Activity In Cord Blood Research Articles

Significant Decreased in Cord Blood (CB) Versus Adult Peripheral Blood (APB) Monocyte (Mo)-Derived Dendritic Cells (DC) Gene Expression Patterns and T Cell Functional Activation: Insight into Immaturity of CB Cellular Immunity.

It has been recognized that dysfunction of CB immunity is in part due to the immaturity of the neonatal immune system (Cairo, Blood, 1997). However, biological pathways and molecular mechanisms associated with the immaturity of CB immunity are still poorly understood. Recently we have utilized oligonucleotide microarray to examine gene expression profiling of CB versus APB Mo and have demonstrated significant differential gene expression patterns, including surface molecules, cytokines, signaling molecules, transcription factors and apoptotic genes (Jiang/Cairo, et al, J. of Immunol., 2004). We sought to examine whether there are differential expressed genes occurred in Mo-derived CB versus APB DC and their impact on DC mediated T cell activity. Briefly, Mo were purified from fresh CB or APB and cultured for 8 days with GM-CSF and IL-4 (immature DC (iDC)) and LPS for mature DC (mDC). mRNA was isolated and oligonucleotide microarray was carried out (Affymetrix, U133A). Data was analyzed by Microarray Suite Version 5.0 (Affymetrix) and GeneSpring 5.0 software (Silicon Genetics). Selected genes were analyzed by RT-PCR (SuperScript, Invetrogen). We identified gene expression patterns that were significantly lower in CB versus APB DC including surface molecules HLA-DQA1 (4F), HLA-DRB3 (5F), HLA-DRB4 (5.5F), CD80 (3F), CD38 (3.8F); cytokine/chemokine genes IL-1b (2.5F), IL6 (2.9F), IL12B (3.5F), CXCL10 (6.6F); immunoregulatory genes ISG20 (11F), IFI27 (7.6F), TNFSF10 (4.5F), SOCS3 (2.5F). Moreover, several transcription factor genes whose proteins may involve in the activation of expression of these immune regulator genes were also differentially expressed (IRF-5 (3F), IRF7 (3F), MAD (6.3F)). We therefore compared CB versus APB DC antigen presentation activity to APB CD8 T cells by ELISPOT assay for interferon-r (IFNr) production (BD Pharmagen). Briefly, the purified CD8 T cells (MHC HLA A2) were incubated with CB or APB DC that were loaded without or with influenza peptide onto ELISPOT plate (Larsson, et al, J. of Immunol., 2000). The ELISPOT plates were developed, scanned and quantitated by an ELISPOT reader (C.T.L. Technology). Our results demonstrated that, although CB or APB mDC had allogeneic effects, influenza peptide loaded CB mDC was not able to induce CD8 T cells to produce IFNr while APB mDC loaded with influenza peptide strongly induced CD8 T cells to produce IFNr. This stimulatory effect of APB mDC on CD8 T cells to produce IFNr was 3.5 fold greater than that of CB mDC. We further examined DC antigen presentation activity to CD4 T cells and observed that APB-DC had stronger effects on CD4 T cell proliferation (3 fold for mDC vs. iDC) compared with CB-DC (only 1.5 fold for mDC vs. iDC) by CFSE assay (Molecular Probe). We postulate that decreased expression of specific surface molecules and other genes resulting in lower surface protein expression in CB DC may in part be responsible for the lack of initiation of signaling events from cell surface to trigger CB-DC to stimulate activation of CD8 and CD4 T cells. The decreased expression of transcription factor genes may also in part be responsible for the lower expressed surface molecule genes. Furthermore, these decreased expressed genes in other molecular categories in LPS-CB vs. APB DC may also partially be responsible for differential innate and adaptive immune function and properties of CB vs. APB.

Immaturity of IL‐18 gene expression and protein production in cord blood (CB) versus peripheral blood (PB) mononuclear cells and differential effects in natural killer (NK) cell development and function*

We have previously demonstrated dysregulation of IL-12 and IL-15 gene and protein expression between activated cord blood (CB) versus peripheral blood (PB) mononuclear cells (MNCs). In the present study, we compared IL-18 gene expression and protein production and IL-18 mRNA half-life in basal versus activated CB versus PB MNCs, the effects of IL-18 +/- IL-12 on MNCs IFN-gamma protein production and ex vivo expansion and activation of CB with IL-12 + IL-2 + anti-CD3 +/- IL-18. Basal and activated levels of IL-18 were significantly higher in PB versus CB MNCs (P < 0.05). IL-18 mRNA was coincidental with protein levels and significantly lower in CB (P < 0.05) and its half-life significantly shorter in CB versus PB MNCs (P < 0.05). IL-18 synergistically with IL-12 induced IFN-gamma production from PB greater than CB MNCs (P < 0.05). NK cells expansion (P < 0.001) and cytotoxicity (P < 0.01) was significantly increased with IL-12 + IL-2 + anti-CD3 and IL-18. In summary IL-18 gene expression and protein production are significantly decreased in activated CB versus PB MNCs, in part secondary to increased degradation of CB IL-18 mRNA. These results may have implications for the mechanism(s) in part responsible for the immaturity of CB T-cell immunity.

IgE dependent activation of cord blood derived mast cells (CBMC) modulates TRAIL induced apoptosis but does not cause the release of intracellular TRAIL

IgE dependent activation of cord blood derived mast cells (CBMC) modulates TRAIL induced apoptosis but does not cause the release of intracellular TRAIL

The Addition of Interleukin (IL)-15 to ANTI-CD3, IL-2 and FLT-3 Results in Significant Increase in Expansion, Activation, Proliferation and Survival of Cord Blood (CB) NK Cells and Cytotoxic T-Lymphocyte (CTL) Subsets: Potential for Adoptive Cellular Immunotherapy (ACI).

A limitation of CB as a source for ACI is the limited number of immunoeffector T and NK cell subsets. IL-15 is an important lymphokine regulating NK activity, T-cell proliferation and CTL activities. We have previously demonstrated reduced IL-15 mRNA expression and protein production from CB which may in part contribute to the immaturity of CB cellular immunity (Qian/Cairo et al, Blood 1997). NK cells are characterized by NK receptors which exist in both activatory and inhibitory forms (Moretta et al, Ann Rev Immun 2001). We have previously demonstrated the ex vivo expansion, maturation and activation of fresh CB derived T lymphocytes with anti-CD3, IL-2, IL-12 and IL-7 (Robinson/Cairo et al, Exp Hem 2001). For this study, we compared the expansion, proliferation, activation and survival of CB mononuclear cells (MNC) with IL-2, anti-CD3, FLT-3 ± IL-15. CB MNC were isolated by Ficoll density centrifugation and incubated overnight in AIM-V media. The non-adherent MNC fraction was cultured in AIM-V media alone and with IL-2 (5ng/ml), anti-CD3 (50ng/ml), FLT-3 (50ng/ml) and IL-15 (10 &100ng/ml) for 48 hrs. Cell proliferation was determined by the colorimetric assay system using WST-1. Expression of T & NK cell subsets were analyzed by three color flow cytometry using CD3, CD8, CD4, CD25, CD45RO, CD16, CD56, CD158a, CD158b, NKB1, CD94 & NKG2A monoclonal antibodies. Apoptosis markers were determined by measuring Annexin-V & PI by flow cytometry. Cytotoxicity was determined by europium release assay using K562 & Daudi target cells at 20:1 E:T ratio. Expansion of lymphocyte subsets of CD4+/25+, CD8+/25+, CD3+/45RO+ & CD94/NKG2A was significantly increased with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to IL-2, anti-CD3, FLT-3 and compared to media (CD4+/25+: 67.2±1.2 vs 39.19±5.3 vs 3.54±0.3%, p<0.01; CD8+/25+: 57.8±8.6 vs 16.94±0.3 vs 0.99±0.4%, p<0.01; CD3+/45RO+: 77.13±1.0 vs 29.96±1.9 vs 6.69±0.3%, p<0.001; CD94/NKG2A: 3.4±0.68 vs 0.29±0.092 vs 0.23±0.09%, p<0.01, respectively). We also noted a significant increase in CD3−/16+/56+ with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to media alone (p<0.01). There was no significant increase in expansion of CD3+/16+/56+, CD3−/56+/158a, CD3−/56+/158b or CD3−/56+/NKB1 in various culture conditions described above. IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) resulted in significant improvement in cell survival compared to media alone (Annexin-V staining) (3.3±0.26 vs 15.51±3.3%, p<0.05). We also noted increase in cell proliferation with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs. media alone (1.11±0.09 vs 0.82±0.01 vs 0.69±0.03 OD, p<0.05). We also noted significant increase in cytotoxicity with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs media alone (K562–57.81±8.1 vs 17.5±1.4 vs 2.68±0.6%, p<0.01; Daudi-79.8±1.7 vs 23.69±1.5 vs 6.38±0.6, p<0.001) The combination of IL-2 + anti-CD3 + FLT-3 + IL-15 appears to enhance expansion and cytotoxicity of CB CTL and NK subsets in short term culture in part secondary to inhibition of apoptosis. Future studies are warranted to determine if the combination of anti-CD3 + IL-2 + FLT-3 + IL-15 can enhance CB CTL and NK cells for ACI to induce GVHM effects following UCBT.

Characterization and Comparison of Cord Blood (CB) vs Adult Peripheral Blood (APB) CD4+CD25+ NAÏVE and Antigen Presentation Cell (APC) Induced T Regulatory (TREG): Significant Increase in APC Induced CB vs APB CD4+/CD25+/CTLA-4+ TREG.

The mechanism(s) responsible for the significant decrease in the incidence of a+cGVHD following UCBT remain to be determined (Cairo et al, 90:4665 Blood 1997). Treg (CD4+CD25+) cells have been demonstrated to be critically important in maintaining self-tolerance, transplantation tolerance, controlling autoimmunity and tumor immunity (Sakaguchi et al, Immuno Rev 2001) and suppressing aGVHD in a rodent model (Hoffman et al, J Exp Med 2002; Taylor et al, Blood 2002). We have previously reported that naïve CB vs APB CD4+CD25+ vs CD4+CD25− selectively express amplified genes including CTLA, GITR, and Foxp3 among others (Oberfield/Cairo et al, Blood 697a, 2003). In this study, we compared naïve and APC induced CB vs APB CD4+CD25− and CD4+CD25+ T cells with respect to gene profiles, immunophenotype, suppressive activity and mechanisms of suppression. CD4+CD25+ and CD4+CD25− cells were purified by negative selection for CD4 and positive selection for CD25 (Miltenyi Biotech), respectively. APC were generated from APB monocyte (M-iDC) or MUTZ-3 stimulated with GM-CSF (100ng/ml), IL-4 (10ng/ml) and TNF-a (2.5ng/ml) for 7d (MUTZ-iDC). Suppression assays were preformed with autologous CD4+CD25− (Th), allo-M-iDC or MUTZ-iDC (stimulators) and CD4+CD25+ cells (effectors) pulsed with 3H-thymidine. Gene expression of for Foxp3, CTLA-4, CD25, GITR, Tnfrsf-1B, IFRF4, SOCS2, Rarg, IL-10, IFN-γ, TGFß-1 and GAPDH were quantified by RT-PCR. MUTZ-iDC expressed significantly increased levels of CD1a, Cd11c, CD83, CD86, CMRF44 and induced similar primary but significantly reduced secondary MLR in naïve CB CD4+ T cells (p<0.02). Naïve CB and APB CD4+CD25+ vs CD4+CD25− expressed similar increased mRNA levels of genes including CTLA-4, GITR, Foxp3, Rarg, Tnfrsf-1B, IFR4 and CD25 and increased protein expression of CTLA4 (p<0.001) and GITR (p<0.001). However, APB but not CB CD4+CD25+ T cells significantly suppressed MUTZ-iDC stimulated Th responses (CB 8±3 % vs APB 52±5%, p<0.02). MUTZ-iDC stimulated CB and APB CD4+ T cells induced similar amplification of Foxp3 and CTLA-4 in CD25+ vs CD25− T cells but no significant difference in GITR, Rarg and Tnfrsf-1B. However, CD4+/CD25+/CTLA-4+ subset was significantly induced following CB vs APB MUTZ-iDC stimulation (CB 27±5% vs APB 10±2%, p<0.0023, n=8) and was CD62L positive. Most importantly, both CB and APB CD4+CD25+ T cells significantly suppress CD4+Th responses with MUTZ-iDC vs Allo-M-iDC stimulation (CB, p<0.001, vs APB, p=0.0012). However, there was no significant difference in either CB or APB IL10 or TGFß1 at both mRNA or protein levels. Taken together, these data suggest that activated CB CD4+CD25+ T cells induced similar primary but decreased secondary MLR and have similar Foxp3, GITR, CTLA-4 gene expression compared to APB but that MUTZ-iDC vs allo-M-iDC induced CB CD4+CD25+ T cells are associated with a significant intrinsic suppression activity with increased CTLA-4+/CD25+ expansion in CB vs APB, suggesting the importance of alloantigen specificity. This suppression does not appear to be mediated by IL10 or TGFß1. Our results provide insight into the mechanism(s) in alloantigen recognition and activation of CB vs APB CD4+Treg and these findings may in part responsible for the decrease in a+cGVHD seen post UCBT.

Cytokine Production and Hematopoiesis Supporting Activity of Cord Blood Derived Unrestricted Somatic Stem Cells.

Particularly within the last several years cord blood (CB) has become a well established hematopoietic source for unrelated transplantation in children. Assuming, however, a threshold of 2x107 nucleated cells (NC)/kg body weight required for transplantation in adults over 70kg, an average of only 25% of the CB units presently stored in the major CB banks contain sufficient NC to safely engraft patients. Thus far the majority of studies have assessed selective cytokine driven expansion of progenitors for transplantation, which however may be associated with exhaustion of stem cells and an increase of GvHD in adult patients. Therefore, we assessed cytokine production and hematopoiesis supporting stromal activity of CB derived unrestricted somatic stem cells (USSC, Koegler et al. JEM 2004: 200: 2: 1–13) in comparison to bone marrow mesenchymal stem cells (BMMSC) and hematopoietic progenitor expansion solely driven by recombinant cytokines. Initiation of USSC generation was initiated from fresh (n=325) and cryopreserved CB (n=66). In the presence of H5100 medium/10−7M dexamethasone, USSC cultures from fresh CB were initiated from 57.1% of CB samples (n= 52 out of 91 CB samples), whereas in DMEM-medium/10−7M dexamethasone the frequency was 31.6% (n=74 out of 234 CB samples). The generation frequency from frozen HES separated CB was only 19.5%. Cytokine production by USSC and BMMSC was determined qualitatively by cytokine mRNA expression array analyses or quantitatively by Luminex or ELISA analyses. To analyze hematopoiesis supporting activity, CB CD34+ cells were expanded in co-cultures with USSC, BMMSC or in the presence of Flt3-L, SCF, TPO. Expansion of CD34+ cells, total cells and colony-forming cells (CFC) were determined after 1, 2, 3 and 4 weeks of culture. USSC constitutively produced SCF, LIF, TGF-1ß, M-CSF, GM-CSF, VEGF, IL-1ß, IL-6, IL-8, IL-11, IL-12, IL-15, SDF-1a and HGF. When USSC were stimulated with IL-1ß, G-CSF was released. Production of SCF (p= 0.0104) and LIF (p= 0.046) was significantly higher in USSC compared to BMMSC. At 1, 2, 3 and 4 weeks, respectively co-cultivation of CD34+ cells on the USSC layer resulted in a 14.62±1.1-fold, 110.1±17.9-fold, 151.8±39.7-fold and 183.6±40.4-fold amplification of total cells and in a 30.56±4.4-fold, 101.4±27.5-fold, 64.67±15.8-fold and 29.4±3.1-fold amplification of CFC. Expansion achieved was significantly higher for USSC compared to BMMSC feeder layers. USSC produce functionally significant amounts of hematopoiesis supporting cytokines and are able to expand CD34+ cells from CB in significantly higher amounts compared to BMMSC. Therefore the USSC is a suitable candidate to direct ex-vivo expansion of hematopoietic CB cells for short term reconstitution.

Nuclear factor kappa B activation in human cord blood mononuclear cells.

The immunologic signals participating in immune responses early in life have not been completely elucidated. Regarding the characterization of neonatal cells, little is known concerning the activity of transcription factor nuclear factor kappa B (NF-kappaB), which regulates inflammatory genes and cytokine production. The aim of this study was to characterize NF-kappaB activation in cord blood mononuclear cells (CBMC). We analyzed the potential association of NF-kappaB activity with lymphocyte proliferation and influences on cytokine secretion in the early immune system. To determine the contribution of a disease whereby inheritance may impact neonatal immunity, we assessed the influence of maternal allergic disease on NF-kappaB regulation and cytokine secretion. CBMC from healthy newborns were isolated and stimulated with mitogen (n = 28). Nuclear extracts were analyzed by electrophoretic mobility shift assay, cytokine secretion by ELISA. FISH analysis excluded relevant maternal contamination of CBMC. All samples showed a positive lymphoproliferative response, and NF-kappaB activity was both increased and decreased after mitogen stimulation. Increased NF-kappaB activation was significantly associated with decreased TNF-alpha secretion (median 6.1 versus 50.3 pg/mL) in unstimulated CBMC. Mitogen stimulation resulted in increased NF-kappaB activity with a trend to increased IL-13 production. Maternal allergic disease was associated with higher TNF-alpha (median 982 versus 173 pg/mL) and IL-13 secretion (median 1328 versus 1120 pg/mL) after mitogen stimulation. Together, NF-kappaB activity is differentially activated in cord blood and associated with a distinct cytokine pattern. Whether differential NF-kappaB activity in cord blood is related to the subsequent development of immune diseases requires further investigation.

Unique character and metabolism of high density lipoprotein (HDL) in fetus

Lipid and lipoprotein profiles, and enzymes for the lipid metabolism were compared between cord and adult blood. Consistent with previous reports, the major lipoprotein in cord blood was high-density lipoprotein (HDL), and that in adult blood was low-density lipoprotein (LDL). The level of apolipoprotein E (apo E) in cord blood was almost equivalent to that in adult blood, while other apolipoproteins and lipids were all lower than the adult level. In cord blood, apo E-rich HDL cholesterol represented more than 30% of total HDL cholesterol (around 11% in adult), and the concentration was about twice of that in adult blood. This apo E-rich HDL cholesterol was poorly esterified (E/T 56%) compared with that in adults (93%). The lecithin:cholesterol acyltransferase (LCAT) activity in cord blood was extremely low, while the activity and mass of cholesteryl ester transfer protein (CETP) were higher than those in adult blood. The apo E genotype did not show influences on total cholesterol, LDL cholesterol, total HDL cholesterol, and apo E rich HDL cholesterol levels in cord blood, as opposed to those in adult blood. The association of D442G mutation of the CETP gene with the increased HDL cholesterol in adult blood was not seen in cord blood. Rather, the mutation was associated with low total cholesterol and LDL cholesterol levels in cord blood. These results indicate that, in fetus, the character and metabolism of HDL, especially of apo E-rich HDL cholesterol, are distinct from those in adults.

Maternal immunization with pneumococcal polysaccharide vaccine in the third trimester of gestation

In a randomized, double blinded study, 23-valent pneumococcal polysaccharide vaccine (PSV) or conjugate Haemophilus influenzae type b (HbOC) vaccine was administered to 60 healthy women in the third trimester of gestation. Total IgG, IgG1, and IgG2 antibodies to pneumococcal serotypes 6B, 14, 19F and 23F were measured by ELISA in mothers prior to immunization, at delivery and 7 months after delivery, and in infants at birth (cord blood), 2 and 7 months after delivery. IgA was evaluated in breast milk at 2 and 7 months, and opsonophagocytic activity in cord blood. PSV was safe and immunogenic in pregnant women. Transplacental transmission of vaccine-specific antibodies was efficient. Maternal immunization with PSV resulted in significantly higher concentrations of pneumococcal antibodies in infants at birth and at 2 months of age, and greater functional opsonophagocytic activity of passively acquired IgG antibody.

A study of prolidase in mothers, their newborn babies and in non-pregnant controls.

The prolidase activity in serum and in erythrocytes was measured in 28 healthy mothers and in the cord blood of their newborn babies in using a modified Chinard method. 45 healthy non-pregnant women aged between 15-36 years formed a control group. Biochemical parameters (CK, BUN, C-peptid, AFP, Uric acid) were also measured. The serum and erythrocyte prolidase activities in maternal blood were 45.8+/-13.4 U/L and 37.8+/-2.7 U/g Hb respectively. There was no significant difference in the enzyme activities between pregnant women and the control group (p>0.05). However serum and erythrocyte prolidase activity in cord blood (20.3+/-8.2 U/L and 31.6+/-7.3 U/g Hb) was significantly different when compared with control group (53.4+/-14.7 U/L in serum and 42.3+/-10.3 U/g Hb in erythrocyte, p<0.001). There was a significant correlation between maternal and cord blood serum enzyme activity (r: 0.76 p<0.01). This correlation was also shown in erythrocyte prolidase activities of both groups (r: 0.49, p<0.05). Cord blood prolidase activity was positively correlated with birth weight (r: 0.89, p<0.01). Prolidase activity in cord blood was low even though collagen turnover is increased in fetal growth.

EFFECT OF TWO-ROUND FICOLL-HYPAQUE DENSITY GRADIENT CENTRIFUGATION ON LYMPHOCYTE SUBSETS AND NATURAL KILLER ACTIVITY OF UMBILICAL CORD BLOOD MONONUCLEAR CELLS

Umbilical cord blood (CB) mononuclear cells (MNCs) obtained from Ficoll-Hypaque density gradient centrifugation (FDGC) are frequently contaminated with erythrocytes and nucleated erythroid precursors. The authors investigated the effect of two-round (2-r) FDGC on lymphocyte subsets and natural killer activity of CB and adult peripheral blood (APB) MNCs, in comparison with those obtained from conventional one-round (1-r) separation. The percentage of CD45-expressing CB MNCs was greatly increased after the second density step (p < .01), indicating the efficacy in purification. The percentages of CD3/CD4, CD3/CD8, and CD16/56 double-positive staining CB MNCs were significantly increased after 2-r FDGC, as compared to those after 1-r separation. However, the percentages of CD34+ stemcells and CD19+ B cells were not affected by 2-r FDGC. MNCs obtained from 2-r FDGC had higher natural killer (NK) activity than did MNCs obtained from 1-r separation (p < .01). In contrast, 2-r FDGC did not affect lymphocyte subsets and NK activity of APB MNCs as compared to 1-r FDGC. Thus, 2-r FDGC are recommended to obtain CB MNCs for flow cytometric analysis and NK cytotoxicity assays.

Effects of insulin-like growth factor i on the regulation of telomere length and telomerase activity in cord blood t cells

Insulin-like growth factor-I (IGF-I) has been shown to enhance the PHA-induced increase in telomerase activity for cord blood (CB) mononuclear cells. It is hypothesized that the IGF-I on telomerase activity may have an impact on the telomere length and replicative potential of cells. In this study, we have cultured isolate CB T cells (CD3+ purity = 98%) in a similar serum free liquid medium containing: (a) NoGF; (b) IGF-I, (c) PHA, and (d) IGF-I + PHA. The telomere length of cells harvested on Day 0, 3, 5, 8, 14 and 21 was measured by a quantitative flow cytometry based fluorescence in situ hybridisation technique (Q-Flow FISH). The corresponding telomerase activity was determined by the non-radioactive PCR-based Telomeric Repeat Amplification Protocol (TRAP). The telomere length of PHA stimulated T cells increased to a peak level on Day 8 and decreased back to the initial level by Day 21. The addition of IGF-1 increased the telomere length of PHA stimulated T cells throughout the course of the culture. Interestingly, this later increase in telomere length was not accompanied by a high level of telomerase in the cells at later time points in the culture. The heterogeneous relationship between telomerase and telomere length suggested that the effect of IGF-I on telomere length elongation may be due to the involvement of alternative pathways other than the initial enhancement in telomerase activity in the cells. Further experiments are needed to clarify the mechanisms involved and the impact on replicative potential of cells. This data may be useful for the ex vivo expansion of T cells for adoptive immunotherapy and transplantation.

Erythrocyte glutathione S transferase as a marker of oxidative stress at birth

AIMSTo determine the level of oxidative stress and cell damage as a result of exposure to O2 at birth.METHODSUsing glutathione S transferase (GST) as an indicator of oxidative stress, GST...

Fetal natural killer cell function is suppressed.

Previous studies have reported reduced natural killer (NK) cell activity in cord blood (CB) compared with adult blood mononuclear cell populations. Using a non-radioactive killing assay, we have verified these findings suggesting that either the fetal NK cell function is suppressed or that these cells are functionally immature. We have shown that CB NK cells are functional, since activating them with cytokines known to activate adult NK cells [interleukin-2 (IL-2), IL-12 and IL-15] increased activation. However, resting the cells, which enhanced adult NK cell activity (P < 0.01), had no effect on fetal NK cells (P = 0.2). These results suggested that fetal NK cells have the capacity to kill, but this is suppressed in vitro. This hypothesis was strengthened by our observation that eight of nine CB mononuclear cell populations had their NK activity restored by freeze-thawing, whereas four of five adult peripheral blood mononuclear cells had a reduced killing ability on freeze-thawing. Freeze-thawing removes a population of cells that suppresses CB NK cell function. To determine which was the case we performed extensive phenotypic analysis of the CB populations pre- and post-freezing and found that the percentage of the CD3- CD56+ population within CB increased significantly (P < 0.0005 by paired t-test) with freezing, whereas freeze-thawing had no effect on this population within a normal adult peripheral blood mononuclear cell population. Our data suggest that within CB there is a population of cells, as yet undefined, which may be inhibiting NK cell function. This report therefore shows clear differences between NK cells within the adult periphery and in CB, and may lead to a better understanding of events occurring in vivo.

Opsonic activity in cord blood: Comparison between normal and elevated immunoglobulin M infants

In order to clarify the relation between opsonic activity (OA) in newborn infants and their gestational age, we determined OA by Cypiridina luciferin analog-dependent chemiluminescence (MCLA-CL). In addition, complement C3 which plays the major role in OA, was determined by the two-dimensional immunodiffusion method. Although OA and C3 in newborn infants increased in proportion to their gestational age, the levels were significantly lower in full-term infants than in adults. In neonates with IgM levels of more than 30 mg/dL at birth, both OA and C3 levels were significantly higher than in normal neonates, yet these levels were significantly lower than in adults. These findings suggest that low opsonin activity is an important factor in neonatal susceptibility to infection. When OA is measured by chemiluminescence, MCLA-CL is the preferred technique to luminol-dependent chemiluminescence.

Plasma xanthine oxidase activity and lipid hydroperoxide levels in preterm infants.

Ischemia-reperfusion injury may affect morbidity and mortality in preterm and asphyxiated term infants. Reoxygenation of hypoxic tissues leads to the formation of free oxygen radicals by xanthine oxidase that may induce lipid peroxidation, enzyme inhibition, and DNA strand breakage. We measured arterial cord blood samples from 36 healthy term infants for baseline values and arterial blood sampled at 1 and 4 h after birth from 45 preterm infants admitted for intensive care for serial estimates of plasma xanthine oxidase activity and lipid hydroperoxide levels. Mean +/- SEM plasma xanthine oxidase activity in cord blood of term infants was 2.3 +/- 0.4 mU/mL and lipid hydroperoxide levels were 2.6 +/- 0.3 nmol/mL. Eighteen of the 45 preterm infants met the criteria defining poor outcome (poor outcome group) and had lower umbilical arterial pH and base excess than the 27 preterm infants in the control group. Mean plasma xanthine oxidase activity increased from 2.7 +/- 0.4 at 1 h to 4.7 +/- 0.6 mU/mL at 4 h of age (p < 0.001) in the poor outcome group and decreased from 2.1 +/- 0.3 to 1.1 +/- 0.2 mU/mL (p = 0.004) in the control group. Lipid hydroperoxide levels in the poor outcome group increased from 2.8 +/- 0.6 nmol/mL at 1 h to 4.3 +/- 0.6 nmol/mL at 4 h of age (p < 0.001) and decreased from 2.1 +/- 0.6 to 1.6 +/- 0.2 nmol/mL (p = 0.008) in the control group. At 4 h of age, xanthine oxidase activity and lipid hydroperoxide levels were significantly higher in the poor outcome group than in the controls (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose-6-phosphate dehydrogenase, hexokinase and pyruvate kinase activities in erythrocytes of neonates and adults in Basrah

The mean activities (95% confidence interval; number of patients) for erythrocyte glucose-6-phosphate dehydrogenase (G6PD), hexokinase (HK), and pyruvate kinase (PK) in haemolysate of clinically normal neonates from Basrah, Iraq were found to be 3.29 (0.10; n = 456), 0.61 (0.02; n = 219), and 5.10 (0.07; n = 500), respectively, all expressed as U/10(10) RBC at 37 degrees C. Comparative values for apparently healthy adults were: 2.14 (0.06; n = 186), 0.41 (0.02; n = 46), and 3.61 (0.07; n = 243). Differences between sexes in the mean activities of each of the three enzymes were not significant (p > 0.5), being several times less than the corresponding 95% confidence intervals. However, differences were highly significant between neonates and adults (p < 0.001), presumably reflecting the greater proportion of 'young' cells and/or their programming for higher activity in cord blood. The neonate-adult difference (95% confidence interval) for each enzyme was: G6PD, 1.15 (0.43); HK, 0.20 (0.14); and PK, 1.49 (0.64). Some degree of G6PD-deficiency was observed in 7.9% of male and 9.7% of female neonates, whereas the frequency in adults was 9.2% and 11.8% for males and females, respectively. The higher frequency in females accords with expectations based on gene frequency estimations.

Hemopoietic and lymphoid progenitor cells in human umbilical cord blood.

Human umbilical cord blood, which in the past was discarded with the placental tissue, provides a convenient source of fetal hemopoietic cells for scientific analysis and clinical use. Cord blood cells are immature compared to analogous populations in adult peripheral blood. Cord blood B lymphocytes display unique phenotypic and functional characteristics. The antigens CD1C, CD38, CD5, and CD23, although normally expressed on only a small percentage of circulating B cells in adults, are highly expressed on cord blood B cells. Recent studies have demonstrated that whereas cord blood B cells are functionally naive, their potential is similar to that of adult B cells if optimal T-cell help is available. Thus, the failure of B-cell responses in cord blood is due to the T cells. The functional abnormalities of T cells from newborns can be summarized as a dominance of the effects of TH0 cells. Thus, the cytokines produced are immunosuppressive rather than mediating helper activity for B cells. NK activity in cord blood is also depressed compared to that in adults. Cord blood is a very rich source of hemopoietic progenitor cells. The spectrum of progenitors shows a predominance of early progenitor cells when compared with bone marrow. These cells provide an alternative source to adult bone marrow for stem cells to use for hemopoietic reconstitution and as targets in the treatment of hereditary deficiencies by gene therapy. These features make cord blood a unique research tool to investigate hemopoietic ontogeny and a unique clinical tool for transplantation.

Cord blood serum creatine kinase isoenzymes with placental dysfunction.

It has been suggested that perinatal asphyxia is not generally followed by neurological impairment unless there is preexisting chronic fetal distress. In cases of brain damage one can observe elevated levels of CK-BB. The purpose of our study was to evaluate CK isoenzymes in umbilical cord blood sera of newborns affected by chronic fetal distress. Fetal distress reflected by placental dysfunction was characterized by a diminished HPL level and decreased activity of CAP. We estimated CK isoenzymes with the use of DEAE-sepharose CL-6B column chromatography. Total CK activity was measured using kits supplied by Boehringer-Mannheim (Monotest CK-NAC aktiviert). The clinical state of examined newborns was estimated. Investigations were carried out in the group of 57 infants delivered after 37 weeks of gestation. Total CK activity in cord sera ranged from 40 to 400 U/l. Our results showed a significant rise of CK-BB activity in cord sera of newborns delivered from pregnancies with placental dysfunction (figure 2) as well as in cases of asphyxiated infants (figure 3). We were unable to demonstrate differences in total CK, CK-MM and CK-MB activities in all examined groups of newborns. Other authors have confirmed that severe asphyxia results in increase in CK-BB activity in cord blood. Infants with ominous fetal heart rate patterns have higher CK-BB activity. There are several possible sources for CK-BB activity in umbilical cord blood sera, i.e. fetal brain, lung, gastrointestinal tract, placenta and uterus. It appears that the brain is most likely the source of elevated CK-BB activity found in cord blood in cases of placental dysfunction.

Alkaline ribonuclease activity in maternal serum and in serum of newborns from birth up to thirty days of age. A study made with full-term appropriate-, full-term small- and preterm appropriate-for-gestational-age infants.

We have studied the alkaline ribonuclease (RNase) activity in maternal serum and serum of full-term small- (T-SGA), full-term appropriate- (T-AGA) and preterm appropriate-for-gestational age (PT-AGA) newborns. A significantly lower level of RNase was observed in T-AGA and T-SGA newborns on the 30th day of age and in PT-AGA newborns on the 15th and 30th days of age, as compared to other T-AGA, T-SGA and PT-AGA groups of infants at birth. RNase activity was significantly higher in cord blood than in the maternal blood in all categories studied. Moreover, in preterm newborns, RNase activity in cord blood was significantly higher in those presenting a lower gestational age. We did not observe any significant difference in RNase levels in the cord blood of newborns from the 3 categories studied. The same results were observed concerning maternal blood. We, therefore, conclude that RNase activity in cord blood or in maternal blood is not a very satisfactory indicator of fetal malnutrition.