Eosinophil Basophil Research Articles

Diagnostic insights from chemometric analysis of hemogram inflammatory indexes in male smokers with and without asthma or chronic obstructive pulmonary disease.

Diagnosis of asthma and chronic obstructive pulmonary disease (COPD) becomes difficult in a primary healthcare center due to ambiguous interpretation of spirometry and lack of facility to access established biomarkers. While routine hematological indices are easily available and accessible. The study aimed to evaluate the role of different hemogram indexes in males in COPD, asthma, and healthy smokers. Lung function tests and complete blood count (CBC) were done for 50 male subjects each from asthma, COPD, and healthy smokers. Multivariate analysis (MVA) was performed on blood indices data set. Receiver operating characteristic (ROC) curve was plotted to observe the performance of indexes. Pearson correlation was used to establish association between the lung function and blood indices. Most of the indices were elevated in COPD. Whereas, asthma patients showed a significant increase in eosinophil basophil ratio (EBR), lymphocyte-monocyte ratio (LMR), and mean platelet volume-platelet count ratio (MPR). Orthogonal (O)- Partial Least-Squares Discriminant Analysis (PLSDA) and variable importance in projection (VIP) score established EBR, neutrophil-lymphocyte ratio (NLR) and LMR, as discriminants for asthma. Whereas, Systemic Inflammatory Response Index (SIRI), NLR and EBR were the key variables for COPD. NLR (r = -0.73, p < 0.001) and SIRI (r = -0.71, p < 0.001) were found to be negatively correlated with forced expiratory volume in 1 s (FEV1) percentage of the predicted value (%pred) in asthma and COPD, respectively. EBR showed the sensitivity and specificity of 96% and 86% respectively in asthma. NLR was having sensitivity of 82% and 90% specificity in COPD. Our study in males shows routine hematological indices as being cost-effective, feasible, and seem to have tremendous potential as screening markers among chronic respiratory diseases in a primary healthcare center.

Determination of the hematological values and detection of Chlamydophila psittaci antibodies in captive Palawan Hill Mynah at the Ninoy Aquino Parks and Wildlife Nature Center

Background: Gracula religiosa palawanensis is a subspecies of the avian species Gracula religiosa under the order Passeriformes. Birds belonging to this order are famously and distinctly known for their unique ability to imitate sounds. The particular subspecies is endemic to the Philippines, solely found in the elevated points of Balabac, Busuanga, Culion, and the Mainland Palawan. Aims and Objectives: Being indigenous to the Philippines, the initial reports of hematological values and the common diseases of this subspecies are significant to be determined and studied. The study aimed to provide an initial report on the hematological values and to detect the presence of antibodies against Chlamydophila psittaci in the blood serum of Palawan hill mynah held captive at the Ninoy Aquino Parks and Wildlife Nature Center. Materials and Methods: Captive birds at the Ninoy Aquino Parks and Wildlife Nature Center in the Philippines were used as samples. Blood was collected and subjected to hematology and serum analysis. Results: The packed cell volume (PCV), total red blood cell (RBC) count, RBC indices, hemoglobin concentration, total and differential white blood counts, and total thrombocyte counts were accounted for the hematological values of the three sampled G. religiosa palawanensis. Values of 45.00 ± 3.00%, 3.61 ± 0.20 × 1000000 cells/mm3, 13.47 ± 0.78 g/dL, and 14.86 ± 2.05 × 1000 cells/mm3 were, respectively, observed for the PCV, total RBC counts, hemoglobin concentration, and total white blood cell (WBC) count. The mean corpuscular hemoglobin (MCH), MCH concentration, and mean cell volume values, the RBC indices, were found to be 37.35 ± 2.40 pg, 29.77 ± 2.08%, and 126.05 ± 14.76 femtoliter, respectively. The following showed the differential WBC count with their mean relative values and their mean absolute values enclosed in parentheses: 60.00 ± 8.00 % (8.95 ± 2.19 × 1000 cells/mm3), 1.00 ± 1.00 % (0.15 ± 0.16 × 1000 cells/mm3), 1.00 ± 1.00 % (0.20 ± 0.10 × 1000 cells/mm3), 37.00 ± 8.00% (5.33 ± 0.60 × 1000 cells/mm3), and 0.00 ± 1.00 % (0.05 ± 0.09 × 1000 cells/mm3). The values correspond to the heterophil, eosinophil basophil, lymphocyte, and monocyte counts accordingly. The presence of C. psittaci was observed from the avian specimens but was merely observed at low concentrations, having a low positive ImmunoComb® reading. Conclusion: The blood values of the Palawan Hill Mynah do not differ from their close relatives. Furthermore, the presence of Chlamydophila psittaci may show the previous infection to the bacteria.

Thymic Stromal Lymphopoietin (TSLP) promotes human eosinophil‐basophil in situ hemopoieisis, and its secretion from human nasal epithelium is a function of TSLP genotype

Background Allergic diseases are characterized by tissue eosinophilic and basophilic inflammation. There is substantial evidence that this particular inflammatory profile results from the migration to tissues of a common eosinophil-basophil (Eo/B) progenitor that undergoes a differentiative process regulated by local cytokines, termed in situ hemopoiesis. We therefore investigated the role and mechanisms of TSLP involvement in human Eo/B in situ hemopoiesis in relation to atopy. Also, since candidate gene and genomewide association studies have identified ‘protective’ associations between the single nucleotide polymorphism (SNP) rs1837253 in the TSLP gene and risk for allergy, asthma and airway hyper-responsiveness, we evaluated the secretion of TSLP protein from primary nasal epithelial cells (NEC) in relation to rs1837253 genotype.

Microbiome Effects on Hematopoietic Eosinophil/Basophil Progenitor Phenotype: Implications for the Pathogenesis of Allergic Inflammation

CD34+hematopoietic eosinophil/basophil (Eo/B) progenitors express TLRs and cytokine receptors (CyR) differentially in both newborns and adults, depending on allergic risk or disease, thus providing evidence for allergy as a systemic disease. We hypothesized that Eo/B progenitor phenotype would be affected by the host’s gut microbiome, examining this in ulcerative colitis (UC), a prototypical systemic inflammatory disease.

IL-4 and IL-13 regulate TLR expression and eosinophil-basophil differentiation of cord blood CD34+ progenitor cells

Background Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent allergic disease development [1]. We have previously shown that cord blood (CB) progenitor cells of high atopic risk infants have reduced toll-like receptor (TLR) expression and produce fewer lipopolysaccharide (LPS)-stimulated eosinophil-basophil (Eo/B) colonies, compared to lowatopic risk infants. In the present study, we investigated whether a surrogate ex vivo TH2 milieu (i.e., either IL-4 or IL-13), could represent an underlying mechanism to explain our previous findings.

IL-4 and IL-13 differentially regulate TLR-induced eosinophil-basophil differentiation of cord blood CD34+ progenitor cells.

Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent allergic disease development. We have previously shown that fewer lipopolysaccharide (LPS)-stimulated eosinophil-basophil (Eo/B) colonies grow from cord blood (CB) of high-atopic risk infants, compared to low-atopic risk infants. In the present study, we investigated whether a surrogate ex vivo TH2 milieu (i.e., either IL-4 or IL-13) could represent an underlying mechanism to explain our previous findings. CB CD34+ cells from healthy donors were cultured with IL-4 or IL-13 (in combination with LPS) and assessed for Eo/B differentiation using methylcellulose cultures and flow cytometry for related intracellular signalling pathways. Pharmacological inhibitors were added to the methylcellulose cultures to determine the effect of blocking intracellular signalling in CB CD34+ cells in relation to Eo/B colony forming unit (CFU) formation. Stimulation of CD34+ cells with IL-4, but not IL-13, reduced Eo/B CFU formation in the presence of LPS; this was found to be dependent on IL-4Rα and not IL-13Rα1. Additionally, IL-4 reduced the expression of ERK 1/2 after LPS stimulation, which was recovered by inhibition of IL-4Rα. While IL-13 did not have an inhibitory effect on ERK 1/2 expression, inhibition of ERK 1/2 significantly reduced Eo/B CFU formation. Thus, the responsiveness of CB CD34+ progenitor cells to LPS is differentially regulated by the TH2 cytokines, IL-4 and IL-13. This may have implications for in utero interactions between placental-derived pro-allergic cytokines and neonatal progenitor cells influencing Eo/B-mediated inflammatory responses in early life.

The effects of thymic stromal lymphopoietin and IL-3 on human eosinophil-basophil lineage commitment: Relevance to atopic sensitization.

An important immunopathological hallmark of allergic disease is tissue eosinophilic and basophilic inflammation, a phenomenon which originates from hemopoietic progenitors (HP). The fate of HP is determined by local inflammatory cytokines that permit “in situ hemopoiesis,” which leads to the accumulation of eosinophils and basophils (Eo/B). Given that recent evidence supports a critical immunomodulatory role for thymic stromal lymphopoietin (TSLP) in allergic inflammation, as well as TSLP effects on CD34+ progenitor cytokine and chemokine secretion, we investigated the role of TSLP in mediating eosinophilo- and basophilopoiesis, the mechanisms involved, and the association of these processes with atopic sensitisation. In the studies presented herein, we demonstrate a direct role for TSLP in Eo/B differentiation from human peripheral blood CD34+ cells. In the presence of IL-3, TSLP significantly promoted the formation of Eo/B colony forming units (CFU) (including both eosinophils and basophils) from human HP (HHP), which was dependent on TSLP–TSLPR interactions. IL-3/TSLP-stimulated HHP actively secreted an array of cytokines/chemokines, key among which was TNFα, which, together with IL-3, enhanced surface expression of TSLPR. Moreover, pre-stimulation of HHP with IL-3/TNFα further promoted TSLP-dependent Eo/B CFU formation. HHP isolated from atopic individuals were functionally and phenotypically more responsive to TSLP than those from nonatopic individuals. This is the first study to demonstrate enhanced TSLP-mediated hemopoiesis ex vivo in relation to clinical atopic status. The capacity of HHP to participate in TSLP-driven allergic inflammation points to the potential importance of “in situ hemopoiesis” in allergic inflammation initiated at the epithelial surface.

The effects of poly I:C stimulation of primary bronchial epithelial cells and TSLP secretion on CD34+ progenitor cell eosinophil and basophil differentiation

Background In asthmatic lungs, elevated levels of thymic stromal lymphopoetin (TSLP) are linked to eosinophilic inflammation and disease severity [1]. TSLP is involved in initiating a TH2 inflammatory response, through activation of T cells, and recently, CD34+ hemopoietic progenitor cells [2,3]. However, the biological effects of epithelial-derived TSLP on human peripheral blood (PB) CD34+ progenitor eosinophil-basophil (Eo/B) lineage commitment have not been described. The aim of the current study is to examine the effects of primary bronchial epithelial cell-derived TSLP on CD34+ hemopoietic progenitor differentiation.

IL-4 and IL-13 Differentially Regulate TLR-Induced Eosinophil-Basophil Differentiation Of Cord Blood CD34+ Progenitor Cells

Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent atopy. The effect of “pro-allergic” TH2 cytokines on neonatal cord blood (CB) hematopoietic progenitor cells, the phenotype and differentiation of which are associated with atopic risk, is currently unknown. Since we have previously shown that high-atopic risk infants have decreased eosinophil-basophil (Eo/B) differentiation after stimulation with LPS, we investigated whether a TH2 milieu (IL-4 or IL-13) could influence LPS-induced Eo/B colony forming units (CFU), and the mechanism(s) through which this might occur.

Toll‐like receptor‐mediated eosinophil–basophil differentiation: autocrine signalling by granulocyte–macrophage colony‐stimulating factor in cord blood haematopoietic progenitors

Eosinophils are multi-functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil-basophil (Eo/B) differentiation-inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll-like receptor (TLR)-mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony-forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34-enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive, as opposed to interleukin-5-responsive, Eo/B CFU, which also correlated with significant increases in CD34(+) cell GM-CSFRα expression. Functionally, CB CD34(+) cells secrete abundant amounts of GM-CSF following LPS stimulation, via a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK-dependent GM-CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.

Effect of Maternal Allergic Sensitization and Smoking during Pregnancy on Eosinophil- Basophil Lineage Commitment

RATIONALE: We have previously shown that intrauterine environmental exposures (e.g., microbial) can influence eosinophil-basophil (Eo/B) lineage commitment in infants at risk for atopy. However, it is unknown how maternal smoking affects Eo/B lineage commitment in neonates at risk for allergy. Since cigarette smoke may modulate eosinophilopoiesis, we investigated the effects of maternal smoking during pregnancy on Eo/B lineage priming.

Circulating Eosinophil/Basophil (Eo/B) Progenitors at One Year of Age Positively Correlate with Atopic Eczema and Indoor Chemical Exposure in the LINA Study

Circulating Eosinophil/Basophil (Eo/B) Progenitors at One Year of Age Positively Correlate with Atopic Eczema and Indoor Chemical Exposure in the LINA Study

Maternal allergy modulates cord blood hematopoietic progenitor Toll-like receptor expression and function

Little is known regarding the prenatal determinants of innate immune responses in relation to infant allergic risk. Environmental exposures, including microbial stimuli, might predispose susceptible subjects to atopy and asthma in early infancy or even in utero. Because Toll-like receptors (TLRs) recognize microbial products and because cord blood (CB) progenitor alterations have been observed in neonates at risk for atopy, we investigated the expression and function of TLRs on CB hematopoietic progenitors in relation to atopic risk, as defined by maternal allergic sensitization. Thirty-two (15 with low and 17 with high atopic risk) infant CB samples were assessed for phenotypic and functional alterations in CD34(+) cells by means of flow cytometry and methylcellulose culture, respectively. CD34(+) hematopoietic progenitors were stained for TLR-2, TLR-4, TLR-9, GM-CSF receptor α, IL-5 receptor α, and IL-3 receptor α or cultured in methylcellulose assays for hematopoietic cytokine-stimulated eosinophil-basophil (Eo/B) colony-forming units (CFUs) with or without LPS. High-atopic-risk infants had significantly lower CB CD34(+) cell TLR-2, TLR-4, and TLR-9 expression (P = .009). High-risk infant progenitors gave rise to significantly more Eo/B CFUs (P = .002) with hematopoietic cytokine (IL-3, IL-5, or GM-CSF) stimulation ex vivo. Although LPS costimulation induced Eo/B CFUs from both low- and high-risk infant CB CD34(+) cells, this response was significantly (P = .020) muted in the high-risk CB progenitors. Neonatal CB CD34(+) hematopoietic progenitor cell TLR expression and functional responsiveness are altered in CB from atopic at-risk infants. Maternal allergic sensitization might modulate hematopoietic progenitor TLR expression and function in utero; specifically, Eo/B "lineage priming" at birth might be circumvented through engagement of TLR pathways in early life.

Cord blood molecular biomarkers of eosinophilopoiesis: kinetic analysis of GATA-1, MBP1 and IL-5R alpha mRNA expression.

Eosinophil/basophil (Eo/B) progenitor phenotype and function in cord blood (CB) are associated with atopic risk at birth and infant clinical outcomes. Molecular analyses of eosinophil-basophil differentiation events could identify clinically predictive biomarkers. To determine CB kinetic patterns of Eo/B lineage-associated gene expression (GATA-1, MBP1 and IL-5R alpha) after IL-5 stimulation, CB non-adherent mononuclear cells were isolated from random fresh and frozen samples and incubated in the presence of recombinant human interleukin-5. Some underwent CD34+ positive selection using magnetic cell separation. At various time-points, mRNA expression of GATA-1, MBP1 and IL-5R alpha (total transcripts) was determined utilizing multiplex quantitative polymerase chain reaction (Q-PCR). Relative expression levels of the IL-5R alpha soluble vs. transmembrane isoforms were also analyzed. Stimulation of the non-adherent mononuclear cells with IL-5 resulted in early up-regulation of GATA-1, peaking at 48 h, followed by decreasing expression and down-regulation by 96 h. The CD34+ enriched population demonstrated an equivalent expression pattern (r = 0.963, p = 0.0349). MBP1 mRNA expression [non-adherent mononuclear cells (NAMNCs) and CD34+ alike; r = 0.988, p = 0.012] was slowly up-regulated in response to IL-5, maximal at 96 h. Total IL-5R alpha expression appeared stable over the time-course, mediated by differential expression of the soluble and transmembrane isoforms (i.e., initial increase in the transmembrane contribution followed by a predominance of the soluble isoform by 48-72 h). Multiplex Q-PCR analysis of mRNA from CB demonstrates expression of critical eosinophil-basophil lineage-specific events that are consistent with current understanding of eosinophil differentiation and maturation. The non-adherent mononuclear cell population provides a surrogate signal for the CD34+ progenitor population.

Eosinophil Progenitors in Airway Diseases: Clinical Implications

Asthma, allergic rhinitis, nasal polyposis, chronic rhinosinusitis, and related forms of upper and lower airway diseases are often characterized by eosinophilic and basophilic inflammation, involving systemic processes. Eosinophil/basophil (Eo/B) lineage-committed progenitor cells in cord blood, peripheral blood, bone marrow, lung tissue, and sputum are up-regulated in the above conditions, and respond to allergen and other stimuli with increased differentiative and migratory capacity. A considerable body of evidence now exists showing that activation of such Eo/B-selective hemopoietic processes is not only associated with the onset and maintenance of allergic inflammation in atopic adults, but also with the development of the allergic diathesis. Moreover, eosinophilopoietic processes within hemopoietic compartments and, importantly, at mucosal tissue sites during an allergic inflammatory response provide novel targets for the treatment of allergy as a systemic process and disease.

Cord blood hemopoietic progenitor profiles predict acute respiratory symptoms in infancy

Atopy is characterized by eosinophilic inflammation associated with recruitment of eosinophil/basophil (Eo/B) progenitors. We have previously shown that Eo/B progenitor phenotypes are altered in cord blood (CB) in infants at high risk of atopy/asthma, and respond to maternal dietary intervention during pregnancy. As respiratory tract viral infections have been shown to induce wheeze in infancy, we investigated the relationship between CB progenitor function and phenotype and acute respiratory illness (ARI), specifically wheeze and fever. CB from 39 high‐risk infants was studied by flow cytometry for CD34+ progenitor phenotype and by ex vivo Eo/B‐colony forming unit (CFU) responses to cytokine stimulation in relation to ARI in the first year of life. A consistent relationship was observed between increased numbers of granulocyte/macrophage (GM)‐colony‐stimulating factor (CSF)‐ and IL‐3‐responsive Eo/B‐CFU in CB and the frequency/characteristics of ARI during infancy. Comparable associations were found between ARI and CB IL‐3R+ and GM‐CSFR+CD34+ cell numbers. Conversely, a reciprocal decrease in the proportion of CB IL‐5R+ cells was found in relation to the clinical outcomes. The elevation of IL‐3/GM‐CSF‐responsive Eo/B progenitors in high‐risk infants in relation to ARI outcomes suggests a mechanism for the increased severity of inflammatory responses in these subjects following viral infection.

The effects of montelukast on tissue inflammatory and bone marrow responses in murine experimental allergic rhinitis: interaction with interleukin‐5 deficiency

The cysteinyl leukotrienes (cysLTs) are potent lipid mediators in allergic disease, acting through the receptors, cysLT1R and cysLTR2, and are produced by eosinophils derived from eosinophil/basophil (Eo/B) bone marrow (BM) progenitors. We have demonstrated the suppressive effects of either interleukin-5 (IL-5) deficiency or montelukast on eosinophil recruitment in murine allergic rhinitis, but neither of them fully abrogated the symptoms caused by residual inflammation and cytokine redundancy in eliciting BM Eo/B responses. We hypothesized that IL-5 deficiency and montelukast act synergistically to suppress tissue inflammatory and BM responses. Our objective was to investigate the effects of the cysLT1R antagonist, montelukast, on in vivo tissue inflammatory and BM responses in murine experimental allergic rhinitis with or without IL-5 deficiency. Three groups of age-matched BALB/c mice with or without IL-5 deficiency were tested: controls (ovalbumin sensitization and challenge, placebo treatment) and two montelukast-treated groups (2.5 mg/kg or 5 mg/kg). Nasal symptoms, BM and nasal mucosal eosinophils, basophils, and BM Eo/B colony-forming units (CFU) were evaluated. Montelukast decreased nasal symptoms in a dose-dependent manner, and significantly decreased the number of eosinophils in both BM and nasal tissue in IL-5-replete mice compared to controls. In IL-5-deficient mice, in which eosinophilia was absent, montelukast significantly decreased both nasal symptoms and basophils in BM and nasal mucosal tissue, and lowered IL-5-responsive Eo/B-CFU ex vivo, compared to controls. The addition of cysLT1R blockade to IL-5 deficiency more fully attenuates symptoms and upper airway inflammation than either factor alone, providing evidence of systemic, BM mechanisms in allergic rhinitis.

Molecular Markers of Eosinophilopoiesis: Multiplex Q-PCR Analysis of GATA-1, MBP and IL-5 Receptor mRNA Expression in Peripheral Blood

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Meeting program

Meeting program

Molecular Markers of Eosinophilopoiesis at Birth: Kinetics of Cord Blood GATA-1, MBP and IL-5 Receptor Expression

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.