The purpose of this study is to evaluate the eligibility of gene therapy in treating glaucoma by constructing a replication deficient recombinant adenovirus carrying cDNA of stromelysin and by observing its expression in the rat eye. Stromelysin cDNA was obtained by RT-PCR using total RNA extracted from the cultured human trabecular cells. The cDNA was cloned into TA vector and subsequently into p deltaA. CMV. pA. The adenovirus vector containing stromelysin cDNA was constructed by co-transfection of pJM17 and p delta A. CMV-str. pA into the 293 cells. The expression of stromelysin in the trabecular meshwork and uveoscleral outflow channel of the rat eyes was detected by in situ hybridization and immunohistochemistry. In conclusion, this study showed the possibility of gene therapy in glaucoma by showing the expression of stromelysin with recombinant adenovirus vector containing stromelysin cDNA in the rat eye.
Adenoviridae* ; Animals ; Clone Cells ; DNA, Complementary ; Gene Expression* ; Genetic Therapy ; Glaucoma ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Matrix Metalloproteinase 3* ; Rats* ; RNA ; Trabecular Meshwork

Herein, we report a case of unusually elevated serum insulin level as a result of increased anti-insulin antibody (IA)-bound insulin after continuous subcutaneous insulin infusion therapy. Detecting free insulin (unbound IAs) levels after polyethylene glycol pre-treatment could be useful to assess functional insulin levels in diabetic patients receiving insulin therapy. The E170 insulin assay can estimate total insulin (bound IAs and free insulin) levels, but it does not measure the levels of exogenous insulin analogues.
Humans ; Insulin ; Insulin Antibodies ; Polyethylene Glycols

PURPOSE: To investigate subcellular localizations of wild type myocilin and trunctated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: GFP and GFP tagged truncated myocilin, full-length myocilin, and stromelysin were expressed in TM cells using adenoviral vectors, and their secretory properties were examined by western blotting. To determine the subcellular localizations of myocilins, cellular organelles of the infected TM cells were stained with antibodies or orgenelle specific fluorescent indicators and examined under confocal microscope. RESULTS: Wild type myocilin was expressed as discrete fine vesicles in the perinuclear region of TM cells as well as in ER, but not in microtubules or mitochondria. Colocalization of wild type and truncated myocilin, indicative of in vivo interaction of the proteins, was also observed in cells co-expressing the proteins. Truncated myocilin was found to be accumulated as aggregates in ER, and inhibited the secretion of normal myocilin. CONCLUSIONS: Our result suggests that intracellular accumulation of truncated myocilin may cause a dysfunction of the cells, resulting in alterations in structural compartmentalization of trabecular ECM and obstruction of aqueous outflow.
Antibodies ; Blotting, Western ; Humans ; Matrix Metalloproteinase 3 ; Microtubules ; Mitochondria ; Organelles ; Trabecular Meshwork*

PURPOSE: To investigate the cytotoxicities of wild type myocilin and trunctated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: GFP tagged truncated myocilin and DsRED1 tagged wild type myocilin were expressed in TM cells using adenoviral vectors and observed colocalization by confocal microscope. Cytopathic effects in the cells were examined by light microscope and WST-1 cell proliferation assay. RESULTS: Colocalization of wild type and truncated myocilin was observed in cells co-expressing the proteins. Truncated myocilin was found to be toxic to cells, leading to deformed cellular morphology and diminished cell proliferation. CONCLUSIONS: The intracellular accumulation of truncated myocilin exhibited cytotoxicity in trabecular meshwork cells, and eventually resulted in diminished number and dysfunction of trabecular meshwork cells, which might be involved in the pathogenesis of glaucoma.
Cell Proliferation ; Glaucoma ; Humans ; Trabecular Meshwork*

In the present study, we have evaluated the mutations of the TIGR[Trabecular meshwork Inducible Glucocorticoid Response] gene, which has been reported to be associated with the glaucoma, in primary open angle glaucoma[POAG], normal tension glaucoma[NTG], and steroid-induced glaucoma[SIG]. We have analyzed the TIGR gene in 18 members of 3 families affected with POAG, 28 familially unrelated patients with POAG, 32 patients with NTG, 30 patients with SIG, and 45 normal subjects.DNA was extracted from the blood samples of each patient, exon 2 and exon 3 of the TIGR gene were amplified by PCR and DNA sequencing was performed.No mutation was found in familially unrelated patients with POAG.Two kinds of mutation[Ser341Pro, Gly367Arg]were found in 3 families affected with POAG.Another mutations, located in exon 3, were detected in one NTG patient and in one SIG patient, but they were silent substitution.Identification of TIGR gene mutation will provide early diagnosis of POAG before irreversible visual impairment develops in cases of positive family history of glaucoma.
Early Diagnosis ; Exons ; Glaucoma* ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Vision Disorders

PURPOSE: We investigated whether TIGR/MYOC, a candidate gene for the primary open angle glaucoma(POAG) is also involved in the pathogenesis of normal tension glaucoma(NTG) and steroid-induced glau-coma(SIG). METHODS: Genomic DNA was extracted from the peripheral blood samples collected from 72 normal volunteers and 60 POAG, 47 NTG, 61 SIG patients. The genotype distribution of dinucleotide repeat polymorphism, (G-T)n microsatellite located 249 bp upstream of transcription start site was determined by direct sequencing of the Polymerase Chain Reaction(PCR) product. RESULTS: We found 6 alleles in the (G-T)n microsatellite of TIGR/MYOC ranging from 12 to 17, which differ slightly from that of previous reports. There was no obvious difference in the genotype distribution and allele frequency between the POAG group and the control group. However, a significant association of the microsatellite marker with SIG and, to a lesser extent, with NTG was observed. A significant increase in the frequency of (G-T)13/(G-T)13 genotype and a concomitant decrease in the frequency of (G-T)13/(G-T)14 genotype was seen in both the NTG and SIG group compared to that of the control group. In the SIG group, a significant decrease in the frequency of (G-T)14 allele was also observed compared to the control group, although the decrease did not contribute to the increase in the frequency of the allele. CONCLUSIONS: These findings suggest that a polymorphism in the 5 flanking region of the TIGR/MYOC is associated with patients with NTG and SIG.
Alleles ; Dinucleotide Repeats* ; DNA ; Gene Frequency ; Genotype ; Glaucoma* ; Healthy Volunteers ; Humans ; Microsatellite Repeats ; Transcription Initiation Site

BACKGROUND/AIMS: In countries with a higher risk of gastric atrophic gastritis, noninvasive tests are helpful for a more reliable diagnosis of Helicobacter pylori infection. The aim of this study was to evaluate the characteristics of seropositive subjects according to their stool H. pylori antigen test, serum pepsinogen (PG) assay, and endoscopic findings. METHODS: Consecutive subjects who visited Konkuk University Medical Center for upper gastrointestinal endoscopy for a regular check-up were included in a prospective setting if the serum anti-H. pylori immunoglobulin G assay was positive. A H. pylori antigen stool test was measured using a stool H. pylori antigen enzyme-linked immunosorbent assay kit on the same day as a serum PG assay and endoscopy. RESULTS: Of 318 seropositive subjects, 256 (80.5%) showed positive stool test findings. Subjects with a negative stool test result showed lower serum PG I (p < 0.001) and PG II (p < 0.001) levels and higher PG I/II ratio (p < 0.001) than those with a positive stool test. Chronic atrophic gastritis was more common in the positive stool test group than the negative stool test group on endoscopic finding (p = 0.009). A higher serum PG I level (p = 0.001) and a lower serum PG I/II ratio (p = 0.001) were independent risk factors for the presence of H. pylori antigen in stool. CONCLUSIONS: A high serum PG level denotes an ongoing current H. pylori infection with positive stool H. pylori antigen test findings. Seropositive subjects with increased gastric secreting ability tend to have H. pylori in their fecal material as reflected by a positive stool H. pylori antigen test finding.
Academic Medical Centers ; Diagnosis ; Endoscopy ; Endoscopy, Gastrointestinal ; Enzyme-Linked Immunosorbent Assay ; Gastritis, Atrophic ; Helicobacter pylori ; Helicobacter* ; Immunoglobulin G ; Pepsinogen A ; Prospective Studies* ; Risk Factors

Red cell antigens, A, B, and H can be weakened or lost especially in patients with hematologic malignancies. We report a 42-year-old female patient with acute myeloid leukemia, who showed loss of A antigen on her red cells. She showed the persistence of leukemia in spite of three cycles of induction chemotherapy. Her ABO blood group showed a discrepancy: the cell type was O and the serum type was A. Adsorption/elution test could not identify the presence of A antigen on her red cells, and the test for A and B transferases was negative. ABO genotyping using PCR/restriction fragment length polymorphism and sequencing of exons 6 and 7 of the ABO gene demonstrated 467 C>T substitution in exon 7 and confirmed the genotype of A102/O01. She was transfused with leukapheresis products collected from donors with blood group A, but expired of severe sepsis. This is the first Korean case, in which red cell A antigen loss was genetically proven using sequencing, and underscores the necessity of ABO genotyping to solve the ABO discrepancy and to transfuse effectively.
Adult ; Exons ; Female ; Genotype ; Hematologic Neoplasms ; Humans ; Induction Chemotherapy ; Leukapheresis ; Leukemia ; Leukemia, Myeloid, Acute ; Sepsis ; Tissue Donors ; Transferases

BACKGROUND: Carbohydrate-deficient transferrin (CDT) levels have rarely been determined in an Asian population. We evaluated the analytical performance of a test for measuring CDT levels by using capillary electrophoresis (EP). METHODS: We determined the precision of CDT measurement by using capillary EP and nephelometry and compared the CDT values obtained using both the methods. We included healthy control subjects, abstinent patients with liver disease, and individuals consuming varying amounts of alcohol. RESULTS: The CDT measurement by using capillary EP were correlated well with those CDT measurement by using nephelometry, N Latex CDT assay, Y=0.5706X+1.581, R=0.930. The results obtained from both methods showed good qualitative agreement with each other (kappa coefficient=0.61). Genetic variants of transferrin isoforms were detected in 4.1% of the tested population. Both the CDT and gamma-glutamyl transpeptidase (GGT) levels in the abstinent patients with liver disease were significantly higher than those in healthy abstinent individuals (0.9% vs. 0.5%, 109.5 mg/dL vs. 28.5 mg/dL, respectively), but the difference in CDT values in the 2 groups was less pronounced for the CDT values. Individuals who had a mean daily alcohol intake of more than 60 g/day showed significantly higher CDT levels than those who had a mean daily alcohol intake of less than 60 g/day (1.9% vs. 0.7%, P=0.03). CONCLUSIONS: The CDT test using capillary EP showed good performance, and this method has several advantages such as automation and detection of variant forms. Thus, CDT can be a more useful marker than GGT for monitoring alcohol abstinence, especially in patients with liver disease.
Adolescent ; Adult ; Aged ; Automation ; Electrophoresis, Capillary/*methods ; Female ; Gene Frequency ; Humans ; Liver Diseases, Alcoholic/diagnosis ; Male ; Middle Aged ; Nephelometry and Turbidimetry/methods ; Protein Isoforms/analysis ; ROC Curve ; Republic of Korea ; Transferrin/*analogs & derivatives/analysis ; gamma-Glutamyltransferase/analysis

BACKGROUND: Liver biopsies have been partially replaced by noninvasive methods for assessing liver fibrosis. We explored the usefulness of four novel biomarkers, enhanced liver fibrosis (ELF), glycosylation isomer of Mac-2 binding protein (M2BPGi), galectin-3, and soluble suppression of tumorigenicity 2 (sST2), in association with liver fibrosis. METHODS: ELF, M2BPGi, galectin-3, and sST2 were assayed in 173 patients with chronic liver diseases. The results were analyzed according to fibrosis grade (F0/1, F2, and F3/4) by transient elastography (TE). RESULTS: ELF, M2BPGi, galectin-3, and sST2 values differed significantly according to TE grade; ELF and M2BPGi values were higher in F2 and F3/4 than in F0/1 (P≤0.001, all), sST2 values were higher in F3/4 than in F0/1 and F2 (P < 0.05), and galectin-3 values were higher in F3/4 than in F0/1 (P=0.0036). ELF and M2BPGi showed good TE fibrosis detection performance (area under the curves [AUC], 0.841 and 0.833 for ≥F2; and 0.837 and 0.808 for ≥F3). The sensitivity and specificity for predicting TE grade F≥2 were 84.1% and 76.7% for ELF and 63.6% and 91.5% for M2BPGi. CONCLUSIONS: This is the first study to compare the liver fibrosis assessment of four novel biomarkers: ELF, M2BPGi, galectin-3, and sST2. The biomarkers varied significantly according to TE grade, and each biomarker showed a different trend. ELF and M2BPGi seem to have comparable good performance for detecting liver fibrosis.
Biomarkers ; Biopsy ; Carrier Proteins* ; Elasticity Imaging Techniques ; Fibrosis ; Galectin 3* ; Glycosylation* ; Humans ; Liver Cirrhosis* ; Liver Diseases* ; Liver* ; Sensitivity and Specificity