Biological quality control for extracorporeal photochemotherapy: Assessing mononuclear cell apoptosis levels in ECP bags of chronic GvHD patients

Extracorporeal photochemotherapy (ECP) is a treatment approved by the FDA for cutaneous T‐cell lymphoma, and it is currently used off‐label for graft‐versus‐host disease (GvHD) and other conditions. In agreement with good practices for the therapeutic use of human cells, quality control has to be performed to validate the ECP procedure with the off‐line technique. Since no gold‐standard biological test is available, we assessed the apoptosis generated in the ECP bag using a flow cytometric analysis. Thirty‐one ECP procedures performed on 13 patients with chronic GvHD were studied by monitoring the induction of mononuclear cell (MNC) apoptosis using annexin V/propidium iodide double staining; residual lymphocyte proliferation to standard mitogens was also measured in 17 of the procedures. The kinetics of apoptosis was analyzed at different times in MNCs untreated or treated with 8‐methoxy‐psoralen plus ultraviolet A; the variation (Δ_APOPTOSIS) after 24 h revealed the efficacy of the treatment. In 88.6% of the 31 ECP procedures, Δ_APOPTOSIS was >15% (the “alerting” threshold for Δ_APOPTOSIS was set at 15% on the basis of our data); in the remainder (19.4%), the increment in apoptosis was lower. In four procedures, the proliferation assay was useful for assessing the effect of ECP on the apheretic bag. In conclusion, both flow cytometric assays enabled a biologically significant result to be obtained. In our opinion, the apoptosis test—being faster and easier than the proliferation test—could be a reliable way to validate ECP procedures. J. Clin. Apheresis 30:162–170, 2015. © 2014 Wiley Periodicals, Inc.     

Eosinophil activation and cysteinyl leukotriene production in infants with respiratory syncytial virus bronchiolitis

BACKGROUND: It has been suggested that acute infantile bronchiolitis associated with respiratory syncytial virus (RSV) may share some pathogenic features with atopic asthma in that virus-specific IgE is produced and cysteinyl leukotrienes (cLTs) and eosinophil cationic protein (ECP) have been detected in airway secretions. ECP is a specific marker of eosinophil activation although leukotrienes can be released from a variety of cells including mast cells, eosinophils and monocytes. OBJECTIVE: To test the association between eosinophil activation and cysteinyl leukotriene production in the upper airway secretions of infants with RSV positive (RSV+ve) bronchiolitis. METHODS: Nasal lavage samples were performed in 78 infants (0.0-11.5 months) admitted to hospital with RSV+ve bronchiolitis soon after admission (0-48 h). Leukotriene C4 (LTC4) was assayed by enzyme immunoassay (EIA) and eosinophil cationic protein (ECP) by fluoroimmunoassay (FIA). RESULTS: LTC4 was detectable in 51 and ECP in 57 of 78 samples with a significant positive relationship between LTC4 and ECP (r=0.557, P<0.001). CONCLUSION: In the majority of our subjects with RSV+ve bronchiolitis ECP and LTC4 were detectable in upper airway secretions and were significantly associated with each other. In this clinical setting much of the detected LTC4 within upper airway secretions is likely to originate from the eosinophil, an observation that may have implications for clinical management and for delineation of the underlying mechanisms associated with this illness.     

过敏性鼻炎患者血清SIgE和ECP水平的关系探讨

目的：分析过敏性鼻炎（ Ａ Ｒ） 患者血清特异性 Ｉｇ Ｅ（ Ｓ Ｉｇ Ｅ） 和嗜酸粒细胞阳离子蛋白（ Ｅ Ｃ Ｐ） 的关系。方法：采用荧光酶联法对３８ 例 Ａ Ｒ 患者进行吸入性过敏原过筛试验、 Ｓ Ｉｇ Ｅ 和血清 Ｅ Ｃ Ｐ 含量测定。结果：吸入性过敏原过筛试验阳性率为９４ ．７ ％ 。１０例对单一变应原有反应,２６ 例检出２ ～６ 种变应原（ 户尘螨、屋尘、普通豚草、蒿草、藜草和动物皮毛屑） 的 Ｓ Ｉｇ Ｅ,测定值为０ ．３５～６３ ．６ Ｋ Ｕ／ Ｌ,血清 Ｅ Ｃ Ｐ水平为２４ ．８７ ±１０ ．５５ μｇ／ Ｌ,健康对照组为９ ．９３ ±５ ．６２ μｇ／ Ｌ,两组有显著性差异（ Ｐ＜ ０ ．０１） 。临床观察, Ｓ Ｉｇ Ｅ 含量与患者症状及体征无明显相关性,但患者的咳喘程度与血清 Ｅ Ｃ Ｐ 的浓度有关,血清中 Ｓ Ｉｇ Ｅ 含量与 Ｅ Ｃ Ｐ浓度间相关性分析没有统计学意义（ｒ＝ ０ ．１０４７ , Ｐ＞ ０ ．０５） 。结论：检查血清 Ｉｇ Ｅ 对过敏性鼻炎特异性病因诊断是非常重要的,也对该病病程发展和治疗监测有一定的参考意义。     

中药封脐疗法治疗慢性荨麻疹的临床观察及对ECP的影响

目的探讨中药封脐疗法对慢性荨麻疹(CU)临床近期、远期疗效,以及对患者细胞免疫功能的影响。方法对照组口服氯雷他定分散片(原研),治疗组加用中药封脐,观察疗效和治疗前后外周血T淋巴细胞亚群、外周血EOS计数、血清免疫球蛋白E(IgE)和血清嗜酸性阳离子蛋白(ECP)的变化。结果治疗后,治疗组CD4+升高、CD8+下降,CD4+/CD8+比值升高,IgE、ECP计数下降。治疗后6个月随访,治疗组复发率明显低于对照组。结论中药封脐疗法可增强慢性荨麻疹患者细胞免疫功能,提高疗效,减少复发。     

Subcellular fractionation of human eosinophils: Isolation of functional specific granules on isoosmotic density gradients

Subcellular fractionation has been an important tool in investigating human eosinophil structure and function, including localizing of cytokine/chemokines within granules, investigating granule protein translocation and intracellular transport during eosinophil secretion, and studying secretory mechanisms of granules. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nonionic iodinated density-gradient metrizamide and Nycodenz media that, unlike sucrose, exhibit relatively low tonicity throughout the gradient. However, the structure and membrane preservation of isolated organelles were still compromised due to the lack of gradient isoosmolarity. This paper describes a detailed protocol of subcellular fractionation of nitrogen cavitated eosinophils on an isoosmotic iodinated density gradient (iodixanol - OptiPrep) and the isolation of well preserved and functional membrane-bound specific granules.     

支气管哮喘患儿血清IL-17、ECP及IgE的水平测定及临床意义

目的 探讨支气管哮喘患儿血清白介素17(IL-17)、嗜酸细胞阳离子蛋白(ECP)及免疫球蛋白E(IgE)水平与哮喘发生、发展的关系及相关性研究。方法 对82例轻中度哮喘患儿血清IL-17、ECP及IgE水平检测,并与健康对照组的20例儿童进行比较分析。结果 与对照组比较,哮喘组血清IL-17、ECP及IgE水平显著升高,差异有统计学意义(t=70.594、19.202 、44.824,P均<0.01);与缓解组比较,急性发作组血清ECP、IgE水平显著升高,差异有统计学意义(P均<0.01),IL-17水平升高,但差异无明显统计学意义(P>0.05);哮喘组血清ECP与IgE呈正相关(r=0.337,P<0.01),IL-17与ECP、IgE之间无明显相关,但在急性发作组,IL-17与ECP、IgE呈正相关(r=0.314和0.325,P均<0.05)。结论 IL-17是一个复杂的细胞因子,参与介导哮喘的嗜酸粒细胞性炎症,ECP及IgE水平对哮喘病情判断及治疗具有重要临床意义。     

43例急性荨麻疹患者血清ECP和TIgE的水平及相关性分析

采用UniCAP-100全自动变态反应分析仪,对43例急性荨麻疹患者发疹时进行血清TIgE和ECP检测,并设20例正常人对照。结果:43例患者中血清TIgE水平为(220.6±234.3)ku/L,高于对照组(P<0.05);血清ECP为19.9±14.6,显著高于对照组(P<0.01);两者间略趋正相关(r=0.0376,t=0.241,P>0.05);发疹组血清TIgE水平与病情严重程度略趋正相关(r=0.2840,t=1.8966,P>0.05),发疹组血清ECP水平与病情严重程度呈正相关(r=0.3658,t=2.5167,0.01ECP值有助于了解急性荨麻疹的发病机制、病因、病情严重程度,对治疗有一定帮助。     

Eosinophil cationic protein- and eosinophil-derived neurotoxin/eosinophil protein X-immunoreactive eosinophils in prurigo nodularis

Abstract It is known that eosinophils are actively involved in allergy and inflammation. The granular components of eosinophils, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin/eosinophil protein X (EDN/EPX), play an important role in such allergic and inflammatory processes. Prurigo nodularis is a chronic inflammatory skin disease with obvious cutaneous nervous involvement. To detect ECP and EDN/ EPX expression in the eosinophils and their relation to nerve fibres in prurigo nodularis, ECP and EDN/EPX single-labelling immunofluorescence, and ECP and PGP 9.5 double-labelling immunofluorescence, were performed. In prurigo nodularis lesional skin, the ECP- and EDN/EPX-containing cells, which were mainly distributed in the upper dermis, were significantly increased in number compared to their numbers in uninvolved and normal skin. The immunoreactivity of ECP and EDN/EPX in prurigo lesional skin was stronger than in uninvolved skin or control skin. The PGP 9.5-immunoreactive nerves were also increased in number in the areas where there were increased eosinophils. The nerves were in close proximity to eosinophils, and occasionally even seemed to be in contact. The present results indicate that the cutaneous nerves and the ECP- and EDN/EPX-containing eosinophils are possibly involved in the pathogenesis of the disease. The close relationship of nerves and eosinophils indicates that the cutaneous nerves may influence eosinophil function in the chronic inflammatory states of prurigo nodularis. ECP and EDN/EPX could thus be released to the local tissue and modulate the inflammation of the prurigo nodularis lesion. Received: 28 August 1999 / Received: 2 January 2000 / Accepted: 20 March 2000     

Comparative immunoreactivity of the eosinophil constituents MBP and ECP in different types of urticaria

In order to elucidate the role of eosinophil constituents in urticaria, we investigated major basic protein expression immunohistologically in comparison with that of eosinophilic cationic protein and the low-affinity IgE receptor in lesional and uninvolved skin of different types of urticaria. Eosinophil activation was studied with the markers EG1 and EG2. Different eosinophil constituents were found in all urticarial lesions except those of urticaria pigmentosa. MBP staining tended to be distributed diffusely throughout the tissue, whereas EG1 and EG2 antibodies were located at or close to individual cells. Staining with the low affinity IgE receptor antibody was rare. In uninvolved skin, major basic protein and particularly eosinophilic cationic protein reactivity was found in chronic recurrent urticaria, delayed pressure urticaria and, to a minor degree, in cholinergic urticaria. No correlation was found between antibody reactivity and eosinophil counts. Reactivity with either of the eosinophil constituents is thus a better marker for eosinophil involvement than routine H&E staining of the cells. The demonstration of eosinophil constituents in nonlesional skin of some urticaria patients suggests generalized eosinophil activation in certain subtypes of the disease.     

Effects Of Theophylline Withdrawal in Well-Controlled Asthmatics Treated with Inhaled Corticosteroid

We examined effects of theophylline withdrawal in 17 adult asthmatics whose symptoms were well controlled under a treatment of a combination of theophylline and inhaled beclomethasone dipropionate (iBDP). We measured daily symptoms, daily peak flow values, spirometry, peripheral blood eosinophil count (EOS), and serum eosinophil cationic protein (sECP) at intervals of 1-3 weeks for 3 months after theophylline withdrawal. Twelve patients experienced exacerbation of asthma (exacerbation group), whereas the remaining 5 patients exhibited no symptoms (stationary group). In the exacerbation group, forced expiratory volume in 1 sec (FEV₁) and percent vital capacity (%VC) gradually decreased until exacerbation of asthma, and the extent of these decreases within the first week after the withdrawal was greater compared with that at later than the third week. decreased in both the exacerbation and stationary groups. In particular, the extent of the velocity of expiratory flow at 25% of the vital capacity/height () decrease in the exacerbation group was much greater than that of FEV₁ or %VC in this group. Neither EOS nor sECP changed significantly during the clinical course in any patient. The rapid decrease in FEV₁ and %VC after the withdrawal suggests that under treatment with iBDP, theophylline causes direct bronchodilating effects on smooth muscle, rather than anti-inflammatory effects. These results also suggest the importance of theophylline on peripheral as well as central airways.