• Clinical and Experimental Pediatrics
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• v.51 no.9
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• pp.987-991
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• 2008

Purpose : Kikuchi-Fujimoto disease (KFD), also known as histiocytic necrotizing lymphadenitis (HNL), is a self-limited disease characterized by cervical lymphadenopathy and fever. The etiology of KFD remains unknown; however, the self-limiting nature of HNL suggests the cause of this disease could be viral infection. For this reason, several viruses have been evaluated as possible etiologies of HNL, including Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), human herpesvirus 8 (HHV8), and cytomegalovirus (CMV). The aim of this study was to examine the relationship of EBV and HHV6 to HNL. Methods : Data pertaining to 51 cases with biopsy-confirmed HNL were collected between January 1999 and December 2005, from the Department of Pathology, College of Medicine, Pusan National University, Busan, Korea. The clinical records-including data regarding age, gender, duration of fever, and lymph node involvementwere reviewed retrospectively. The in situ hybridization (ISH) assay was performed by EBER PNA probe (Dako, Capinteria, CA, USA), and immunohistochemistry testing was performed with anti-HHV type 6 monoclonal antibodies (Chemicon, Temecula, CA, USA). Results : The HNL patients in this study were 24 males and 27 females, ranging in age from seven to 61 years (median: 25.9). ISH for EBV was positive in 8/51 (15.7%) biopsies, and immunohistochemistry for HHV6 was positive in 15/51 (29.4%) biopsies. Serologic analysis of EBV IgM was performed in 23 cases; only one patient was positive for EBV IgM and EBV ISH. Conclusion : Our study could not provide supportive evidence of a viral pathogenesis for HNL; therefore, cases of HNL may not have a dominant viral cause. However, some rare exceptional cases may have been caused by viral infection.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.5
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• pp.587-594
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• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.

• IMMUNE NETWORK
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• v.11 no.3
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• pp.182-189
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• 2011

Background: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-${\gamma}$ ELISPOT assay, cytotoxicity assay and tetramer staining. Results: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of $CD8^+$ and $CD4^+$ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

• IMMUNE NETWORK
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• v.8 no.4
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• pp.130-136
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• 2008

Background: Human cytomegalovirus UL18, a MHC class I homologue, has been considered a natural killer (NK) cell decoy. It ligates LIR-1/ILT2 (CD85j), an NK inhibitory receptor, to prevent lysis of infected target cells. However, precise role of UL18 to NK cell cytotoxicity is yet elusive. Difficulty in clarifying the function of UL18 lies in complication in detecting UL18 mainly due to low level expression of UL18 on the surface and gradual loss of its expression. Methods: To overcome this hurdle, cDNA of cytoplasmic tail-less UL18 was constructed and expressed in swine endothelial cell (SEC). The expression level and its stability in the cell surface were monitored with FACS analysis. Results: Surface expression of UL18 is up-regulated by removing cytoplasmic tail portion from UL18F (a full sequence of UL18). SECs transfected with a cDNA of UL18CY (a cytoplasmic tail-less UL18) stably expressed UL18 molecule on the surface without gradual loss of its expression during 6 week continuous cultures. In the NK cytotoxicity assay, UL18 functions either inhibiting or activating NK cell cytotoxicity according to the source of NK cells. We found that there is individual susceptibility in determining whether the engagement of NK cell and UL18 results in overall inhibiting or activating NK cell cytotoxicity. Conclusion: In this study, we found that cytoplasmic tail is closely related to the regulatory function for controlling surface expression of UL18. Furthermore, by constructing stable cell line in which UL18 expression is up-regulated and stable, we provided a useful tool to clarify exact functions of UL18 on various immune cells having ILT2 receptor.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.3
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• pp.262-267
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• 2007

Implantation of allografts has increased widely with not only the availability of many allogenic bone but also allogenic soft tissues. The aim of tissue banking is to provide surgeons with safe tissues compatible with their intended clinical application. The incidence of tissue transplant-transmitted infection is unknown and can only be inferred from prospective studies. The possibility of donor-to-recipient disease transmission through soft tissue transplantation can be considered by reviewing the risk associated with other transplanted hard tissues. Viral, bacterial, and fungal infections have been transmitted via transplantation of soft tissue allografts such as skin, cornea, dura, pericardium. fascia lata, and heart valves. Corneas have transmitted rabies, Creutzfeldt-Jakob disease (CJD), hepatitis B (HBV), cytomegalovirus (CMV), herpes simplex virus (HSV), bacteria, and fungi. Heart valves have been implicated in transmitting tuberculosis, hepatitis B. HIV-1 and CMV. CJD has been transmitted by dura and pericardium transplants. Skin has transmitted CMV, bacteria, and fungi. Cadaveric skin, pericardium, dura, and fascia lata have been used in dental patients with intra-oral soft tissue injuries and GBR. This study is review of the considering transmission of infectious disease in allogenic soft tissues and guidelines of reducing the risk. Prior to use, many tissues are exposed to antibiotics, disinfectants, and sterilants, which further reduce or remove the risk of transmitted disease. Because some soft tissue grafts cannot be subjected to sterilization steps, the risk of infectious disease transmission remains and thorough donor screening and testing is especially important.

• Journal of Microbiology
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• v.39 no.4
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• pp.300-304
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• 2001

Epstein-Barr Virus(EBV)-transformed lymphoblastoid B cell lines, BLCL which expresse antigens, are potential antigen-presenting cells(APCs) for the induction of CTL in vitro. However transfection of BLCLs with subsequent selection by antibiotics is notoriously difficult because plating efficiencies of BLCLsare reported to be 1% or less. To generated stable transfectants of BLCLs we produced high titers of retroviruess encoding pp 65 antigen of human cytomegalovirus of foreign antigens and trans-duced them of BLCLs. The pp 65 gene was cloned into the retroviral vector pLXSN. The recombinant retroviral vector was transfected to ecotropic packaging cell line, CP&E86, and this polyclonal recom-binant retrovirus was transduced to PA317 that is amphotropic pakaging cell line. The titers of colned PA317 amphotropic retroviruses ranged from 5 to $\times$10$^{6}$ colony forming units (CFU)per ml (CFU/ml) We performed three rounds of consecutive transductions to BLCLs in order to improve the clon-ing effieiencies. The expression of recombinant HCMV-pp65 antigen was more than 20% after the final transduction. THe third-transduced BLCLs were easily selected in optimal concentration of G418. BLCLs expressing foreign antigens could be used as target cells for CTL assay and/or as APCs for induction of in vitro CTL responses specific for viral and tumor antigens.

• Tuberculosis and Respiratory Diseases
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• v.45 no.4
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• pp.805-812
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• 1998

Background: Among the variety of opportunistic infections, pneumonia comprises the major morbidity in immunocompromised patients. Pneumocystis carnii pneumonia (PCP) and cytomegalovirus (CMV) pneumonia are common infectious illness of immunocompromised hosts. Although there are many reports regarding to the co-infection of PCP and CMV diagnosed by bronchoalveolar lavage (BAL) fluid examination, the effects of CMV co-infection on the outcome of PCP is still controversial. The purpose of this investigation is to evaluate the effects of CMV detected by BAL fluid examination on the clinical course of PCP in the immunocompromised patients other than human immunodeficiency virus infection. Method: Ten patients with PCP were enrolled and retrospective analysis of their medical records were done. HIV infected persons were excluded. The PCP was diagnosed by BAL fluid examination with Calcofluor-White staining. CMV was detected in BAL fluid by Shell-vial culture system. Chest radiographic findings were reviewed. We used Fisher's exact test and Mann-Whitney U test for statistical analysis of data. Results: The underlying disorders of patients were idiopathic pulmonary fibrosis (n=1), renal transplantation (n=4), necrotizing vasculitis (n=l), systemic lupus erythematosus (n=1), brain tumor (n=1), chronic myelogenous leukemia (n=1), unidentified (n=1). There were no difference in clinical course, APACHE III score, arterial blood gas analysis, white blood cell count, lymphocyte count, serum albumin concentration, chest radiographic findings and mortality between patients with PCP alone (n=4) and those with CMV co-infection (n=6). Univariate analysis regarding to the factors that associated with mortality of PCP were revealed that the application of mechanical ventilation (p=0.028), the level of APACHE III score (p=0.018) and serum albumin concentration (p=0.048) were related to the mortality of patients with PCP. Conclusion: The clinical course of PCP patients co-infected by CMV were not different from PCP only patients. Instead, accompanied respiratory failure, high APACHE III score and poor nutritional status were associated with poor outcome of PCP.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

• Clinical and Experimental Pediatrics
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• v.52 no.3
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• pp.324-329
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• 2009

Purpose : Acute interstitial pneumonia (AIP) is a rare disease, but its prognosis is fatal because of lack of efficient treatment modality. Recently, it has been reported that there was epidemic AIP in Korea. This study aims to investigate the past and current status of AIP in Korea. Methods : We performed a nationwide survey and a prospective study. From August 6 to 15, 2008, a questionnaire survey was conducted to identify the prevalence, local distribution, and response to current treatments. The questionnaire was answered by pediatrician working in 23 referral centers in Korea. In addition, 5 referral centers in Seoul performed a preliminary prospective observational study by obtaining clinical data and specimens from appropriate patients. The Korea Centers for Disease Control and Prevention analyzed the samples for possible pathogens. Results : The survey showed 78 AIP cases had occurred and 36 patients had died. Lung biopsy was performed only on 20 patients. In 2008, 9 AIP cases developed. In a prospective study, 9 (M:F=5:4) patients developed AIP in spring and 7 (78%) died, with the mean rate of death occurring 46 days after diagnosis. Human corona virus 229E, cytomegalovirus, influenza A virus, influenza B virus, and parainfluenza virus were isolated from the respiratory specimens. Conclusion : This study showed nationwide prevalence of AIP in Korea. In addition, because of the high mortality rate and rapid progress, pediatricians need to be aware of the disease. Further studies and a nationwide network are required for reducing the morbidity and mortality rates related to AIP.

• Tuberculosis and Respiratory Diseases
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• v.45 no.3
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• pp.587-595
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• 1998

Background: Recombinant adenovirus hold promise as vectors to carry therapeutic genes for several reasons: 1) they can infect both dividing and non-dividing cells; 2) they have the ability to directly transduce tissues in vivo; 3) they can easily be produced in high titer; and 4) they have an established record of safety as vaccination material. However, one of the major limitation in the use of adenoviruses is that transgene expression is quite short because adenovirusees insert their DNA genome episomally rather than by chromosomal integration, and an immune response against the virus destroys cells expressing the therapeutic gene. Since sodium butyrate has been reported to induce adenovirus-mediated gene expression, we hypothesized that treatment of tumor cells, transduced with herpes simples virus thymidine kinase(HSVtk) gene using adenoviral vector, with butyrate could augment the effect of gene therapy. Methods: We transduced HSVtk gene, driven by the cytomegalovirus promoter, into REN cell line(human mesothelioma cell line). Before proceeding with the comparison of HSVtk/ganciclovir mediated bystander killing, we evaluated the effect of butyrate on the growth of tumor cells in order to rule out a potential antitumor effect of butyrate alone, and also on expression of HSVtk gene by Western blot analysis. Then we determined the effects of butyrate on bystander-mediated cell killing in vitro. Results: There was no inhibition of growth of cells exposed to butyrate for 24 hours at a concentration of 1.5mM/L. Toxic effects were seen when the concentration of butyrate was greater than 2.0mM/L. Gene expression was more stable and bystander effect was augmented by butyrate treatment of a concentration of 1.5mM/L. Conclusion: These results provide evidence that butyrate can augment the efficiency of cell killing with HSVtk/GCV system by inducing transgene expression and may thus by a promising new approach to improve responses in gene therapy using adenoviral vectors.