An efficient and cost-effective method for the purification of human basophils.

Human basophils are terminally differentiated granulocytes that are least abundant in the peripheral blood but play important roles in allergic diseases. Studies on human basophils are limited by the high cost on the isolation of human basophils by magnetic-activated cell sorting (MACS) for negative depletion of non-basophils, followed by CD123-based positive selection of basophils. Moreover, such CD123-based purification of basophils may be limited by blocking of the binding of IL-3/anti-CD123 to the surface CD123. Here we identified SSClow CD4- CD127- HLA-DR- CRTH2high as unique markers for the identification of human basophils through stringent flow cytometric analysis of leukocytes from buffy coat. We established an efficient and cost-effective method for isolating human basophils from buffy coat based on positive magnetic selection of CRTH2+ cells followed by flow cytometric sorting of SSClow CD4- CD127- HLA-DR- CRTH2high cells. Approximately 1 to 1.5 million basophils were isolated from one buffy coat with a purity of >97%. Basophils purified by this method were viable and efficiently responded to key regulators of basophils including IL-3 and anti-IgE. This method can be used for purifying human basophils for subsequent functional studies.

Induction of the apoptosis, degranulation and IL-13 production of human basophils by butyrate and propionate via suppression of histone deacetylation.

Allergic diseases are caused by dysregulated Th2 immune responses involving multiple effector cells including basophils. Short chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, exert immunomodulatory functions via activation of its receptors GPR41 and GPR43, and inhibition of the histone deacetylases (HDACs) activity. In allergic diseases, SCFAs suppress the activity of mast cells, eosinophils and type 2 innate lymphoid cells (ILC2) but enhance the function of Th2 cells. Here, we aimed to elucidate the function of SCFAs on human basophils. Human basophils were purified from healthy donors by flow cytometric sorting. The surface proteins, apoptosis and degranulation of basophils were analyzed by flow cytometric analysis. The mRNA expression was assayed using real-time PCR. Interleukin 4 (IL-4) and IL-13 were measured by ELISA. Histone acetylation was examined by western blot. GPR41 was expressed by basophils and was enhanced by IL-3. Acetate induced intracellular calcium influx in basophils which was suppressed by blocking GPR41. Propionate and butyrate, but not acetate, induced the expression of CD69 and IL-13. In addition, propionate and butyrate enhanced IgE-mediated basophil degranulation but inhibited basophil survival and IL-4 secretion. Propionate and butyrate induced histone acetylation of basophils and suppression of HDACs activity mimicked the effects of propionate and butyrate on human basophils. Our findings demonstrate that propionate and butyrate may play a complex role in regulating basophil apoptosis, activation and degranulation via inhibiting HDACs activity. The in vivo effects of SCFAs on the regulation of basophil-associated allergic diseases need to be further explored.

Interleukin 2 regulates the activation of human basophils.

Basophils are important effector cells in allergic disorders and anti-parasitic immune response. A number of activators including interleukin 3 (IL-3) and IgE have been identified in the regulation of human basophils expressing mediators such as histamine and leukotriene C4 (LTC4) and cytokines, including IL-4 and IL-13. Human basophils express high levels of IL-2 receptors. However, the function of the IL-2 pathway in basophils remains unknown. Here, we identified that IL-2 induced the activation of human basophils in vitro to express a variety of inflammatory cytokines and chemokines including IL-5, IL-13, GM-CSF and CCL-17. This effect by IL-2 is confirmed by an upstream regulator analysis using Ingenuity pathway analysis. Of note, one of the top regulated cytokines, IL-5, was for the first time identified to be induced by IL-2 in human basophils rather than IL-3 or anti-IgE. Immunofluorescence analysis of skin specimens from bullous pemphigoid and eczema revealed that infiltrating basophils in skin lesions widely expressed IL-5 and GM-CSF. Together, our findings reveal IL-2 as a novel regulator of human basophils. This adds a new layer to support the importance of basophils in allergic disorders.

Interleukin 3-induced GITR promotes the activation of human basophils.

Human basophils regulate allergic reactions by secreting histamine, interleukin 4 (IL-4) and IL-13 through key surface receptors FcεRI as well as IL-3R, which are constitutively expressed on basophils. IL-3/IL-3R signaling axis plays key roles in regulating the development and activation of basophils. We and others have shown that IL-3-induced surface receptors e.g. ST2, IL-17RB and IL-2 receptors regulate the biology of basophils. However, the expression and function of IL-3-induced surface proteins on human basophils remain to be elucidated. We in this study aimed to identify new basophil activation regulators by transcriptomic analysis of IL-3-stimulated basophils. Gene expression microarray analysis of IL-3-treated basophils revealed 2050 differentially expressed genes, of which 323 genes encoded surface proteins including GITR. We identified that GITR was preferentially induced by IL-3 rather than anti-IgE, IL-33, fMLP and C5a. IL-3-induced GITR was suppressed by inhibitors targeting JAK2, PI3K and MEK1/2. Stimulation of IL-3-treated basophils by GITR enhanced the expression of IL-4 and IL-13. Moreover, IgE-mediated degranulation was enhanced by GITRL in the presence of IL-3. This transcriptomic analysis of IL-3-activated basophils helps to identify novel activation regulator. IL-3-induced GITR promoted the activation of basophils, adding new evidence supporting GITR as an important player in Th2-associated immune responses.

Bayesian evaluation of the human immunodeficiency virus antibody screening strategy of duplicate enzyme-linked immunosorbent assay in Xuzhou Blood Center, China.

BACKGROUND: Accurate estimation of the risk of human immunodeficiency virus (HIV) infection through transfusion is essential for monitoring blood safety. The risk, however, is so low that it can only be estimated by mathematical modeling. With the Bayesian dependence model, this study evaluates the HIV antibody screening strategy of duplicate enzyme-linked immunosorbent assay (ELISA) in Xuzhou Blood Center and therefore estimates part of the total risks of transfusion-transmitted HIV infection. STUDY DESIGN AND METHODS: Data from Xuzhou Blood Center between 2004 and 2008 were used. Information was obtained on donor profiles and screening and confirmatory test results. The portion of the risks of HIV infection through transfusion concerned was estimated by evaluating the screening algorithm in terms of its accuracy and predictive power with the Bayesian dependence model. RESULTS: A total of 234,602 donations from voluntary blood donors in Xuzhou Blood Center were screened for HIV antibody. For the study screening algorithm, its sensitivity, specificity, false-positive predictive value (FPPV), and false-negative predictive value (FNPV) were 0.9951 (95% Bayesian credible interval [BCI], 0.9763-0.9997), 0.9991 (95% BCI, 0.9990-0.9992), 0.9647 (95% BCI, 0.9018-0.9923), and 1.52 × 10(-7) (95% BCI, 7.31 × 10(-9) -1.15 × 10(-6) ), respectively. For the positive detection rate (9.60 × 10(-4) ) and FPPV (0.9647), the differences between their own Bayesian median estimates and real values were 2.70 × 10(-5) and -0.0033, respectively. CONCLUSIONS: The HIV antibody screening algorithm of duplicate ELISA is well evaluated in its accuracy and predictive power with the Bayesian dependence model. The FNPV measures the part of the risks of transfusion-associated HIV transmission concerned.

The false-positive and false-negative predictive value of HIV antibody test in the Chinese population.

OBJECTIVES: To analyse the relationship between the false-positive/false-negative predictive value (FPPV/FNPV) of the HIV-antibody (HIV-Ab) test and prevalence in different Chinese population groups. METHODS: HIV prevalence among different population groups was obtained by a screening survey of blood donors and the national HIV/AIDS surveillance programme in China. Given the sensitivity and specificity of a test kit and the prevalence of HIV infection, the estimated values of FPPV/FNPV were calculated using Bayes' formula. The actual value of FPPV of blood donors was obtained by screening 1,195,286 blood donors. RESULTS: This study indicates that the FPPV of HIV-Ab enzyme-linked immunosorbent assay (ELISA) assays varies widely in different Chinese populations: about 99.5% in the blood donor population, but only 3.2% in the injecting-drug users in high-risk areas. In 1,195,286 sera specimens from the blood donors, 2439 specimens were HIV-Ab positive by third ELISA, and 11 HIV cases were confirmed by Western blot. The HIV prevalence of the blood donor population in this survey was 0.0009% (11/1,195,286), but the HIV-Ab positive rate of third ELISA is 0.2% (2439/1,195,286) and 222 times higher than the prevalence. CONCLUSIONS: Evaluation of HIV prevalence through the HIV-Ab positive rate by third ELISA will significantly overestimate the true prevalence in a low-prevalence population. Individual HIV-infection status should be taken into consideration when analysing the results of HIV-Ab tests in a population with low infection.

Estimation of the sensitivity and specificity of assays for screening antibodies to HIV: a comparison between the frequentist and Bayesian approaches.

Bayesian and frequentist methods have been applied rarely to the same sets of data for evaluating assays for screening antibodies to HIV, especially for assays with relatively high sensitivities and/or specificities of 100% compared with reference assays. In this study, 95% confidence intervals and 95% Bayesian credible intervals were calculated for sensitivity and specificity for the evaluation of the accuracy of HIV antibody assays using data from China, WHO UNAIDS, USA, Australia, Tanzania, and India. When the sensitivity and/or specificity were 100%, a Bayesian approach obtained reasonable interval estimates of assays for screening antibodies to HIV, whereas frequentist methods express objectively the accuracy of each individual assay. It is suggested that the two types of estimates be reported simultaneously to evaluate more comprehensively a set of highly accurate antibodies for HIV assays.

Evaluation of 30 commercial assays for the detection of antibodies to HIV in China using classical and Bayesian statistics.

The purpose of this study was to evaluate the 30 commercial HIV-antibody (HIV-Ab) assays in the nationwide assessment program of China using classical and Bayesian statistical methods. The classical estimates of sensitivity and specificity varied from 95.9% to 100% and from 94.6% to 100%, respectively. The proportions of assays with 100% sensitivity and with 100% specificity reached 63.3% (19/30) and 3.3% (1/30), respectively. Using the Bayesian logit hierarchical model, the overall estimates of sensitivity and specificity were 99.8% (95% Bayesian credible interval [BCI]: 99.4-100%) and 98.1% (95% BCI: 97.4-98.7%), respectively, for the 17 ELISAs under evaluation. For the 13 rapid assays, the corresponding overall estimates were reported to be 99.2% (95% BCI: 98.5-99.8%) and 98.4% (95% BCI: 97.8-98.9%), respectively. In addition, given the prevalences of HIV infection among the general population of China and the intravenous drug user group in China, the positive predictive values were estimated for each individual assay in the framework of the two schools of statistical thought. Furthermore, by comparing the two types of estimates, it is concluded that the two types of statistical methods were complementary for the evaluation of very accurate HIV-Ab assays.