Jeong Wan Seo

Adherent Cells Generated During Long‐Term Culture of Human Umbilical Cord Blood CD34+ Cells Have Characteristics of Endothelial Cells and Beneficial Effect on Cord Blood Ex Vivo Expansion

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34+ cells were cultured for 5 weeks in a stroma‐free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte‐colony stimulating factor. By week 4‐5, we found that firmly adherent fibroblast‐like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule‐1, human intracellular adhesion molecule‐1, human CD31, E‐selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34+ cells, and colony‐forming units (CFUs)‐granulocyte‐macrophage and CFUs‐granulocyte‐erythroid‐megakaryocyte‐macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34+ cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34+ cells after 5 weeks of culture. These results suggest that the UCB CD34+ cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.

Role of Treg and TH17 cells of the gastric mucosa in children with Helicobacter pylori gastritis.

Objective: The aim of the present study was to examine the expression of FOXP3, interleukin (IL)-10, transforming growth factor (TGF)-&bgr;1, IL-17A, and T helper 17 (TH17) cells/FOXP3+ regulatory T (Treg) cells balance in the gastric mucosa of children with Helicobacter pylori infection, in relation to the gastric histopathology. Methods: Antral mucosal biopsies were obtained from 20 children with H pylori(+) gastritis and 20 age- and sex-matched normal controls. Histopathology was assessed by the updated Sydney classification. Gene expression of FOXP3, IL-10, and TGF-&bgr;1 was analyzed by quantitative real-time polymerase chain reaction. Immunohistochemical staining for FOXP3+ Treg and TH17 cells was performed. Results: The gene expression levels of FOXP3, TGF-&bgr;1, and IL-10 messenger RNA (mRNA) and the number of FOXP3+ Treg were significantly higher in the H pylori(+) gastritis group than in the control group (P < 0.01). FOXP3 mRNA levels were correlated positively with TGF-&bgr;1 and IL-10 mRNA levels in the H pylori(+) gastritis group (P < 0.05). Furthermore, FOXP3 mRNA levels were correlated positively with the bacterial density, infiltration of polymorphonuclear cells, and mononuclear cells in the H pylori(+) gastritis group (P < 0.05). The number of TH17 cells was significantly higher in the H pylori(+) gastritis group than in the control group (P < 0.05). In addition, the number of TH17 cells was correlated negatively with the bacterial density and positively with the inflammatory scores of polymorphonuclear cells and mononuclear cells in the H pylori(+) gastritis group (P < 0.05). A negative correlation between the TH17 cells/FOXP3+ Treg ratio and the bacterial density was demonstrated in the H pylori(+) gastritis group (P < 0.05). Conclusions: This study suggested that a TH17/Treg balance toward a Treg-biased response favors the persistence of bacteria, causing chronic active gastritis.

FOXP3+ Regulatory T Cells in Children with Helicobacter pylori Infection

Despite an intensive pro-inflammatory response, the immune system is unable to clear the organism in Helicobacter pylori gastritis. Regulatory T (Treg) cells, which suppress the immune response of antigen-specific T cells, have recently been demonstrated to play a key role in chronic inflammation by immunologic tolerance. The purpose of our study was to investigate the histopathology, FOXP3+CD4+CD25highTreg (FOXP3+ Treg) cell expression, and immune responses in children with H. pylori infection. Twenty-four H. pylori–positive and 24 H. pylori–negative children who underwent esophagogastroduodenoscopy for dyspeptic symptoms were included. Histopathologic grading according to the updated Sydney classification and immunohistochemical stains for FOXP3, transforming growth factor–β1 (TGF-β1), and CD4 were performed. Histopathologic bacterial density score, gastritis activity score, and chronic inflammation score were higher in the H. pylori–positive group than in the negative group (P < 0.01). The number of FOXP3+Treg cells and CD4+T cells and the grade of TGF-β1 expression were significantly increased in the H. pylori–positive group compared to the negative group (P < 0.01). The number of FOXP3+Treg cells correlated positively with the grade of TGF-β1 expression regardless of H. pylori status (P < 0.05). The number of FOXP3+Treg cells and the grade of TGF-β1 expression correlated positively with H. pylori density, gastritis activity score, and chronic inflammation score regardless of H. pylori status (P < 0.01). These findings indicate that FOXP3+Treg cells could play a role in persistent H. pylori infection.