We compared lymphocyte subsets and cytokine responses to bacteria among term, preterm infants, and adults. Lymphocyte subset percentages in cord blood (22 preterm, 27 term neonates) and peripheral blood from 21 adults and cytokine/chemokine interleukin (IL)-6, IL-8, IL-10, IL-12, interferon gamma (IFN gamma) responses to Escherichia coli, group B Streptococcus (GBS), Staphylococcus epidermidis, and Lactobacillus plantarum (Lp299v) were assessed by flow cytometry. Preterm compared with term infants had increased CD8 (+) T cells (p = 0.02) and reduced naïve CD4 (+) T cells (p < 0.0001). Memory T and natural killer (NK) T cells were reduced (p < 0.001) in neonates; NK and CD56 (+)161 (+) NK cells were increased (p < 0.001). CD56 (+)CD8 (+) NK cells were higher in preterm compared with term infants. Despite individual exceptions, cytokine responses in neonates were weaker than adults except for IL-8 response to E. coli in preterm and IL-12 response to Lp299v in term infants. IL-10 responses were weaker in preterm (p = 0.01) and term (p = 0.005) infants to S. epidermidis and to E. coli (p = 0.03 for both) compared with adults. Differences in regulatory subpopulations of NK and T cells between neonates and adults and term compared with preterm infants were observed. These differences rather than intrinsic functional deficiency may account for neonatal cytokine responses to bacteria.
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.
We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and (2)H(2)O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 2-8 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to (2)H(2)O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from (2)H(2)O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.33-0.90% per day (half-life of 77-210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis.
Maitake beta-glucan (MBG) is an extract from the fruit body of the Grifola frondosa mushroom that is being widely used to treat cancer in Asia. We have previously reported that MBG enhances mouse bone marrow cell (BMC) hematopoiesis in vitro and protects BMC from doxorubicin (DOX) toxicity. In the current study, we investigated the ability of MBG to enhance hematopoiesis and to reduce the toxic effects of DOX on fresh human umbilical cord blood (CB) cells. MBG treatment significantly enhanced the colony formation unit (CFU) response of granulocytes-macrophages (CFU-GM response) over the whole dose range of 12.5 to 100 g/ml (P < 0.05). The addition of MBG to DOX-treated CB cells significantly protected granulocyte-macrophage colony formation from the toxicity of DOX, which otherwise produced strong hematopoietic repression. MBG also partially replaced recombinant human granulocyte colony-stimulating factor (rhG-CSF), as shown by a significant augmentation of the CFU-GM response in the absence of rhG-CSF. We found that MBG induces granulocyte colony-stimulating factor (G-CSF) production in CB CD33؉ monocytes, as detected by intracellular cytokine flow cytometric assessment. In contrast, we found that adult peripheral blood monocytes did not produce a significant G-CSF response to MBG, whereas both adult and CB monocytes produced G-CSF in response to lipopolysaccharide. These studies provide the first evidence that MBG induces hematopoietic stem cell proliferation and differentiation of CFU-GM in umbilical CB cells and acts directly to induce G-CSF.Maitake beta-glucan (MBG) is an extract from the fruit body of the Grifola frondosa mushroom that is widely used in Asia for the treatment of cancers, although the mechanism(s) of action is unclear (4). MBG contains glucan polysaccharide compounds that have a beta-1,6-glucopyranoside main chain with branches of beta-1,3-linked glucose (24). The results of experimental studies with animals suggest that MBG administered orally activates the host antitumor response through effects on the immune system rather than by direct cytostatic or cytotoxic effects on tumor cells (19,37). However, dose-response relationships have not been shown (15, 21). Since other beta-glucans have been found to reduce myelosuppression and to enhance hematopoiesis in vitro and the mobilization of stem cells in vivo in animal models (16, 27, 33), we initially tested the effects of MBG on mouse bone marrow cells. Those studies showed for the first time that MBG enhances murine bone marrow cell proliferation and differentiation into granulocytesmacrophages (GMs) in a dose-dependent manner (22). In the presence of the chemotherapy drug doxorubicin (DOX), MBG promoted bone marrow cell viability and protected the bone marrow stem cell colony formation unit response of GMs (CFU-GM response) from DOX-induced hematopoietic toxicity. On the basis of the results of our studies with the mouse, we are interested in the possible use of MBG for myelosuppression secondary to cancer chemotherapy and for ex vivo expa...
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