Biomedical subjects

Pedro Romero

Publications and source records attributed to Pedro Romero.

At least 19 recordsLinked to original sources

PubMed2006

Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor.

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-gamma secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-gamma production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.

CD8-Positive T-Lymphocytes↗

PubMed2006

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Amino Acid Sequence↗

PubMed2006

[Immunotherapy for head and neck squamous cell carcinoma].

In the past decades, prognosis of head and neck squamous cell carcinoma (HNSCC) has not improved despite substantial progress in treatment options. Since antitumoral immunity was described, immunotherapy has shown promising results as an adjunctive treatment in various cancer types. Tumor-associated antigens (TAAs) have been identified and shown to stimulate selective T-cell-mediated antitumoral immune response. This article briefly reviews the work done in the field of immunotherapy of HNSCC in the past few years. It gives confidence that immunotherapy may play an important role in the treatment of head and neck squamous cell carcinoma. Among various TAAs, the family of cancer testis antigens (CTAs) may be promising candidates for specific immune therapy in HNSCC. Ongoing studies will confirm whether CTAs may generate an immune response in clinical vaccine trials.

Carcinoma, Squamous Cell↗

PubMed2006

[Role of atherogenic dyslipidemia in the development of metabolic syndrome].

BACKGROUND AND OBJECTIVE: Our goal was to know if the diagnosis of metabolic syndrome (MS) as defined by International Diabetes Federation (IDF) criteria 2005 identifies more subjects with atherogenic dyslipidemia and high cardiovascular risk than the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP-III) criteria. PATIENTS AND METHOD: Cross-sectional study in an adult population (20-70 years) of 344 subjects (158 males and 186 females), who visited an out-patient clinic in the metropolitan area of Valencia over a period of one year. Opportunistic search method was used. We studied: anthropometrics parameters, blood pressure, plasma lipids -non-high-density lipoprotein cholesterol (no-HDL-c), and total cholesterol:high-density lipoprotein cholesterol index (TC-HDL-c)-, glucose and insulin, and HOMA (Homeostasis Model Assessment) index. RESULTS: We have separated the subjects on the basis to the IDF or ATP-III criteria to define the MS. The MS define a group of subjects with important plasma lipids alterations and cardiovascular risk: increase of no-HDL-c, of TC:HDL-c index and of apolipoprotein B. The TC:HDL-c index was > or = 6 in 28.6% of the subjects with MS and only in 9.4% of the subjects without MS (p < 0.0001) by IDF criteria and in 34.6% of the subjects with MS-ATP-III, and in 8.6% without MS-ATP-III (p < 0.0001). The apolipoprotein B > or = 1.2 g/l was observed in 44% of the subjects with MS and only in 26% without MS IDF criteria (p < 0.0001), and only in 47.6% and 28.6%, respectively, by ATP-III criteria (p < 0.0001). CONCLUSIONS: The MS defined by the IDF or ATP-III criteria equally identifies subjects with atherogenic dyslipidemia, with increase of no-HDL-c, of the TC: HDL-c index and of the apolipoprotein B and, for this reason, with high cardiovascular risk.

Adult↗

PubMed2006

Molecular and immunological evaluation of the expression of cancer/testis gene products in human colorectal cancer.

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8(+) T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses.

Antigens, Neoplasm↗

PubMed2006

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site.

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

Adult↗

PubMed2006

Rational drug design via intrinsically disordered protein.

Despite substantial increases in research funding by the pharmaceutical industry, drug discovery rates seem to have reached a plateau or perhaps are even declining, suggesting the need for new strategies. Protein-protein interactions have long been thought to provide interesting drug discovery targets, but the development of small molecules that modulate such interactions has so far achieved a low success rate. In contrast to this historic trend, a few recent successes raise hopes for routinely identifying druggable protein-protein interactions. In this Opinion article, we point out the importance of coupled binding and folding for protein-protein signalling interactions generally, and from this and associated observations, we develop a new strategy for identifying protein-protein interactions that would be particularly promising targets for modulation by small molecules. This novel strategy, based on intrinsically disordered protein, has the potential to increase significantly the discovery rate for new molecule entities.

Chemistry, Pharmaceutical↗

PubMed2006

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination.

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Antigen Presentation↗

PubMed2006

LiveCount Assay: concomitant measurement of cytolytic activity and phenotypic characterisation of CD8(+) T-cells by flow cytometry.

Tumour immunologists strive to develop efficient tumour vaccination and adoptive transfer therapies that enlarge the pool of tumour-specific and -reactive effector T-cells in vivo. To assess the efficiency of the various strategies, ex vivo assays are needed for the longitudinal monitoring of the patient's specific immune responses providing both quantitative and qualitative data. In particular, since tumour cell cytolysis is the end goal of tumour immunotherapy, routine immune monitoring protocols need to include a read-out for the cytolytic efficiency of Ag-specific cells. We propose to combine current immune monitoring techniques in a highly sensitive and reproducible multi-parametric flow cytometry based cytotoxicity assay that has been optimised to require low numbers of Ag-specific T-cells. The possibility of re-analysing those T-cells that have undergone lytic activity is illustrated by the concomitant detection of CD107a upregulation on the surface of degranulated T-cells. To date, the LiveCount Assay provides the only possibility of assessing the ex vivo cytolytic activity of low-frequency Ag-specific cytotoxic T-lymphocytes from patient material.

Antigens, Neoplasm↗

PubMed2006

Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells.

Direct identification as well as isolation of antigen-specific T cells became possible since the development of "tetramers" based on avidin-fluorochrome conjugates associated with mono-biotinylated class I MHC-peptide monomeric complexes. In principle, a series of distinct class I MHC-peptide tetramers, each labelled with a different fluorochrome, would allow to simultaneously enumerate as many unique antigen-specific CD8(+) T cells. Practically, however, only phycoerythrin and allophycocyanin conjugated tetramers have been generally available, imposing serious constraints for multiple labeling. To overcome this limitation, we have developed dextramers which are multimers based on a dextran backbone bearing multiple fluorescein and streptavidin moieties. Here we demonstrate the functionality and optimization of these new probes on human CD8(+) T cell clones with four independent antigen specificities. Their applications to the analysis of relatively low frequency antigen-specific T cells in peripheral blood, as well as their use in fluorescence microscopy, are demonstrated. The data show that dextramers produce a stronger signal than their fluoresceinated tetramer counterparts. Thus, these could become the reagents of choice as the antigen-specific T cell labeling transitions from basic research to clinical application.

Antigens, Neoplasm↗

PubMed2006

Ex vivo detectable human CD8 T-cell responses to cancer-testis antigens.

Clinical trials have shown that strong tumor antigen-specific CD8 T-cell responses are difficult to induce but can be achieved for T-cells specific for melanoma differentiation antigens, upon repetitive vaccination with stable emulsions prepared with synthetic peptides and incomplete Freund's adjuvant. Here, we show in four melanoma patients that ex vivo detectable T-cells and thus strong T-cell responses can also be induced against the more universal cancer-testis antigens NY-ESO-1 and Mage-A10. Interestingly, all patients had ex vivo detectable T-cell responses against multiple antigens after serial vaccinations with three peptides emulsified in incomplete Freund's adjuvant. Antigen-specific T-cells displayed an activated phenotype and secreted IFNgamma. The robust immune responses provide a solid basis for further development of human T-cell vaccination.

Antigens, Neoplasm↗

PubMed2006

Distinct mechanisms control human naive and antigen-experienced CD8+ T lymphocyte proliferation.

Human Ag-specific CD8(+) T lymphocytes are heterogeneous and include functionally distinct populations. In this study, we report that at least two distinct mechanisms control the expansion of circulating naive, memory, and effector CD8(+) T lymphocytes when exposed to mitogen or Ag stimulation. The first one leads to apoptosis and occurs shortly after in vitro stimulation. Susceptibility to cell death is prominent among primed T cell subsets, and it is inversely correlated with the size of the ex vivo Bcl-2(high) population within these subsets. Importantly, the Bcl-2(high) phenotype is associated to the proportion of responsive CD8(+) T cells, independently of their differentiation stage. The second one depends on the expression of newly synthesized cyclin-dependent kinase inhibitor p16(INK4a) that occurs in a significant fraction of T cells that had been actively cycling, leading to their cell cycle arrest upon stimulation. Strikingly, accumulation of p16(INK4a) protein preferentially occurs in naive as opposed to primed derived T lymphocytes and is not related to apoptosis. Significant levels of p16 are readily detectable in a small number of ex vivo CD8(+) T cells. Our observations reveal that activation-induced p16 expression represents an alternative process to apoptosis, limiting the proliferation potential of activated naive derived T lymphocytes.

Adult↗

PubMed2006

Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex.

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.

Adult↗

PubMed2006

EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming.

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

Amino Acid Sequence↗

PubMed2006

Decreased specific CD8+ T cell cross-reactivity of antigen recognition following vaccination with Melan-A peptide.

The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8(+) T cells following vaccination of HLA-A2(+) melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8(+) T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8(+) T cell clones. While Melan-A-reactive CD8(+) T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.

Antigens, Neoplasm↗

PubMed2006

Intracameral cefazolin as prophylaxis against endophthalmitis in cataract surgery.

PURPOSE: To evaluate the efficacy of prophylactic intracameral cefazolin in preventing endophthalmitis in cataract surgery. SETTING: Service of Ophthalmology, St. Joan Hospital, Reus, Spain. METHODS: In this noncontrolled retrospective observational study, all patients had uncombined cataract surgery from January 2001 to December 2004. All cases of postoperative endophthalmitis were related. The patients were classified into 2 groups. The first (n = 3650) had surgery between January 2001 and December 2002, and the second (n = 3618) had surgery between January 2003 and December 2004; the second group received a bolus of intracameral cefazolin instilled at the end of surgery. RESULTS: The rate of postoperative endophthalmitis was lower in the cases with intracameral injection at the end of surgery (0.055%) than in those without cefazolin injection (0.63%). No patients in the group with intracameral cefazoline developed corneal endothelium or retinal toxicity. CONCLUSIONS: Intracameral bolus injection of cefazolin (1 mg in 0.1 mL solution) in cataract surgery demonstrated prophylactic efficacy in diminishing the rate of postoperative endophthalmitis without toxic effects on the cornea or retina.

Aged↗

PubMed2006

The human T cell response to melanoma antigens.

The cornerstone of the concept of immunosurveillance in cancer should be the experimental demonstration of immune responses able to alter the course of in vivo spontaneous tumor progression. Elegant genetic manipulation of the mouse immune system has proved this tenet. In parallel, progress in understanding human T cell mediated immunity has allowed to document the existence in cancer patients of naturally acquired T cell responses to molecularly defined tumor antigens. Various attributes of cutaneous melanoma tumors, notably their adaptability to in vitro tissue culture conditions, have contributed to convert this tumor in the prototype for studies of human antitumor immune responses. As a consequence, the first human cytolytic T lymphocyte (CTL)-defined tumor antigen and numerous others have been identified using lymphocyte material from patients bearing this tumor, detailed analyses of specific T cell responses have been reported and a relatively large number of clinical trials of vaccination have been performed in the last 15 years. Thus, the "melanoma model" continues to provide valuable insights to guide the development of clinically effective cancer therapies based on the recruitment of the immune system. This chapter reviews recent knowledge on human CD8 and CD4 T cell responses to melanoma antigens.

Antigens, Neoplasm↗

PubMed2006

Conservation of intrinsic disorder in protein domains and families: I. A database of conserved predicted disordered regions.

Many protein regions have been shown to be intrinsically disordered, lacking unique structure under physiological conditions. These intrinsically disordered regions are not only very common in proteomes, but also crucial to the function of many proteins, especially those involved in signaling, recognition, and regulation. The goal of this work was to identify the prevalence, characteristics, and functions of conserved disordered regions within protein domains and families. A database was created to store the amino acid sequences of nearly one million proteins and their domain matches from the InterPro database, a resource integrating eight different protein family and domain databases. Disorder prediction was performed on these protein sequences. Regions of sequence corresponding to domains were aligned using a multiple sequence alignment tool. From this initial information, regions of conserved predicted disorder were found within the domains. The methodology for this search consisted of finding regions of consecutive positions in the multiple sequence alignments in which a 90% or more of the sequences were predicted to be disordered. This procedure was constrained to find such regions of conserved disorder prediction that were at least 20 amino acids in length. The results of this work included 3,653 regions of conserved disorder prediction, found within 2,898 distinct InterPro entries. Most regions of conserved predicted disorder detected were short, with less than 10% of those found exceeding 30 residues in length.

Amino Acid Sequence↗