Background: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. Methods: We have used CHO-$dhfr^-$ cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. Results: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. Conclusion: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection.
Kim, Kwang-Dong;Kim, Jin-Koo;Kim, Se-Jin;Choe, In-Seong;Chung, Tae-Hwa;Choe, Yong-Kyung;Lim, Jong-Seok
Archives of Pharmacal Research
/
v.22
no.4
/
pp.340-347
/
1999
Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with none marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocutured DCs was able to induce complete protectiv immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DC s induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be used for designing tumor vaccines using DCs when the information about tumor antigens is limited.
Ha, Eun-Suk;Hwang, Soo-Hyun;Shin, Kwang-Soon;Yu, Kwang-Won;Lee, Keyong-Ho;Choi, Joo-Sun;Park, Woo-Mun;Yoon, Taek-Joon
Archives of Pharmacal Research
/
v.27
no.2
/
pp.217-224
/
2004
Previously, we reported that water-extracted Acanthopanax senticasus exhibited anti-meta-static activity by stimulating the immune system. In this study, we fractionated glycoproteins (EN-SP) from the soluble protein layer (GF-AS) of A. senticasus and determined their basic chemical properties. We also investigated the anti-tumor and immunostimulating activities of the fractionated glycoprotein, EN-SP. We found that intravenous (i.v.) administration of GF-AS dramatically inhibited metastasis of colon26-M3.1 carcinoma cells to the lung in a dose-dependent manner. In vitro analysis showed GF-AS to enhance the proliferation of splenocytes. GF-AS also stimulated peritoneal macrophage, which was followed by the production of various cytokines such as IL-1$\beta$, TNF-$\alpha$, IL-12 and IFN-${\gamma}$. Furthermore, the production of these cytokines was partially blocked when peritoneal macrophage was cultured with the polyclonal antibodies against GF-AS. The depletion of NK cells by rabbit anti-asialo GM1 serum partly abolished the inhibitory effect of GF-AS on lung metastasis of colon26-M3.1 cells. Using gel filtration, EN-SP, an active glycoprotein fraction, is isolated from GF-AS. While both GF-AS and EN-SP stimulated the proliferatation of splenocytes of normal mice, EN-SP showed higher anti-metastatic activity and more potently stimulated the proliferation of splenocytes compared to GF-AS. These results suggest the use of EN-SP, the fractionated glycoprotein from A. senticasus, can be used as a therapeutical reagent to prevent or inhibit tumor metastasis.
Seo, Seung-Ho;Park, Seong-Eun;Moon, Yang-Seon;Lee, Yu-Mi;Na, Chang-Su;Son, Hong-Seok
Korean Journal of Food Science and Technology
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v.48
no.5
/
pp.515-520
/
2016
This study was carried out to investigate the component and immunoregulatory effects of Sparassis crispa stipe. Herein, S. crispa was divided into the pileus and stipe to compare their ingredients (${\beta}-glucan$), antioxidant activity (in vitro), and the immunoregulatory function (cytokines, leukocytes, and spleen weight). The ${\beta}-glucan$ content in each part showed about 1.8 times higher content in the stipe than that in the pileus. The stipe also showed a higher total phenol content and antioxidant activity than the pileus. The cytokines $TNF-{\alpha}$, IL-2, and IL-10 have adjusted in the S. crispa extract-injected groups. In addition, the number of leukocytes was also significantly elevated in the rats administered with the S. crispa stipe extract. These results suggest that the stipe of S. crispa has great potential as an ingredient in functional foods.
Jae In, Jung;Hyun Sook, Lee;So Mi, Kim;Soyeon, Kim;Jihoon, Lim;Moonjea, Woo;Eun Ji, Kim
Nutrition Research and Practice
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v.16
no.6
/
pp.685-699
/
2022
BACKGROUND/OBJECTIVES: Platycodon grandiflorum (PG) has long been known as a medicinal herb effective in various diseases, including bronchitis and asthma, but is still more widely used for food. Fermentation methods are being applied to increase the pharmacological composition of PG extracts and commercialize them with high added value. This study examines the hydrolyzed and fermented PG extract (HFPGE) fermented with Lactobacillus casei in RAW 264.7 cells, and investigates the effect of amplifying the immune and the probable molecular mechanism. MATERIALS/METHODS: HFPGE's total phenolic, flavonoid, saponin, and platycodin D contents were analyzed by colorimetric analysis or high-performance liquid chromatography. Cell viability was measured by the MTT assay. Phagocytic activity was analyzed by a phagocytosis assay kit, nitric oxide (NO) production by a Griess reagent system, and cytokines by enzyme-linked immunosorbent assay kits. The mRNA expressions of inducible nitric oxide synthase (iNOS) and cytokines were analyzed by reverse transcription-polymerase chain reaction, whereas MAPK and nuclear factor (NF)-κB activation were analyzed by Western blots. RESULTS: Compared to PGE, HFPGE was determined to contain 13.76 times and 6.69 times higher contents of crude saponin and platycodin D, respectively. HFPGE promoted cell proliferation and phagocytosis in RAW 264.7 cells and regulated the NO production and iNOS expression. Treatment with HFPGE also resulted in increased production of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, C-X-C motif chemokine ligand10, granulocyte-colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1, and the mRNA expressions of these cytokines. HFPGE also resulted in significantly increasing the phosphorylation of NF-κB p65, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. CONCLUSIONS: Taken together, our results imply that fermentation and hydrolysis result in the extraction of more active ingredients of PG. Furthermore, we determined that HFPGE exerts immunostimulatory activity via the MAPK and NF-κB signaling pathways.
Proceedings of the Korean Society for Food Science of Animal Resources Conference
/
2001.11a
/
pp.43-56
/
2001
Colostrum contains various kinds of cytokines including TGF-${\beta}$ which is known to be multifunctional in immune response and act as an anti-inflammatory agent. First, we measured the amount of TGF-${\beta}$ in bovine and human colostrum. Expression pattern of TGF-${\beta}$ isotypes was dramatically different between human and bovine colostrial samples. Bovine colostrum collected on day 1 post-delivery retained $41.79{\pm}16.96ng/ml$ of TGF-${\beta}$ 1 and $108.4{\pm}78.65ng/ml$ of TGF-${\beta}$ 2 while in human, $284{\pm}124.75ng/ml$ of TGF-${\beta}$ 1 and $29.75{\pm}6.73ng/ml$ of TGF-${\beta}$ 2. Thus, TGF-${\beta}$ is the predominant TGF-${\beta}$ isotype in bovine colostrum and vice versa in human colostrum. Both TGF-${\beta}$ isotypes diminished significantly in human and bovine colostrum with time. Next, biological activity of colostrial samples was examined in vitro. Both human and bovine colostrum increased IgA synthesis by LPS-activated mouse spleen B cells, which is a typical effect of TGF-${\beta}$ on the mouse B cell differentiation. Futhermore, we found that anti-proliferative activity in MV1LU cells by colostrum samples disappeared by addition of anti-TGF-${\beta}$ 1 and anti-TGF-${\beta}$ 2 antibody. In conclusion, there are substantial amounts of biologically active TGF-${\beta}$ 1 and TGF-${\beta}$ 2 in bovine and human colostrum. The results that the colostrum can increase IgA expression has important implications since IgA is the major Ig class produced in the gastrointestinal tract. We have previously shown that the stimulatory effect of Bifidobacteria bifidum on spllen B cells was quite similar to that of LPS which is a well-known polyclonal activator for murine B cells. In the present study, we further asked whether B. bifidum regulate the synthesis of IgA by mucosal lymphoid cells present in Peyers patches (PP) and mesenteric lymph nodes (MLN). B. bifidum alone, but not C. perfringens, significantly induced overall IgA and IgM synthesis by both MLN and PP cells. This observation indicates that B. bifidum possesses a modulatory effect on the mucosal antibody production in vivo. We, therefore, investigated the mucosal antibody prodduction following peroral administration of B. bifidum to mice. Ingested B. bifidum significantly increased the numbers of Ig (IgM, IgG, and IgA) secreting cells in the culture of both MLN and spleen cells, indicating that peroally introduced B. bifidum enhances mucosal and systemic antibody response. Importantly, however, B. bifidum itself does not induce the own specific antibody responses, implying that B. bifidum do not incite any unwanted immune reaction. Subsequently, it was found that excapsulation of B. bifidum further augments the total IgA production by increasing the number of IgA-secreting cells in the culture of both MLN and spleen cells. Finally, we found that the immuno-stimulating activity of B. bifidum is due to its cell wall components but not due to any actively secreting component(s) from bacteria. Thus our data reveal that peroral administration of B. bifidum can enhance intestinal IgA production and that encapsulation of B. bifidum further reinforces the IgA production.
Ginsan, a polysaccharide isolated from Panax ginseng, has been shown to be a potent immunomodulator, producing a variety of cytokines such as TNF-a, IL-1$\beta$, IL-2, IL-6, IL-12, IFN-${\gamma}$ and GM-CSF, and stimulating lymphoid cells to proliferate. In the present study, we analyzed some immune functions 1$^{st}$-5$^{th}$ days after ginsan i.p. injection, including the level of non-protein thiols (NPSH) as antioxidants, heme oxygenase (HO) activity as a marker of oxidative stress, zoxazolamine-induced paralysis time and level of hepatic cytochrome P-450 (CYP450) as indices of drug metabolism system, and activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, and albumin level as indicators of hepatotoxicity. Ginsan in the dose of 100 mg/kg caused marked elevation (1.7-2 fold) of HO activity, decrease of total CYP450 level (by 20-34%), and prolongation of zoxazolamine-induced paralysis time (by 65-70%), and showed some differences between male and female mice. Ginsan treatment did not seem to cause hepatic injury, since serum AST, ALT, and ALP activities and levels of total bilirubin and albumin were not changed.d.
Journal of the Korean Society of Food Science and Nutrition
/
v.43
no.1
/
pp.9-15
/
2014
To characterize novel biologically-active ingredients in traditional Korean soy sauces, polysaccharide fractions were isolated from two different soy sauces made either commercially (CSP-0) or through a traditional Korean process (KTSP-0), after which their intestinal immune-modulating activities were examined. CSP-0 and KTSP-0 showed enhanced production of interleukine-6 (IL-6) in culture supernatant of Peyer's patch cells. However, KTSP-0 activity was more potent than that of CSP-0. Only KTSP-0 increased in vitro immunoglobulin A (IgA) production by Peyer's patch cells in a dose-dependent manner. KTSP-0 also showed the higher bone marrow cell proliferation activity through Peyer's patch cells than that of the CSP-0 group. To investigate the in vivo effects on the intestinal immune system, CSP-0 and KTSP-0 were administered orally to four experimental groups of mice (0.0, 0.5, 1.0, and 5.0 mg/mouse/day, 30 days). Oral administration of CSP-0 and KTSP-0 induced IgA production by Peyer's patch cells and increased IgA excretion into mouse stools in a dose-dependent manner. Peyer's patch cells from the mice administered both CSP-0 and KTSP-0 showed significantly higher IL-6 production than that of the untreated or CSP-0 groups. However, oral administration of KTSP-0 was more effective at the same dosage. KTSP-0 administration augmented IL-6 content in mouse sera, whereas CSP-0 did not show any effect on IL-6 induction. The above data lead us to conclude that the intestinal immune-stimulating activities of polysaccharides from Korean traditional soy sauce are much better than those of commercial ones.
Journal of the Korean Society of Food Science and Nutrition
/
v.45
no.7
/
pp.948-957
/
2016
We previously developed an herbal composition (HemoHIM) based on the water extracts of Angelica gigas radix, Cnidium officinale rhizoma, and Paeonia japonica radix to protect and recover hematopoietic and intestinal tissues against radiation injuries. In this study, to develop a composition with improved activities based on enhanced fat-soluble polyphenol contents, we prepared a new herbal composition, MH-30, from the above three herbs by 30% ethanol extraction and hot water extraction. HPLC analysis of the ethanol fractions of MH-30 and HemoHIM revealed that MH-30 had higher contents of many fat-soluble polyphenol compounds than HemoHIM (8.7-fold increase for decursin), whereas contents of water-soluble polyphenol compounds showed little differences between the two compositions. Then, we evaluated MH-30 and HemoHIM for their in vitro antioxidant and immune cell-stimulating activities as well as in vivo protective effects against radiation injuries in hematopoietic and self-renewal tissues. In antioxidant activity assays, MH-30 showed higher hydroxyl radical scavenging activity than HemoHIM (1.4- to 1.9-fold for compositions and 2.3- to 4.5-fold for ethanol fractions). On the other hand, MH-30 and HemoHIM exhibited similar immune cell-stimulating activities as measured by in vitro lymphocyte proliferation. MH-30 increased endogenous spleen colony formation, decreased bone marrow cell apoptosis, and enhanced survival of intestinal crypts in irradiated mice, demonstrating effective protection of MH-30 against radiation-induced injuries in hematopoietic and self-renewal tissues. The 30-day survival rate of lethally irradiated mice, a comprehensive index for radioprotective efficacy, was also elevated by MH-30. Noticeably, MH-30 showed higher protective effects than HemoHIM in all mouse experiments. These results demonstrate that MH-30 can protect hematopoietic and self-renewal tissues against radiation injuries more effectively than HemoHIM. Therefore, MH-30 can be a good candidate to reduce radiation injuries in hematopoietic and self-renewal tissues incurred by radiation accidents or cancer radiation therapy.
This study was conducted to determine the effects of dietary supplementation of either 100 mg/kg chito-oligosaccharide (COS) or chlortetracycline (CTC) with corn-soybean-fish meal on immunity in broiler chickens. A total of 147 one-day old male broiler chicks were randomly allocated to 3 treatments with 7 replicate pens per treatment and 7 birds per pen. The experimental diets consisted of a control diet based on corn, soybean and fish meal without COS and any antibiotic supplement and similar diets supplemented with either CTC (80 mg/kg from d 1 to 21 and 50 mg/kg from d 22 to 42) or COS (100 mg/kg from d 1 to 42). During the entire experimental period, all birds had ad libitum access to diets and water. The main immune organ indices, T-lymphocyte proliferation, serum cytokine concentrations, serum NO level and serum iNOS activity were measured on d 21 and d 42. On d 21, broilers fed 100 mg/kg COS had improved (p<0.01) indices of spleen, thymus, and bursa of Fabricius compared with the control and CTC birds. Birds receiving 100 mg/kg COS had higher (p<0.05) serum concentrations of $IL-1{\beta}$, IL-6, IgM, NO and iNOS than birds on the control treatment. Serum $Ca^{2+}$ level of birds fed 100 mg/kg COS tended to be higher (p = 0.049) than in birds fed CTC. On d 42, the birds fed 100 mg/kg COS had higher (p<0.05) concentrations of TNF-${\alpha}$ and IgM in serum than birds in both the CTC and control treatments. Birds fed 100 mg/kg COS had a higher concentration of IFN-$\gamma$ than the control group. In conclusion, dietary supplementation of COS appeared to improve the immunity of broilers by promoting the weight of the main immune organs, increasing IgM secretion, stimulating microphages to release $TNF-{\alpha}$, $IL-1{\beta}$, IL-6 and IFN-$\gamma$, and activating iNOS to induce NO.
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