硫酸镁联合低分子肝素治疗早发型子痫前期的病例对照研究

目的探讨硫酸镁联合低分子肝素治疗早发型子痫前期的疗效及其妊娠结局。方法将50例早发型子痫前期患者分为硫酸镁治疗组（n=22）和硫酸镁联合低分子肝素治疗组（n=28）,并进行病例对照分析,观察治疗前后的凝血功能、肾功能变化及妊娠结局。结果两组患者治疗后收缩压及舒张压均明显下降,但下降幅度差异无统计学意义（P〉0.05）。排除治疗前凝血功能或肾功能基础值的影响,硫酸镁联合低分子肝素治疗使凝血酶原时间（PT）明显延长、凝血酶原时间国际标准化比值（PT-INR）明显增大、凝血酶时间（TT）明显延长,差异有统计学意义（P〈0.05）,但上述各指标改变后仍在正常范围以内。硫酸镁联合低分子肝素治疗对尿蛋白及血尿酸的减少影响更显著,差异有统计学意义（P〈0.05）。两个治疗组之间的血清尿素氮和血清肌酐的变化差异无统计学意义（P〉0.05）。两组之间的分娩孕周、期待治疗时间、新生儿体重及新生儿出生Apgar评分及母儿并发症发生率差异均无统计学意义（P〉0.05）。结论在传统治疗的基础上加用低分子肝素辅助治疗早发型子痫前期是安全的,且可以对肾功能起到保护作用,明显减少尿蛋白量。尚未观察到低分子肝素对妊娠结局的影响。     

Clinical cure of severe, early onset preeclampsia with low molecular weight heparin therapy in primigravida with hyperreactio luteinalis and thrombophilia

Inherited thrombophilias, suggested to be risk factors for ovarian hyperstimulation syndrome and known to be associated with venous thromboembolism during pregnancy, may also increase the risk for preeclampsia (PE). We describe the case of a 29-year-old woman with primary infertility with no history of thrombosis, hypertension or renal disorders. In her first pregnancy, achieved by frozen embryo transfer, she developed severe early-onset (23rd gestational week) PE with heavy proteinuria, and at the same time was found to have enlarged ovaries with hyperreactio luteinalis. After admission we found that she was a heterozygotic carrier of the factor V Leiden mutation. After administering low molecular weight heparin (LMWH) therapy, her blood pressure normalized, proteinuria diminished and her d-dimer values returned to that of a normal pregnant level. The fetus grew normally. Her ovaries normalized during the pregnancy, as determined by ultrasound examinations. At term she delivered spontaneously a normal weight, healthy girl. Previously, only prophylactic LMWH, in subsequent pregnancy, have been administered in patients with thrombophilia and a history of severe PE. We describe a case of spontaneous hyperreactio luteinalis, where the clinical characteristics of PE improved after beginning LMWH therapy in severe, very early onset PE. Inherited thrombophilia, spontaneous hyperreactio luteinalis and PE may be associated phenomena.     

低分子肝素对URSA患者外周血免疫细胞的影响

目的探讨低分子肝素对不明原因复发性流产（URSA）患者外周血免疫细胞的影响。方法研究组包括28例URSA患者,予低分子肝素（Fraxiparine 0.4-0.6ml/d或Enoxaparin 0.4-0.6ml/d iH）治疗至妊娠12w,于治疗前、3w后采用流式细胞术检测患者外周血中淋巴细胞亚群及血常规;15例未接受低分子肝素治疗的URSA患者为对照组,于首次就诊（血HCG确认妊娠）及2~3w后（流产清宫前）检测以上项目。结果研究组保胎成功率为86%,明显高于对照组33%（P〈0.05）;使用低分子肝素后USRA患者外周血中总T细胞数比例为（66.9±5.6）%,较使用前（61.2±7.4）%升高（P〈0.05）;NK细胞比例为（16.6±4.4）%,较使用前（19.4±7.3%）下降（P〈0.05）,中性粒细胞比例为（67.1±7.9）%,红细胞压积为（36.1±3.9）%,较使用前（70.7±7.8%）、（33.8±7.8%）均下降（P〈0.05）。结论低分子肝素对URSA有显著治疗效果,其作用机理除抗凝外,还可能直接降调URSA外周血中的NK细胞及中性粒细胞。     

Protective effect of ultra low molecular weight heparin on glutamate-induced apoptosis in cortical cells

PURPOSE: To investigate the effect of ultra low molecular weight heparin (ULMWH) on glutamate induced apoptosis in rat cortical cells and to explore the possible mechanisms. MATERIALS AND METHODS: Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was first analyzed with Hoechst 33258 and then confirmed by DNA fragmentation. The concentration of free intracellular calcium ([Ca(2+)](i)) was determined with fura-2/AM fluorometry. The expression of Bcl-2 family protein and caspase-3 were evaluated with Western blot. RESULTS: Typical apoptotic morphological change in rat cortical cells treated with 100 micromol/L glutamate for 24h was detected by Hoechst 33258 staining, which was then confirmed by the DNA ladder of agarose gel electrophoresis. The apoptotic rate of the glutamate treated cells was up to 33.21%, and 24 h of treatment with glutamate increased [Ca(2+)](i), down-regulated Bcl-2 expression, up-regulated Bax expression, and increased caspase-3 activation in rat cortical cells. Our research demonstrated that ULMWH pretreatment can prevent the glutamate-induced apoptosis, attenuate the increase of [Ca(2+)](i) not only in medium containing Ca(2+) but also in Ca(2+)-free medium, up-regulate the expression of Bcl-2, down-regulate the expression of Bax, and decrease caspase-3 activation. CONCLUSION: ULMWH has neuroprotective capacity to antagonize glutamate-induced apoptosis in cortical cells, through decrease of Ca(2+) release and modulation of apoptotic processes.     

Successful pregnancy with low molecular weight heparin in two women with recurrent miscarriage of unknown etiology

We report here two cases of recurrent miscarriages that were successfully treated with continuous intravenous administration of low molecular weight heparin (LMWH). One patient experienced 11 spontaneous abortions, and the other eight abortions. Previous treatments including prednisone, aspirin and mononuclear-cell immunization were all unsuccessful. They were negative for anticardiolipin antibodies and lupus anticoagulant, and had no inherited thrombophilic disorder. Intravenous administration of LMWH, 4800 units of dalteparin, was started as soon as the conception was confirmed, and was continued until 34 weeks of gestation. They were delivered of live born infants.     

In vivo treatment of rats with unfractionated heparin (UFH) or low molecular weight heparin (LMWH) does not affect experimentally induced colon carcinoma metastasis

Recent randomized trials have suggested that treatment with low molecular weight heparin (LMWH) improves survival of cancer patients with venous thromboembolism, as compared to treatment with unfractionated heparin (UFH). Experimental studies have shown that UFH has activities besides its anticoagulant function which may affect progression of malignancy, including stimulation of new blood vessel formation. In contrast, LMWH has been suggested to inhibit angiogenesis. In the present study, we compared quantitatively the effects of treatment with UFH, LMWH or placebo on the development of experimentally induced colon carcinoma metastases in rat liver and on tumor-associated angiogenesis. It is shown that UFH and LMWH in therapeutic dosages neither affect development of metastases nor tumor blood vessel formation in this animal model. These results indicate that heparins do not affect colon cancer metastasis in liver. Further studies in other animal models are required to establish the mechanisms by which heparins potentially affect cancer.     

低分子肝素钙预防腰椎退行性疾病术后深静脉血栓形成的有效性和安全性

目的探讨低分子肝素钙预防腰椎退行性疾病术后深静脉血栓形成(DVT)的有效性和安全性。方法回顾性分析广安市广安区人民医院68例腰椎退行性疾病患者的临床资料,术后均采用抗凝预防血栓形成,按照术后预防DVT所采用方式的不同分为对照组和低分子肝素钙组。分析比较2组患者术后引流量、切口愈合情况、DVT发生率、皮下瘀斑情况、血小板数值、凝血功能、D-二聚体。结果术后2组患者引流量、切口愈合情况、皮下瘀斑情况、凝血功能相关指标比较,差异无统计学意义(P>0.05)。术前和术后1、10 d 2组患者血小板数量的变化差异无统计学意义(P>0.05)。DVT发生率2组患者比较差异有统计学意义(P<0.05)。术前及术后1 d 2组患者D-二聚体比较,差异无统计学意义(P>0.05);术后10 d,对照组患者D-二聚体显著增加,2组比较差异有统计学意义(P<0.05)。结论腰椎退行性疾病术后使用低分子肝素钙进行抗凝,可以显著降低DVT的发生率,具有良好的安全性。     

Inhibitory effects of budesonide,desloratadine and dexamethasone on cytokine release from human mast cell line (HMC-1)

Objective: To determine the inhibitory potency of budesonide on interleukin (IL)-4, 6 and 8, GM-CSF and TNF- release from the human mast cell line (HMC-1) in comparison with the systemic glucocorticosteroid, dexamethasone, and H₁ antagonist, desloratadine.Methods: HMC-1 was stimulated with 25 ng/ml phorbol 12- myristate 13-acetate (PMA) and 2.5 × 10^–7 M ionomycin (A23187) for 6, 12 and 24 h in both the presence and absence of 10^–6–10^–10 M concentrations of the test drugs. Culture supernatants were collected and assayed by ELISAs.Results: HMC-1 produced substantial amounts of GM-CSF and IL-8 and smaller amounts of TNF-, IL-4 and IL-6 after being stimulated with PMA together with A23187. Budesonide and dexamethasone had potent inhibitory effects and desloratadine had modest inhibitory effects on the release of these cytokines. Budesonide was more potent than dexamethasone at most concentrations and time points. IL-4 was the cytokine which was most susceptible to inhibition by the three tested drugs. The inhibitory effects, in some cases, were time- and concentration-dependent.Conclusion: Budesonide had a potent inhibitory effect on cytokine release from HMC-1. Its potency was greater than that of both dexamethasone and desloratadine.Received 2 January 2004; returned for revision 22 April 2004; returned for final revision 10 June 2004; accepted by N. Boughton-Smith 15 July 2004     

低分子肝素治疗重度子痫前期患者外周血vWF、AT的变化

目的探讨低分子肝素能否改变重度子痫前期患者血浆中vWF含量,AT的活性水平,为低分子肝素用于重度子痫前期的治疗提供新的理论基础。方法选择2010年3月1日～2010年11月30日在天津医科大学宝坻临床学院、天津医科大学总医院分娩的重度子痫前期病人39例为研究组。根据入院顺序随机分成2组,传统治疗组20人,给予传统常规的治疗。低分子肝素治疗组19人,在传统常规治疗的基础上给予低分子肝素钙0.4m1,每天一次,皮下注射,两组分别于入院时及治疗3～5天后抽取孕妇静脉血,检测血浆vWF：Ag和AT：A及常规凝血试验。结果两组重度子痫前期患者治疗前血浆中vWF：Ag与同期对照组比较明显升高,而AT：A与同期对照组比较明显减少,低分子肝素治疗后血浆中vWF：Ag与治疗前比较明显降低,而AT：A与治疗前比较明显增高,传统方法治疗后血浆vWF：Ag和AT：A与治疗前比较差异无统计学意义。结论低分子肝素结合传统疗法治疗重度子痫前期患者,可改变患者血浆中vWF含量,AT的活性水平。     

Pathogenicity of human anti-platelet factor 4 (PF4)/heparin in vivo: generation of mouse anti-PF4/heparin and induction of thrombocytopenia by heparin

Heparin-induced thrombocytopenia/thrombosis (HIT) is a severe thrombotic disorder that occurs in ≈1% of patients treated with heparin. Affected patients commonly develop antibodies that recognize PF4/heparin complexes that may form on the surface of activated platelets and on the endothelium. However, it has not been established that anti-PF4/heparin antibodies are responsible for the clinical manifestations of HIT. To address this issue, we employed a recently developed model of active immunity to study the effect of IgG anti-PF4/heparin antibody in vivo. In previous studies we have shown that it is possible to induce autoimmune diseases such as systemic lupus erythematosus (SLE), anti-phospholipid syndrome (APS) or vasculitis in naive mice by active immunization with anti-DNA, anti-cardiolipin and anti-neutrophil cytoplasmic antibodies, respectively. Immunized animals develop anti-idiotypic antibodies (Ab₂) and, after 2–4 months, anti-anti-idiotypic antibodies (Ab₃). Ab₃s generated in this manner often simulate the binding activity of Ab₁ and their expression correlates with the development of specific clinical manifestations typical of the respective human disease. Based on this experience, naive BALB/c mice were immunized with IgG anti-PF4/heparin antibodies isolated from two patients with HIT. The actively immunized mice developed mouse anti-PF4/heparin antibody (Ab₃). Administration of unfractionated heparin, but not low molecular weight heparin (LMWH), to the actively immunized animals induced thrombocytopenia by day 4 of drug exposure. There was no evidence of thrombosis. The results of this study support the importance of anti-PF4/heparin antibodies in the pathogenesis of HIT. Further, this model may help to elucidate the factors responsible for thrombosis as well as providing means to assess new treatment options for patients with this disorder.     

Dexamethasone inhibits maturation, cytokine production and FcεRI expression of human cord blood-derived mast cells

BACKGROUND: Mast cells are responsible for eliciting the early phase and for contributing to the development of the late phase of allergic reactions, through the release of cytokines and other inflammatory mediators. OBJECTIVE: To assess whether the glucocorticoid dexamethasone has a direct effect on mast cell progenitor maturation and on mature cord blood-derived mast cell properties. METHODS: Mast cells were obtained by culturing human umbilical cord blood mononuclear cells with stem cell factor, IL-6 and prostaglandin E2. Mast cell numbers were assessed by Toluidine Blue staining and immunocytochemistry of tryptase positive cells. The expression of Fc epsilon RI, CD49d and c-kit was assessed by flow cytometry. Histamine release was determined by a radioenzymatic assay. Cys-LT, GM-CSF and TNF-alpha production and release were determined by ELISA. RESULTS: Dexamethasone (10(-6) M-10(-9) M) time- and dose-dependently inhibited the maturation of the mast cell progenitors. Dexamethasone did not affect the basal expression of Fc epsilon RI, CD49d and c-kit, but it inhibited the IgE-dependent enhanced expression of Fc epsilon RI. Dexamethasone (10(-6) M-10(-9) M) had no significant effect on Fc epsilon RI-dependent histamine release or the synthesis and release of Cys-LT from the mature mast cells. However, pre-incubation of the mast cell cultures with dexamethasone for 1 h, prior to cross-linking of Fc epsilon RI, dose-dependently inhibited the production and secretion of both GM-CSF and TNF-alpha. CONCLUSIONS: From these in vitro data we propose that glucocorticosteroids are effective drugs in the management of allergic inflammation due to their capacity to inhibit mast cell development, IgE-dependent Fc epsilon RI expression and mast cell production of GM-CSF and TNF-alpha.     

FK506 inhibition of histamine release and cytokine production by mast cells and basophils

Histamine release and cytokine production by mast cells and basophils are thought to be closely involved in the pathogenesis of allergic diseases. Some reports show that FK506 (tacrolimus hydrate) inhibited histamine release and cytokine production by mast cells and basophils. However, as the effects of FK506 has not been compared with those of clinically used drugs in those reports, the clinical relevancy of FK506 inhibition remained unclear. In this paper, we compared the actions of FK506 with those of steroids or disodium cromoglycate (DSCG) which has been clinically used. FK506 inhibited histamine release by Brown-Norway rat peritoneal mast cells more potently than steroids and especially DSCG. FK506 also inhibited histamine release by a mast rat basophilic leukemia (RBL)-1 cell line and human peripheral blood basophils, whereas steroids failed to inhibit histamine release by human basophils. FK506 as well as steroids inhibited TNF-alpha and IL-4 production by RBL-1 cells. FK506 was therefore more effective than steroids and DSCG in inhibiting histamine release, and it also had the ability of inhibiting cytokine production by mast cells as steroids do. We concluded that FK506 might regulate allergic diseases via these actions, judging from the viewpoint of clinical relevancy.     

Prospective randomized clinical study in general surgery comparing a new low molecular weight heparin with unfractionated heparin in the prevention of thrombosis

A prospective, randomized, controlled clinical trial was performed comparing the antithrombotic efficacy of the low molecular weight heparin LMWH 21–23, (Braun) with an unfractionated heparin in elective general surgical patients over an observation period of 7 postoperative days. A total of 230 patients were admitted: 103 (group I) received low molecular weight heparin and 100 (group II) low-dose unfractionated heparin treatment given subcutaneously. In group I 41 patients (46%) were operated on for malignant disease and in group II 54 patients (54%). Due to the large amount of great abdominal procedures the intra- and perioperative application of hydroxyethyl starch was allowed for volume substitution. None of the patients died due to fatal pulmonary embolism. In group I four patients revealed positive ¹²⁵I-labeled fibrinogen uptake (3.9%); two patients belonged to the hydroxyethyl starch subgroup. In group II five patients displayed a positive fibrinogen uptake (5%); two belonged to the hydroxyethyl starch subgroup. The results of the hemostaseological investigations (e.g., prothrombin time, activated partial thromboplastin time, thrombin clotting time, fibrinogen, antithrombin III, protein C, plasminogen, ₂-antiplasmin , tissue-type plasminogen activator, plasminogen activator inhibitor) revealed no statistically significant differences between groups I and II or their subgroups, although a tendency to prolonged clotting times was observed. The antifactor Xa activity values, however, displayed a statistically significant difference between the two groups (P < 0.05). The antifactor Xa activity measured up to 0.16 U/ml for the low molecular weight heparin (group I) and 0.05 U/ml for the unfractionated heparin (group II) in the postoperative period. Major bleeding complications were not encountered in this study. Fundamental for the low incidence of thrombosis, the additive administration of hydroxyethyl starch appeared in almost one-third of the patients in both study groups.Abbreviations APTT activated partial thromboplastin time - aXa anti-factor Xa - DVT deep venous thrombosis - FUT ¹²⁵I-labeled fibrinogen uptake test - HES hydroxyethyl starch - LMWH low molecular weight heparin - PE pulmonary embolism - UFH unfractioned heparin     

低分子肝素联合表皮细胞生长因子对吸入性损伤大鼠肺治疗效果的研究

目的探讨低分子肝素（LMWH）联合表皮细胞生长因子（EGF）对吸入性损伤后大鼠肺水清除及怖功能恢复的治疗作用。方法将40只Wistar大鼠随机分为4组：正常对照组、致伤埘照绀、EGF组和EGF联合LMWH组。正常对照组不做致伤处理,气管内滴入生理盐水,其余3组致伤后分别于气管内滴注生理盐水、EGF液、EGF和LMWH混合液。各组大鼠气管内淌入的液体总量均为2ml／kg。各组大鼠分别于致伤72h后取右肺做肺组织干湿比,左肺做组织病删切片和Brdu免疫组化观察肺卜皮细胞的增生情况。结果显微镜下观察,正常对照绀大鼠忡组织结卡句正常。敛伤对照组肺泡壁破坏严重,肺泡腔内可见大量粒细胞聚集,肺含水芾升高。EGF组和EGF联合LMWH组的上述病埋变化较轻,Brdu标记的肺上皮细胞数增多,忡禽水量降低。与EGF组相比,EGF联合LMWH组肺泡壁较完整,有较多增生的肺上皮细胞（P〈0．05）,且肺含水量下降（P〈0．05）。结论LMWH与EGF联合气管内给药可以减轻吸入性损伤后引起的肺水肿,促进肺上皮细胞增牛,有利于伤后肺功能的恢复。     

Differential modulation of mediator release from human basophils and mast cells by mizolastine

BACKGROUND: Basophils and mast cells play a major role in the pathogenesis of allergic disorders by releasing several proinflammatory mediators. Some histamine H1 receptor antagonists exert anti-inflammatory activities by modulating mediator release from basophils and mast cells. OBJECTIVE: To study the in vitro effects of mizolastine, an H1 receptor antagonist, on the release of eicosanoids, histamine and IL-4 from human basophils and lung mast cells. METHODS AND RESULTS: Mizolastine (10(-7)-10(-5) M) concentration-dependently inhibited the release of cysteinyl leukotriene C4 from anti-IgE-stimulated basophils (IC(50): 3.85+/-0.28 microM) and mast cells (IC(50): 3.92+/-0.41 microM). The same concentrations of mizolastine did not affect anti-IgE-induced prostaglandin D2 release from lung mast cells. In contrast, mizolastine enhanced up to 80% IgE-mediated histamine release (EC(50): 4.63+/-0.14 microM) from basophils, but not from mast cells and it significantly potentiated IL-4 release from basophils induced by anti-IgE. Mizolastine did not affect histamine release from basophils induced by formyl peptide, whereas it inhibited cysteinyl leukotriene C4 release (IC(50): 1.86+/-0.24 microM). Blockade of cytosolic phospholipase A2 and arachidonic acid mobilization by pyrrolidine-1 did not alter the effect of mizolastine on histamine release from basophils, thereby excluding accumulation of arachidonic acid metabolic intermediates as the cause of this effect. Mizolastine did not influence anti-IgE-induced activation of extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) in human basophils. CONCLUSIONS: Mizolastine efficiently inhibits LTC4 synthesis in human basophils and mast cells presumably by interfering with 5-lipoxygenase. In contrast, it enhances histamine and IL-4 release only from anti-IgE-stimulated basophils. Therefore, mizolastine differentially regulates the production of mediators from basophils and mast cells in a cell- and stimulus-specific fashion.     

弹力绷带加压包扎联合低分子肝素对全髋关节置换患者下肢深静脉血栓的影响

目的探讨弹力绷带下肢加压包扎联合低分子肝素(Low molecular weight heparin,LMWH)预防全髋关节置换(Total hip arthroplasty,THA)患者下肢深静脉血栓(Deep venous thrombosis,DVT)的临床效果。方法回归分析2014年1月~2015年10月初次行全髋关节置换病例120例,所有患者皆使用低分子肝素抗凝以预防血栓形成,根据术后下肢是否采用弹力绷带加压包扎分为观察组和对照组。记录并观察术前血小板(Platelet,PLT)、活化部分凝血活酶时间(Activated partial thromboplastin time,APTT)、凝血酶原时间(Prothrombin time,PT)、甘油三酯(Triglyceride,TG)、纤维蛋白原(Fibrinogen,FIB)、总胆固醇(Cholesterol,CHOL)、手术时间及术后引流量;采用彩色多普勒检查评估深静脉血栓形成情况。结果两组患者基线特征一致,具有可比性。术前两组PLT、APTT、PT、TG、FIB、CHOL及手术时间比较,差异无显著性意义(0.05)。观察组术后引流量高于对照组,但两组比较,差异无显著性意义(0.05)。术后深静脉血栓形成观察组少于对照组,但两组经卡方检验,差异无显著性意义(0.05)。结论弹力绷带下肢加压包扎联合低分子肝素预防THA术后深静脉血栓形成与单独使用低分子肝素比较无明显统计学差异,其是否能够明显减少DVT数量尚需扩大样本量进行深入研究。     

Protease–proteoglycan complexes of mouse and human mast cells and importance of their β-tryptase–heparin complexes in inflammation and innate immunity

Summary:  Approximately 50% of the weight of a mature mast cell (MC) consists of varied neutral proteases stored in the cell's secretory granules ionically bound to serglycin proteoglycans that contain heparin and/or chondroitin sulfate E/diB chains. Mouse MCs express the exopeptidase carboxypeptidase A3 and at least 15 serine proteases [designated as mouse MC protease (mMCP) 1–11, transmembrane tryptase/tryptase γ/protease serine member S (Prss) 31, cathepsin G, granzyme B, and neuropsin/Prss19]. mMCP-6, mMCP-7, mMCP-11/Prss34, and Prss31 are the four members of the chromosome 17A3.3 family of tryptases that are preferentially expressed in MCs. One of the challenges ahead is to understand why MCs express so many different protease–proteoglycan macromolecular complexes. MC-like cells that contain tryptase–heparin complexes in their secretory granules have been identified in the Ciona intestinalis and Styela plicata urochordates that appeared approximately 500 million years ago. Because sea squirts lack B cells and T cells, it is likely that MCs and their tryptase–proteoglycan granule mediators initially appeared in lower organisms as part of their innate immune system. The conservation of MCs throughout evolution suggests that some of these protease–proteoglycan complexes are essential to our survival. In support of this conclusion, no human has been identified that lacks MCs. Moreover, transgenic mice lacking the β-tryptase mMCP-6 are unable to combat a Klebsiella pneumoniae infection effectively. Here we summarize the nature and function of some of the tryptase–serglycin proteoglycan complexes found in mouse and human MCs.     

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) induced in human CD4⁺ and CD8⁺ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4⁺ cells can make IL-2 than the other two cytokines, and that there are more TNF-α-positive CD4⁺ cells than cells with IFN-γ. For normal CD8⁺ cells the highest production of cytokine is of IFN-γ (up to 50% of the cells) especially at longer times (10–20h) of stimulation. For CD8⁺ cells, IL-2-positive cells exceed those with TNF-α. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4⁺ or CD8⁺ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-α but significantly raised levels of production of IFN-γ in both CD4⁺ and CD8⁺ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.