• Archives of Plastic Surgery
• /
• v.32 no.4
• /
• pp.529-532
• /
• 2005

Expanding cells ex-vivo is very important in tissue-engineering. Culture medium is usually supplemented with fetal bovine serum(FBS) in most of the experiments. However, cells grown in bovine serum media may posses the possibilities of disseminating bovine diseases and/or stimulating the patient's immune reactions. To overcome these problems, autologous or homologous serum should be used instead of the FBS. The purpose of this study is to compare cell proliferation and collagen synthesis depending on the kind of sera mixed on media and to provide a guideline on applying established experimental data to clinical cases. Human dermal fibroblasts were obtained from four patients. Five thousand cells per well in 96-well plates were incubated DMEM/F-12 Nutrient with varying serum mixture; 10% autologous serum, 10% homologous serum, and 10% FBS. Five days after incubation fibroblast proliferation and collagen production were determined by MTT assay and CICP enzyme immunoassay. The mean cell number were; $3.95{\times}10^4/well$, $2.97{\times}10^4/well$ and $2.30{\times}10^4/well$, respectively. The average amounts of collagen synthesized were; 238.13 ng/ml, 204.88 ng/ml, and 163.88 ng/ml in each. These results show that the use of human serum mixture may contribute to, not only preventing disseminated infection of bovine diseases. but also increase cell proliferation and collagen synthesis without simulating the patient's immune reactions.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.5
• /
• pp.696-703
• /
• 2014

Clostridium difficile causes mucosal damage and diarrhea by releasing two exotoxins: toxin A and toxin B. C. difficile colitis is associated with alterations in bowel flora and the failure to mount an effective antibody response. The aim of the current study was to investigate whether antitoxin sera prevent toxin-A-induced apoptosis, cytoskeletal disaggregation, cell detachment, and tight junction loss in cultured colonic epithelial cells. Serum samples were isolated from mice that survived a C. difficile infection following antibiotic treatment, and the antitoxin effects of these samples were investigated in toxin-A-exposed HT29 colonic epithelial cells and a toxin-A-induced animal model of gut inflammation. Unchallenged mice did not produce IgG against toxin A, whereas serum (antiserum) from C. difficile-challenged mice showed significant IgG responses against toxin A. Treatment with the antiserum markedly inhibited mucosal damage and inflammation in the toxin-A-treated mouse model. In contrast to control mouse serum, the antiserum also markedly inhibited toxin-A-induced DNA fragmentation, dephosphorylation of paxillin and Epo receptor (EpoR), deacetylation of tubulin, and upregulation of p21(WAF1/CIP1) and p53. Taken together, these results reveal that the generated antitoxin serum has biotherapeutic effects in preventing various C. difficile toxin-A-induced cellular toxicities.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.4
• /
• pp.537-543
• /
• 2003

The mouse interferon gene (MuIFN-${\gamma}$) was cloned and then used to transform Saccharomyces cerevisiae. Expressed MuIFN-$\{gamma}$ protein (MuIFN-${\gamma}$) was successfully secreted into culture medium due to the presence oi the signal peptide of rice amylase 1A. Two different promoters fused to MuIFN-${\gamma}$ were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD (AG) promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Using the hybrid promoter, the accumulation of MuIFN-${\gamma}$transcript was the highest after the 24 h cultivation, and then gradually decreased as the cultivation proceeded. However, both cell growth and recombinant MuIFN-${\gamma}$production reached their peaks after the 4-day cultivation. It was possible to produce 6.5 mg/l of MuIFN-${\gamma}$ without any changes in cell growth. Using GPD promoter, the MuIFN-${\gamma}$ transcript accumulation and the recombinant MuIFN-${\gamma}$ production followed the same pattern as the cell growth. However. compared to that of the hybrid promoter, the production of recombinant MuIFN-${\gamma}$ was 0.2 mg/l. The secreted MuIFN-${\gamma}$ had estimated molecular masses of 21 kDa and 23 kDa, which were larger than that of the encoded size due to glycosylation. The protection assay against the viral infection indicated that the recombinant MuIFN-${\gamma}$ was bioactive.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.12
• /
• pp.1330-1335
• /
• 2011

N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.8
• /
• pp.1154-1158
• /
• 2013

Ganciclovir resistance of human cytomegalovirus is associated with mutations in the viral UL97 gene and poses severe problems for immunocompromised patients. In this study, PCR-based restriction fragment length polymorphism and sequencing analyses detected the UL97 D605E mutation in all five clinical isolates from patients with ganciclovir-resistant human cytomegalovirus infection during prolonged ganciclovir therapy, whereas the M460V mutation was only present in 1 of 5 isolates. On the other hand, the detection rates of the D605E mutation in the stored available DNA samples from the donor and allogeneic stem cell transplantation recipients were 66.7% and 93.7%, respectively, suggesting that the presence of D605E mutation was not associated with the ganciclovir exposure. Although the D605E mutation may not be related to ganciclovir resistance, we suggest that this mutation could be an important molecular marker of human cytomegalovirus evolution in East Asian countries. Moreover, the restriction fragment length polymorphism method using the restriction enzyme HaeIII, which is generally used to detect the UL97 A591V mutation, could also detect the D605E mutation and may therefore be a useful tool for future research on the investigation of UL97 gene mutations.

• Asian-Australasian Journal of Animal Sciences
• /
• v.25 no.7
• /
• pp.1038-1044
• /
• 2012

In the present study, the impact of Salmonella Typhimurium on cell-mediated immunity (CMI) was investigated in 5 week-old immuno divergent broiler lines selected for the high and low response to phytohemagglutinin-P. The immune response was assessed in peripheral-blood mononuclear cells (PBMCs) induced with Salmonella Typhimurium at different time intervals (0 h, 0.5 h, 2 h, 4 h, 6 h, 12 h and 24 h). The differential mRNA expression patterns of IFN-${\gamma}$, IL-2 and iNOS were evaluated by quantitative real time PCR. In-vitro production of nitric oxide (NO) was also estimated in the culture supernatant and correlated with iNOS mRNA expression. Present study showed higher production of NO in the high cell-mediated line (HCMI) as compared to the low cell-mediated line (LCMI) upon stimulation with Salmonella Typhimurium. Correspondingly, higher mRNA expression of iNOS and IFN-${\gamma}$ were observed in high response birds (HCMI); but IL-2 was down regulated in this line compared to the low response birds (LCMI). Significantly (p<0.05) higher expression of iNOS, IFN-${\gamma}$ and higher production of NO in high line indicated that the selection for PHA-P response might be employed for increasing the immune competence against Salmonella Typhimurium in chicken flocks.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.6
• /
• pp.911-920
• /
• 2006

[ $CD8^+$ ] T Iymphocytes with the cytotoxic activity and capability to release various cytokines are the major players in immune responses against viral infection and cancer. To identify the proteins specific to resting or activated human CD8$^+$ T cells, human CD8$^+$ T cells were activated with anti-CD3+anti-CD28 mAb in the presence of IL-2. The solubilized proteins from resting and activated human CD8$^+$ T cells were separated by high-resolution two-dimensional polyacrylamide gel electrophoresis, and their proteomes were analyzed. Proteomic analysis of resting and activated T cells resulted in identification of 35 proteins with the altered expression. Mass spectrometry coupled with Profound and SWISS-PROT database analysis revealed that these identified proteins are to be functionally associated with cell proliferation, metabolic pathways, antigen presentation, and intracellular signal transduction pathways. We also identified six unknown proteins predicted from genomic DNA sequences specific to resting or activated CD8$^+$ T cells. Protein network studies and functional characterization of these novel proteins may provide new insight into the signaling transduction pathway of CD8$^+$ T cell activation.

• Journal of Veterinary Clinics
• /
• v.28 no.1
• /
• pp.113-121
• /
• 2011

Foot-and-mouth disease (FMD) is a severe vesicular disease of cloven-hoofed animals including domesticated ruminants and pigs. Acute clinical signs may be mild in sheep and goats but are associated with lameness in pigs and mouth lesions with vesicles in cattle. The required condition for a successful pathogen appears to be the ability to counteract both the host innate and adaptive immune response. FMD virus (FMDV) inhibits the induction of antiviral molecules and interferes with the secretory pathway in the infected cell. The surface expression of Major Histocompatibility Complex (MHC) class I molecules is reduced in infected cells. Thus, the ability of the host to recognize and eliminate virus infected cells is decreased. Furthermore, FMDV infection results in a rapid, but transient lymphopenia, reducing the number of T and B cells, and affecting T cell function. The virus appears to premature apoptosis-mediated cell death because it has a very short replication cycle and is able to rapidly produce large amounts of virus. FMDV engages the host protective response at multiple steps to ensure its effective replication and pathogenesis. This review describes the recent pathological and immunological studies to overcome the powerful abilities of FMDV to counteract defense mechanism of host.

• IMMUNE NETWORK
• /
• v.10 no.3
• /
• pp.104-108
• /
• 2010

CD137 (4-1BB/tnfrsf9) has been shown to co-stimulate T cells. However, agonistic anti-CD137 monoclonal antibody (mAb) treatment can suppress $CD4^+$ T cells, ameliorating autoimmune diseases, whereas it induces activation of $CD8^+$ T cells, resulting in diverse therapeutic activity in cancer, viral infection. To investigate the CD137-mediated T cell suppression mechanism, we examined whether anti-CD137 mAb treatment could affect $CD11b^+Gr-1^+$ myeloid-derived suppressor cells (MDSCs). Intriguingly, anti-CD137 mAb injection significantly increased $CD11b^+Gr-1^+$ cells, peaking at days 5 to 10 and continuing for at least 25 days. Furthermore, this cell population could suppress both $CD8^+$ T cells and $CD4^+$ T cells. Thus, this study demonstrated that, for the first time, anti-CD137 mAb treatment could induce $CD11b^+Gr-1^+$ MDSCs under normal conditions, suggesting a possible relationship between myeloid cell induction and CD137-mediated immune suppression.

• BMB Reports
• /
• v.40 no.3
• /
• pp.358-367
• /
• 2007

A novel mannose-binding tuber lectin with in vitro antiproliferative activity towards human cancer cell lines and antiviral activity against HSV-II was isolated from fresh tubers of a traditional Chinese medicinal herb, Typhonium divaricatum (L.) Decne by a combined procedure involving extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE-SEPHAROSE, CM-SEPHAROSE and gel-filtration on sephacryl S-200. The apparent molecular mass of the purified Typhonium divaricatum lectin (TDL) was 48 kDa. TDL exhibits hemagglutinating activity toward rabbit erythrocytes at 0.95 $\mu$g/ml, and its activity could be strongly inhibited by mannan, ovomucoid, asialofetuin and thyroglobulin. TDL showed antiproliferative activity towards some well established human cancer cell lines, e.g. Pro-01 (56.7 $\pm$ 6.8), Bre-04 (41.5 $\pm$ 4.8), and Lu-04 (11.4 $\pm$ 0.3). The anti-HSV-II activity of TDL was elucidated by testing its HSV-II infection inhibitory activity in Vero cells with $TC_50$ and $EC_50$ of 5.176 mg/ml and 3.054 $\mu$g/ml respectively. The full-length cDNA sequence of TDL was 1145 bp and contained an 813-bp open reading frame (ORF) encoding a 271 amino acid precursor of 29-kDa. Homology analysis showed that TDL had high homology with many other mannose-binding lectins. Secondary and three-dimensional structures analyses showed that TDL is heterotetramer and similar with lectins from mannose-binding lectin superfamily, especially those from family Araceae.