An investigation of methylation pattern changes in the IKZF1 promoter in patients with childhood B-cell acute lymphoblastic leukemia

BACKGROUND: Ikaros family zinc finger 1 (IKZF1) is a transcription factor with an important role in controlling hematopoietic proliferation and function, particularly lymphoid cell differentiation. It was previously shown that various mechanisms and expression patterns of Ikaros are linked to a variety of cancers. We hypothesized that aberrant methylation (hypomethylation) of the IKZF1 promoter region might be one of the causes of B-cell acute lymphoblastic leukemia (B-ALL). In B-ALL patients, an increased expression of this gene is a potential cause of B-cell differentiation arrest and proliferation induction. Therefore, as more than 90% of patients with ALL are <15 years old, we investigated the methylation pattern of the IKZF1 promoter in childhood B-ALL. METHODS: Twenty-five newly diagnosed B-ALL cases were included (all younger than 15 yr). In addition, we selected 25 healthy age- and sex-matched children as the control group. We collected the blood samples in EDTA-containing tubes and isolated lymphocytes from whole blood using Ficoll 1.077 Lymphosep. Next, we extracted genomic DNA with the phenol/chloroform method. Two microgram of DNA per sample was treated with sodium bisulfite using the EpiTect Bisulfite Kit, followed by an assessment of DNA methylation by polymerase chain reaction (PCR) analysis of the bisulfite-modified genomic DNA. RESULTS: Our data highlighted a hypomethylated status of the IKZF1 promoter in the ALL cases (96% of the cases were unmethylated). In contrast, the control group samples were partially methylated (68%). CONCLUSION: This study demonstrated a hypomethylated pattern of the IKZF1 promoter region in childhood B-ALL, which might underlie the aberrant Ikaros expression patterns that were previously linked to this malignancy.

Angiopoietin-1 and -2 and vascular endothelial growth factor expression in ovarian grafts after cryopreservation using two methods / 대한생식의학회지

OBJECTIVE: The favored method of preserving fertility in young female cancer survivors is cryopreservation and autotransplantation of ovarian tissue. Reducing hypoxia until angiogenesis takes place is essential for the survival of transplanted ovarian tissue. The aim of this study was to investigate the role of angiopoietin-1 (Angpt-1), angiopoietin-2 (Angpt-2), and vascular endothelial growth factor (VEGF) in ovarian tissue grafts that were cryopreserved using two methods. METHODS: Ovarian tissues harvested from ICR mice were divided into three groups: group I (control), no cryopreservation; group II, vitrification in EFS (ethylene-glycol, ficoll, and sucrose solution)-40; and group III, slow freezing in dimethyl sulfoxide. We extracted mRNA for VEGF, Angpt-1, and Angpt-2 from ovarian tissue 1 week following cryopreservation and again 2 weeks after autotransplantation. We used reverse transcriptase-polymerase chain reaction to quantify the levels of VEGF, Angpt-1, and Angpt-2 in the tissue. RESULTS: Angpt-1 and Angpt-2 expression decreased after cryopreservation in groups II and III. After autotransplantation, Angpt-1 and Angpt-2 expression in ovarian tissue showed different trends. Angpt-1 expression in groups II and III was lower than in group I, but Angpt-2 in groups II and III showed no significant difference from group I. The vitrified ovarian tissues had higher expression of VEGF and Angpt-2 than the slowfrozen ovarian tissues, but the difference was not statistically significant. CONCLUSION: Our results indicate that Angpt-2 may play an important role in ovarian tissue transplantation after cryopreservation although further studies are needed to understand its exact function.

Effects of lycopene on number and function of human peripheral blood endothelial progenitor cells cultivated with high glucose

BACKGROUND/OBJECTIVES: The objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs. MATERIALS/METHODS: Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and 50 microg/mL of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK. RESULTS: The proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and 50 microg/mL of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury. CONCLUSIONS: Lycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.

Effects of lycopene on number and function of human peripheral blood endothelial progenitor cells cultivated with high glucose

BACKGROUND/OBJECTIVES: The objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs. MATERIALS/METHODS: Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and 50 microg/mL of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK. RESULTS: The proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and 50 microg/mL of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury. CONCLUSIONS: Lycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.

Vitrification of mouse embryos using the thin plastic strip method / 대한생식의학회지

OBJECTIVE: The aim of this study was to compare vitrification optimization of mouse embryos using electron microscopy (EM) grid, cryotop, and thin plastic strip (TPS) containers by evaluating developmental competence and apoptosis rates. METHODS: Mouse embryos were obtained from superovulated mice. Mouse cleavage-stage, expanded, hatching-stage, and hatched-stage embryos were cryopreserved in EM grid, cryotop, and TPS containers by vitrification in 15% ethylene glycol, 15% dimethylsulfoxide, 10 microg/mL Ficoll, and 0.65 M sucrose, and 20% serum substitute supplement (SSS) with basal medium, respectively. For the three groups in which the embryos were thawed in the EM grid, cryotop, and TPS containers, the thawing solution consisted of 0.25 M sucrose, 0.125 M sucrose, and 20% SSS with basal medium, respectively. Rates of survival, re-expansion, reaching the hatched stage, and apoptosis after thawing were compared among the three groups. RESULTS: Developmental competence after thawing of vitrified expanded and hatching-stage blastocysts using cryotop and TPS methods were significantly higher than survival using the EM grid (p<0.05). Also, apoptosis positive nuclei rates after thawing of vitrified expanded blastocysts using cryotop and TPS were significantly lower than when using the EM grid (p<0.05). CONCLUSION: The TPS vitrification method has the advantages of achieving a high developmental ability and effective preservation.

Cultivo de células mesenquimais do sangue de cordão umbilical com e sem uso do gradiente de densidade Ficoll-Paque / Blood mesenchymal stem cell culture from the umbilical cord with and without Ficoll-Paque density gradient method

OBJETIVOS: Implantação de técnicas de isolamento e cultivo de células-tronco mesenquimais do sangue de cordão umbilical humano, com e sem uso de gradiente de densidade Ficoll-Paque (d=1,077g/ml). MÉTODOS: Dez amostras de sangue de cordão umbilical humano de gestação a termo foram submetidas a dois procedimentos de cultivo de células-tronco mesenquimais: sem gradiente de densidade Ficoll-Paque e com gradiente de densidade. As células foram semeadas em frascos de 25cm² a uma densidade de 1x10(7)células nucleadas/cm² (sem Ficoll) e 1,0x10(6) células mononucleares/cm² (com Ficoll). As células aderentes foram submetidas a marcação citoquímica com fosfatase ácida e reativo de Schiff. RESULTADOS: No procedimento sem gradiente de densidade Ficoll, foram obtidas 2,0-13,0x10(7) células nucleadas (mediana=2,35x10(7)) e, no procedimento com gradiente de densidade Ficoll, foram obtidas 3,7-15,7x10(6) células mononucleares (mediana=7,2x10(6)). Em todas as culturas foram observadas células aderentes 24 horas após o início de cultivo. As células apresentaram morfologias fibroblastóides ou epitelióides. Na maioria das culturas houve proliferação celular nas primeiras semanas de cultivo, mas após a segunda semana, somente três culturas provenientes do método sem gradiente de densidade Ficoll-Paque mantiveram crescimento celular, formando focos confluentes de células. Essas culturas foram submetidas a várias etapas de tripsinização para espalhamento ou subdivisão e permaneceram em cultivo por períodos que variaram de dois a três meses. CONCLUSÃO: Nas amostras estudadas, o isolamento e cultivo de células-tronco mesenquimais do sangue de cordão umbilical humano pelo método sem gradiente de densidade Ficoll-Paque foi mais eficiente do que o método com gradiente de densidade Ficoll-Paque.

OBJECTIVES: Implantation of cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood with and without using the Ficoll-Paque gradient density method (d=1.077g/ml). METHODS: Ten samples of the umbilical cord blood obtained from full-term deliveries were submitted to two different procedures of mesenchymal stem cell culture: a) Method without the Ficoll-Paque density gradient, which concentrates all nucleated cells; b) Method with the Ficoll-Paque density gradient, which selects only low-density mononuclear cells. Cells were initially plated into 25 cm² cultures flasks at a density of 1x10(7) nucleated cells/cm² and 1x10(6) mononuclear cells/cm². RESULTS: It was obtained 2-13x10(7) (median = 2.35x10(7)) nucleated cells/cm² by the method without the Ficoll-Paque gradient density, and 3.7-15.7x10(6) (median = 7.2x10(6)) mononuclear cells/cm² by the method with the Ficoll-Paque gradient density. In all cultures adherent cells were observed 24 hours after being cultured. Cells presented fibroblastoid and epithelioid morphology. In most of the cultures, cell proliferation occurred in the first week, but after the second week only some cultures - derived from the method without the Ficoll-Paque gradient density - maintained the growth rate reaching confluence. Those cultures were submitted to trypsinization with 0.25 percent trypsin/EDTA solution and cultured for two to three months. CONCLUSION: In the samples analyzed, cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood by the method without the Ficoll-Paque density gradient was more efficient than the method with the Ficoll-Paque density gradient.

Comparison of Intra-gall Bladder and Intra-common Bile Duct Injection of Collagenase Solution for Mouse Pancreatic Islet Isolation

PURPOSE: A high yield of viable pancreatic islets is an essential prerequisite for the study of pancreatic islet transplantation. The purpose of this study is to compare the yield between intra-gall bladder (intra-GB) and intra-common bile duct (intra-CBD) injection of collagenase solution for isolation of mouse pancreatic islets. METHODS: The mice were divided into two groups, the intra-GB and intra-CBD groups, and each group included twelve mice, respectively. Collagenase solution was injected via the gallbladder in the intra-GB group mice, while this was done via the common bile duct in the intra-CBD group. After removal and digestion of the mouse pancreases, the pancreatic islets were isolated by Ficoll density gradient centrifugation and hand picking. RESULTS: The intra-GB group yielded 121.67+/-39.86 IEQs, and the intra-CBD group reveled 168.17+/-29.23 IEQs. There was a statistically significant difference in islet yield between the two groups (P=0.005, Mann-Whitney Test). The purities of the isolated islets were 86.42+/-3.99% for the intra-GB group and 87.17+/-4.47% for the intra-CBD group, and there was no difference between the two groups (P=0.755, Mann- Whitney Test). CONCLUSION: Both the intra-GB and intra-CBD groups yielded an average of >120 IEQs. However, the intra-CBD group revealed a higher yield than the intra-GB group for isolating mouse pancreatic islets.

Portal Vein Transfusion Increases Pancreatic Islet Allograft Survival in Mice

PURPOSE: Considering the complications of nonspecific immunosuppression, as well as the availability of insulin, heavy immunosuppressive treatment to pancreatic islet transplantation patients is not justified. Antigen administration via the portal vein has been demonstrated to induce immunosuppression, and may present a possible mechanism for the induction of tolerance. Using a mouse model, without any immunosuppressive treatment, the islet allograft survivals were compared between portal venous transfusion and portal venous saline injection groups. METHODS: Six C57BL/6J mice were used as pancreatic islet donors per Balb/c recipient mouse. Islets were harvested by digestion of the pancreata with collagenase, with subsequent Ficoll density gradient separation. Recipient mice were divided into two groups: seven mice received a portal venous injection of 0.1 cc saline (PVS) and eight a portal venous transfusion of 0.1 cc donor blood (PVT). Islets were transplanted into the subcapsular space of the left kidney. Transplantation failure was determined if the transplanted mouse failed to show a blood glucose level less than 200 mg/dl at 24 hours after the transplantation; these mice were not included in the statistics. Rejection was determined when the normalized blood glucose level (<200 mg/dl) returned to above 300 mg/dl. RESULTS: The mean islet equivalent numbers (IEQ) of the seven PVS and eight PVT mice were 893+/-262 and 911+/-288, respectively. The islet allograft survival of the PVS group ranged between 1 to 9 days; whereas, that of the PVT group ranged between 6 to 16 days. The PVT group showed significantly higher islet allograft survival than the PVS group (P=0.0443). CONCLUSION: A portal venous transfusion prolonged the islet allograft survival.

Um novo modelo de isolamento do tumor de Walker utilizando o gradiente de Ficoll-Hypaque / A novel model for isolation of Walker's tumoral cells using the Ficoll-Hypaque gradient

OBJETIVO: Analisar uma nova técnica de isolamento das células do tumor de Walker, utilizando o gradiente de ficoll-hypaque. MÉTODOS: Vinte ratos Wistar machos foram divididos em 2 grupos (G1, G2). O tumor do animal doador foi dissecado, triturado em solução de ringer lactato e filtrado. A suspensão celular obtida foi dividida em duas alíquotas (A1 e A2) e a alíquota A2 foi centrifugada em gradiente de ficoll-hypaque. As duas alíquotas foram ajustadas para uma concentração de 1X10(6) células/ml. Um ml da suspensão A1 ou A2 foi injetado por via subcutânea na axila direita dos ratos dos seus respectivos grupos. Os diâmetros tumorais, peso tumoral e análise histológica foram avaliados. RESULTADOS: Não houve diferença no volume tumoral (G1=17,9±3,8cm³; G2=17,2±4,4cm³; p=0,190) e no peso tumoral (G1=7,0±1,8g; G2=7,3±2,8g; p=0,569). A análise histológica evidenciou padrões semelhantes de infiltração de células pequenas indiferenciadas e de necrose nos dois grupos. Entretanto, exsudato granulocítico leve a moderado foi mais acentuado no G2 do que no grupo G1 e hemorragia leve a moderada foi observada somente no grupo com ficoll. CONCLUSÃO: O gradiente de ficoll-hypaque é uma técnica adequada de isolamento das células do tumor de Walker e fornece uma suspensão celular menos contaminada com outros tipos celulares.

A Feasibility Study of Adenosine Triphosphate-based Chemotherapy Response Assay (ATP-CRA) as a Chemosensitivity Test for Lung Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: A chemosensitivity test can reflect the differences in responses of individual cancer patients to chemotherapeutic agents. The adenosine triphosphate-based chemotherapy response assay (ATP-CRA)is an accurate method, which does not require a large amount of tissue specimen. So far, no studies have evaluated the utility of the ATP-CRA in Korea. Therefore, we investigated the clinical usefulness of the ATP-CRA in 53 patients with lung cancer. MATERIALS AND METHODS: Tumor tissues were obtained from bronchoscopic biopsies or surgical resections. The validity of ATP-CRA was assessed focusing on the success rate, experimental error level (intraassay mean coefficient of variation [CV]) and reproducibility. RESULTS: The overall success rate of ATP-CRA was 90.6% (48/53). Normal cells were effectively eliminated from the tumor tissues with the use of ficoll gradient centrifugation and immunomagnetic separation, which was confirmed using loss of heterozygosity analysis of the 3p deletion. The mean CV of ATP assays was 10.5+/-4.6%. The reproducibility of ATP assays was 94+/-3.8%. The results of the ATP assays were reported to physicians within 7 days of specimen collection. More than 6 anticancer drugs were tested on the tumor specimens obtained from bronchoscopic biopsies. CONCLUSION: The ATP-CRA is a stable, accurate and potentially practical chemosensitivity test in patients with lung cancer.

Vitrification of Mouse Embryos in Ethylene Glycol-based Solutions / 대한불임학회지

OBJECTIVE: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. MATERIALS AND METHODS: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. RESULTS: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. CONCLUSION: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

Immune Tolerance in Murine Islet Transplantation Across HY Disparity

BACKGROUND: Minor histocompatibility HY antigen, as a transplantation antigen, has been known to cause graft rejection in MHC (major histocompatibility complex) matched donor-recipient. The aim of our study is to investigate the role of male antigen (HY) disparity on MHC matched pancreatic islet transplantation and to examine the mechanism of the immune reaction. METHODS: Pancreatic islets were isolated and purified by collagen digestion followed by Ficoll gradient. The isolated islets of male C57BL6/J were transplanted underneath the kidney capsule of syngeneic female mice rendered diabetic with streptozotocine. Blood glucose was monitored for the rejection of engrafted islets. After certain period of time, tail to flank skin transplantation was performed either on mouse transplanted with HY mismatched islets or on sham treated mouse. The rejection was monitored by scoring gross pathology of the engrafted skin. RESULTS: HY mismatched islets survived more than 300 days in 14 out of 15 mice. The acceptance of second party graft (male B6 islets) and the rejection of third party graft (male BALB/c islets) in these mice suggested the tolerance to islets with HY disparity. B6 Skin with HY disparity was rejected on day 25 +/- 7. However, HY mismatched skin transplanted on the mice tolerated to HY mismatched islets survived more than 240 days. Tetramer staining in these mice indicated the CTL recognizing MHC Db/Uty was not deleted or anergized. CONCLUSION: The islet transplantation across HY disparity induced tolerance to HY antigen in C57BL6 mouse, which in turn induced tolerance to HY mismatched skin, which otherwise would be rejected within 25 days. The MHC tetramer staining suggested the underlying mechanisms would not be clonal deletion or anergy.

Vitrification of Mouse Blastocyst / 대한산부인과학회잡지

OBJECTIVE: The aim of this study was to compare the survival and developmental rate of two vitrification solutions for the vitrification of mouse expanded blastocysts. METHODS: Mouse embryos were obtained at 2 cell stage and cultured to expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% serum substitute supplement (SSS). The vitrification solutions used were EFS40 and VS. EFS40 consisted of 40% ethylene glycol, 18% ficoll, and 0.5 M sucrose while VS consisted of 20% ethylene glycol, 20% DMSO, and 10% 1,3-butanediol diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% calf serum (CS). Toxicity was tested by exposing expanded blastocysts to vitrification solution. The vitrification procedure used for EFS40 was performed in three steps, after which they were warmed in 5 steps with 0.5 M sucrose. VS was performed in two steps, after which they were warmed with 1.0 M trehalose. Recovery, survival and hatching rate per expanded blastocysts recovered were compared between two groups. RESULTS: In toxicity test, survival and hatching rate of EFS40 group were 95% and 100%, respectively. In contrast, survival and hatching rate of VS group were 100% and 87.5%, respectively. After vitrification and warming in solution, recovery rate for EFS40 group was 73.7% whereas recovery rate for VS group was 66.5%. After 24 h culture, survival and hatching rate were 80.5% and 20.7% for EFS40 and 66% and 0% for VS group, respectively. After 48 h culture, survival and hatching rate were 69% and 33.3% for EFS40 and 58.3% and 1.9% for VS group, respectively. Survival and hatching rate in EFS40 group were significantly higher than those found in VS group. CONCLUSION: The EFS40 solution was better than VS solution for vitrification of mouse expanded blastocysts.

The Role of Uteroglobin in the Immunomodulation of Nonsmall Cell Lung Cancer Cells / 결핵및호흡기질환

BACKGROUND: Immunotherapy for cancer has not been successful because of several obstacles in tumor and its environment. Inappropriate secretions of cytokines and growth factors by tumors cause substantial changes in the immune responses against tumors, affording the tumors some degree of protection from immune attack. Uteroglobin (UG, Clara cell secretory protein) has been known to have anti-inflammatory, immunomodulatory and anti-cancer activities. However, in lung cancer cells, UG expression is decreased. This study investigated the role of UG in the immunomodulation of lung cancer. METHODS: The UG protein was overexpressed by Adenovirus(Ad)-UG transduction in non-small cell lung cancer cell lines. The concentration of Prostaglandin E2 (PGE2) was measured by Enzyme Immunoassay (EIA). Peripheral blood mononuclear cells (PBMC) from whole blood were prepared with Ficoll. PBMC were cultured in RPMI 1640, supernatant of A549, or A549 with UG or NS-398. Concentration of Th 1 type and Th 2 type cytokines from PBMC were measured by ELISA. RESULTS: UG suppressed PGE2, Cyclooxygenase-2 (COX-2) product. Both Th1 type such as Interleukin-2 (IL-2), Interferon-gamma (IFN-gamma) and Tumor necrosis factor-alpha (TNF-alpha) and Th2 type cytokines such as IL-10 and Tumor growth factor-beta (TGF-beta) were increased when PBMC were cultured with supernatant of non small lung cancer cells. UG and COX-2 inhibitor, NS-398 induced normal immune response of PBMC. Although Th 1 type cytokines were increased, Th 2 type cytokines were reduced by UG. CONCLUSION: UG suppressed PGE2, COX-2 product. Supernatant of NSCLC induced imbalance of immune response of PBMC. However, UG reversed this imbalance. These results suggest that UG may be used in the development of immunotherapy for lung cancer.

IL-1 and TNF-alpha release in human polymorphonuclear leukocytes after exposure to calcium hydroxide treated Porphyromonas endodontalis lipopolysaccharide / 대한치과보존학회지

Bacterial lipopolysaccharide (LPS) plays a major role in stimulating the synthesis and release of the principal osteoclast-activating cytokines, namely, interleukin 1 and tumor necrosis factor-alpha from immune cells. Although monocytes/macrophages are the main producers of these cytokines, recent evidence has indicated that polymorphonuclear leukocytes (PMN) have the ability to release IL-1 and TNF-alpha. Calcium hydroxide has been shown to be an effective medicament in root canal infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)2 may also be the result of direct inactivation of LPS. The purpose of this study was to investigate whether treatment of Porphyromonas endodontalis LPS with calcium hydroxide alters its biological action as measured by human PMN secretion of IL-1 and TNF-alpha, and it was compared with Escherichia coli LPS. P. endodontalis ATCC 35406 was cultured in anaerobic condition, and LPS was extracted using the hot-phenol water extraction method and purified. Purchased E. coli LPS was also purified. 100 microg/ml of each LPS in pyrogen free water were incubated with 25mg/ml Ca(OH)2 at 37degrees C for 7 days. The supernatants were subjected to ultrafiltration, and the isolates were lyophilized and weighed. PMNs were obtained from peripheral blood by centrifugation layered over Lymphoprep. The cells were resuspended (4x106 cells/ml) in RPMI 1640 followed by treatment with various concentrations of LPS (0, 0.1, 1, 10microg/ml) for 24 hours at 37degrees C in 5% CO2 incubator. The supernatants of cells were collected and the levels of IL-1alpha, IL-1beta and TNF-alpha were measured by enzyme-linked immunosorbent assay. The results were as follows; 1. The levels of IL-1alpha, IL-1beta, TNF-alpha from PMN treated with each LPS were significantly higher than those released from unstimulated PMN of the control group (p0.05). 3. The levels of secretion for all three cytokines were affected in a dose-dependent manner in PMN stimulated with each LPS (p0.05). 4. The levels of all three cytokines released from PMN stimulated with P. endodontalis LPS were significantly lower than those released from PMN stimulated with E. coli LPS (p<0.05).

Establishment and Management of Pediatric Tumor Tissue Bank / 대한소아혈액종양학회지

PURPOSE: Current pediatric cancer research requires an organized pediatric tumor tissue bank with standardized guidelines for preparation and storage of human tumor tissue samples, white cells, serum, genomic DNA, RNA, cDNA and proteins.. Our institution established and managed pediatric tumor tissue bank for the last one year, and we want to present an overview of our experiences and guidelines. METHODS: From leukemia patients, peripheral blood and bone marrow aspirates were collected at initial diagnosis. Leukemic cells were prepared by Ficoll density-gradient centrifugation and stored at 196oC liquid nitrogen. For solid tumors, tissue cultures were performed as soon as possible after surgical excision or needle biopsy. Serum free media and primary cultured cells were collected and stored at 20degrees C and at 196degrees C, respectively. Genomic DNA, RNA and cDNA were isolated from leukemic cells and cultured solid tumor cells, and stored at 20degrees C. We also isolated genomic DNA from white blood cells of solid tumor patients and stored at 20degrees C. Finally we collected serum samples from all pediatric cancer patients at diagnosis and stored at 20degrees C. RESULTS: Among the 41 cases of leukemia and 100 cases of solid tumor patients who were diagnosed at department of pediatrics, Samsung Medical Center, from August 2000 to July 2001, 26 cases (63%) of leukemia and 59 cases (59%) of solid tumor patients were registered to Pediatric Tumor Tissue Bank. Primary cell cultures were performed in 21 cases of solid tumors and were successful in 19 cases (90%). The isolated genomic DNA, RNA and cDNA were all in high quality confirmed by electrophoresis in agarose gel. CONCLUSION: The problem of tissue sample size obtained by needle biopsy could be overcome by primary cell cultures. For the effective management of pediatric tumor tissue bank, fresh tissue collection with active cooperation of surgeons, organized personnel structure, and multidisciplinary standardized guidelines are necessary.

Stimulation of Bactericidal and Fungicidal Activity of Neonatal Monocytes by Immunoactivating Peptide

PURPOSE: To evaluate the function of Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) in human neonatal monocytes. METHODS: The peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), was synthesized, purified, and prepared in the Peptide Library Support Facility at Pohang University of Science and Technology. Female Sprague-Dawley rats (200+/-10 g) were preinfected with S. aureus and treated with WKYMVm through femoral vein. At various time points, blood samples were obtained by puncture of femoral artery and the serum was plated on the nutrient agar plate. The number of viable bacteria was determined by counting the number of bacterial colonies. In addition, using S. aureus and C. albicans, we evaluated the bactericidal and fungicidal activities of neonatal monocytes, which were separated from umbilical cord blood by Ficoll gradient. RESULTS: The numbers of bacteria in the blood of WKYMVm-treated rats were rapidly decreased with time, as compared with those of the untreated rats. The peptide treatment enhanced the bactericidal activity in vivo within 10 minutes. In neonatal monocytes, WKYMVm stimulated the intracellular killing of S. aureus in a dose dependent manner, showing the maximum effect at 100 nM. WKYMVm stimulated the phagocytic and fungicidal activities against C. albicans in a dose dependent manner, with the maximum effect at the 100 nM. CONCLUSION: These results suggest that WKYMVm may be an effective agent against the neonatal infections.

Thrombopoietin Level and Bone Marrow Megakaryocyte Colony Formation in Various Diseases with Thrombocytopenia / 대한소아혈액종양학회지

PURPOSE: Thrombocytopenia is a serious life threatening consequence in patients with bone marrow failure syndrome. Thrombopoietin (TPO), recently cloned by several groups has been shown to be a key regulation of megakaryopoiesis and thrombopoiesis. Recent studies have demonstrated a positive or negative relationship between TPO levels and platelet counts due to underlying disease states. To clarify the role of TPO in thrombocytopenic condition we determined plasma TPO levels and megakaryocyte colony assay. METHPDS: TPO levels were measured in thrombocytopenic patient with aplastic anemia, chemotherapy induced bone marrow failure, idiopathic thrombocytopenic purpura (ITP) and in newborn by ELISA (QuantikineTM, R&D System, USA). Controls were short statured normal children with normal platelet counts. Plasma was preserved in 20oC until test. CFU-mega was determined by MegaCultTM (Stem Cell Tech. Inc., Canada). Ficoll separated mononuclear cells were cultured for 10~12 days with TPO or stem cell factor (SCF) in 37degrees C 5% CO2 atmosphere, colonies were fixed, stained and examined with inverted microscope. Results were analysed by Student-t test. RESULTS: TPO levels were markedly increased in aplastic anemia and chemotherapy induced thrombocytopenia compared to those of normal controls. Patients with ITP had decreased level of plasma TPO. There was inverse relationship between platelet count and TPO levels for patients with aplastic anemia and chemotherapy induced thrombocytopenia. There was no definite relationship between platelet counts and TPO levels but inverse relationship between platelet counts and PDW levels in neonates was noted. The levels of TPO were increased after improvement of platelet in thrombocytopenic neonate. Megakaryocyte colonies were increased in the mononuclear cells of the patients with ITP and chemotherapy induced thrombocytopenia. There was little colony formation in aplastic anemia. TPO had no definite effect in megakaryocyte colony formation but SCF increased colony formation. CONCLUSION: TPO levels were increased in aplastic anemia and chemotherapy induced thrombocytopenia but decreased in ITP. There was inverse relationship between platelet count and TPO levels in aplastic anemia and chemotherapy induced thrombocytopenia. Thus TPO could be useful for differentiate the etiology of thrombocytopenia. Megakaryocyte colony was increased in ITP and chemotherapy induced thrombocytopenia, but decreased in aplastic anemia. SCF was effective in megakaryocyte colony formation. TPO and SCF will be helpful to increase platelet in thrombocytopenic patients. However, further study will be needed.

Chemosensitivity Test of Childhood Leukemic Cells

PURPOSE: The purpose of the present study is to determine the relation between in vitro resistance to 9 drugs, measured with colorimetric methylthiazol tetrazolium(MTT) assay and prognostic factors. METHODS: Thirty children with leukemia were evaluated at the pediatric department of Dongsan Medical Center. All samples tested with the MTT assay contained 80% or more leukemic cells, which were isolated by Ficoll density gradient centrifugation, and were incubated with 9 drugs for 4 days. The optical density(OD) of the wells was measured with microplate spectrophotometer. Leukemic cell survival(LCS) was calculated by OD treated well/OD control wellsx100(%). LD50 was calculated from the dose-response curve and used as a measure of resistance. RESULTS: Among the 30 children with leukemia, 16 were ALL, 14 were AML. Seventeen boys and thirteen girls ranged in age from 9 months to 16 years. Comparing LD50 values according to leukemic type, AML revealed relatively high LD50 values for all drugs, except VCR. But there were no significant differences between ALL and AML(P>0.05). Male showed high LD50 values for ASP, DET, ARA-C, VP16, ADR and 6TG. Age10 years children showed high LD50 values for all drugs, except 6TG. Patients with a leukocyte count>100,000/mm3 at diagnosis showed high LD50 values for VCR, ASP, DET, MTX, ARA-C, ADR, and 6TG. Patients with normal chromosome showed higher LD50 values. CONCLUSION: Our study showed higher LD50 values at AML, male, ageyears old, leukocyte count>100,000/mm3, and normal karyotype. The MTT test may contribute to the selection of effective chemotherapeutic agent for children with acute leukemia.

Cytokine Gene Expression According to Cell Separation of Cord Blood and Bone Marrow / 대한소아혈액종양학회지

PURPOSE: Umbilical cord blood (CB) transplantation is an alternative method instead of allogeneic bone marrow (BM) transplantation. CB transplantation has used grafts with red blood cell (RBC) depletion but previous reports in CB investigated the immune reaction about mononulcear cell separated by density gradient. To study the real immune response in CB transplantation, the experimental group designed the total nuclear cell (TNC) groups by RBC depletion and the mononuclear cell (MNC) groups by Ficoll Hypaque in CB and BM. METHODS: We evaluated the various cytokine gene expression by semiquantitative RT- PCR method after immune stimulation with phytohemagglutinin in cord blood and bone marrow according to cell separation method. RESULTS: 1) All samples of CB and BM expressed IL-2 mRNA. There was no difference in amounts of IL-2 mRNA between CBTNC and CBMNC, between BMTNC and BMMNC. 2) All samples of CB and BM expressed IL-10 mRNA. There was no difference in amounts of IL-10 mRNA between the CBTNC and CBMNC but significantly different between the BMTNC and BMMNC (P<0.05). The amounts of IL-10 mRNA in the BMMNC and CBTNC group showed larger than in the BMTNC group (P<0.05). 3) The expression of IL-4, IFN-gamma was not shown in this study. These results suggest that no difference of IL-2 mRNA between CB and BM may reveal IL-2 as major cytokine gene in CB after immune stimulation. The expression of IL-10 mRNA in CBTNC showed more than in BMTNC group. Conclusion: Our results suggest that CBTNC may contain a lot of cells producing IL-10, as a cytokine of immunoregulatory function, and therefore the immune reaction in CB transplantation may be less apparent than in BM transplantation. The cell component producing IL-10 in CB may be T regulator cell. There were not showed the IFN-gamma and IL-4 mRNA due to short duration of immune stimulation. Therefore, further studies will identify the IL-4 and IFN-gamma mRNA expression after long time stimulation and various cytokine gene expression to certificate the precise immune response after flow cytometry to composed of immune cells in CB and BM.