Cows may suffer impaired ovarian function, often accompanied by reduced conception rates and increased embryonic loss. Cystic ovarian disease (COD) is one of the most frequently diagnosed gynecological findings in dairy cattle. It causes temporary infertility and is likely to affect reproduction as well as production parameters in cattle. Therefore, the purpose of this study was to determine the expression patterns of apoptosis (Bcl-2, Bax), implantation (E-cadherin) and immune related proteins (TNF-α, IL-10) in uterine endometrium of Hanwoo (Korean native cattle) with ovarian cyst and normal ovarian follicles. In the Western blot analysis, the expression of anti-apoptotic Bcl-2 protein was significantly higher in endometrium with normal ovarian follicles, whereas expression of pro-apoptotic Bax protein was significantly lower. Also, the expressions of E-cadherin and TNF-α proteins were significantly higher in uterine endometrium with normal ovarian follicles. On the other hand, the expression of IL-10 protein was significantly lower in uterine endometrium with normal ovarian follicles. Taken together, our results provided that the expressions of apoptosis, adhesion and immune related proteins in uterine endometrium with ovarian cyst were showed the aberrant patterns, and we suggest that different expression changes of these proteins may be affect to pregnancy ability of cattle.

시스타틴(cystatin: CST)은 C1A류 시스테인 단백질분해효소에 대한 경쟁적 가역억제자로서 동식물류에서 파파인과 같은 캐셉신을 억제 대상으로 작용하게 된다. 바이러스 유래 CST (CpBV-CST1)이 폴리드나바이러스의 일종인 CpBV (Cotesia plutellae bracovirus)에서 동정되었 다. 기존 연구는 이 유전자의 과발현이 배추좀나방(Plutella xylostella) 유충의 면역 및 발육을 교란한다는 것을 보여 주었다. 본 연구는 이 유전자 의 단백질 기능을 분석하기 위해 세균발현시스템을 이용하여 재조합단백질(rCpBV-CST1)을 형성하여 단백질분해효소에 대한 활성억제효과를 결정하고, 곤충의 면역과 발육에 대한 생리적 억제효과를 분석했다. 이 유전자 번역부위는 138 개 아미노산으로 약 15 kDa 크기의 단백질로 추 정되었다. CpBV-CST1이 먼저 pGEX 발현벡터에 재조합되고, BL21 STAR (DE3) competent cells에 형질전환된 후 0.5 mM IPTG로 4 시 간동안 과발현되었다. 분리된 재조합단백질은 파파인에 대한 뚜렷한 억제효과를 나타냈다. 이 재조합단백질은 파밤나방(Spodoptera exigua)에 대 해서 혈구소낭형성의 세포성 면역반응을 억제하고, 경구로 처리할 때 배추좀나방의 유충발육을 처리 농도에 비례하여 제한시켰다. 이상의 결과 는 CpBV-CST1이 해충 밀도 억제에 응용될 수 있음을 제시하고 있다.

한우 송아지의 생후 환경스트레스에 대한 면역반응성 및 신바이오틱제제의 급여효과를 구명하기 위해서 출생 후부터 각 처리구별로 5두씩 배치하여 총10두(2처리×5두)를 대상으로 대조구(신바이오틱제제미투여)와 처리구(신바이오틱제제 투여구)로 배치하여 총 65일간(포유기간) 시험하였다. 송아지 체중은 대조구(21.4±2.51 kg - 57.0±12.83 kg)에 비해 신바이오틱제제 급여 처리구(22.0±2.12 kg - 53.2±4.32 kg) 개체간의 체중의 편차를 줄이는 역할을 하는 것으로 나타났다. Nitric oxide는 신바이오틱제제 처리구(12.52 μM/L-39.72 μM/L)가 대조구(19.68 μM/L-64.80uM/L)보다는 낮았으며, 코티졸 농도는 한우 송아지 신바이오틱제제 처리구에서 7일령, 45일령, 그리고 65일령에서 유의적으로 감소하였고(P<0.05), 혈중 Glutathione는 65일령에서 유의적으로 증가하였다(P<0.05). SOD의 농도는 신바이오틱제제 처리구의 28일령과 65일령에서 유의적으로 감소하였고(P<0.05), H2O2의 농도는 신바이오틱제제 처리구 45일령과 65일령에서 유의적으로 감소하였다(P<0.05). Interlukin-1β 농도는 대조구에서 28일령(43.26±4.40 pg/mL)과 처리구 21일령(24.68±3.20 pg/mL)에 가장 높았으며, 14일령, 28일령, 65일령의 신바이오틱제제 처리구에서는 유의적으로 낮았다. IL-2 농도는 대조구 28일령 (218±16.94 pg/mL)과 신바이오틱제제 처리구 7일령(174.60±11.60 pg/m)에서 각각 가장 높았고, 21일령 이후 처리구에서 유의적으로 감소하였다. IL-6 농도 또한 대조구 28일령(403.20±48.19 pg/mL)과, 신바이오틱제제 처리구 65일령( 238.20±15.63 pg/mL)에서 가장 높았으며, 21일령 이후 신바이오틱제제 처리구에서 유의적으로 낮았다. PGE2 농도는 대조구 45일령(3660±463.25 pg/mL)과 신바이오틱제제 처리구 45일령(1070±141.92 pg/mL)에서 각각 가장 높았으며, 14일령 이후 신바이오틱 제제 처리구에서 유의적으로 낮았다. 앞의 결과를 종합해보면, 신바이오틱제제의 경구투여로 생체내의 싸이토카인의 체내 균형조절 및 면역 염증반응을 억제하는 데 관여하는 것으로 보인다. 그리고 신바이오틱제제의 급여로 분만 직후 한우 송아지의 체내의 변화가 체외의 변화로 이어져 가축의 성장촉진 및 열악한 주변환경 극복 등에 보다 더 확실한 효과를 기대하기 위해서는 신바이오틱제제의 급여기간을 늘릴 필요가 있을 것으로 사료된다.

Cotesia plutellae known as an endoparasitoid parasitizes larvae of the diamondback moth, Plutella xylostella which is a major pest in cruciferous crops. For the successful parasitization, maternal and embryonic factors of C. plutellae such as polydnavirus, ovarian proteins, teratocytes and venom are required. In this study, we identified calreticulin (Cp-CRT) gene from transcriptome data of the venom gland in C. plutellae. cDNA of CRT was cloned from total RNA of the venom gland via PCR and encodes 403 amino acids harboring several structural motifs such as a signal peptide sequence, a repetitive sequence, a putative coiled-coil sequence encompassing, and endoplasmic reticulum-recognizing domain (-KDEL). Phylogenetic analysis showed that the Cp-CRT gene formed a unique cluster with other hymenopteran CRT genes, indicating that the Cp-CRT belongs to the CRT family. To examine the physiological function of Cp-CRT, recombinant Cp-CRT, fused with 6X-His at N-terminal was constructed and expressed in E. coli. Recombinant Cp-CRT was successfully expressed via Western blot analysis and suppressed significant nodule formation when co-injected with E. coli as immune response inducer. These results suggest that the Cp-CRT involves in suppression of cellular immune response in the host

‘급속면역금나노입자막대 (RIGS) 키트’라 명명된 RIGS 키트는 간단한 면역 흡착막대 방식으로 사용자가 빠르고 편리하게 이용할 수 있으며 토마토덤불위축바이러스 (TBSV)에 대한 현장진단을 하기 위하여 개발되었다. 토끼의 TBSV 항 혈청에서 정제된 면역글로블린G (IgG)는 단백질A 크로마토그래피법으로 정제되었으며 이후 금 나노입자와 결합하여 니트로셀룰로스막에서 진단 선을 표시하도록 고안되었다. TBSV 항체와 비특이적으로 결합하는 단백질A를 같은 진단막대에서 대조선으로 이용되었다. RIGS-TBSV 키트를 이용한 진단은 의심 식물 시료를 완충액이 들어간 플라스틱 봉지에 넣고 착즙후 진단막대기를 넣으면 5-10분 후 결과를 알 수 있도록 고안되었다. TBSV가 감염된 토마토 즙액에 RIGS 막대기를 넣고 진단한 결과 TBSV 농도에 비례하여 진단 선이 형성됨이 관찰되었으며, 이들 키트들은 TBSV와 연관되지 않은 다른 고추 바이러스들에서는 비특이적 반응이 형성되지 않았다. 이런 결과들은 RIGS-TBSV 키트가 TBSV 진단에 다른 어려운 실험이나 기술이 필요하지 않고 쉽게 병원균을 진단 할 수 있다는 것을 의미한다. 그러므로, RIGS-44 TBSV 키트는 TBSV 감염이 의심되는 식물체들의 현장 진단 뿐만 아니라 실험실에서 의 TBSV 진단에 효과적이라 할 수 있다.

Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in Protaetia brevitarsis seulensis. we identified the professional phagocytes, granulocytes (GRs), which mediate encapsulation and phagocytosis of pathogens. The GRs were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo.

Prostaglandins (PGs) mediate insect immune responses. However, their biosynthesis in insects is little understood due to lack of cyclooxygenase (COX) ortholog. This study aimed to identify PG-biosynthetic factor(s) in Spodoptera exigua, which has been a well-known insect in possessing immune responses mediated via PGs. Peroxidases (POXs) are a sister group of COX genes. Ten putative POXs (POX-A ∼POX-J) were expressed in S. exigua. Especially, expressions of POX-F and POX-H were inducible to bacterial challenge and expressed in hemocytes and fat body. Individual RNA interference (RNAi) of each of ten POXs was performed by hemocoelic injection of their specific double-strnaded RNAs (dsRNAs). Only RNAi of POX-F or POX-H specifically suppressed hemocyte-spreading behavior and nodule formation. Addition of PGE2 significantly nescued the immunosuppression in either dsRNA treatment of POX-F or POX-H. Structural analysis indicated that both POX-F and POX-H have conserved domain and residues corresponding to peroxinectin of Drosophila melanogaster, which mimics COX-like activity. These results suggest that POX-E and POX-H are involved in PG biosynthesis in S. exigua.

Polydnavirus are well known to interfere with the host endocrine system, causing immune suppression and other physiological disorders. The Cotesia rubculla polydnavirus gene products, CrV1, are known to be a potent immunosuppressive agent. CrV1 protein express within 12 h after viral infection at oviposition during deposition of parasitoid eggs and are mainly secreted in to host hemocyte, where it functions like phagocytosis and cell spreading. This study identified its homolog in CpBV and analyzed its molecular characteristics motif called “coiled-coil. A point mutation of Alanine to Proline of CpBV-CrV1 could lose the coiled-coil motif from in silico assay. The coiled type CpBV-CrV1 could inhibit host cellular immunity, however, interestingly the mutant CpBV-CrV1 lacking in coiled-coil motif completely lost the immunosuppressive activity. This study suggests that the coiled-coil motif is functional to inhibit host cellular immune responses. RNA interference against CrV1 significantly loses the inhibitory activity and thus further supporting the immunosuppressive activity of CrV1. In this study we also have analyzed the localization of CrV1 by transient transfection in HiFive Cell lines by in situ hybridization.

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2,517 bp) of a integrin subunit β1 (HaITGβ1) was cloned from the oriental tobacco budworm, Helicoverpa assulta. Phylogenetic analysis showed that HaITGβ1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (61%) to β1 of other Noctuidae such as Spodoptera exigua and S. litura. Structural analysis of the HaITGβ1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that HaITGβ1 was expressed in all developmental stages and all tested tissues of H. assulta. Injection of double-stranded HaITGβ1 RNA (dsHaITGβ1) into third instar of H. assulta suppressed HaITGβ1 expression and resulted in significant delay from last larval stage to pupal stage. The dsHaITGβ1 injection significantly impaired nodule formation of H. assulta in response to bacterial challenge and hemocyte adherence. These results suggest that HaITGβ1 plays crucial roles in cellular immune responses as well as development in H. assulta.

Brucellosis is a zoonotic infectious disease of domestic animals, wild animals and humans. Innate immunity is a rapid and non-specific immune response that occurs during the early stages of Brucella invasion. Physical barriers such as epithelial cells and gastric juice secretions form the first line of defense. Humoral components such as complement and lysozyme can remove microorganisms by opsonization and bactericidal actions. Cellular components of the immune system, including macrophages, dendritic cells, neutrophils and innate T cells, have major roles in innate immunity. They recognize invading Brucella spp. by various cell surface receptors and then kill both the invading microorganisms and infected cells owing to their phagocytic or cytotoxic activity. In addition, they present Brucella antigens or produce cytokines to trigger adaptive immunity. Activated adaptive immunity consists of T helper cells, cytotoxic T cells and antigen-specific antibody-producing B cells. These can eliminate Brucella spp. effectively via antigen-specific mechanisms and by immunological memory. T cells activate bactericidal functions in macrophages by producing cytokines such as IFN-γ and by exerting cytotoxic effects on the infected cells. B cells produce antigen-specific antibodies that neutralize or opsonize the antigen. Because Brucella spp. can survive in macrophages and other host cells, Th-1 cellular immunity that enhances the bactericidal effects of phagocytic cells and the cytotoxic effects of lymphocytes is more important than humoral immunity in Brucella infection.

Pathologic chronic inflammation, such as that seen with microbial infection and autoimmune diseases, creates microenvironmental conditions that promote cancer. Therefore, if chronic inflammation can be alleviated, the risk of carcinoge- nesis may decrease. Turmeric is a dried rhizome powder from Curcuma longa. Curcumin, the major constituent of turmeric, presents anti-inflammatory, antioxidant, antimicrobial and chemopreventive activities. In the present study, we investigated immune responses to dietary turmeric in ICR mice to determine the effects of turmeric when used as a dietary chemoprevention agent. After dietary turmeric was given for three or six weeks to ICR male mice, the immune responses were characterized. The methods of characterization involved; the determination of the T cell subpopulation in the spleen, relative mRNA expression levels of IFN-γ and TNF-α and serum lysozyme activity. Dietary turmeric was found to decrease spleen weight, decrease the proportion of CD4-CD8+ T cells, and decrease phagocytic activity. These results suggest that turmeric might alleviate abnormal chronic inflammation by the action of immune suppression.

‘급속면역금나노입자막대 (RIGS) 키트’라 명명된 빠르며 사용자 편의 및 간단한 면역흡착막대 방식의 키트가 오이모자이크바이러스 (CMV)의 현장진단을 하기 위하여 개발되었다. 토끼의 CMV항혈청에서 정제된 면역글로블린G (IgG)는 단백질A 크로마토그래피법으로 정제되었으며 이후 금나노입자와 결합하여 니트로셀룰로스막에서 진단선 표시하도록 고안되었다.CMV항체와 비특이적으로 결합하는 단백질A를 같은 진단막대에서 대조선으로 이용되었다. RIGS-CMV 키트를 이용한 진단은 의심 식물 시료를 완충액이 들어간 플라스틱 봉지에 넣은 후 착즙 후 진단막대기를 넣으면 된다. 결과는 5-10분 후알 수 있다. CMV가 감염된 고추, 오이 및 멜론의 즙액에RIGS 막대기를 넣고 진단한 결과 CMV 농도에 비례하여 진단선이 형성됨이 관찰되었으며, 이들 키트들은 CMV와 연관되지 않은 다른 고추바이러스들에서 비특이적 반응이 형성되지 않았다. 이런 결과들은 RIGS-CMV 키트가 매우 민감하며,진단에 별 다른 실험실 기술이나 경험이 필요하지 않는 다는것을 의미한다. 그러므로, RIGS-CMV 키트는 CMV 감염이의심되는 식물체들의 현장 진단 뿐만 아니라 실험실에서의CMV 진단에 효과적이다.

Platycodon grandiflorum have been used as a traditional remedy and food source. This study was performed to investigate the immunomodulating effects of Platycodon grandiflorum in mouse, using ex vivo experiments. Six to seven-week old mice were fed ad libitum on a chow diet, and water extract of Platycodon grandiflorum was orally administrated at two different concentractions (50 and 500 ㎎/㎏ B.W./day) every other day for four weeks. In ex vivo experiments, the highest proliferation of splenocytes and levels of cytokine (IL-1β, IL-6, TNF-α) production were observed in 500 ㎎/㎏ BW/day supplementation group for all three cytokines stimulated by LPS. In conclusion, this study suggests that Platycodon grandiflorum extracts may enhance the immune function by regulating the splenocytes proliferation and cytokine production capacity by activating macrophages in mice.

김치에서 Weissella속, Leuconostoc속, Lactobacillus속 등의 154주의 젖산균을 분리할 수 있었다. 분리한 24주의 젖산균을 시료화하여 RAW264.7 cell의 세포 생존률을 기준으로 선별된 24주의 젖산균의 면역활성능을 확인한 결과IL-1α는 FBT215주가 676.9±22.2pg/mL로, TNF-α는 FBT202주가 1105.3±30.5pg/mL로 가장 많이 분비량이 높았다.종합적으로 확인하였을 때 FBT215가 cytokine 분비를 촉진하였다. 또, 내산성을 확인하기 위해 인공 위액에서 배양한결과 대체적으로 산성에 잘 견디는 것을 알 수 있었고, 내담즙성을 확인하기 위하여 oxgall을 첨가한 배지에 FBT215주를 접종한 후 3시간까지는 증식을 하였으나, 그 뒤는 담즙으로 인하여 생균수가 감소하는 것을 확인했다. BSH 활성을 통해 FBT215주는 BSH 활성이 양성으로 나타났으므로 담즙 분해 효소가 있는 것을 확인할 수 있었다. API 50CH kit와 16S rRNA sequencing을 통하여 FBT215를 동정한 결과 Lactobacillus plantarum 임을 확인하였다.

Matrix 2 protein ectodomain (M2e) of influenza virus appears to be a promising vaccine candidate because its sequence is highly conserved among virus strains. However, M2e is too meager to induce a strong immune response by itself. Several approaches are being used to increase the antigenicity of M2e. In an effort to enhance the M2e-specific immune response, we generated a TAT-conjugated M2e recombinant protein. Seven-week-old BALB/c mice were divided into three groups and transcutaneously immunized with 100 μg TAT-8×M2e (TAT conjugated with eight copies of M2e) and 8×M2e (eight copies of M2e) proteins on days 1, 15 and 29. The control mice were injected with PBS on the same days. Antibody titers specific for M2e were measured using indirect ELISA. Mice immunized with the TAT-8×M2e and 8×M2e proteins developed almost the same levels of M2e-specific total IgG and IgG1 antibodies. However, a higher level of M2e-specific IgG2a was observed in mice immunized with TAT-8×M2e than in those immunized with 8×M2e. These results suggest that TAT has an adjuvant effect that induces a Th1-type immune response. Therefore, the TAT-M2e vaccine can be applied to animals as a new influenza vaccine for enhancement of Th1-type immune responses.

The purpose of the study was to investigate an effect of water temperature on a non-specific immune response and mortality of tilapia, Oreochromis niloticus, following a bacterial infection. Seventy five tilapia acclimated to 25℃ were then transferred at 16 and 36℃, and examined for non-specific immune responses over 12-96 h. Respiratory burst activity was reduced significantly in the group of fish cultured at 16 and 36℃ over 24-96 h, whereas phagocytic activity decreased significantly in the group of fish reared at a low temperature (16℃) over 12 and 24 h and high temperatures (36℃) over 12-96 h. Lysozyme activity diminished significantly in the group of fish transferred to 16℃ over 12-48 h, but increased significantly in the group of fish at 36℃ over 48 and 96 h. Alternative complement pathway (ACH50) decreased significantly when transferred to 16℃ after 12 h, but increased significantly when transferred to 36℃ after 24 h. In a challenging test, 30 tilapia reared at 25℃ were injected intraperitoneally with Streptococcus iniae at a dose of 2x107 cfu/fish, and then reared onward at water temperatures of 15, 25 (control), and 36℃. Over 12-96 h, the cumulative mortality of S. iniae-injected fish held in 16 and 36℃ was significantly higher than that of injected-fish held in 25℃ In conclusion, transfer of tilapia from 25℃ to low temperature (16℃) after 12 h, and transfer of fish from 25℃ to high temperature (35℃) reduced their immune capability. Furthermore, tilapia under temperature stress at 16 and 36℃ from 25℃ decreased its resistance against S. iniae

Immune mediators play crucial roles in amplifying the emergency signals with massive amounts of de novo synthesized mediators and relaying the specific recognition signals to the immune-associated target tissues. Eicosanoids are the representative immune mediators and synthesized from a polyunsaturated fatty acid (PUFA), arachidonic acid. Compared to mammalian systems, insects have relatively low levels of arachidonic acid in the biological membranes. This has raised a fundamental issue that eicosanoids may be not significant in insect system. Our previous chemical analysis suggests that the hemocytes of Spodoptera exigua have less than 5% arachidonic acid. We postulated that S. exigua may store arachidonic acid in other tissues, such as fat body. This analysed fatty acid compositions of two immune-associated tissues using a gas chromatography (GC) eguipped with FID detector or GC-MS. Our analysis of PUFA in the immune tissues suggests that insects maintain a low level of PUFA including arachidonic acid due to its evolutionary origin from the paleozoic era at which the oxygen level was 35%, compared to the present era 21%.

The present study is designed to explore an anti-tumor activity on crude extracts of Oplopanax elatus. Water extractions of Oplopanax elatus were performed at 100℃(OeE-100). OeE-100 doses up to 62.5 ㎍/㎖ had no cytotoxicity on the tumor cell lines in vitro. In experimental lung metastasis of colon26-M3.1 carcinoma or B16-BL6 melanoma, the prophylactic intravenous (4~100 ㎍/mouse) or oral (2 ㎎/mouse) administration of OeE-100 significantly inhibited tumor metastasis as compared with tumor controls. Peritoneal macrophages stimulated with OeE-100 produced various cytokines such as TNF-α, IL-6 and IL-12. In an analysis of NK-cell activities, i.v. administration of OeE-100 (10~100 ㎍/mouse) significantly augmented the cytotoxicity to YAC-1 tumor cells. Vaccination of mice with boiling-treated tumor cells (BT-vaccine) in combination with OeE-100 (100 ㎍/mouse) showed higher inhibitions in tumor metastasis when compared with the mice of BT-vaccine treatment. In addition, the splenocytes from OeE-100 admixed BT-vaccine immunized mice secreted a higher concentration of Th1 type cytokine such as IFN-γ. These results suggested that the OeE-100 stimulated immune system and was a good candidate adjuvant of anti-tumor immune responses.