Systemic Review for Efficacy of Human Papillomavirus Vaccines

Two human papillomavirus (HPV) vaccines (Gardasil(R) and Cevarix(TM)) were launched between 2006~2007. Clinical trials have been performed in several countries. However, it takes few decades to measure HPV vaccine efficacy for the protection of cervical cancer. Therefore, several surrogate markers such as seroconversion rate, presence of HPV DNA, and cytological/ histological abnormalities have been evaluated. Until now, long-term follow-up data for 5 years (Gardasil) and for 8.4 years (Cevarix) were available from international trials. However, only seroconversion rate at 4 weeks after vaccination and safety were evaluated in Korea. It is necessary to establish a reference laboratory and long-term follow-up monitoring system for the proper evaluation of HPV vaccines in Korea.

Seroepidemiologic Survey of Varicella-Zoster Virus in Korean Adults Using Glycoprotein Enzyme Immuno Assay and Fluorescent Antibody to Membrane Antigen Test

BACKGROUND: Herpes zoster (HZ) occurs mainly in the elderly and Korea is rapidly becoming an aging society. Therefore, it is important to know the immune status against varicella-zoster virus (VZV) in Korean adults to prevent the disease. OBJECTIVE: The aim of this study was to survey the immune status of Korean adults over 40 years of age against VZV. METHODS: Antibody titer was measured using a VaccZyme(TM) VZV glycoprotein enzyme immunoassay (gpEIA) (Binding Site, UK). Fluorescent antibody to membrane antigen (FAMA) test was performed to measure the seropositive rate. RESULTS: HZ incidence in the 214 adults enrolled in this study was 10.3%. The gpEIA geometric mean titer (GMT) was 490 mIU/ml and 90.2% of the subjects had a protective level of gpEIA antibody titer against varicella. The average gpEIA GMT of adults who previously had HZ was 1,122 mIU/ml, which was higher than the average gpEIA GMT of 457 mIU/ml in adults who had not had HZ. The FAMA positive rate was 98.6%. CONCLUSION: Most (90.2%) Korean adults > or =40-years-of-age have a protective level of gpEIA antibody against varicella and 98.6% were FAMA seropositive. The GMT of gpEIA antibody was significantly increased with age, and was higher in adults with a history of HZ.

The Effects of Hantaan Virus on the Expression of Platelet Activating Factor Receptor and on the Activity of Platelet Activating Factor Acetylhydrolase / 영남의대학술지

BACKGROUND: The central physiological derangement of hemorrhagic fever with renal syndrome (HFRS) caused by hantaan virus (HTNV) is a vascular dysfunction, manifested by hemorrhage, impaired vascular tone and increased vascular permeability. Platelet activating factor (PAF), whose actions are mediated through a specific receptor, is a potent bioactive lipid. PAF has diverse biological functions in the vascular system, such as increasing vascular permeability, adhesion of leukocytes to the endothelium and reduction of cardiac output, which result in hypotension and shock. The goal of the present study was to investigate whether PAF is involved in the pathogenesis of HFRS. For this purpose, we evaluated the effect of HTNV on the expression of PAF receptor (PAF-R) and on the activity of PAF-acetylhydrolase (PAF-AH) instead of PAF because PAF is rapidly degraded by PAF-AH in vivo. MATERIALS AND METHODS: To evaluate the expression of PAF-R, we performed reverse-transcription PCR, western blot and FACS analyses using HTNV-infected human umbilical vein endothelial cells (HUVECs) and non-infected (control) HUVECs. In addition, we measured the activity of plasma PAF-AH in HFRS patients and normal healthy persons. RESULTS: The mRNA and protein expression of PAF-R was increased in HTNV-infected HUVECs compared with control HUVECs at 2 and 3 days post-infection (d.p.i.). FACS analysis showed that HTNV induced the surface expression of PAF-R in HUVECs from 2 d.p.i. The activity of plasma PAF-AH was 2.5-fold lower in HFRS patients than in normal healthy persons. CONCLUSION: Increased PAF-R expression by HTNV might increase the responsiveness to PAF in endothelial cells. Reduced PAF-AH activity in the blood of HFRS patients might delay PAF degradation. These results suggest that changes in PAF-R and PAF-AH by HTNV might influence to PAF activity and might be involved in the vascular dysfunction of HFRS.

Interferon-Stimulated Genes Response in Endothelial Cells Following Hantaan Virus Infection

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta , IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta??(3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.

Hantaan Virus Reduces the von Willebrand Factor in Human Umbilical Vein Endothelial Cells

To understand the molecular mechanism of hemorrhagic tendency represented in hemorrhagic fever with renal syndrome (HFRS), the effect of Hantaan virus (HTNV) on the von Willebrand factor (vWF) was observed in human umbilical vein endothelial cells (HuVECs). An immunofluorescence assay (IFA) showed a significant reduction of the vWF in the cytoplasm of HTNV-infected HuVECs. The amount of vWF protein in HTNV-infected HuVECs was reduced to 86, 49, 67, and 42% of those in control HuVECs at 1(st), 3(rd), 5(th), and 7(th) days of post infection (d.p.i.), respectively. However, there were no significant differences in the vWF mRNA expression in both groups at all time courses by reverse transcriptase polymerase chain reaction (RT-PCR). The amounts of secreted vWF in the culture supernatants of the HTNV-infected HuVECs were 79, 87, 83, and 82% of those in control HuVECs at 1(st), 3(rd), 5(th), and 7(th) d.p.i., respectively. These results indicated that the reduction of vWF by HTNV was regulated at post-transcriptional level and this might delay the coagulation process on the site of HTNV infection, thus leading to hemorrhage in HFRS.

Colocalization of Interferon Regulatory Factor 7 (IRF7) with Latent Membrane Protein 1 (LMP1) of Epstein-Barr Virus

Interferon regulatory factor 7 (IRF7) is one of the transcriptional factors for the activation of type I Interferon (IFN) genes. It is known that IRF7 and the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) are highly expressed in EBV type III latency cells, and LMP1 induces mRNA expression of IRF7. In this study, the expression pattern of endogenous IRF7 was observed in several B cell lines with or without EBV infection by immunofluorescence staining. IRF7 was localized in the cytoplasm of EBV-negative B cells and EBV type I latency B cell lines. However, IRF7 was located both in the cytoplasm and nucleus of EBV type III latency cell lines. In the Jijoye cell (type III latency cell), IRF7 was colocalized with LMP1 in the cytoplasm in a capping configuration, and their interaction was confirmed by co-immunoprecipitation of LMP1 and IRF7. This colocalization was confirmed by co-transfection of IRF7 and LMP1 plasmids in EBV-negative B cells. These results suggest that the IRF7 and LMP1 interact with each other, and this may relate to the mechanism whereby LMP1 exerts functional effects in B-lymphocytes.

Two Cases of Brugian Filariasis Diagnosed Incidentally on Peripheral Blood Smears / 대한진단검사의학회지

The Brugian filariasis in Korea was once endemic in several areas including Jeju-do and small remote islands located in the southwestern part of the Korean peninsula. But recently it has almost been controlled. The first patient was a 42-year-old man, who visited the hospital with the chief complaints of fatigue, jaundice and edema on the right low extremity. Examination of a peripheral blood smear revealed non-megaloblastic macrocytic anemia, thrombocytopenia and eosinophilia, and a parasite, which was identified as microfilaria of Brugia malayi on the glass slide of blood obtained from the patient at midnight. The second patient was a 23-year-old man, who visited the hospital with the chief complaints of cough, sputum and fever. A parasite resembling that of the first patient was found in a peripheral blood smear accompaning neutrophilia. No more parasites, however, were found in the peripheral blood obtained from the patient at midnight. These two clinical cases of Brugian filariasis indicate that the clinical laboratory in Korea should be able to detect microfilariae for the diagnosis of filariasis.

Renoprotective Effect of Angiotensin Converting Enzyme Inhibitor and Angiotensin Receptor Blocker in Chronic Uremic Rats: Comparison of Early Treatment with Delayed Treatment / 대한신장학회잡지

BACKGROUND: We compared the renoprotective effect of angiotensin converting enzyme inhibitor (ACEi) and angiotensin receptor blocker (ARB) between early treatment and delayed treatment in chronic uremic rats. METHODS: Male Sprague-Dawley rats underwent 5/6 nephrectomy or sham operation (sham) and received no treatment (UC), enalapril (E) 100 mg/L, candesartan cilexetil (C) 10 mg/L or combination of E and C in drinking water. Some rats began to be treated from the next day of 5/6 nephrectomy (early group) and the others began from 12 weeks of 5/6 nephrectomy (delayed group). Blood pressure, renal function, proteinuria, histological changes and renal cortical TGFbeta1 expression were observed for 24 weeks. RESULTS: Blood pressure was significantly lower in E, C or E+C of both early and delayed group than in UC. Particularly blood pressure in E+C of early group was lower than in E or C. In E, C or E+C of both early and delayed group, BUN was lower and creatinine clearance was higher compared with UC. Proteinuria was also significantly lower in E, C or E+C of both early and delayed group compared with UC. In addition, remnant kidney weight in E, C or E+C of early and delayed group was significantly lower compared with UC, representing less renal hypertrophy. On the histological examination, glomerulosclerosis score, interstitial fibrosis score and the number of infiltrated glomerular macrophage were significantly lower in E, C or E+C of both early and delayed group compared with UC. Furthermore, renal cortical TGF-beta1 expression was also lower in E, C or E+C of both early and delayed group compared with UC. CONCLUSION: ACEi and ARB have renoprotective effect biochemically and histologically even though the treatment was delayed until moderate chronic renal failure had occurred.

The Synergistic Effect of LMP1 and IRF7 on the Expression of TAP1 in DG 75 Cell Line

In this study, the author explored the role of interferon regulatory factor 7 (IRF7) and latent membrane protein 1 (LMP1) on the regulation of antigen presenting molecules in B-lymphoblastoid cell lines. First, the author observed the endogenous expression of IRF7 and LMP1 in paired EBV-positive B-lymphoblastoid cell lines, Sav I and Sav III, which represent EBV type I and type III latency, respectively. The Sav I cell, which does not express LMP1, showed very low levels of endogenous IRF7 in the cytoplasm. However, Sav III cells, which express large amounts of LMP1, contained high levels of endogenous IRF7 in both the cytoplasm and the nucleus. The expression of surface MHC class I antigen was 7.8-fold higher in Sav III compared with Sav I cells when measured by fluorescence activated cell sorter (FACS) analysis. To understand whether IRF7 is involved in regulation of MHC I and TAP1, LMP1 or IRF7 were expressed by cotransfection in DG75 cells. Levels of TAP1 protein were up-regulated by LMP1 and IRF7 alone, and by LMP1 co expression with IRF7, the expression level was highest after co-transfection of LMP1 with IRF7. TAP1 promoter activity was also up-regulated to 2.4, 2.0, 3.2-fold by LMP1, by IRF7, and by LMP1 plus IRF7, respectively. Surface expression of MHC class I antigen was up-regulated by LMP1 alone and LMP1 plus IRF7, but not by IRF7 alone. These results suggest that IRF7 induces the expression of TAP1, but not MHC class I antigen and that LMP1 and IRF7 have additive effects on the expression of TAP1 protein.

The Synergistic Effect of LMP1 and IRF7 on the Expression of TAP1 in DG 75 Cell Line

In this study, the author explored the role of interferon regulatory factor 7 (IRF7) and latent membrane protein 1 (LMP1) on the regulation of antigen presenting molecules in B-lymphoblastoid cell lines. First, the author observed the endogenous expression of IRF7 and LMP1 in paired EBV-positive B-lymphoblastoid cell lines, Sav I and Sav III, which represent EBV type I and type III latency, respectively. The Sav I cell, which does not express LMP1, showed very low levels of endogenous IRF7 in the cytoplasm. However, Sav III cells, which express large amounts of LMP1, contained high levels of endogenous IRF7 in both the cytoplasm and the nucleus. The expression of surface MHC class I antigen was 7.8-fold higher in Sav III compared with Sav I cells when measured by fluorescence activated cell sorter (FACS) analysis. To understand whether IRF7 is involved in regulation of MHC I and TAP1, LMP1 or IRF7 were expressed by cotransfection in DG75 cells. Levels of TAP1 protein were up-regulated by LMP1 and IRF7 alone, and by LMP1 co expression with IRF7, the expression level was highest after co-transfection of LMP1 with IRF7. TAP1 promoter activity was also up-regulated to 2.4, 2.0, 3.2-fold by LMP1, by IRF7, and by LMP1 plus IRF7, respectively. Surface expression of MHC class I antigen was up-regulated by LMP1 alone and LMP1 plus IRF7, but not by IRF7 alone. These results suggest that IRF7 induces the expression of TAP1, but not MHC class I antigen and that LMP1 and IRF7 have additive effects on the expression of TAP1 protein.

Pirfenidone Suppressed the Development of Glomerulosclerosis in the FGS/Kist mouse

Pirfenidone(PFD) is a newly developed anti-fibrotic agent. We evaluated the effect of PFD for the prevention of renal fibrosis using a spontaneous progressive glomerulosclerosis animal model, FGS/Kist mice. Male and female FGS/Kist mice were fed a diet containing 0.5% PFD or the same control diet (CD) without PFD, for 1, 2, or 3-month periods. Body weight was monitored for the general effect of PFD on the mice. Proteinuria and glomerular filtration rate (GFR) were evaluated for renal function. The sclerosis index was examined for the morphological changes. There were no significant changes in body weight between the PFD and control groups in both sexes. Proteinuria levels were low in all the PFD groups compared to the corresponding CD groups. The sclerosis scores were also reduced in both sexes of the 3-month PFD groups (p<0.05), and glomerular filtration rates were increased in both sexes of the 3-month PFD groups compared to the CD groups. The treatment of PFD for 1 or 2-month periods did not have statistic significances but the treatment for 3 months had statistic significances in sclerosis and GFR compared to CD groups. These results suggested that long-term administration of PFD sup-pressed the progression of glomerulosclerosis and improved renal function of the renal function of the FGS/Kist mice.

Effect of Leptin on the Expression of Chemokine Genes in THP-1 Cells / 영남의대학술지

BACKGROUND: Leptin is a 16-KDa non-glycosylated peptide hormone synthesized almost exclusively by adipocytes. The well-known function of leptin is regulation of food intake and energy expenditure. Leptin also plays a regulatory role in immune and inflammatory process including cytokine production. The purpose of this study was to investigate the effect of leptin on the expression of several chemokine genes(RANTES, IL-8, MCP-1, IP-10, Mig, MIP-1alpha, MIP-1beta, and GRO-alpha) in THP-1 cells. MATERIALS AND METHODS: Total RNA of THP-1 cells were prepared by Trizol method, and then stimulated with the leptin(250 ng/microliter) or LPS(100 ng/microliter). We examined the expression patterns of various chemokine mRNAs in THP-1 cell lines by RT-PCR and Northern blot. RESULTS: Leptin did not induce the expression of chemokine mRNAs in THP-1 cells. The expression patterns of RANTES, IL-8, MCP-1, IP-10, and Mig mRNAs in THP-1 cells stimulated with leptin and LPS simultaneously was almost same to the patterns of LPS alone-induced chemokine mRNAs. RANTES mRNA expression was independent on the concentrations of leptin. Although leptin did not have strong effect on the expression of RANTES, IL-8, MCP-1, IP-10, Mig, MIP-1alpha, MIP-1beta, and GRO-alpha mRNAs in THP-1 cells, leptin could induce the expression of long isoform of leptin receptor(OB-RL) mRNA, and its expression was elevated in simultaneous stimulation of leptin and LPS. CONCLUSION: These data suggest that leptin is able to induce OB-RL in THP-1 cells, however, leptin has little effect on the expression of pro-inflammatory chemokine genes.

Effect of Leptin on the Expression of Chemokine Genes in THP-1 Cells / 영남의대학술지

BACKGROUND: Leptin is a 16-KDa non-glycosylated peptide hormone synthesized almost exclusively by adipocytes. The well-known function of leptin is regulation of food intake and energy expenditure. Leptin also plays a regulatory role in immune and inflammatory process including cytokine production. The purpose of this study was to investigate the effect of leptin on the expression of several chemokine genes(RANTES, IL-8, MCP-1, IP-10, Mig, MIP-1alpha, MIP-1beta, and GRO-alpha) in THP-1 cells. MATERIALS AND METHODS: Total RNA of THP-1 cells were prepared by Trizol method, and then stimulated with the leptin(250 ng/microliter) or LPS(100 ng/microliter). We examined the expression patterns of various chemokine mRNAs in THP-1 cell lines by RT-PCR and Northern blot. RESULTS: Leptin did not induce the expression of chemokine mRNAs in THP-1 cells. The expression patterns of RANTES, IL-8, MCP-1, IP-10, and Mig mRNAs in THP-1 cells stimulated with leptin and LPS simultaneously was almost same to the patterns of LPS alone-induced chemokine mRNAs. RANTES mRNA expression was independent on the concentrations of leptin. Although leptin did not have strong effect on the expression of RANTES, IL-8, MCP-1, IP-10, Mig, MIP-1alpha, MIP-1beta, and GRO-alpha mRNAs in THP-1 cells, leptin could induce the expression of long isoform of leptin receptor(OB-RL) mRNA, and its expression was elevated in simultaneous stimulation of leptin and LPS. CONCLUSION: These data suggest that leptin is able to induce OB-RL in THP-1 cells, however, leptin has little effect on the expression of pro-inflammatory chemokine genes.

The Effects of Hantaan Virus on Fibronectin and Matrix Metalloproteinase-3

The clinical symptoms and signs of hemorrhagic fever with renal syndrome (HFRS) are well known, but the pathophysiology caused by Hantaan virus is not fully understood yet. This study was designed to evaluate the effect of Hantaan virus on the regulation of extracellular matrix proteins. The changes of fibronectin, fibronectin receptor (alpha5beta1 integrin), the matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in Vero E6 cell line were observed. Hantaan virus induced the mRNA expression of fibronectin at day 7 after the infection but the surface expression of fibronectin receptor (alpha5beta1 integrin) was reduced. The mRNA expression of MMP-3 was increased at day 5 by 8-fold in the virus infected cells compared with the control cells, and continued to increase until day 9. However, the mRNA expression of TIMP-1 did not show any significant changes. The concentration of plasma fibronectin in the HFRS patients was measured by ELISA. The fibronectin levels were variable and could be divided into three groups: low (14.8%), normal (62.9%) and high (22.2%) groups in comparison with normal range value. These results suggest that Hantaan virus should affect not only the infected cell itself but also the extracelluar matrix, which might play an important role in the pathogenesis.

Development of a Mycobacterial Gene Knock-out System using Sequence-specific Recombinase FLP/FRT and its Application to the Construction of a Rhamnose Biosynthetic Gene rmlD Deletion Mutant

Development of a new and effecient tuberculosis vaccine is very important since the efficacy of the only available vaccine against tuberculosis, BCG, is variable among races and different ages. Attempts to develop attenuated vaccines by disrupting virulence gene(s) specifically in Mycobacterium tuberculosis are now actively being tried after the release of whole genome sequence of M. tuberculosis in 1998. However, disruption of specific genes in M. tuberculosis is still very difficult due to the lack of effective gene knock-out system(s) in mycobacteria. In this study, we developed a novel method to delete specific genes in both Escherichia coli and mycobacteria. This knock-out system is operated by a sequence-specific recombinase FLP and its recognition sequence FRT (FLP/FRT system). Two shuttle vectors (an FLP expressing vector and a gene targeting vector) between Escherichia coli and Mycobacteria were developed. The gene targeting vector contains a kanamycin resistance gene (KmR) flanked by two neighboring genes and two FRTs (FLPrecognition targets). We applied this system to knock-out the rhamnose biosynthetic gene rmlD of Escherichia coli. The upstream and downstream genes of rmlD, rmlB and rmlA, were cloned into the gene targeting vector. After and allelic exchange of E. coli chromosomal rmlB, rmlD, rmlA with vectoral rmlB, FRT-KmR-FRT, rmlA by homologous recombination, FLP-expressing plasmid was introduced to induce the excision of KmR cassette remaining one FRT sequence between rmlB and rmlA. We also demonstrated our shuttle vector could disrupt a target gene (kanamycin resistance gene) in M. smegmatis. These results suggest that our gene knock-out system can be used for the development of an attenuated tuberculosis vaccines and for the functional genomic study of mycobacteria.

Effect of Interleukin-10 on Lipopolysaccahride/Interferon-gamma- Induced Chemokine Mig Gene Expression

Interferon-gamma (IFN-gamma) is well known as a potent inducer in monokine induced by IFN-gamma (Mig) mRNA expression. Although lipopolysaccharide (LPS) alone is weakly effective on Mig mRNA expression. the stimulation of LPS and IFN-gamma (LPS/IFN-gamma simultaneously has been shown to synergize to produce a high level of Mig mRNA in mouse peritoneal macrophages. In this study, interleukin-10 (IL-10) was found to suppress the LPS/IFN-gamma- induced Mig mRNA expression in cell type- and mouse strain-specific fashion, but IFN-gamma alone-induced Mig mRNA was unaffected by IL-10 under identical experimental conditions. The IL-10-mediated suppression of LPS/IFN-gamma-stimulated Mig mRNA expression was dependent on the concentration of IL-10, and was prevented when the agent was added 2 hours after LPS/IFN-gamma treatment. The suppressive action of IL-10 was dependent on a protein synthesis. However, IL-10 did not reduce the stability of LPS/IFN-gamma-induced Mig mRNA. These data may have important implications for a previously unrecognized role for IL-10 as a regulator of synergistic effect of LPS on the IFN-gamma-induced expression of the Mig gene in macrophages.

The Supressive Effects of Integrin Antibodies on the Infection of Hantaan Virus in Fibroblasts / 영남의대학술지

Pathophysiological mechanism of hemorrhagic fever with renal syndrome (HFRS) is not fully understood. Major clinical findings of HFRS patients are widespread hemorrhage, acute renal failure and shock. Basic lesion is vascular injury with microvascular hemorrhage and relatively little inflammation. According to autopsy findings, renal medulla shows focal hemorrhage, tubular necrosis and interstitial mononuclear infiltrates. The predominant cell type in the renal and pulmonary interstitium is a fibroblast and it participates in the healing process at the injury site by secreting a large amount of extracellular matrix proteins. Cultured human lung fibroblasts and Mongolian gerbil fibroblasts were known to be good host cells for the hantaan virus. It is possible that not only the endothelial cell but also the fibroblast is a target of Hantaan virus and the fibroblast might be involved in the pathogenesis and the healing process in HFRS. Integrins are adhesion molecules, and act as receptors for many extracellular matrix proteins. Recently, there are many reports that cell surface integrins influence on some viral infections or reversely viruses influence on the expression of integrins. The alpha5beta1 integrin is a major receptor for the fibronectin which is an important extracellular matrix protein secreted by fibroblasts. In this study, the role of alpha5beta1 integrin in the infection of Hantaan virus was examined by using anti-alpha5beta1 integrin, anti-alpha5 integrin and anti-beta1 integrin antibodies in chicken embryo fibroblasts (CEF) and Mongolian gerbil fibroblasts(MGF). The treatment of anti-alpha5beta1 integrin antibody in CEF reduced the virion titers 26.8% and the amount of nucleocapsid N protein 32.6% when compared with control CEF. When MGF were treated with anti-alpha5, anti-beta1 and anti-alpha5beta1 integrin antibodies, virion titers were reduced by 26.5%, 29.4% and 28.7% and the amount of nucleocapsid N protein were reduced by 65.2%, 59.7% and 72.6%. These results suggested that alpha5beta1 integrin might act as a receptor for the Hantaan virus or blocking of alpha5beta1 integrin influences on the viral replication in CEF and MGF. It is also possible that the blocking of only one subunit of integrin represents similar results in that of whole molecule.

Dose effect of LPS on Cytokine mRNA Expressions by Mouse Peritoneal Macrophages

Many studies for LPS-induced cytokines, chemokines gene expressions have been reported, but results are highly diverse. The dose of LPS used, cell type studied or other technical factors may contribute to the differences. We investigated the cytokine mRNA expressions, interleukin-2 (IL-2), IL-6, IL-10 and IFNr, on thioglycollate (TG) elicited-mouse peritoneal macrophages stimulated with various concentrations, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1 ug/ml and 10 ug/ml, of LPS used by reverse transcription polymerase chain reaction (RT-PCR) and northern blot analysis. TG- elicited peritoneal macrophages expressed IL-6 and IFN-r mRNA in a dose-dependent manner. The expressions of IL-2 and IL-10 mRNA were dose-independent manner. In northern blot analysis, IL-6 mRNA expression was detected only in 10 ug/ml LPS concentration for short stimulation time (0.5 h), while IL-10 mRNAs were expressed evenly in all LPS concentrations. The expression of IL-6 mRNA was sustained until 72 h at 10 ug/ml concentration only, but IL- 10 mRNAs of all cases of LPS concentrations were sustained evenly until then. These results suggest that the concentration of LPS required for cytokine induction must be determined differently from gene to gene types, and although LPS concentration 10 ug/ml has not been used as an ordinary concentration in vitro cytokine study, 10 ug/ml of LPS might be more appropriate concentration in the study of IL-6 expression on mouse peritoneal macrophages.

In Vitro Culture of Human Nasal Epithelial Cells by Monolayer Culture of Dissociated Cells / 영남의대학술지

Different techniques for culturing respiratory epithelial cells have been developed to overcome the limitations of studies on in vivo and on bioptic material. Traditionally, culture systems are divided into organ cultures, explant cultures and dissociated cell cultures. The first two contain both epithelial and non-epithelial cells. However, in monolayer cultures of dissociated cells only epithelial cells are present, the effects observed are caused by a pure epithelial responses. The purpose of this study is to establish primary culture method of human nasal epithelium (HNEC) by monolayer culture of dissociated cells to evaluate the role of the epithelial cells in the allergic and non-allergic nasal inflammatory reactions. HNEC was prepared by primary culture method of monolayer culture of dissociated cells from human inferior nasal turbinate mucosa of septal deviation patients. Primary cultured cells were characterized by indirect immunofluorescence assay and transmission electron microscopy. The immunoreactivities of cytokeratin-pan and cytokeratin No. 8 were observed in cultured HNEC. However, the immnoreactivities of vimentin and von Willebrand factor were not observed in cultured HNEC. The tonofilaments and desmosome were observed in cultured HNEC. The cultured epithelial cells were identified to be pure nasal epithelial cells. The monolayer culture of dissociated cells could successfully be employed for further study to investigate the role of the epithelial cells in allergic or non-allergic nasal inflammatory diseases.

Effects of Hantaan Virus and IFN-gammaon Induction of Surface ICAM-1 in Primary Cultured Buman Nasal Epithelial Cells and Human Lung Fibroblasts

The primary culture of human nasal epithelial cells was performed using the inferior nasal turbinate tissues, and infected with Hantaan virus to examine the hypothesis of airborne transmission of Hantaan virus in humans. The primary culture cells were identified as epithelial cells by morphologic and immunologic analyses. The viral antigens were detected in the primary human nasal epithelial cells infected with Hantaan virus by immunofluorescence staining. The ICAM-1 induction by Hantaan virus or IFN-gammawas examined in the primary human nasal epithelial cells and human lung fibroblasts (WI-38). Hantaan virus induced the surface ICAM-1 in Wl-38 cells in a time-dependent manner, and IFN-gammainduced the surface ICAM-1 in a dose-dependent manner in HNEC and WI-38 cells. These results revealed that the human nasal epithelial cells are susceptible to Hantaan viral infection supporting the hypothesis of airborne transmission of Hantaan virus in humans. The human lung fibroblasts also might have an important role in the pathogenesis of Hantaan virus through the induction of ICAM-1.