Preterm Cord Research Articles

823: Magnesium decreases inflammatory cytokine production: a novel innate immunomodulatory mechanism

ObjectiveTo determine if magnesium sulfate (MgSO4) influences inflammatory cytokine production.Study DesignMgSO4 exposure before preterm birth reduces the risk of cerebral palsy and major motor dysfunction. As neonatal inflammatory cytokine levels strongly correlate with neurologic outcome, we assessed the effect of MgSO4 on cytokine production within monocytic cell lines, cord blood mononuclear cells and maternal blood from women receiving parenteral magnesium sulfate for clinical indications.ResultsIn vivo MgSO4 treatment substantially reduced maternal TNF-α and IL-6 production. THP-1 cells exposed to TLR4 ligand in the presence of a clinically-effective MgSO4 concentration (6mg/dl) demonstrated reduced IL-1β, IL-6, IL-8 and TNF-α production. We next exposed term and preterm cord blood mononuclear cells to MgSO4 in vitro and found a significantly reduced frequency of neonatal monocytes producing cytokines under constitutive and TLR-stimulated conditions, as measured by intracellular cytokine staining. Total cellular magnesium content rapidly increased upon MgSO4 exposure, and the immunomodulatory effect was mediated by magnesium and not the sulfate moiety of the compound. Decreased cytokine production was linked to lower cytokine and IκBα gene expression and diminished NF-κB activation. Reduced cytokine production was seen following exposure to different TLR ligands, suggesting that magnesium acts intracellularly, directly influencing NF-κB or IκBα activity.ConclusionOur findings potentially elucidate the mechanism by which magnesium sulfate reduces the risk of cerebral palsy and establishes a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis. ObjectiveTo determine if magnesium sulfate (MgSO4) influences inflammatory cytokine production. To determine if magnesium sulfate (MgSO4) influences inflammatory cytokine production. Study DesignMgSO4 exposure before preterm birth reduces the risk of cerebral palsy and major motor dysfunction. As neonatal inflammatory cytokine levels strongly correlate with neurologic outcome, we assessed the effect of MgSO4 on cytokine production within monocytic cell lines, cord blood mononuclear cells and maternal blood from women receiving parenteral magnesium sulfate for clinical indications. MgSO4 exposure before preterm birth reduces the risk of cerebral palsy and major motor dysfunction. As neonatal inflammatory cytokine levels strongly correlate with neurologic outcome, we assessed the effect of MgSO4 on cytokine production within monocytic cell lines, cord blood mononuclear cells and maternal blood from women receiving parenteral magnesium sulfate for clinical indications. ResultsIn vivo MgSO4 treatment substantially reduced maternal TNF-α and IL-6 production. THP-1 cells exposed to TLR4 ligand in the presence of a clinically-effective MgSO4 concentration (6mg/dl) demonstrated reduced IL-1β, IL-6, IL-8 and TNF-α production. We next exposed term and preterm cord blood mononuclear cells to MgSO4 in vitro and found a significantly reduced frequency of neonatal monocytes producing cytokines under constitutive and TLR-stimulated conditions, as measured by intracellular cytokine staining. Total cellular magnesium content rapidly increased upon MgSO4 exposure, and the immunomodulatory effect was mediated by magnesium and not the sulfate moiety of the compound. Decreased cytokine production was linked to lower cytokine and IκBα gene expression and diminished NF-κB activation. Reduced cytokine production was seen following exposure to different TLR ligands, suggesting that magnesium acts intracellularly, directly influencing NF-κB or IκBα activity. In vivo MgSO4 treatment substantially reduced maternal TNF-α and IL-6 production. THP-1 cells exposed to TLR4 ligand in the presence of a clinically-effective MgSO4 concentration (6mg/dl) demonstrated reduced IL-1β, IL-6, IL-8 and TNF-α production. We next exposed term and preterm cord blood mononuclear cells to MgSO4 in vitro and found a significantly reduced frequency of neonatal monocytes producing cytokines under constitutive and TLR-stimulated conditions, as measured by intracellular cytokine staining. Total cellular magnesium content rapidly increased upon MgSO4 exposure, and the immunomodulatory effect was mediated by magnesium and not the sulfate moiety of the compound. Decreased cytokine production was linked to lower cytokine and IκBα gene expression and diminished NF-κB activation. Reduced cytokine production was seen following exposure to different TLR ligands, suggesting that magnesium acts intracellularly, directly influencing NF-κB or IκBα activity. ConclusionOur findings potentially elucidate the mechanism by which magnesium sulfate reduces the risk of cerebral palsy and establishes a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis. Our findings potentially elucidate the mechanism by which magnesium sulfate reduces the risk of cerebral palsy and establishes a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis.

Neonatal T-cell maturation and homing receptor responses to Toll-like receptor ligands differ from those of adult naive T cells: relationship to prematurity

Inflammation and infection are associated with premature birth and with activation of the fetal immune system. We hypothesized that exposure to microbial Toll-like receptor (TLR) ligands plays an important role in neonatal T-cell maturation and that early exposure to microbial products may result in early T-cell maturation and a tendency for these matured effector cells to change their homing receptor patterns. Expression of the CD45RO marker was induced in term neonatal T cells after in vitro exposure to TLR ligands for 7 days. Interestingly, naive T cells from adult blood were unaffected by TLR ligand exposure. In addition, neonatal T cells had more cells with decreased expression of the α4β7 integrins and increased expression of CCR4 after in vitro exposure of TLR ligands-similar to the expression of these molecules in adult naive T cells. These findings are relevant for the understanding of neonatal T-cell maturation and may contribute to our understanding of multiorgan inflammatory complications of prematurity. Cord blood was obtained from term and preterm infants. Using flow cytometry, we identified a mature (CD45RO(+)) phenotype in preterm infant cord blood (CB) T cells that had decreased expression of the α4β7 integrins and increased expression of the C-C chemokine receptor 4 (CCR4) as compared with term infant CB.

Early Postnatal Surge of Serum Clara Cell Secretory Protein in Newborn Infants

Background: Clara cell secretory protein (CCSP) is an anti-inflammatory mediator, but its role in neonatal lung adaptation and diseases is uncertain. Objective: To characterize postnatal changes in serum CCSP in relation to gestation, respiratory disease (RDS) and bronchopulmonary dysplasia (BPD) in comparison with other anti-inflammatory cytokines (IL-4, -10 and -13). Methods: Blood was collected from 76 infants (26 of 23–29 weeks’ gestation, 33 of 30–36 weeks’ gestation and 17 term infants) at birth (preterm cord blood); on admission; at 12, 24 and 48 h; and on days 3–4 and 7 of life. CCSP was assayed by ELISA and cytokines by Bio-Plex. Results: Median serum CCSP in extremely and moderately preterm infants rose from a baseline of 13.6 and 15.9 to 33.4 ng/ml (p = 0.04) and 59.8 ng/ml (p = 0.03) at 12 h of age, respectively. CCSP levels were highest in term infants (80.7 ng/ml at 12 h). CCSP then decreased to 22.5 ng/ml on days 3–4 (p = 0.001). CCSP of 37 RDS infants fell to a lower baseline on days 4 and 7 than that of the 22 non-RDS preterms. The 8 infants who developed BPD had persistently low serum CCSP (12.7 ng/ml at 12 h). In contrast, early postnatal changes were not seen in IL-4, -10 and -13 levels, but low IL-10 and -13 levels were found on day 7 in BPD infants. Conclusions: Serum CCSP levels were characterized by an early postnatal surge. This apparent gestation-influenced surge may represent an initiation of a protective cascade against postnatal lung injury during extrauterine adaptation.

Outcome of twin deliveries at the University of Maiduguri Teaching Hospital: A 5-year review

The objectives of the study were to determine the outcome of twin births at the University of Maiduguri Teaching Hospital (UMTH), Maiduguri, in terms of morbidity and mortality and to recommend possible measures to curtail or reduce some of the preventable complications. This is a 5-year retrospective study, from January 2000 to December 2004, of twin births at the UMTH. There were 196 twin deliveries in 8431 total deliveries, with a twin incidence of 2.3%. Dizygotic twins accounted for 63.4%. The increasing maternal age and positive family history of multiple pregnancies were associated with the increasing twinning rate. The main complications encountered were preterm labor, pregnancy-induced hypertension, and cord prolapse. The perinatal mortality rate of 107.5/1000 births was higher than that observed for singleton pregnancies in the same institution. Similarly, there was a higher Cesarian section rate of 24.7% compared to singletons within the same period. There were significantly higher perinatal mortality rates among the preterm (P = 0.000002) and low-birth-weight (P = 0.000004) fetuses. Considering that fetal prematurity and low birth weight, sequelae to preterm labor, are the commonest causes of perinatal death in this study, efforts should be geared during the antenatal period toward the prevention of a premature birth.

Polychromatic flow cytometry analysis of CD34+ hematopoietic stem cells in cryopreserved early preterm human cord blood samples

During the last decades, extended characterizations were performed of human full-term cord blood (hTCB) cells, but little information is available on human early preterm cord blood (hEPCB) hematopoietic stem cells (HSCs). In our study, we analyzed by flow cytometry 19 hEPCB and 17 hTCB samples. First, we observed that the percentage of CD34(Pos)CD45(Dim) cells was higher in hEPCB compared with hTCB and that it decreased during 16th-20th week of pregnancy. Within the CD34(Pos)CD45(Dim) population, we examined the expression of CD29, CD31, CD38, CD90, CD117, CD133, CD135, CD200, CD243, and CD338. We found that CD135 intensity and CD243(Pos) cells percentage were lower in hEPCB compared with hTCB. As to CD38, we observed that hEPCB samples were richer in undifferentiated CD34(Pos)CD45(Dim)CD38(Neg) HSCs compared with hTCB counterparts. We also compared the expression of the above-mentioned molecules in undifferentiated and committed HSCs residing in hEPCB and hTCB. In particular, although CD34(Pos)CD45(Dim)CD38(Pos) HSCs from both hEPCB and hTCB expressed relatively higher amounts of CD29, CD71, and CD135 compared with CD34(Pos)CD45(Dim)CD38(Neg) cells, a higher expression of CD31 was restricted to CD34(Pos)CD45(Dim)CD38(Pos) cells from hEPCB samples, and a higher expression of CD117 was demonstrated in CD34(Pos)CD45(Dim)CD38(Pos) cells from hTCB samples. Moreover, our data showed that CD34(Pos)CD45(Dim) cell population from hEPCB displayed higher percent of undifferentiated CD38(Neg)CD133(Pos) cells compared with hTCB samples. Finally, analyzing monocytes and lymphocytes within the two samples, we observed that T-cell percentages were higher in hTCB, whereas B-cell percentages were higher in hEPCB. We, therefore, studied the B-cell lineage maturation and found a higher percent of pro-B and pre-B cells in hEPCB compared with hTCB samples. Taken together, these results evidence the hematopoietic peculiarity of hEPCB, potentially useful for highlighting early steps of human hematolymphopoiesis as well as for developing novel strategies of stem cell-based therapy.

Placental surface shape, function, and effects of maternal and fetal vascular pathology

Goal In clinical practice, variability of placental surface shape is common. We measure the average placental shape in a birth cohort and the effect deviations from the average have on placental functional efficiency. We test whether altered placental shape improves the specificity of histopathology diagnoses of maternal uteroplacental and fetoplacental vascular pathology for clinical outcomes. Materials and methods 1225 Placentas from a prospective cohort had chorionic plate digital photographs with perimeters marked at 1–2 cm intervals. After exclusions of pre-term ( n = 202) and velamentous cord insertion ( n = 44), 979 (95.7%) placentas were analyzed. Median shape and mean perimeter were estimated. The relationship of fetal and placental weight was used as an index of placental efficiency termed “ β”. The principal placental histopathology diagnoses of maternal uteroplacental and fetoplacental vascular pathologies were coded by review of individual lesion scores. Acute fetal inflammation was scored as a “negative control” pathology not expected to affect shape. ANOVA with Bonferroni tests for subgroup comparisons were used. Results The mean placental chorionic shape at term was round with a radius estimated at 9.1 cm. Increased variability of the placental shape was associated with lower placental functional efficiency. After stratifying on placental shape, the presence of either maternal uteroplacental or fetoplacental vascular pathology was significantly associated with lower placental efficiency only when shape was abnormal. Conclusions Quantifying abnormality of placental shape is a meaningful clinical tool. Abnormal shapes are associated with reduced placental efficiency. We hypothesize that such shapes reflect deformations of placental vascular architecture, and that an abnormal placental shape serves as a marker of maternal uteroplacental and/or fetoplacental vascular pathology of sufficiently long standing to impact placental (and by extension, potentially fetal) development.

Hyperoxia disrupts vascular endothelial growth factor-nitric oxide signaling and decreases growth of endothelial colony-forming cells from preterm infants

Exposure of preterm infants to hyperoxia impairs vascular growth, contributing to the development of bronchopulmonary dysplasia and retinopathy of prematurity. Disruption of vascular endothelial growth factor (VEGF)-nitric oxide (NO) signaling impairs vascular growth. Endothelial progenitor cells (EPCs) may play an important role in vascular growth. Endothelial colony-forming cells (ECFCs), a type of EPC, from human preterm cord blood are more susceptible to hyperoxia-induced growth impairment than term ECFCs. Therefore, we hypothesized that hyperoxia disrupts VEGF-NO signaling and impairs growth in preterm ECFCs and that exogenous VEGF or NO preserves growth in hyperoxia. Growth kinetics of preterm cord blood-derived ECFCs (gestational ages, 27-34 wk) were assessed in room air (RA) and hyperoxia (40-50% oxygen) with or without VEGF, NO, or N(omega)-nitro-l-arginine. VEGF, VEGF receptor-2 (VEGFR-2), and endothelial NO synthase (eNOS) protein expression and NO production were compared. Compared with RA controls, hyperoxia significantly decreased growth, VEGFR-2 and eNOS expression, and NO production. VEGF treatment restored growth in hyperoxia to values measured in RA controls and significantly increased eNOS expression in hyperoxia. NO treatment also increased growth in hyperoxia. N(omega)-nitro-l-arginine treatment inhibited VEGF-augmented growth in RA and hyperoxia. We conclude that hyperoxia decreases growth and disrupts VEGF-NO signaling in human preterm ECFCs. VEGF treatment restores growth in hyperoxia by increasing NO production. NO treatment also increases growth during hyperoxia. Exogenous VEGF or NO may protect preterm ECFCs from the adverse effects of hyperoxia and preservation of ECFC function may improve outcomes of preterm infants.

Hematopoietic capacity of preterm cord blood hematopoietic stem/progenitor cells

Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38− cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.

Very low-density lipoprotein in the cord blood of preterm neonates

Human fetuses have markedly low levels of serum lipids and a unique lipoprotein profile with respect to quality, with low-density lipoprotein (LDL)–like particle as the dominant cholesterol carrier. However, little is known about triglyceride (TG) distribution. In addition, lipid metabolism is important in lung development, with indications that TG from very low-density lipoprotein (VLDL) is essential for surfactant synthesis. We investigated TG distribution in preterm neonate cord blood and the relationship of VLDL-TG levels with respiratory distress syndrome (RDS). The study included 103 appropriate-for-gestational-age neonates (61 males). We performed serum lipoprotein analyses in cord blood by high-performance liquid chromatography with gel permeation columns. Term neonates had low cord blood TG concentrations distributed equally to the LDL and VLDL fractions. However, preterm neonates had even lower TG concentrations, with VLDL as the dominant carrier. The LDL-TG and high-density lipoprotein–TG concentrations in cord blood increased gradually with gestational age, but cord blood VLDL-TG concentrations increased dramatically from 32 to 34 weeks of gestational age. Neonates with RDS exhibited no RDS-specific lipoprotein profile; however, most were born before the timing of the dramatic VLDL-TG increase. Our results suggest that 34 weeks of gestation is a critical period for TG metabolism, indicating the need for evaluation of the lipid nutritional state in preterm neonates.

Dilation and Evacuation of a Single Fetus after Midtrimester PROM in Previable Twin Pregnancy

Midtrimester premature rupture of membranes (PROM) is an unusual complication of multiple gestation that most often results in poor obstetric outcome. Presented are four cases of twin pregnancy complicated by preterm PROM and umbilical cord prolapse at 16 to 21 weeks' gestation. After a period of expectant management, dilation and evacuation of only the presenting fetus was performed with careful ultrasound guidance, to attempt delayed interval delivery. This resulted in pregnancy prolongation of 42 to 133 days for the remaining cotwins. Dilation and evacuation of a single demised fetus in twin pregnancy complicated by PROM and cord prolapse can be accomplished with favorable clinical outcome.

Cytokine Expression in Response to Bacterial Antigens in Preterm and Term Infant Cord Blood Monocytes

Background: Neonatal susceptibility to bacterial infection is associated with an immature immune system, but the role of different bacterial antigens in specific responses is largely unknown. Objective: To evaluate differences in intracellular cytokine response to physiologically relevant bacterial antigens in term and preterm infants as compared with adults. Methods: Cord blood samples from preterm and term neonates and adult peripheral blood samples were cultured ex vivo with and without whole heat-killed bacteria. Intracellular leukocyte production of interleukin (IL)-6, IL-10, IL-12, and IL-8 responses was assessed by flow cytometry. Results: Monocytes were the primary producers of all mediators. Escherichia coli was the most potent stimulant. Lactobacillus plantarum299v activated fewer monocytes as compared with E. coli for all responses (p < 0.05), except for IL-12 in term neonates. IL-6 response to Staphylococcus epidermidis was lower in both groups of neonates as compared with adults (p = 0.023 and p = 0.001). IL-8 response to S. epidermidis was lower in term as compared with preterm neonates and adults (p = 0.003). IL-10 response to group B streptococci was lower in term neonates as compared with adults and higher in preterm as compared with term neonates (p = 0.015). Conclusions: Monocytes from term neonates compared to preterm neonates show a downregulated anti-inflammatory response to specific bacteria. High neonatal response to pathogenic E. coli in the preterm infant could cause uncontrolled inflammatory response, while lower IL-6 response to S. epidermidis in neonates may indicate a basis for vulnerability to S. epidermidis infection.

A Comparative Analysis of Cartilage Engineered from Different Perinatal Mesenchymal Progenitor Cells

We sought to compare engineered cartilaginous constructs derived from different perinatal mesenchymal progenitor cell (MPC) sources. Ovine MPCs isolated from amniotic fluid (AF, n = 8), neonatal bone marrow (BM, n = 6), and preterm umbilical cord blood (CB, n = 12) were expanded and comparably seeded onto synthetic scaffolds. Constructs were maintained in chondrogenic media containing transforming growth factor-beta. After 12-15 weeks, specimens were compared with native fetal hyaline and elastic cartilage by gross inspection, histology, immunohistochemistry, and quantitative extracellular matrix (ECM) assays. MPCs from AF proliferated significantly faster ex vivo when compared to MPCs from the other sources. Chondrogenic differentiation was evident in all groups, as shown by toluidine blue staining and expression of aggrecan, cartilage proteoglycan link protein, and collagen type II. Quantitatively, all engineered specimens had significantly lower levels of glycosaminoglycans than native hyaline cartilage. Elastin levels in AF-based constructs (156.0 +/- 120.4 microg/mg) were comparable to that of native elastic cartilage (235.8 +/- 54.2 microg/mg), both of which were significantly higher than in BM- and CB-based specimens. We conclude that the ECM profile of cartilage engineered from perinatal MPCs is highly dependent on cell source. ECM peculiarities should be considered when designing the optimal cartilaginous bioprosthesis for use in perinatal surgical reconstruction.

Perivascular Human Umbilical Cord Cells Are Capable of Long Term Culture and Respond to Chemotactic Gradient in an In Vitro-Model of Alveolar Damage.

Bronchopulmonary dysplasia is a chronic lung disease that occurs in very premature infants and is characterized by impaired alveologenesis. Despite the use of surfactant, antenatal glucocorticoid treatments and new ventilation strategies, mortality in these subjects is still high and the impairment of alveolar formation in the surviving premature infants leads to abnormal lung function that induces neurological and growth dysfunction. In this context innovative cell-based lung repair approaches are highly needed. In particular, it would be important to develop a system that could regenerate the alveolar type I cells (ATI), that cover about 95% of the alveolar surface, and the alveolar type II cells (ATII). ATII cells ensure surfactant production, which decreases the surface tension of the alveolar surface, allowing the alveoli to expand during inspiration and preventing the collapse during expiration. Moreover, a sufficient ATII cell number is important because they serve as stem cells for ATI cells. A very promising, still poorly known and easily available, stem cell source is the human umbilical cord (HUC). We isolated and characterized a cell population within the HUC of pre-term newborns (HUC perivascular cells) to evaluate their capability to induce or improve pneumocyte repair after damage. With this aim we processed the HUCs (n=14) from deliveries of consenting mothers (23–32weeks of gestation). Pieces of cord were dissected to expose the Wharton's Jelly and the vein and the two arteries were exposed. The ends of each dissected vessel were tied together and digested with Collagenase A for 18 hours. After digestion the cells were extensively characterized by flow cytometry. The median (and range) percentage of the cells with a phenotype consistent with perivascular/pericyte-like cells (CD146+/CD45−/CD34−) was 17.5 (7.6–39). Moreover, these cells were positive for alpha-SMA (median and range: 90.7%, 46.9–94), NG2 (median and range: 23.2%, 13.05–31.8), VeCad (median and range:10.8%, 2.6–32.4), KDR (median and range: 15.25%, 1.5–26.2), CD31 (median and range: 3%, 0.8–8.7) and negative for CD45 and CD105. The HUC perivascular cells were cultured in the presence of EGM2 on a pre-coated gelatin layer for 1 week, and then with DMEM +20%FBS and maintained in long-term culture (11 passages). The cells were then stained with the fluorescent dye PKH26 and co-cultured in transwell with the ATII epithelial rat cellular line damaged by bleomycin. Control cultures were set up with not-damaged cell line. After 4-day co-culture, as a result of the chemiotactic migration, we observed PKH26 positive cells in contact with the damaged alveolar cell layer and not in the control cultures. By immunocytochemistry these cells showed to co-express PKH26 and the typical markers (pro-surfactant protein C, cytokeratin 18) of the ATII cells. Our results suggest that it is possible to isolate and to maintain in long-term culture HUC perivascular cells from pre-term cords. Moreover, this population is able to migrate towards alveolar cell layer previously damaged with bleomycin and to express the markers of the ATII cells. Our data encourage further investigations in order to evaluate the feasibility of therapeutic applications of these cells.

The impact of mode of delivery and gestational age on cord blood hematopoietic stem/progenitor cells

Human cord blood has been successfully used as an alternative source of hematopoietic stem cells suitable for transplantation. The aim of this study was to assess the impact of gestational age and the mode of delivery on cord blood hematopoietic stem/progenitor cell characteristics. The mode of delivery does not seem to affect either the replating capacity of hematopoietic progenitors colony-forming unit-granulocyte-macrophage or the cord blood content in CD34(+) cells. The higher percentage of CD34(+) cells in cord blood from preterm deliveries compared to full-term ones indicates that hematopoietic progenitors from preterm cord blood may be suitable for transplantation. These findings should be taken into consideration when selection of cord blood units is required for potential use in transplantation.

Effects of Hypoxia on Megakaryocyte Progenitors Obtained from the Umbilical Cord Blood of Term and Preterm Neonates

Background: Placental insufficiency is associated with early-onset thrombocytopenia in preterm neonates. Prior studies demonstrated a reduction in circulating megakaryocyte (Mk) progenitors, suggesting decreased platelet production. We hypothesized that decreased Mk production is the result of a direct inhibitory effect of hypoxia on the proliferation of Mk progenitors, or a hypoxia-induced change in the fetal hematopoietic environment. Objective: To test the effects of hypoxia on the clonogenic maturation of Mk progenitors obtained from term and preterm cord blood CD34^pos cells, either cultured alone or in conjunction with CD34^neg light density mononuclear cells (LDMCs). Methods: CD34^pos cells and CD34^neg LDMCs were isolated from the cord blood of term and preterm deliveries, and mobilized peripheral blood CD34^pos cells were obtained from healthy adults. CD34^pos cells were then cultured alone or co-cultured with CD34^neg LDMCs in a semisolid, serum-free media containing rTpo, IL-3, and IL-6. Cultures were exposed to 20%, 5%, or 1% oxygen for 10–12 days. Mk colonies were then quantified following immunohistochemical staining. Results: Pure CD34^pos cells from preterm (n = 5) and term (n = 5) neonates and from adults (n = 4) generated similar numbers of Mk colonies in all three oxygen concentrations. However, the number of Mk colonies in preterm co-cultures was progressively lower with decreasing O₂ concentrations. Conclusions: Hypoxia did not appear to directly inhibit colony formation of Mk progenitors from preterm and term cord blood CD34^pos cells. However, co-culture studies showed a decrease in Mk colony formation with hypoxia, suggesting an indirect inhibitory effect of hypoxia on Mk clonogenic maturation mediated by non-progenitor cells in the hematopoietic microenvironment.

Flow cytometric assessment of hematopoietic cell subsets in cryopreserved preterm and term cord blood, influence of obstetrical parameters, and availability for transplantation

The aim of this study was to characterize the lymphocyte and the hematopoietic stem and progenitor cell (HPC) subsets of cryopreserved premature cord blood (PCB) compared to term cord blood (TCB) by flow cytometry, to study the influence of birth conditions, and to assess its availability for transplantation. Four-color flow cytometric analysis was performed on 43 PCB and 40 TCB cryopreserved samples using a panel of 24 different mAbs, directed against lymphoid and HPC surface markers. The CB volume was estimated by the weight of the newborn to determine the absolute MNC and CD34(+) cell content/CB sample. Clinical and obstetrical data were recovered. Statistical comparisons and a multiple regression analysis were performed. No consistent differences were found in the mononuclear cell (MNC) or CD34(+) cell concentration (x10(6)/L) between PCB and TCB. The percentage of primitive HPC (CD34(+)CD38(-), CD34(+)CD38(-)CD90(-)HLA-DR(-), CD34(+)CD38(-)CD90(-)HLA-DR(+)) and primitive lymphoid progenitors (CD34(+)CD7(+), CD34(+)CD7(+)CD19(-)CD117(-)) were higher in PCB than in TCB. Correspondingly, TCB had an increased percentage of committed HPC. No sample of PCB contained >2 x 10(7) MNC/kg (and only 48% had >1 x 10(5) CD34(+) cells) for a recipient of 20 kg body wt, as the minimum threshold recommended for CB transplantation. Obstetrical factors modulated mainly lymphocyte subsets and fewer HPC subpopulations. Acute fetal distress increased CD34(+) cells, especially the immature subsets. Maternal treatment with dexamethasone and intrauterine growth retardation decreased CD3(+) cells. No other obstetrical factors played a detrimental effect on CB cells if used for transplantation. PCB is richer in immature cells both in lymphocyte and HPC populations, and its use for transplantation, at least in special cases, should be reconsidered.

The Measurement of Dendritic Cells in Umbilical Cord Blood: A Novel Technique Using Small Volumes of Whole Blood

To develop a technique that would permit the identification of dendritic cells (DC) in small volumes of preterm neonatal cord blood in order to investigate neonatal immune response in relation to infection and preterm labor. We used three-color flow cytometry and a fixed quantity of fluorospheres to determine absolute cell numbers. Four red cell lysis techniques, sample dilution, time delay experiments, and a comparison with traditional Ficoll cell separation techniques were performed. Absolute numbers of DC recovered using each technique were calculated and compared. With increasing time delay, there was a statistically significant reduction in the numbers of leukocytes in adult blood. In contrast, there was a significant increase in leukocytes in umbilical cord blood. Sample dilution did not significantly affect the total number of leukocytes or DC. The use of the reagent Optilyse B (Immunotech, Oxford, UK) combined with an additional washing step produced the best discrimination of all populations based on light scatter properties. For the same blood sample, antibody labeling of whole blood resulted in a greater recovery of DC when compared to prior cell purification using a Ficoll density gradient. Using traditional Ficoll cell separation, cell manipulation requires large volumes of blood and leads to cell loss and alteration in phenotype. We have validated a novel method using small volumes of whole blood, diluted if necessary, and using red cell lysis to enable analysis of small volumes of preterm neonatal cord blood. This may permit further analysis of the contribution of the fetal immune response in the development of spontaneous preterm labor and preterm birth due to infection.

130 The Effect of Serum from Preeclampsia Mother and Her Neonatal Cord Blood on The Activity of Soluble Adhesion Molecules and Apo-Ptosis in Cultured Huvec

Purpose: In this study we tested the effect of serum from preeclampsia mother and her neonatal umbilical cord bood on the activity of soluble intercellular adhesion molecule-1(sICAM-1) and apoptosis in cultured human umbilical venous endothelial cells(HUVEC). We also determined if unknown circulating factors in preeclamptic women may influence to the endothelial cells of her fetus. Study design: Maternal serum samples were collected before delivery from women with preterm and term preeclampsia (preeclamptic term mother group, n= 14, preeclamptic preterm mother group, n= 12) and uncomplicated control pregnancies (control term mother group, n= 12, control preterm mother group, n= 11). Neonatal serum samples of these women were also collected from the umbilical veins(preeclamptic term cord group, n= 13, preeclamptic preterm cord group, n= 12, control term cord group, n= 12, control preterm cord group, n= 11). The sICAM-1 were measured with a sandwich ELISA technique in isolated Results: 1) In sICAM-1 levels: Each mother groups determined significantly higher levels compared to each neonatal cord groups(p< 0.001). Preeclamptic mother groups showed increased levels compaired to control mother groups but have no statistical differences(p0.05). However, the preterm mother groups determined increased levels compaired to term mother groups(p< 0.001). In between each mother groups, preterm preeclampsia mother groups were significantly elevated compared to other mother groups(p< 0.001). In between neonatal cord groups, there were no statistical differences in each groups. 2) In TUNEL stain: Preterm preeclampsia mother group showed significantly increased number of apoptotic nuclei and decreased number of cells compaired to other groups. Especially, the neonatal cord groups of preterm preeclampsia showed no apoptotic body on cultured HUVEC. Conclusions: Unknown circulating factors in preterm preeclampsia mother activate expression of ICAM-1 and apoptosis in cultured human umbilical venous endothelial cells. But the fetal circulation may not be affected by the factor(s) that lead to disturbed endothelial cell function in women with preterm preeclampsia.

Necrotic cell injury in the preterm and near-term ovine fetal brain after intermittent umbilical cord occlusion

ObjectiveThis study was undertaken to determine the extent of necrotic cell death as a measure of neurologic injury in the preterm and near-term ovine fetal brain in response to intermittent umbilical cord occlusion (UCO) with severe, but limited hypoxia and no cumulative acidosis to ensure longer-term survival. Study designFetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days with UCOs performed in the experimental group animals by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then euthanized and the fetal brain perfusion-fixed and prepared for subsequent histology and assessment of necrotic cell injury by using standard staining with hematoxylin and eosin (H&E), and with a novel fluorescent marker, Fluoro-Jade B, that targets degenerating neurons. ResultsIn both preterm and near-term animal groups, UCO caused a large decline in arterial Po2 (to ∼7 mm Hg), a modest decline in pH (to ∼7.30), and a modest rise in Pco2 (to ∼61 mm Hg) (all P<.01), but with a return to control values after the occluder release and no cumulative acidosis over each day of study. Overall, very low levels of H&E-stained necrotic-appearing cells and Fluoro-Jade B–stained positive cells were observed across all brain regions studied with values not significantly different from zero, excepting that for the gray matter of the preterm control (by H&E staining), preterm and near-term cord occlusion (by H&E and Fluoro-Jade B staining), and the thalamus of the near-term cord occlusion (by H&E staining) animals. Although there were no differences in the levels of H&E-stained necrotic-appearing cells and Fluoro-Jade B–stained positive cells between respective control and cord occlusion group animals for most of the brain regions studied, a significant increase in Fluoro-Jade B–stained positive cells was observed in the gray matter of both the preterm and near-term cord occlusion animals (P<.05). ConclusionIntermittent cord occlusion insult with severe but limited fetal hypoxemia and no cumulative acidosis, was generally well tolerated in both the preterm and near-term animal groups as assessed by measures of necrotic cell injury throughout the brain with minimal evidence for such. However, compensatory mechanisms which are protective for the brain may become limited with repetitive hypoxia insult over time as suggested by the low level of Fluoro-Jade B–stained positive cells noted in the gray matter tissues for both occlusion groups.

Expression and regulation of B7 family molecules on macrophages (MPhi) in preterm and term neonatal cord blood and peripheral blood of adults.

Macrophage (MPhi) receptors of the B7 family (CD80, CD86) play a crucial role in T cell activation: the lack of costimulation leads to anergy or apoptosis of reactive T cells. MPhi may differentiate into different subsets, the balance of which defines MPhi-dependent T cell reactions. The aim of this study was to examine neonatal and adult T cell response with respect to the costimulatory MPhi-potential in order to identify molecular predictors for the neonatal immune defense. MPhi from peripheral blood (PBMPhi) or cord blood (CBMPhi) were stimulated with interferon-gamma (IFN-gamma), cyclic adenosine monophosphate (cAMP), CD40 ligand (CD40L), or alphaCD3. As compared to PBMPhi, CBMPhi showed a significantly decreased upregulation of CD80 and/or CD86 after stimulation with IFN-gamma, cAMP, CD40L, and alphaCD3. Accordingly, the proliferative T cell response was impaired in the presence of CBMPhi. The fraction of T cells that underwent cell death was higher, and blast formation was significantly lower than that observed in the presence of PBMPhi. CBMPhi, as compared to PBMPhi, delivered fewer costimulatory but more cytotoxic signals to T cells. These observations suggest that MPhi are one factor explaining the suboptimal immune defense of neonates and their increased susceptibility to infection. Using the costimulatory MPhi-potential as a predictor for immune responses requires a separate reference value system in neonatology.