A Novel Immunomodulatory Mechanism Dependent on Acetylcholine Secreted by Human Bone Marrow-derived Mesenchymal Stem Cells

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are used to treat autoimmune or inflammatory diseases. Our aim was to determine the immunomodulatory mechanisms elicited by MSCs during inflammation. METHODS AND RESULTS: We cocultured MSCs with peripheral blood mononuclear cells for a mixed lymphocyte reaction or stimulated them by phytohemagglutinin. Morphological changes of MSCs and secretion of acetylcholine (ACh) from MSCs were measured. The effects of an ACh antagonist and ACh agonist on lymphocyte proliferation and proinflammatory-cytokine production were determined. The inflammatory milieu created by immune-cell activation caused MSCs to adopt a neuronlike phenotype and induced them to release ACh. Additionally, nicotinic acetylcholine receptors (nAChRs) were upregulated in activated peripheral blood mononuclear cells. We observed that ACh bound to nAChR on activated immune cells and led to the inhibition of lymphocyte proliferation and of proinflammatory-cytokine production. MSC-mediated immunosuppression through ACh activity was reversed by an ACh antagonist called α-bungarotoxin, and lymphocyte proliferation was inhibited by an ACh agonist, ACh chloride. CONCLUSIONS: Our findings point to a novel immunomodulatory mechanism in which ACh secreted by MSCs under inflammatory conditions might modulate immune cells. This study may provide a novel method for the treatment of autoimmune diseases by means of MSCs.

Allogeneic clonal mesenchymal stem cell therapy for refractory graft-versus-host disease to standard treatment: a phase I study

Severe graft-versus-host disease (GVHD) is an often lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). The safety of clinical-grade mesenchymal stem cells (MSCs) has been validated, but mixed results have been obtained due to heterogeneity of the MSCs. In this phase I study, the safety of bone marrow-derived homogeneous clonal MSCs (cMSCs) isolated by a new subfractionation culturing method was evaluated. cMSCs were produced in a GMP facility and intravenously administered to patients who had refractory GVHD to standard treatment resulting after allogeneic HSCT for hematologic malignancies. After administration of a single dose (1x10(6) cells/kg), 11 patients were evaluated for cMSC treatment safety and efficacy. During the trial, nine patients had 85 total adverse events and the rate of serious adverse events was 27.3% (3/11 patients). The only one adverse drug reaction related to cMSC administration was grade 2 myalgia in one patient. Treatment response was observed in four patients: one with acute GVHD (partial response) and three with chronic GVHD. The other chronic patients maintained stable disease during the observation period. This study demonstrates single cMSC infusion to have an acceptable safety profile and promising efficacy, suggesting that we can proceed with the next stage of the clinical trial.

Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the beta-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

Molecular Characterization of Neurally Differentiated Human Bone Marrow-derived Clonal Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, with the ability to differentiate into different cell types. Additionally, the immunomodulatory activity of MSCs can downregulate inflammatory responses. The use of MSCs to repair injured tissues and treat inflammation, including in neuroimmune diseases, has been extensively explored. Although MSCs have emerged as a promising resource for the treatment of neuroimmune diseases, attempts to define the molecular properties of MSCs have been limited by the heterogeneity of MSC populations. We recently developed a new method, the subfractionation culturing method, to isolate homogeneous human clonal MSCs (hcMSCs). The hcMSCs were able to differentiate into fat, cartilage, bone, neuroglia, and liver cell types. In this study, to better understand the properties of neurally differentiated MSCs, gene expression in highly homogeneous hcMSCs was analyzed. Neural differentiation of hcMSCs was induced for 14 days. Thereafter, RNA and genomic DNA was isolated and subjected to microarray analysis and DNA methylation array analysis, respectively. We correlated the transcriptome of hcMSCs during neural differentiation with the DNA methylation status. Here, we describe and discuss the gene expression profile of neurally differentiated hcMSCs. These findings will expand our understanding of the molecular properties of MSCs and contribute to the development of cell therapy for neuroimmune diseases.

Erratum: Mesenchymal Stem Cell Lines Isolated by Different Isolation Methods Show Variations in the Regulation of Graft-versus-host Disease

Typographical error has been detected in acknowledgements.

Mesenchymal Stem Cell Lines Isolated by Different Isolation Methods Show Variations in the Regulation of Graft-versus-host Disease

Since the discovery of the immunomodulation property of mesenchymal stem cells (MSCs) about a decade ago, it has been extensively investigated whether MSCs can be used for the treatment of immune-related diseases, such as graft-versus-host disease (GvHD). However, how to evaluate the efficacy of human MSCs for the clinical trial is still unclear. We used an MHC-mismatched model of GvHD (B6 into BALB/c). Surprisingly, the administration of the human MSCs (hMSCs) could reduce the GvHD-related mortality of the mouse recipients and xenogeneically inhibit mouse T-cell proliferation and IFN-gamma production in vitro. We recently established a new protocol for the isolation of a homogeneous population of MSCs called subfractionation culturing methods (SCM), and established a library of clonal MSC lines. Therefore, we also investigated whether MSCs isolated by the conventional gradient centrifugation method (GCM) and SCM show different efficacy in vivo. Intriguingly, clonal hMSCs (hcMSCs) isolated by SCM showed better efficacy than hMSCs isolated by GCM. Based on these results, the MHC-mismatched model of GvHD may be useful for evaluating the efficacy of human MSCs before the clinical trial. The results of this study suggest that different MSC lines may show different efficacy in vivo and in vitro.

Analysis of Primary Squamous Cell Carcinoma of Thyroid / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Squamous cell carcinoma of thyroid is uncommon and accounts for less than 1% of all primary thyroid malignancies. Clinical features mimic the natural course of anaplastic carcinoma. This study reviewed the clinical course of six cases of primary squamous cell carcinoma of thyroid. SUBJECTS AND METHOD: We diagnosed six cases of primary squamous cell carcinoma of thyroid diagnosed from 1999 to 2006 at the College of Medicine Department of Pathology. Clinical data, treatment modality, and pathologic test results from medical records were retrospectively analyzed. RESULTS: We found five women and one man (with the mean age of 52.1 years) with squamous cell carcinoma of thyroid. The main presenting features were abruptly enlarging neck swelling and obstructive symptom. Pre-operative needle aspiration biopsy revealed papillary carcinoma in five cases. Only one patient was diagnosed as squamous cell carcinoma through pre-operative needle aspiration biopsy. Three patients had massive adjacent organ invasion, and four patients had lymph node metastasis according to the pathology review. There were no cases of distant metastasis at the time of treatment. All patients received surgery and adjuvant therapy (radiation therapy, chemotherapy, radioiodine therapy). Three patients are still alive with a mean follow up period of 47.3 months (range, 44-49 months). The other three patients died within one year post-operatively. CONCLUSION: Primary squamous cell carcinoma of thyroid should be considered in patients diagnosed with papillary carcinoma and who exhibit aggressive clinical behavior. Complete tumor resection and radiotherapy should be performed if thyroid squamous cell carcinoma is confirmed.

The Correlation between TGF-beta 1 Blood Levels and the Formation of Bullae in Patients with Spontaneous Pneumothorax / 대한흉부외과학회지

BACKGROUND: The overexpression of transforming growth factor-beta 1 receptor II (TGF-beta1RII) and transforming growth factor-beta 1 (TGF-beta1) ligand may be involved in the formation of a bulla. In this study, we tested if serum TGF-beta1 ligand levels correlated with the expression level of TGF-beta1RII and TGF-beta1 in bullous tissues from patients with spontaneous pneumothorax. MATERIAL AND METHOD: Bullous lung tissues and blood samples were obtained from 19 patients with spontaneous pneumothorax, 18 males and 1 female, aged 17 to 35 years old. The bullous tissues were obtained by video-assisted thoracic surgery (VATS), fixed in formalin, embedded in paraffin, and cut into 5~6micrometer thick slices. Sections were immunohistochemically stained with primary antibodies against TGF-beta1 or TGF-beta1RII, and serum levels of TGF-beta1 in patients and normal controls was measured by enzyme-linked immunosorbent assay (ELISA). RESULT: Of the 19 patients, 16 were TGF-beta1 positive and 10 were TGF-beta1RII positive. Among the 16 TGF-beta1 positives, 9 were also TGF-beta1RII positive. As seen previously, strong immunohistochemical staining of TGF-beta1RII and TGF-beta was detected in the boundary region between the bullous and normal lung tissues. Average TGF-beta1 blood levels of both TGF-beta1 and TGF-beta1RII positive patients was 38.36+/-16.2 ng/mL, and that of five controls was 54.06+/-15 ng/mL. CONCLUSION: These results suggest that overexpression of TGF-beta1 and TGF-beta1RII expression may be involved in the formation of bullae. TGF-beta1 blood levels in patients with primary spontaneous pneumothorax is lower than normal people, suggesting that the high level of local TGF-beta1 expression in the bullous tissue region, but not in the whole blood, may contribute more in the formation of bullae.

TGF-beta1 Protein Expression in Bullae of Patients with Spontaneous Pneumothorax / 대한흉부외과학회지

BACKGROUND: In our previous study, we demonstrated that transforming growth factor-beta 1 receptor II (TGF-beta1RII) may have a role in the formation of bullae. In this study, we investigated if expression of transforming growth factor-beta 1 (TGF-beta1) ligand was altered in a bullous lung tissue by immunohistochemical staining of bullous tissues from patients with primary spontaneous pneumothorax. MATERIAL AND METHOD: Bullous lung tissues were obtained from 36 patients with primary spontaneous pneumothorax, including 34 males and 2 females aged 14 to 38 years old. RESULT: Of the 36 patients, 19 were TGF-beta1 positive and 24 were transforming growth factor-beta 1 receptor II (TGF-beta1RII) positive. Among the 19 TGF-beta1 positives, 15 were also TGF-beta1RII positive, observation at high magnification showed that strong immunohistochemical stain was detected in the boundary region between the bullous and normal lung tissues. CONCLUSION: These results suggest that overexpression of TGF-beta1 may be involved in the formation of a bulla as well as the alteration of TGF-beta1RII expression. Further molecular studies are needed to elucidate the more detailed molecular mechanisms of the bulla formation.

Relationship between XRCC1 Polymorphism and Acute Complication of Chemoradiation Therapy in the Patients with Colorectal Cancer / 대한방사선종양학회지

PURPOSE: It is well known from clinical experience that acute complications of chemoradiation therapy vary from patients to patients. However, there are no known factors to predict these acute complications before treatment starts. The human XRCC1 gene is known as a DNA base excision repair gene. We investigated the possibilities of XRCC1 gene polymorphisms as a predictor for the acute complications of chemoradiation therapy in colorectal cancer patients. MATERIALS AND METHODS: From July 1997 to June 2003, 86 colorectal cancer patients (71 rectal cancer, 13 sigmoid colon cancer and 2 colon cancer patients) were treated with chemoradiation therapy at the Department of Radiation Oncology, Inha University Hospital. Twenty-two patients were in stage B, 50 were in stage C, 8 were in stage D and 6 patients were unresectable cases. External radiation therapy was delivered with 10MV X-ray at a 1.8 Gy fraction per day for a total dose of radiation of 30.6~59.4 Gy (median: 54 Gy). All the patients received 5-FU based chemotherapy regimen. We analyzed the acute complications of upper and lower gastrointestinal tract based on the RTOG complication scale. The initial and lowest WBC and platelet count were recorded during both the RT period and the whole treatment period. Allelic variants of the XRCC1 gene at codons 194, 280 and 399 were analyzed in the lymphocyte DNA by performing PCR-RFLP. Statistical analyses were carried out with the SAS (version 6.12) statistical package. RESULTS: When all the variables were assessed on the multivariate analysis, recurrent disease revealed the factors that significantly correlated with upper gastrointestinal acute complications. Arg399Gln polymorphisms of the XRCC1 gene, the radiation dose and the frequencies of chemotherapy during radiation therapy were significantly correlated with lower gastrointestinal complications. Arg399Gln polymorphisms also affected the decrease of the WBC and platelet count during radiation therapy. CONCLUSION: Although the present sample size was too small for fully evaluating this hypothesis, this study suggests that Arg399Gln polymorphisms of the XRCC1 genes may be used as one of the predictors for acute complications of chemoradiation therapy in colorectal cancer patients.

Combined Treatment of an Intratumoral Injection of Dendritic Cells and Systemic Chemotherapy (Paclitaxel) for Murine Fibrosarcoma

A novel combined treatment of conventional chemotherapy with an intratumoral injection of syngeneic dendritic cells (DCs) has emerged as a potent cancer treatment strategy. In this study, we evaluated the synergistic effect of an intraperitoneal (i.p.) injection of a chemotherapeutic drug, paclitaxel, and an intratumoral (i.t.) injection of syngeneic bone marrow- derived DCs for the treatment of pre-existing fibrosarcoma. Subcutaneous tumors were established using MCA102 fibrosarcoma cells in syngeneic C57BL/6 mice. The results demonstrated that the combined treatment of paclitaxel chemotherapy and the injection of DCs led to complete tumor regression, in contrast to only partial eradication of the tumors with chemotherapy or DCs alone. Furthermore, the tumor-free mice were able to resist a repeat challenge with the same type of tumor. These findings suggest that a combination therapy of systemic chemotherapy along with the intratumoral administration of DCs is a potent treatment strategy for fibrosarcoma.

Ex vivo Expansion and Clonal Maintenance of CD34+ Selected Cells from Cord Blood and Peripheral Blood / 소아과

PURPOSE: Because of the unavailability of marrow transplantation, umbilical cord blood (CB) is increasingly being used. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from cord blood source and mobilized peripheral blood (PB) in a serum-free media. METHODS: The CD34+ cells, selected from CB and mobilized PB, were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at culture days 0, day 4, day 7, and day 14 with various growth factors. RESULTS: The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the PB at day 7 (2 fold increase than PB). The CB-selected CD34+ cells produced more BFU-E colonies than did the PB on culture at days 7 and at day 14. Also, the CB-selected CD34+ cells produced more CFU-Mk colonies than did the PB on culture at day 4 and at day 7. CONCLUSION: The ex vivo expansion of the CB cells may be promising in producing total cellular expansion, CFU-Mk and BFU-E compared with PB for 7 to 14 days. The growth factors combination including megakaryocyte growth and development, flt3-ligand and interleukin-3 showed more expansion in the view of total cells and clonal maintenance compared with less combination.

Plasma transforming growth factor-beta1 levels in patients with erectile dysfunction / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To evaluate the plasma TGF-beta1 level in erectile dysfunction (ED) patients of various causes.</p><p><b>METHODS</b>Sixty-two patients with ED and 26 potent men were subjected to the study. Based on multidisciplinary work-ups, including medical history, physical examinations, blood tests with lipid profile and hormones, penile duplex Doppler ultrasonogram and neurophysiological tests, causes for ED were classified as psychogenic (n=15), neurogenic (n=16) and vasculogenic (n=31). The plasma TGF-beta1 level was measured by the ELISA method.</p><p><b>RESULTS</b>The plasma TGF-beta1 level was significantly increased in the ED group (6.7+/-4.9 ng/mL), compared to the control (4.0 +/-2.1 ng/mL) (P<0.01). In the ED groups, there was a significant increase in the vasculogenic group (9.0 +/-5.5 ng/mL), compared to the psychogenic (3.8 +/-1.8 ng/mL) and neurogenic groups (4.8+/-3.2 ng/mL) (P<0.01). Of the vascular risk factors, both the smoking (7.5 +/-4.7 ng/mL) and dyslipidemia groups (7.4+/-4.4 ng/mL) showed significantly increased plasma TGF-beta1 levels, compared to the non-smokers (5.5+/-2.8 ng/mL), and those without dyslipidemia (4.8+/-2.8 ng/mL) (P<0.05).</p><p><b>CONCLUSION</b>Vascular risk factors are associated with an elevated plasma TGF-beta1 level, which may contribute to cavernous fibrosis and ED.</p>

Normal Serum Macrophage Migration Inhibitory Factor Concentrations of Korean people According to Age

BACKGROUND: Macrophage migration inhibitory factor(MIF) was discovered in 1960s as a T-cell cytokine which inhibited random migration of macrophages. MIF is a multifunctional protein acting as cytokine, hormone, or enzyme. It plays a pivotal role in innate and adaptive immune responses and early phase of inflammatory response, as well as cell proliferation, differentiation, and tumor progression. Many inflammatory diseases and cancers show increased activity and serum concentration. The purpose of this study was to measure the normal serum MIF concentration of Korean people to be utilized as base data for future MIF research. METHODS: Sera of 20 healthy adults from each groups of 20's to 60's(total 100 persons) who visited the Health Promotion center of Inha university Hospital were collected. The MIF concentration in each serum was measured by enzyme-linked immunosorbent assay(ELISA). RESULTS: The average serum MIF concentration was 1.49 ng/ml(ranging from 0 to 3.33), and there was no significant difference between age groups. CONCLUSION: The normal serum MIF concentration of Korean people is 1.49 ng/ml, and seems to be unchanged with aging.

Immunohistochemical Analysis of the Bullae in Patients with Primary Spontaneous Pneumothorax / 대한흉부외과학회지

BACKGROUND: Bulla is an air-filled space within the lung parenchyma resulting from deterioration of the alveolar tissue. Molecular mechanism of the formation of the bulla is not well described. Fibroblast growth factor(FGF)-7, bone morphogenetic protein(BMP) receptor, and transforming growth factor(TGF)-beta receptor are known to have a stimulatory or inhibitory role in the lung formation. We investigated to see if these growth factor or cytokine receptors are involved in the bulla formation by immunohistochemical staining of bullous lung tissues from patients with primary spontaneous pneumothorax. MATERIAL AND METHOD: Bullous lung tissues were obtained from 31 patients with primary spontaneous pneumothorax, including 30 males and 1 female from 15 to 39 years old. The bullous tissues were obtained by video-thoracoscopic surgery and/or mini-thoracotomy and fixed in formalin. Blocks of the specimens were embedded with paraffin and cut into 5~6 micrometer thick slices. The sections were deparaffinized and hydrated and then incubated with primary antibodies against FGF-7, BMP-RII, or TGF-RII. RESULT: Of the 31 patients, 24 were TGF-RII positive including 18 strong and 6 weak positives. Observation with high magnification showed that strong immunostaining was detected in the boundary region between bullous and normal lung tissues. In contrast, all of the sections were negative with FGF-7 or BMP-RII antibodies. CONCLUSION: These results suggest that overexpression of TGF-beta RII may be involved in the formation of bulla, although further molecular studies are needed to find out more detailed molecular mechanisms.

Combination treatment for osteosarcoma with baculoviral vector mediated gene therapy (p53) and chemotherapy (adriamycin)

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been evaluated as a vector for gene delivery to human tumor cells. A human osteogenic sarcoma cell line, Saos-2, was found to be highly susceptible to infection with a baculoviral vector, with nearly 100% of Saos-2 cells being able to express a lacZ reporter gene after a brief exposure to the virus at a m.o.i. of 30 pfu/cell. The production of beta-galactosidase protein was 18-times greater than that in HepG2 cells which were previously thought to be the mammalian cells most susceptible to the baculovirus. The possibility of developing a baculovirus as a cytotoxic vector for p53-defective cancer was tested by destruction of Saos-2 cells (p53-/-) with a recombinant baculovirus containing the wild type p53 gene (BV-p53) in vitro. The p53 baculovirus induced apoptotic cell death in tumor cells in a dose-dependent manner with approximately 60% killing at an m.o.i. of 160 pfu/cell. Combined treatments of gene therapy (p53) and chemotherapy (adriamycin) resulted in synergistic and potent killing of the osteogenic sarcoma cells. For example, greater than 95% of Saos-2 cells were killed by the combination of BV-p53 (m.o.i. of 100) and adriamycin (35 ng/ml), whereas approximately 50% and approximately 55% cells were killed by BV-p53 and adriamycin alone, respectively. These results indicate that a baculoviral gene delivery vector can be used to efficiently target certain types of mammalian cells and the combination treatment of gene-therapy mediated by a baculovirus and chemotherapy may enhance induction of apoptosis in cancer cells.