Immunologic properties of differentiated and undifferentiated mesenchymal stem cells derived from umbilical cord blood

The expression of immunogenic markers after differentiation of umbilical cord blood (UCB)-derived mesenchymal stem cells (MSC) has been poorly investigated and requires extensive in vitro and in vivo testing for clinical application. The expression of human leukocyte antigen (HLA) classes on UCB-derived MSC was tested by Fluorescence-activated cell sorting analysis and immunocytochemical staining. The undifferentiated MSC were moderately positive for HLA-ABC, but almost completely negative for HLA-DR. The MSC differentiated to chondrocytes expressed neither HLA-ABC nor HLA-DR. The proliferation of MSC was not significantly affected by the allogeneic lymphocytes stimulated with concanavalin A. The responder lymphocytes showed no significant decrease in proliferation in the presence of the MSC, but the apoptosis rate of the lymphocytes was increased in the presence of MSC. Taken together, these findings indicate that UCB-derived MSC differentiated to chondrocytes expressed less HLA class I and no class II antigens. The MSC showed an immunomodulatory effect on the proliferation and apoptosis of allogeneic lymphocytes. These data suggest that the differentiated and undifferentiated allogeneic MSC derived from umbilical cord blood can be a useful candidate for allogeneic cell therapy and transplantation without a major risk of rejection.

Cortical Activity Measured with EEG during Stepping on a Recumbent Stepper

OBJECTIVE: Recent neuroimaging studies have shown a strong involvement of the cortex as well as brainstem locomotor center in locomotion. In the present study, cortical activity during constant stepping was investigated using electroencephalography (EEG). METHOD: Five healthy volunteers participated in this study. Sixty-four channel EEG was measured for 10 min while the participants performed constant stepping on a cadence of 100 rpm (50 cycles per 1 min) using a recumbent stepper. Surface electromyography (EMG) was also measured at the bilateral vastus medialis muscles. Each stepping cycle was epoched from the onset of EMG signal of the right vastus medialis muscle. Averaged event-related potentials (ERP) and event-related spectral perturbations (ERSP) of all subjects were calculated at all EEG channels. RESULTS: Periodic change of ERP was centered on the CZ and FPZ electrodes. While ERP at the CZ started to increase at 200 and 800 ms, ERP at the FPZ started to increase at -50 and 600 ms. ERSP was remarkable at the CZ during stepping cycles. According to the stepping cycle, power increases were pronounced at low-gamma frequency band and also observed at beta band. CONCLUSION: This study showed cortical activity during constant stepping using EEG in healthy participants. Periodic cortical activities were remarkable at the sensorimotor cortex area, and precedent activities were observed at the prefrontal area. EEG measurement during stepping on a recumbent stepper may be a valuable tool in investigating cortical activates related to walking in patients with gait disorders.

Ciglitazone, a Peroxisome Proliferator-Activated Receptor Gamma Ligand, Inhibits Proliferation and Differentiation of Th17 Cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) was identified as a cell-intrinsic regulator of Th17 cell differentiation. Th17 cells have been associated with several autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), inflammatory bowel disease (IBD), and collagen-induced arthritis. In this study, we confirmed PPARgamma-mediated inhibition of Th17 cell differentiation and cytokine production at an early stage. Treatment with ciglitazone, a PPARgamma ligand, reduced both IL-1beta-mediated enhancement of Th17 differentiation and activation of Th17 cells after polarization. For Th17 cell differentiation, we found that ciglitazone-treated cells had a relatively low proliferative activity and produced a lower amount of cytokines, regardless of the presence of IL-1beta. The inhibitory activity of ciglitazone might be due to decrease of CCNB1 expression, which regulates the cell cycle in T cells. Hence, we postulate that a pharmaceutical PPARgamma activator might be a potent candidate for treatment of Th17-mediated autoimmune disease patients.

Characteristics of Abdominal Obesity in Persons With Spinal Cord Injury

OBJECTIVE: To investigate the characteristics of community-dwelling spinal cord injury (SCI) persons with obesity, including diet, socioeconomic factors, weight reduction method, and frequency of body weight and abdominal circumference measurements. METHODS: We developed a questionnaire based on 'the Fourth Korea National Health and Nutrition Examination Survey, 2009'. A total of 371 community-dwelling SCI persons were enrolled in this study. Inclusion criteria were SCI persons older than 20 years with more than 1 year elapsed since the injury. Trained investigators visited SCI persons' home to complete the questionnaire and measure abdominal obesity (AO) as defined by the waist circumference. RESULTS: Prevalence of AO was 29.2% in SCI persons and 27.4% in the general population (GP), showing no significant difference. Education showed correlation with AO in both SCI persons and the GP. The injury level, type of injury and income did not show any correlation with AO in SCI persons. Only 28.8% and 48.8% of SCI persons measured their waist circumference and body weight within the past year, respectively. Also, SCI persons with AO thought that their body was less obese compared to persons with AO in the GP (p<0.001). The method of weight reduction was diet modification in 53.6% of SCI persons with AO, which was higher than 37.1% of persons with AO in the GP. CONCLUSION: In SCI persons, obesity perception as well as socioeconomic factors correlated with AO, but these were not relevant factors in the GP. Therefore, development of a specific and intensive weight control program for SCI persons is necessary.

ICF Based Comprehensive Evaluation for Post-Acute Spinal Cord Injury

OBJECTIVE: To evaluate the feasibility of the ICF for initial comprehensive evaluation of early post-acute spinal cord injury. METHOD: A comprehensive evaluation of 62 early post-acute spinal cord injury (SCI) patients was conducted by rehabilitation team members, such as physicians, physical therapists, occupational therapists, nutritionists, medical social-workers, and nurses. They recorded each of their evaluation according to the ICF first level classification. The contents of the comprehensive evaluation were linked to the ICF second level categories, retrospectively. The linked codes were analyzed descriptively and were also compared with the brief ICF core set for early post-acute SCI. RESULTS: In the evaluation of early post-acute SCI patients based on the ICF first level categories, 19 items from the body functions domain, such as muscle power functions (b730) and urination functions (b620), 15 items from the body structures domain, including spinal cord and related structures (s120), 11 items from the activities and participation domain, such as transferring oneself (d420) and walking (d450), and 9 items from the environmental factors domain, e.g., health professionals (e355), were linked to the ICF second level categories. In total, 82.4% of all contents were linked to the brief ICF core set. Prognosis insight, a personal factor not linkable to an ICF code, was mentioned in 29.0% of all patients. CONCLUSION: First level ICF categories can provide a structural base for a comprehensive evaluation in early post-acute spinal cord injury. However, frequently linked items, including the brief core set, as well as personal factors should be considered via a checklist in order to prevent the omission of significant contents.

Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors / 대한진단검사의학회지

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.

Inflammatory Mediators Modulate NK Cell-stimulating Activity of Dendritic Cells by Inducing Development of Polarized Effector Function

BACKGROUND: It is well established that cross talk between natural killer (NK) cells and myeloid dendritic cells (DC) leads to NK cell activation and DC maturation. In the present study, we investigated whether type 1-polarized DC (DC1) matured in the presence of IFN-gamma and type 2-polarized DC (DC2) matured in the presence of PGE2 can differentially activate NK cells. METHODS: In order to generate DC, plastic adherent monocytes were cultured in RPMI 1640 containing GM-CSF and IL-4. At day 6, maturation was induced by culturing the cells for 2 days with cytokines or PGE2 in the presence or absence of LPS. Each population of DC was cocultured with NK cells for 24 h. The antigen expression on DC was analyzed by flow cytometry and cytokine production in culture supernatant was measured by ELISA or a bioassay for TNF-alpha determination. NK cell-mediated lysis was determined using a standard 4 h chromium release assay. RESULTS: DC2, unlike DC1, had weak, if any, ability to induce NK cell activation as measured by IFN-gamma production and cytolytic activity. DC2 were weakly stimulated by activated NK cells compared to DC1. In addition, IFN-gamma-primed mature DC appeared to be most resistant to active NK cell-mediated lysis even at a high NK cell/DC ratio. On the other hand, PGE2-primed DC were less resistant to feedback regulation by NK cells than IFN-gamma-primed mature DC. Finally, we showed that the differential effect of two types of DC population on NK cell activity is not due to differences in their ability to form conjugates with NK cells. CONCLUSION: These results suggest that different combinations of inflammatory mediators differentially affect the effector function of DC and, as a result, the function of NK cells, eventually leading to distinct levels of activation in adaptive immunity.

Impaired responses of leukemic dendritic cells derived from a human myeloid cell line to LPS stimulation

Several myeloid leukemia-derived cells have been reported to possess the ability to differentiate into dendritic cells (DC). MUTZ-3, a myeloid leukemia cell line, responds to GM-CSF, IL-4 and TNF-alpha, and acquires a phenotype similar to immature monocyte-derived DC (MoDC). In the present study, MUTZ-3-derived DC (MuDC) showed high level expression of HLA class II molecules, CD80 and CD86, and were able to function as potent antigen presenting cells as previously reported. Interestingly, MuDC maturation was induced by CD40-mediated stimulation, but not by LPS stimulation. We analyzed CCR1, CCR7 and Toll-like receptor (TLR) expressions in MuDC, and measured IL-10 and IL-12 production after maturation stimuli. Although MuDC expressed the mRNA for TLR4, a major component of the LPS receptor system, they did not show an enhanced level of CCR7 or cytokine production after LPS stimulation. In contrast, they responded to CD40 stimulation, which resulted in increased levels of CD83, CD86 and CCR7. Moreover, while LPSstimulated MoDC could potently stimulate NK cells in a DC-NK cell co-culture, LPS-stimulated MuDC failed to stimulate primary NK cells. Taken together, our findings suggest that, although MuDC express TLR4, unlike TNF-alpha and IL-1beta, LPS does not stimulate MuDC to acquire mature phenotypes, and they may have impaired activity to initiate innate immune response.

Activated natural killer cell-mediated immunity is required for the inhibition of tumor metastasis by dendritic cell vaccination

Immunization with dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL), which is responsible for tumor protection and regression. In this study, we examined whether DCs pulsed with necrotic tumor lysates can efficiently prevent malignant melanoma tumor cell metastasis to the lung. DCs derived from mouse bone marrow were found to produce remarkably elevated levels of IL-12 after being pulsed with the tumor lysates. Moreover, immunization with these DCs induced CTL activation and protected mice from metastasis development by intravenously inoculated tumor cells. In addition, these DCs activated NK cells in vitro in a contact-dependent manner, and induced NK activities in vivo. Furthermore, NK cell depletion before DC vaccination significantly reduced the tumor-specific CTL activity, IFN-g production, and IFN-gamma- inducible gene expression, and eventually interfered with the antitumor effect of tumor-pulsed DCs. Finally, similar findings with respect to NK cell dependency were obtained in the C57BL/ 6J-bg/bg mice, which have severe deficiency in cytolytic activity of NK cells. These data suggest that the antitumor effect elicited by DC vaccination, at least in a B16 melanoma model, requires the participation of both cytolytic NK and CD8+ T cells. The findings of this study would provide important data for the effective design of DC vaccines for cancer immunotherapy.

IL-12 Production and Subsequent Natural Killer Cell Activation by Necrotic Tumor Cell-loaded Dendritic Cells in Therapeutic Vaccinations

BACKGROUND: Immunization of dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. In this study, we examined whether the uptake of necrotic tumor cells could modulate DC phenotypes and whether the immunization of necrotic tumor cell-loaded DCs could elicit efficient tumor specific immune responses followed by a regression of established tumor burdens. METHODS: We prepared necrotic tumor cell-pulsed DCs for the therapeutic vaccination and investigated their phenotypic characteristics, the immune responses induced by these DCs, and therapeutic vaccine efficacy against colon carcinoma in vivo. Several parameters including phagocytosis of tumor cells, surface antigen expression, chemokine receptor expression, IL-12 production, and NK as well as CTL activation were assessed to characterize the immune response. RESULTS: DCs derived from mouse bone marrow efficiently phagocytosed necrotic tumor cells and after the uptake, they produced remarkably increased levels of IL-12. A decreased CCR1 and increased CCR7 expression on DCs was also observed after the tumor uptake, suggesting that antigen uptake could induce DC maturation. Furthermore, co-culturing of DCs with NK cells in vitro enhanced IL-12 production in DCs and IFN-gamma production in NK cells, which was significantly dependent on IL-12 production and cell-to-cell contact. Immunization of necrotic tumor cell-loaded DCs induced cytotoxic T lymphocytes as well as NK activation, and protected mice against subsequent tumor challenge. In addition, intratumoral or contra-lateral immunization of these DCs not only inhibited the growth of established tumors, but also eradicated tumors in more than 60% of tumor-bearing mice. CONCLUSION: Our data indicate that production of IL-12, chemokine receptor expression and NK as well as CTL activation may serve as major parameters in assessing the effect of tumor cell-pulsed DC vaccine. Therefore, DCs loaded with necrotic tumor cells offer a rational strategy to treat tumors and eventually lead to prolonged survival.

A Case of Light Chain Deposition Disease Associated with Renal Amyoidosis / 대한신장학회잡지

Light chain deposition disease of kidney is characterized by deposition of monoclonal immunoglobulin light chain and electron-dense material in glomerular and tubular basement membrane and usually associated with multiple myeloma or other plasma cell dyscrasia. With light chain deposition disease affecting kidney, three clinical patterns have been recognized; nephrotic syndrome, rapidly progressive renal failure and slowly progressing chronic renal failure. The majority of patients present proteinuria and renal insufficiency. Cytotoxic therapy has been considered as treatment of choice. Favorable effect of melphalan given together with prednisone has been reported in a few cases. A 64-year-old male was admitted with generalized edema and exertional dyspnea, and was presumptively diagnosed as congestive heart failure and hypertension. He also presented increased serum creatinine and nephrotic range proteinuria. Urine protein electrophoresis and urine and serum immunoelectrophoresis revealed monoclonal gammopathy of IgG kappa type. Work up for multiple myeloma including bone marrow biopsy showed results compatible with smoldering myeloma. Renal biopsy showed findings of light chain deposition disease and Congo-red positive amyloidosis. After we treated the patient with melphalan and predinsone for two cycles, amount of proteinuria and serum creatinine were decreased.

A Study of Treatment in Adult Patients with Idiopathic Membranous glomerulonephritis / 대한내과학회지

OBJECTIVE: We aimed to compare efficacy of treatment between steroid therapy and steroid-chlorambucil combination therapy in patients with adult-onset idiopathic membranous glomerulonephritis (MN). METHODS: A series of 31 biopsy-proved idiopathic MN patients was analyzed retrospectively to estimate effect of treatment with steroid and/or cytotoxic agent. All patients (male 15, female 16, mean age of 37 years old) presented a full-blown nephrotic syndrome (proteinuria >3.0gm/day, serum albumin <3.0mg/dL, edema) at the initiation of treatment and were observed for at least 6 months (mean follow up period: 28 +/- 23 months). Clinical and laboratory information were obtained at the time of presentation and at last follow up. Each patient was assigned to one of the following protacols. 1) Steroid therapy prednisolone 40 or 60mg/day (single dose) for 16 weeks. 2) Steroid-Chlorambucil combination therapy: for 6 months with three cycles of methylprednisolone pulse therapy (lgm 1V for 3 days), prednisolone 0.5mg/kg/day for 27 days, then chlorambucil 0.2mg/kg/day for 28 days. 3. Cyclophosphamide 2mg/kg/day for 28 days. RESULTS: 1) Final status in total 31 cases irrespective of therapeutic modality were complete remission in 5 (16%) cases, partial remission in 9 (29%) cases, no response in 12 (41%) cases, spontaneous complete remission in 1 cases, and spontaneous partial remission in 1 case. 2) After initial steroid therapy (in 25 cases), We observed no response in 17 (68%) case, partial remission in 3 (12%) cases, complete remission in 3 (12%) cases, and spontaneous partial remission in 2 (8%) cases. 3) The combination therapy (steroid and chlorambucil) tried in 10 cases results in 5 (50%) cases of partial remission, 2 (20%) cases of complete remission, 1 (10%) case of spontaneous partial remission, and 2 (20%) cases of no response. 4) During follow up period, renal functional deterioration was absent in any case and final albumin levels were significantly increased (p<0.05). CONCLUSION: Steroid-chlorambucil combination protocol is considered to be a more effective treatment with higher rate of overall remission compared to steroid therapy. Idiopathic MN itself seems to have a relatively benign course when considering that renal function was preserved in all cases without progression to chronic renal failure and that a few cases of spontaneous remission could be observed. For the limitation in number of cases and duration of follow up in this study, it needs prospective controlled study of more larger scale with long-term follow up to get a more reliable results.

Myocardial Contrast Echocardiography for the Assessment of Coronary Blood Flow Reserve

BACKGROUND: The assessment of coronary blood flow reserve measured by intracoronary Dopper syudy is a useful method for evaluation of functional impairment of coronary artery disease irrespective of significant anatomic stenosis. To validate the usefullness of myocardial contrast echocardiography in clinical assessment of coronary blood flow reserve, several variables analysed by myocardial contrast echocardiography were compared with coronary flow reserve measured by Dopper catheter study. METHODS: During the coronary angiography, coronary flow reserve was measured by intracoronary Dopper-tipped guidewire with coronary blood flow velocity ratio in 16 patients without angiographically significant coronary artery disease. For the measurement of coronary flow reserve, we analyzed the time-video intensity curve of short axis image of the left ventricle follwing infusion of sonicated hexabrix before and after intracoronary administration of adenosine. RESULTS: 1) There was no significiant difference or correlation between peak intensity, peak intensity ratio, washout time, half time of washout, and washout time ratio analysed by contrast echocardiography and coronary blood flow reserve measured by intracoronary Doppler study(p>0.05). But inverse correlation was observed between half time ratio of contrast washout and coronary flow reserve(r=0.63,p<0.05). 2) There was no significiant difference between non myocardial infarction group and myocardial infartion group in coronary flow reserve and half time ratio of contrast washout. 3) No significant difference was observed before and after administrantion of sonicated hexabrix in hemodynamic variables. CONCLUSION: Among several variables of myocardial contrast echocardiography analysis half time ratio of washout was significantly correlated with coronary flow reserve. Thus assesssment of coronary flow reserve with myocardial contrast echocardiography is promising method in the evaluation of dymamic coronary perfusion and myocardial viability.

Intravascular Ultrasonographic Detection of Atherosclerotic Lesions in Spastic Segments of the Coronary Arteries in Patients with Variant Angina

Coronary artery spasm plays an important role for evoking myocardial ischemia and infarction as well as sudden cardiac death in patients with variant angina. The coronary anatomy in patients with variant angina has been defined both at autopsy and during coronary arteriography. Severe porximal coronary atherosclerosis of at least one major vessel occurs in 3/4 of patients and the remainder have normal coronary arteries. Coronary angiography is a relatively insensitive diagnostic tool especially in the early stages of coronary artery disease. Due to arterial remodelling, angiographic luminogram may show little or no narrowing even though a large part of the total vessel area is occupied by plaque. Intravascular ultrasonography(IVUS) enables accurate determination of vessel dimensions and wall characteristics and is more sensitive in delineating early intimal changes than angiography. We experienced 2 cases that IVUS showed focal or diffuse atherosclerosis in spastic segments of the coronary arteries, even though they appeared angiographically normal.