Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions

We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecular-weight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission.     

S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFα, and IL-1β were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA–DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.     

Enhanced receptor–clathrin interactions induced by N-glycan–mediated membrane micropatterning

Glycan–protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN–clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.Glycans are fundamental cellular components ubiquitously present in the extracellular matrix and cell membrane as glycoproteins or glycolipids. Glycan-binding proteins such as galectins, siglecs, and selectins are mostly multivalent and thus thought to cross-link glycoproteins into higher-order aggregates, creating a cell surface glycan-based connectivity also called glycan lattice or network (1–3). By concentrating specific glycoproteins or glycolipids while excluding other cell surface molecules, surface glycan-based connectivity can organize the plasma membrane into specialized domains that perform unique functions (1, 3–6). Nevertheless, direct observation of glycan-mediated ligand cross-linking in living cells remains challenging (7). Notwithstanding, there is no doubt that surface glycan-based connectivity is essential in the control of multiple biological processes including immune cell activation and homeostasis, cell proliferation and differentiation, and receptor turnover and endocytosis (1, 5, 6, 8).Clathrin-mediated endocytosis (CME) constitutes the primary pathway of cargo internalization in mammalian cells regulating the surface expression of receptors (9). Formation of clathrin-coated pits (CCPs) starts by nucleation of coat assembly at distributed positions in the inner surface of the plasma membrane, where it continues to grow or dissolve rapidly unless coat stabilization occurs (10, 11). One event that clearly correlates with successful CCP stabilization is cargo loading (11). Recent studies show that cargo molecules diffuse randomly on the cell membrane until they meet growing CCPs, with the extent of cargo interactions regulating CCP maturation (12). As such, factors that affect cargo mobility within/at the cell surface will inevitably impact on CCP maturation and successful internalization. In the context of surface glycan–protein interactions, it has been shown that glycoproteins with an intact glycan-based connectivity exhibit reduced lateral mobility and this correlates with compromised endocytosis (3, 13–17). How this precisely occurs is poorly defined, although fluorescence recovery after photobleaching on the EGF receptor (EGFR) suggested that cell surface glycan-based interactions restrict EGFR dynamics and localization into membrane regions away from endocytic platforms (14, 17). Whether this is a general mechanism for glycosylated proteins or specific to EGFR is not known. Moreover, visualization of receptor interactions with the endocytic machinery under the influence of the glycan network has not yet been attained.In this work we applied superresolution nanoscopy and developed a dedicated dual-color single-molecule spatio-dynamic exploration approach to visualize the impact of glycan-based interactions on the spatiotemporal organization and clathrin interaction of a glycosylated membrane receptor involved in pathogen recognition and uptake. We focused on the transmembrane glycoprotein dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) given its importance in supporting primary immune responses such as pathogen recognition and uptake on immature dendritic cells (imDCs), signaling, and cell adhesion (6, 18–20). Moreover, DC-SIGN contains a single N-glycosylation site, organizes in nanoclusters at the cell membrane (19, 21–23), and internalizes bound antigens via CPPs for subsequent processing and presentation to T cells (20, 24–26). Our work provides insights on how surface glycan-mediated interactions tune spatiotemporal micropatterning of receptors on the cell membrane, potentially regulating interactions with the endocytic machinery and underscoring the importance and complex role of surface glycans on cell membrane organization and function.     

口腔白斑组织中树突状细胞亚群的免疫组化分析

目的：分析口腔白斑（OLK）组织中树突状细胞的特征性表型。方法：应用免疫组化 SP 法检测10例正常黏膜、10例单纯增生白斑和20例异常增生白斑中树突状细胞特异性标记 CD1 a、CD209、CD123、CD83的表达状况,并进行统计学分析。结果：3组样本中 CD1 a、CD209均有表达。固有层内 CD1 a 阳性郎格汉斯细胞（LCs）数量随病变程度逐渐升高,且异常增生白斑组高于单纯增生白斑组及正常黏膜组（P ＜0．01）。异常增生白斑及单纯增生白斑中 CD209阳性 DCs 被大量召集,其数量显著高于正常黏膜。正常黏膜中未见 CD123及 CD83表达,但在单纯增生及异常增生白斑中 CD123阳性和 CD83阳性细胞数逐渐升高（P ＜0．01）。结论：朗格汉斯细胞、间质 DC 及浆细胞样树突细胞在 OLK 间质的表达水平增加逐渐成熟与机体的免疫监视功能密切相关,在 OLK 发展过程中均可能起到重要作用。     

人DC-SIGN真核表达载体的构建及表达

目的构建含有FLAG标签的人DC—SIGN真核表达载体,并在人胚肾293T细胞中进行表达。方法通过PCR方法获得含有FLAG标签的人DC—SIGN分子基因,将其插入到真核表达载体pIRES—neo中。构建的重组质粒pIRES—neO—FLAG—DC—SIGN转染293T细胞,利用流式细胞仪、免疫荧光及WesternBlot检测其表达情况。结果含FLAG标签的人DC—SIGN基因测序正确,PCR和酶切鉴定证明FLAG—DC—SIGN基因已成功连入真核表达载体pIRES—neo中;检测结果显示重组质粒pIRES—neo—FLAG—DC—SIGN在293T细胞中得到表达。结论成功构建了重组质粒pIRES—neo—FLAG—DC—SIGN,且在293T细胞中能有效表达,为后续DC靶向性疫苗研究奠定了基础。     

Single nucleotide polymorphism in the promoter region of the CD209 gene is associated with human predisposition to severe forms of tick-borne encephalitis

Tick-borne encephalitis virus (TBEV) is a neurotropic, positive-sense RNA virus of the genus Flavivirus (family Flaviviridae) which can cause a variety of clinical manifestations in humans. Previously the severity and outcome of dengue fever and hepatitis C (diseases caused by viruses from the family Flaviviridae) were associated with the rs4804803 single nucleotide polymorphism (SNP) located in the promoter region of the human CD209 gene. This gene encodes dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN), a C-type lectin pathogen-recognition receptor expressed on the surface of dendritic cells and some types of macrophages. In the current study, a possible association between two SNPs in the promoter region of the CD209 gene (rs4804803 and rs2287886) and predisposition to severe forms of TBEV-induced disease was investigated. The genotypic, allelic and haplotypic frequencies of these SNPs were analyzed in 136 non-immunized Russian patients with different clinical manifestations of tick-borne encephalitis (TBE) and in a control group. An increase in the frequency of the rs2287886 SNP AA homozygotes and the A allele was detected among patients with severe central nervous system disease compared with the group of patients with meningitis (P = 0.003 and 0.019), or a combined group of patients with mild forms (fever and meningitis) (P = 0.003 and 0.026), or the control group (P = 0.007 and 0.035). Thus, our results suggest that the CD209 gene promoter region rs2287886 SNP is associated with predisposition to severe forms of TBE in the Russian population.     

慢性乙型肝炎患者DC-SIGN启动区变异分析

目的:了解慢性乙型肝炎(chronic hepatitis B,CHB)患者与既往乙型肝炎病毒(hepatitis B virus,HBV)感染者体内树突状细胞特异性细胞间黏附分子-3结合非整合素因子(dendritic cell-specific intercellular adhension molecule-3-...     

The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4(+) cells

It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4⁺ cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4⁺ cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides.     

Mucin 6 in seminal plasma binds DC-SIGN and potently blocks dendritic cell mediated transfer of HIV-1 to CD4 T-lymphocytes

Many viruses transmitted via the genital or oral mucosa have the potential to interact with dendritic cell-specific intercellular adhesion molecule-3 grabbing non integrin (DC-SIGN) expressed on immature dendritic cells (iDCs) that lie below the mucosal surface. These cells have been postulated to capture and disseminate human immunodeficiency virus type-1 (HIV-1) to CD4⁺ lymphocytes, potentially through breaches in the mucosal lining. We have previously described that BSSL (bile salt-stimulated lipase) in human milk can bind DC-SIGN and block transfer. Here we demonstrate that seminal plasma has similar DC-SIGN blocking properties as BSSL in human milk. Using comparative SDS-PAGE and Western blotting combined with mass spectrometry we identified mucin 6 as the DC-SIGN binding component in seminal plasma. Additionally, we demonstrate that purified mucin 6 binds DC-SIGN and successfully inhibits viral transfer. Mucin 6 in seminal plasma may therefore interfere with the sexual transmission of HIV-1 and other DC-SIGN co-opting viruses.     

人DC-SIGN绿色荧光融合蛋白真核表达与功能鉴定

目的:构建人DC-SIGN绿色荧光融合蛋白的真核表达载体,在COS7细胞表面表达鉴定.方法:用RT-PCR从人外周血单个核细胞扩增DC-SIGN蛋白基因片段,连接到T载体.测序鉴定后切下相应片段,连接到pEGFP-C1表达载体,重组质粒(命名为:DS-pEGFP-C1)转染COS7细胞,表达DC-SIGN绿色荧光融合蛋白(命名为:DS-EGFP).荧光定量PCR检测融合基因mRNA拷贝量,激光扫描共焦显微镜鉴定DS-pEGFP-C1的表达和摄取减毒牛型分枝杆菌BCG功能.结果:用RT-PCR扩增得到正确的目的基因片段并构建成真核表达载体DS-pEGFP-C1,转染COS7细胞,荧光定量PCR检测DS-pEGFP-C1转录mRNA拷贝量是4.52×1011 copies/ml,激光扫描共焦显微镜检测证实DS-EGFP在COS7细胞表面表达,同时具有摄取BCG的功能.结论:本研究成功构建人DC-SIGN绿色荧光融合蛋白的真核表达载体DS-pEGFP-C1,表达了融合蛋白,后者能够摄取BCG.