Prebiotic oligosaccharides: In vitro evidence for gastrointestinal epithelial transfer and immunomodulatory properties

Eiwegger T, Stahl B, Haidl P, Schmitt J, Boehm G, Dehlink E, Urbanek R, Szépfalusi Z. Prebiotic oligosaccharides: In vitro evidence for gastrointestinal epithelial transfer and immunomodulatory properties.
Pediatr Allergy Immunol 2010: 21: 1179–1188.
© 2010 John Wiley & Sons A/S Prebiotic oligosaccharides are present in breast milk and evidence is pointing toward immunomodulatory properties of the acidic fraction. Recently, prebiotic supplements of infant formula [short‐chain galacto (scGOS)‐, long‐chain fructo (lcFOS)‐oligosaccharides] showed preventive effects on atopic disease development. We aimed to define the direct immunologic effects of these oligosaccharides and of human (aHMOS) and cows’ milk (aCMOS) acidic oligosaccharides and to investigate the systemic uptake of prebiotic supplements of infant formula and a specific pectin‐derived acidic oligosaccharide hydrolysate (pAOS) in vitro. After assurance of LPS‐free conditions (limulus assay, toll like receptor‐2, ‐4 transfected human embryonic kidney‐cells), in vitro‐transfer through a CaCo‐2 cell monolayer was measured using high‐pH anion exchange chromatography with pulsed amperometric detection. Direct effects on proliferation, cytokine‐induction of cord blood mononuclear cells and modulation of allergen‐specific CD4+ T‐cell cytokine profiles from allergic and non‐allergic individuals were investigated. Transfer of scGOS/lcFOS and pAOS in‐vitro was detected with a rate of transfer of 4–14%, depending on the molecular size and structure. AHMOS induced IFN‐γ and IL‐10 but not the Th‐2 cytokine IL‐13 at physiologic concentrations (10–100 μg/ml) in cord blood, whereas aCMOS did not induce any of these cytokines. AHMOS significantly suppressed Th‐2 type cytokine‐production by Ara h1‐specific CD4+ T cells (CFSE^low CD3⁺CD4⁺cells) from peanut allergic patients. In conclusion, human milk‐derived acidic oligosaccharides may modulate postnatal allergen‐specific immune responses by the suppression of Th‐2‐type responses in atopy‐prone individuals. Moreover, there is in vitro evidence for epithelial transport of prebiotic oligosaccharides.     

Neonatal formula feeding leads to immunological alterations in an animal model of type 1 diabetes

Neonatal diet may influence the development of type 1 diabetes (T1D) in susceptible individuals through an intestinal mucosal inflammatory response, resulting in loss of self-tolerance. We tested the hypothesis that formula feeding during the neonatal period accelerates the development of T1D in diabetes-prone BioBreeding (BBDP) rats through regulation of CD4+CD25+ regulatory T lymphocytes (T(reg)) and anti-inflammatory cytokines. BBDP rat pups fed rat milk substitute (RMS) via a "pup-in-the cup" system were compared with mother-fed (MF) rats. The spleen and thymus were analyzed for Foxp3-expressing CD4+/CD25+ T cells. Multiplex enzyme-linked immunosorbent assays (ELISAs) were performed to measure cytokine-induced neutrophil chemoattractant (CINC), tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-4, IL-10, and IL-18. Diabetes-free survival, time of disease onset, and T(reg)/total T lymphocyte ratios were not different. MF pups had higher ileal CINC (p < 0.001) and IL-18 (p = 0.002), but no differences in the liver. There were no differences in ileal cytokine concentrations of 75-d-old rats, but the formula-fed rats had greater liver TNF-alpha (p < 0.001), IFN-gamma, and IL-4 (p < 0.01) and lower IL-10 (p = 0.002) compared with MF animals. Formula versus maternal milk altered the hepatic cytokine profile at 75 d toward an inflammatory pattern but did not result in altered T(reg) cell frequencies or the development of T1D.     

Dysregulation of IL-13 production by cord blood CD4+ T cells is associated with the subsequent development of atopic disease in infants.

Early intervention strategies in allergic diseases will be dependent on identification of newborns at high risk for later development of atopic disease. In this cohort study of 106 neonates, we investigated whether cytokine production property and responsiveness to IL-12 of neonatal CD4(+) T cells were associated with the subsequent development of atopic disease and whether a skewed cytokine production property was intrinsic to helper T cells. To exclude the effects of contaminating cells, highly purified cord blood CD4(+) T cells were stimulated with anti-CD3 MAb and recombinant B7-2 molecule in the presence or absence of IL-12. Production of IL-13 and interferon-gamma was determined by ELISA. The infants were assessed at 12 mo for the development of atopic diseases. CD4(+) T cells of neonates who manifested allergic symptoms (atopic group) produced higher levels of IL-13 compared with those of the nonatopic group in both the presence and absence of IL-12. No significant difference was noted between the two groups with respect to interferon-gamma production. Moreover, higher IL-13 production was also observed in neonates with chronic eczema than those with short-term eczema. Our data suggest that increased production of IL-13 by neonatal CD4(+) T cells is a useful marker of newborns at high risk for subsequent development of atopic diseases and that an intrinsic abnormality of CD4(+) T cell is associated with the pathogeneses of atopic disease, especially atopic dermatitis in infants.     

CD30 on stimulated CD4+ T lymphocytes in newborns regarding atopic heredity

CD30 was initially described as Ki-1 Ag on Reed-Sternberg cells of Hodgkin's lymphoma and its and CD30L(+) expression on T cells in placenta were equally frequent in the atopic and non-atopic women. In this article we present a study of CD30 mean fluorescence intensity (MFI) on CB T CD4(+) cells. We tested the hypothesis that in newborns with atopy family history there is a changed CB T cells response after antigen stimulation comparing with those without atopy family history. The study population consisted of 31 newborn babies (29-breastfed, two non-breastfed) and their mothers. Eleven of them had positive and 20 had negative atopy family history. Performed tests included cord blood, which was a subject to flowcytometry analysis and was cultured for 24 h, cytokine production was measured (IFN- gamma, IL-4 and IL-12). Secondly, we measured total maternal and cord blood IgE levels. We studied CD30 MFI as in our studies in larger group of newborns, CD30 expression on CD4(+) T cells appeared to be very low. MFI of CD4(+) CD30(+) after PHA-stimulation (213.55: range: 41.77-434.51) was significantly increased compared to MFI of CD4(+) CD30(+) before PHA-stimulation (43.63: range 28.67-134.67)(p 0.05). After PHA stimulation MFI of CD4(+) CD30(+) in non-atopic (273.05 (range: 42.9-434.51) was significantly increased compared with the atopic newborns to MFI of 87.1 (range: 41.78-241.42) (p = 0.00). We have not found any correlation between MFI of CD4(+) CD30(+) and total maternal and total CB IgE levels. The role of CD30 in immunological response needs further research studies.     

SOCS低甲基化在儿童过敏性紫癜Th17/Treg细胞失衡中的作用研究

目的 通过研究细胞因子信号转导抑制因子（SOCS）低甲基化与过敏性紫癜（HSP）患儿Th17/Treg细胞失衡的关系,探讨HSP的免疫发病机制。方法 选取2014年5月至2015年1月32例急性期HSP住院患儿为研究对象,另选取行健康体检的28例儿童作为健康对照组。采用ELISA法检测血浆IL-6水平;流式细胞术检测外周血CD4⁺IL-17A⁺T细胞（Th17细胞）比例、CD4⁺CD25⁺调节性T细胞（Treg）比例和CD4⁺T细胞磷酸化STAT3（pSTAT3）蛋白平均荧光强度（MFI）;实时荧光定量PCR（RT-qPCR）技术检测CD4⁺T细胞SOCS1、SOCS3基因mRNA表达;高分辨率熔解曲线（HRM）分析法检测外周血单个核细胞SOCS1基因外显子2、SOCS3基因5'端非翻译区（5'-UTR）可能的STAT3结合位点CpG岛甲基化水平。结果 与健康对照组比较,HSP组血浆IL-6浓度、CD4⁺T细胞pSTAT3的MFI显著增加;HSP组Th17细胞比例显著上调,Treg细胞比例显著下调（P < 0.05）。HSP组患儿急性期外周血单个核细胞SOCS1 mRNA和SOCS3 mRNA水平均显著高于健康对照组（P < 0.05）;HSP组SOCS1 mRNA及SOCS3 mRNA表达均与Th17/Treg比值呈负相关（P < 0.05）。HSP组患儿急性期SOCS1基因外显子2、SOCS3基因5'-UTR区可能的STAT结合位点CpG岛呈低甲基化,而健康对照组呈完全去甲基化状态。结论 SOCS1、SOCS3基因低甲基化所致其相对表达不足可能是HSP患儿Th17/Treg失衡的因素之一。     

Responses of CD4(+) CD25(+) Foxp3(+) and IL-10-secreting type I T regulatory cells to cluster-specific immunotherapy for allergic rhinitis in children

We investigated the effects of cluster specific immunotherapy (SIT) with Dermatophagoides pteronyssinus (Der p) on CD4(+) CD25(+) Foxp3(+) Treg cells and IL-10-secreting type I T regulatory (Tr1) cells in Der p-sensitized children with allergic rhinitis (AR). We performed a prospective randomized study involving 46 children (aged 8-13 yr), of whom 25 children received Der p-SIT + pharmacotherapy and 21 received only pharmacotherapy, over a period of 1 yr. Prior to and at end of treatment, CD4(+) CD25(+) Foxp3(+) Treg cells and allergen-specific IL-10(+) IL-4(-) , IFN-γ(+) IL-4(-) , and IL-4(+) IFN-γ-CD4(+) T cells were measured by flow cytometry. Similarly, IL-4, IFN-γ, and IL-10 in supernatants from allergen-stimulated peripheral blood mononuclear cell (PBMC) cultures were measured by ELISA, and the suppressive effect of CD4(+) CD25(high) T cells on cell proliferation and cytokine release was estimated from both groups. Allergen-specific serum IgE and IgG4 were also assessed at the beginning and end of treatment by RAST and ELISA, respectively. The levels of allergen-specific Tr1 cells, IgG4, and allergen-induced IL-10 synthesis from PBMC cultures were significantly increased after SIT for 1 yr compared with baseline levels (p < 0.001 for all), with significant correlation between increased levels of Tr1 cells and improvements in nasal symptoms (r = 0.48, p < 0.05). In contrast, the levels of CD4(+) CD25(+) Foxp3(+) T cells, allergen-specific Th1 and Th2 cells, the production of IL-4 and IFN-γ, and the function of CD4(+) CD25(high) T cells were not altered in either group at the end of treatment. These data suggest that the up-regulation of Tr1 cells may play an important role in SIT and be a useful marker of successful SIT in AR patients.     

Variations in oligosaccharides and lactose in human milk during the first week of lactation

Variations in oligosaccharides and lactose in human milk were studied in 15 mothers during the first week of lactation. The neuraminyloligosaccharides and heavy neutral oligosaccharides increased slightly from days 2 to 5 postpartum and appeared to decrease until day 7. The lacto-N-difucohexaoses, lacto-N-fucopentaoses, and lacto-N-tetraose increased until day 5 and then decreased. Lactodifucotetraose and the fucosidolactoses decreased substantially until day 5 (p less than 0.05) and appeared to stablize in the following days. Lactose increased until day 5 (p less than 0.05) and continued to increase thereafter. Lactose was negatively correlated with total oligosaccharides (p less than 0.10). The fluctuations observed in total oligosaccharides from days 2 to 5 postpartum and their subsequent stabilization and regular decrease during lactation confirm the hypothesis of Kulski and Hartmann that mammary secretion occurs in three periods: colostrum for the first 36 h postpartum, transitional milk from days 2 to 5 postpartum, and mature milk after day 5. The oligosaccharide variations we found corresponded to those of other milk constituents observed by other authors. The significance of the oligosaccharide variations is discussed.     

哮喘儿童IgE T细胞亚群细胞因子的动态观察及临床意义

目的：探讨哮喘儿童血IgE和T细胞亚群、细胞因子的动态变化及临床意义。方法：应用免疫萤光法及双抗夹心酶联免疫吸附试验(ELISA)分析方法对45例哮喘儿童发作期和缓解期分别测定IgE,T细胞亚群和细胞因子。对照组为20例健康儿童。结果：哮喘发作期、缓解期CD_3~+,CD_4~+ T细胞及CD_4~+/CD_8~+高于对照组,差异有显著性(P<0.05或0.01),CD_8~+ T细胞与对照组比差异无显著性(P>0.05)。发作期CD_4~+ T细胞及CD_4~+/CD_8~+高于缓解期(P<0.05)。发作期IL-2,EFN-γ低于对照组(P<0.01或0.05),IL-4,IL-6,IL-8和IgE高于对照组(P<0.01或0.05);缓解期IL-2,IFN-γ低于对照组(P<0.01),IL-4,IL-8,IgE高于对照组(P<0.05或0.01),缓解期IL-6与对照组比较差异无显著性(P>0.05)。结论：儿童哮喘在发作期和缓解期均存在着免疫功能紊乱,提示儿童哮喘应长期抗变应性炎症治疗。 [中国当代儿科杂志,2003,5(1):23-26]     

Peripheral blood intracellular cytokine analysis in children newly diagnosed with inflammatory bowel disease

Patterns of cytokine profiles have emerged for different forms of inflammatory bowel disease with a predominance of type 1 cytokines in patients with Crohn disease and type 2 cytokine expression in patients with ulcerative colitis. Most of these studies have involved older patients with long-standing disease or after various therapeutic interventions, and patterns of cytokine expression were hypothesized to be influenced by these factors. To evaluate for these possibilities, we studied 23 patients (15 boys) with newly diagnosed Crohn disease (n = 14) or ulcerative colitis. Their mean age at diagnosis was 13.1 +/- 2.9 y (mean +/- SD). Healthy control subjects (n = 9) were previously obtained. Peripheral blood intracellular cytokine analysis was performed within 24 h using a modification of Becton Dickinson's FastImmune Cytokine system. Multiparametric flow cytometry and phenotyping of lymphocytes was performed. T-cell populations were defined as type 1 being CD69(+), CD3(+), and interferon-gamma(+) and type 2 being CD69(+), CD3(+), and IL-4(+). The median percent of type 1 T cells from normal subjects (2.8%) was similar to that of ulcerative colitis subjects (1.8%, p > 0.20) but greater than that of Crohn disease subjects (0.55%, p = 0.05). The median percent of type 2 lymphocytes in normal subjects (1.8%) was greater than that of ulcerative colitis subjects (0.35%, p = 0.02) but was similar to that of Crohn disease subjects (1.1%, p > 0.20). Serial determinations showed the median percent of type 2 T cells increased in ulcerative colitis patients as remission was induced. Reduced activated peripheral type 1 T cells of newly diagnosed, untreated children are similar to interferon-gamma expression in mucosa of adults with postoperative recurrence. Reduced type 2 cytokine expression patterns in subjects with ulcerative colitis are similar to lamina propria T-cell expression levels in adults and improve with disease remission.     

Lower proportion of CD45R0+ cells and deficient interleukin-10 production by formula-fed infants, compared with human-fed, is corrected with supplementation of long-chain polyunsaturated fatty acids

BACKGROUND: The immune consequences of adding 20:4n-6 and 22:6n-3 fatty acids to preterm infant formula are not known. METHODS: The effect of feeding preterm infants (14-42 days of age) human milk (Human Milk group), infant formula (Formula group), or formula with added long-chain polyunsaturated fatty acids 20:4n-6 and 22:6n-3 (Formula + LCP group) on isolated peripheral blood lymphocytes (by flow cytometry) and lipid composition (by gas-liquid chromatography) was determined. Lymphocytes were stimulated in vitro with phytohemagglutinin to measure soluble interleukin (sIL)-2R and IL-10 production (by enzyme-linked immunosorbent assay). RESULTS: With age, the percentage of CD3+ CD4+ T cells and the percentage of CD20+ cells increased in the Human Milk and Formula + LCP groups (P < 0.05), but not in the unsupplemented Formula group. Compared with the Formula group, CD4+ cells from the Formula + LCP and Human Milk groups expressed more CD45R0 (antigen mature) and less CD45RA (antigen naive) at 42 days of age (P < 0.05). At 42 days, IL-10 production was lower (P < 0.05) in cells of the Formula group than in cells of the Human Milk group. Production of IL-10 by the cells of the Formula + LCP group was not different from that produced by the Human Milk group cells. An age-related decrease (P < 0.05) in sIL-2R production by Formula + LCP lymphocytes was observed, but sIL-2R production at 42 days in the Formula + LCP group did not differ significantly from that in the Human Milk group. Compared with Formula alone, adding LCP to formula resulted in a lower C18:2n-6 and higher C20:4n-6 content in lymphocyte phospholipids (P < 0.05). CONCLUSIONS: Adding LCP to a preterm infant formula resulted in lymphocyte populations, phospholipid composition, cytokine production, and antigen maturity that are more consistent with that in human milk-fed infants. This may affect the ability of the infant to respond to immune challenges.     

Differential modulation of the immune response by breast- or formula-feeding of infants

Spontaneous integrin expression on CD4+, CD8+ and CD19+ lymphocytes at 6 months was significantly lower in breastfed than formula-fed infants ( p < 0.05). In another study of 59 formula-fed and 64 breastfed 12-month-old children blast transformation and cytokine production by lymphocytes, and T cell changes were measured before and after measles-mumps-rubella vaccination (MMR). Before vaccination, lymphocytes of breastfed children had lower levels of blast transformation without antigen ( p < 0.001), with tetanus toxoid ( p < 0.02) or Candida ( p < 0.04), and lower interferon-γ production ( p < 0.03). Fourteen days after the live viral vaccination, only the breastfed children had increased production of interferon-γ ( p < 0.02) and increased percentages of CD56+ ( p < 0.022) and CD8+ cells ( p < 0.004). These findings are consistent with a Thl type response by breastfed children, not evident in formula-fed children. Feeding mode has an important long-term immunomodulating effect on infants beyond weaning.     

Cytokine production, activation marker, and skin homing receptor in children with atopic dermatitis and bronchial asthma

T cells are known to develop a critical role in the pathogenesis of atopic dermatitis (AD) and bronchial asthma. T cells involved in AD express the skin homing receptor CLA, but no lung homing receptor has been identified in bronchial asthma. We compared different cell markers and the cytokine production in T cells from children with AD or bronchial asthma. We studied the involvement of CLA+ and CLA- T-cell subpopulations in these diseases. We studied 20 children with acute AD lesions, 15 with mild persistent asthma, and 15 non-atopic controls. All patients were sensitized to house dust mite (DP) and evaluated during the acute phase. Total and specific IgE were measured by immunoassay and the expression of different cell markers and the cytokine production was analyzed by flow cytometry in peripheral blood mononuclear cells. Total IgE was significantly higher in AD children and IgE to DP in the asthmatic children. There was a significant increase in CD25+ CD4+ cells in asthmatic children and in HLA-DR+ CD4+ and HLA-DR+ CD8+ cells in AD. In the CD4+ subsets, there was an increase in IL-13, IL-5 and TNF-alpha in AD compared to controls, a decrease in IFN-gamma in asthmatic children compared to controls, and an increase in IL-13, IL5, IL2, TNF-alpha, and IFN-gamma in the AD compared to asthmatic children. Changes in cytokine production were mainly detected in CLA+ cells in AD and in CLA- cells in asthma. Differences exist in total and specific IgE, activation markers, and cytokine patterns between AD children and children with asthma, with the former expressing a Th2 pattern whereas in asthmatic children we only detected a decrease in IFN-gamma. Moreover, the subpopulations (CLA+ vs. CLA-) expressing these changes were different, indicating that the underlying mechanisms in the two diseases are not exactly the same.     

Disease progression markers during asymptomatic phase of HIV-1 infected children with unimpaired CD4+ cell values: evaluation of repeat CD4+ cell evaluation vs. other immunological parameters

The availability of a marker that could predict the course of disease progression in HIV-infected individuals would be of considerable relevance during the asymptomatic stage in order to undertake timely prophylactic measures. A prospective study was undertaken in a group of 42 children suffering from thalassemia major with HIV-1 infection to assess the status of immune parameters, such as peripheral CD4+ T lymphocyte (CD4+ cell) percentage, delayed type of hypersensitivity (DTH) response to recall antigens, detection rate and levels of p24 antigen, and levels of beta-2 microglobulin and cytokines in serum. All were assessed at an interval of 2 years during the asymptomatic period, (baseline and follow-up assessments) in relation to the development of AIDS defining illness within a follow-up period of 3 years. No difference could be observed in the mean CD4+ cell percentage at baseline between those who progressed subsequently to develop AIDS within the follow-up period (progressors) and those who did not (non-progressors). However, at the point of follow-up assessment the progressor group showed significantly lower CD4+ cell percentage compared to the non-progressor group (33 +/- 4.9 vs. 22 +/- 5.6; p < 0.05), although in the progressor group there was no correlation of the baseline and follow-up CD4+ cell percentage with the duration of the AIDS-free interval. However, in the progressor group there was a strong negative correlation between the rate of decline in CD4+ cell percentage and subsequent duration of the AIDS-free interval (r = -0.859). Analysis of additional immune parameters at baseline revealed that the progressor group, despite having CD4+ cell values comparable to non-progressors, showed impaired DTH response (number and total induration of positive responses being 2.0 +/- 1.23 and 6.2 +/- 1.4 in the former group vs. 3.2 +/- 0.76 and 12.6 +/- 3.80 in the later group; p < 0.05 for both the parameters), and elevated levels (mg/l) of serum beta-2 microglobulin (2.92 +/- 0.89 vs. 1.38 +/- 0.43; p < 0.05). The serum cytokine profile at baseline in the progressor group showed a T helper type-2 (Th2) dominant pattern, i.e. elevation of interleukin-4 (IL-4) and interleukin-10 (IL-10) levels with decreased levels of interleukin-2 (IL-2) and gamma interferon (gamma-IFN) compared to the non-progressor group that showed a T helper type-1 (Th1) dominant profile, i.e., elevation of IL-1 and gamma-IFN level with decreased levels of IL-4 and IL-10 (p < 0.05 for all four cytokines). The present study points out that rate of decline rather than single point of assessment of CD4+ cell values is a more reliable predictor for disease progression in HIV-1 infected children. In addition, parameters such as DTH response, serum levels of beta-2 microglobulin and serum cytokine profile, may provide valuable predictors of subsequent fall in CD4+ cell value.     

Association of cytokine single nucleotide polymorphisms with B7 costimulatory molecules in kidney allograft recipients

African-American race is associated with an increased risk of allograft loss, suggesting that African-American patients may form an immunologically higher risk group. Previously, we demonstrated that immune cell costimulatory molecule expression is significantly higher in African-Americans than in Caucasians. Polymorphic variations in the genes for cytokines have been associated with a number of immunological conditions, and with transplant rejection. This study was performed to determine the distribution, in African-American and Caucasian renal transplant recipients, of single nucleotide polymorphisms (SNPs) in the following cytokine genes: tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-6, IL-10, and transforming growth factor-beta (TGF-beta). Cytokine production from blood cells was determined, and cell-surface B7 (CD80, CD86) expression was measured. There was a significant link between IL-10 genotype and acute rejection episodes, but only in African-American patients (p < 0.01). Also, African-American patients had a significantly higher probability of having the IL-6 G-allele (p < 0.0001), which is associated with a high production of IL-6 protein. Incubation of blood cells with IL-6 resulted in increased expression of surface CD80 and CD86, while IL-10 decreased CD80 expression. This study demonstrated a clear correlation of the IL-6 G-allele with increased cellular CD80 expression and the IL-10 G-allele with decreased CD80 expression. These data raise the possibility that specific genotypes are associated with local cytokine regulation of cell-surface costimulatory molecule expression. African-American patients may have a genetically determined, quantitatively different immune response than Caucasian patients, contributing to adverse transplant outcomes.     

Expression of CD21 and CD23 during human fetal development

Neonates produce lower levels of IgE compared with adults. Diminished IL-4 production and impaired up-regulation of CD40L by neonatal T cells could explain this, however other regulators of IgE production, such as CD21 and CD23, could contribute to reduced circulating IgE levels during fetal development. Heparinized blood samples were collected from adults and from the umbilical cord at premature and term births. Whole blood flow cytometry was used to assess the percentage of T (CD3(+)) and B (CD19(+)) lymphocytes expressing CD21 and/or CD23 at 26-29 (n = 3), 30-33 (n = 7), 34-37 (n = 5), and >37 (n = 11) wk of gestation, as well as in adults (n = 15). Plasma-soluble CD21 was also measured. At term, the percentage of CD21(+) and CD23(+) B cells was comparable to the adult, however, the percentage of cells positive for each of these surface antigens was decreased significantly before term. The percentage of T cells expressing CD21 from all gestations was significantly higher than the adult and the percentage positive decreased with increasing gestational age. Conversely, soluble CD21 levels increased with increasing gestation to be comparable to the adult by term. Thus, it is unlikely that altered expression of CD21 and CD23 on B cells contributes to the low level of IgE in the neonatal circulation unless functional differences occur or a lack of processing to the soluble form is important in regulating IgE production. However the abundance of CD21-positive T cells could alter the T- and B-cell interaction necessary for IgE switching by B cells and, thereby, especially with impaired IL-4 production, limit IgE production.     

Induced apoptosis of TH2 lymphocytes in asthmatic children treated with Dermatophagoides pteronyssinus immunotherapy

Allergen-specific immunotherapy (IT) has been effectively used for the treatment of asthma. Allergen specific IT induced immune tolerance with induction of TH2 cells anergy remain to be clarified. The aim of this study was to evaluate whether the mite allergen Dermatophagoides pteronyssinus (Dpt) specific IT serially decreased IL-4+/CD4+ (TH2) lymphocytes and induced apoptosis of TH2 lymphocytes in asthmatic children. Sixty Dpt-sensitive asthmatic children were randomly assigned to a received IT and an untreated group. Dermatophagoides pteronyssinus specific IT treated patients were examined at three time points: before IT, after 6 months of an increased dose phase and with maximum tolerated doses after 1 yr. Peripheral blood mononuclear cells (PBMC) were isolated and cultured for 48 h for cellular staining with CD4+, CD45RO cell phenotypes and interleukin (IL)-4 and interferon-gamma expression by fluorescence monoclonal antibodies. Apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method. A simultaneous flow cytometric study using the same permeabilized cell was examined to determine whether apoptosis occurred preferentially in TH2 lymphocytes. The data demonstrated that Dpt specific IT decreased Dpt-specific IgE levels (p < 0.01) after 1 yr of treatment. In addition, decreased CD4+IL-4+ TH2 cells with increased CD4+IFN-gamma+ TH(1) cells were observed at 6 months and 1 yr after IT treatment (p < 0.05). At the same time, apoptosis of CD4+IL-4+ TH2 lymphocytes in the IT group had increased after 1 yr of treatment when compared with the results before treatment (p < 0.001) and after 6 months of treatment (p = 0.046). In addition, CD45RO cells apoptosis mainly occurred after 6 months of IT treatment and after 1-year period of IT treatment (p < 0.05). All of the data suggested that Dpt specific IT decreased Dpt specific IgE and CD4+IL-4+ TH2 lymphocytes with induction apoptosis of CD4+IL-4+ TH2 lymphocytes subsets serially.     

儿童急性B前体淋巴细胞白血病iTR35调节性T细胞亚群改变及意义初探

目的探讨急性B前体淋巴细胞白血病(BCP-ALL)患儿i TR35调节性T细胞改变及其在BCP-ALL免疫发病机制中的作用。方法以2012年7月至2013年12月深圳市儿童医院血液肿瘤科诊断并住院治疗的BCP-ALL初诊患儿为BCP-ALL组,并分为高危、中危和标危;同期体检的健康儿童为对照组。分离外周血CD4+T细胞,采用流式细胞术检测外周血CD4+FOXP3-IL-10-TGF-β-IL-12p35+IL-27EBI3+(i TR35)、CD4+CD25high FOXP3+(Treg)细胞比例及IL-12p35、IL-27EBI3、p STAT1、p STAT4蛋白表达水平;实时荧光定量PCR检测CD4+T细胞IL-12Rβ2、gp130 mRNA表达;ELISA检测血浆IL-35、IL-10水平。比较BCP-ALL组和对照组上述指标的差异。结果 1BCP-ALL组48例(男29例),年龄2.3~11.0岁,平均5.2岁;高危11例,中危21例,标危16例。对照组32例(男21例),年龄2.6~10.8岁,平均5.1岁。两组年龄和性别构成差异均无统计学意义。2BCP-ALL组外周血i TR35细胞比例显著高于对照组(P0.001),其胞内IL-12p35、IL-27EBI3表达水平亦显著高于对照组(P0.001);3BCP-ALL组Treg细胞比例及其胞内IL-12p35、IL-27EBI3表达水平较对照组明显增高(P0.001),血浆IL-35水平及CD4+T细胞IL-12Rβ2、gp130、p STAT1、p STAT4表达均上调(P0.001),且血浆IL-35水平与i TR35细胞比例及其IL-12p35、IL-27EBI3表达呈正相关(r0.63,P0.05)。4BCP-ALL组高、中危患儿血浆IL-35水平和i TR35细胞比例显著高于标危患儿(P分别为0.001和0.002),高危和中危患儿间差异无统计学意义(P0.05)。结论 i TR35细胞数量及功能异常可能是导致BCP-ALL患儿免疫功能低下的重要因素之一。     

哮喘发作患儿CD30分子表达及与Th2源细胞因子和血浆免疫球蛋白E水平的相关性研究

目的　探讨CD3 0 在哮喘发病中的作用。方法　随机选择哮喘急性发作期患儿 2 7例 ,急性上呼吸道感染 (上感 )患儿 16例 ,对照组 19例。采用直接免疫荧光流式细胞术检测外周血单核细胞 (PBMC)中CD4 细胞表达CD3 0 百分率 ,采用ELISA法检测培养上清IL 4、IL 13及血浆IgE水平。 结果　 1.哮喘患儿PBMC中CD4 细胞表达CD3 0 百分率较对照组和上感组明显增高 ,差异有显著性 (P均 <0 .0 5) ;2 .哮喘患儿PBMC培养上清IL 4、IL 13和血浆总IgE水平均较对照组和上感组增高 ,上感组血浆IgE水平亦较对照组增高 ,差异均有显著性 ;3 .哮喘组CD4 细胞表达CD3 0 百分率与培养上清IL 4、IL 13和血浆IgE水平呈显著正相关。 结论　分泌IL 4和IL 13的Th2类细胞活化、增殖的克隆可能主要是由CD3 0 阳性细胞克隆组成 ,Th2细胞表面CD3 0 与CD3 0 L结合后导致Th2细胞分化成熟及释放Th2源细胞因子 ,IL 4和IL 13增加可诱导B细胞分泌较多的IgE。说明CD3 0 信号传导在哮喘发生发展过程中起重要作用     

1 alpha,25(OH)2D3 inhibits not only Th1 but also Th2 differentiation in human cord blood T cells

Human naive CD4+ T helper (Th) and CD8+ cytotoxic (Tc) T cells, which only produce IL-2, may differentiate into Th1/Tc1- or Th2/Tc2-like lymphocytes, characterized by their cytokine production profile. 1 alpha,25-dihydroxyvitamin D3 (1 alpha, 25(OH)2D3) has been reported to inhibit Th1/Tc1-related, but increase Th2/Tc2-associated cytokines in T cells from adults. In industrialized countries, vitamin D supplementation for prevention of rickets is initiated within the first days of life and continued throughout the entire first year. Epidemiologic studies suggest an association of vitamin D exposure in newborns with the incidence of allergic diseases in later life. This study addresses the effects of 1 alpha, 25(OH)2D3 on Th1/Tc1 versus Th2/Tc2 differentiation in long term cell cultures of (naive) cord blood T lymphocytes. Our results show that in CD4+ as well as CD8+ cord blood cells, 1 alpha, 25(OH)2D3 inhibits not only IL-12-generated IFN-gamma production, but also suppresses IL-4 and IL-13 expression induced by IL-4. Thus, in cord blood 1 alpha, 25(OH)2D3 induces a T cell population without predominance of Th2 related cytokines.     

Intracellular production of IL-2, IL-4, IFN-γ, and TNF-α by peripheral blood CD3+ and CD4+ T cells in children with atopic dermatitis

The role of the type-2 T helper (Th2) cell-mediated immune response in the immunopathogenesis of atopic dermatitis (AD) is well documented. Whether polarized immunoresponse is confined to antigen-specific T cells or is distributed among all T cell subsets is still controversial. We investigated frequencies of interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) producing CD3⁺ and CD4⁺ T cells in peripheral blood from children with atopic dermatitis and healthy subjects with and without in vitro stimulation. Children with severe AD had a significantly lower percentage of CD4⁺ T cells spontaneously expressing IL-4 compared with healthy controls (p <0.01). Polyclonal stimulation significantly increased cytokine production in both AD patients and healthy individuals. Frequencies of CD3⁺ and CD4⁺ producing IL-2, IL-4, IFN-γ, and TNF-α after in vitro stimulation with phorbol-12-myristate 13-acetate (PMA) + ionomycin were comparable in the AD and control groups. In response to PMA/ionomycin, children with AD and asthma symptoms had a significantly lower percentage of CD3⁺ T cells producing TNF-α. We failed to demonstrate evidence of an imbalance with respect to type-2 cytokine productions in children with AD. Comparable induction of Th1 and Th2 cytokines in polyclonally stimulated peripheral CD3⁺ and CD4⁺T cells from AD patients and controls puts into question the polarized Th2 immune response as a general characteristic of T cells in children with atopic dermatitis.