheral blood mononuclear cells from patients with atopic dermatitis

Atopic dermatitis (AD) is an inflammatory, chronically relapsing, pruritic skin disease and lesions associated with AD are frequently colonized with Staphylococcus aureus (S. aureus). Activation of T cells by staphylococcal enterotoxins (SE) plays a crucial role in the pathogenesis of AD. Previous studies have demonstrated that lidocaine could attenuate allergen-induced T cell proliferation and cytokine production in peripheral blood mononuclear cells (PBMCs) from asthma patients. The purpose of this study was to investigate the effects of lidocaine on SE-stimulated activation of PBMCs from AD patients. PBMCs were isolated from ten AD patients and stimulated by staphylococcal enterotoxin A (SEA) or staphylococcal enterotoxin B (SEB) in the presence or absence of lidocaine in various concentrations. Cellular proliferation and the release of representative T_H1- and T_H2-type cytokines were measured. The effect of lidocaine on filaggrin (FLG) expression in HaCaT cells co-cultured with SE-activated PBMCs was also examined. Our results demonstrated that lidocaine dose-dependently inhibited the proliferative response and the release of IL-4, IL-5, IL-13, TNF-α, and IFN-γ from SEA- and SEB-stimulated PBMCs and also blocked the down-regulation of FLG expression in HaCaT cells co-cultured with SEA- and SEB-activated PBMCs. These results indicate that lidocaine inhibited SEA- and SEB-stimulated activation of PBMCs from patients with AD. Our findings encourage the use of lidocaine in the treatment of AD.     

Decreased monocyte production of interleukin-1 and impaired lymphocyte proliferation in atopic dermatitis

Summary We studied lymphocyte proliferation and subsets in ten atopic dermatitis (AD) patients and ten healthy controls. In addition, monocyte production of interleukin 1 (IL-1) was investigated. Mean numbers of total T cells, T-cells subsets, and B cells did not significantly differ between AD patients and controls even though the patients had slightly decreased amounts of suppressor T lymphocytes. Proliferative responses of AD patients to purified protein derivative of tuberculin (PPD), concanavalin A (ConA), or allogeneic cells did not significantly differ from those of healthy controls at optimal stimulant concentrations. In contrast, at suboptimal concentrations, AD patients showed a diminished response to all of these stimulants. Monocytes from AD patients elaborated clearly less IL-1 than those from healthy controls.     

Relative impact of CD4+CD25+ regulatory T cells and tacrolimus on inhibition of T-cell proliferation in patients with atopic dermatitis

BACKGROUND: It has been established recently that CD4+CD25+ regulatory T cells (Tregs) play an important role in controlling various immune responses. Immunosuppressive drugs are often used to treat immune dysregulation but are frequently associated with undesirable side-effects. OBJECTIVES: We examined the suppressive capacity of circulating Tregs in patients with atopic dermatitis (AD). Combined effects of Tregs and tacrolimus on the inhibition of T-cell proliferation in vitro were also assessed. METHODS: CD4+CD25+ and CD4+CD25- T cells were isolated from peripheral blood mononuclear cells using immunomagnetic beads. CD4+CD25- T cells were stimulated with purified protein derivative (PPD) or house dust mite allergen (Der p1) for 6 or 7 days, respectively. A dose range of tacrolimus and CD4+CD25+ T cells were added separately, or together. Proliferation was measured by (3)H-thymidine incorporation. RESULTS: CD4+CD25+ T cells from normal controls and patients with AD are anergic and inhibit the proliferation of CD4+CD25- T cells in response to PPD and Der p1 in vitro in a dose-dependent manner. Addition of tacrolimus and Tregs together showed significantly stronger inhibition of proliferation than either on their own. This was true for both antigens and both in normal controls and in patients with AD. CONCLUSIONS: CD4+CD25+ T cells in patients with AD have normal suppressive activity compared with healthy controls. Tregs and tacrolimus have additive effects on the inhibition of proliferation in response to PPD and Der p1.     

特应性皮炎患者外周血T细胞磷酸肌醇3激酶信号转导通路的表达

目的 探讨特应性皮炎（AD）患者外周血T细胞磷酸肌醇3激酶（PI3K）信号转导通路活化情况及其临床意义。 方法 检测38例AD患者的T细胞PI3K通路活性,健康对照组38例。外周血T淋巴细胞分离采用Rosettsep T细胞提取试剂盒提取,PI3K活性采用免疫沉淀和酶联免疫吸附试验（ELISA）,Akt及其磷酸化蛋白采用免疫印迹法,T细胞增殖采用噻唑蓝（MTT）,白细胞介素（IL）6和IL-10水平采用ELISA检测。 结果 新鲜分离的AD患者外周血T细胞PI3K和Akt活性均显著高于健康对照组（P < 0.05）。AD患者T细胞在体外培养24 h后PI3K和Akt活性与健康对照组比较,差异无统计学意义（P ＞ 0.05）,健康对照组T细胞用AD患者血清孵育24 h后PI3K和Akt活性显著增高（P < 0.05）。PI3K特异性抑制剂LY294002显著抑制AD患者T细胞增殖［（123 ± 25）和（195 ± 28）,P < 0.05］及分泌IL-6和IL-10分别为［（431 ± 64） ng/L和（823 ± 128） ng/L,P < 0.05］,［（120 ± 21） ng/L和（213 ± 35） ng/L,P < 0.05］。新鲜分离的AD患者外周血T细胞PI3K和Akt活性与疾病严重程度指数（EASI）无相关。 结论 AD患者外周血T细胞存在磷酸肌醇3激酶信号转导通路活化异常,并与T细胞增殖和细胞因子分泌有关,提示AD患者外周血中可能存在激活该通路的血清因子。     

Lack of correlation between in vitro immunological alterations and the development of scleroderma-like skin lesions in toxic oil syndrome patients.

We examined whether immunological disturbances could influence the development of scleroderma-like skin lesions in patients affected by the Spanish toxic oil syndrome (TOS). To this end, peripheral blood mononuclear cells (PBMC) were collected from 13 chronic patients and 8 control subjects. All patients had suffered a toxic-induced severe neuromyopathy, and 6, in addition, had developed sclerodermoid skin manifestations. The phenotypic profile and the concentrations of interleukin-2 (IL-2) and of molecules with B cell differentiation factor IgG activity (BCDF-IgG) in supernatants of phytohemagglutinin-stimulated lymphocytes were analyzed both in patients and in normal controls. Molecules with BCDF-IgG activity were found increased in supernatants of mitogen-stimulated lymphocytes from TOS patients. Concentrations of IgG secreted by staphylococcus aureus-SAC-B blasts in the presence of TOS PBMC supernatant was 88 +/- 32.62 ng/ml (mean +/- 1 SD) versus 53 +/- 5.34 ng/ml in the presence of control supernatant (p less than 0.01). Levels of BCDF-IgG activity in TOS PBMC supernatants positively correlated with IgG serum levels (r = 0.69, p less than 0.01). The phenotypic profile of lymphocyte populations and the production of IL-2 were not altered in TOS subjects. No statistically significant differences were observed in the lymphocyte distribution nor in the IL-2 and BCDF-IgG production when comparing patients with or without scleroderma-like skin lesions. The results indicate that there was a dysfunction of the immune response in TOS subjects, which, however, was not sufficient for the development of the sclerodermoid lesions.