• IMMUNE NETWORK
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• v.3 no.2
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• pp.96-102
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• 2003

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccines induce very potent systemic anti-tumor immunity in preclinical and clinical models. Our previous phase I clinical trial in patients with metastatic renal cell carcinoma (RCC) has demonstrated both immune cell infiltration at vaccine sites and T cell-mediated delayed-type hypersensitivity (DTH) response to whole tumor cell vaccines. Methods: To investigate the immune responses to autologous genetically- modified tumor cell vaccines, tumor-specific $CD8^+$ T cell lines were generated from peripheral blood lymphocytes (PBL) of a RCC patient 1.24 by repeated in vitro stimulation with either B7.1-transduced autologous RCC tumor cells or B7.1-transduced autologous tumor cells treated with interferon gamma ($IFN{\gamma}$), and cloned by limiting dilution. Results: Among several RCC-specific cytotoxic T lymphocytes (CTLs), a $CD4^+/CD8^+$ double positive T cell clone (17/A2) appeared to recognize $IFN{\gamma}$-treated autologous RCC restricted by HLA-B39. The 17/A2 also recognized other HLA-B39 positive RCC tumor cells after $IFN{\gamma}$ treatment. Conclusion: These results demonstrate that autologous RCC vaccination successfully generates the tumor-specific CTL 17/A2, and suggest that the presentation and recognition of the tumor antigen by the 17/A2 might be upregulated by $IFN{\gamma}$.

• KSBB Journal
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• v.16 no.6
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• pp.568-572
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• 2001

Light is one of the most important environmental factors controlling plant physiology. The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was produced from cell suspension cultures of transgenic tobacco under different light conditions (24 hr light, 18 hr light/dark cycle, dark). Under 24 hr light condition, cell growth was best and dry cell weight reached 14.4 g/L. Light did not influenced the secretion of total proteins. However, in the dark condition, the ratio of secreted total protein/dry cell weight was 1.5 fold higher than those of ethel conditions. Production of hGM-CSF was highest with 18 hr light condition and reached 496.5 ug/L. In addition, the content of hGM-CSf in secreted total proteins was 1.8 fold higher than that of 24 hr light condition, which is beneficial for the purificationof the protein.

• KSBB Journal
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• v.30 no.3
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

• Tuberculosis and Respiratory Diseases
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• v.81 no.4
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• pp.305-310
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• 2018

Background: Mannose-binding lectin (MBL) deficiency leads to increased susceptibility to infection. We investigated whether serial changes in MBL levels are associated with the prognosis of patients diagnosed with septic shock, and correlated with cytokine levels. Methods: We enrolled 131 patients with septic shock in the study. We analyzed the serum samples for MBL and cytokine levels at baseline and 7 days later. Samples on day 7 were available in 73 patients. Results: We divided the patients with septic shock into four groups according to serum MBL levels (< $1.3{\mu}g/mL$ or ${\geq}1.3{\mu}g/mL$) on days 1 and 7. Patients with low MBL levels on day 1 and high MBL levels on day 7 showed a favorable prognosis for 28-day survival (odds ratio, 1.96, 95% confidence interval, 1.10-2.87; p=0.087). The high MBL group on day 7 showed a significant decrease in monocyte chemoattractant protein 1, interleukin (IL)-$1{\beta}$, IL-6, IL-8, interferon-${\gamma}$, and granulocyte macrophage colony-stimulating factor levels compared with the low MBL group on day 7. Conclusion: The increase in MBL levels of patients with septic shock may suggest a favorable prognosis and attenuate pro-inflammatory and anti-inflammatory responses.

• The Journal of Korean Society of Virology
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• v.30 no.2
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• pp.141-150
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• 2000

The effectiveness of noninfectious recombinant adenovirus encoding murine granulocyte-macrophage colony stimulating factor (mGM-CSF) for the treatment of Meth A fibrosarcoma was investigated in syngeneic BALB/C model. Meth A and HeLa cells transduced with the recombinant adenovirus (Ad.mGM-CSF) produced substantial amounts of mGM-CSF, while WEH1164 cells transduced with the virus did not produce mGM-CSF. Mice inoculated subcutaneously with $1{\times}10^6$ Meth A cells, followed by injection of Ad.dE1 as a control, developed large tumors that reached a mean tumor size of 22 mm by day 30. However, tumor development and tumorigenicity were significantly inhibited in mice with a single intratumoral injection of Ad.mGM-CSF at $1{\times}10^8\;pfu$. Histological examination of the tumors injected with Ad.mGM-CSF revealed dense infiltrates of neutrophils, histiocytes, lymphocytes, and eosinophils associated with apoptotic cell death. The results suggest that the recombinant adenovirus encoding GM-CSF have a potential use for cancer gene therapy.

• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.97-102
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• 2003

Lettuce (Lactuca sativa) was transformed with Agrobacterium tumefacience LBA4404 containing human granulocyte macrophage colony stimulating factor (hGM-CSF) gene to produce in cell suspension cultures. Cell suspension culture was established using callus from transgenic lettuce plant. Integration of hGM-CSF gene into plant chromosome was confirmed through genomic PCR and Southern blot analysis. In addition, Northern blot analysis indicated the expression of the introduced hGM-CSF gene in transformed lettuce. The recombinant hGM-CSF was expressed in transgenic cell cultures derived from transgenic plants as a yield of about 149.0 $\mu\textrm{g}$/L in culture filtrate, which was determined by ELISA. These results demonstrated that transformed lettuce cell suspension cultures could be used as a production system of therapeutic proteins such as hGM-CSF.

• KSBB Journal
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• v.16 no.4
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• pp.376-380
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• 2001

The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was produced from cell suspension culture of transgenic tobacco which was transformed by using Agrobacterium harboring the hGM-CSF gene. To improve the production of hGM-CSF in batch culture system, the effects of initial sucrose concentration and inoculum size were investigated. The results show that the hGM-CSF production was not affected by small inoculum size in medium containing either low or high concentration of sucrose. However, the production of hGM-CSF was increased under increasing of the inoculum sizes and sucrose concentration. Under the combination of inoculum and sucrose concentration, the maximum hGM-CSF production of 720 $\mu$g/L was obtained at 90 g/L of initial sucrose concentration and 110 g/L of inoculum size.

• Journal of Microbiology and Biotechnology
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• v.34 no.4
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• pp.920-929
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• 2024

As a pivotal defensive line against multitudinous malignant tumors, natural killer (NK) cells exist in the tumor microenvironment (TME). RAD18 E3 Ubiquitin Protein Ligase (RAD18) has been reported to foster the malignant progression of multiple cancers, but its effect on NK function has not been mined. Here, the study was designed to mine the mechanism by which RAD18 regulates the killing effect of NK cells on colorectal cancer (CRC) cells. Expression of E2F Transcription Factor 7 (E2F7) and RAD18 in CRC tissues, their correlation, binding sites, and RAD18 enrichment pathway were analyzed by bioinformatics. Expression of E2F7 and RAD18 in cells was assayed by qRT-PCR and western blot. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay verified the regulatory relationship between E2F7 and RAD18. CCK-8 assay was utilized to assay cell viability, colony formation assay to detect cell proliferation, lactate dehydrogenase (LDH) test to assay NK cell cytotoxicity, ELISA to assay levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and immunofluorescence to detect expression of toxic molecules perforin and granzyme B. High expression of RAD18 and E2F7 was found in CRC tissues and cells. Silencing RAD18 could hamper the proliferation of CRC cells, foster viability and cytotoxicity of NK cells, and increase the secretion of GM-CSF, TNF-α, IFN-γ as well as the expression of perforin and granzyme B. Additionally, ChIP and dual-luciferase reporter assay ascertained the binding relationship between RAD18 promoter region and E2F7. E2F7 could activate the transcription of RAD18, and silencing RAD18 reversed the inhibitory effect of E2F7 overexpression on NK cell killing. This work clarified the inhibitory effect of the E2F7/RAD18 axis on NK cell killing in CRC, and proffered a new direction for immunotherapy of CRC in targeted immune microenvironment.

• Applied Microscopy
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• v.40 no.2
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• pp.73-80
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• 2010

Liver regeneration is a result of highly coordinated proliferation of hepatocytes and nonparenchymal liver cells. Partial hepatectomy (PH) is the most often used stimulus to study liver regeneration because, compared with other methods that use hepatic toxins, it is not associated with the tissue injury and inflammation, and the initiation of the regenerative stimulus is precisely defined. Granulocyte macrophage-colony stimulating factor (GM-CSF), which is a cytokine able to regulate the proliferation and differentiation of epithelial cells, was first identified as the most potent mitogen for bone marrow. Particularly, rrhGM-CSF, which is highly glycosylated and sustained longer than any other types of GM-CSF in the blood circulation, was specifically produced from rice cell culture. In this experiment, effects of rrhGM-CSF administration were evaluated in the regenerating liver after 78% PH of rats. Morphological changes induced by PH were characterized by destroyed hepatocyte plate around the central vein and enlarged nuclear cytoplasmic ratio and increased hepatocytes with two nuclei. And then, proliferation of liver cells (parenchymal and nonparenchymal) and rearrangement of plates and lobules seemed to be carried out during liver regeneration. These alterations in the experimental group preceded those of the control. Since proliferating cell nuclear antigen (PCNA) is known to be a nuclear protein maximally elevated in the S phase of proliferating cells, the protein was used as a marker of liver regeneration after PH in rats. PCNA levels by western blot analysis and immunohistology were compared between the two groups. PCNA protein expression of two groups at 12 hr and 24 hr after injury showed similar pattern. The protein expression showed the peak at 3 days in both groups, however, the protein level of the experimental group was higher than that of the control. On immunohistochemical observations, the reaction product of PCNA was localized at the nuclei of proliferating cells and the positive reaction in experimental group at 3 days was clearly stronger than that in control group. The results by Western blotting and immunohistology for PCNA showed similar pattern in terms of the protein levels. In conclusion, rrhGM-CSF administration during liver regeneration after 78% PH accelerated breakdown and restoration of the hepatic plate and expression of PCNA. These results suggest that rrhGM-CSF might play an important role during liver regeneration in rats.

• Journal of Periodontal and Implant Science
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• v.24 no.2
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• pp.321-339
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• 1994

The cytokines released by osteoblasts induce bone resorption via the differentiation of osteoclast precursors. In this process, $interleukin-1{\beta}$($IL-1{\beta}$)-induced bone resorption is mediated by granulocyte macrophage-colony stimulation factor(GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor ${\alpha}$($TNF-{\alpha}$) released from osteoblasts. Since these cytokines (GM-CSF, IL-6, $TNF-{\alpha}$) are produced by not only osteoblasts but also monocytes, and interleukin-10(I1-10) inhibits the secretion of these cytokines from monocytes, it may be speculated that IL 10 could modulate the production of GM-CSF, IL-6, and $TNF-{\alpha}$ by osteoblasts, then control $IL-1{\beta}-induced$ bone resorption. Therefore, the aims of the present study were to examine the effects of IL-10 on bone resorption. The sixten or seventeen-day pregnant ICR mice were injected with $^{45}Ca$ and sacrificed one day after injection. Then fetal mouse calvaria prelabeled with $^{45}Ca$ were dissected out. In order to confirm the degree of bone resorption, mouse calvaria were treated with Lipopolysaccharide(LPS), $TNF-{\alpha}$, $IL-1{\alpha}$, IL-8, $IL-1{\beta}$, and $IL-1{\alpha}$, Then, IL-10 and $interferon-{\gamma}$ ($IFN-{\gamma}$) were added to calvarial medium, in an attempt to evaluate the effect of $IL-1{\beta}-induced$ bone resorption. In addition, osteoclasts formation in bone marrow cell cultures, and the concentration of IL-6, $TNF-{\alpha}$, and GM-CSF produced from mouse calvarial cells were investigated in response to $IL-1{\beta}$ alone and simultaneously adding f $IL-1{\beta}$ and IL-10. The degree of bone resorption was expressed as the ratio of $^{45}Ca$ release(the treated/the control). The osteoclasts in bone marrow cultures were indentified by tartrate resistant acid phosphatase(TRAP) stain and the concentration of the cytokines was quantified using enzyme linked immunosorbent method. As results of these studies, bone resorption was induced by LPS(1 ng/ml ; the ratio of $^{45}Ca$ release, $1.14{\pm}0.07$). Also $IL-1{\beta}$(1 ng/ml), $IL-1{\alpha}$(1 ng/ml), and $TNF-{\alpha}$(1 ng/ml) resulted in bone resorption(the rations of $^{45}Ca$ release, $1.61{\pm}0.26$, $1.77{\pm}0.03$, $1.20{\pm}0.15$ respectively), but IL-8 did not(the ratio of $^{45}Ca$ release, $0.93{\pm}0.21$). The ratios of $^{45}Ca$ release in response to IL-10(400 ng/ml) and $IFN-{\gamma}$(100 ng/ml) were $1.24{\pm}0.12$ and $1.08{\pm}0.04$ respectively, hence these cytokines inhibited $IL-1{\beta}$(1 ng/ml)-induced bone resorption(the ratio of $^{45}Ca$ release $1.65{\pm}0.24$). While $IL-1{\beta}$(1 ng/ml) increased the number of TRAP positive multinulcleated cells in bone marrow cultures($20{\pm}11$), simultaneously adding $IL-1{\beta}$(1 ng/ml) and IL-10(400 ng/ml) decreased the number of these cells($2{\pm}2$). Nevertheless, IL-10(400 ng/ml) did not affect the IL-6, GM-CSF, and $TNF-{\alpha}$ secretion from $IL-1{\beta}$(1 ng/ml)-activated mouse calvarial cells. From the above results, it may be suggested that IL-10 inhibites $IL-1{\beta}-induced$ osteoclast differntiation and bone resorption. However, the inhibitory effect of IL-10 on the osteoclast formation seems to be mediated not by the reduction of IL-6, GM-CSF, and $TNF-{\alpha}$ production, but by other mechanisms.