Screening of Sera from Patients with Pancreatitis by an Apoptosis Assay of Skin-derived Cells / 대한소화기학회지

BACKGROUND/AIMS: An excessive inflammatory response is typical in acute pancreatitis and a significant cause of early mortality in severe acute pancreatitis. This is believed to be caused by inflammatory molecules or upregulated cytokine levels in the serum of patients. The aim of this study was to identify the serum-mediated apoptosis-inducing effects in acute pancreatitis patients.METHODS: A skin tissue-derived cell line, BJ, was treated for 24 hours with the sera of 22 healthy volunteers (control) and 71 acute pancreatitis patients (22 with gallstone pancreatitis, 16 with alcoholic pancreatitis, and 11 with pancreatitis with other causes) collected at the time of hospital admission (active) and discharge (resolved). Apoptosis was analyzed by flow cytometry.RESULTS: The average percentage of living cells, early apoptotic cells, and late apoptotic cells ranged from 78.8% to 85.0%, 5.5% to 7.3%, and 7.7% to 13.1%, respectively. The number of live cells increased significantly using the serum from the resolved state of gallstone-induced pancreatitis. In addition, the number of early apoptotic cells increased significantly using the serum from the resolved state of pancreatitis with other causes. The number of late apoptotic cells decreased significantly with the serum from the resolved state compared to the active state of gallstone- and alcohol-induced pancreatitis.CONCLUSIONS: Serum samples from patients with pancreatitis induced a change in the apoptosis profiles of skin-derived cells. These results indicate changes in the serum components in patients with acute pancreatitis.

Screening of Sera from Patients with Pancreatitis by an Apoptosis Assay of Skin-derived Cells / 대한소화기병학회잡지

BACKGROUND/AIMS: An excessive inflammatory response is typical in acute pancreatitis and a significant cause of early mortality in severe acute pancreatitis. This is believed to be caused by inflammatory molecules or upregulated cytokine levels in the serum of patients. The aim of this study was to identify the serum-mediated apoptosis-inducing effects in acute pancreatitis patients. METHODS: A skin tissue-derived cell line, BJ, was treated for 24 hours with the sera of 22 healthy volunteers (control) and 71 acute pancreatitis patients (22 with gallstone pancreatitis, 16 with alcoholic pancreatitis, and 11 with pancreatitis with other causes) collected at the time of hospital admission (active) and discharge (resolved). Apoptosis was analyzed by flow cytometry. RESULTS: The average percentage of living cells, early apoptotic cells, and late apoptotic cells ranged from 78.8% to 85.0%, 5.5% to 7.3%, and 7.7% to 13.1%, respectively. The number of live cells increased significantly using the serum from the resolved state of gallstone-induced pancreatitis. In addition, the number of early apoptotic cells increased significantly using the serum from the resolved state of pancreatitis with other causes. The number of late apoptotic cells decreased significantly with the serum from the resolved state compared to the active state of gallstone- and alcohol-induced pancreatitis. CONCLUSIONS: Serum samples from patients with pancreatitis induced a change in the apoptosis profiles of skin-derived cells. These results indicate changes in the serum components in patients with acute pancreatitis.

N-glycoproteomic analysis of human follicular fluid during natural and stimulated cycles in patients undergoing in vitro fertilization / 대한생식의학회지

OBJECTIVE: Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. METHODS: For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. RESULTS: We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. CONCLUSION: We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.

Proteomic Analysis of Serum from Patients with Major Depressive Disorder to Compare Their Depressive and Remission Statuses

OBJECTIVE: Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. METHODS: Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. RESULTS: The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. CONCLUSION: This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation.

VEGF Expression of Cultured Keloid Fibroblasts from Different Donor Sites Under Normoxic and Hypoxic Conditions / 대한피부과학회지

BACKGROUND: The pathophysiological events resulting in keloid formation remain unclear. Overabundant levels of VEGF have been reported to contribute to excessive wound healing. There have been many studies describing the relationship between keloids and VEGF expression. However, there have been no reports about VEGF expression related to donor sites. OBJECTIVE: We investigated VEGF expression of cultured normal and keloid fibroblasts obtained from different body areas under normoxic and hypoxic culture conditions. METHODS: Normal fibroblasts from the earlobe (n=2), shoulder (n=2) and chest (n=2) as well as keloid fibroblasts from the earlobe (n=3), shoulder (n=3) and chest (n=3) were collected and cultured. VEGF expression of fibroblasts at 6 hours, 12 hours, 24 hours and 48 hours for cells maintained under normoxic and hypoxic conditions was measured by the use of RT-PCR. Paraffin-embedded tissues (normal and keloid tissue) were assayed by immunohistochemical staining. RESULTS: For the cultured normal fibroblasts, VEGF expression for cells in the hypoxic condition was higher as compared to VEGF expression in cells in the normoxic condition, irrespective of the donor site and time. However, for the cultured keloid fibroblasts, VEGF expression for cells in the hypoxic condition was higher as compared to VEGF expression in cells in the normoxic condition for cultured shoulder fibroblasts. For each donor site, VEGF expression was highest in the shoulder, followed by the chest and earlobe for cultured normal fibroblasts, irrespective of time. For the cultured keloid fibroblasts, the highest VEGF expression occurred at 6 hours for cells in the normoxic condition and the highest VEGF expression occurred at 6 hours and 12 hours for cells in the hypoxic condition. Based on immunohistochemical staining, VEGF expression of paraffin-embedded normal tissue was lower as compared to paraffin-embedded keloid tissue. For each donor site in paraffin-embedded keloid tissue, VEGF expression was highest in the shoulder, followed by the chest and earlobe. CONCLUSION: Oxygen tension and the nature of fibroblasts from different donor sites are involved in keloid pathogenesis.

Toxic Effect of Cryoprotectants on Embryo Development in a Murine Model / 대한불임학회지

OBJECTIVES: The aim of this study was to assess toxicities of cryoprotectants. METHODS: Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and 1,2-propanediol (PROH), were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or PROH. Embryo development was evaluated up to the blastocyst stage. Blastocysts were stained with bis-benzimide to evaluate the cell count and with terminal deoxynucleotidyl transferase mediated dUTP nick labeling (TUNEL) to assess apoptosis. RESULTS: The total cell count of blastocysts that were treated with DMSO at the 2-cell stage was significantly lower than that were treated with PROH (75.9+/-27.0) or the control (99.0+/-18.3) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the PROH treated group (p<0.05). Both DMSO (14.2+/-1.5) and PROH (11.2+/-1.4) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.2+/-0.9, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the PROH treated group (p<0.001). CONCLUSIONS: The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or PROH at room temperature. When comparing two cryoprotective agents, PROH appeared to be less toxic than DMSO at least in a murine embryo model.

Effect of GM-CSF on the Embryonic Development and the Expression of Implantation Related Genes of Mouse Embryos / 대한불임학회지

OBJECTIVE: The purpose of the current series of experiments were to assess the effect of GM-CSF, as a medium supplement, on the development of mouse embryos and the expression of LIF and IL-1beta mRNA. MATERIALS AND METHODS: Mouse 2-cell embryos were collected from the oviducts of 6 weeks old ICR mice at 48 hours after hCG injection. Embryos were cultured in P-1 medium supplemented with mouse GM-CSF(0,1,5,10 ng/ml). The embryo development to blastocysts and hatching blastocysts was assessed and the cell number in blastocyst was also examined. Using RT-PCR, the expressions of LIF and IL-1beta mRNA in blastocyst were evaluated in the GM-CSF supplemented group and control group. RESULTS: In mouse, the addition of GM-CSF increased the percentage of blastocysts(65.5%, 68.6%, 73.0% and 76.1% for control and 1, 5 and 10 ng/ml, respectively), and increased the proportion of hatching blastocysts(35.2%, 36.4%, 43.2% and 53.0% for control and 1, 5 and 10 ng/ml, respectively). The mean cell numbers in blastocyst were significantly increased in GM-CSF supplemented groups compared to control group. LIF and IL-1beta expression in blastocysts were significantly higher in GM-CSF supplemented group than in control group. CONCLUSION: The results of experiment by mouse embryos showed beneficial effects of GM-CSF as a medium supplement. Furthermore, the addition of GM-CSF significantly increased the expression of LIF and IL-1beta in mouse embryos. These results suggest that GM-CSF might be a important molecule in embryo implantation.

Influence of Maternal Age on Embryo Quality and the Frequency of Multiple Pregnancy in IVF-ET Program / 대한불임학회지

OBJECTIVE: This study was performed to evaluate the influence of maternal age on embryo quality and the frequency of multiple pregnancy in IVF-ET program. METHOD: 86 conventional IVF-ET cycles were divided into three groups according to the age by 5 year (group A: 26-30, group B: 3135, group C: 36-40 yrs). The in vitro fertilization and development outcome (fertilization, cleavage and high quality embryo rate) and the pregnancy outcome (pregnancy, implantation, G-sac/high quality embryo and multiple pregnancy rate) were examined. And then, these results were compared among the groups. RESULTS: The rates of fertilization (62.7, 68.5 and 65.4%, respectively) and cleavage (95.6, 97.6 and 98.0%, respectively) were not different among the groups. And the high quality embryo (HQE) rate also was not different among the groups (61.8, 62.9 and 62.8%, respectively). The pregnancy rate of group C (23.3%) was significantly lower than that of group A (41.2%) and B (48.7%). And the implantation rate was significantly decreased to group B (32.2%) and C (14.3%) when compared to group A (71.4%) and B (36.8%). CONCLUSION: The pregnancy rate was significantly decreased over 35 years. The G-sac/HQE and multiple pregnancy rate were significantly high below 31 years. Thus, these results suggest that the number of high quality embryo transferred should be limited by the age and another criteria for embryo quality evaluation were required for single embryo transfer.

Expression of Testis Specific Genes of Male Germ Cells in Pre-pubertal Mouse using in vitro Culture System / 대한남성과학회지

PURPOSE: To establish cultures of premeiotic mouse spermatogenic cells that could allow study of the meiotic process in vitro and to investigate expression of sperm-specific genes in premeiotic and postmeiotic division. MATERIALS AND METHODS: Male ICR mice were used. To obtain the premeiotic spermatogenic cells, we examined the histologic appearance of the germ cells in hematoxylin-eosin-stained testis sections during prepubertal development. To confirm the identity of the premeiotic cells, we preformed reverse transcriptase-polymerase chain reaction (RT-PCR) for the genes for LDH-C4, acrosin, protamine-2 (P-2), and SP-10, which are testis-specific marker proteins. In order to establish the culture system, the premeiotic spermatogenic cells were cocultured with mouse Sertoli cells in DMEM containing 10% fetal bovine serum (FBS), amino acids, FSH, and testosterone at 32 degrees C for 6 days, After 2 days of culture, RT-PCR was done to detect the sperm-specific genes. RESULTS: The four genes was not expressed in 10-day-old mice, but expression was detectable in 17-day-old mice. Thus, mice of this age have initiated the meiotic process, and round spermatids were seen in the testis. Premeiotic germ cells isolated from 15-day-old mice expressed LDH-C4 and acrosin but not P-2 and SP-10, which are post-meiotic marker proteins. Beginning after 2 days of culture, expression of the P-2 and SP-10 genes was detected in cultured premeiotic germ cells as in 15-day-old mice. CONCLUSIONS: P-2 and SP-10 may be marker genes for the premeiotic stage of spermatogenesis. Premeiotic male germ cells are able to differentiate into postmeiotic forms during coculture with Sertoli cells.

Effect of Fertilization Promoting Peptide on Kinematic Parameters, Capacitaion and Acrosome Reaction in Human Spermatozoa / 대한불임학회지

OBJECTIVE: This study has been carried out to evaluation the effect of fertilization promoting peptide (FPP) on the kinematic parameters, capacitation and acrosome reaction of the frozen-thawed human spermatozoa. METHODS: After FPP treatment, we examined kinematic parameters, capacitation and acrosome reaction, using the methods of computer-aided sperm analysis (CASA) and chlortetracycline (CTC) fluorescence analysis. RESULTS: We have obtained the evidence that FPP can promote the capacitation and inhibit the spontaneous acrosome reaction of frozen-thawed human spermatozoa in vitro. Fpp (25~100 nM) induced a significant increase in the proportion of B-pattern capacitated spermatozoa, and a significant decrease in the proportion of F-pattern uncapacitated ones without significant stimulation of acrosomal exocytosis. In the kinematic parameters treatment, FPP treated groups maintained higher LIN, BCF and STR than those of control. The VAP, VSL, VCL and ALH were not different. Therefore it is suggested that FPP in human seminal plasma may play a positive role in promoting human sperm function.

The Effect of Uterine Environment during Peri-implantation Period on theUltrastructure of Zona Pellucida in Mouse Oocytes and Embryos / 대한불임학회지

In the studies on the hatching mechanisms in mammals, many investigators focused on the embryonic intrinsic factor(s) in in vitro culture, but the uterine environment as the extrinsic factor(s) is thought to play an important role in hatching mechanism. Therefore, to evaluate the effect of uterine environment on the hatching event in vivo, the immature(GV) and ovulated(MII) oocytes, and the late 2-cell embryos of mouse were transferred to pseudopregnant foster mother's uterus during peri-implantation period. So it was verified whether there would happen hatching by only uterine environment independently on embryonic stage. The ultrastructural changes of the zona surface of transferred group were compared with those of in vivo and vitro group by SEM. 36 hrs after transfer, the immature and ovulated oocytes almost degenerated, and the late 2-cell embryos developed to various embryonic stages. However, the embryos which didn't develop to blastula stage did not hatch. The ultrastructural network of ZP in transferred group seemed to be smoothed uniformly, which was different from in vitro group. In conclusion, it is suggested that the uterine environment during peri-implantation period enhances the embryo hatching by provoking the structural change of ZP.