Biomedical subjects

Robert W Finberg

Publications and source records attributed to Robert W Finberg.

24 records · Page 2Linked to original sources

PubMed2004

Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity.

Toll-like receptors (TLRs) are critically involved in the innate immune response to bacterial, viral and fungal pathogens. We have studied human peripheral blood mononuclear cells, murine embryonic fibroblasts (MEFs) and a panel of human cell lines, including HEK, HeLa, AGS, ECV304 and U373 cells, for expression of TLR-specific mRNAs and for TLR-ligand dependent cytokine secretion. Peripheral blood cells expressed multiple TLRs; however, many studies have shown that blood contains multiple, heterogeneous cell populations with distinct patterns of TLR expression. Cell lines had variable expression of TLRs, and in most cases lacked TLR2 and TLR8 expression and only weakly expressed mRNAs for TLR5, TLR7 and TLR9. In contrast, MEFs expressed high levels of mRNA for TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9. MEFs were highly responsive to TLR-ligand activation and secreted high levels of both IL-6 and MCP-1 in response to TLR ligands. MEFs from mice with targeted deletions of TLR2, TLR4 and MyD88 demonstrated profound defects in their IL-6 response to their specific ligands, consistent with studies of macrophages and tissues from adult knockout animals. MEF cultures are homogenous and amenable to biochemical analysis and should allow rigorous studies of the contribution of individual TLRs to the innate immune response.

Adjuvants, Immunologic↗

PubMed2003

Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling.

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.

Antibodies, Monoclonal↗

PubMed2003

Structure and chromosomal localization of the murine coxsackievirus and adenovirus receptor gene.

We analyzed BAC genomic clones encoding the murine coxsackievirus and adenovirus receptor (mCAR). The mCAR gene is situated on the distal portion of murine chromosome 16, and is composed of at least eight exons, with intron-exon boundaries similar to those reported for the human CAR gene. We previously described two cDNAs encoding mCAR isoforms: the extracellular and transmembrane portions of both are encoded by exons 1-6; the cytoplasmic domain of mCAR 1 is encoded by exon 7, whereas mCAR 2 results from an RNA splice linking the proximal portion of exon 7 to an alternative exon 8. RT-PCR analysis of the mCAR RNA 5'-terminus suggests that transcription may begin 141-161 nucleotides upstream of the ATG translational start site.

5' Flanking Region↗

PubMed2003

Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2.

Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen that causes significant morbidity and mortality in immunocompromised people. An understanding of how CMV induces and circumvents host immunity is of critical importance in efforts to design effective therapeutics. It was recently discovered that mere cell contact by CMV particles leads to profound modulation of cellular gene expression, including induction of inflammatory cytokines and interferon-stimulated genes characteristic of innate immune detection. These findings suggest that a membrane receptor recognizes a CMV envelope protein(s), leading to innate immune activation. Here, we show that the pattern recognition receptors Toll-like receptor 2 (TLR2) and CD14 recognize CMV virions and trigger inflammatory cytokine production. Induction of inflammatory cytokines is mediated via TLR2-dependent activation of NF-kappa B. Since many of the pathological processes associated with CMV disease are facilitated or directly mediated by inflammatory cytokines, identification of the host membrane detection machinery may ultimately lead to improved therapeutics.

Animals↗

PubMed2002

Role of toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils.

In vitro studies as well as clinical trials indicate that the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) enhance the ability of neutrophils (polymorphonuclear leukocytes) to eliminate microbial organisms. Toll-like receptor (TLR) proteins, homologs of the Drosophila protein Toll, have been found on the surface of mammalian cells and are important in the responses of macrophages to bacterial, viral, and fungal antigens. TLR4 is critical for the response to lipopolysaccharide (LPS) of gram-negative bacteria, while TLR2 is important for response to gram-positive bacteria, bacterial peptides, and yeast zymosan. We demonstrate that TLR2, but very little TLR4, is present on the surface of human neutrophils. In addition we demonstrate that GM-CSF and G-CSF dramatically up-regulate TLR2 and CD14 surface expression. GM-CSF treatment also up-regulates TLR2 and CD14 mRNA levels in neutrophils. In addition to increasing receptor expression, GM-CSF treatment enhanced the interleukin 8 (IL-8) secretion and superoxide priming responses of neutrophils to stimulation with TLR2 ligands, including zymosan, peptidoglycan, and lipoarabinomannan. The human monocyte response to crude bacterial LPS is composed of a TLR4-specific response to the pure LPS component and a TLR2-dependent response to associated lipopeptides. The removal of TLR2 lipopeptide components from LPS by phenol re-extraction substantially reduced both the IL-8 and superoxide response of the stimulated neutrophils, indicating that, unlike monocytes, the neutrophil response is preferentially directed to TLR2 ligands. Thus, our studies demonstrate that GM-CSF dramatically enhances the functional response of neutrophils to TLR2 ligands, including LPS-associated lipopeptides.

Cells, Cultured↗

PubMedDate not supplied

Toll like receptors and viruses.

Toll like receptors (TLRs) are 'pattern recognition proteins' that discriminate between self and non-self. They interact with products of infectious agents to activate cells of the innate immune system and also stimulate the adaptive immune system. Intracellular and extracellular TLRs recognise a wide range of viruses leading to the production of different cytokines. In this paper, we summarise the types of TLRs that recognise viruses, the cytokines produced, the specific cell types involved and the activation pathways that have been characterised. We also provide an overview of the viruses demonstrated to interact with TLRs. A greater understanding of relationships between viruses or viral products and TLRs should improve understanding of pathogenesis and lead to the development of new anti-viral therapies.

Animals↗