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To investigate differences of tear neuromediators between subjects with and without dry eye (DE) depending on the ocular sensitivity. Thirty-one subjects with DE and 29 subjects without DE were recruited in this study. The eyes were stimulated by exposure to an irritating product applied to the periocular region. Both DE and non-DE subjects were divided into the high sensitivity and low sensitivity groups based on the degree of ocular sensitivity to ocular irritation. Baseline tear film break-up time (TBUT) and corneal staining score were examined, and tear samples were collected. The concentrations of the tear neuromediators, including nerve growth factor (NGF), serotonin, calcitonin gene-related peptide (CGRP), substance P, neuropeptide Y, and vasoactive intestinal peptide were measured using the enzyme-linked immune sorbent assay. The baseline neuromediator concentrations were compared between subjects with and without DE based on ocular sensitivity. In both DE and non-DE subjects, baseline TBUT was significantly lower in the high sensitivity group than in the low sensitivity group. In the high sensitivity group, baseline tear NGF levels were higher in subjects with DE than in those without DE. In the low sensitivity group, baseline levels of tear CGRP were lower in subjects with DE than in those without DE. Tear neuromediators associated with DE had differences in their concentrations depending on ocular sensitivity. In patients with DE, tear NGF levels increased with high ocular sensitivity to ocular irritation, whereas tear CGRP levels decreased with low ocular sensitivity.
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The eye irritation potential of drug candidates or pharmaceutical ingredients should be evaluated if there is a possibility of ocular exposure. Traditionally, the ocular irritation has been evaluated by the rabbit Draize test. However, rabbit eyes are more sensitive to irritants than human eyes, therefore substantial level of false positives are unavoidable. To resolve this species difference, several three-dimensional human corneal epithelial (HCE) models have been developed as alternative eye irritation test methods. Recently, we introduced a new HCE model, MCTT HCETM which is reconstructed with non-transformed human corneal cells from limbal tissues. Here, we examined if MCTT HCETM can be employed to evaluate eye irritation potential of solid substances. Through optimization of washing method and exposure time, treatment time was established as 10 min and washing procedure was set up as 4 times of washing with 10 mL of PBS and shaking in 30 mL of PBS in a beaker. With the established eye irritation test protocol, 11 solid substances (5 non-irritants, 6 irritants) were evaluated which demonstrated an excellent predictive capacity (100% accuracy, 100% specificity and 100% sensitivity). We also compared the performance of our test method with rabbit Draize test results and in vitro cytotoxicity test with 2D human corneal epithelial cell lines.
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Humanos , Córnea , Células Epiteliais , Irritantes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Carcinoma-associated fibroblasts (CAFs) contribute to carcinogenesis and cancer progression, although their origin and role remain unclear. We recently identified and investigated the in situ identity and implications of gastric submucosa-resident mesenchymal stem cells (GS-MSCs) in the progression of gastric carcinogenesis. METHODS: We isolated GS-MSCs from gastric submucosa using hydrogel-supported organ culture and defined their identity. Isolated cells were assessed in vitro by immunophenotype and mesengenic multipotency. Reciprocal interactions between GS-MSCs and gastric cancer cells were evaluated. To determine the role of GS-MSCs, xenografts were constructed of gastric cancer cells admixed with or without GS-MSCs. RESULTS: Isolated cells fulfilled MSCs requirements in regard to plastic adherence, stromal cell immunophenotype, and multipotency. We demonstrated a paracrine loop that gastric cancer cells enhanced the migration, proliferation, and differentiation of GS-MSCs; additionally, GS-MSCs promoted the proliferation of gastric cancer cell in vitro. Xenograft experiments showed that GS-MSCs significantly promoted cancer growth and angiogenesis. GS-MSCs that integrated into gastric cancer became not only CAFs but also rarely endothelial cells which contributed to the formation of cellular and vascular cancer stroma. CONCLUSIONS: Endogenous GS-MSCs play an important role in gastric cancer progression.
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Carcinogênese , Células Endoteliais , Fibroblastos , Xenoenxertos , Células-Tronco Mesenquimais , Técnicas de Cultura de Órgãos , Plásticos , Neoplasias Gástricas , Células Estromais , Transplante HeterólogoRESUMO
In the treatment of intertrochanteric fractures, most of intramedullary nailings are performed on a fracture table in supine position. In supine position, however, soft tissue mass of the patients and drapes make it difficult to access to the piriformis fossa and to straighten the trajectory of reamer and nail insertion. To resolve these problems, we have treated twenty intertrochanteric fractures in lateral position on the general operation table with IM nail. Adjustment of the position of lag screw in femoral head was done with the technique that overlaps the shadows of the femoral head, nail and targeting guide in the lateral view. Because the entire injured limb can be moved readily, it was easy to reduce fracture and to convert to open procedure. In cases likely that the fracture table is unavailable in which patients are obese, have short stature or are amputated, and that open procedure is strongly likelihood, lateral position will be helpful technique in the treatment of intertrochanteric fractures with IM nail.
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Humanos , Extremidades , Fêmur , Fixação Intramedular de Fraturas , Cabeça , Fraturas do Quadril , Unhas , Mesas Cirúrgicas , Decúbito DorsalRESUMO
PURPOSE: The lack of reliable in vitro infection systems or convenient animal models has hindered the progress of hepatitis B virus (HBV) research and the development of new treatment options. We established an in vitro model of hepatitis B, using recombinant HBV encoding baculovirus, which provided HBV replication and antigens expression in HepG2 cells. The objectives of this study were to characterize the magnitude of HBV expression and the level of replication obtainable in HepG2 cells, to establish the optimum infection and culture conditions of HBV expression and replication. METHODS: Replication of a competent HBV genome encoding the baculovirus, RC-HBV-Bac, was generated for delivering the HBV genome to HepG2 cells. HBV replication and antigens expression were determined in relation to the infection and culture conditions. RESULTS: In RC-HBV-Bac infected HepG2 cells, HBsAg, HBeAg and HBcAg were expressed in the cytoplasm and nuclei, and secreted into the medium. HBV replication was evidenced by the presence of a replication complex and covalently closed circular (ccc) DNA in the cytoplasmic fraction of infected cells. The level of HBV expression was directly proportional to the multiplication of RC-HBV-Bac infection. Polyethylene glycol was able to enhance the infection efficiency of the baculovirus to HepG2 cells. High levels of HBV replication were achieved under culture conditions supplemented with dimethyl sulfoxide and a low serum concentration. CONCLUSION: This in vitro model of hepatitis B, generated by baculovirus gene delivery, represents a simple and flexible system for the study of HBV replication and drug testing.
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Baculoviridae , Citoplasma , Dimetil Sulfóxido , DNA , Técnicas de Transferência de Genes , Genoma , Células Hep G2 , Antígenos do Núcleo do Vírus da Hepatite B , Antígenos E da Hepatite B , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B , Hepatite B , Hepatite , Modelos Animais , PolietilenoglicóisRESUMO
The Kinesin superfamily proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF1Balpha is a monomeric motor that conveys mitochondria and plays an important role in cellular function. Here, we used the yeast two-hybrid system to identify the proteins that interacts with KIF1Balpha and found a specific interaction with the mammalian LIN-7 (MALS)-3/vertebrate homology of LIN-7 (Veri) and synaptic scaffolding molecule (S-SCAM). MALS-3 protein bound to the tail region of KIF1Balpha but not to other kinesin family members in the yeast two-hybrid assay. The "T-X-V" motif at the C-terminal end of KIF1Balpha is essential for interaction with MALS-3. In addition, this protein showed specific interactions in the Glutathione S-transferase (GST) pull-down assay. An antibody to MALS-3 specifically coimmunoprecipitated KIF1Balpha associated with MALS-3 from mouse brain extracts. These results suggest that MALS-3, as KIF1Balpha receptor, is involved in the KIF1Balpha-mediated transport.
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Animais , Humanos , Camundongos , Encéfalo , Glutationa Transferase , Cinesinas , Microtúbulos , Mitocôndrias , Organelas , Domínios PDZ , Técnicas do Sistema de Duplo-HíbridoRESUMO
PURPOSE: The pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), is a central mediator of the immune response involved in a wide range of immuno-inflammatory and infectious diseases. There is increasing evidence that TNF-alpha may promote the development and spread of the cancer. Polymorphisms in the TNF-alpha promoter have been related to TNF-alpha production. Therefore, we investigated the potential association of TNF-alpha genotypes with gastric cancer in the Korean population. METHODS: The study included 66 patients with gastric adenoma, 75 patients with gastric carcinoma, and 551 healthy controls. The -308 and -238 polymorphisms in the TNF-alpha promoter were analyzed by PCR- restriction fragment length polymorphism (RFLP). Distributions of TNF-alpha promoter polymorphisms were compared between groups by chi2 test. P values smaller than 0.05 were considered to be significant. RESULTS: The proportion of individuals carrying the TNF-alpha -308A allele was higher in the carcinoma group compared to controls and adenomas, but the differences were not significant (P=0.124). However, the TNF-alpha -308A allele was significantly associated with advanced gastric carcinoma (P=0.026), serosa invasion (P=0.004), neural invasion (P= 0.021), and lymph node metastasis (P=0.005). On the other hand, the TNF-alpha -238G/A polymorphism was not associated with the development of gastric adenoma and carcinoma and the severity of gastric carcinoma. CONCLUSION: These results suggest that the TNF-alpha -308A allele is associated with the severity of gastric carcinoma in terms of invasion and metastasis in the Korean population. Therefore, TNF-alpha promoter polymorphism could be used as a predictive marker of the severity of gastric carcinoma.
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Humanos , Adenoma , Alelos , Doenças Transmissíveis , Genótipo , Mãos , Linfonodos , Necrose , Metástase Neoplásica , Polimorfismo de Fragmento de Restrição , Membrana Serosa , Neoplasias Gástricas , Fator de Necrose Tumoral alfaRESUMO
The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF1A is a monomeric motor that conveys synaptic vesicle precursors and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein (s) that interacts with the tail region of KIF1A and found a specific interaction with synaptotagmin XI. The amino acid residues between 830 and 1300 of KIF1A are required for the interaction with synaptotagmin XI. KIF1A also bound to the tail region of synaptotagmin IV but not to other synaptotagmin in the yeast two-hybrid assay. KIF1A interacted with GST-synaptotagim XI fusion proteins, but not with GST alone. An antibody to synaptotagmin XI specifically co-mmunoprecipitated KIF1A associated with synaptotagimin from mouse brain extracts. These results suggest that KIF1A motor protein transports of synaptotagmin XI-containing synaptic vesicle precursors along microtubule.
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Animais , Camundongos , Encéfalo , Cinesinas , Microtúbulos , Neurônios , Organelas , Transporte Proteico , Vesículas Sinápticas , Sinaptotagminas , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND AND OBJECTIVES: Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs in the world. NSAIDs are known to be potent inhibitors of the cyclooxygenase (COX) enzymes, a family of enzymes that catalyze the conversion of arachidonic acid to prostagladins. Expression of the gene encoding COX-2 might be regulated by hypoxia. Hypoxiainducible factors (HIFs) are activated by hypoxia. HIFs function in the hypoxic environment to orchestrate adaptational adjustments of vascular homeostasis through the activation of several dozens of target genes. The purpose of this study was to investigate whether a selective COX-2 inhibitor inhibits HIF-1alpha in human nasal polyps. SUBJECTS AND METHOD: Seven patients with nasal polyps with chronic sinusitis were selected. After the first biopsy, all patients were treated with selective cyclooxygenase inhibitor (Celebrax(R), 100mg, twice daily) for 7 days. At the end of the treatment period, a second set of biopsies was taken. HIF-1alpha messenger RNA (mRNA) production was measured by reverse transcriptase polymerase chain reaction and the protein expression of HIF-1alpha was determined by immunohistochemical staining. RESULTS: The expression of HIF-1alpha mRNA and protein were detected in nasal polyps. There was no significant difference in the mean level of HIF-1alpha mRNA between selective COX-2 inhibitoruntreated and treated nasal polyps (p>0.05). Immunohistochemistry shows diffuse and increased expression of HIF-1alpha in the nuclei of pseudostratified columnar epithelial cells. Endothelial cells and inflammatory cells including lymphocytes and histiocytes were expressed with HIF-1alpha in the stroma. Subcellular localization of HIF-1alpha were found mostly in the nucleus, but were occasionally observed in the cytoplasm of histiocytes. The expression of HIF-1alpha protein was not significantly different between selective COX-2 inhibitor-treated and selective COX-2 inhibitor-untreated nasal polyps (p>0.05). CONCLUSION: Selective COX-2 inhibitor did not inhibit HIF-1alpha expression in nasal polyps. Further studies are needed to find out the effect of selective COX-2 inhibitor on nasal polyps.
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Humanos , Hipóxia , Anti-Inflamatórios não Esteroides , Ácido Araquidônico , Biópsia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase , Citoplasma , Células Endoteliais , Células Epiteliais , Histiócitos , Homeostase , Imuno-Histoquímica , Linfócitos , Pólipos Nasais , Prostaglandina-Endoperóxido Sintases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro , SinusiteRESUMO
The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF5 is a heterotetrameric motor that conveys vesicles and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein (s) that interacts with the tail region of KIF5 and found a specific interaction with betaIII spectrin. The amino acid residues between 1394 and 1774 of betaIII spectrin were required for the interaction with KIF5C. betaIII spectrin also bound to the tail region of neuronal KIF5A and ubiquitous KIF5B but not to other kinesin family members in the yeast two-hybrid assay. In addition, these proteins showed specific interactions, confirmed by GST pull-down assay and co-immunoprecipitation. betaIII spectrin interacted with GST-KIF5 fusion proteins, but not with GST alone. An antibody to betaIII spectrin specifically co-immunoprecipitated KIF5s associated with betaIII spectrin from mouse brain extracts. These results suggest that KIF5 motor proteins transport vesicles or organelles that are coated with betaIII spectrin.
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Animais , Humanos , Camundongos , Encéfalo , Imunoprecipitação , Cinesinas , Microtúbulos , Neurônios , Organelas , Espectrina , Vesículas Transportadoras , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND AND OBJECTIVES: The function of hypoxia-inducible factors (HIFs) in the hypoxic environment is to orchestrate adaptational adjustments of vascular homeostasis through the activation of several dozens of target genes including vascular en-dothelial growth factors (VEGF). It has been suggested that VEGF is involved in the pathogenesis of nasal polyp. The purpose of this study is to determine and correlate concentrations of HIF-1alpha and VEGF in nasal polyps. MATERIALS AND METHOD: Twenty-five nasal polyps were collected at the time of endoscopic sinus surgery. The production of HIF-1alpha and VEGF messenger RNA (mRNA) was measured by reverse transcriptase polymerase chain reaction (RT-PCR) and HIF-1alpha and VEGF proteins were determined by immunohistochemical staining. RESULTS: The expressions of HIF-1, VEGF mRNA and proteins were detected in nasal polyps. RT-PCR demonstrated that the level of mRNA expression of HIF-1alpha and VEGF were 1.12+/-0.33 and 1.11+/-0.42, respectively. A positive correlation was observed between HIF-1alpha and VEGF mRNA (correlation coefficient [r]=0.49, p<0.05). The immunohistochemical studies revealed that HIF-1alpha was predominantly expressed in surface epithelial cells, submucosal glandular cells, endothelial cells and inflammatory cells in the stroma and VEGF was more strongly and diffusely expressed in subglandular epithelial cells, vascular endothelial cells, and inflammatory cells than in surface epithelial cells. The expressions of HIF-1alpha and VEGF proteins were 3.24+/-1.80 and 3.52+/-1.89, respectively. A positive correlation was observed between HIF-1alpha and VEGF proteins (r=0.76, p<0.05). CONCLUSION: We suggest that HIF-1alpha has a role in inducing VEGF in nasal polyps, and hypoxia is an important factor in the growth of nasal polyps.
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Hipóxia , Células Endoteliais , Células Epiteliais , Homeostase , Peptídeos e Proteínas de Sinalização Intercelular , Pólipos Nasais , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro , Fator A de Crescimento do Endotélio VascularRESUMO
BACKGROUND/AIMS: There have been much debates on the effects of HBV mutants on the clinical course of HBV-associated chronic liver diseases. The purpose of this study was to define the relationship among HBV mutants, severity of hepatitis and expression patterns of HBcAg METHODS: HBV DNA was extracted from the liver tissue of 31 patients who had been HBsAg positive for more than 6 months. The amplification of X was performed as well as core promoter/precore region of HBV DNA by polymerase chain reaction and then direct sequencing of them. Pathologic severity was classified utilizing Scheuer's scoring system and immunohistochemical staing for HBcAg in hepatocytes was performed. The expression patterns of HBcAg were divided into four types according to expression location of HBcAg: Type I as a nuclear predominant expression of HBcAg; Type II as mixed patterns, combined expression of cytoplasmic and nuclear localization of HBcAg; Type III as a diffuse cytoplasmic expression of HBcAg; and Type II as an inclusive body-like expression in cytoplasm. RESULTS: In investigating the relationship between HBV mutants and clinical findings, ALT, HBV DNA and hepatitis activity index (HAI) in hepatitis with wild HBV were normal to high but those in hepatitis with core promoter or precore mutants were high . There were no statistically significant differences (p=0.062). In terms of the relationship between HBV mutants and the expression pattern of HBcAg, type I, II, IV were noticed in hepatitis with wild HBV but in almost all mutants cases type III, II were noticed (p<0.01). The score of HAI increased as the number of the expression pattern of HBcAg increased from type I to type III or IV(p<0.05). No relationships among the mutation in X region, the mutations in other regions and clinicopathological severity could be found. CONCLUSION: The mutation in X, core promoter and precore region had little association with the severity of hepatitis. And the relationship did not exist between precore mutants and X mutants. The expression pattern of HBcAg could be a useful indicator in determining what stage of chronic hepatitis B is in and whether mutant strains exist.
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Humanos , Citoplasma , DNA , Hepatite , Antígenos do Núcleo do Vírus da Hepatite B , Antígenos de Superfície da Hepatite B , Hepatite B Crônica , Hepatócitos , Fígado , Hepatopatias , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Recently, much attention has been focused on the role of protease inhibitors such as acid stable trypsin inhibitor (ASTI) in the invasive growth of malignant tumors. However, there are no report on expression of ASTI from premalignant and/or malignant skin tumors. OBJECTIVES: In the present study the expression of ASTI was investigated in the different type of premalignant and/or, malignant skin tumors in attempt to clarify the relation between the expression of the ASTI and malignancy. METHODS: For the detection of ASTI in the tumor tissue, the immunoperoxidase techniques that used mouse antibody raised against highly purified ASTI. The degree of ASTI immunoreactivity was semiquantitatively assessed for staining intensity as the percentage of ASTI-positive cells. RESULTS: ASTI immunoreactivity was detected in most of the premalignant and malignant skin tissues. Especially, ASTI expression was present widespread in squamous cell carcinoma(SCC) with strong cytoplasmic membrane, where as in normal epidermis they were primarily present in the horny layer. The strong staining was the SCC, keratoacanthoma, Bowen's disease, actinic keratosis, basal cell epithelioma, Paget's disease in decreasing order. The significant difference in the staining intensity was observed between SCC and other groups. CONCLUSION: Results of immunohistochemical studies suggest that the tumor cells themselves could produce ASTI. Considering the suggestion that ASTI is a self protector, inhibitor to proteolytic protease as well as growth-stimulating factor. The present findings may indicate that ASTI expressed in malignant cells may play a role possibly closely associated with tumor development.