The increasing demand for scar treatments has led to various innovative methods. This paper presents two cases involving the treatment of distinct scar types using a single monopolar microneedle radiofrequency (SMNR) device. The first case involved a 52-year-old male with 2-cm linear depressed facial scar of unknown etiology. The patient underwent two sessions of SMNR spaced two weeks apart and showed scar improvement after four weeks. The second case involved a 35-year-old male presenting with a combination of icepick and boxcar acne scars on both cheeks. He received seven SMNR treatments every two weeks and showed scar improvement after 12 weeks. In both cases, scar improvement was achieved through subcisions of fibrous septa combined with dermal remodeling using microneedle radiofrequency. This case report shows that SMNR is an effective and safe modality for treating various types of scars, including acne scars, with minimal downtime and no significant adverse effects

BACKGROUND: Controlling inflammation is a therapeutic goal of various autoimmune/autoinflammatory diseases including Behçet's disease (BD). The immunomodulatory effect of metabolites or metabolic analogs such as butyrate and 3-bromopyruvate has been observed in animal disease models. OBJECTIVE: We attempted to evaluate the effect of butyrate and 3-bromopyruvate on the inflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) isolated from patients with mucocutaneous involvement of BD. METHODS: PBMCs isolated from 11 patients with BD and 10 healthy controls were stimulated with lipopolysaccharide in the presence of butyrate or 3-bromopyruvate. Butyrate receptor and cytokine messenger ribonucleic acid (mRNA) expression was analyzed by real-time reverse transcription polymerase chain reaction. Cytokine secretion was assessed by enzyme-linked immunosorbent assay. PBMCs survival was analyzed by flow cytometry. RESULTS: Bromopyruvate or butyrate treatment suppressed inflammatory cytokine production in PBMCs from all our subjects. Bromopyruvate also reduced PBMCs survival while butyrate did not. As the effect of butyrate was slightly greater in BD patients than in healthy controls, we analyzed butyrate receptor expression and found that lipopolysaccharide-induced free fatty acid receptor 2 mRNA level in PBMCs was higher in BD patients than in controls. CONCLUSION: We propose bromopyruvate and butyrate as supplementary therapeutic candidates to control inflammation in patients with BD.
Autoimmune Diseases ; Butyrates* ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Glycolysis ; Humans ; Inflammation ; Polymerase Chain Reaction ; Reverse Transcription ; RNA ; RNA, Messenger

PURPOSE: Gastric cancer has the highest incidence rate among cancers in Asia. The advanced type of signet ring cell carcinoma has poor prognosis compared to other types of gastric cancer. The immuno-gene therapy with cytokine-based tumor vaccines has not yet been investigated for gastric cancer. The granulocyte macrophage colony-stimulating factor (GM-CSF)-based tumor vaccine has been demonstrated as the most potent stimulator for specific and long-lasting systemic tumor immunity. MATERIALS AND METHODS: In the present study, KATO III cells, the human signet ring cell gastric carcinoma cell line, were genetically modified by the transduction with the human GM-CSF cDNA or the modified hGM-CSF in replication-deficient retroviruses. The genomic integrations and mRNA expressions of the transgenes were determined by Southern and Northern blot analyses. RESULTS: Wild type (wt) or modified hGM-CSF was integrated into the genome of KATO III cells. The modified hGM-CSF mRNA was more stable than that of wt. The KATO III cells with the modified hGM-CSF produced higher level of hGM-CSF (12.4-19 ng/10(6)cells/48hrs) than that with wt hGM-CSF, when determined by enzyme-linked immunosorbent assay (ELISA). The secreted recombinant hGM-CSF could support the proliferation of the GM-CSF-dependent cell line, indicating that the hGM-CSF secreted by the transduced KATO III cells has biological activities. Irradiated, transduced KATO III cells continued to secret hGM-CSF without proliferation. CONCLUSION: Our results suggest that GM-CSF secreting KATO III cells could be tested for the treatment of gastric cancer as an allogeneic tumor vaccine as a part of immunotherapeutic treatment.
Base Sequence ; Blotting, Northern ; Blotting, Southern ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Granulocyte Macrophage Colony-Stimulating Factors, ; Humans ; Mutagenesis ; RNA, Messenger/genetics/metabolism ; Recombinant Proteins/metabolism/*secretion ; Stomach Neoplasms/genetics/metabolism/pathology ; Transduction, Genetic

BACKGROUND: Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known. OBJECTIVE: To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs). METHODS: Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) β was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPβ and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs. CONCLUSION: We found differential expression of C/EBPβ, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.
Activating Transcription Factor 3 ; Behcet Syndrome* ; Blotting, Western ; CCAAT-Enhancer-Binding Proteins ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Interleukin-6 ; Interleukins ; RNA, Small Interfering ; Transcription Factors* ; Tumor Necrosis Factor-alpha

Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.
Virus Replication/*drug effects ; Trans-Activators/*antagonists & inhibitors/immunology ; Immunoglobulin Variable Region/genetics/metabolism/*pharmacology ; Hepatitis B virus/*drug effects/physiology ; Hepatitis B e Antigens/metabolism ; Cell Line

BACKGROUND: T cell immunoglobulin and mucin domain containing 3 protein (Tim-3) expressed on terminally differentiated Th1 cells plays a suppressive role in Th1-mediated immune responses. Recently, it has been shown that N-glycosylation affects the binding activity of the Tim-3-Ig fusion protein to its ligand, galectin-9, but the binding properties of non-glycosylated Tim-3 on CD4+CD25+ T cells has not been fully examined. In this study, we produced recombinant Tim-3-Ig fusion proteins in different cellular sources and its N-glycosylation mutant forms to evaluate their binding activities to CD4+CD25+ T cells. METHODS: We isolated and cloned Tim-3 cDNA from BALB/C mouse splenocytes. Then, we constructed a mammalian expression vector and a prokaryotic expression vector for the Tim-3-Ig fusion protein. Using a site directed mutagenesis method, plasmid vectors for Tim-3-Ig N-glycosylation mutant expression were produced. The recombinant protein was purified by protein A sepharose column chromatography. The binding activity of Tim-3-Ig fusion protein to CD4+CD25+ T cells was analyzed using flow cytometry. RESULTS: We found that the nonglycosylated Tim-3-Ig fusion proteins expressed in bacteria bound to CD4+CD25+ T cells similarly to the glycosylated Tim-3-Ig protein produced in CHO cells. Further, three N-glycosylation mutant forms (N53Q, N100Q, N53/100Q) of Tim-3-Ig showed similar binding activities to those of wild type glycosylated Tim-3-Ig. CONCLUSION: Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on CD4+CD25+ T cells.
Animals ; Bacteria ; CHO Cells ; Chromatography ; Clone Cells ; Cricetinae ; DNA, Complementary ; Flow Cytometry ; Immunoglobulins ; Ligands ; Mice ; Mucins ; Mutagenesis, Site-Directed ; Plasmids ; Proteins ; Sepharose ; Staphylococcal Protein A ; T-Lymphocytes ; Th1 Cells

BACKGROUND: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than 109 members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to 1014 molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. METHODS: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. RESULTS: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. CONCLUSION: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.
Animals ; Bacteriophages ; DNA ; Genotype ; Hepatitis B virus ; Mice ; Phenotype ; Polymerase Chain Reaction ; Radioimmunoassay ; Ribosomes* ; RNA ; RNA, Messenger ; Translating

BACKGROUND: T cell immunoglobulin mucin containing molecule (TIM)-3 is expressed in differentiated Th1 cells and is involved in the suppression of the cytokine production by these cells. However, the regulation of the expression of other T cell genes by TIM-3 is unclear. Herein, we attempted to identify differentially expressed genes in cells abundantly expressing TIM-3 compared to cells with low expression of TIM-3. METHODS: TIM-3 overexpressing cell clones were established by transfection of Jurkat T cells with TIM-3 expression vector. For screening of differentially expressed genes, gene fishing technology based on reverse transcription-polymerase chain reaction (RT-PCR) using an annealing control primer system was used. The selected candidate genes were validated by semi quantitative and real-time RT-PCR. RESULTS: The transcription of TIMP-1, IFITM1, PAR3 and CCL1 was different between TIM-3 overexpressing cells and control cells. However, only CCL1 transcription was significantly different in cells transiently transfected with TIM3 expression vector compared with control cells. CCL1 transcription was increased in primary human CD4+ T cells abundantly expressing TIM-3 but not in cells with low expression of TIM-3. CONCLUSION: CCL1 was identified as a differentially transcribed gene in TIM-3-expressing CD4+ T cells.
Clone Cells ; Gene Expression ; Genes, vif ; Humans ; Immunoglobulins ; Mass Screening ; Mucins ; T-Lymphocytes ; Th1 Cells ; Tissue Inhibitor of Metalloproteinase-1 ; Transfection

BACKGROUND: CD8+ T cells contribute to the clearance of Hepatitis B virus (HBV) infection and an insufficient CD8+ T cell response may be one of the major factors leading to chronic HBV infection. Since the HBx antigen of HBV can up-regulate cellular expression of several immunomodulatory molecules, we hypothesized that HBx expression in hepatocytes might affect CD8+ T cell activity. METHODS: We analyzed the activation and apoptosis of CD8+ T cells co-cultured with primary hepatocytes rendered capable of expressing HBx by recombinant baculovirus infection. RESULTS: Expression of HBx in hepatocytes induced low production of interferon-gamma and apoptosis of CD8+ T cells, with no effect on CD8 T cell proliferation. However, transcriptional levels of H-2K, ICAM-1 and PD-1 ligand did not correlate with HBx expression in hepatocytes. CONCLUSION: Our results suggest that HBx may inhibit CD8+ T cell response by regulation of interferon-gamma production and apoptosis.
Apoptosis ; Baculoviridae ; Cell Proliferation ; Hepadnaviridae ; Hepatitis ; Hepatitis B ; Hepatitis B virus ; Hepatocytes ; Intercellular Adhesion Molecule-1 ; Interferon-gamma ; T-Lymphocytes ; Trans-Activators ; Viral Proteins

T cell immunoglobulin mucin domain (TIM)-3 is an immunomodulatory molecule and upregulated in T cells by several cytokines. TIM-3 also influences mast cell function but its transcriptional regulation in mast cells has not been clarified. Therefore, we examined the transcript level and the promoter activity of TIM-3 in mast cells. The TIM-3 transcript level was assessed by real-time RT-PCR and promoter activity by luciferase reporter assay. TIM-3 mRNA levels were increased in HMC-1, a human mast cell line by TGF-beta1 stimulation but not by stimulation with interferon (IFN)-alpha, IFN-lambda, TNF-alpha, or IL-10. TIM-3 promoter -349~+144 bp region relative to the transcription start site was crucial for the basal and TGF-beta1-induced TIM-3 promoter activities in HMC-1 cells. TIM-3 promoter activity was increased by overexpression of Smad2 and Smad4, downstream molecules of TGF-beta1 signaling. Our results localize TIM-3 promoter activity to the region spanning -349 to +144 bp in resting and TGF-beta1 stimulated mast cells.
Cytokines ; Humans ; Immunoglobulins ; Interferons ; Interleukin-10 ; Luciferases ; Mast Cells ; Mucins ; RNA, Messenger ; T-Lymphocytes ; Transcription Initiation Site ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha