Biomedical subjects

Pilar Martinez

Publications and source records attributed to Pilar Martinez.

14 recordsLinked to original sources

PubMed2006

Impact of Gag sequence variability on level, phenotype, and function of anti-HIV Gag-specific CD8(+) cytotoxic T lymphocytes in untreated chronically HIV-infected patients.

The role of cytotoxic T lymphocyte responses in controlling viral replication during chronic HIV infection remains controversial. Viral escape mutations driven by immune pressure have been postulated to be an important mechanism contributing to the evasion of CD8(+) T cell responses. To explore this issue in more detail, HIV-1 p17 sequence variability was examined in chronically HIV-infected patients, in parallel with the level, phenotype, and function of HIV-SL9-specific CD8(+) T cell. Thirty-one HLA-A*0201(+) (A2(+)) and 10 HLAA* 02() (A2()) patients were included. The phenotype of SL9-specific CD8(+) T cell and their ability to produce IFN-gamma were analyzed by multiparameter flow cytometry. The HIV Gag p17 was sequenced and the mean variability score for each residue within SL9 and the two epitope flanking regions were calculated using Shannon entropy. The mean variability of SL9 and the proportion of patients with amino acid changes within SL9 were similar in A2(+) and A2() patients. Patients without Tet(+) cells had a significantly higher prevalence of aminoacid changes in SL9 than patients with Tet(+) cells. Interestingly, in patients with Tet(+) cells, the Y79F mutation within SL9 tended to be associated with lower levels of Tet(+) cells. We did not find any association between amino acid changes within SL9 and the differentiation stage of Tet(+) cells, or with IFN-gamma production. A similar analysis within the epitope flanking sequences did not reveal differences in the variability of these regions. These results suggest that viral mutations driven by immune selection pressure may play an important role in evading the immunological response in chronically HIV-infected individuals.

CD8-Positive T-Lymphocytes↗

PubMed2006

Lobular neoplasia in breast core needle biopsy specimens is associated with a low risk of ductal carcinoma in situ or invasive carcinoma on subsequent excision.

To address the significance of lobular neoplasia (LN) in breast core needle biopsy specimens, we prospectively obtained LN cases and correlated results of subsequent tissue sampling. LN was diagnosed by core needle biopsy in 467 women; in 101 (21.6%), invasive carcinoma (IC) or ductal carcinoma in situ (DCIS) was diagnosed concurrently. Two patients (0.4%) had previous diagnoses of IC or DCIS, and 17 (3.6%) had a concurrent diagnosis of contralateral IC or DCIS. Of 366 patients without a concurrent diagnosis of IC or DCIS, subsequent tissue diagnoses were available for 156 cases (42.6%). Of 60 cases of LN and atypical ductal hyperplasia on the biopsy, 5 had IC and 10 had DCIS on the excision (total, 25%). Of 4 women with LN and a mucocele-like lesion on the biopsy, none had IC or DCIS on excision. Of 92 with LN alone on the biopsy, 7 had IC (6) or DCIS (1) on excision. Two cases were in sites away from the biopsy site, 3 in subsequent excisions of the biopsy site, and 2 after previous excision of the biopsy site without finding IC or DCIS. Although LN is associated with a high overall rate of IC and DCIS (30%), excision of the biopsy site for women with LN alone on core needle biopsy has a very low rate of IC and DCIS in our center. Women in whom biopsy sites are excised are still at risk for subsequent DCIS and IC.

Biopsy, Needle↗

PubMed2005

CD4+ T cell recovery beyond the first year of complete suppression of viral replication during highly active antiretroviral therapy is not influenced by CD8+ T cell activation.

CD38 expression on CD8(+) T cells was longitudinally assessed in 31 human immunodeficiency virus (HIV)-infected persons with undetectable plasma viremia who had undergone highly active antiretroviral therapy (HAART) for 12 months and were followed for a mean of 30 months thereafter. Overall, CD4(+)T cell counts increased during follow-up, whereas CD38 expression remained stable. However, a subset of patients showed declines in CD38 expression, and, conversely, another subset showed increases in CD38 expression. No association could be found between long-term gains in CD4(+) T cells and evolution of CD38 expression. Thus, activation of CD8(+) T cells does not seem to be associated with the extent of CD4(+) T cell recovery beyond the first year of successful HAART.

ADP-ribosyl Cyclase 1↗

PubMed2005

Identifying and characterising the Plasmodium falciparum merozoite surface protein 10 Plasmodium vivax homologue.

Plasmodium vivax malaria is one of the most prevalent parasitic diseases in Asia and Latin-America. The difficulty of maintaining this parasite culture in vitro has hampered identifying and characterising proteins implied in merozoite invasion of red blood cells. We have been able to identify an open reading frame in P. vivax encoding the Plasmodium falciparum merozoite surface protein 10 homologous protein using the partial sequences from this parasite's genome reported during 2004. This new protein contains 479 amino-acids, two epidermal growth factor-like domains, hydrophobic regions at the N- and C-termini, being compatible with a signal peptide and a glycosylphosphatidylinositol anchor site, respectively. The protein is expressed during the parasite's asexual stage and is recognised by polyclonal sera in parasite lysate using Western blot. P. vivax-infected patients' sera highly recognised recombinant protein by ELISA.

Amino Acid Sequence↗

PubMed2005

Differential upregulation of CD38 on different T-cell subsets may influence the ability to reconstitute CD4+ T cells under successful highly active antiretroviral therapy.

BACKGROUND: Immune activation is an independent surrogate marker of CD4 T-cell depletion in HIV-infected patients. Highly active antiretroviral therapy (HAART) reduces disease progression as a direct consequence of suppressing HIV replication. Immune function does not normalize completely in most subjects on HAART, however, perhaps reflecting residual HIV replication. So far, it is unclear to what extent immune activation may influence the evolution of CD4 T-cell counts in patients on HAART. PATIENTS AND METHODS: The expression of CD38 on naive and memory subsets of CD4+ and CD8+ T cells was measured quantitatively by flow cytometry in 62 drug-naive HIV-positive and 30 HIV-uninfected controls. In addition, the evolution of this marker as well as that of some virologic parameters (plasma viremia and proviral load) and CD4 counts were assessed in 25 HIV-infected individuals who initiated HAART and were followed for 12 months. RESULTS: The mean level of CD38 on memory CD4+ and CD8+ T cells as well as in naive CD8+ cells was significantly higher in drug-naive HIV-positive subjects than in HIV-negative controls. Moreover, it was highly correlated with viral load titers. In patients on successful HAART, immune activation declined in all T-cell subsets, particularly among memory CD8+ cells. It remained elevated with respect to HIV-negative controls, however, even after 12 months of HAART. There was a significant correlation between the CD8+ T-cell activation decay and the increase of CD4+ T cells on HAART. Patients with the highest decline in CD8 activation were those showing the highest CD4 T-cell gains after 12 months of therapy. CONCLUSIONS: The level of CD38 expression on different T-cell subsets is differentially upregulated in drug-naive HIV-infected patients. After successful HAART, immune activation decreases in all T-cell subsets, although it still remains elevated in most cases after 12 months of HAART. The extent of immune deactivation under successful HAART correlates with the ability to reconstitute CD4 counts.

ADP-ribosyl Cyclase↗

PubMed2005

High level of conservation in Plasmodium vivax merozoite surface protein 4 (PvMSP4).

Plasmodium vivax merozoite surface protein 4 (PvMSP4) has been considered to be a promising malarial vaccine candidate. The antigenic diversity displayed by parasite populations is one of the main factors limiting the efficacy of asexual-stage anti-malarial vaccine candidates. The present study is the first characterising PvMSP4 polymorphism. P. vivax isolates were collected from endemic areas in Colombia and diversity and selection pattern studies were carried out. Overall conservation in this protein was remarkable. Changes were only found in exons I and II, the former only having single nucleotide polymorphisms (SNPs) whilst the latter showed variations in tandem repeat number caused by exon II slippage. The remaining regions (EGF-like domain, GPI anchor and intron) were completely conserved. Selection and neutrality tests carried out over variant exons indicated negative selective forces acting on them. No evidence of intragenic recombination was found. The strong conservation displayed in this molecule by isolates from geographically different regions (Colombia, Salvador and Thailand) stresses its potential importance as a candidate for a vaccine against P. vivax malaria.

Amino Acid Sequence↗

PubMed2005

MHC class I genes in the owl monkey: mosaic organisation, convergence and loci diversity.

The MHC class I molecule plays an important role in immune response, pathogen recognition and response against vaccines and self- versus non-self-recognition. Studying MHC class I characteristics thus became a priority when dealing with Aotus to ensure its use as an animal model for biomedical research. Isolation, cloning and sequencing of exons 1-8 from 27 MHC class I alleles obtained from 13 individuals classified as belonging to three owl monkey species (A. nancymaae, A. nigriceps and A. vociferans) were carried out to establish similarities between Aotus MHC class I genes and those expressed by other New and Old World primates. Six Aotus MHC class I sequence groups (Ao-g1, Ao-g2, Ao-g3, Ao-g4, Ao-g5 and Ao-g6) weakly related to non-classical Catarrhini MHC were identified. An allelic lineage was also identified in one A. nancymaae and two A. vociferans monkeys, exhibiting a high degree of conservation, negative selection along the molecule and premature termination of the open reading frame at exon 5 (Ao-g5). These sequences' high conservation suggests that they more likely correspond to a soluble form of Aotus MHC class I molecules than to a new group of processed pseudogenes. Another group, named Ao-g6, exhibited a strong relationship with Catarrhini's classical MHC-B-C loci. Sequence evolution and variability analysis indicated that Aotus MHC class I molecules experience inter-locus gene conversion phenomena, contributing towards their high variability.

Amino Acid Sequence↗

PubMed2005

Challenges of DNA profiling in mass disaster investigations.

In cases of mass disaster, there is often a need for managing, analyzing, and comparing large numbers of biological samples and DNA profiles. This requires the use of laboratory information management systems for large-scale sample logging and tracking, coupled with bioinformatic tools for DNA database searching according to different matching algorithms, and for the evaluation of the significance of each match by likelihood ratio calculations. There are many different interrelated factors and circumstances involved in each specific mass disaster scenario that may challenge the final DNA identification goal, such as: the number of victims, the mechanisms of body destruction, the extent of body fragmentation, the rate of DNA degradation, the body accessibility for sample collection, or the type of DNA reference samples availability. In this paper, we examine the different steps of the DNA identification analysis (DNA sampling, DNA analysis and technology, DNA database searching, and concordance and kinship analysis) reviewing the "lessons learned" and the scientific progress made in some mass disaster cases described in the scientific literature. We will put special emphasis on the valuable scientific feedback that genetic forensic community has received from the collaborative efforts of several public and private USA forensic laboratories in assisting with the more critical areas of the World Trade Center (WTC) mass fatality of September 11, 2001. The main challenges in identifying the victims of the recent South Asian Tsunami disaster, which has produced the steepest death count rise in history, will also be considered. We also present data from two recent mass fatality cases that involved Spanish victims: the Madrid terrorist attack of March 11, 2004, and the Yakolev-42 aircraft accident in Trabzon, Turkey, of May 26, 2003.

DNA Fingerprinting↗

PubMed2004

A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization.

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements.

5' Flanking Region↗

PubMed2004

Plasmodium vivax merozoite surface protein 8 cloning, expression, and characterisation.

Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespread throughout the world, leading to nearly 80 million cases per year, mainly in Latin-America and Asia. An open reading frame encoding the Plasmodium falciparum merozoite surface protein 8 P. vivax homologue has been identified in the present study by screening the current data obtained from this parasite's partially sequenced genome. This new protein contains 487 amino-acids, two epidermal growth factor like domains, hydrophobic regions at the N- and C-termini compatible with a signal peptide, and a glycosylphosphatidylinositol anchor site, respectively. This gene's transcription and its encoded protein expression have been assessed, as well as its recognition by P. vivax-infected patients' sera. Based on this recognition, and a previous study showing that mice immunised with the Plasmodium yoelii homologous protein were protected, we consider the PvMSP8 a good candidate to be included in a multi-stage multi-antigen P. vivax vaccine.

Amino Acid Motifs↗

PubMed2004

Identifying and structurally characterizing CD1b in Aotus nancymaae owl monkeys.

This study reports the molecular characterization and tissue expression of the non-human Aotus nancymaae primate CD1b isoform in the search for an experimental animal model to be used in evaluating the role of non-peptide antigen-presentation molecules in the immune response to infectious agents. CD1b expression on the surface of A. nancymaae peripheral blood monocyte-derived dendritic cells, shown by flow cytometry, was made possible by using human CD1b isoform antibodies. Studying the expression of CD1b-encoded transcripts revealed this molecule's broad distribution in several tissues. The A. nancymaae CD1b transcript-encoded amino-acid sequence showed 95.5% identity with the human sequence. Such high sequence homology was reflected in the identical structural conservation of how pockets A', C' and F' and tunnel T' conforming the antigen's binding site are organized, the similar arrangement of those amino-acids interacting with the T-cell receptor (TCR) during antigen presentation, and the conservation of YQNI-motif sequence in the cytoplasmatic tail (responsible for the molecule's intracellular trafficking in humans). Comparing the structure of human CD1a and CD1b and mouse CD1d proteins with CD1b structure in A. nancymaae obtained by minimization revealed that changes in the latter molecule's alpha1 and alpha2 domains imposed a narrowing of the antigen-binding groove in A. nancymaae CD1b. The high structural similarity between A. nancymaae CD1b and that from humans presented in this study leads to A. nancymaae being proposed as a suitable experimental animal model for analyzing CD1b in vivo, mainly in bacterial and parasite infections such as tuberculosis and malaria, respectively.

Amino Acid Sequence↗

PubMed2004

CD38 expression on CD8 T lymphocytes as a marker of residual virus replication in chronically HIV-infected patients receiving antiretroviral therapy.

The level of CD8+ CD38+ T lymphocytes in blood correlates with disease progression in HIV-infected individuals, independently of the CD4 count. Effective antiretroviral therapy reduces this lymphocyte subset in parallel with plasma viremia, although CD38 expression on CD8+ cells does not normalize completely in most subjects, and might reflect residual HIV replication. The expression of CD38 on CD8+ cells (as number of CD38 molecules per CD8+ cell) was measured quantitatively by flow cytometry in 200 individuals, of whom 170 were HIV positive and 30 were HIV-uninfected controls. Forty-six HIV-infected subjects were on antiretroviral therapy and had undetectable viral load. The remaining 124 HIV-positive persons were not on therapy and had detectable plasma viremia. The mean level of CD38 on CD8+ cells was higher in HIV-positive, untreated patients than in subjects on antiviral therapy and controls (5023, 2029, and 1978 molecules per CD8+ cell, respectively, p < 0.01). In HIV-positive, untreated subjects, the higher CD38 expression mainly occurred on CD45RO+ CD8+ cells. The level of CD38 strongly correlated with plasma HIV-RNA (r = 0.63, p < 0.001). The levels of CD38 on CD8+ cells declined steadily in HIV-positive subjects after beginning antiretroviral therapy. A few individuals presented viral blips whereas being on antiviral treatment, levels of CD38 on CD8+ cells increased transiently in parallel with episodes of viral replication. Levels of CD38 on CD8+ cells are increased in chronic HIV infection, and strongly correlate with plasma viremia. The slow decline of CD38 expression on CD8+ cells over time in subjects with undetectable plasma viremia while being on antiretroviral therapy suggests that CD38 expression on CD8+ cells could be used as a marker of residual virus replication.

ADP-ribosyl Cyclase↗

PubMed2003

Phenotype and functional characteristics of HIV-specific cytotoxic CD8+ T cells in chronically infected patients: dual effects of highly active antiretroviral therapy.

BACKGROUND: Cytotoxic CD8+ T cells (CTLs) are critical for the control of viral infections. Although these cells can be recognized in most HIV-infected individuals, they fail to successfully control HIV replication. Distinct functional defects seem to limit their efficacy in HIV infection, although they have been not fully elucidated. PATIENTS AND METHODS: Blood lymphocytes collected from 61 HLA-A0201+, untreated, chronically HIV-infected individuals were examined for the presence of CTLs against epitopes from HIV Gag and Pol proteins, using tetrameric complexes. Several functional aspects of these cells were further analyzed (immunophenotype; ability to produce interferon-gamma (IFN-gamma) in response to the specific peptide; proliferative capacity; and cytolytic activity). Lymphoproliferative responses of these cells confronting different stimulus were also evaluated. A longitudinal analysis was carried out in a subgroup who underwent antiretroviral therapy and were followed for 6 months. RESULTS: CD8+ T cells staining with the tetramer complexes (Tet+) were detected in 44% of patients, with TetGag+ cells being more frequently detected and at higher levels than TetPol+ cells. Most Tet+ cells expressed a memory phenotype, showed an impaired ability to produce IFN-gamma when stimulated with the cognate peptide, and showed a very low expansion when cultured in the presence of the peptide. There was a negative correlation between the proportion of Tet+ cells producing IFN-gamma and plasma HIV-RNA. Although Tet+ cells diminished in most individuals after beginning antiretroviral therapy, some patients showed de novo appearance of Tet+ cells. CONCLUSIONS: Most Tet+ cells in chronic HIV-infected individuals express a memory phenotype and show an impaired production of IFN-gamma and a lower proliferative response to specific HIV antigens. Interestingly, some individuals under successful antiretroviral therapy may show de novo appearance of specific CTLs. The implications of these findings are relevant for a better understanding of virus-host interactions.

Adult↗

PubMed2003

Solubilization of adenylyl cyclase from human myometrium in a alphas-coupled form.

Adenylyl cyclase (AC) was extracted from human myometrium with either non-ionic (Lubrol-PX or Triton X-100) or zwitterionic (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS) detergents. The soluble enzyme was stimulated by forskolin, a hydrophobic activator, in the presence of Mg2+ indicating that the catalytic subunit had not been damaged after solubilization. The enzyme was also activated by 5'-guanylyl imidodiphosphate (Gpp(NH)p) showing that the catalytic unit was not separated from stimulatory guanine nucleotide binding protein (Gs) during the extraction. Both activators showed different effects on the stimulatory efficacy and potency of AC activity solobulized with detergents. Gel filtration of Lubrol-PX and CHAPS extracts over a Sepharose CL-2B column partially resolved AC and its complexes. The chromatographic profile for Lubrol-solubilized AC presented a main peak of about 200 kDa whereas CHAPS-solubilized AC showed a dominant peak of about 1100 kDa. The heterodisperse peaks obtained revealed that the catalytic AC subunit was not separated from Gs proteins after gel filtration, and that AC could be associated with other cellular proteins. When Lubrol extract was submitted to anionic-exchange chromatography, the enzyme was purified about 7.5 fold (enzymatic activity of 48.1 pmol/min/mg of protein). The catalytic subunit was co-eluted with both AC-activating proteins Galphas large (52.2 kDa) and Galphas small (48.7 kDa). This is the first demonstration of the stable physical association of AC with both alphas subunits of G proteins in human myometrium.

Adenylyl Cyclases↗