Biomedical subjects

Pedro Roda-Navarro

Publications and source records attributed to Pedro Roda-Navarro.

8 recordsLinked to original sources

PubMed2006

Transfer of NKG2D and MICB at the cytotoxic NK cell immune synapse correlates with a reduction in NK cell cytotoxic function.

Although transfer of membrane proteins has been shown to occur during immune cell interactions, the functional significance of this process is not well understood. Here we describe the intercellular transfer of NKG2D and MHC class I chain-related molecule (MIC) B proteins at the cytotoxic natural killer cell immune synapse (cNK-IS). MICB expressed on the 721.221 cell line induced clustering of NKG2D at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing F-actin. Moreover, natural killer (NK) cell membrane-connective structures formed during cytotoxic interactions contained F-actin, perforin, and NKG2D. NKG2D transfer depended on binding to MICB and was specific because transfer of other molecules not involved in NK-IS formation was not observed. Transfer of MICB to NK cells also was noted, suggesting a bidirectional exchange of receptor/ligand pairs at cNK-IS. Experiments designed to test the functional significance of these observations revealed that brief interactions between NK cells and MICB expressing target cells led to a reduction in NKG2D-dependent NK cytotoxicity. These data demonstrate interchange of an activating receptor and its ligand at the cNK-IS and document a correlation between synapse organization, intercellular protein transfer, and compromised NK cell function after interaction with a susceptible target cell.

Actins↗

PubMed2006

The human cytomegalovirus glycoprotein UL16 traffics through the plasma membrane and the nuclear envelope.

The human cytomegalovirus (HCMV) UL16 gene encodes a glycoprotein that interferes with the immune response to the virus-infected cell. In vitro, UL16 interacts with MICB and ULBPs that are ligands for the stimulatory receptor NKG2D, expressed on NK cells and CD8(+)T cells. UL16 expression has been shown to promote intracellular accumulation of MICB, ULBP1 and 2 and thus, interfere with the immune response to HCMV-infected cells. The mechanism that has been suggested for UL16-mediated MICB downmodulation is retention in the ER. Here, we studied the intracellular localization and maturation of UL16 and MICB in HCMV-infected cells and transfectant systems. UL16 trafficked through the ER, TGN and progressed to the plasma membrane, after which the protein was internalized. Strikingly, UL16 was also observed in the inner nuclear membrane. MICB was also localized in the TGN in HCMV-infected cells. These data suggest that MICB trafficking might be affected after its transit through the ER.

Cell Line↗

PubMed2006

Interleukin-15 and interferon-gamma participate in the cross-talk between natural killer and monocytic cells required for tumour necrosis factor production.

We have characterized the lymphocyte subset and the receptor molecules involved in inducing the secretion of TNF by monocytic cells in vitro. The TNF secreted by monocytic cells was measured when they were co-cultured with either resting or IL-15-stimulated lymphocytes, T cells, B cells or natural killer (NK) cells isolated from the peripheral blood of healthy subjects and from the synovial fluid from patients with inflammatory arthropathies. Co-culture with IL-15-activated peripheral blood or synovial fluid lymphocytes induced TNF production by monocytic cells within 24 hours, an effect that was mainly mediated by NK cells. In turn, monocytic cells induced CD69 expression and IFN-gamma production in NK cells, an effect that was mediated mainly by beta2 integrins and membrane-bound IL-15. Furthermore, IFN-gamma increased the production of membrane-bound IL-15 in monocytic cells. Blockade of beta2 integrins and membrane-bound IL-15 inhibited TNF production, whereas TNF synthesis increased in the presence of anti-CD48 and anti-CD244 (2B4) monoclonal antibodies. All these findings suggest that the cross-talk between NK cells and monocytes results in the sustained stimulation of TNF production. This phenomenon might be important in the pathogenesis of conditions such as rheumatoid arthritis in which the synthesis of TNF is enhanced.

Antigens, CD↗

PubMed2006

Thymidylate synthase expression pattern, expression level and single nucleotide polymorphism are predictors for disease-free survival in patients of colorectal cancer treated with 5-fluorouracil.

Several variables associated to thymidylate synthase (TS), the biological target of 5-fluorouracil (5FU) have been studied for their possible role as predictors of the clinical outcome and response to chemotherapy in colorectal cancer (CRC) patients. The level of protein expression and the number of variable tandem-repeats of a 28-bp sequence within the gene promoter have been proposed as predictive and/or prognostic factors with variable agreement, while consensus seems to be achieved with respect to the value of a single nucleotide polymorphism (SNP) described within this same region. More recently, an association between TS expression pattern and survival has been disclosed. Paraffin-embedded sections from 140 CRC patients were analyzed by immuno-histochemistry (Mab TS106) for TS levels and expression pattern. Also, VNTR and SNP were determined by polymerase-chain reaction (PCR) and restriction-length-fragment polymorphism (RFLP) in 123 and 112 patients, respectively. Cytoplasmic expression pattern tended to be associated to C SNP (p=0.06). Low TS expression levels, cytoplasmic expression pattern and C SNP arose as variables associated to longer progression-free survival (PFS) in patients treated with 5FU. Accordingly, patients having at least two favourable or unfavourable variables were classified respectively as 'low risk' and 'high risk', the former showing significantly longer PFS (p=0.0299). The possibility for designing a selection method for subsequent therapies is suggested on the basis of a probable combined effect of the above mentioned parameters but further studies in larger populations are needed to confirm these results.

Aged↗

PubMed2004

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs).

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.

Amino Acid Motifs↗

PubMed2004

Dynamic redistribution of the activating 2B4/SAP complex at the cytotoxic NK cell immune synapse.

The 2B4 molecule (CD244) has been described as a coreceptor in human NK cell activation. However, the behavior of 2B4 during the cytotoxic NK cell immune synapse (NK-IS) formation remains undetermined. In this study, we demonstrate the redistribution of 2B4 and the signaling adaptor molecule, signaling lymphocyte activation molecule-associated protein (SAP), to the cytotoxic NK-IS upon formation of conjugates between resting NK cells and EBV-infected 721.221 human cells. Confocal microscopy showed that 2B4 localized at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing talin within NK cell and ICAM-1 on target cells. Videomicroscopy studies with 2B4-GFP-transfected NK cells revealed that 2B4 redistributed to cytotoxic NK-IS as soon as the cell contact occurred. Simultaneously, a SAP-GFP also clustered at the contact site, where it remained during the interaction period. The 2B4 molecular clusters remained bound to the target cell even after NK cell detachment. These results underscore the function of 2B4 as an adhesion molecule and suggest a relevant role in the initial binding, scanning of target cells, and formation of cytotoxic NK-IS. Finally, these findings are indicative of an important role of the activating 2B4/signaling lymphocyte activation molecule-associated protein complex during the recognition of EBV-infected cells.

Antigens, CD↗

PubMed2004

The human C-type lectin CLECSF8 is a novel monocyte/macrophage endocytic receptor.

Cell surface lectin receptors play important roles in the function of macrophages. Herein, we have identified and characterized the human orthologue of the mouse Mcl/Clecsf8. Human CLECSF8 codes for a type II membrane glycoprotein of 215 amino acids that belongs to the human calcium-dependent lectin family (C-type lectin). The cytoplasmic tail of CLECSF8 lacks consensus signaling motifs and its extracellular region shows a single carbohydrate recognition domain (CRD). The CLECSF8 gene has been localized on the telomeric region of the NK gene complex on chromosome 12p13 close to MINCLE. CLECSF8 mRNA shows a monocyte/macrophage expression pattern. Biochemical analysis of CLECSF8 on transiently transfected cells showed a glycoprotein of 30 kDa. Cross-linking of the receptor leads to a rapid internalization suggesting that CLECSF8 constitutes and endocytic receptor.

Alternative Splicing↗

PubMed2004

AM3 inhibits HBV replication through activation of peripheral blood mononuclear cells.

In this report, we have analyzed the effect of AM3, a glycoconjugate of natural origin with immunomodulatory properties, which is available under the commercial name of Inmunoferon, on hepatitis B virus (HBV) replication in HBV-transfected cells. We found that AM3 inhibited HBV RNA expression as well as DNA synthesis and viral antigen expression by an indirect mechanism. We found that AM3 lacked intrinsic antiviral properties, and that the antiviral effect of the glycoconjugate was due to stimulation of secretion of molecules with antiviral properties by peripheral blood mononuclear cells. Our data indicate that the employment of AM3 as an adjuvant administered simultaneously with conventional antiviral drugs may potentiate the endogenous response against viral infection.

Adjuvants, Immunologic↗