11 research outputs found
Cytokine profiles of umbilical cord blood mononuclear cells upon in vitro stimulation with lipopolysaccharides of different vaginal gram-negative bacteria
No full textInflammatory immune responses induced by lipopolysaccharides (LPS) of gram-negative bacteria play an important role in the pathogenesis of preterm labor and delivery, and in neonatal disorders. To better characterize LPS-induced inflammatory response, we determined the cytokine profile of umbilical cord blood mononuclear cells (UBMC) stimulated with LPS of seven vaginal gram-negative bacteria commonly found in pregnant women with preterm labor and preterm rupture of membrane. UBMC from ten newborns of healthy volunteer mothers were stimulated with purified LPS of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter calcoaceticus, Citrobacter freundii, and Pseudomonas aeruginosa. UBMC supernatants were tested for the presence of secreted pro-inflammatory cytokines (IL-6, IL-1β, TNF), anti-inflammatory cytokine (IL-10), TH1-type cytokines (IL-12, IFN-γ), and chemokines (IL-8, MIP-1α, MIP-1β, MCP-1) by Luminex technology. The ten cytokines were differentially induced by the LPS variants. LPS of E. coli and E. aerogenes showed the strongest stimulatory activity and P. aeruginosa the lowest. Interestingly, the ability of UBMC to respond to LPS varied greatly among donors, suggesting a strong individual heterogeneity in LPS-triggered inflammatory response
A novel model to study neonatal<i>Escherichia coli</i>sepsis and the effect of treatment on the human immune system using humanized mice
No full textProblem
Neonatal sepsis is a serious threat especially for preterm infants. As existing in vitro and in vivo models have limitations, we generated a novel neonatal sepsis model using humanized mice and tested the effect of Betamethasone and Indomethacin which are used in the clinic in case of premature birth.
Method of study
Humanized mice were infected with Escherichia coli (E. coli). Subsequently, the effect of the infection itself, and treatment with Betamethasone and Indomethacin on survival, recovery, bacterial burden, leukocyte populations, and cytokine production, was analyzed.
Results
The human immune system in the animals responded with leukocyte trafficking to the site of infection and granulopoiesis in the bone marrow. Treatment with Indomethacin had no pronounced effect on the immune system or bacterial burden. Betamethasone induced a decline of splenocytes.
Conclusion
The human immune system in humanized mice responds to the infection, making them a suitable model to study neonatal E. coli sepsis and the immune response of the neonatal immune system. Treatment with Betamethasone could have potential negative long-term effects for the immune system of the child
Dose response of LPS on the release of pro-inflammatory cytokines by UBMC.
No full textUBMC isolated from three of the ten deliveries (Subjects 1–3; Table 1) were stimulated for 36 hours with increasing amounts (0–3027 ng/ml) of purified LPS derived from the indicated seven gram-negative bacteria (E. coli, E. aerogenes, K. pneumoniae, P. mirabilis, A. calcoaceticus, C. freundii, P. aeruginosa). The levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF) released in the supernatants of UBMC were measured by Luminex, as described in the Methods section. UBMC of Subject 1 released levels of IL-6 above the detection range of the Luminex assay (saturated signal), as indicated by the green dotted line and the (*) sign.</p
Dose response and time course of LPS on the release of cytokines and chemokines by UBMC.
No full textUBMC isolated from Subject 2 (Table 1) were stimulated for either 4 or 36 hours with increasing amounts (0–3027 ng/ml) of purified LPS derived from the indicated seven gram-negative bacteria (E. coli, E. aerogenes, K. pneumoniae, P. mirabilis, A. calcoaceticus, C. freundii, P. aeruginosa). The levels of IL-12, IL-6 and TNF released in the UBMC supernatant were measured by Luminex, as described in the Methods section. Based on these preliminary assays (and reports from the literature; see text), a stimulation duration of 36 h was used for further experiments.</p
Luminex assay using a human cytokine 30-plex panel on UBMC's supernantant of Subject 1, following stimulation for 36 h with 37.5 ng/ml purified LPS obtained from the indicated baterial species (<i>E</i>. <i>coli</i>, <i>E</i>. <i>aerogenes</i>, <i>K</i>. <i>pneumoniae</i>, <i>P</i>. <i>mirabilis</i>, <i>A</i>. <i>calcoaceticus</i>, <i>C</i>. <i>freundii</i>, <i>P</i>. <i>aeruginosa</i>).
No full textAbbreviation: SD, standard deviation.</p
Dose response of LPS on the release of chemokines by UBMC.
No full textUBMC isolated from three of the ten deliveries (Subjects 1–3; Table 1) were stimulated for 36 hours with increasing amounts (0–3027 ng/ml) of purified LPS derived from the indicated seven gram-negative bacteria (E. coli, E. aerogenes, K. pneumoniae, P. mirabilis, A. calcoaceticus, C. freundii, P. aeruginosa). The levels of chemokines (IL-8, MCP-1) released in the supernatants of UBMC were measured by Luminex, as described in the Methods section.</p
Summary of the cytokine profiles in UBMC stimulated with LPS of seven gram-negative bacteria.
No full textMedian cytokine levels in LPS-stimulated UBMC isolated from ten uncomplicated deliveries. Median cytokine levels are directly derived from the data presented in Fig 6. Due to log scale representation, values of 0 pg/ml were replaced by 0.1; thus values depicted at 0.1 pg/ml along the x axis are actually equal to zero. IL-8, IL-6, MIP-1α, MIP-1β and MCP-1 showed the highest median level of induction, followed by TNF, IL-1β, IL-10 and IL-12. IFN-γ showed the lowest median level of secretion.</p
Dose response of LPS on the release of chemokines by UBMC.
No full textUBMC isolated from three of the ten deliveries (Subjects 1–3; Table 1) were stimulated for 36 hours with increasing amounts (0–3027 ng/ml) of purified LPS derived from the indicated seven gram-negative bacteria (E. coli, E. aerogenes, K. pneumoniae, P. mirabilis, A. calcoaceticus, C. freundii, P. aeruginosa). The levels of chemokines (MIP-1α, MIP-1β) released in the supernatants of UBMC were measured by Luminex, as described in the Methods section. Based on the assays presented in Figs 1–4, the concentration of 37.5 ng/ml LPS was used for further experiments.</p
Dose response of LPS on the release of anti-inflammatory and TH1-type cytokines by UBMC.
No full textUBMC isolated from three of the ten deliveries (Subjects 1–3; Table 1) were stimulated for 36 hours with increasing amounts (0–3027 ng/ml) of purified LPS derived from the indicated seven gram-negative bacteria (E. coli, E. aerogenes, K. pneumoniae, P. mirabilis, A. calcoaceticus, C. freundii, P. aeruginosa). The levels of anti-inflammatory cytokine (IL-10) and TH1-type cytokines (IL-12, IFN-γ) released in the supernatants of UBMC were measured by Luminex, as described in the Methods section.</p
Levels of cytokines secreted by LPS-stimulated UBMC.
No full textLevels of pro-inflammatory (IL-6, IL-1ß, TNF) and anti-inflammatory (IL-10) cytokines (A), of the TH1-polarizing cytokines IL-12 (p40/p70) and IFN-γ (B), and of the chemokines IL-8, MIP-1α, MIP-1β and MCP-1 (C) released by LPS-stimulated UBMC. UBMC isolated from ten uncomplicated deliveries of at-term newborns were stimulated for 36 h with 37.5 ng/ml purified LPS derived from the indicated seven gram-negative bacteria (E. coli, E. aerogenes, K. pneumoniae, P. mirabilis, A. calcoaceticus, C. freundii, P. aeruginosa). Unstimulated UBMC served as reference (Control). The TLR1/2 agonist PAM3 served as additional control in seven out of ten donors. Cytokines levels in the respective UBMC supernatants were measured by Luminex. Differences between unstimulated and stimulated conditions were evaluated using the non-parametric Mann-Whitney U-test. Respective P values are indicated above each box plot. Medians (horizontal white line), arithmetic means (horizontal black line) and interquartile ranges are shown. IL-6 levels in Subject 1 were above the detection range after stimulation with six out of seven LPS variants (see also Figs 1–4 and Table 2), and thus the respective concentrations were excluded from the graphic representation and statistical analysis.</p
