The role of cysteinyl leukotrienes (CysLTS) in bone marrow responses in murine experimental allergic rhinitis: blockade of eosinophil production and tissue eosinophilia by a CysLT1 receptor antagonist

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Molecular markers of eosinophilopoiesis at birth: kinetics of cord blood GATA-1, MBP and IL-5 receptor expression

<b>RATIONALE:</b> Using colony assays and flow cytometry, we have recently shown that eosinophil/basophil (Eo/B) progenitor phenotype and function are associated with atopic risk at birth and infant clinical outcomes. The current study aimed to utilize real-time polymerase chain reaction (Q-PCR) to ascertain the kinetic patterns of expression of CB Eo/B-lineage specific genes, GATA-1, MBP and IL-5Rα in order to develop molecular markers of Eo/B differentiation.<p></p> <b>METHODS:</b> CB non-adherent mononuclear cells (NAMNCs) were isolated from random fresh and frozen samples, and incubated in the presence of rhIL-5 (1 ng/mL). At 24, 48 and 72h post-stimulation, RNA was isolated, reverse transcribed, and expression of IL-5Rα, GATA-1, and MBP were determined utilizing comparative Q-PCR in a multiplex reaction. The relative expression ratios between stimulated and un-stimulated cells were calculated using the delta-delta Ct method.<p></p> <b>RESULTS:</b> Stimulation with IL-5 resulted in an up-regulation of GATA-1 expression; this peaked between 24 and 48hrs. In contrast, MBP was up-regulated in a slowly progressive pattern, with maximal up-regulation at 72h, while there was a stable, minor down-regulation of IL-5Rα. In keeping with these molecular kinetic findings, Eo/B colony-forming cells, grown in 14-day methylcellulose culture, were found to be present in relation to timing of GATA-1 expression.<p></p> <b>CONCLUSIONS:</b> Multiplex Q-PCR analysis of mRNA from CB mononuclear cells stimulated with IL-5 demonstrates sequential expression of critical lineage-specific events, and can be used as a surrogate, molecular marker of CB Eo/B differentiation in clinical studies.<p></p&gt

The effect of lipopolysaccharide (LPS) on cord-blood hemopoietic progenitors

RATIONALE: The Hygiene Hypothesis has focused attention on the interaction of the innate immune system and the microbial environment. Since hemopoietic mechanisms are involved in the maintenance and development of atopy, we studied the effect of the gram-negative bacterial endotoxin, lipopolysaccharide, on cord-blood hemopoietic progenitors.<p></p> METHODS: Non-adherent mononuclear (NAMNC) human cord blood cells were enriched for CD34+ hemopoietic progenitors using magnetic activated cell sorting (MACS), either via depletion of CD3+/CD16+ cells (n=9) or via positive CD34+ selection (n=14), then incubated for 24 hours in varying doses of LPS (0-, 0.01, 0.1, 1 and 10 mcg/ml), followed by washing and incubation in methylcellulose colony-forming assays (CFU) in the presence of IL-3, IL-5 or GM-CSF. Eosinophil/basophil (Eo/B) CFU were enumerated at 14 days, and analysis performed using Friedman's ANOVA.<p></p> RESULTS: There was a significantly positive dose-response of Eo/B CFU to LPS using either enrichment method for CD34+ cord blood populations. For CD3/CD16 depleted NAMNC, there were more Eo/B CFU with the optimal LPS dose (10 mcg/ml) compared to control (2.5 compared to 11.5 CFU/2x10<sup>5</sup> cells plated, p= 0.018); overall LPS effect (p=0.047). For CD34-selected cells, there were more Eo/B CFU with the optimal LPS dose (10 mcg/ml) compared to control (3 compared to 8.25 CFU/10<sup>4</sup> cells plated, p= 0.033); overall LPS effect (p=0.048).<p></p> CONCLUSIONS: CB NAMNC, specifically CD34+ cells, are capable of responding to LPS and hemopoietic cytokines functionally, by differentiating into mature eosinophils and basophils. The findings highlight a potential mechanism through which microbial stimuli could directly modulate atopy and/or allergic inflammation.<p></p&gt

Cord blood (BR) progenitor toll- like receptor (TLR) expression: an alternate innate immune pathway in the development of atopy

RATIONALE: Atopy in early life may be modulated by the expression and stimulation of TLRs on immunocompetent cells. Infants at high risk for developing atopy demonstrate phenotypic alterations of their CB progenitor cell hemopoietic cytokine receptors (HCR). Since hemopoietic mechanisms are involved in atopic development and maintenance, we investigated the co-expression of TLR and HCR on CB progenitors.<p></p> METHODS: Fresh CB, enriched for CD34+ cells through magnetic cell separation techniques, were stimulated with 10 μL lipopolysaccharide (LPS) overnight and stained for surface and intracellular expression of TLR-2, TLR-4, TLR-9, IL-5R, IL-3R and GM-CSFR. Median fluorescence intensity (MFI) and mean percent expression were calculated.<p></p> RESULTS: Prior to stimulation, mean expression and MFI were: TLR-2 (5.1 ± 5%, 1.8+/-0), TLR-4 (1.8 ± 0.1%, 1.6+/-0.2), TLR-9 (86 ± 2%, 3.8+/-0.5), IL-5R (18.2 ± 10%, 3.2+/-1.2), IL-3R (4.1 ± 0.08%, 1.2+/-0.1), GM-CSFR (17 ± 13%, 2.4+/-0.1). After stimulation, mean expression of TLR-2 (1.6 ± 0.7%) decreased (p = 0.0009, n = 4); TLR-9 (94.9 ± 0.8%) increased (p = 0.0001, n = 4), while mean expression of IL-5R (10.1 ± 4.3%), IL-3R (0.6 ± 0.4%) and GM-CSFR (8.9 ± 3.7%) decreased (p = 0.01, n = 4).<p></p> CONCLUSIONS: We have demonstrated that CB progenitor cells have significant TLR expression and TLR stimulation directly affects both TLR and HCR expression. These alterations may have functional consequences for CB progenitor cell differentiation and maturation of pro-inflammatory cells previously shown to be important in allergic inflammation (e.g. eosinophils). This may indicate an alternate innate immune pathway of microbial influence on development of atopy in early life.<p></p&gt

The relationship between speech perception and auditory organisation Studies with spectrally reduced speech

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN026092 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Long-term unemployment and labour market flexibility

Available from British Library Document Supply Centre-DSC:q96/28118 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Molecular markers of eosinophilopoiesis: multiplex Q-PCR analysis of GATA-1, MBP and IL-5 receptor mRNA expression in peripheral blood

<b>RATIONALE:</b> Using colony assays and flow cytometry, we have shown that eosinophil/basophil (Eo/B) progenitor phenotype and function are associated with atopic risk at birth and early childhood clinical outcomes. We have also recently demonstrated that real-time polymerase chain reaction (Q-PCR) can reveal kinetic patterns of expression in cord blood (CB) of several Eo/B lineage-specific genes, specifically GATA-1, MBP and IL-5Rα, as surrogate molecular markers of Eo/B differentiation. These same methods have yet to be established in peripheral blood (PB) samples. Our objective was to determine the kinetic patterns of expression of CB Eo/B-lineage specific genes in PB.<p></p> <b>METHODS:</b> PB non-adherent mononuclear cells (PB NAMNC) were isolated from random fresh samples, and incubated in the presence of IL-5. At 24, 48, and 72h post-stimulation, RNA was isolated, reverse transcribed, and expression of IL-5Rα, GATA-1, and MBP was determined utilizing multiplex Q-PCR. Relative expression ratios of stimulated to un-stimulated cells were calculated using the delta-delta Ct method.<p></p> <b>RESULTS:</b> Stimulation of PB NANMC with IL-5 resulted in an up-regulation of GATA-1 expression, peaking at 24h, with a slower return to baseline expression than that observed in CB. MBP expression was minimally altered at all time points, compared to CB, where slow up-regulation, maximal at 72h, had been observed. There was completely stable expression IL-5Rα, similar to that seen in CB.<p></p> <b>CONCLUSIONS:</b> Multiplex Q-PCR analysis of mRNA from PB demonstrates expression of critical Eo/B lineage-specific events. Further investigation of the validity and utility of Q-PCR analyses of PB for surrogate, molecular markers Eo/B differentiation is underway.<p></p&gt