PURPOSE: Recently Wilms tumor gene (WT1) transcripts have been detected in leukemia regardless of the disease subtype and the specific DNA markers suggesting that WT1 gene might be a useful panleukemic marker for monitoring minimal residual disease (MRD). This study was performed to investigate the expression of WT1 gene by a quantitative methods and to find the prognostic value of WT1 gene in childhood acute leukemia. METHODS: From the mononuclear cells isolated from bone marrow aspirates and peripheral bloods of 22 childhood acute and chronic leukemia patients, mRNA were extracted for the reverse transcriptase-polymerase chain reactions (RT-PCR). Relative levels of WT1 gene expression was calculated by using the value in K562 cell line to be 1.00 as a positive control. RESULTS: The sensitivity of detection of MRD with WT1 primers was 10 4 and comparable to that of bcr/abl expression in K562 cells and a patient with CML in blast crisis. WT1 gene expression was detected in 17 of 22 (77%) patients; 9/10 of acute lymphoblastic leukemia (ALL), 6/10 acute myelogenous leukemia (AML), 1 acute mixed lineage leukemia (AMLL) and 1 chronic myelogenous leukemia (CML) in blast crisis. In 4 AML patients who received autologous peripheral blood stem cell transplantation (PBSCT), two patients relapsed after reappearance of WT1 gene expression in bone marrow aspirates and the remaining two were in complete remission without expression of WT1 gene. CONCLUSION: These results show that WT1 gene expression is frequently noted in childhood acute leukemia and can be a useful sensitive marker for the detection of MRD comparable to bcr/abl transcripts. WT1 gene can be used as a panleukemic marker for the MRD monitoring for the evaluation of the remission status and in predicting early relapse in children with acute leukemia in the molecular levels. It may also be a useful tool for the detection of leukemic cell contamination in the process of peripheral blood stem cell transplantation.
Blast Crisis ; Bone Marrow ; Cell Line ; Child ; Gene Expression ; Genetic Markers ; Humans ; K562 Cells ; Leukemia* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukemia, Myeloid, Acute ; Neoplasm, Residual ; Peripheral Blood Stem Cell Transplantation ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Recurrence ; RNA, Messenger ; Wilms Tumor

Background/Aims: Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events, including endocrine dysfunctions, which can have serious consequences on patient health and quality of life. The clinical course and characteristics of immune-related hypophysitis (irH) are not well established. This study aimed to analyze the clinical course and characteristics of irH. Methods: This single-center, retrospective study analyzed data from electronic medical records of Asan Medical Center, spanning January 2017 through June 2021. It included adult patients with solid tumors who underwent thyroid and adrenal function tests, along with gonadotropin and/or growth hormone evaluations, following the initiation of ICI treatment within the same period. The study explored the clinical characteristics of ICI-treated patients with and without irH, the incidence of irH, the time to irH onset, and the associated hormonal changes. Results: Twenty-one patients were included in this analysis. Clinical characteristics did not differ significantly between the irH (n = 13) and non-irH (n = 8) groups. Deficiency rates in the irH group were 23.1% for thyroid-stimulating hormone (n = 3), 76.9% for adrenocorticotropic hormone (n = 10), 61.5% for gonadotropin (n = 8), and 15.4% for growth hormone (n = 2). The overall incidence was 0.9 per person-year, with 6-month and 1-year cumulative incidences of 38.8% and 57.1%, respectively. The median time from ICI initiation to irH diagnosis was 7.7 months. Time to levothyroxine replacement was shorter in the irH group. Conclusions The findings provide evidence that could facilitate the prediction of ICI-induced irH based on clinical course and characteristics.

Background/Aims: Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events, including endocrine dysfunctions, which can have serious consequences on patient health and quality of life. The clinical course and characteristics of immune-related hypophysitis (irH) are not well established. This study aimed to analyze the clinical course and characteristics of irH. Methods: This single-center, retrospective study analyzed data from electronic medical records of Asan Medical Center, spanning January 2017 through June 2021. It included adult patients with solid tumors who underwent thyroid and adrenal function tests, along with gonadotropin and/or growth hormone evaluations, following the initiation of ICI treatment within the same period. The study explored the clinical characteristics of ICI-treated patients with and without irH, the incidence of irH, the time to irH onset, and the associated hormonal changes. Results: Twenty-one patients were included in this analysis. Clinical characteristics did not differ significantly between the irH (n = 13) and non-irH (n = 8) groups. Deficiency rates in the irH group were 23.1% for thyroid-stimulating hormone (n = 3), 76.9% for adrenocorticotropic hormone (n = 10), 61.5% for gonadotropin (n = 8), and 15.4% for growth hormone (n = 2). The overall incidence was 0.9 per person-year, with 6-month and 1-year cumulative incidences of 38.8% and 57.1%, respectively. The median time from ICI initiation to irH diagnosis was 7.7 months. Time to levothyroxine replacement was shorter in the irH group. Conclusions The findings provide evidence that could facilitate the prediction of ICI-induced irH based on clinical course and characteristics.

Background/Aims: Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events, including endocrine dysfunctions, which can have serious consequences on patient health and quality of life. The clinical course and characteristics of immune-related hypophysitis (irH) are not well established. This study aimed to analyze the clinical course and characteristics of irH. Methods: This single-center, retrospective study analyzed data from electronic medical records of Asan Medical Center, spanning January 2017 through June 2021. It included adult patients with solid tumors who underwent thyroid and adrenal function tests, along with gonadotropin and/or growth hormone evaluations, following the initiation of ICI treatment within the same period. The study explored the clinical characteristics of ICI-treated patients with and without irH, the incidence of irH, the time to irH onset, and the associated hormonal changes. Results: Twenty-one patients were included in this analysis. Clinical characteristics did not differ significantly between the irH (n = 13) and non-irH (n = 8) groups. Deficiency rates in the irH group were 23.1% for thyroid-stimulating hormone (n = 3), 76.9% for adrenocorticotropic hormone (n = 10), 61.5% for gonadotropin (n = 8), and 15.4% for growth hormone (n = 2). The overall incidence was 0.9 per person-year, with 6-month and 1-year cumulative incidences of 38.8% and 57.1%, respectively. The median time from ICI initiation to irH diagnosis was 7.7 months. Time to levothyroxine replacement was shorter in the irH group. Conclusions The findings provide evidence that could facilitate the prediction of ICI-induced irH based on clinical course and characteristics.

Background/Aims: Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events, including endocrine dysfunctions, which can have serious consequences on patient health and quality of life. The clinical course and characteristics of immune-related hypophysitis (irH) are not well established. This study aimed to analyze the clinical course and characteristics of irH. Methods: This single-center, retrospective study analyzed data from electronic medical records of Asan Medical Center, spanning January 2017 through June 2021. It included adult patients with solid tumors who underwent thyroid and adrenal function tests, along with gonadotropin and/or growth hormone evaluations, following the initiation of ICI treatment within the same period. The study explored the clinical characteristics of ICI-treated patients with and without irH, the incidence of irH, the time to irH onset, and the associated hormonal changes. Results: Twenty-one patients were included in this analysis. Clinical characteristics did not differ significantly between the irH (n = 13) and non-irH (n = 8) groups. Deficiency rates in the irH group were 23.1% for thyroid-stimulating hormone (n = 3), 76.9% for adrenocorticotropic hormone (n = 10), 61.5% for gonadotropin (n = 8), and 15.4% for growth hormone (n = 2). The overall incidence was 0.9 per person-year, with 6-month and 1-year cumulative incidences of 38.8% and 57.1%, respectively. The median time from ICI initiation to irH diagnosis was 7.7 months. Time to levothyroxine replacement was shorter in the irH group. Conclusions The findings provide evidence that could facilitate the prediction of ICI-induced irH based on clinical course and characteristics.

PURPOSE: For separation of RBC from cord blood, it is important to minimize RBC contamination without significant loss of nucleated cells using sedimentation agent that is safe for human use. This study was performed to investigate the possibility of replacing 6% hydroxyethylstarch (HES) with 10% pentastarch (PS) which is a lower molecular weight hetastarch-analog that is cleared from the circulation rapidly. METHODS: After dilution of cord blood till hematocrit 25%, PS or HES were added by the ratio of 7:1 and 5:1 respectively. Sedimentation was performed for 2 hours by gravity. RESULTS: PS was used in 14 cases with volume of 72.4+/-22.3 mL (45~126 mL) and HES in 8 cases with volume of 58.4+/-8.0 mL (50~70 mL). Sedimentation rate has reached at plateau by 90 minutes in PS group and it was slightly faster than in HES group. Recovery rate of nucleated cells and residual RBC were 82.9+/-10.7%, 7.6+/-5.4% in PS group, and 84.0+/-4.7%, 10.7+/-2.3% in HES group. There were no significant differences between the two groups (P=0.657, 0.219). Cell viabilities were high in both groups; 92+/-3% before separation and 97+/-2% in PS group and 98+/-3% in HES group. CD34+ cells were 0.75+/-0.28% before separation and 0.64+/-0.21% in PS group and 0.60+/-0.30% in HES group (P=0.690). CFU-GM after 2 week culture were 27.4+/-20.0 per 1 105 mononuclear cells in PS group and 22.9+/-8.6 in HES group (P=0.856). CONCLUSION: These results demonstrated that PS has similar efficacy to HES for separation of RBC from umbilical cord blood. Considering its rapid clearance and faster sedimentation rate, PS can replace HES for RBC separation in cord blood banking.
Cell Survival ; Fetal Blood* ; Granulocyte-Macrophage Progenitor Cells ; Gravitation ; Hematocrit ; Humans ; Hydroxyethyl Starch Derivatives* ; Molecular Weight

PURPOSE: CD34+ cells from umbilical cord blood (UCB) were cultured with stromal cells and growth factors, and cell proliferation and megakaryocytic differentiation were observed. The purposes of this study are to find out the optimal culture condition for the megakaryocytic differentiation of CD34+ cells from UCB. METHODS: CD34+ cells of mobilized peripheral blood (PB) and UCB were cultured in IMDM at a concentration of 1x105 cells/mL for 11 days. Thrombopoietin (TPO) 5 ng/mL and 50 ng/mL, flt-3 ligand (FL) 50 ng/mL and stem cell factor (SCF) 50 ng/mL were used as growth factors. Stromal cells cultured from bone marrow (BM) mononuclear cells appeared as a single layer of fusiform adherent cells with expression of SH-2 and ASMA. RESULTS: When PB CD34+ cells were cultured with growth factors only, the cell number increased in TPO 5 ng/mL and 50 ng/mL, TPO and FL, TPO, FL and SCF group increased upto mean 2.0 (in TPO 5 ng/mL), 2.9 (in TPO 50 ng/mL), 2.4 (TPO and FL), 2.8 (TPO, FL and SCF) fold, and the expression of CD61a were mean 11.5%, 12.5%, 13.2% and 17.0%, respectively. When the stromal cells were added to the growth factors, the cell number increased upto mean 4.6, 4.9, 9.2 and 68.5 fold and CD61a were expressed in mean 43.1%, 48.4%, 35.6%, and 6.2% of cultured cells. For UCB CD34+ cells, the cell number were increased upto 7.5, 7.6, 8.2 and 23.6 fold with growth factors and stromal cells. Expression of CD61a were mean 26.0%, 34.3%, 31.5% and 23.5%, respectively. CONCLUSION: Stromal cell enhanced the cellular proliferation and megakaryocytic differentiation of UCB CD34+ cells. The combination of TPO, FL and SCF increased total cell number upto the highest value but the proportion of the CD61a+ cells were relatively low. The combination of TPO and FL induced both cellular proliferation and megakaryocytic differentiation in a large amount.
Bone Marrow ; Cell Count ; Cell Proliferation ; Cells, Cultured ; Fetal Blood* ; Intercellular Signaling Peptides and Proteins* ; Stem Cell Factor ; Stromal Cells* ; Thrombopoietin ; Umbilical Cord*

Bcr-abl antisense oligodeoxynucleotides (AS-ODNs) have provided evidence of an antileukemia effect when tested in vitro against Philadelphia-positive cells. In order to investigate the efficacy of AS-ODNs as purging agents in chronic myeloid leukemia (CML) patients, K562 cells, a human CML cell line, were treated in vitro with various types of AS-ODNs and interferon-alpha. Cells were treated in vitro for 0 and 36 hr with 40 microgram/mL of AS-ODNs, respectively, and incubated at 37 degrees C for 36 hr. Cytotoxic effects were measured by counting the number of viable cells as well as by MTT test. Clonogenic activities were evaluated by methylcellulose culture for 2 weeks. The effects of purging agents on the rearrangement of bcrabl gene were evaluated by RT-PCR. AS-ODNs inhibited the proliferation of K562 cells with time in cell count assay and MTT test. AS-ODNs were superior to INF-alpha in inhibiting clonogenic activity (recovery rate; 26.3% vs 64.0%). After incubation with bcr-abl AS-ODNs primers and mRNA isolated from K562 cells, positive bands were abolished, especially of b3a2 type and phosphorothioate type. Our results suggest that AS-ODNs mediated purging may be one of the efficient methods and that autograft may be an alternative treatment for allograft in high-risk group patients of CML if they do not have a stem cell donor.
Bone Marrow Purging* ; Colony-Forming Units Assay ; Fusion Proteins, bcr-abl/genetics ; Fusion Proteins, bcr-abl/metabolism ; Hematopoietic Stem Cells/physiology* ; Human ; Leukemia, Myeloid, Chronic/therapy ; Neuroblastoma/therapy* ; Oligonucleotides, Antisense/metabolism* ; Oligonucleotides, Antisense/therapeutic use ; Transplantation, Autologous* ; Tumor Cells, Cultured

Cyclooxygenase(COX)-1 and COX-2 expression in dermal wound healing of mouse was detected by immunohistochemistry and Western blot analysis. In order to gain more information on the functional importance of COX-1 and COX-2 in dermal wound healing, we analysed COX-1 and COX-2 protein levels using the Western blotting technique. In addition, we used immunohistochemistry to determine the cellular localization of the protein products. The collected skins were rapidly frozen and kept at -70degrees Cuntil assayed. Each frozen skin was lysed with 0.5 ml of ice-cold solution. Large tissue debris and nuclear fragments were removed by two low-speed centrifugations and the resulting supernatant fraction was used for blots. The skin extracts were stored below -20degrees Cfor further experiments. By Western blotting, compared to the activity of COX-2 in normal skin, its activity was increased at days 1, 4, 8, and 12 and was maximal at 1 day after incisional wound of mouse skin whereas COX-1 was barely detectable. In normal skin, COX-1 immunostaining was observed among the basal cells of epidermis whereas COX-2 immunostaining was detected in the more differentiated, suprabasal keratinocytes. At post-incision 1-4 days, COX-2 staining was particularly prominent in the inflammatory cells, and at day 8, many macrophage-like cells were stained positively. COX-2 immunoreactive fibroblast, macrophage-like cells, and newly formed vascular endothelial cells were increased in number at 12 days after incision. These data suggest that COX-2 is constitutively expressed, just as is COX-1, in epidermis and is associated with keratinocyte differentiation. In addition, these findings support the well-established role for COX-2, the prostaglandins that they generate, as mediators of inflammatory response.
Animals ; Blotting, Western ; Endothelial Cells ; Epidermis ; Fibroblasts ; Immunohistochemistry ; Isoenzymes* ; Keratinocytes ; Mice* ; Prostaglandin-Endoperoxide Synthases* ; Prostaglandins ; Skin ; Wound Healing* ; Wounds and Injuries*

PURPOSE: This study was performed to induce proliferation and differentiation of erythroid progenitors from cord blood CD34 cells using extracellular matrix (ECM) and stromal cells. METHODS: Cord blood mononuclear cells were separated using Ficoll-Hypaque and CD34 cells were purified by Mini-MACS column. Cells were cultured in IMDM medium including 10% FBS and several cytokines (EPO, flt3-ligand, SCF and TPO) upto 14 days. ECM like laminin, collagen IV, fibrinogen and fibronectin were used alone or in combination. Stromal cells were derived from BM mononuclear cells for 4 weeks of culture and irradiated before use. On day 14 of stromal coculture of cord blood CD34 cells, flow cytometric analysis were done with fluorescence isothiocyanate-conjugated (FITC) antihuman glycophorin A antibody for erythroid cells. RESULTS: ECM-treated group showed 16.3~31.3 fold increase of the cell numbers on day 14 and there were no difference from cytokines-treated group. Stromal cells induced great amount of fold increase compared with ECM-treated group on day 7 (25.4 fold vs. 2.2~5.4 fold). The cell numbers increased upto 16.4 fold on day 7, 92.8 fold on day 10, and 198.4 fold on day 14 with stromal cells. Erythroid progenitors expressing glycophorin A increased from 2.78% on day 0 to 21.57% on day 7 and 43.87% on day 14. CONCLUSION: Stromal coculture of cord blood CD34 cells induced marked proliferation and differentiation of erythroid cells compared with cytokines or ECM-treated group. Efficient in vitro erythroid culture might have implications for gene therapy in RBC defects or developing blood substitutes for transfusions.
Blood Substitutes ; Cell Count ; Coculture Techniques ; Collagen ; Cytokines ; Erythroid Cells ; Extracellular Matrix* ; Fetal Blood* ; Fibrinogen ; Fibronectins ; Fluorescence ; Genetic Therapy ; Glycophorin ; Laminin ; Stromal Cells*