BACKGROUND: To determine the molecular structure of type II collagen-specific T-cell receptors associated with rheumatoid arthritis (RA). METHODS: We generated CII-specific T-cell lines of 8 RA patient s by prolonged in vitro culture with bovine CII (bCII) and the immunogenic peptide (256-270) of human CII. The proliferation response towards CII stimulation was measured from the uptake of 3 H-thymidine. Changes in the secretion of Th 1 and Th2 cytokines in the culture supernatent were measured by ELISA. The TCR clonotypes of these T-cells were examined by RT-PCR/ SSCP analyses of all 22 V beta chains. RESULTS: T-cells from patients' tissue exhibited strong proliferation index upon CII stimulation, which was maintained up to 6 months in the culture. The secretion of INF-gamma from these T-cells increased along with the duration of culture time, while the amount of IL-4 production did not show significant changes. The SSCP band patterns of patients' T-cells appear as discrete bands unlike the smeary streak produced from normal samples. Some SSCP bands, each representing selected expansion of a TCR containing certain subtype of V beta peptides, appeared to be identical in more than one patients. Among these, the expansion of SSCP band representing the V beta 14 CDR3 region persisted after switching the antigen to the immunogenic human peptide (256-270). CONCLUSION: CII-reactive T-cells expressing distinct CDR3 motifs are selectively expanded in the peripheral blood and synovial fluid of RA patients, and their persistent proliferation upon CII stimulation, as well as the production Th 1-type cytokines, may play pivotal roles in RA pathogenesis.
Arthritis, Rheumatoid* ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-4 ; Molecular Structure ; Peptides ; Polymorphism, Single-Stranded Conformational ; Receptors, Antigen, T-Cell ; Synovial Fluid ; T-Lymphocytes*

No abstract available.
Interleukin-4* ; Interleukins* ; Receptors, Interleukin-4* ; Rheumatic Diseases*

BACKGROUND: Mycoplasma spp. occasionally colonize the genital tract and these organisms are some of the most important contaminants in cell culture laboratories and cell banks. We analyzed the Mycoplasma contamination rates in the donated cord blood units (CBUs) before cell processing. METHODS: A total of 151 CBUs that were donated with informed consent (November 3rd~December 28th, 2006) were randomly selected and enrolled in the study. We performed blood culture and Mycoplasma DNA PCR assay with using samples from the collection bags before processing. RESULTS: All of the CBUs were obtained from full-term (gestational age 37~42 weeks) deliveries. Two units showed positive results on blood culture however, Mycoplasma DNA is not found in the tested samples. CONCLUSION: The contamination rates of Mycoplasma in the CBUs, which are donated from the mothers who have full-term delivery and no pregnancy complications, are extremely low. The donated CBUs could be used in culture and for an expansion process without concern of incurring pre-processing Mycoplasma contamination. The rate of Mollicute contamination in the CBUs could become clear with the results of performing Ureaplasma assay.
Cell Culture Techniques ; Colon ; DNA ; Fetal Blood ; Humans ; Informed Consent ; Mothers ; Mycoplasma ; Polymerase Chain Reaction ; Pregnancy Complications ; Ureaplasma

Clostridium difficile is a significant nosocomial and community-acquired pathogen, and is the leading cause of antibiotic-induced diarrhea associated with high morbidity and mortality. Given that the treatment outcome depends on the severity of C. difficile infection (CDI), we aimed to establish an efficient method of assessing severity, and focused on the stool biomarker fecal calprotectin (FC). FC directly reflects the intestinal inflammation status of a patient, and can aid in interpreting the current guidelines, which requires the integration of indirect laboratory parameters. The distinction of 80 patients with CDI versus 71 healthy controls and 30 severe infection cases versus 50 mild cases was possible using FC as a marker. The area under the receiver operating characteristic curves were 0.821 and 0.746 with a sensitivity of 75% and 70% and specificity of 79% and 80%, for severe versus mild cases, respectively. We suggest FC as a predictive marker for assessing CDI severity, which is expected to improve the clinical management of CDI.
Aged ; Area Under Curve ; Biomarkers/analysis ; Clostridium difficile/*isolation & purification ; Enterocolitis, Pseudomembranous/diagnosis/microbiology/*pathology ; Enzyme-Linked Immunosorbent Assay ; Feces/*chemistry ; Female ; Humans ; Leukocyte L1 Antigen Complex/*analysis ; Male ; Middle Aged ; ROC Curve ; Severity of Illness Index

PURPOSE: Simple and noninvasive methods for the diagnosis of transitional cell carcinoma of the bladder are needed for the prevention of invasive tumor. A proteomic technology has recently been developed to facilitate protein profiling of biological mixtures. We investigated the role of this proteomic approach as a possible tool to detect the marker protein during the initiation stages on BBN-induced bladder carcinogenesis in rats. MATERIALS AND METHODS: Ten rats group A were given 0.05% BBN in drinking water for 12 weeks. Ten rats in group B were designated as a control group and were not given BBN. Whole urinary bladders of all rats were excised at 12 weeks from the beginning of the experiment. Conventional proteomics was performed with high resolution 2-D gel electrophoresis followed by computational image analysis and protein identification using mass spectrometry. RESULTS: A comparison of urinary bladder hyperplasia tissue with control tissue showed that five proteins; actin gamma2 propeptide, cytokeratin-20, proapolipoprotein, alpha2 actin(alpha-cardiac actin) and heat shock 27kDa protein 1 were over-expressed in hyperplastic tissues. Three protein; transcription factors, seminal vesicle secretory protein VI precursor and hypothetical protein RMT-7 were under-expressed in hyperplastic tissues. CONCLUSIONS: In an animal model system, BBN-induced, urinary bladder mucosal hyperplasia resulted in an increase in five proteins and a decrease in three proteins. Of these altered proteins, CK-20 and SVS-VI seem to be important. The proteomic approach may be a simple and noninvasive method for monitoring and follow-up of bladder cancer patients. However more information is needed regarding CK-20 expression in nonmalignant urological disease and in human tumor tissue, and regarding SVS-VI expression in other organs, for clinical usage.
Actins ; Animals ; Carcinogenesis ; Carcinoma, Transitional Cell ; Diagnosis ; Drinking Water ; Electrophoresis, Gel, Two-Dimensional ; Follow-Up Studies ; Hot Temperature ; Humans ; Hyperplasia ; Keratin-20 ; Mass Spectrometry ; Models, Animal ; Precancerous Conditions* ; Proteomics ; Rats ; Seminal Vesicles ; Shock ; Transcription Factors ; Urinary Bladder Neoplasms ; Urinary Bladder* ; Urologic Diseases

OBJECTIVES: The purpose of this study is to isolate a population of multipotent cells from human amnion and decidua, respectively. METHODS: Human placentas (gestational age, 30~42 weeks) were obtained after vaginal or cesarean deliveries. Amnions and deciduas were divided mechanically. The collected cells from the amnion and decidua were cultured. Cultured cells were immunophenotypically characterized. The adipogenic, osteogenic and neurogenic differentiation capacities were tested, and their growth kinetics were analyzed. RESULTS: We successfully isolated MSCs from both the amnion and decidua. The phenotype of MSCs cultured from different fetal and maternal parts of the placenta was comparable. The growth kinetics of MSCs derived from amnions and deciduas were similar. Isolated MSCs were differentiated into various cell lines such as adipogenic, osteogenic, myogenic and neurogenic cells. CONCLUSIONS: The human amnion and decidua could be an excellent source of MSC because they are easily obtainable after delivery and showed a higher expansion capacity than that of MSCs from adult bone marrow.
Adult ; Amnion ; Cell Line ; Cells, Cultured ; Decidua ; Durapatite ; Female ; Humans ; Kinetics ; Mesenchymal Stromal Cells ; Phenotype ; Placenta

PURPOSE: Epigenetic alterations of specific genes have recently been identified as diagnostic biomarkers for human cancers. However, there are currently no standardized epigenetic biomarkers for drug sensitivity in human gastrointestinal cancer. Therefore, the aim of this study is to identify a novel epigenetic biomarker in gastrointestinal cancer. MATERIALS AND METHODS: Using bisulfite sequencing and pyrosequencing analysis, DNA methylation patterns of gastric, colon primary tissues and their cancer cells were analyzed, and histone modifications were analyzed using chromatin immunoprecipitation assay. In addition, cancer cells were exposed to cisplatin and treated with a DNA methyltransferase inhibitor. RESULTS: We report that in human gastric and colon cancers, latrophilin 2 (LPHN2) is silenced by epigenetic modifications, including CpG island methylation and aberrant histone modifications. We also confirmed that LPHN2 was silenced by DNA hypermethylation in primary gastric and colon tumor tissues compared to their normal counterparts. Interestingly, we found that cancer cells with methylated LPHN2 showed higher sensitivity to cisplatin. Also, 5-aza- 2′-deoxycytidine combined with cisplatin decreased the cytotoxicity of cisplatin in cancer cells with methylated LPHN2. In addition, LPHN2 knockdown in cancer cells with high LPHN2 expression sensitized these cells to the anti-proliferative effects of cisplatin. CONCLUSION: In human gastrointestinal cancer, we found that LPHN2 is regulated by epigenetic modifications, and that cancer cells with lower LPHN2 expression show higher sensitivity to cisplatin. Therefore, the methylation status of LPHN2 is a potential novel epigenetic biomarker for cisplatin treatment in human gastric and colon cancers.
Biological Markers ; Chromatin Immunoprecipitation ; Cisplatin ; Colon ; Colonic Neoplasms ; CpG Islands ; DNA ; DNA Methylation ; Epigenomics* ; Gastrointestinal Neoplasms* ; Histones ; Humans* ; Methylation

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade the extracellular matrix and other extracellular proteins. Upregulation of MMPs activity is known to be required for the inflammatory cell infiltration after spinal cord injury (SCI) and most likely contributes to early blood spinal barrier disruption and inflammation, thereby leading to the impairment of functional recovery. Here, we examined the effect of ethanol extract of Bupleurum falcatum (BF) on functional recovery by inhibiting MMP-2 and -9 activation and inflammation after SCI. Rats received a moderate, weight-drop contusion injury to spinal cord were administered orally with BF at a dose of 100 mg/kg for 14 d and functional recovery was measured by Basso-Beattie-Bresnahan locomotor open field behavioral rating test, inclined plane test and foot print analysis. To examine the neuroprotective effect of BF, TUNEL staining and counting were also performed. In addition, the expression and/or activation of MMP-2, MMP-9 and inflammatory mediators such as TNF-alpha, IL-1beta, COX-2, and iNOS were examined by RT-PCR and gelatin zymography using spinal cord tissue from 1 d after injury. Our data showed that BF significantly inhibited the expression and activation of both MMP-2 and MMP-9 after SCI. The mRNA expressions of TNF-alpha, IL-1beta, COX-2, and iNOS were also significantly attenuated by BF. Furthermore, BF reduced apoptotic cell death at 1 d after injury, thereby significantly reduced lesion volume and improved functional recovery. Taken together, these results suggest that BF can be used as a potential therapeutic agent for treating acute spinal injury.
Animals ; Blood-Brain Barrier ; Bupleurum ; Cell Death ; Contusions ; Endopeptidases ; Ethanol ; Extracellular Matrix ; Foot ; Gelatin ; In Situ Nick-End Labeling ; Inflammation ; Matrix Metalloproteinases ; Neuroprotective Agents ; Proteins ; Rats ; RNA, Messenger ; Spinal Cord ; Spinal Cord Injuries ; Spinal Injuries ; Tumor Necrosis Factor-alpha ; Up-Regulation

Objective: Despite extensive research on implantation failure, little is known about the molecular mechanisms underlying the crosstalk between the embryo and the maternal endometrium, which is critical for successful pregnancy. Profilin 1 (PFN1), which is expressed both in the embryo and in the endometrial epithelium, acts as a potent regulator of actin polymerization and the cytoskeletal network. In this study, we identified the specific role of endometrial PFN1 during embryo implantation. Methods: Morphological alterations depending on the status of PFN1 expression were assessed in PFN1-depleted or control cells grown on Matrigel-coated cover glass. Day-5 mouse embryos were cocultured with Ishikawa cells. Comparisons of the rates of F-actin formation and embryo attachment were performed by measuring the stability of the attached embryo onto PFN1-depleted or control cells. Results: Depletion of PFN1 in endometrial epithelial cells induced a significant reduction in cell-cell adhesion displaying less formation of colonies and a more circular cell shape. Mouse embryos co-cultured with PFN1-depleted cells failed to form actin cytoskeletal networks, whereas more F-actin formation in the direction of surrounding PFN1-intact endometrial epithelial cells was detected. Furthermore, significantly lower embryo attachment stability was observed in PFN1-depleted cells than in control cells. This may have been due to reduced endometrial receptivity caused by impaired actin cytoskeletal networks associated with PFN1 deficiency. Conclusion These observations definitively demonstrate an important role of PFN1 in mediating cell-cell adhesion during the initial stage of embryo implantation and suggest a potential therapeutic target or novel biomarker for patients suffering from implantation failure.

BACKGROUND: The sagittal alignment of the spine and pelvis is not only closely related to the overall posture of the body but also to the evaluation and treatment of spine disease. In the last few years, the EOS imaging system, a new low-dose radiation X-ray device, became available for sagittal alignment assessment. However, there has been little research on the reliability of EOS. The purpose of this study was to evaluate the intrarater and interrater reliability of EOS for the sagittal alignment assessment of the spine and pelvis. METHODS: Records of 46 patients were selected from the EOS recording system between November 2016 and April 2017. The exclusion criteria were congenital spinal anomaly and deformity, and previous history of spine and pelvis operation. Sagittal parameters of the spine and pelvis were measured by three examiners three times each using both manual and EOS methods. Means comparison t-test, Pearson bivariate correlation analysis, and reliability analysis by intraclass correlation coefficients (ICCs) for intrarater and interrater reliability were performed using R package “irr.” RESULTS: We found excellent intrarater and interrater reliability of EOS measurements. For intrarater reliability, the ICC ranged from 0.898 to 0.982. For interrater reliability, the ICC ranged from 0.794 to 0.837. We used a paired t-test to compare the values measured by manual and EOS methods: there was no statistically significant difference between the two methods. Correlation analysis also showed a statistically significant positive correlation. CONCLUSIONS: EOS showed excellent reliability for assessment of the sagittal alignment of the spine and pelvis.
Congenital Abnormalities ; Humans ; Pelvis ; Postural Balance ; Posture ; Reproducibility of Results ; Spine ; Whole Body Imaging