Dendritic Cell-specific Intercellular Adhesion Research Articles

Sequencing of DC-SIGN promoter indicates an association between promoter variation and risk of nasopharyngeal carcinoma in cantonese

BackgroundThe dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) is an important pathogen recognition receptor of the innate immune system. DC-SIGN promoter variants play important role in the susceptibility to various infectious diseases. Nasopharyngeal carcinoma (NPC) is a malignancy that is common in southern China and whether DC-SIGN promoter variants have effects on susceptibility to NPC is still unknown. The aim of this study is to ascertain the potential involvement of DC-SIGN promoter single nucleotide polymorphisms (SNPs) in NPC susceptibility.MethodsWe conducted a case control study based on Cantonese population including 444 NPC patients and 464 controls matched on age and sex. The 1041 bp of DC-SIGN promoter region was directly sequenced for all samples. Sequence alignment and SNP search were inspected using DNAStar analysis programs and haplotype frequencies were estimated in Haploview V 4.0. The associations between the SNPs and the risk of NPC were analyzed using chi-square test and non-conditional logistic regression analysis with SPSS 13.0 software.ResultsA total of six variants were observed in the DC-SIGN promoter region and DC-SIGN -139 GG and -939 AA were significantly associated with NPC risk with adjusted Odds Ratios (ORs) of 2.10 (95% confidence interval [CI] = 1.23-3.59; P = 0.006) and 2.52 (1.29-4.93; P = 0.007) respectively and subjects carrying the risk allele DC-SIGN -871 G had 1.47-fold (95% CI = 1.14-1.90) increased risks of developing NPC (P = 0.003). Haplotype analysis revealed that h1 'AAAG' was significantly associated with protection against NPC (OR = 0.69; P = 0.0002) and the association was still significant when using 1000 permutation test runs (P = 0.001).ConclusionsOur study indicated that DC-SIGN promoter variants appear to be involved in the susceptibility to NPC and the detailed mechanism of this effect need further studies.

Cigarette Smoke-Induced Changes to Alveolar Macrophage Phenotype and Function Are Improved by Treatment with Procysteine

Defective efferocytosis may perpetuate inflammation in smokers with or without chronic obstructive pulmonary disease (COPD). Macrophages may phenotypically polarize to classically activated M1 (proinflammatory; regulation of antigen presentation) or alternatively activated M2 (poor antigen presentation; improved efferocytosis) markers. In bronchoalveolar lavage (BAL)-derived macrophages from control subjects and smoker/ex-smoker COPD subjects, we investigated M1 markers (antigen-presenting major histocompatibility complex [MHC] Classes I and II), complement receptors (CRs), the high-affinity Fc receptor involved with immunoglobulin binding for phagocytosis (Fc-gamma receptor, FcγR1), M2 markers (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin [DC-SIGN] and arginase), and macrophage function (efferocytosis and proinflammatory cytokine production in response to LPS). The availability of glutathione (GSH) in BAL was assessed, because GSH is essential for both M1 function and efferocytosis. We used a murine model to investigate macrophage phenotype/function further in response to cigarette smoke. In lung tissue (disaggregated) and BAL, we investigated CRs, the available GSH, arginase, and efferocytosis. We further investigated the therapeutic effects of an oral administration of a GSH precursor, cysteine l-2-oxothiazolidine-4-carboxylic acid (procysteine). Significantly decreased efferocytosis, available GSH, and M1 antigen-presenting molecules were evident in both COPD groups, with increased DC-SIGN and production of proinflammatory cytokines. Increased CR-3 was evident in the current-smoker COPD group. In smoke-exposed mice, we found decreased efferocytosis (BAL and tissue) and available GSH, and increased arginase, CR-3, and CR-4. Treatment with procysteine significantly increased GSH, efferocytosis (BAL: control group, 26.2%; smoke-exposed group, 17.66%; procysteine + smoke-exposed group, 27.8%; tissue: control group, 35.9%; smoke-exposed group, 21.6%; procysteine + smoke-exposed group, 34.5%), and decreased CR-4 in lung tissue. Macrophages in COPD are of a mixed phenotype and function. The increased efferocytosis and availability of GSH in response to procysteine indicates that this treatment may be useful as adjunct therapy for improving macrophage function in COPD and in susceptible smokers.

Actinohivin, a Broadly Neutralizing Prokaryotic Lectin, Inhibits HIV-1 Infection by Specifically Targeting High-Mannose-Type Glycans on the gp120 Envelope

The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydrate-binding sites. AH strongly interacts with gp120 derived from different X4 and R5 human immunodeficiency virus (HIV) strains, simian immunodeficiency virus (SIV) gp130, and HIV type 1 (HIV-1) gp41 with affinity constants (KD) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (alpha1,2-mannose)3 oligosaccharide but not by alpha1,3/alpha1,6-mannose- or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of alpha1,2-mannose-specific monoclonal antibody 2G12, and AH covers a broader epitope on gp120 than 2G12. Prolonged exposure of HIV-1-infected CEM T-cell cultures with escalating AH concentrations selects for mutant virus strains containing N-glycosylation site deletions (predominantly affecting high-mannose-type glycans) in gp120. In contrast to 2G12, AH has a high genetic barrier, since several concomitant N-glycosylation site deletions in gp120 are required to afford significant phenotypic drug resistance. AH is endowed with broadly neutralizing activity against laboratory-adapted HIV strains and a variety of X4 and/or R5 HIV-1 clinical clade isolates and blocks viral entry within a narrow concentration window of variation (approximately 5-fold). In contrast, the neutralizing activity of 2G12 varied up to 1,000-fold, depending on the virus strain. Since AH efficiently prevents syncytium formation in cocultures of persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T lymphocytes, inhibits dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated capture of HIV-1 and subsequent virus transmission to CD4+ T lymphocytes, does not upregulate cellular activation markers, lacks mitogenic activity, and does not induce cytokines/chemokines in peripheral blood mononuclear cell cultures, it should be considered a potential candidate drug for microbicidal use.

Expression of Vascular Notch Ligand Delta-Like 4 and Inflammatory Markers in Breast Cancer

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

Influence of polymorphism in dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-related ( DC-SIGNR) gene on HIV-1 trans-infection

The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGN-related (DC-SIGNR) molecules on the cell surface are known to enhance human immunodeficiency virus type 1 (HIV-1) infection by capturing the virions and transmitting them to CD4+ T-cell, a process termed trans-infection. The neck region and carbohydrate recognition domain of the two proteins are important for efficient binding to the HIV-1 envelope protein. DC-SIGNR is polymorphic in Exons 4 and 5 that encode the neck region and carbohydrate recognition domain, respectively; the former contains a variable number of tandem repeats, and the latter the SNP (rs2277998). Since it remains unclear whether the DC-SIGNR polymorphism is related to the risk of HIV-1 infection, we tested possible effects of the polymorphism on HIV-1 trans-infection efficiency, by constructing six kinds of cDNAs encoding DC-SIGNR variants with various numbers of repeat units and various SNP. We were able to express the variants on the surface of Raji cells, a human B cell line. Flow cytometry showed that all the tested DC-SIGNR molecules were efficiently expressed on the cell surface at various levels; the assay for HIV trans-infection efficacy showed that all the tested variants had that activity with different efficacy levels. We found a correlation between the HIV trans-infection efficiency and the mean fluorescent intensity of DC-SIGNR expression ( R 2 = 0.95). Thus, our results suggest that the variation of the tested DC-SIGNR genotypes affects the efficacy of trans-infection by affecting the amounts of the protein expressed on the cell surface.

Genetic susceptibility in tuberculosis

The importance of host genetic factors in determining susceptibility to tuberculosis (TB) has been studied extensively using various methods, such as case-control, candidate gene and genome-wide linkage studies. Several important candidate genes like human leucocyte antigen/alleles and non-human leucocyte antigen genes, such as cytokines and their receptors, chemokines and their receptors, pattern recognition receptors (including toll-like receptors, mannose binding lectin and the dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin), solute carrier family 11A member 1 (formerly known as natural resistance-associated macrophage protein 1) and purinergic P2X7 receptor gene polymorphisms, have been associated with differential susceptibility to TB in various ethnic populations. This heterogeneity has been explained by host-pathogen and gene-environment interactions and evolutionary selection pressures. Although the achievements of genetics studies might not yet have advanced the prevention and treatment of TB, researchers have begun to widen their scope of investigation to encompass these practical considerations.

Pradimicin S, a highly soluble nonpeptidic small-size carbohydrate-binding antibiotic, is an anti-HIV drug lead for both microbicidal and systemic use.

Pradimicin S (PRM-S) is a highly water-soluble, negatively charged derivative of the antibiotic pradimicin A (PRM-A) in which the terminal xylose moiety has been replaced by 3-sulfated glucose. PRM-S does not prevent human immunodeficiency virus (HIV) adsorption on CD4(+) T cells, but it blocks virus entry into its target cells. It inhibits a wide variety of HIV-1 laboratory strains and clinical isolates, HIV-2, and simian immunodeficiency virus (SIV) in various cell culture systems (50% and 90% effective concentrations [EC(50)s and EC(90)s] invariably in the lower micromolar range). PRM-S inhibits syncytium formation between persistently HIV-1- and SIV-infected cells and uninfected CD4(+) T lymphocytes, and prevents dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-mediated HIV-1 and SIV capture and subsequent virus transmission to CD4(+) T cells. Surface plasmon resonance (SPR) studies revealed that PRM-S strongly binds to gp120 in a Ca(2+)-dependent manner at an affinity constant (K(D)) in the higher nanomolar range. Its anti-HIV activity and HIV-1 gp120-binding properties can be dose-dependently reversed in the presence of an (alpha-1,2)mannose trimer. Dose-escalating exposure of HIV-1-infected cells to PRM-S eventually led to the isolation of mutant virus strains that had various deleted N-glycosylation sites in the envelope gp120 with a strong preference for the deletion of the high-mannose-type glycans. Genotypic resistance development occurred slowly, and significant phenotypic resistance occurred only after the sequential appearance of up to six mutations in gp120, pointing to a high genetic barrier of PRM-S. The antibiotic is nontoxic against a variety of cell lines, is not mitogenic, and does not induce cytokines and chemokines in peripheral blood mononuclear cells as determined by the Bio-Plex human cytokine 27-plex assay. It proved stable at high temperature and low pH. Therefore, PRM-S may qualify as a potential anti-HIV drug candidate for further (pre)clinical studies, including its microbicidal use.

Targeting DC-SIGN with carbohydrate multivalent systems

DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin), a C-type lectin mainly present at the surface of immature dendritic cells, plays a relevant role activating and tailoring adaptive immune responses against different pathogens. This lectin recognizes, in a multivalent and calcium-dependent manner, highly glycosylated proteins present at the surface of pathogens. Several groups have devoted remarkable efforts to develop carbohydrate multivalent compounds targeting this lectin to modulate its role in pathogen capture and in the generation of an immune response. Most of these approaches have been based on mannosylation of immunogenic proteins such as ovalbumin but new strategies have been envisaged to achieve these goals. Although mannosylated systems cannot provide the required selectivity for a specific lectin at dendritic cells, fucosylated compounds have overcome this problem specifically targeting DC-SIGN and avoiding interferences with other lectins, such as the mannose receptor. The use of these carbohydrate multivalent compounds to target DC-SIGN can be considered a promising strategy to inhibit pathogen entry and to develop new vaccines against pathogen infection or cancer. New studies are required to provide more insights into the complex immune pathway involving DC-SIGN.

Identification of Novel Contributions to High-affinity Glycoprotein–Receptor Interactions using Engineered Ligands

Engineered receptor fragments and glycoprotein ligands employed in different assay formats have been used to dissect the basis for the dramatic enhancement of binding of two model membrane receptors, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the macrophage galactose lectin, to glycoprotein ligands compared to simple sugars. These approaches make it possible to quantify the importance of two major factors that combine to enhance the affinity of single carbohydrate-recognition domains (CRDs) for glycoprotein ligands by 100-to 300-fold. First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity. Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans. In contrast, when solution-phase networking is avoided, enhancement due to binding of multiple branches of a glycan to multiple CRDs in the oligomeric forms of these receptors is minimal and binding of a receptor oligomer to multiple glycans on a single glycoprotein makes only a twofold contribution to overall affinity. Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity. These findings, combined with considerations of membrane receptor geometry, are consistent with the idea that further enhancement of the binding to multivalent glycoprotein ligands requires interaction of multiple receptor oligomers with the ligands.

The relationship of interacting immunological components in dengue pathogenesis

The World Health Organization (WHO) estimates that there are over 50 million cases of dengue fever reported annually and approximately 2.5 billion people are at risk. Mild dengue fever presents with headache, fever, rash, myalgia, osteogenic pain, and lethargy. Severe disease can manifest as dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF). Symptoms of DSS/DHF are leukopenia, low blood volume and pressure encephalitis, cold and sweaty skin, gastrointestinal bleeding, and spontaneous bleeding from gums and nose. Currently, there are no therapeutics available beyond supportive care and untreated complicated dengue fever can have a 50% mortality rate. According to WHO DSS/DHF is the leading cause of childhood mortality in some Asian countries. Dendritic cells are professional antigen presenting cells that are primary targets in a dengue infection. Dengue binds to Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN). DC-SIGN has a high affinity for ICAM3 which is expressed in activating T-cells. Previous studies have demonstrated an altered T-cell phenotype expressed in dengue infected patients that could be potentially mediated by dengue-infected DCs.Dengue is enhanced by three interacting components of the immune system. Dengue begins by infecting dendritic cells which in immature dendritic cells is mediated by DC-SIGN. In mature dendritic cells, antibodies can enhance dengue infection via Fc receptors. Downstream of dendritic cells T-cells become activated and generate the very cytokines implicated in vascular leak and shock in addition to activating effector cells. Both the virus and the antibodies are involved in release of complement and anaphylatoxins which can cause or exacerbate DHF/DSS. These systems are inextricable and strongly associated with dengue pathogenesis.

DC-SIGN and mannosylated surface structures of Mycobacterium tuberculosis: a deceptive liaison

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is recognized by pattern recognition receptors on macrophages and dendritic cells, thereby triggering phagocytosis, antigen presentation to T cells and cytokine secretion. The dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN) is a calcium-dependent carbohydrate-binding protein with specificity for mannose-containing glycoconjugates and fucose-containing Lewis antigens. Mannosylated moieties of the mycobacterial cell wall, such as mannose-capped lipoarabinomannan (manLAM) or higher-order phosphatidylinositol-mannosides (PIMs) of Mtb, were previously shown to bind to DC-SIGN on immature dendritic cells and macrophage subpopulations. This interaction reportedly impaired dendritic cell maturation, modulated cytokine secretion by phagocytes and dendritic cells and was postulated to cause suppression of protective immunity to TB. However, experimental Mtb infections in mice transgenic for human DC-SIGN revealed that, instead of favoring immune evasion of mycobacteria, DC-SIGN may promote host protection by limiting tissue pathology. Furthermore, infection studies with mycobacterial strains genetically engineered to lack manLAM or PIMs demonstrated that the manLAM/PIM–DC-SIGN interaction was not critical for cytokine secretion in vitro and protective immunity in vivo. The dominant Mtb-derived ligands for DC-SIGN are presently unknown, and a major role of DC-SIGN in the immune response to Mtb infection may lie in its capacity to maintain a balanced inflammatory state during chronic TB.

Activation of p38 MAPK Pathway by Hepatitis C Virus E2 in Cells Transiently Expressing DC-SIGN

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a cellular receptor for hepatitis C virus for the binding of viral envelope glycoprotein E2. Interaction of DC-SIGN with the E2 may evoke cellular signal transduction implicated in viral pathogenesis. We developed a cell model with DC-SIGN transient transfection to study p38 mitogen-activated protein kinase (MAPK) signaling pathway in response to the E2 treatment. HEK293T and HeLa were DC-SIGN-deficient cell lines. DC-SIGN was detectable at the surface of HEK293T and HeLa transfected with DC-SIGN, and the levels of DC-SIGN were high in transfected-HEK293T as compared with HeLa. The transfected-HEK293T displayed ability for the E2 binding. In the transfected-HEK293T, level of p38 MAPK phosphorylation was increased upon the E2 treatment and reduced following blockage of DC-SIGN with an antibody against DC-SIGN. Phosphorylation of downstream transcription factor activating transcription factor (ATF)-2 was also up-regulated by the E2 via DC-SIGN. Similar results were obtained with NIH3T3 cells stably expressing DC-SIGN and Huh7 cells. Our results indicate that DC-SIGN transient expression in HEK293T is a useful cell model for investigating p38 MAPK pathway triggered by the E2, which may provide information for understanding cellular receptors-mediated signaling events and the viral pathogenesis.

Angiogenic DC‐SIGN+ cells are present at the attachment sites of epitheliochorial placentae

Spontaneous early and mid-gestation fetal losses occur in swine. At both stages, endometrial lymphocytes associated with smaller, paler conceptuses have fewer pro-angiogenic and more pro-inflammatory cytokine transcripts compared with robust conceptuses. We hypothesized that similar differences occur in conceptus-associated dendritic cells (DCs). Using laser capture-microdissection, dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN)(+) cells were isolated from attachment sites of healthy and arresting conceptuses at gestation day (gd)20 and 50. DC-SIGN(+) cells were screened using real-time PCR for vascular endothelial cell growth factor (Vegf), its receptors, semaphorins (Sema) and plexins (Plxn), and for toll-like receptor (Tlr) transcripts to address potential activation pathways. Homogenized endometrial and trophoblast biopsies were quantified for type 1/type 2 cytokine transcripts/proteins. DC-SIGN(+) cells from healthy and arresting conceptuses had more Vegf transcripts at early than mid gestation whereas transcripts for Vegfr1 and Vegfr2 were stable. In gd20 arresting site DC-SIGN(+) cells, Neuropilin-2 transcripts were elevated, whereas at gd50 arresting sites, Plxn-A2 increased and Sema3A transcripts were lost. Tlr-1, Tlr-4 and Tlr-6 transcript abundance was independent of conceptus health. At gd20, type 1 cytokines were prevalent, whereas at gd50 type 2 cytokines predominated in endometrium and trophoblast. Thus, gestational features, characteristic of haemochorial placentation, are present in species with distinctly different placentation.

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.

Model for Local Skin Defense Against Dengue Virus Infection

Evaluation of: Kwan WH, Navarro-Sanchez E, Dumortier H et al.: Dermal-type macrophage expressing CD209/DC-SIGN show inherent resistance to Dengue virus growth. PLoS Negl. Trop. Dis. 2(10), E311 (2008). Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN/CD209) was recently identified as a molecule that facilitates Dengue virus entry, leading to productive cellular infection. Initially, DC-SIGN expression was thought to be restricted to dendritic cells, however, it is now known that tissue macrophages also express it. Here we review a paper that used an ex vivo and an in vitro model of skin-dwelling macrophages expressing DC-SIGN, demonstrating that they are nonproductively infected with the Dengue virus. The authors reveal a novel type I interferon-independent mechanism whereby the virus enters these DC-SIGN-bearing cells but traffics to poorly acidified phagosomes, thereby limiting viral replication. We discuss this new model to study early infection and local host defenses against viral replication using IL-10-derived dermal macrophages expressing DC-SIGN.

Oligomerization of the Macrophage Mannose Receptor Enhances gp120-mediated Binding of HIV-1

C-type lectin receptors expressed on the surface of dendritic cells and macrophages are able to bind glycoproteins of microbial pathogens via mannose, fucose, and N-acetylglucosamine. Langerin on Langerhans cells, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin on dendritic cells, and mannose receptor (MR) on dendritic cells and macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120 principally via high mannose oligosaccharides. These C-type lectin receptors can also oligomerize to facilitate enhanced ligand binding. This study examined the effect of oligomerization of MR on its ability to bind to mannan, monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry analysis of cross-linked MR showed homodimerization on the surface of primary monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric MR were precipitated by mannan, but only the dimeric form was co-immunoprecipitated by gp120. These results were confirmed independently by flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a cellular HIV virion capture assay. As expected, mannan bound to the carbohydrate recognition domains of MR dimers mostly in a calcium-dependent fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only partially blocked by mannan, and was also partially inhibited by N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding occurs via the calcium-dependent, non-calcium-dependent carbohydrate recognition domains and the cysteine-rich domain at the C terminus of MR dimers, presenting a much broader target for potential inhibitors of gp120-MR binding.

Antiviral activity of carbohydrate-binding agents and the role of DC-SIGN in dengue virus infection

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is an important binding receptor for dengue virus (DENV) that recognizes N-glycosylation sites on the viral E-glycoprotein. DENV cannot bind nor infect the human B-cell line Raji/0. However, DENV productively infects Raji/DC-SIGN + cells that constitutively express DC-SIGN on their surface. IL-4-treated monocytes, expressing high levels of DC-SIGN, are also susceptible for DENV infection. Several carbohydrate-binding agents (CBAs), such as the plant lectins HHA, GNA (mannose-specific) and UDA (N-acetylglucosamine-specific), inhibited dose-dependently the binding of DENV and subsequently viral replication in Raji/DC-SIGN + cells (EC 50: 0.1–2.2 μM). These CBAs were clearly more active against DENV in IL-4-treated monocytes (EC 50: 4–56 nM). However, the CBAs were devoid of antiviral activity in DENV-susceptible Vero-B (DC-SIGN −) cells, demonstrating cell type-dependent differences in viral entry mechanisms.

Effects of DC-SIGN expression on renal tubulointerstitial fibrosis in nephritis

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is important for dendritic cell (DC) in migrating, recognizing, capturing, presenting antigens and in initiating T cell responses. In the present study, we investigated the role of DC-SIGN in renal tubulointerstitial inflammation and fibrosis. DC-SIGN was mainly expressed in tubular epithelial cells and DC-SIGN+ DCs were primarily distributed in renal tubulointerstitial areas during the early stage of nephritis, which was correlated with the degree of renal tubular interstitial lesions and fibrosis. In vitro, DC-SIGN expression in cultured human renal tubular epithelial cells was elevated when treated by tumor necrosis factor-alpha, and was inhibited by anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb). In a rat model of chronic renal interstitial fibrosis, there was a significant correlation of DC-SIGN expression with DC-SIGN+ DC distribution and the degree of tubulointerstitial lesion. PsL-EGFmAb reduced DC-SIGN expression and DC-SIGN+ DC accumulation in renal tissues in this rat model. These results suggest that DC-SIGN plays an important role in DC-mediated renal tubular interstitial lesions induced by immuno-inflammatory responses.

Effects of DC-SIGN expression on renal tubulointerstitial fibrosis in nephritis

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is important for dendritic cell (DC) in migrating, recognizing, capturing, presenting antigens and in initiating T cell responses. In the present study, we investigated the role of DC-SIGN in renal tubulointerstitial inflammation and fibrosis. DC-SIGN was mainly expressed in tubular epithelial cells and DC-SIGN+ DCs were primarily distributed in renal tubulointerstitial areas during the early stage of nephritis, which was correlated with the degree of renal tubular interstitial lesions and fibrosis. In vitro, DC-SIGN expression in cultured human renal tubular epithelial cells was elevated when treated by tumor necrosis factor-alpha, and was inhibited by anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb). In a rat model of chronic renal interstitial fibrosis, there was a significant correlation of DC-SIGN expression with DC-SIGN+ DC distribution and the degree of tubulointerstitial lesion. PsL-EGFmAb reduced DC-SIGN expression and DC-SIGN+ DC accumulation in renal tissues in this rat model. These results suggest that DC-SIGN plays an important role in DC-mediated renal tubular interstitial lesions induced by immuno-inflammatory responses.

Chemical Synthesis of All Phosphatidylinositol Mannoside (PIM) Glycans from Mycobacterium tuberculosis

The emergence of multidrug-resistant tuberculosis (TB) and problems with the BCG tuberculosis vaccine to protect humans against TB have prompted investigations into alternative approaches to combat this disease by exploring novel bacterial drug targets and vaccines. Phosphatidylinositol mannosides (PIMs) are biologically important glycoconjugates and represent common essential precursors of more complex mycobacterial cell wall glycolipids including lipomannan (LM), lipoarabinomannan (LAM), and mannan capped lipoarabinomannan (ManLAM). Synthetic PIMs constitute important biochemical tools to elucidate the biosynthesis of this class of molecules, to reveal PIM interactions with host cells, and to investigate the function of PIMs as potential antigens and/or adjuvants for vaccine development. Here, we report the efficient synthesis of all PIMs including phosphatidylinositol (PI) and phosphatidylinositol mono- to hexa-mannoside (PIM1 to PIM6). Robust synthetic protocols were developed for utilizing bicyclic and tricyclic orthoesters as well as mannosyl phosphates as glycosylating agents. Each synthetic PIM was equipped with a thiol-linker for immobilization on surfaces and carrier proteins for biological and immunological studies. The synthetic PIMs were immobilized on microarray slides to elucidate differences in binding to the dendritic cell specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) receptor. Synthetic PIMs served as immune stimulators during immunization experiments in C57BL/6 mice when coupled to the model antigen keyhole-limpet hemocyanin (KLH).