The DevOps 2.1 toolkit: Docker Swarm : building, testing, deploying, and monitoring services inside Docker Swarm clusters

The expressions of the CD106 gene (VCAM1), CXCL12, CCL2, and IL-6 genes were detected by quantitative real-time polymerase chain reaction (qRT-PCR). (PDF 1180 kb

Pre-transplantation thymic function is associated with the risk of acute graft versus host disease and cytomegalovirus viremia after allogeneic hematopoietic stem cell transplantation

<p><b>Objectives:</b> To analyze the kinetics of T-cell subsets and thymic function reconstitution after allogeneic hematopoietic stem cell transplantation (AHSCT); to determine whether sjTREC (signal joint TCR rearrangement excision circle) and CD31-positive recent thymic emigrant (CD31 + RTE) are correlated with acute graft versus host disease (aGVHD) or CMV (cytomegalovirus) viremia after AHSCT.</p> <p><b>Methods:</b> Forty-nine patients who underwent AHSCT in our institution were prospectively enrolled. Periphery blood samples were collected before conditioning and at 1, 2, 3 months after AHSCT. T-cell subsets were analyzed with flow cytometry. Genomic DNA was purified from peripheral blood mononuclear cells (PBMCs), and sjTREC was quantified by real-time PCR. Impact of sjTREC and CD31 + RTE on aGVHD and CMV viremia was evaluated by univariate and multivariate Cox regression analyses.</p> <p><b>Results:</b> The analyzed T-cell subsets and sjTREC of patients before AHSCT were all significantly lower than those of healthy donors (<i>p</i> < 0.05). sjTREC and CD31 + RTE were remarkably decreased in 3 months after AHSCT (<i>p</i> < 0.05). Patients with lower pre-transplantation sjTREC and CD31 + RTE level had higher incidence of CMV viremia after AHSCT (<i>p</i> < 0.05). sjTREC/10⁶ PBMCs was negatively correlated with aGVHD (<i>p</i> = 0.024).</p> <p><b>Conclusion:</b> Thymic function was impaired before transplantation, and was consistently decreased in 3 months after AHSCT. Patients who had lower pre-transplantation sjTREC level were at high risk of aGVHD and CMV viremia after AHSCT, low pre-transplantation CD31 + RTE was correlated with CMV viremia after AHSCT.</p

Extracellular Acidification Acts as a Key Modulator of Neutrophil Apoptosis and Functions

<div><p>In human pathological conditions, the acidification of local environment is a frequent feature, such as tumor and inflammation. As the pH of microenvironment alters, the functions of immune cells are about to change. It makes the extracellular acidification a key modulator of innate immunity. Here we detected the impact of extracellular acidification on neutrophil apoptosis and functions, including cell death, respiratory burst, migration and phagocytosis. As a result, we found that under the acid environment, neutrophil apoptosis delayed, respiratory burst inhibited, polarization augmented, chemotaxis differed, endocytosis enhanced and bacteria killing suppressed. These findings suggested that extracellular acidification acts as a key regulator of neutrophil apoptosis and functions.</p></div

Neutrophils in acid medium were more sensitive to the fMLP-induced polarization.

<p>(A) fMLP (concentration ranged from 0nM to 10μM,) induced ruffling in neutrophils cultured in medium of pH 6.0 and pH 7.4. Images were captured every 5 sec for 10 min. (B, C)At 3 min and 5 min of fMLP stimulation, percentage of polarization cells were calculated in each group of different fMLP concentration. Data are from three independent experiments(B, C) or are representative of three experiments(A). (***, <i>p</i><0.001, **, <i>p</i><0.01, *, <i>p</i><0.05).</p

Extracellular acid enhance neutrophil endocytosis but suppress the bacteria killing ability.

<p>(A,B,C,D) Neutrophils endocytose FITC-Zymosan. 2×10⁶ neutrophils were mixed with 1×10⁷ opsonized FITC-Zymosan and co-incubated at 37°C for 30 min. Pictures were captured by a fluorescence microscope(600×). (A) Percentage of neutrophils happened to phagocytose. More than 100 cells were counted from random fields of each group. (B) Phagocytosis index was expressed as the number of the internalized particles per 100 neutrophils. (C) Binding index was expressed as the number of the binding particles per 100 neutrophils. (D) Fluorescence images of neutrophil phagocytosis. (E,F,G)Killing assay using neutrophils incubated with <i>E</i>.<i>Coli</i>.2×10⁶ neutrophils were mixed with 1×10⁷ opsonized <i>E</i>.<i>Coli</i> and co-incubated at 37°C for 30min. (E) Cells containing <i>E</i>.<i>Coli</i> were lysed, diluted and spread on the LB agar. CFUs of this group stand for the bacteria loading. Another two groups of neutrophils containing <i>E</i>.<i>Coli</i> were further incubated in the medium of pH 6.0 and pH 7.4 separately. After incubated for another 30 min, cells were lysed, diluted and spread on the LB agar. (F) The number of CFUs of each group were listed in the table. (G)</p><p></p><p></p><p>killing percentage<mo>=</mo></p><p><mo>(</mo></p><p><mn>1</mn><mo>-</mo></p><p></p><p>CFUs of pH 6.0/7.4</p><p>bacteria loading</p><p></p><p></p><mo>)</mo><p></p><mo>×</mo><mn>100</mn><mi>%</mi><p></p><p></p><p></p>. Data are from three independent experiments(A,B,C) or are representative of three experiments(D,E,F,G,H,I). (***,<i>p</i><0.001, **, <i>p</i><0.01, *,<i>p</i><0.05).<p></p

Freshly sorted normal adult human bone marrow CD34⁺ cells had higher autophagic activity than lymphocytes and neutrophils.

<p><b>(A) and (D):</b> TEM (×10,000). Autophagosomes and the mean number of vacuoles (autophagosomes) in CD34⁺ cells, lymphocytes and neutrophils. CD34⁺ cells contain more vacuoles (autophagosomes) than lymphocytes and neutrophils. In each cell population, the mean number of vacuoles per cell (mean±SEM) is presented. <b>(B) and (E)</b>: Confocal microscopy (×1,000). LC3B fluorescence and mean LC3B fluorescence density is shown. CD34⁺ cells exhibit higher mean fluorescence density than lymphocytes and neutrophils. <b>(C) and (F)</b>: NanoPro1000^TM system. The LC3-I and LC3-II peaks and the ratio of the LC3-II peak area normalized to GAPDH is shown. CD34⁺ cells have higher levels of LC3-II than lymphocytes and neutrophils.</p

Levels of autophagy in healthy adult bone marrow CD34⁺ cells at different time-points of nutrient deprivation.

<p>Freshly sorted CD34⁺ cells were cultured for 12 h and placed in PBS for 0 min, 30 min, 60 min and 120 min after harvest. <b>(A) and (C)</b>: Analysis of LC3 protein using the NanoPro1000^TM system. The LC3-I and LC3-II peaks and the ratio of LC3-II peak area normalized to GAPDH at different time-points is shown. The LC3-II peak and peak area rapidly increased with the duration of nutrient deprivation. <b>(B) and (D)</b>: Analysis of LC3B using confocal microscopy. LC3B fluorescence and mean LC3B fluorescence density is presented. The LC3B fluorescence density in CD34⁺ cells was weak at 0 min and rapidly increased with the duration of nutrient deprivation.</p

Superoxide production of neutrophils was inhibited in acid environment.

<p>(A, E)Fluorescent probe DCFH-DA. 5×10⁵ cells were labeled with DCFH-DA and stimulated by 1μM fMLP for 10 min. Fluorescence intensity were read by the Microplate Reader(A) and images were captured using Leica laser scanning confocal microscope (E). (B)Luminol chemiluminescence assay of neutrophils ROS production. Purified neutrophils in different pH medium were stimulated by 1μM fMLP in the presence of lunimol (50μM) and horseradish peroxidase(HRP, 4U/ml). ROS production was monitored in a luminometer at 37°C. Cells pre-treated with 25μM DPI were as negative control. (C, D) Quantification of F-actin of neutrophils in acid and neutral medium. At the 3^th minute of fMLP(10nM) stimulation, cells were fixed and stained with rhodamine-phalloidin(Red) and DAPI(Blue), the fluorescence intensity of rhodamine-phalloidin was read by the Microplate Reader(D). Pictures were taken by fluorescence microscope(C). (F)Extracellular acid increase the Akt phosphorylation of neutrophils Purified neutrophils were cultured in the medium of pH 6.0 to7.4 for 2h, Protein extracts were resolved on SDS/PAGE. Total and phosphorylated Akt were detected by Western blot using anti-Akt and anti-phospho-Akt (Ser473) antibodies. (G) The glutathionylation of actin was down regulated in neutrohils in acid environment. Neutropihls cultured for 4h were collected and lysed by RIPA. Anti-actin antibody and protein A/G agrose beads were used to pull down and anti-glutathione antibody were used in the western blot to detect the glutathionylated actin. Data are from three independent experiments(A,D) or are representative of three experiments(B,C, E,F,G). (***,<i>p</i><0.001, *,<i>p</i><0.05).</p

Autophagy effects on the survival of CD34⁺ cells.

<p><b>(A) and (B):</b> CD34⁺ cells were treated with 3-MA or CQ, and the number of cells was counted at days 4, 7 and 10. 3-MA and CQ were able to inhibit proliferation of CD34⁺ cells in a concentration-dependent manner. The results shown are the mean±SEM of three independent experiments. <b>(C) and (D):</b> CFC assay. CD34⁺ cells were treated with 3-MA or CQ, and colonies were counted at day 10 and normalized to control conditions. 3-MA and CQ inhibited differentiation of CD34⁺ cells in a concentration-dependent manner. The results shown are the mean±SEM of three independent experiments. <b>(E) and (F):</b> Trypan blue assays revealing the percentage of dead cells. CD34⁺ cells were treated with 3-MA or CQ for 48 h prior to application of trypan blue. 3-MA and CQ induced the death of CD34⁺ cells in a concentration-dependent manner. The data represent the mean±SEM of three independent experiments. <b>(G-I)</b>: Apoptosis detection by AnnexinV-FITC/PI assays. CD34⁺ cells were treated with 3-MA or CQ for 24 h, followed by staining with Annexin-V/PI. 3-MA and CQ induced the apoptosis of CD34⁺ cells in a concentration-dependent manner. <b>G</b> and <b>H</b> represent the mean±SEM of three independent experiments, with the Q2 quadrant (Annexin V+/PI+) and Q3 quadrant (Annexin V+/PI-) in <b>I</b> representing the percentages of early and late apoptotic cells, respectively. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Improved autophagy in CD34⁺ cells from AA patients with the amelioration of disease.

<p>All freshly sorted CD34⁺ cells were analyzed immediately after isolation without culture. <b>(A) and (D)</b>: NanoPro1000^TM system. The LC3-I and LC3-II peak and the ratio of the LC3-II peak area normalized to GAPDH of each sample is presented (NR AA group, n = 9; PR AA group, n = 7; CR AA group, n = 7; control group, n = 4). With the amelioration of AA, the level of LC3-II increased but was still lower than in healthy controls. <b>(B) and (E)</b>: Confocal microscopy (×1,000). The LC3B fluorescence and mean LC3B fluorescence density of CD34⁺ cells is shown (NR AA group, n = 5; PR AA group, n = 7; CR AA group, n = 5; control group, n = 3). With the amelioration of AA, the mean LC3B fluorescence density increased, but was still lower than in healthy controls. <b>(C) and (F)</b>: Confocal microscopy (×1,000). The MDC fluorescence and mean MDC fluorescence density of CD34⁺ cells is shown (NR AA group, n = 5; PR AA group, n = 6; CR AA group, n = 7; control group, n = 3). With the amelioration of AA, the mean MDC fluorescence density increased, but was still lower than in healthy controls. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p