• IMMUNE NETWORK
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• 제13권1호
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• pp.30-33
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• 2013

Previously we showed that biodegradable nanoparticles containing poly-IC or CpG oligodeoxynucleotide (ODN) together with ovalbumin (OVA) were efficient at inducing MHC-restricted presentation of OVA peptides in dendritic cells. The CTL-inducing activities of the nanoparticles were examined in the present study. Nanoparticles containing poly-IC or CpG ODN together with OVA were prepared using biodegradable polymer poly(D,L-lactic acid-co-glycolic acid), and then were opsonized with mouse IgG. The nanoparticles were injected into the tail vein of mice, and 7 days later the OVA-specific CTL activities were measured using an in vivo CTL assay. Immunization of mice with the nanoparticles containing poly-IC or CpG ODN together with OVA elicited potent OVA-specific CTL activity compared to those containing OVA only. In accordance with these results, nanoparticles containing poly-IC or CpG ODN together with OVA exerted potent antitumor activity in mice that were subcutaneously implanted with EG7.OVA tumor cells. These results show that encapsulation of poly-IC or CpG ODN together with antigen in biodegradable nanoparticles is an effective approach for the induction of potent antigen-specific CTL responses in vivo.

• 한국식품영양과학회지
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• 제42권5호
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• pp.697-704
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• 2013

본 실험에서는 primary culture된 연골세포 in vitro 실험모델과 MIA로 유발한 골관절염 in vivo 실험모델을 이용하여 꾸지뽕나무 잎 추출물의 관절염 예방 효과를 확인하였다. 먼저 MTT 시험법을 통해 세포 사용 적정농도를 $500{\mu}g/mL$ 이하로 결정하여 연골세포사멸 억제를 확인하고, 이를 근간으로 골관절염 동물실험 모델에서 골관절염 예방효과를 확인하였다. $H_2O_2$ 처리에 따른 산화적 독성으로 연골세포 사멸을 유도한 실험에서 꾸지뽕 잎 추출물은 정상세포 수준으로 사멸을 억제하였으며, 이러한 효과는 CTL80의 $200{\mu}g/mL$, CTL10의 $300{\mu}g/mL$ 농도에서 비교적 높게 나타났다. 교원질 합성을 억제하고 분해를 촉진시키는 MMPs(MMP-7, MMP-13)의 발현을 실시간 정량 PCR로 측정하여 발현변화를 살펴보았다. 그 결과 앞선 세포실험 결과와 마찬가지로 CTL80과 CTL10 처리군에서 발현이 유의적으로 낮아졌음을 살펴볼 수 있었다. 특히 CTL80에서 MMP-7과 MMP-13의 발현이 농도 유의적으로 감소하였으며, CTL10의 경우 200, $300{\mu}g/mL$ 농도에서 유의적으로 발현이 감소하는 것을 확인하였다. 세포실험 결과를 바탕으로 동물실험에서의 적정농도를 결정하였으며, 동물독성실험 결과 이상이 없음을 확인하고 실험을 진행하였다. 이때 세포실험결과 선정된 두농도(200, $300{\mu}g/mL$) 간의 차이가 미미하여 동물실험에 적용할 경우 비슷한 실험결과가 나타날 것으로 사료되어 두 실험군 간의 결과를 정확히 구분 짓기 위해 200, $500{\mu}g/mL$ 농도를 선정하여 사용하였다. 골관절염 유발 동물모델을 만들기 위해 SD rat의 관절강에 MIA를 injection 하였으며, 꾸지뽕 잎 에탄올 추출물 투여에 따른 관절염 예방 효과를 관찰하기 위해 관절염 유발 2주일 전부터 1일 1회 경구투여를 실시하고, 유발 후 3주간 지속적으로 투여하고 관찰하였다. 동물 관절의 병리학적 변화를 관찰하기 위하여 Micro-CT 촬영 및 분석을 실시한 결과 Control 군은 골의 강도와 밀도가 감소한 반면, 양성대조군인 MTX 투여군에서 정상군과 비슷한 수준으로 회복된 것을 확인하였고, CTL80-200군과 CTL10-500군에서 Control 군에 비해 유의적으로 수치가 감소하여 골관절염에 따른 손상이 감소한 것을 확인하였다. 동물의 관절조직의 H&E 염색을 통한 조직학적인 변화에서는 골관절염 유발로 연골세포의 손상과 뼈조직의 손상을 관찰하였으며 관절형태를 알아볼 수 없을 정도로 손상된 것을 확인하였다. 반면 CTL80과 CTL10에서는 관절강 세포의 형태가 정상군과 비슷한 둥근모양을 띤 양상을 보였으며 연골조직의 형태가 잘 유지되어 Control 군에 비해 꾸지뽕잎의 투여효과가 나타났음을 관찰하였다. 이상의 결과를 통하여 꾸지뽕 잎 에탄올 추출물은 높은 항관절염 효과가 있을 것으로 사료되며, 항관절염 효능을 지니는 기능성 소재로써 개발 가능성을 확인하였다.

• IMMUNE NETWORK
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• 제3권4호
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• pp.295-301
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• 2003

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. In this study, we used a replication-deficient adenovirus containing CEA to study CTL induction in vitro after adenovirus-mediated gene transfer into DC. Methods: DC were obtained from mouse bone marrow and cultured with IL-4 and GM-CSF. For measuring CTL activity, splenocytes were harvested from the mice, which were immunized with DC that had been infected AdV-CEA or pulsed with CEA peptide. Untreated DC was used as a control. Splenocytes were re-stimulated in vitro with DC pulsed with CEA peptide for 7 days and CTL activity with CEA peptide-pulsed EL-4 cells were assessed in a standard $^{51}Cr$-release assay. The frequencies of antigen-specific cytokine-secreting T cell were determined with $mIFN-{\gamma}$ELISPOT. Results: DC infected with recombinant adenovirus expressing CEA induced CEA-specific CTL responses in vivo. Splenocyte induced from mice immunized with AdV-CEA-infected DC increase in the number of $IFN-{\gamma}$ secreting T cells compared with those from mice immunized with CEA peptide-pulsed DC. Conclusion: These results suggested that DC infected with recombinant adenovirus has advantages over other forms of vaccination and could provide an alternative approach vaccination therapies.

• IMMUNE NETWORK
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• 제3권4호
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• pp.302-309
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• 2003

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.

• 대한두경부종양학회지
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• 제19권1호
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• pp.25-33
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• 2003

Background and Objectives: The Herpes Simplex type 2 Defective Infectious Single Cycle virus (DISC virus) is attenuated virus originally produced as viral vaccines but are also efficient gene transfer vehicle. The main goals of this study were to examine the efficiencies of the gene transfer using DISC vectors for various head and neck squamous cell carcinoma cell lines and to evaluate the efficacy of vaccination with DISC virus carrying a immunomodulatory genes (GM-CSF) as cancer therapy in a organotopic oral cavity squamous cell cancer model. Materials and Methods : We determinated the gene transfer efficiency of DISC virus by x-gal stain method and proved gene and protein expression of DISC-GMCSF transfected SCCVII cells by RT-PCR and ELISA method. Also we evaluated the ex vivo vaccination effects of SCCVII/GMCSF (DISC-GMCSF transfected SCCVII vaccine) vaccine on preventing the recurrence of micro-residual tumor. After the vaccination of SCCVII/GMCSF, specific cytotoxic T-cell responses was evaluated by CTL assay. Results: At an MOI of 10 DISC virus showed 64-88% of transfection rates in various head and neck squamous cancer cell lines. SCCVII cells transduced by DISC virus vector (MOI=10) carrying the GM-CSF gene, produced 4.5 nanogram quantities of GM-CSF per $10^6$ cells. In vivo vaccination using tumor cells transduced ex vivo with DISC-GMCSF resulted in better protection rate against subsequent tumor recurrence in organotopic oral cavity cancer model. Although tumor free survival rate was not statistically significantly increased in vaccination group (p=0.078), tumor specific cytotocic T-cell responses were significantly increased in SCCVII/GMCSF vaccination group. Conclusion: These data demonstrate that; 1) The DISC virus vector is capable of efficient gene transfer to various head and neck squamous cancer cell lines, 2) GM-CSF secreting genetically modified tumor vaccine (SCCVII/GMCSF) efficiently protected against tumor recurrence in organotopic micro-residual oral cavity cancer model and produced tumor specific cytotoxic T-cell response. DISC virus-mediated, cytokine gene transfer may prove to be useful as a clinical therapy for head and neck cancers.

• Biomolecules & Therapeutics
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• 제25권2호
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• pp.140-148
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• 2017

Hyperlipidemia, which is closely associated with a fatty diet and aging, is commonly observed in the western and aged society. Therefore, a novel therapeutic approach for this disease is critical, and an immunological view has been suggested as a novel strategy, because hyperlipidemia is closely associated with inflammation and immune dysfunction. In this study, the effects of an aqueous extract of Rubus occidentalis (RO) in obese mice were investigated using immunological indexes. The mice were fed a high-fat diet (HFD) to induce hyperlipidemia, which was confirmed by biochemical analysis and examination of the mouse physiology. Two different doses of RO and rosuvastatin, a cholesterol synthesis inhibitor used as a control, were orally administered. Disturbances in immune cellularity as well as lymphocyte proliferation and cytokine production were significantly normalized by oral administration of RO, which also decreased the elevated serum tumor necrosis factor $(TNF)-{\alpha}$ level and total cholesterol. The specific immune-related actions of RO comprised considerable improvement in cytotoxic T cell killing functions and regulation of antibody production to within the normal range. The immunological evidence confirms the significant cholesterol-lowering effect of RO, suggesting its potential as a novel therapeutic agent for hyperlipidemia and associated immune decline.