Dysregulation of Telomere Lengths and Telomerase Activity in Myelodysplastic Syndrome

BACKGROUND: Telomere shortening is thought to be involved in the pathophysiology of myeloid malignancies, but telomere lengths (TL) during interphase and metaphase in hematopoietic malignancies have not been analyzed. We aimed to assess the TLs of interphase and metaphase cells of MDS and telomerase activity (TA) and to find out prognostic significances of TL and TA. METHODS: The prognostic significance of TA by quantitative PCR and TL by quantitative fluorescence in situ hybridization (QFISH) of interphase nuclei and metaphase chromosome arms of bone marrow cells from patients with MDS were evaluated. RESULTS: MDS patients had shorter interphase TL than normal healthy donors (P<0.001). Average interphase and metaphase TL were inversely correlated (P=0.013, p arm; P=0.029, q arm), but there was no statistically significant correlation between TA and TL (P=0.258). The progression free survival was significantly shorter in patients with high TA, but the overall survival was not different according to average TA or interphase TL groups. Multivariable Cox analysis showed that old age, higher International Prognostic Scoring System (IPSS) subtypes, transformation to AML, no history of hematopoietic stem cell transplantation and short average interphase TL (<433 TL) as independent prognostic factors for poorer survival (P=0.003, 0.001, 0.005, 0.005, and 0.013, respectively). CONCLUSIONS: The lack of correlation between age and TL, TA, and TL, and the inverse relationship between TL and TA in MDS patients reflect the dysregulation of telomere status and proliferation. As a prognostic marker for leukemia progression, TA may be considered, and since interphase TL has the advantage of automated measurement by QFISH, it may be used as a prognostic marker for survival in MDS.

WHIM Syndrome With a Novel CXCR4 Variant in a Korean Child

No abstract available.

Cold Medications Aggravated Rhabdomyolysis Symptoms Induced by Building Construction Work and Strenuous Exercise: a Case Report

SUMMARY: A 21-year-old healthy Korean man worked on a building construction site every day for almost 2 months and exercised every day for 1 or 2 hours after working hard. He felt dizziness, nausea, and experienced vomiting and body aches immediately after exercise and immediately took cold medicines including acetaminophen, cimetidine, bepotastine, and Codenal? complex for the common cold symptoms for 2 days because he was scheduled to participate in navy training at that time. He complained of severe trapezius pain and aches in his left calf 3 days after joining the Navy training. Testing revealed creatine phosphokinase (CPK) 6260 U/L, myogloblin 176 mcg/L in the urine, liver enzymes increased, and oliguria, suggesting rhabdomyolysis. He recovered with intravenous fluids without any complications.

The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding

The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.

Generation of Insulin-Producing Human Mesenchymal Stem Cells Using Recombinant Adeno-Associated Virus

The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet beta-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced with AAV which is containing furin-cleavable human preproinsulin gene to generate insulin-producing cells as surrogate beta-cells for the type 1 diabetes therapy. In the rAAV production procedure, rAAV was generated by transfection of AD293 cells. Human mesenchymal stems cells were transduced using rAAV with a various multiplicity of infection. Transduction of recombinant AAV was also tested using beta-galactosidse expression. Cell viability was determined by using MTT assay to evaluate the toxicity of the transduction procedure. Expression and production of Insulin were tested using reverse transcriptase-polymerase chain reaction and immunocytochemistry. Secretion of human insulin and C-peptide from the cells was assayed using enzyme-linked immunosorbent assay. Production of insulin and C-peptide from the test group represented a higher increase compared to the control group. In this study, we examined generation of insulin-producing cells from mesenchymal stem cells by genetic engineering for diabetes therapy. This work might be valuable to the field of tissue engineering for diabetes treatment.

Effects of Sinusoidal Electromagnetic Field on Structure and Function of Different Kinds of Cell Lines

This study investigated that whether a 2 mT, 60 Hz, sinusoidal electromagnetic field (EMF) alters the structure and function of cells. This research compared the effects of EMF on four kinds of cell lines: hFOB 1.19 (fetal osteoblast), T/G HA-VSMC (aortic vascular smooth muscle cell), RPMI 7666 (B lymphoblast), and HCN-2 (cortical neuronal cell). Over 14 days, cells were exposed to EMF for 1, 3, or 6 hours per day (hrs/d). The results pointed to a cell type-specific reaction to EMF exposure. In addition, the cellular responses were dependent on duration of EMF exposure. In the present study, cell proliferation was the trait most sensitive to EMF. EMF treatment promoted growth of hFOB 1.19 and HCN-2 compared with control cells at 7 and 14 days of incubation. When the exposure time was 3 hrs/d, EMF enhanced the proliferation of RPMI 7666 but inhibited that of T/G HA- VSMC. On the other hand, the effects of EMF on cell cycle distribution, cell differentiation, and actin distribution were unclear. Furthermore, we hardly found any correlation between EMF exposure and gap junctional intercellular communication in hFOB 1.19. This study revealed that EMF might serve as a potential tool for manipulating cell proliferation.

Effects of Sinusoidal Electromagnetic Field on Structure and Function of Different Kinds of Cell Lines

This study investigated that whether a 2 mT, 60 Hz, sinusoidal electromagnetic field (EMF) alters the structure and function of cells. This research compared the effects of EMF on four kinds of cell lines: hFOB 1.19 (fetal osteoblast), T/G HA-VSMC (aortic vascular smooth muscle cell), RPMI 7666 (B lymphoblast), and HCN-2 (cortical neuronal cell). Over 14 days, cells were exposed to EMF for 1, 3, or 6 hours per day (hrs/d). The results pointed to a cell type-specific reaction to EMF exposure. In addition, the cellular responses were dependent on duration of EMF exposure. In the present study, cell proliferation was the trait most sensitive to EMF. EMF treatment promoted growth of hFOB 1.19 and HCN-2 compared with control cells at 7 and 14 days of incubation. When the exposure time was 3 hrs/d, EMF enhanced the proliferation of RPMI 7666 but inhibited that of T/G HA- VSMC. On the other hand, the effects of EMF on cell cycle distribution, cell differentiation, and actin distribution were unclear. Furthermore, we hardly found any correlation between EMF exposure and gap junctional intercellular communication in hFOB 1.19. This study revealed that EMF might serve as a potential tool for manipulating cell proliferation.

Tissue Engineered Intervertebral Disc by Atelocollagen Scaffolds and Growth Factors / 대한척추외과학회지

STUDY DESIGN: In vitro experimental study. OBJECTIVES: To examine the cellular proliferation, synthetic activity and phenotypical expression of intervertebral disc (IVD) cells seeded on types I and II atelocollagen scaffolds, with the stimulation of TGF-beta1 and BMP-2. SUMMARY OF LITERATURE REVIEW: Recently, tissue engineering is regarded as a new experimental technique for the biological treatment of degenerative IVD diseases, and has been highlighted as a promising technique for the regeneration of tissues and organs in the human body. Research on cell transplantation in artificial scaffolds has provided that the conditions for tissue engineering have to be equilibrated, including the cell viability and proliferation, maintenance of characteristic phenotype, suitable scaffolds in organisms and biologically stimulated growth factor. MATERIAL AND METHOD: Lumbar IVD cells were harvested from 10 New Zealand white rabbits, with the nucleus pulposus cells isolated by sequential enzymatic digestion. Each of 1% types I and II atelocollagen dispersions were poured into a 96-well plate (diameter 5 mm), frozen at -70 degrees C, and then lyophilized at -50 degrees C. Fabricated porous collagen matrices were made using the cross-linking method. Cell suspensions were imbibed by surface tension into a scaffold consisting of atelocollagen. The cell cultured scaffolds were then treated with TGF-beta1 (10 ng/ml) or BMP-2 (100 ng/ml) or both. After 1 and 2 week culture periods, the DNA synthesis was measured by [3H] thymidine incorporation, and newly synthesized proteoglycan by incorporation of [35S] sulphate. Reverse transcription-polymerase chain reactions for the mRNA expressions of type I and II collagen, aggrecan and osteocalcin were performed. The inner morphology of the scaffolds was determined by scanning electron microscopy (SEM). RESULTS: The IVD cultures in collagen type II with TGF-beta1 demonstrated an increase in proteoglycan synthesis and up regulation of aggrecan and types I and II collagen mRNA expressions compared to the control. IVD cultures in the type I atelocollagen scaffold with growth factors exhibited an increase in DNA synthesis and up regulation of the type II atelocollagen mRNA expression. With all combinations of growth factor, the IVD cultures in types I and II atelocollagen scaffolds showed no up regulation of the osteocalcin mRNA expression. Furthermore, there was no synergistic effect of TGF-beta1 and BMP-2 in the matrix synthesis or for the mRNA expression of the matrix components. CONCLUSIONS: Nucleus pulposus cells from rabbit were viable in atelocollagen types I and II atelocollagen scaffolds. The type I atelocollagen scaffold was suitable for cell proliferation, but the type II atelocollagen scaffold was more suitable for extracellular matrix synthesis. The IVD cells in both scaffolds were biologically responsive to growth factors. Taken together, nucleus pulposus cells in atelocollagen scaffolds, with anabolic growth factors, provide a mechanism for tissue engineering of IVD cells.

Tissue Engineered Intervertebral Disc by Atelocollagen Scaffolds and Growth Factors / 대한척추외과학회지

STUDY DESIGN: In vitro experimental study. OBJECTIVES: To examine the cellular proliferation, synthetic activity and phenotypical expression of intervertebral disc (IVD) cells seeded on types I and II atelocollagen scaffolds, with the stimulation of TGF-beta1 and BMP-2. SUMMARY OF LITERATURE REVIEW: Recently, tissue engineering is regarded as a new experimental technique for the biological treatment of degenerative IVD diseases, and has been highlighted as a promising technique for the regeneration of tissues and organs in the human body. Research on cell transplantation in artificial scaffolds has provided that the conditions for tissue engineering have to be equilibrated, including the cell viability and proliferation, maintenance of characteristic phenotype, suitable scaffolds in organisms and biologically stimulated growth factor. MATERIAL AND METHOD: Lumbar IVD cells were harvested from 10 New Zealand white rabbits, with the nucleus pulposus cells isolated by sequential enzymatic digestion. Each of 1% types I and II atelocollagen dispersions were poured into a 96-well plate (diameter 5 mm), frozen at -70 degrees C, and then lyophilized at -50 degrees C. Fabricated porous collagen matrices were made using the cross-linking method. Cell suspensions were imbibed by surface tension into a scaffold consisting of atelocollagen. The cell cultured scaffolds were then treated with TGF-beta1 (10 ng/ml) or BMP-2 (100 ng/ml) or both. After 1 and 2 week culture periods, the DNA synthesis was measured by [3H] thymidine incorporation, and newly synthesized proteoglycan by incorporation of [35S] sulphate. Reverse transcription-polymerase chain reactions for the mRNA expressions of type I and II collagen, aggrecan and osteocalcin were performed. The inner morphology of the scaffolds was determined by scanning electron microscopy (SEM). RESULTS: The IVD cultures in collagen type II with TGF-beta1 demonstrated an increase in proteoglycan synthesis and up regulation of aggrecan and types I and II collagen mRNA expressions compared to the control. IVD cultures in the type I atelocollagen scaffold with growth factors exhibited an increase in DNA synthesis and up regulation of the type II atelocollagen mRNA expression. With all combinations of growth factor, the IVD cultures in types I and II atelocollagen scaffolds showed no up regulation of the osteocalcin mRNA expression. Furthermore, there was no synergistic effect of TGF-beta1 and BMP-2 in the matrix synthesis or for the mRNA expression of the matrix components. CONCLUSIONS: Nucleus pulposus cells from rabbit were viable in atelocollagen types I and II atelocollagen scaffolds. The type I atelocollagen scaffold was suitable for cell proliferation, but the type II atelocollagen scaffold was more suitable for extracellular matrix synthesis. The IVD cells in both scaffolds were biologically responsive to growth factors. Taken together, nucleus pulposus cells in atelocollagen scaffolds, with anabolic growth factors, provide a mechanism for tissue engineering of IVD cells.