Biomedical subjects

Clara Bueno

Publications and source records attributed to Clara Bueno.

10 recordsLinked to original sources

PubMed2006

Molecular basis of TCR selectivity, cross-reactivity, and allelic discrimination by a bacterial superantigen: integrative functional and energetic mapping of the SpeC-Vbeta2.1 molecular interface.

Superantigens activate large fractions of T cells through unconventional interactions with both TCR beta-chain V domains (Vbetas) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vbeta2(+) T cells. Herein, we have analyzed the SpeC-Vbeta2.1 interaction by mutating all SpeC residues that make contact with Vbeta2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vbeta2.1(+) Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC "functional epitope" for TCR engagement. Although only two SpeC residues (Tyr(15) and Arg(181)) are critical for activation of virtually all human CD3(+) T cells, a larger cluster of four hot spot residues are required for interaction with Vbeta2.1. Three of these residues (Tyr(15), Phe(75), and Arg(181)) concentrate their binding energy on the CDR2 loop residue Ser(52a), a noncanonical residue insertion found only in Vbeta2 and Vbeta4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vbeta2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vbeta2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vbeta domain subfamily.

Alleles↗

PubMed2006

Wilms' tumor 1-associating protein regulates the proliferation of vascular smooth muscle cells.

Smooth muscle cells (SMCs) are called on to proliferate during vascular restructuring but must return to a nonproliferative state if remodeling is to appropriately terminate. To identify mediators of the reacquisition of replicative quiescence, we undertook gene expression screening in a uniquely plastic human SMC line. As proliferating SMCs shifted to a contractile and nonproliferative state, expression of TIMP-3, Axl, and KIAA0098 decreased whereas expression of complement C1s, cathepsin B, cellular repressor of E1A-activated genes increased. Wilms' tumor 1-associating protein (WTAP), a nuclear constituent of unknown function, was also upregulated as SMCs became nonproliferative. Furthermore, WTAP in the intima of injured arteries was substantially upregulated in the late stages of repair. Introduction of WTAP complementary DNA into human SMCs inhibited their proliferation, with a corresponding decrease in DNA synthesis and an increase in apoptosis. Knocking down endogenous WTAP increased SMC proliferation, because of increased DNA synthesis and G(1)/S phase transition, together with reduced apoptosis. WTAP was found to associate with the Wilms' tumor-1 protein in human SMCs and WTAP overexpression inhibited the binding of WT1 to an oligonucleotide containing a consensus WT1 binding site, whereas WTAP knockdown accentuated this interaction. Expression of the WT1 target genes, amphiregulin and Bcl-2, was suppressed in WTAP-overexpressing SMCs and increased in WTAP-deficient SMCs. Moreover, exogenous amphiregulin rescued the antiproliferative effect of WTAP. These findings identify WTAP as a novel regulator of the cell cycle and cell survival and implicate a WTAP-WT1 axis as a novel pathway for controlling vascular SMC phenotype.

Amphiregulin↗

PubMed2006

Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

Antigens, Bacterial↗

PubMed2005

Long-term immune recovery of patients undergoing allogeneic stem cell transplantation: a comparison with their respective sibling donors.

We have addressed whether patients' immune system status after allogeneic stem cell transplantation, assessed more than 1 year after the procedure, recovers normal function as compared with that of their respective donors. An additional aim was to compare the status of the immune system between patients receiving reduced-intensity conditioning regimens and those undergoing myeloablative transplantations. For this purpose, we analyzed not only the different subsets of peripheral blood (PB) lymphocytes, but also circulating dendritic cell (DC) subpopulations, together with cytokine production by PB T cells, in a series of 38 patients undergoing allogeneic stem cell transplantation. We compared these patients with their respective HLA-matched donors by performing a simultaneous patient/donor paired study. Complete bone marrow chimerism status and normal PB cell counts were demonstrated in all recipients. The most relevant numeric differences found between patients and donors were related to the distribution of the distinct subsets of PB DCs (CD16+ DCs were increased, whereas myeloid and plasmacytoid DC subsets were decreased in the patient group). This was associated with an increased number of B cells, an inverted CD4/CD8 T-cell ratio, and a decrease in CD4+/CD8+ double-positive T cells in the patient group. In addition, a predominance of a T-helper 1 pattern of cytokine production (interferon gamma and tumor necrosis factor alpha) with decreased secretion of T-helper 2-associated cytokines (interleukin 5 and interleukin 10) was also observed at the single-cell level. No significant differences were found in any of the parameters analyzed between patients receiving reduced-intensity conditioning regimens and those undergoing myeloablative transplantations.

Adolescent↗

PubMed2005

Human embryonic stem cells: potential tool for achieving immunotolerance?

The derivation of human embryonic stem cells (hESCs), whose in vitro differentiation might be directed toward different cell types, has raised the hope for cell replacement therapies. Despite the emerging reports to differentiate hESCs into specific lineages and then to distinct mature cell subsets, there are still several issues that need to be resolved before transplantation of these cells can be realized. In this context, immune rejection by the host immune system has been considered to be one of the greatest hurdles for cellular transplantation. However, recent data support the concept that hESCs and/or their differentiated derivatives possess immune-privileged properties, suggesting that cells derived from hESC may provide a potential tool for induction of immunetolerance. Currently, our understanding of the tolerogenic potential of hESCs is limited to assessment by in vitro assays or xenogenic transplantation approaches in vivo. Human ESCs express low levels of major histocompatability complex (MHC)-I antigens and lack expression of MHC-II antigens and costimulatory molecules, and are not recognized by natural killer cells and inhibit T-cell induced-stimulation by third-party antigen-presenting cells. Upon injection into immunocompetent mice, hESCs are unable to induce an immune response as demonstrated by their inability to induce an inflammatory response. Based on these initial observations, further studies in hESCs immunobiology are warranted and may reveal unique mechanisms that account for the immunological properties of hESCs. Here, we explore the prospect of using hESCs and their derivatives for immunomodulation and tolerance induction.

Animals↗

PubMed2004

Mechanism of modulation of T cell responses by N-palmitoylated peptides.

Small structural changes in the antigenic peptides recognized by TCR can alter the biological properties of those peptides and convert them into weak agonists, partial agonists, or antagonists of these receptors. These altered peptide ligands (APL) are usually generated by conservative amino acid substitutions at TCR contact residues. Here, we show that APL with therapeutic properties can also be generated by attachment of palmitic acid at the N terminus of the peptide without the need to modify the peptide's primary sequence. Using N-palmitoylated pigeon cytochrome-c peptide 81-104 (PALPCC(81-104)), we were able to induce T cell hyporesponsiveness to the wild-type peptide in vitro. More importantly, administration of the PALPCC(81-104 )to mice reduced the responsiveness to the native peptide when tested ex vivo. Biochemical and functional experiments indicated that the action of N-palmitoylated peptides was due to the conversion of the native peptide into a weak agonist that could then induce T cell anergy. Our results demonstrate that N-palmitoylation of antigenic peptides is a feasible strategy to generate APL, as it avoids the need to screen multiple amino acid variants of each specific antigen to identify those with therapeutic properties.

Animals↗

PubMed2004

A new simple whole blood flow cytometry-based method for simultaneous identification of activated cells and quantitative evaluation of cytokines released during activation.

The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor alpha (TNFalpha) at the cell surface through the use of a specific inhibitor of the TNFalpha-converting enzyme. This inhibitor does not affect the release of cytokines other than TNFalpha and makes it possible to assess, in the same measurement, the phenotype of TNFalpha(+)-secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNFalpha+ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3 h with PMA plus ionomycin, cellular responses were associated with surface TNFalpha expression on the majority of CD3+ T cells and secretion of Th1-associated cytokines: interferon gamma, interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4 h of LPS-stimulation, the majority of CD14+ monocytes showed surface TNFalpha expression; in parallel, high amounts of soluble IL1beta, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viral-specific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNFalpha+ monocytes, TNFalpha+/CD8+ T cells and TNFalpha+/CD8- T lymphocytes in association with an increased secretion of IFNgamma, IL6 and TNFalpha.

Adult↗

PubMed2004

Incidence and characteristics of CD4(+)/HLA DRhi dendritic cell malignancies.

BACKGROUND AND OBJECTIVES: Recent reports suggest that CD4(+)/CD56(+)/lineage(-) hematopoietic neoplasias are aggressive types of malignancies involving lymphoplasmacytoid/DC2 dendritic cells (DC). Here, we report on the incidence of DC malignancies and their clinical, biological, phenotypic and cytogenetic characteristics. DESIGN AND METHODS: From a large series of 392 patients with acute myeloblastic leukemia (AML) and 739 with non-Hodgkin's lymphoma (NHL), five cases (three presenting as acute leukemia and two as NHL) showed clinical, morphologic and phenotypic features compatible with a DC malignancy. RESULTS: The overall incidence of DC malignancies among all AML and NHL cases was 0.76% and 0.27%, respectively. At presentation, these patients displayed cutaneous nodules, splenomegaly and lymph node involvement with variable levels of peripheral blood (PB) and/or bone marrow (BM) infiltration in association with anemia and thrombocytopenia. Cytologic studies showed immature appearing blast cells with negative cytochemistry reactions for both myeloperoxidase and esterases. A highly suggestive DC phenotype based on co-expression of CD123(hi)/HLADR(+)/lin(-)/CD56(+)/CD45(dim) associated with a germline configuration of both the IgH and TCRgamma genes was found in all except one patient who was CD56(-). Expression of other markers compatible with a DC origin was found in all cases. INTERPRETATION AND CONCLUSIONS: We show that DC-derived malignancies can present as either cutaneous lymphoma or acute leukemia, although their incidence is extremely low (<< >1%). While most of these DC neoplasias probably correspond to the malignant counterpart of DC2/lymphoplasmacytoid DC, neoplasias of myeloid DC might also exist, chemotherapy followed by consolidation with ASCT is apparently the most effective strategy for achieving a durable remission in these individuals.

Acute Disease↗

PubMed2003

TCRalphabeta+/CD4+ large granular lymphocytosis: a new clonal T-cell lymphoproliferative disorder.

Large granular lymphocyte (LGL) leukemia is a well-recognized disease of mature T-CD8(+) or less frequently natural killer cells; in contrast, monoclonal expansions of CD4(+) T-LGL have only been sporadically reported in the literature. In the present article we have explored throughout a period of 56 months the incidence of monoclonal expansions of CD4(+) T-LGL in a population of 2.2 million inhabitants and analyzed the immunophenotype and the pattern of cytokine production of clonal CD4(+) T cells of a series of 34 consecutive cases. Like CD8(+) T-LGL leukemias, CD4(+) T-LGL leukemia patients have an indolent disease; however, in contrast to CD8(+) T-LGL leukemias, they do not show cytopenias and autoimmune phenomena and they frequently have associated neoplasias, which is usually determining the clinical course of the disease. Monoclonal CD4(+) T-LGLshowed expression of TCRalphabeta, variable levels of CD8 (CD8(-/+dim)) and a homogeneous typical cytotoxic (granzyme B(+), CD56(+), CD57(+), CD11b(+/-)) and activated/memory T cell (CD2(+bright), CD7(-/+dim), CD11a(+bright), CD28(-), CD62L(-) HLA-DR(+)) immunophenotype. In addition, they exhibited a Th1 pattern of cytokine production [interferon-gamma(++), tumor necrosis factor-alpha(++), interleukin (IL-2)(-/+), IL-4(-), IL-10(-), IL-13(-)]. Phenotypic analysis of the TCR-Vbeta repertoire revealed large monoclonal TCR-Vbeta expansions; only a restricted number of TCR-Vbeta families were represented in the 34 cases analyzed. These findings suggest that monoclonal TCRalphabeta(+)/CD4(+)/NKa(+)/CD8(-/+dim) T-LGL represent a subgroup of monoclonal LGL lymphoproliferative disorders different from both CD8(+) T-LGL and natural killer cell-type LGL leukemias. Longer follow-up periods are necessary to determine the exact significance of this clonal disorder.

Adult↗

PubMed2002

A new method for detecting TNF-alpha-secreting cells using direct-immunofluorescence surface membrane stainings.

In the present study, a new flow cytometric method for the identification of TNF-alpha-secreting cells based on the use of a TNF-alpha converting enzyme (TACE) inhibitor compound (BB3103) is described. TNF-alpha secreting cells were measured in parallel in stimulated peripheral blood samples (n=4), using the BB3103 TACE inhibitor or brefeldin A as secretion blocking agents. To induce TNF-alpha production by PB T-cells and monocytes, whole blood samples were stimulated either for 4 h with PMA plus ionomycin or for 6 h with LPS plus IFNgamma, respectively. Interestingly, slightly higher percentages of TNF-alpha(+) CD4(+) (65+/-11% versus 49+/-11.4%, p=0.06) and TNF-alpha(+) CD8(+) (60+/-9.9% versus 47+/-27.7% p=0.46) T-cells together with a greater amounts of TNF-alpha/cell-mean fluorescence intensity (MFI) of 1050+/-230 versus 258+/-112 for CD4(+), p=0.06 and 424+/-169 versus 266+/-201 for CD8(+), p=0.27-were found for activated T-lymphocytes cultured with BB3103 as compared to those treated with brefeldin A. Kinetic analysis of surface TNF-alpha expression under these stimulatory conditions showed detectable surface TNF-alpha levels on both T-cells and monocytes after 30 min. Thereafter, surface TNF-alpha expression on both T-cells and monocytes progressively increased for up to 3 and 4 h, respectively. From this time on, a decrease in the membrane levels of TNF-alpha was observed in the monocytes, presumably due to the occurrence of cell death. In order to show that the BB3103 inhibitor was also active on other TACE-associated molecules, CD62L expression on PMA-stimulated PB lymphocytes, monocytes and neutrophils was analyzed by flow cytometry in the presence and absence of BB3103. The TACE inhibitor proved to be active in stabilizing CD62L expression on PMA-stimulated PB leukocytes. In summary, our results show that stimulation of PB T-cells and monocytes in the presence of the TACE inhibitor BB3103 followed by surface staining for TNF-alpha provides a new, simple and rapid method for the identification of intact TNF-alpha producting cells present in a sample without the need for prior cell fixation and permeabilization. In addition, this approach could also be applied in order to stabilize the expression of other metalloprotease-sensitive molecules such as CD62L on the surface of PB leukocytes.

ADAM Proteins↗