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Biomedical subjects

D Vercelli

Publications and source records attributed to D Vercelli.

At least 19 recordsLinked to original sources
PubMed2001

A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhances transcriptional activity.

CD14 is a pattern recognition receptor that plays a central role in innate immunity through recognition of bacterial lipoglycans, primarily LPS. Recently, our group has identified a common single nucleotide polymorphism, -159C-->T, in the CD14 proximal promoter. Homozygous carriers of the T allele have a significant increase in soluble CD14, but a decreased total serum IgE. This epidemiologic evidence led us to investigate the molecular basis for the effects of CD14/-159C-->T on CD14 regulation in monocytes and hepatocytes, the two major cell types known to express this gene in vivo. EMSA analysis showed that the T allele results in decreased affinity of DNA/protein interactions at a GC box that contains a binding site for Sp1, Sp2, and Sp3 transcription factors. In reporter assays, the transcriptional activity of the T allele was increased in monocytic Mono Mac 6 cells, which express low levels of Sp3, a member of the Sp family with inhibitory potential relative to activating Sp1 and Sp2. By contrast, both alleles were transcribed equivalently in Sp3-rich hepatocytic HepG2 cells. Our data indicate that the interplay between CD14 promoter affinity and the [Sp3]:[Sp1 + Sp2] ratio plays a critical mechanistic role in regulating transcription of the two CD14 alleles. Variation in a key gene of innate immunity may be important for the pathogenesis of allergy and inflammatory disease through gene-by-gene and/or gene-by-environment interactions.

Base Sequence↗
PubMed2001

Immunoglobulin E and its regulators.

Novel paradigms are emerging in the field of IgE regulation. Gene-environment interactions are increasingly considered to be major determinants of the type and amplitude of allergen-induced antibody responses. At the molecular level, the mechanistic connection between transcription and switching has been strengthened by the recent discovery that class switch recombination requires activation-induced deaminase, a novel RNA editing enzyme. These findings will be critical for our understanding of the mechanisms that determine immunoglobulin isotype expression during immune responses.

Animals↗
PubMed2001

CD14: a bridge between innate immunity and adaptive IgE responses.

Total IgE levels are known to be under genetic control. Linkage studies have indicated that one or more loci on chromosome 5q may control total IgE, as well as asthma and bronchial hyperresponsiveness to non-specific stimuli. Our group has undertaken a systematic analysis of chromosome 5q, and has recently characterized five single nucleotide polymorphisms at position -1619, -1359, -1145, -809, and -159 in the promoter of the gene encoding CD14, the myeloid pattern recognition receptor that is critical for efficient innate immune responses to lipopolysaccharide and bacterial ligands. Individuals homozygous for the three major CD14 haplotypes found in the Children Respiratory Study population (n = 390) were analyzed for serum levels of total IgE and soluble CD14. A strong inverse correlation was found between these two parameters, i.e. carriers of the -1359T/-1145A/-159C haplotype had the highest levels of IgE, and the lowest levels of sCD14. Conversely, carriers of the -1359G/-1145G/-159T haplotype had the highest levels of sCD14 and the lowest IgE values. Our results suggest that genetic variation in CD14, a key gene of innate immunity, may modulate the effects that exposure to bacterial ligands has on the development of Th2 responses.

Child↗
PubMed2000

IgE isotype determination: epsilon-germline gene transcription, DNA recombination and B-cell differentiation.

Immunoglobulin class switching is the process which determines whether a B-cell secretes antibodies of the IgM, IgG, IgA or IgE class (or isotype). IgE is the antibody that mediates the allergic response by sensitising mast cells to allergens at the mucosal barrier. Class switching proceeds by three successive steps, culminating in the synthesis and secretion of antibody: these are germline gene transcription, DNA recombination and B-cell differentiation. We review here the present state of knowledge concerning the mechanisms involved in each of these steps, with particular reference to IgE. Intervention in the mechanisms that specify the selection of IgE may offer a means to combat allergy.

Antibodies↗
PubMed1999

Role of CD4 and CCR5 levels in the susceptibility of primary macrophages to infection by CCR5-dependent HIV type 1 isolates.

Macrophages are a preferred target for sexually transmitted human immunodeficiency virus type 1 (HIV-1) isolates that use CCR5 as a coreceptor in combination with CD4. To assess whether the susceptibility of MDMs to infection by an R5 isolate was influenced by CD4 and/or CCR5 expression, levels of membrane CD4 or CCR5 transcripts at the time of infection and ID50 values 15 days postinfection were measured in cultures of primary macrophages infected with HIV-1(10005). To analyze the data, subjects were divided so as to maximize differences in the levels of CD4 or CCR5 expression between groups. Indeed, the difference in CD4 expression between the CD4high (MFI, 16.7 +/- 2.2) and CD4low (MFI, 6.7 +/- 0.7) groups attained high significance (p < 0.005). Of note, susceptibility to infection of MDMs isolated from CD4high donors was strikingly enhanced as compared with CD4low subjects, as shown by a fourfold increase in ID50 titers at day 15 postinfection (p < 0.002). In contrast, no significant difference in ID50 was apparent when the subjects were grouped according to the levels of CCR5 transcripts, even though CCR5 expression in the two groups differed significantly (p = 0.01). These results suggest that, regardless of variations among individuals, the intensity of CD4 expression in macrophages is such that CCR5 levels are above the threshold required for efficient HIV-1 infection. Consistent with this hypothesis, macrophages from three additional donors selected for high CD4 expression and low CCR5 transcripts were found to be highly susceptible to HIV-1 infection.

CD4 Lymphocyte Count↗
PubMed1999

To E or not to E?

Explore the source record for details and available documents.

Binding, Competitive↗
PubMed1998

Soluble CD14(1-152) confers responsiveness to both lipoarabinomannan and lipopolysaccharide in a novel HL-60 cell bioassay.

CD14 is a pattern recognition receptor involved in the interaction with multiple ligands, including LPS from gram-negative bacteria and lipoarabinomannan (LAM) from mycobacteria. While the interactions between LPS and soluble CD14 (sCD14) have been analyzed in detail, LAM/CD14 interactions remain uncharacterized due to the lack of suitable functional assays. We describe herein a novel bioassay for the analysis of CD14/ligand interactions. CD14-negative myeloid HL-60 cells up-regulate endogenous CD14 gene expression when stimulated with LPS in the presence of recombinant soluble CD14(1-348). Using the HL-60 bioassay, we showed that sCD14(1-348) confers responsiveness not only to LPS, but also to LAM. The response to LAM, but not that to LPS, was highly dependent on LPS binding protein (LBP). The N-terminal half of CD14 was sufficient to mediate HL-60 responses to LAM, since HL-60 cells responded with similar efficiency when stimulated with LAM and LBP in the presence of sCD14(1-348) or sCD14(1-152). Thus, the N-terminal 152 amino acids of CD14 contain the site(s) involved in the interaction with LAM and LBP, as well as the residues required for LAM-dependent CD14 signaling.

Gene Expression Regulation↗
PubMed1998

CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates.

The identification of HIV-1 coreceptors has provided a molecular basis for the tropism of different HIV-1 strains. CXC chemokine receptor-4 (CXCR4) mediates the entry of both primary and T cell line-adapted (TCLA) syncytia-inducing strains. Although macrophages (M phi) express CXCR4, this coreceptor is assumed to be nonfunctional for HIV-1 infection. We addressed this apparent paradox by infecting human monocyte-derived M phi with primary and TCLA isolates that were rigorously characterized for coreceptor usage and by adding the natural CXCR4 ligand, stem cell differentiation factor-1, to specifically block CXCR4-mediated entry. Our results show that primary HIV-1 isolates that selectively use CXCR4 productively infected both normal and C-C chemokine receptor-5-null M phi. By contrast, M phi supported the entry of CXCR4-dependent TCLA strains with variable efficiency but were not productively infected. Thus, the tropism of HIV isolates results from complex virus/host cell interactions both at the entry and postentry levels.

Anti-HIV Agents↗
PubMed1998

To E or not to E? Can an IL-4-induced B cell choose between IgE and IgG4?

Parasite immunologists had known for some time that IgE-mediated hypersensitivity reactions are rare in patients with chronic helminth infections, even though basophils and mast cells in these patients are sensitized with antiparasite IgE and exposed, often continuously, to parasite antigens. The inhibition of allergic reactivity in chronic helminth infections is mainly due to IgG4 'blocking antibodies' in the serum of the infected individual. IgG4 do not fix complement and bind weakly to Fcgamma receptors. Thus, antigen binding by IgG4, unlike IgE, is likely to have no or minimally harmful consequences. The discovery that, similar to IgE, expression of IgG4 is IL-4-dependent and is an intermediate step in sequential switching from IgM to IgE makes it imperative to understand how the two isotypes are coregulated and whether the two responses can be uncoupled, selectively boosting IgG4 over IgE. The ultimate goal is to apply to allergy the lesson we learnt from helminth infections.

Animals↗
PubMed1997

The transcription factor B cell-specific activator protein (BSAP) enhances both IL-4- and CD40-mediated activation of the human epsilon germline promoter.

Induction of isotype switching to a particular C(H) gene correlates with the transcriptional activation of the same gene in germline configuration. Induction of correctly spliced germline transcripts is necessary to target a switch region for recombination and switching. Different cytokines activate transcription at different germline promoters. Because binding sites for the B cell-specific transcription factor BSAP are located upstream of several switch regions in the Ig locus, BSAP might play a role in isotype switching by regulating germline transcription. We investigated whether BSAP plays a role in the transcriptional regulation of the epsilon germline promoter in human B cells. We identified human EBV-negative B cell lines that express epsilon germline transcripts upon stimulation with IL-4. Electrophoretic mobility shift assay analysis showed that the human epsilon germline promoter binds BSAP. BSAP activity was expressed constitutively and was not affected by stimulation with IL-4 and/or anti-CD40 mAb. Reporter assays with constructs containing a luciferase gene driven by the epsilon germline promoter, with or without mutations in the BSAP binding site, showed that BSAP plays a role in both IL-4-dependent induction and CD40-mediated up-regulation of human epsilon germline transcription. Furthermore, epsilon germline promoter activity was abrogated in REH cells that express a BSAP polypeptide truncated in the trans-activation domain. Among the transcription factors that regulate epsilon germline expression, BSAP is unique, in that it is B cell-specific and is at the merging point of two signaling pathways that are distinct but both critical for the induction of IgE switching.

Base Sequence↗
PubMed1997

C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells.

Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C-C chemokines (RANTES, MIP-1 alpha, and MIP-1 beta) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

Cells, Cultured↗
PubMed1995

IL-13 down-regulates CD14 expression and TNF-alpha secretion in normal human monocytes.

CD14, a glycosylphosphatidylinositol (GPI)-linked protein expressed on monocytes and neutrophils, regulates monocyte-lymphocyte interactions and serves as the LPS receptor. We showed previously that IL-4 down-regulates the expression of human CD14 by acting at the transcriptional level. We now investigate whether CD14 expression could also be regulated by IL-13, another member of the chromosome 5 cytokine gene family. IL-13 dose-dependently inhibited CD14 expression on human monocytes. By contrast, expression of CD23 and CD11b was enhanced strongly. Down-regulation of CD14 involved neither shedding nor activation of endogenous GPI anchor-cleaving enzymes. Indeed, soluble CD14 was not increased in the supernatants of IL-13-stimulated monocytes, and expression of CD55/DAF, another GPI-linked protein, was unaffected by IL-13. CD14 transcript levels were reduced sixfold in IL-13-treated monocytes. These results suggest that IL-13 down-regulates membrane CD14 by suppressing CD14 RNA expression. IL-13-dependent down-regulation of CD14 resulted in the inhibition of CD14-mediated events. Indeed, CD14-mediated release of TNF-alpha was inhibited markedly (approximately 75%) in monocytes stimulated with LPS (100 ng/ml) after a 72-h preincubation with IL-13. However, IL-13 also directly inhibited monokine secretion, because it blocked PMA-induced, CD14-independent TNF-alpha release. Down-regulation of CD14 and TNF-alpha secretion may play a major role in the anti-inflammatory effects of IL-13 on LPS-stimulated monocytes.

Antigens, CD↗
PubMed1995

Expression of IL-2 and IL-4 in T lymphocytes transformed by herpesvirus saimiri.

Herpesvirus saimiri (H. saimiri) is a highly oncogenic lymphotropic herpesvirus which can immortalize T lymphocytes and cause tumors in rabbits and New World monkeys. T cells infected with strain 484-77 of group C express four viral U-like small RNAs (HSUR1-4) and a 1.2-kb mRNA which encodes open reading frames ORF-1 and ORF-2. ORF-1 encodes a collagen-like oncoprotein. Deletion mutation analysis showed that ORF-1 and ORF-2 are essential for IL-2 independent growth of human T cells infected with H. saimiri. An earlier study also demonstrated that H. saimiri-immortalized cells carry functional IL-2 receptors. The work presented in this report investigated whether IL-2 and IL-4 is produced by H. saimiri-immortalized T lymphocytes. Both IL-2 mRNA and IL-4 mRNA were detected in various monkey T cells as well as human peripheral blood lymphocytes infected with wild-type H. saimiri. Secretion of IL-2 was suggested by cyclosporin A inhibition. IL-4 secretion by monkey T cell cultures was demonstrated by a bioassay and inhibition of bioactivity by an antibody to IL-4. The data also show that recombinant IL-4 stimulate H. saimiri-immortalized T cells; thus, IL-4 receptors are expressed. However, antibodies to human IL-4, IL-4 receptor, or soluble IL-4 receptor did not curtail growth of transformed cells. T cells infected with ORF-1 and ORF-2 deletion mutants expressed no detectable IL-2 mRNA. ORF-1, ORF-2, HSUR1, and HSUR2, were all essential for expression of IL-4 mRNA. These data are consistent with the hypothesis that H. saimiri-immortalized monkey and human T lymphocytes proliferate through autocrine secretion of IL-2 and that ORF-1, ORF-2, and HSUR sequences of the virus are involved in expression of lymphokines.

Animals↗