Immunostimulatory effect by aqueous extract of <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> in RAW 264.7 macrophage and whole spleen cells

Hizikia fusiforme is a commonly used food that possesses potent anti-bacterial, anti-fungal, and anti-inflammatory activities. The immunostimulatory activities of aqueous extract of Hizikia fusiforme (HFAE) in RAW 264.7 macrophages and whole spleen cells were investigated. HFAE activated RAW 264.7 macrophages to produce cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a dose-dependent manner. In addition, HFAE induced the mRNA expression of TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages. Moreover, HFAE stimulated proliferation of whole spleen cells and reference mitogen. Taken together, the results demonstrate that HFAE potently activates the immune function by regulating NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage and promoting spleen cell proliferation.     

Innate immunity triggers IL-32 expression by fibroblast-like synoviocytes in rheumatoid arthritis

Introduction  

Interleukin-32 (IL-32) is a recently described cytokine that is a strong inducer of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IL-8. The expression of this cytokine is highly increased in the rheumatoid synovium and correlated with the severity of joint inflammation. Little is known regarding the innate immune-related regulation of IL-32 by fibroblast-like synoviocytes (FLSs). We therefore investigated the effect of innate immune stimulation by ligands of Toll-like receptor (TLR)2, TLR3, and TLR4, and cytokines such as TNF-α and interferon (IFN)-γ, on IL-32 expression by FLSs.     

感染GCRV的草鱼miRNA测序与比较分析

为探索基于高通量筛选抗性主效miRNA功能分子的方法应用于抗性草鱼(Ctenopharyngodon idellus)的选育,研究借助高通量测序鉴定分析感染GCRV前后的草鱼头肾组织中miRNAs.测序结果共鉴定出821个成熟miRNAs,其中118个在GCRV攻毒组特异表达,82个为未攻毒组特有;差异分析结果显示,G...     

Identification of immune-relevant genes by expressed sequence tag analysis of head kidney from grass carp (Ctenopharyngodon idella)

Grass carp is the third largest aquaculture species in global production. However, genomic research of this species has been limited. To identify immune-related genes in grass carp, a normalized full-length cDNA library was constructed from head kidney tissues, and 6432 randomly selected clones were sequenced. 5289 high quality expressed sequence tags (EST) were generated and assembled into 2687 unigenes. Among them, 1585 unigenes showed significant similarity with known sequences in public databases, whereas the remaining 1102 unigenes appeared to be novel sequences with unknown functions. In particular, 136 immune-related genes were identified to encode immunoglobulins, FcRγ, IFN-related proteins, various CD markers, MHCs, complements and other important immune-related factors; a majority of these genes are reported in grass carp for the first time. Sequence analysis indicated that grass carp has at least three subtypes of immunoglobulin light chains, namely L1, L2 and L3. Furthermore, FCRγ was found to broadly express in different tissues. Our study constitutes the first EST analysis of lymphatic tissue in grass carp, and could pave the way for further research of immune-related genes and functional genomics in grass carp.     

草鱼免疫相关基因Prkrip1的克隆和表达分析

随着草鱼养殖规模的扩大, 草鱼的病毒性疾病极大地影响着草鱼的产量。开展鱼类病毒免疫反应相关功能基因的研究意义重大。研究首先通过同源克隆的方法从草鱼中克隆到了一段Prkrip1基因的EST序列, 进一步通过RACE、长片段PCR和Genome walking的方法获得了该基因的全长cDNA序列、基因组DNA序列和启动子区序列。氨基酸序列分析显示, Prkrip1含有3个核定位信号和一个双链RNA结合区, 并具有与PKR结合的保守N端区; 荧光报告基因的表达证实我们所克隆到的启动子区是有活性的, 可用于后续该基因的转录调控分析; Real-time PCR分析发现, Prkrip1 基因在草鱼的肝和血中表达量最高, GCRV感染后在大部分免疫组织中均上调表达, 说明该基因确实与病毒感染相关。研究结果为Prkrip1基因在硬骨鱼类的功能研究提供了线索, 也为鱼类天然免疫反应中调控PKR信号通路的系统研究提供了理论依据。       

Cell contact interactions in rheumatology, The Kennedy Institute for Rheumatology, London, UK, 1-2 June 2000

The intricate interactions that regulate relationships between endogenous tissue cells and infiltrating immune cells in the rheumatic joint, particularly in rheumatoid arthritis (RA), were the subject of the meeting. A better understanding of these interactions might help to define intervention points that could be used to develop specific therapies. The presentations and discussions highlighted the fact that, once chronic inflammation is established, several pro-inflammatory loops involving tumour necrosis factor (TNF)-α and interleukin (IL)-1β can be defined. Direct cellular contact with stimulated T lymphocytes induces TNF-α and IL-1β in monocytes which in turn induce functions in fibroblast-like synoviocytes. The latter include the production of stromal cell-derived factor-1α (SDF-1α) which enhances the expression of CD40L in T cells, which stimulates SDF-1α production in synoviocytes, which in turn protects T and B cells from apoptosis and enhances cell recruitment thus favoring inflammatory processes. IL-1β and TNF-α also induce IL-15 in fibroblast-like synoviocytes, which induces the production of IL-17 which in turn potentiates IL-1β and TNF-α production in monocyte-macrophages. This underlines the importance of TNF-α and IL-1β in RA pathogenesis, and helps explain the efficiency of agents blocking the activity of these cytokines in RA. Factors able to block the induction of cytokine production (such as apolipoprotein A-I [apo A-I] and interferon [IFN]-β) might interfere more distally in the inflammatory process. Furthermore, stimulated T lymphocytes produce osteoclast differentiation factor (ODF), which triggers erosive functions of osteoclasts. Therefore, factors capable of affecting the level of T lymphocyte activation, such as IFN-β, IL-15 antagonist, or SDF-1α antagonist, might be of interest in RA therapy.     

二龄草鱼脾脏、肝脏组织高表达甘露糖结合凝集素mRNA

Innate immunity is expected to be very important in fish. Mannose-bingding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. In this experiment, total mRNA was isolated from spleen, liver, gills, thymus, head kidney and kidney of adult and immature grass carp Ctenopharygodon idllus. The cDNA of MBL was obtained by RT-PCR using total mRNA from the spleen of carp as template. Such cDNA was labled with ^32p and used as probe for Northern analysis, and autoradiographic signals were quantified by densitometry analysis. The results showed that MBL was high expressed in the spleen and liver and low in gills, thymus, head kidney and kidney of adult grass carp, and MBL was much lower expressed in spleen and liver of immature grass carp than those of adult grass carp. The results might partially explain why immature grass carp are vulnerable to grass carp hemorrhage virus (GCHV) whereas adult grass carp are not.This suggested that MBL mav be an imoortant anti-GCHV factor [Acta Zoologica Sinica 50 (1): 137 - 140. 2004].     

Elevated β1,4-galactosyltransferase-I induced by the intraspinal injection of lipopolysaccharide

β1,4-Galactosyltransferase-I (β1,4-GalT-I) is one of the best studied glycosyltransferases. Previous studies demonstrated that β1,4-GalT-I was a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of β1,4-GalT-I deficient mice were impaired. In this study, we investigate the expression of β1,4-GalT-I in lipopolysaccharide (LPS)-induced neuroinflammatory processes. The results of this study demonstrated that β1,4-GalT-I was strongly induced by intraspinal administration of LPS. More than 90% galactose-containing glycans and β1,4-GalT-I were expressed in immune cells. The ELISA assay shows focal injection LPS also induces TNF-α alteration. Double staining indicated β1,4-GalT-I overlapped with TNF-α. Moreover, RT-PCR for β1,4-GalT-I mRNA showed that β1,4-GalT-I mRNA in microglia in vitro was affected in a dose- and time dependent manner in response to LPS or TNF-α stimulation. All these results indicated that the increase of β1,4-GalT-I might attribute to the effect of TNF-α excreting during inflammation. E-selectin, which ligand was modified by β1,4-GalT-I, was correlated with galactose-containing glycans following injecting LPS into spinal cord. We therefore suggest that β1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site. Aiguo Shen and Jianping Chen contributed equally to this work.     

Grass carp (Ctenopharyngodon idella) TRAF6 and TAK1: Molecular cloning and expression analysis after Ichthyophthirius multifiliis infection

Ichthyophthirius multifiliis, a pathogenic ciliate parasite, infects almost all freshwater fish species and causes significant economic losses. Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) are two important signaling molecules involved in toll-like receptor (TLR) signal transduction. To date, the roles of TRAF6 and TAK1 in host defense against fish parasites are still poorly understood. In the present study, TRAF6 (CiTRAF6) and TAK1 (CiTAK1) were identified from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiTRAF6 (2250 bp) includes an open reading frame (ORF) of 1629 bp, which shows a high similarity to that of Cyprinus carpio TRAF6 and encodes a putative protein of 542 amino acids containing one RING domain, two zinc fingers, one coiled-coil region, and one MATH domain. The full-length CiTAK1 cDNA sequence is 2768 bp and includes an ORF of 1626 bp that encodes a putative protein of 541 amino acids containing a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. Phylogenetic analysis showed that CiTRAF6 and CiTAK1 were clustered with TRAF6 and TAK1 of other teleosts, respectively. CiTRAF6 and CiTAK1 were both constitutively expressed in all examined tissues but with varied expression levels. The highest expressions of CiTRAF6 and CiTAK1 were in the head kidney and spleen, respectively. The expression profiles of CiTRAF6 and CiTAK1 were detected in grass carp after I. multifiliis infection. Expressions of both genes were significantly up-regulated in the skin, gill, head kidney, and spleen at most time points after infection, indicating that CiTRAF6 and CiTAK1 may play essential roles in grass carp defense against I. multifiliis.     

病毒感染草鱼胸腺的EST分析和免疫相关基因的鉴定

以感染草鱼(Ctenopharyngodon idellus)出血病病毒(GCHV)的草鱼胸腺为材料,构建了草鱼胸腺的SMARTcDNA文库.筛选文库获得到1933条有效EST序列.BLASTX分析显示,583条序列在公共数据库中能找到同源基因(E-value≤1.00E 10-3,Identities≥30%),另外1350条序列则找不到显著同源性.已知基因按具体功能可划分为6类,大部分与细胞内的各种生理过程、细胞结构以及免疫防御相关.研究结果从分子水平上表明鱼类的胸腺在机体感染病毒的免疫反应中发挥重要作用,同时也表明胸腺组织在病毒感染后可能表达很多目前还不清楚功能的新基因.     

The Role of TNF-<Emphasis Type="Italic">α</Emphasis> and its Receptors in the Production of <Emphasis Type="Italic">β</Emphasis>-1,4-galactosyltransferase I mRNA by Rat Primary Type-2 Astrocytes

β-1,4-galactosyltransferase I (β-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte–endothelial cell interaction. The expression of β-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-α (TNF-α). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-α when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of β-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-α and LPS. Real-time PCR showed that TNF-α or LPS affected β-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-α, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-α or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed β-1,4-GalT I mRNA expression induced by TNF-α or LPS. From these results, we conclude that TNF-α signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce β-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by type-2 astrocytes affected β-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I mRNA in response to inflammation. Chunlin Xia is the co-first author.     

Interferon-α/β upregulate IL-15 expression in vitro and in vivo: analysis in human hepatocellular carcinoma cell lines and in chronic hepatitis C patients during interferon-α/β treatment

Type I interferon (IFN) possesses antiviral and antitumor activities and also having an immune regulatory effect, activating cellular immune response and upregulating several cytokines. Recent study has shown that type I IFN upregurates the dendritic cell production of IL-15 capable of activating natural killer cells and CD8⁺ memory T lymphocytes. However, it is still unknown if type I IFN induces IL-15 production in non-immune cells and if type I IFN affects IL-15 production in vivo. The present study investigated the effect of type I IFNs on IL-15 expression in hepatocellular carcinoma (HCC) cell lines in vitro and in patients with chronic hepatitis C in vivo. When three HCC cell lines, Huh7, HepG2, and JHH4 were cultured in vitro, IFN upregulation of IL-15 expression was observed at both the mRNA and protein levels. In experiments using Huh7 cells, upregulation of IL-15 expression occurred within 24 h of the start of IFN stimulation, and both IFN-α and -β dose-dependently increased IL-15 production in the range from 100 U/ml to 10,000 U/ml of concentration. IFN-β showed stronger activity in IL-15 production induction in vitro than IFN-α. For in vivo examination, sera were obtained from 21 chronic hepatitis C patients treated with IFN and 29 healthy individuals, and the serum IL-15 level was quantified by ELISA. The serum IL-15 level of chronic hepatitis C patients before IFN treatment was similar to that of the healthy controls and significantly increased only during the IFN administration period. These results confirm that IFN-α/β induce IL-15 production and also suggest that IL-15 may be associated with type I IFN-induced immune response.     

Effect of TNF-α and IL-1β genetic variants on the development of myocardial infarction in Turkish population

Tumor necrosis factor-alpha (TNF-α) and interleukin 1 beta (IL-1β) genetic variants which resulting in TNF-α and IL-1 overproduction may increase susceptibility to autoimmune diseases such as atherosclerosis. We have studied the association of TNF-α G308A and IL-1β (+3953) C/T polymorphism with myocardial infarction in Turkish population. 143 patients with myocardial infarction and 213 age-matched healthy controls were included in the study. In univariant analysis, the frequencies of IL-1β, TNF-α genotype or allele, and haplotype of C:A and T:A were significantly elevated in patients when compared with those of controls. GA genotype of TNF-α, T allele of IL-1β and A of TNF-α allele seem to be risk factors for myocardial infarction. In contrast, CC genotype of IL-1β and GG genotype of TNF-α have protective effect against myocardial infarction. In multivariate logistic regression analysis, TNF-α A allele, gender and smoking were associated with myocardial infarction. In conclusion, we can state that TNF-α A allele might be associated with myocardial infarction.