融合表达DEC205单链抗体和NS3蛋白的HCV DNA疫苗的免疫原性研究

为研发新型HCV DNA疫苗并探讨优化其免疫原性的策略,我们分析靶向树突状细胞(Dendritic cells,DC)的分子对HCV DNA疫苗免疫原性的影响。我们基于抗小鼠DC细胞表面分子DEC205/CD205的单克隆抗体DEC205的单链分子,构建可单独表达DEC205单链抗体或者与HCV非结构蛋白NS3融合表达的DNA表达质粒,并构建单独表达HCV非结构蛋白NS3的DNA表达质粒;经瞬时转染法鉴定HCV NS3及其与DEC205单链抗体融合蛋白的表达;随后采用注射结合电转的方式免疫Balb/C小鼠并研究各疫苗的体液(NS3特异性IgG抗体)与细胞免疫(IFN-γELISPOT)效果。结果表明:DEC205单链抗体基因与HCV NS3编码基因的融合可显著增强NS3特异的免疫应答;采用皮内注射加卡钳电极电转的方式可以产生最强的NS3特异性抗体和T细胞免疫反应。因此,通过DEC205单链抗体与HCV DNA疫苗靶抗原融合可明显增强免疫应答效果。该策略为HCV及其他类似病原的新型DNA疫苗研制提供重要依据。     

mTOR regulates TLR-induced c-fos and Th1 responses to HBV and HCV vaccines

Although IL-12 plays a critical role in priming Th1 and cytotoxic T lymphocyte(CTL) responses, Toll-like receptor(TLR) signaling only induces low amounts of IL-12 in dendritic cells and macrophages, implying the existence of stringent regulatory mechanisms. In this study, we sought to uncover the mechanisms underlying TLR-induced IL-12 expression and the Th1 response. By systemic screening, we identified a number of protein kinases involved in the regulation of TLRinduced IL-12 expression. In particular, PI3 K, ERK, and m TOR play critical roles in the TLR-induced Th1 response by regulating IL-12 and IL-10 production in innate immune cells. Moreover, we identified c-fos as a key molecule that mediates m TOR-regulated IL-12 and IL-10 expression in TLR signaling. Mechanistically, m TOR plays a crucial role in c-fos expression, thereby modulating NFκB binding to promoters of IL-12 and IL-10. By controlling the expression of a special innate gene program, m TOR can specifically regulate the TLR-induced T cell response in vivo. Furthermore, blockade of m TOR by rapamycin efficiently boosted TLR-induced antigen-specific T and B cell responses to HBV and HCV vaccines. Taken together, these results reveal a novel mechanism through which m TOR regulates TLR-induced IL-12 and IL-10 production, contributing new insights for strategies to improve vaccine efficacy.     

HCV NS3/4A蛋白酶抑制剂细胞筛选模型

丙型肝炎病毒(hepatitis C virus,HCV)持续感染可导致慢性丙型肝炎,并可发展为肝硬化和肝细胞癌。HCV NS3/4A蛋白酶在HCV复制和致病机制中起着非常关键的作用,已成为HCV研究热点。由于候选药物分子必须具有易进入细胞、稳定性好等特征,建立一种细胞水平上酶活性的测定系统对于筛选抗NS3/4A药物无疑有着重要意义。目前已有多种NS3/4A蛋白酶筛选系统开发出来,本文将对此作一综述。     

Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8⁺ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.     

Fusion of HBsAg and prime/boosting augment Th1 and CTL responses to HCV polytope DNA vaccine

Correlation of hepatitis C virus (HCV) spontaneous resolution with Th1 and CD8⁺CTL responses during natural infection implies the potentiality of poly-CTL-epitopic HCV vaccines. We recently reported in silico design and construction of DNA vaccines (pcPOL-plasmids) harboring HCV CTL epitopes. Herein, we provide data of mice immunization by pcPOL, (encoding; core_132-142 [C], E2_405-414 [E₄], E2_614-622 [E₆] and NS3_1406-1415 [N] CD8⁺CTL epitopes as CE₄E₆N polytope) and its HBsAg-fused counterpart (pcHPOL), compared to the adjuvant-formulated (Montanide + CpG) CE₄E₆N synthetic-peptide immunization. All vaccinated groups developed different levels of cellular responses, however, only the pcHPOL-immunized mice elicited strong CTLs and IFN-γ-secreting cells that were further augmented towards a Th1 response and partial tumor protection by DNA-prime/peptide-boosting regimen. Priming with HBsAg alone could not afford its augmenting effect indicating the importance of priming by polytope itself. Hence, fusion of immunocarriers like HBsAg conjoined with DNA-prime/peptide-boost immunization regimen seems a strategy to enhance the epitope-specific immune responses towards poly-CTL-epitopic vaccines.     

Mtv-1 Superantigen Trafficks Independently of Major Histocompatibility Complex Class II Directly to the B-Cell Surface by the Exocytic Pathway

Presentation of the Mtv-1 superantigen (vSag1) to specific V_β-bearing T cells requires association with major histocompatibility complex class II molecules. The intracellular route by which vSag1 trafficks to the cell surface and the site of vSag1-class II complex assembly in antigen-presenting B lymphocytes have not been determined. Here, we show that vSag1 trafficks independently of class II to the plasma membrane by the exocytic secretory pathway. At the surface of B cells, vSag1 associates primarily with mature peptide-bound class II αβ dimers, which are stable in sodium dodecyl sulfate. vSag1 is unstable on the cell surface in the absence of class II, and reagents that alter the surface expression of vSag1 and the conformation of class II molecules affect vSag1 stimulation of superantigen reactive T cells.

T lymphocytes respond to peptide antigens presented by either major histocompatibility complex (MHC) class I or class II molecules. Many viruses have evolved sophisticated strategies that interfere with antigen presentation by infected cells in order to escape recognition by T lymphocytes. Most strategies studied rely on disrupting MHC class I presentation, either by affecting components of the processing machinery that generate and transport viral peptides into the endoplasmic reticulum (ER) or by retarding transport or targeting class I molecules into the degradation pathway (for a review, see reference 73).In contrast, mouse mammary tumor virus (MMTV) utilizes T-cell stimulation to promote its life cycle. MMTVs encode within their 3′ long terminal repeat a viral superantigen (vSag), and coexpression of the Sag glycoprotein with MHC class II molecules on the surface of virally infected B cells induces V_β-specific T-cell stimulation, generating an immune response which is critical for amplification of MMTV and ensures vertical transmission of virus to the next generation (13, 29, 30). In the absence of B cells, MHC class II, or Sag-reactive T cells, the infection is short-lived (5, 6, 24, 28). The assembly and functional expression of vSag-class II complexes are therefore essential to the viral life cycle. When inherited as germ line elements, Mtv proviruses expressing vSags during ontogeny trigger V_β-specific clonal elimination of immature T cells and profoundly shape the T-cell repertoire (for a review, see reference 1).vSags are type II integral membrane glycoproteins (14, 36). They possess up to six potential N-linked glycosylation sites, and carbohydrate addition is essential for vSag stability and activity (45). Their protein sequence is highly conserved among all MMTV strains except at the C-terminal 29 to 32 residues, which vary and confer T-cell V_β specificity (77). Biochemical analyses of vSag7 (minor lymphocyte stimulating locus 1, Mls-1^a) molecular forms after transfection into a murine B-cell line have identified a predominant 45-kDa endo-β-N-acetylglucosaminidase H (endo H)-sensitive ER-resident glycoprotein, as well as multiple highly glycosylated forms (74). It is thought that an 18-kDa C-terminal fragment binds MHC class II products (75). It has also been suggested that vSags associate weakly with class II in the ER and that proteolytic processing is required for the efficient assembly of vSag-class II complexes for presentation to T cells (46, 49, 75). As yet, the intracellular route that vSags take to the cell surface, the compartment in which they bind class II, and whether they associate with peptide-loaded class II dimers have been enigmatic.Newly synthesized MHC class II αβ heterodimers assemble with invariant chain (Ii), a type II integral membrane protein, to form an oligomeric complex in the ER (37). Ii prevents class II heterodimers from binding peptides in the ER and Golgi complex (55), and signals in its cytoplasmic tail sort the complex into the endocytic pathway (4, 42). In this acidic, protease-rich compartment, Ii is degraded and class II binds antigenic peptides. After the formation of peptide-class II dimers, the complexes are exported to the plasma membrane (8, 48). In the absence of Ii, class II αβ heterodimers exhibit defective post-ER transport, and their conversion into functionally mature, sodium dodecyl sulfate (SDS)-stable compact dimers by peptide antigens is affected (7, 16, 22, 70).A specialized endosomal compartment where class II peptide loading occurs, termed the MHC class II-enriched compartment (MIIC or CIIV), has been found recently in antigen-presenting cells (2, 50, 53, 58, 68, 71). Whether nascent Ii-class II complexes traffic directly to the MIIC from the trans-Golgi network (TGN) or transit first to early endosomes, either directly or via the cell surface, before entering late endocytic vesicles and MIIC is still under debate (26, 56, 57). Transport by all these routes most probably occurs to ensure the capture and loading of antigenic peptides throughout the endocytic pathway (12). MIIC vesicles are positive for lysosome-associated membrane proteins (LAMPs) and cathepsin D and are enriched for HLA-DM or H-2M (18, 32, 59), proteins that facilitate the catalytic exchange of class II-associated invariant peptide chain (CLIP) for antigenic peptides (19, 61, 62). The ultrastructural colocalization of DM with intracellular peptide-class II complexes suggests that the MIIC is a main site where class II dimers bind exogenous and endogenous peptide antigens (47, 58).Determining the route by which vSag protein(s) trafficks to the cell surface and the cellular location where vSag1 processing and assembly with class II molecules occurs is central to understanding the mechanism whereby vSags activate T cells to maintain the viral life cycle. It has been unclear whether vSags traffic independently by the constitutive exocytic pathway or with class II and Ii to the MIIC before reaching the cell surface. Reagents that alter class II expression have been shown to affect vSag presentation (43, 46). Furthermore, mice lacking Ii show reduced intrathymic V_β-specific T-cell deletion (70), suggesting that Ii may play a role, either by ensuring proper maturation of class II dimers or by targeting vSag-class II complexes to the MIIC, in promoting efficient vSag-induced immune responses.To investigate these issues, we used immunochemical detection of vSag1 protein in combination with subcellular fractionation and surface reexpression assays. We show that class II is required for stable vSag1 surface expression. vSag1 trafficks directly to the cell surface independently of class II, and reagents that alter the conversion of newly synthesized class II into peptide-loaded SDS-stable dimers affect functional vSag1 surface expression.     

抗丙肝病毒NS5蛋白单克隆抗体的研制及鉴定

用基因重组的丙肝病毒 ＮＳ５蛋白免疫Ｂａｌ ｂ／ｃ ／小鼠,制备免疫脾Ｂ淋巴细胞,与小鼠骨髓瘤Ｓｐ２／０细胞系融合,筛选获得了１株能分泌抗 ＮＳ５蛋白单克隆抗体的杂交瘤细胞株,该株单克隆抗体为 ＩｇＧ１。ＥＬＩＳＡ及 Ｗｅｓｔｅｒｎ Ｂｌｏｔ证实该株单抗对 ＮＳ５蛋白有较好的特异性。     

Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2

The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.     

Visualizing ATP-Dependent RNA Translocation by the NS3 Helicase from HCV

The structural mechanism by which nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3′ end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a “spring-loading” mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.     

LXR-alpha/SREBP-1c通路参与HCV NS5A诱导的肝细胞脂质代谢紊乱

肝细胞脂肪变性是丙型肝炎患者的突出病理特征,但丙肝病毒（HCV）诱导脂肪变性的分子机制尚不清楚.为探究HCV非结构蛋白5A（NS5A）参与诱导脂肪变性的可能分子机制,本研究以HCV NS5A转基因小鼠为研究对象,采集3～16月龄NS5A转基因小鼠和同窝非转基因小鼠的肝组织,进行病理学检测,并用气相色谱 质谱（GC-MS）法分析脂质主要成分胆固醇酯的含量.采用RT-PCR法检测肝细胞中与脂质代谢密切相关基因肝X受体（LXR-alpha）、固醇调节元件结合蛋白（SREBP-1c）、脂肪酸合成酶（FAS）、硬脂酰辅酶A去饱和酶1（SCD1）、过氧化物酶体增殖物受体alpha（PPAR-alpha）的mRNA表达水平.结果表明,与同窝非转基因对照小鼠相比,3～5月龄NS5A转基因小鼠的肝组织没有发生显著的病理性变化,但6～16月龄的NS5A转基因小鼠的肝脏发生了显著的脂肪变性（47.1% vs 130%;P=0.003）.与此相一致,胆固醇酯的含量在NS5A转基因小鼠的肝脏中显著升高（P < 0.01）.RT PCR检测结果表明,与对照小鼠相比,14月龄NS5A转基因小鼠肝组织中与脂质代谢相关的基因LXR.alpha、SREBP.1c、FAS、SCD1的mRNA表达水平显著增高（P < 0.05）,而PPAR alpha的表达则没有显著变化（P > 0.05）. 以上结果提示,NS5A在小鼠肝细胞中可能通过调高LXR.alpha/SREBP.1c信号通路相关基因的表达,进而促进脂质重新合成,诱导脂肪变性.     

Th1类细胞因子对pHCV—C重组体诱生免疫应答的增强作用

为了探索Th1类细胞因子IL 2和IL 12对含丙型肝炎病毒 (HCV)核心 (C)基因重组体诱生的免疫应答的增强作用 ,本文构建了包含HCVC基因片段的重组质粒pHCV C ,将其单独或与Th1类细胞因子表达质粒 pIL 2或pIL 12共免疫BALB/c小鼠 ,ELISA法检测免疫小鼠血清中的HCVC特异性抗体滴度 ;以pHCV C转染SP2 / 0细胞 ,经筛选稳定表达HCVC抗原者 (SP2 / 0 HCV C)为靶细胞 ,51Cr释放试验检测细胞毒T淋巴细胞 (CTLs)的体外特异性杀伤功能。结果发现 ,pIL 2能够增加 pHCV C诱导的抗体产生 ,对CTLs的杀伤作用增强却不明显 ;而 pIL 12对 pHCV C诱导的抗体和细胞免疫应答均有增强作用 (p <0 .0 5 )。由此推断 ,佐以Th1类细胞因子 ,不仅可以增强基因疫苗诱生的免疫应答强度 ,而且可能使机体对基因疫苗的免疫应答朝着有利于优先诱导CTLs免疫应答清除病原体的方向发展。     

丙型肝炎病毒 NS3 蛋白促进人源肝细胞的增殖及其相关机制研究

丙型肝炎病毒 (HCV) NS3 蛋白与肝癌细胞 (HCC) 的发生密切相关,但其机制尚不清楚 . 既往体外研究极少采用 HCV 的自然宿主细胞———人肝细胞作为研究体系,故所获研究结果有待进一步探讨和证实 . 构建了表达 HCV NS3 蛋白的真核质粒,通过稳定转染人源永生化肝细胞系 QSG7701 ,建立了稳定表达 HCV NS3 蛋白的人源永生化肝细胞系 QSG7701/NS3 ,以此为实验平台,检测了细胞增殖的变化,丝裂原蛋白激酶 (MAPK) 通路激酶磷酸化水平的改变及转录因子 AP-1 、 NF-κB 和 STAT3 的活性变化 . 结果表明： HCV NS3 蛋白可促进人源永生化肝细胞 QSG7701 的增殖, HCV NS3 蛋白激活 ERKs/AP-1 可能是其促进细胞增殖的重要机制,并通过上调转录因子 NF-κB 和 STAT3 的活性,诱导宿主细胞急性炎症损伤 .     

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-α (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers⁺ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IA^s/PLP 139-151 dextramers (specific)/anti-CD4 and IA^s/Theiler’s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IA^k/Myhc 334-352 dextramers/anti-CD4 and IA^k/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer⁺ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the ‘Z’ serial images.     

Efficient Class II Major Histocompatibility Complex Presentation of Endogenously Synthesized Hepatitis C Virus Core Protein by Epstein-Barr Virus-Transformed B-Lymphoblastoid Cell Lines to CD4+ T Cells

The induction of an efficient CD4⁺ T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4⁺ T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10⁴ transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.Hepatitis C virus (HCV) has been known as a major etiologic agent of posttransfusion and sporadic community-acquired non-A, non-B hepatitis. Like the other members in the family Flaviviridae, HCV contains a single, positive-strand RNA genome with a single long open reading frame (ORF) coding for a polyprotein precursor of about 3,000 amino acids (aa) (12). HCV infection is frequently persistent in the majority of patients and is closely associated with the later development of liver cirrhosis and hepatocellular carcinoma (3, 12, 16, 32). The effective control of HCV infection has been limited by the high frequency of viral genetic heterogeneity (7), the low rate of response to alpha interferon (46), and inadequate production of protective immunity (44, 45). These features strongly suggest that there is a great need to establish a new, highly effective therapy.CD4⁺ T cells are considered to play a central role in the generation of protective immunity against infections, because they can provide help to B cells for antibody production (42) and to cytotoxic precursor T cells for their maturation to effectors (21). Some CD4⁺ T cells may also act as cytotoxic effectors (30). It has been recognized that CD4⁺ T-cell response to HCV antigens is important for determining the clinical course of HCV infection (17, 37). Generally, T-cell proliferation is more frequent and stronger in patients with a benign course (6, 17, 20, 33, 37) that is accompanied by the normalization of serum alanine aminotransferase and, in some cases, the clearance of viral RNA (17, 37). In contrast, patients who have a poor T-cell response tend to develop persistent infection (17, 37). These findings support the hypothesis that a sufficient CD4⁺ T-lymphocyte response is critical for limiting HCV infection.Activation of T lymphocytes depends on the recognition of processed viral peptides, but not native antigens, in the context of major histocompatibility complex (MHC) molecules that are presented by antigen-presenting cells (APCs) (56). The B cell is an important professional APC, and its role in mediating antigen-specific immune response has been described extensively (11). Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells are frequently used as APCs in in vitro analyses for antigen processing and presentation to T cells. These cells are characterized by high-level expression of class I and class II MHC molecules, along with accessory molecules such as ICAM-1, B7/BB1, and LFA-3, known to be important costimulatory molecules for T-cell activation (9, 15, 24). Importantly, transfected EBV-immortalized B cells expressing a tumor antigen have been shown to be capable of eliciting both T-helper and cytotoxic-T-lymphocyte (CTL) responses following in vivo inoculation (40). Nevertheless, dendritic cells have been shown to be critical for initiating responses by naive T cells (53), and in some situations presentation by B cells has been suggested to be toleragenic (35). To date, the role of B cells in processing and presenting HCV antigens has not been studied in detail and the mechanisms underlying T-cell–B-cell interaction are still being worked out.In the present study, EBV-transformed B-lymphoblastoid cell lines (B-LCLs) established from HCV-infected patients were transfected with an expression vector coding for the whole structural region and part of the NS2 region of the HCV genome. The capacity of transfected B-LCL APCs for presenting intracellularly synthesized peptides was assessed by in vitro induction of the HCV-specific lymphoproliferative response of autologous T-cell lines. Our results indicated that core protein was properly expressed and efficiently presented by B-LCL APCs to CD4⁺ T cells. We demonstrated that the endogenous core peptides were presented through the class II MHC pathway and that they need B7/BB1 for providing costimulatory signals.     

Dectin-1-triggered Recruitment of Light Chain 3 Protein to Phagosomes Facilitates Major Histocompatibility Complex Class II Presentation of Fungal-derived Antigens

Dectin-1 is a pattern recognition receptor that is important for innate immune responses against fungi in humans and mice. Dectin-1 binds to β-glucans in fungal cell walls and triggers phagocytosis, production of reactive oxygen by the NADPH oxidase, and inflammatory cytokine production which all contribute to host immune responses against fungi. Although the autophagy pathway was originally characterized for its role in the formation of double-membrane compartments engulfing cytosolic organelles and debris, recent studies have suggested that components of the autophagy pathway may also participate in traditional phagocytosis. In this study, we show that Dectin-1 signaling in macrophages and bone marrow-derived dendritic cells triggers formation of LC3II, a major component of the autophagy machinery. Further, Dectin-1 directs the recruitment of LC3II to phagosomes, and this requires Syk, activation of reactive oxygen production by the NADPH oxidase, and ATG5. Using LC3-deficient dendritic cells we show that whereas LC3 recruitment to phagosomes is not important for triggering phagocytosis, killing or Dectin-1-mediated inflammatory cytokine production, it facilitates recruitment of MHC class II molecules to phagosomes and promotes presentation of fungal-derived antigens to CD4 T cells.     

HCV基因组NS1区的分子克隆及序列测定

对广东省一名慢性丙型肝炎病人血清中的ＨＣＶ基因组ＮＳ１区进行分子克隆及序列测定。采用微粒吸附法提取ＨＣＶ ＲＮＡ,随机引物逆转录后进行聚合酶链反应。所用引物位于ＮＳ１区,扩增产物７８０ｂｐ在低熔点琼脂糖中电泳,加嘏相应条带处凝胶,与ｐＵＣ１８的连接批应直接在低熔点琼脂糖中完成。重组体转化ＪＭ１０９,挑取菌落增殖后提取的质粒采用ＰＣＲ和酶切法鉴定阳性克隆。将其中３２０ｂｐ的片段亚克隆到ｐＵＣ１８和ｐ     

湖南地区HBV患者MHC—I类链相关基因A第五外显子微卫星多态性研究

分别提取湖南汉族人群108例慢性乙肝病毒（hepatitis B virus,HBV）患者、96例HBV携带者和142例健康对照者外周血基因组DNA,利用聚合酶链反应和基因扫描技术分别对它们的主要组织相容性复合体I类链相关A（major histocompatibility complex class I chain related A, MICA）基因第5外显子进行微卫星多态性分析;应用DNA测序分析对不同的基因型进行验证,同时应用PCR／SSP技术进行MICA＊Del检测,确定MICA基因第5外显子基因型。发现本研究的三组中分别检出44、A5、A5．1、A6、A9五种等位基因,且以A5和A5．1为主;在HBV携带组和健康对照组中分别检出MICA＊Del基因。结果同时显示慢性HBV患者组MICA＊A5．1／A9基因型频率、慢性HBV患者组的MICA＊A9等位基因频率、慢性HBV患者组MICA＊A9表型频率和HBV携带组MICA＊A5．I／A9基因型频率均低于相应的健康对照组;而湖南地区汉族人群HBV携带者和慢性HBV患者问的基因型频率、等位基因频率和表型频率无显著性差异;因而推测MICA＊A5．1／A9基因型和MICA$A9等位基因可能是抗HBV感染的一种保护性等位基因。为今后进一步研究HBV的感染、预防和治疗提供参考依据。