Neonatal Cord Blood Mononuclear Cells Research Articles

Mitochondrial implications in human pregnancies with intrauterine growth restriction and associated cardiac remodelling.

Intrauterine growth restriction (IUGR) is an obstetric complication characterised by placental insufficiency and secondary cardiovascular remodelling that can lead to cardiomyopathy in adulthood. Despite its aetiology and potential therapeutics are poorly understood, bioenergetic deficits have been demonstrated in adverse foetal and cardiac development. We aimed to evaluate the role of mitochondria in human pregnancies with IUGR. In a single‐site, cross‐sectional and observational study, we included placenta and maternal peripheral and neonatal cord blood mononuclear cells (PBMC and CBMC) from 14 IUGR and 22 control pregnancies. The following mitochondrial measurements were assessed: enzymatic activities of mitochondrial respiratory chain (MRC) complexes I, II, IV, I + III and II + III, oxygen consumption (cell and complex I‐stimulated respiration), mitochondrial content (citrate synthase [CS] activity and mitochondrial DNA copy number), total ATP levels and lipid peroxidation. Sirtuin3 expression was evaluated as a potential regulator of bioenergetic imbalance. Intrauterine growth restriction placental tissue showed a significant decrease of MRC CI enzymatic activity (P < 0.05) and CI‐stimulated oxygen consumption (P < 0.05) accompanied by a significant increase of Sirtuin3/β‐actin protein levels (P < 0.05). Maternal PBMC and neonatal CBMC from IUGR patients presented a not significant decrease in oxygen consumption (cell and CI‐stimulated respiration) and MRC enzymatic activities (CII and CIV). Moreover, CS activity was significantly reduced in IUGR new‐borns (P < 0.05). Total ATP levels and lipid peroxidation were preserved in all the studied tissues. Altered mitochondrial function of IUGR is especially present at placental and neonatal level, conveying potential targets to modulate obstetric outcome through dietary interventions aimed to regulate Sirtuin3 function.

Imbalance in mitochondrial dynamics and apoptosis in pregnancies among HIV-infected women on HAART with obstetric complications.

HIV infection and HAART trigger genetic and functional mitochondrial alterations leading to cell death and adverse clinical manifestations. Mitochondrial dynamics enable mitochondrial turnover and degradation of damaged mitochondria, which may lead to apoptosis. To evaluate markers of mitochondrial dynamics and apoptosis in pregnancies among HIV-infected women on HAART and determine their potential association with obstetric complications. This controlled, single-site, observational study without intervention included 26 HIV-infected pregnant women on HAART and 18 control pregnancies and their newborns. Maternal PBMCs and neonatal cord blood mononuclear cells (CBMCs) were isolated at the first trimester of gestation and at delivery. The placenta was homogenized at 5% w/v. Mitochondrial dynamics, fusion events [mitofusin 2 (Mfn2)/β-actin] and fission events [dynamin-related protein 1 (Drp1/β-actin)] and apoptosis (caspase3/β-actin) were assessed by western blot analysis. Obstetric complications were significantly more frequent in pregnancies among HIV-infected women [OR 5.00 (95% CI 1.21-20.70)]. Mfn2/β-actin levels in PBMCs from controls significantly decreased during pregnancy (202.13 ± 57.45%), whereas cases maintained reduced levels from the first trimester of pregnancy and no differences were observed in CBMCs. Mfn2/β-actin and Drp1/β-actin contents significantly decreased in the placenta of cases. Caspase3/β-actin levels significantly increased during pregnancy in PBMCs of cases (50.00 ± 7.89%), remaining significantly higher than in controls. No significant differences in caspase 3/β-actin content of neonatal CBMCs were observed, but there was a slight increased trend in placenta from cases. HIV- and HAART-mediated mitochondrial damage may be enhanced by decreased mitochondrial dynamics and increased apoptosis in maternal and placental compartments but not in the uninfected fetus. However, direct effects on mitochondrial dynamics and implication of apoptosis were not demonstrated in adverse obstetric outcomes.

Intracellular DNA sensing pathway of cGAS-cGAMP is decreased in human newborns and young children

Newborns are highly susceptible to DNA virus infections, which may result from the characteristics of neonatal innate immune systems. Here we analyzed for the first time the development of innate immune sensing and signaling of intracellular DNA virus infection in human newborns and young children. Both mRNA and protein expression of cGAS, an intracellular DNA sensor, were shown to be significantly reduced in neonatal peripheral blood mononuclear cells (PBMCs). In addition, cGAS expression in neonatal PBMCs could be induced upon herpes simplex virus type 1 (HSV-1) or interferon-α (IFNα) stimulation. Furthermore, production of the second messenger cGAMP and activation of the transcriptional factor IRF3 was severely decreased in neonatal cord blood mononuclear cells (CBMCs) or PBMCs compared with adults. In contrast, the downstream signaling STING-TBK1-IRF3 appeared to be functional in neonatal PBMCs, as demonstrated by the fact that IRF3 phosphorylation and IFNβ production in these cells could be activated by cGAMP. Intriguingly, decreased expression of cGAS in neonatal cells can be rescued by DNA demethylation, with concomitant enhancement in IFNβ induction by HSV-1. Thus, cGAS restoration or STING stimulation by small molecules during infancy might improve the age-dependent susceptibility to DNA virus infection.

The expression and function of human CD300 receptors on blood circulating mononuclear cells are distinct in neonates and adults.

Neonates are more susceptible to infections than adults. This susceptibility is thought to reflect neonates’ qualitative and quantitative defects in the adaptive and innate immune responses. Differential expression of cell surface receptors may result in altered thresholds of neonatal immune cell activation. We determined whether the expression and function of the lipid-binding CD300 family of receptors are different on neonatal immune cells compared to adult immune cells. A multiparametric flow cytometry analysis was performed to determine the expression of CD300 receptors on adult peripheral blood mononuclear cells and neonatal cord blood mononuclear cells. The expression of the CD300a inhibitory receptor was significantly reduced on cells from the newborn adaptive immune system, and neonatal antigen presenting cells exhibited a different CD300 receptors expression pattern. We also found differential LPS-mediated regulation of CD300 receptors expression on adult monocytes compared to cord blood monocytes, and that CD300c and CD300e-mediated activation was quantitatively different in neonatal monocytes. This is the first complete study examining the expression of CD300 receptors on human neonatal immune cells compared with adult immune cells. Significant differences in the expression and function of CD300 receptors may help to explain the peculiarities and distinctness of the neonatal immune responses.

The effect of Indomethacin and Betamethasone on the cytokine response of human neonatal mononuclear cells to gram-positive bacteria

Intrauterine infections with gram-positive bacteria pose a serious threat to neonates since they can result in neonatal sepsis, induce a fetal inflammatory response and also cause preterm birth. Despite intensive care, prematurity remains a leading cause of neonatal death, and is often accompanied by a number of morbidities. In order to prevent premature birth, tocolytic agents like Indomethacin are administered. Betamethasone is used to promote lung maturation and prevent respiratory distress syndrome. A combination of both drugs is assumed to prevent premature delivery while simultaneously facilitating lung maturation. This study investigates the effect of Betamethasone, Indomethacin and a combination of both on the cytokine production of neonatal cord blood mononuclear cells (CBMC) after stimulation with lysates of the gram-positive pathogens Streptococcus agalactiae and Enterococcus faecalis. The aim of the study is to determine the impact of these drugs on the function of the neonatal immune system which should aid clinicians in choosing the optimal therapy in case of preterm birth associated with intrauterine infection. Betamethasone reduced the production of the pro-inflammatory cytokines IL-6, IL-12p40, MIP-1α and TNF and increased the expression of the anti-inflammatory cytokine IL-10, depending on the pathogen used for stimulation. In contrast to Betamethasone, Indomethacin almost exclusively increased IL-10 production. The combination of both drugs decreased the expression of IL-6, IL-12p40, MIP-1α and TNF while increasing IL-10 production, depending on the concentration of Indomethacin and the pathogen used for stimulation. Based on our results, the combination therapy with Indomethacin and Betamethasone has a similar effect on cytokine production as Betamethasone alone, which is generally administered in case of impending preterm birth. However, the combination therapy has the advantage of promoting lung maturation while simultaneously blocking preterm labor effectively.

Expression of Toll-like receptor 4 in neonatal cord blood mononuclear cells in patients with preeclampsia

The expression of Toll-like receptor 4 (TLR4) in neonatal cord blood mononuclear cells (MNCs) and serum TNF-α were investigated in order to explore the roles of TLR4 in the pathogenesis of preeclampsia. The study enrolled 27 patients suffering from preeclampsia (experimental group) and 21 normal pregnancy patients (control group). After MNCs were separated, the expression of TLR4 mRNA and protein was detected by using real-time quantitative PCR and Western blotting respectively, and the expression of TNF-α by using ELISA. The results showed the TLR4 mRNA level in cord blood MNCs (2(-ΔCT): 0.07±0.17), TLR4 protein expression level (absorbance ratio: 0.81%±0.15%) and TNF-α level (9.5±1.73 pg/mL) were all increased in experimental group as compared with control group with the differences being statistically significant (P<0.05). There was a positive correlation between the expression of TLR4 mRNA and TNF-± in both experimental group and control group (r=0.54 and 0.53, respectively, P<0.05). It was concluded that TLR4 expression in the experimental group of cord blood MNCs was increased and there was a positive correlation between the expression of TLR4 mRNA and TNF-α in both groups. TLR4-mediated release of inflammatory cytokines may be one of the important reasons leading to preeclampsia.

Effect of melatonin on proliferation of neonatal cord blood mononuclear cells

Pineal melatonin (MLT) is a neuroendocrine hormone that possesses a wide variety of biological effects. MLT regulation of the immune system has been studied in recent years. But very little is known about MLT interaction with neonatal cord blood mononuclear cells (CBMCs) and the lymphocyte immune system in neonates. This study was designed to investigate the proliferative effects of MLT on CBMCs and peripheral blood mononuclear cells (PBMCs). Cord blood samples were collected from 10 normal full-term infants at the Guangzhou Maternal and Infant Hospital, China. Ten samples of adult peripheral blood were also collected from healthy volunteers. (3)H-thymidine ((3)H-TdR) incorporation was used to analyze the influence of MLT on proliferation of CBMCs. The effects of MLT on proliferation of CBMCs and PBMCs were compared. (3)H-TdR incorporation increased in a dose-dependent manner with varying MLT concentrations (50 pg/ml-50 ng/ml), but peaked at 5 ng/ml. After incubation with MLT (5 ng/ml), interleukin-2 (IL-2, 50 ng/ml), MLT+phytohemagglutinin (PHA, 5 microg/ml), and MLT+IL-2, respectively in CBMCs media, (3)H-TdR incorporation rates were 114 327+/-52 863, 16 087+/-9006, 118 360+/-59 207, and 17 682+/-7391. Compared to the control cell suspension (14 133+/-8688), (3)H-TdR incorporation rates of the MLT and MLT+PHA groups were significantly increased (t=5.9143, P<0.001; t=5.5078, P<0.001). (3)H-TdR incorporation was not different between the IL-2 and MLT+IL-2 groups (t=0.4983, P>0.05; t=0.9839, P>0.05). PHA treatment (110 397+/- 48 663) presented no difference in (3)H-TdR incorporation compared to the MLT or MLT+PHA groups (t=0.1730, P>0.05; t=0.3286, P>0.05). (3)H-TdR incorporation was significantly greater in CBMCs than in PBMCs cultures after addition of various stimulators to the culture media. MLT promoted proliferation of PBMCs and also enhanced proliferation of CBMCs. The proliferative effects of MLT were greater on CBMCs than on PBMCs.

Effects of melatonin on the cytokines production of neonatal cord blood mononuclear cell

Objective To investigate the effects of melatonin(MLT)on the cytokines production of neonatal cord blood mononuclear cells(CBMC)in vitro. Methods Ten blood specimens from placenta vena umbilicalis of 10 healthy term delivery newborns were obtained,and 10 healthy adult volunteers'peripheral blood mononuclear cells(PBMC)specimens were taken as controls.CBMC and PBMC suspensions were cultivated in the system without serum and hormone,and then 200 μl PBMC/CBMC suspensions(2 105 cells)were co-cultured with MLT with different concentrations(50 ng/ml,5 ng/ml,500 pg/ml,50 pg/ml)for 72 hours.The concentrations of cytokines including IL-1β,IL-2,IL-4,IL-6,IL-8,IL-12,IFN-γ and TNF-α in the cell supernatant were detected.Results The 500 pg/ml MLT induced secretions of IL-2[(12.18 7.31)pg/ml],IL-6[(2091.08±968.58)pg/ml],IL-8[(2779.04±1168.49)pg/ml],IL-12[(15.29±6.88)Pg/ml]and IFN-γ[(52.22 17.77)pg/ml]of CBMC were more than those of the control group,which were(6.99±4.94)pg/ml,(1017.06±452.60)pg/ml,(1293.33±791.34)pg/ml,(6.84±2.26)pg/ml and(22.24±10.38)pg/ml(P<0.05).Compared with control group[(175.63±62.10)Pg/ml],all concentrations of MLT could inhibit the TNF-α secretion[(24.87 11.21)pg/ml,(18.14±9.44)pg/ml,(31.38±15.28)pg/ml and(28.18±17.35)pg/ml]of CBMC significantly.The 50 ng/ml MLT could induce the PBMC's secretion of IL-2, IL-12, IFN-γ,and inhibit the secretion of TNF-α. The best effect of MLT on CBMC was at the concentration of 500 pg/ml, while that on PBMC was 5 ng/ml. Comparing the best effects of MLT, the levels of IL-12 and IFN-γ of CBMC were higher than those of PBMC, and IL-2 level of CBMC was lower than that of PBMC (P<0.01). Conclusions MLT could promote the cytokine secretion of PBMC and CBMC, and the effects of MLT on IL-12 and IFN-γ secretion of CBMC were stronger than those of PBMC. The results suggested that MLT may contribute to promote the immune function in newborn infants, and provide experimental evidence for clinical application of MLT. Key words: Melatonin; Fetal blood; Leukocytes, mononuclear; Cytokines

A Comparison of the Effect of Vitamin A on Cytokine Secretion by Mononuclear Cells of Preterm Newborns and Adults

Background: For a long time vitamin A has been known to be essential for immune defense of the organism and protection against infections. Vitamin A deficiency in children is associated with morbidity and mortality from infectious diseases which could be prevented and even alleviated by vitamin A supplementation. Moreover, this vitamin is involved in the modulation of immunological and inflammatory responses by regulation of cytokine production. The aim of the study was to compare the in vitro effect of vitamin A on the production of pro-inflammatory (IL-1β and IL-6) and anti-inflammatory (IL-1 receptor antagonist (ra) and IL-10) cytokines, as well as IL-2 and IFNγ by cord blood mononuclear cells (CBMC) of preterm newborns to that of peripheral blood mononuclear cells (PBMC) from adults. Methods: Mononuclear cells (MC) from individuals of the two age groups were incubated with vitamin A (retinyl palmitate) at various concentrations in the presence of phytohemagglutinin for IL-2 and IFNγ production or LPS for IL-1β, IL-1ra, IL-6 and IL-10 secretion. The level of the cytokines in the supernatants was tested by ELISA. Results: Vitamin A exerted an in vitro inhibitory effect on the production of the anti-inflammatory cytokine IL-1ra by MC of preterm newborns and adults, but did not affect the secretion of the pro-inflammatory cytokines IL-1β, IL-6 and IFNγ. Vitamin A caused inhibition of IL-10 secretion by cells from adults, but it did not significantly affect this function in cells from newborns except when high unphysiological doses were applied. In addition vitamin A stimulated the secretion of IL-2 by cells isolated from adults but had no effect on those derived from premature neonates. Conclusions: The results indicate that vitamin A may affect the immune function of premature infants via inhibition of IL-1ra secretion. It is suggested that the beneficial effect of vitamin A on the clinical course of bronchopulmonary dysplasia (BPD) may be due to the reduced production of anti-inflammatory cytokines by neonatal CBMC. This may indicate the importance of the pro-inflammatory cytokines in the management of severe lung diseases and BPD.

Interleukin-18 enhances the production of interferon-gamma (IFN-gamma) by allergen-specific and unspecific stimulated cord blood mononuclear cells.

IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-gaamma and IL-13 release of CBMC of healthy neonates. CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-gamma, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 microg/ml) and an allergen-specific stimulus (BLG 25 microg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-gamma and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-gamma, 6154; IL-13, 4357; BLG: IFN-gamma, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-gamma release (PHA: 6154; PHA + IL-18: 13474, p = 0.0001; BLG: 801; BLG + IL-18: 1077, p = 0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p = 0.16) or BLG + IL-18 (124, p = 0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-gamma release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.

Neonates at risk of atopy show impaired production of interferon-gamma after stimulation with bacterial products (LPS and SEE)

Recent studies demonstrate reduced interferon-gamma (IFN-gamma) secretion in neonates who became atopic later in life. The underlying pathomechanism is still unknown. We therefore examined the effects of bacterial products on neonatal IFN-gamma production acting through different T-cell- or antigen-presenting-cell (APC)-stimulating mechanisms: cord-blood mononuclear cells (CBMC) were incubated with lipopolysaccharide (LPS), staphylococcal enterotoxin E (SEE), or a combination of both and restimulated with PMA and ionomycin. LPS and SEE as single stimuli induced IFN-gamma production to the same extent in CBMC of neonates with high and low risk of atopy. In contrast, a combination of LPS and SEE had a multiplying effect on IFN-gamma secretion only in CBMC of neonates with low risk of atopy. Phenotype analysis revealed that only memory T cells showed impaired IFN-gamma synthesis (median 3.6% IFN-gamma-producing cells vs 14.2% in controls: P < 0.01), whereas IFN-gamma production by naive T cells did not differ in either group. Taken together, these results point to the existence of a disturbed function of costimulatory mechanisms in neonates at high risk of atopy, provoking reduced memory T-cell IFN-gamma production.

Human cord blood mononuclear cells are preferentially infected by non-syncytium-inducing, macrophage-tropic human immunodeficiency virus type 1 isolates.

Identification of the factors which impact on the transmission of human immunodeficiency virus type 1 (HIV-1) from an infected mother to her infant is essential for the development of effective strategies to prevent perinatal HIV-1 infection. The current study was designed to determine if unstimulated human neonatal cord blood mononuclear cells (CBMC) differ from adult peripheral blood mononuclear cells (PBMC) in susceptibility to HIV-1 infection. Both cell populations were challenged with two laboratory and two clinical HIV-1 isolates with different phenotypic properties. Infection was evaluated by quantitation of p24 antigen production and p24 antigen expression by an enzyme immunoassay and immunofluorescence, respectively. T-cell markers were determined by flow cytometry. Unstimulated CBMC were preferentially infected by macrophage-tropic, non-syncytium-inducing (non-SI) laboratory and clinical isolates, whereas PBMC were more susceptible to T-lymphotropic, SI HIV-1 strains. The macrophage-tropic strain HIV-1Ba-L replicated to 100-fold higher titers in CBMC than a similar inoculum of the SI isolate HIV-1LAI. The opposite occurred in unstimulated PBMC, which replicated HIVLAI to eightfold higher titers than the macrophage-tropic isolate. These findings indicate that a selection of viral phenotype may occur with unstimulated CBMC displaying a predominant susceptibility to infection by macrophage-tropic, non-SI HIV-1 strains and that this selection may influence mother-infant transmission of HIV-1.

Fetal mononuclear cells show a comparable capacity with maternal mononuclear cells to produce IL-8 in response to lipopolysaccharide in chorioamnionitis

IL-8 is a chemotactic and activating cytokine for neutrophils which eliminate invading bacteria by releasing bactericidal metabolites. Cord blood mononuclear cells (CBMCs) obtained from neonates born to mothers with chorioamnionitis actively produced a significantly higher amount of IL-8 than those of neonates without chorioamnionitis, suggesting that the mononuclear cells of fetuses with chorioamnionitis had been activated in utero. As lipopolysaccharide (LPS) can often be detected in the uteroplacental space in chorioamnionitis, the LPS-mediated activation mechanism of neonatal mononuclear cells was analyzed in vitro to produce IL-8. Neonatal mononuclear cells stimulated with LPS increased IL-8 production in a time- and dose-dependent manner. The ability of term or preterm neonatal mononuclear cells to produce IL-8 was comparable with that of adult (maternal) mononuclear cells, suggesting functional maturity of the neonatal or fetal mononuclear cells to produce IL-8. However, IL-8 production by neonatal CBMCs was down-regulated by dexamethasone, a glucocorticoid which is clinically administered to mothers to promote fetal lung maturity in preterm delivery. Our present study revealed a regulatory mechanism of fetal IL-8 production, suggesting that functionally mature fetal mononuclear cells produce IL-8 in response to LPS in chorioamnionitis and activate the fetal defense mechanism against infection.