Application of single-cell real-time imaging flow cytometry in rapid detection of pathogenic fungi in clinical liquid specimens.

Rapid and direct observation of fungal spores or hyphae in clinical liquid specimens poses a challenge for the diagnosis of invasive fungal infection. To allow rapid detection of fungal pathogens, we designed a new method of fungal cell detection involving double fluorescence staining with calcium fluorescent white (CFW) and SYTOX green combined with single-cell real-time imaging flow cytometry (IFC). IFC allowed quick detection and analysis of detailed morphology of the spores and pseudohyphae of Candida albicans, and small hyphae and typical truncated large mycelia of Aspergillus fumigatus. Further, cell sorting based on fluorescence, the width-to-height ratio and bright-field parameters preferentially identified spores or hyphae with a typical cell wall. The specificity and overall coincidence rate of IFC for fungi detection in common clinical samples were 100% and 98.18%, respectively. Moreover, the detection rate by IFC (102/105, 97.14%) was significantly higher (P = 0.002) than that by wet mount method (89/105, 84.5%). Therefore, IFC is a reliable diagnostic method with a high potential for application for rapid diagnosis of fungal infection in the clinic.

Deficient IL-2 Produced by Activated CD56+ T Cells Contributes to Impaired NK Cell-Mediated ADCC Function in Chronic HIV-1 Infection.

Background: Antibody-dependent cellular cytotoxicity (ADCC), which mainly mediated by natural killer (NK) cells, may play a critical role in human immunodeficiency virus type-1 (HIV-1) disease progression. However, the potential mechanisms that affecting NK-mediated ADCC response are still not well-elucidated. Methods: Antigen-antibody complex model of Ab-opsonized P815 cells was adopted to induce a typical non-specific ADCC response. The capacities of HIV-1 specific NK-ADCC were measured by using the combination model of gp120 protein and plasma of HIV-1 elite controllers. The levels of plasma cytokine were measured by ELISA. Anti-IL-2 blocking antibody was used to analyze the impact of activated CD56+ T cells on NK-ADCC response. Results: IL-2, IL-15, IFN-α, and IFN-ß could effectively enhance the non-specific and HIV-1-specific NK-ADCC responses. Compared with healthy controls, HIV-1-infected patients showed decreased plasma IL-2 levels, while no differences of plasma IFN-α, IL-15, and IFN-ß were presented. IL-2 production was detected from CD56+ T cells activated through antibody-dependent manner. The capability of NK-ADCC could be weakened by blocking IL-2 secretion from activated CD56+ T cells. Although no difference of frequencies of CD56+ T cells was found between HIV-1-infected patients and healthy controls, deficient IL-2 secretion from activated CD56+ T were found in chronic HIV-1 infection. Conclusions: The impaired ability of activated CD56+ T cells to secreting IL-2 might contribute to the attenuated NK cell-mediated ADCC function in HIV-1 infection.

Intracellular cytokine staining (ICS) for detection of NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) / 中华微生物学和免疫学杂志

Objective To analyze the possibility of using intracellular cytokine staining (ICS) to evaluate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and to detect the changes in ADCC activity among patients with chronic HIV and/or HCV infection. Methods Flow cytometry was per-formed to determine the percentages of NK cells and the expression of NK cell receptors. ImageStreamX MarkⅡ system was used to identify the expression of CD3, CD56, CD16 and CD32 on CD56brightNK and CD56dimNK subsets. Degranulation process and cytokine production in NK cells were detected using an anti-gen-antibody complex model of P815/Ab in combination with ICS. Differences in NK cell-mediated ADCC were evaluated among patients infected with HIV and/or HIV and healthy subjects by flow cytometry. Re-sults The percentages of CD107a+and IFN-γ+NK cells were positively correlated with the decrease of mean fluorescence intensity (MFI) of CD16. ICS assay revealed a positive correlation between the secretion of CD107a and IFN-γ by NK cells. CD16 was highly expressed in CD56dimNK cells. The ADCC mediated by CD56dimNK cells was stronger than that mediated by CD56brightNK cells. The rate of target cell lysis detected by rapid fluorescence assay was positively correlated with the percentage of CD107a+/IFN-γ+NK cells meas-ured by ICS. NK cell-mediated ADCC was suppressed in patients with chronic HIV and/or HCV infection. Conclusion This study suggests that ICS assay could be used to evaluate NK cell-mediated ADCC. It also reveals that NK cell-mediated ADCC is suppressed in patients with chronic HIV and/or HCV infection.