Hae-Kyung Park

Human Eosinophils Show Chemotaxis to Lymphoid Chemokines and Exhibit Antigen-Presenting-Cell-Like Properties upon Stimulation with IFN-γ, IL-3 and GM-CSF

Background: Eosinophils are multifunctional leukocytes. Under physiological conditions, they circulate in the blood and through the tissues to serve their functions. In certain inflammatory states, they enter the T-cell areas of lymph nodes (LNs) that drain the inflamed tissue and communicate with T cells in LNs, but the underlying mechanism that regulates their trafficking to LNs is not yet fully explored. Here, we report that a human eosinophilic leukemia cell line, EoL-1, and human peripheral blood (PB) eosinophils become reactive to the lymphoid chemokines CCL21 and CCL25 upon stimulation. Methods: EoL-1 cells were differentiated with dibutyryl cyclic AMP (dEoL-1) and subsequently pulsed with IFN-γ, IL-3 and GM-CSF. The eosinophil fraction was purified from normal human adult PB and incubated for 1 day with the same cytokine combination. Results: Upon cytokine stimulation, dEoL-1 cells expressed chemokine receptors CCR7, CCR9 and CCR3 and developed chemotactic responsiveness to CCL21, CCL25 and CCL11, which bind to the respective receptors. Human PB eosinophils also showed chemotactic responsiveness to CCL21 and CCL25 upon stimulation with IFN-γ, IL-3 and GM-CSF. In addition, the cytokine-stimulated dEoL-1 cells expressed costimulatory molecules, including CD40, CD80, CD86 and HLA-DR, and also expressed a tolerogenic and Th2-polarizing enzyme, indoleamine 2,3-dioxygenase. Conclusions: These in vitro observations raise the possibility that eosinophils may utilize lymphoid chemokines to enter LNs and serve antigen-presenting functions in the LN under certain inflammatory conditions.

Comparison of Escherichia coli uropathogenic genes (kps, usp and ireA) and enteroaggregative genes (aggR and aap) via multiplex polymerase chain reaction from suprapubic urine specimens of young children with fever

Objective. Escherichia coli is the most frequently identified microbiological agent in childhood urinary tract infections (UTIs). However, the pathogenic role of this organism in young children remains to be clearly elucidated. So far, no studies have been conducted in which multiplex polymerase chain reaction (PCR) has been applied to determine the association between childhood UTIs and E. coli and urovirulent genes. Material and methods. Altogether, 330 suprapubic urine specimens from febrile young children were cultured. In 33 of the cases, E. coli was identified; among these cases, 18 had a UTI (>104–105 cfu/ml), four had a suspected UTI (>102–103 cfu/ml) and 11 did not have UTIs (102 cfu/ml). Using multiplex PCR, three uropathogenic E. coli (UPEC) genes and two enteroaggregative E. coli (EAEC) genes were detected. Results. In the UTI-UPEC cases, the kps gene was detected in 18 of 22 cases (82%) and the usp gene in 16 of 22 cases (73%). Among the 18 cases of children with UTIs characterized by 104–105 E. coli cfu/ml, urinary tract abnormalities were identified via dimercaptosuccinic acid scans in seven of 18 cases (39%) and via voiding cystourethrograms in four of the 18 cases (22%). Conclusions. The UPEC kps and usp genes were clearly associated with childhood UTIs, and may also be associated with kidney or urinary tract dysfunctions in young children. Escherichia coli colony count numbers in excess of 104–105 cfu/ml in the suprapubic urine were considered to be strong evidence of UTI in infants.

Megakaryothrombopoiesis during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin.

Thrombopoietin (TPO) is one of the most promising stimulants for ex vivo expansion of haematopoietic stem cells. Previously, we have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human cord blood (CB) CD34+ cells and that the TPO‐induced apoptotic cells belong to megakaryocyte (MK) lineage. In this study, we have examined the maturation of MK and platelet production in association with the TPO‐induced apoptosis. CD34+ cells, purified from human CB, were expanded in serum‐free conditions stimulated with TPO. Apoptosis was confirmed by terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end labelling (TUNEL) assay and electron microscopy (EM). Simultaneous measurement of DNA content and immunophenotyping revealed that the cells with higher DNA content (>8 N) constituted less than 5% of the CD41+ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, EM observation showed not only platelet territories but also newly produced platelets in which granules and microfilaments could be identified. Furthermore, flow cytometry demonstrated that the platelet fraction expressed P‐selectin and an activation motif on GPIIb/IIIa recognized by monoclonal antibody PAC‐1 upon stimulation with adenosine diphosphate (ADP). In addition, periodic acid‐Schiff (PAS)‐positive materials and nonspecific esterase activities could be demonstrated. Therefore, it is suggested that platelet production and the accompanying processes, rather than apoptosis only, be hastened during the ex vivo expansion of CB CD34+ cells when using TPO.

Pim-1 induced polyploidy but did not affect megakaryocytic differentiation of K562 cells and CD34+ cells from cord blood.

In a previous study, we determined the gene expression profile of both megakaryocytic and non‐megakaryocytic lineage cells via serial analysis of gene expression and microarray methods, and demonstrated that Pim‐1 was expressed more abundantly in megakaryocytic lineage cells. In this study, we knocked down Pim‐1 in K562 cells, as well as in CD34+ cells from cord blood, via RNA interference, in order to analyze the effects of Pim‐1 expression on the megakaryocytic differentiation of these cells. We then additionally overexpressed the Pim‐1 genes in K562 cells, and conducted a comparison of these effects with those of RNAi cells on the course of megakaryocytic differentiation. The results of this study revealed that Pim‐1 knockdown exerted no effects on commitment or differentiation toward megakaryocytic lineage, as evidenced by the detected CD41+ or CD61+ cells, or on the number of megakaryocytic colony forming units. However, Pim‐1 knockdown was found to elicit a reduction in CD41+ cells with >4n DNA content, and a concomitant increase in the fraction of cells achieving a ploidy of >4n in the Pim‐1 overexpressing population of K562 cells. Collectively, the findings of these studies indicate that the expression of Pim‐1 expression is both necessary and sufficient for polyploidization, but is not critical to cytoplasmic differentiation on megakaryopoiesis.