Biomedical subjects

Geraldo A S Passos

Publications and source records attributed to Geraldo A S Passos.

At least 19 recordsLinked to original sources

PubMed2006

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells.

BACKGROUND: Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. RESULTS: In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation - cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-beta-glucosidase) in mycelium cells; and ags (an alpha-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport - two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells - isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. CONCLUSION: Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.

Biological Transport↗

PubMed2006

Onset of promiscuous gene expression in murine fetal thymus organ culture.

T-cell differentiation and induction of tolerance to self-antigens occurs mainly in the thymus. Thymic stromal cells, specifically medullary thymic epithelial cells, express a diverse set of genes encoding parenchymal organ-specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has been implicated in preventing organ-specific autoimmunity by inducing T-cell tolerance to self antigens. Early thymopoiesis and the critical factors involved in T-cell differentiation can be reproduced in vitro by murine fetal thymus organ culture (FTOC), which mimics the natural thymic microenvironment. To evaluate the occurrence of PGE in FTOC, gene expression profiling during in vitro thymic development in BALB/c mice was performed using a set of nylon cDNA microarrays containing 9216 sequences. The statistical analysis of the microarray data (sam program) revealed the temporal repression and induction of 57 parenchymal and seven lymphoid organ-specific genes. Most of the genes analysed are repressed during early thymic development (15-17 days post-coitum). The expression of the autoimmune regulator (AIRE) gene at 16 days post-coitum marks the onset of PGE. This precedes the induction of parenchymal organ genes during the late developmental phase at 20 days post-coitum. The mechanism of T-cell tolerance induction begins during fetal development and continues into adulthood. Our findings are significant because they show a fine demarcation of PGE onset, which plays a central role in induction of T-cell tolerance.

Animals↗

PubMed2006

Using cDNA microarrays to identify human CD19(+) B cell gene products (ESTs) originated from systemic lupus erythematosus susceptibility loci.

Systemic lupus erythematosus (SLE) is a prototype of autoimmune disease which arises from interactions between susceptibility genes and environmental factors. Despite the heterogeneous manifestations in this disease, all SLE patients present plasma autoantibodies recognizing nuclear components. Thus, auto reactive B cells represent key effectors to be investigated. Human linkage analysis is providing the localization of susceptibility loci distributed in chromosomes contributing to elucidate the manner in which interactions between these loci mediate SLE pathogenesis. We associate the cDNA microarray technology to investigate the differential gene expression of CD19(+) B cells with genetic linkage data. Bioinformatics programs served to evidentiate the differentially expressed sequences and the design of the microarray allowed hierarchical clustering of patients and controls. Sequencing allowed the identification of 8 new gene products differentially expressed (ESTs) that were co-localized in SLE or other autoimmune diseases susceptibility loci on chromosome 1p21, 2q21, 13q33, 16p12.1 and 16q12.1. These findings strongly suggest that chromosomal regions previously identified as SLE susceptibility loci are in fact transcribed in CD19(+) B cells of patients. In this review, we delineate a new possibility for the use of cDNA microarrays in studies focusing the control of gene expression of disease susceptibility loci identified by genetic linkage.

Antigens, CD19↗

PubMed2006

Metabolism genes are among the differentially expressed ones observed in lymphomononuclear cells of recently diagnosed type 1 diabetes mellitus patients.

The large-scale differential gene expression in lymphomononuclear cells of six patients with recently diagnosed type), and six normal individuals matched to patients for sex and age were studied. Glass slides containing 4608 cDNAs from the IMAGE library were spotted using robotic technology. Statistical analysis was carried out by the SAM program, and gene function assessed by the FATIGO program. Thirty differentially expressed genes (21 induced and 9 repressed) were disclosed when DM-1 patients were compared with controls. Although presenting with distinct biological function, most of the induced or repressed genes were related with protein, phosphate, DNA, RNA, carboxylic acid, and fatty acid metabolism. Although some of these genes have been previously associated with the pathogenesis of T1DM, many other genes were identified for further studies.

Adolescent↗

PubMed2006

TNFa-e microsatellite, HLA-DRB1 and -DQB1 alleles and haplotypes in Brazilian patients presenting recently diagnosed type 1 diabetes mellitus.

TNF microsatellite and HLA class II polymorphisms were studied in 28 recently diagnosed Brazilian patients presenting type 1 diabetes mellitus (T1DM) and in 120 healthy controls. TNFa-e and HLA-DRB1/DQB1 alleles were identified using sets of sequence-specific primers. Compared to controls, the DRB1*03 and DQB1*02 allele groups, TNFa1 allele, and the TNFa4-b5-c1-d4-e3 and TNFa10-b5-c1-d4-e3 haplotypes were overrepresented in patients. TNF microsatellite together with HLA polymorphisms is associated with type 1 diabetes in Brazilian patients, corroborating the participation of the MHC genes in disease susceptibility.

Adolescent↗

PubMed2006

Is HLA class II profile relevant for the study of large-scale differentially expressed genes in type 1 diabetes mellitus patients?

We have previously identified 30 differentially expressed genes when comparing recently diagnosed type 1 diabetes mellitus (DM-1) patients and controls paired for sex, age, and ethnic background. In this article we performed the hierarchical clustering of these genes taking into account the human-leukocyte-antigen (HLA)-DRB1/DQB1 profile. The dendrogram obtained using the Cluster program grouped patients and controls into three clusters, one including individuals with no susceptibility alleles, another including individuals with at least three susceptibility alleles, and a third intermingling susceptibility/protective alleles. In addition to other variables, the results of the present article suggest that the major histocompatibility complex (MHC) class II profile may be of relevance for the study of a large-scale differentially expressed genes.

Algorithms↗

PubMed2005

Transcriptional profiles of the human pathogenic fungus Paracoccidioides brasiliensis in mycelium and yeast cells.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

Blotting, Northern↗

PubMed2005

Hybridization signatures of gamma-irradiated murine fetal thymus organ culture (FTOC) reveal modulation of genes associated with T-cell receptor V(D)J recombination and DNA repair.

In this study, we observed the occurrence of TRBV8.1-DB2.1 V(D)J recombination in murine fetal thymus organ culture (FTOC), in which the thymic microenvironment is mimicked. Since ionizing radiation affects T-cell development, we irradiated FTOCs with gamma rays to evaluate the modulation of genes implicated in TRBV8.1-BD2.1 rearrangements. The nylon cDNA microarray method was employed to monitor the expression of 9216 genes, which were organized in coexpression clusters. Clustering analysis showed similar expression profiling of genes implicated in the V(D)J recombination and DNA double strand break (DSB) repair processes such as XRCC4, RAG-2, Artemis and DNA-PK-cs, thus suggesting overlap between the two processes. The RUNX3 gene, whose coded protein binds to the enhancers of TR genes, was also modulated and the DNA cross-linking LR1 gene, which plays a role in the opening of hairpin DNA structures and whose expression pattern is similar to Artemis, may play a role in the control of V(D)J recombination. Furthermore, our data demonstrate that the FTOC model system and cDNA microarray method are useful tools to evidentiate genes that may play a role in both processes V(D)J recombination and DNA repair.

Animals↗

PubMed2005

Changes in the gene expression profiling of the thymus in response to fibrosarcoma growth.

The aim of this study was to determine whether subcutaneous injection of tumor cells into Balb-c mice, which induces a fibrosarcoma at the site of injection, produced a differential expression profile in the thymus that could be correlated with tumor growth. A dynamic transcriptional profile of the thymus in response to tumor development was observed using nylon cDNA microarrays. The Cluster-Tree View and the SAM programs were used to reveal induced and repressed genes during tumor growth. This experimental model-system showed that this approach is adequate to detect the presence of tumor cells in vivo.

Aminopeptidases↗

PubMed2004

Hybridization signatures during thymus ontogeny reveals modulation of genes coding for T-cell signaling proteins.

Non-manipulated inbred mouse strains constitutes an interesting model-system for in vivo studies on thymus ontogeny due to the possibility to observe the molecular events of the thymocyte maturation. In previous studies, using RT-PCR method, we have found that several immune system genes such as interleukins and MHC are differentially expressed during ontogeny of the thymus whose genes act as modulators of T-cell differentiation. To determine which other genes are modulated on a large-scale basis, we measured the levels of mRNA expression in mouse fetal thymus (14-17 days of gestation) by hybridization with cDNA microarrays containing 1,576 cDNA sequences derived from the IMAGE MTB library. T-cell maturation was monitored by detection of the T-cell receptor beta TRBV8.1-BD2.1 rearranged DNA segment. Each developmental phase of thymus, displayed a characteristic expression profile, as evaluated by the Cluster and Tree-View softwares. Genes differentially and significantly expressed were selected on the basis of significance analysis of the microarray data (SAM program). With the reclustering of only significantly expressed genes, it was possible to characterize the phases of thymus ontogeny, based on the differential profile of expression. Our method provided the detection of genes implicated in the cell signaling, such as the hematopoietic cell signal transducer gene, genes implicated in T-cell calcium influx (tyrosine phosphatase) and calcium signaling proteins (vesicle transport binding protein 3, proline rich Gla, casein kinase alpha 1 and Down syndrome homolog protein 1) and a gene important for the protein transport, including T-cell receptors chains, towards the cell membrane (Golgi SNAP receptor complex member 2). The results demonstrate that the cDNA microarray used to explore the gene expression was useful for understanding the modulation of several cell-signaling genes, including the calcium cascade pathway, which is important for individual stages of T-cell maturation and control of anergy during thymus ontogeny.

Animals↗

PubMed2004

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

Alternative Splicing↗

PubMed2004

Large scale gene expression analysis of CBA/J mouse strain fetal thymus using cDNA-array hybridizations.

The CBA/J inbred mouse strain constitutes an interesting in vivo model-system for studies on molecular genetics of thymus ontogeny. Using RT-PCR method we have found previously that several immune system related genes as interleukins and MHC are differentially expressed. During this period the onset of T-cell receptor beta rearrangements also occur. To know which other genes are modulated during the ontogeny of the thymus, the mRNA expression levels of fetal thymus (15 and 16 days gestation) of CBA/J mouse strain were measured by hybridization with a set of four macroarrays containing a panel of 6,144 IMAGE cDNA clones from MTB thymus library. We found 145 differentially expressed sequences; 44 were up- and 101 down-regulated in the thymus at 15-16 days gestation. Among these sequences, only 20 are identified as genes whose functions are known and 125 are still unknown. Our data demonstrated that, despite intense research on maturation of the immune system focusing on the activity of several well-characterized genes, the large scale expression profile during thymus ontogeny is still an open matter. The use of cDNA-array technology is an affordable method to identify new genes that may play a role in this phenomenon.

Animals↗

PubMed2004

Immunosuppressive therapy modulates T lymphocyte gene expression in patients with systemic lupus erythematosus.

To evaluate the T-cell large-scale differential gene expression in systemic lupus erythematosus (SLE) patients presenting with glomerulonephritis we studied SLE patients before and after immunosuppressive treatment. Large-scale gene expression of peripheral blood mononuclear T cells was evaluated using cDNA microarray nylon membranes containing 5184 cDNAs. Data were analysed using the SAM and Cluster and Treeview software. When untreated patients were compared to healthy individuals, 38 genes, most of them located on chromosomes 1, 3, 6, 17 and 19, were repressed, and when untreated patients were compared to treated ones, 154 genes, located on chromosomes 1, 6, 7, 12 and 17, were induced. In terms of biological function of coded proteins, the differentially expressed genes were associated with apoptosis, cell cycle, chromosomal scaffold, cytokine/chemokine, DNA repair/replication, Golgi/mitochondrial proteins, mRNA processing, signalling molecules and tumour suppressors. Two autoantigen genes related to RNA splicing (small nuclear riboprotein 70,000 MW-U1 SNR, and splicing factor 3a, 60,000 MW), and the tetranectin-plasminogen-binding protein were repressed. The Fc fragment of immunoglobulin G low affinity IIb, apoptotic protease activating factor-1, two subunits of cytochrome c, caspase 8, complement C5a, HLA-DRA, HLA-DQB1, transforming growth factor-beta receptor II, small nuclear ribonucleoprotein polypeptide N (Sm protein N) genes, heterogeneous nuclear riboprotein-C, and argininosuccinate lyase genes, among others, were induced. A total of 10 genes were repressed in untreated patients and induced in treated ones, among them tumour necrosis factor (ligand) superfamily member 9, tumour protein p53, mannosidase alpha class IA, and CD22. Although some of these differentially expressed genes are typically expressed in B cells, CD22 and CD32 have also been reported in T cells and may provide regulatory signals to B cells. Assessment of differential gene expression may provide hybridization signatures that may identify susceptibility, diagnostic and prognostic markers of SLE.

Adult↗

PubMed2003

Genomic EcoRI polymorphism and cosmid sequencing reveal an insertion/deletion and a new IGLV5 allele in the human immunoglobulin lambda variable locus (22q11.2/IGLV).

The human immunoglobulin lambda locus ( IGL) is mapped at Chromosome 22q11.2, spanning about 1 Mb of DNA, and directs the synthesis of lambda-type immunoglobulin light chains. The positions of about 73-74 germline V-lambda genes, depending on the haplotypes, are known, with 29-33 of them being functional IGLV genes. These genes were divided into 11 subgroups ( IGLV1 to IGLV11) distributed into three gene clusters ( VA, VB, and VC). We constructed a high-resolution restriction map of a 37-kb cosmid clone (cosmid 8.3) harboring genes of the IGLV1, IGLV7, and IGLV5 families and the non-coding sequences IGLV(I)-42 and IGLV(VII)-41-1, located at cluster VB of the IGL locus. These IGLV genes were associated with unique EcoRI fragments detectable in Southern blots of genomic DNA. Population RFLP has revealed new IGLV alleles and haplotypes. We used the restriction map of cosmid 8.3 and the IMGT database as a reference for RFLP studies. EcoRI Southern blot hybridizations with subgroup-specific probes of the functional and open reading frame sequences present in cosmid 8.3 revealed different frequencies of IGLV gene fragments, as well as deletions of IGLV1-50 and IGLV5-39 genes and RFLP involving IGLV5-45 and IGLV5-48 genes. All members of the IGLV7 subgroup were monomorphic. Sequencing of the genes present in cosmid 8.3 revealed a new allelic variant of the IGLV5 subgroup. These data contribute to a better understanding of the contribution of the germline IGLV genes to the human genetic background and polymorphism.

Alleles↗

PubMed2003

Gene expression profiles in human cells submitted to genotoxic stress.

Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). In this report, we present three approaches to document gene expression profiles, dealing with the evaluation of cellular responses to genotoxic agents (gamma-rays from 60Cobalt and cyclophosphamide). We used the method of cDNA arrays to analyze the differential gene expression profiles that were displayed by lymphocytes from radiation-exposed individuals, a human fibroblast cell line, and T lymphocytes from systemic lupus erythematosus (SLE) patients who were treated with cyclophosphamide. A preliminary analysis performed in lymphocytes from three radiation-workers showed that several induced genes can be associated with cell response to ionizing radiation: TRRAP (cell cycle regulation), Ligase IV (DNA repair), MAPK8IP1 and MAPK10 (signal transduction), RASSF2 (apoptosis induction/tumorigenesis), p53 (damage response/maintenance of genetic stability). The in vitro irradiated normal VH16 cell line (primary) showed a complex response to the genotoxic stress at the molecular level. Many apoptotic pathways were concomitantly induced. In addition, several genes involved in signaling and cell cycle arrest/control were significantly modulated after irradiation. Many genes involved in oxidative damage were also induced, indicating that this mechanism seems to be an important component of cell response. After treatment of the SLE patients with cyclophosphamide, 154 genes were differentially and significantly induced. Among them, we identified those associated with drug detoxification, cell cycle control, apoptosis, and tumor-suppressor. These findings indicate that at least two apoptotic pathways were induced after cyclophosphamide treatment. The induction of APAF1 and two genes coding for two subunits of cytochrome c supports a previous report showing increased apoptosis in lymphocytes from SLE patients. The present study provides new information on the molecular mechanism underlying the cell response to genotoxic stress, with relevance to basic and clinical research.

Blood Donors↗

PubMed2003

Gene expression profiling during thymus ontogeny and its association with TCRVbeta8.1-Dbeta2.1 rearrangements of inbred mouse strains.

The V(D)J recombination of TCRalpha and beta in early developing T-cells is a highly modulated phenomenon initiated and completed by recombinase complex (RAG-1 and RAG-2), and regulated by other gene products such as interleukins. To further evaluate the association of several other gene products with the evolution of TCRVbeta8.1 V(D)J rearrangements in vivo, the mRNA expression levels of seven interleukins, three cytokines, receptors TCRVbeta8.1 and IL-2Rbeta, MHC-I/MHC-II, RAG-1/ RAG-2 and retroviral superantigen MMTV(SW) were measured by RT-PCR during the fetal development of the thymus of three inbred mouse strains (Balb-c, C57B1/6 and CBA/J). Clustering using the Tree View software, was used to organize these genes based on similarity of expression patterns. Each strain displayed a different expression profile during thymus ontogeny. During the late developmental stage the most evident association was the kinetics of MMTV(SW) retrovirus, IL-2Rbeta and IL-7 overexpression with reduction of TCRVbeta8.1-D1beta2.1 rearrangement in the thymus of CBA/J mice. These data suggest a susceptibility of this strain to expression of MMTV(SW) upon reduction of the rearranged TCRVbeta8.1-Dbeta2.1 segment in developing thymocytes, with parallel IL-7 overexpression.

Animals↗

PubMed2002

Systemic lupus erythematosus patients under immunosuppressive treatment express high levels of the immunoglobulin lambda variable IGLV8S1 gene with silent somatic mutations.

Systemic lupus erythematosus (SLE) patients express high titers of somatically mutated serum autoantibodies against nuclear structures including double-stranded DNA. These somatic mutations accumulate codons for basic amino acids in the immunoglobulin variable regions of both, heavy and light chains, facilitating binding to nucleic acids. The variable (V) immunoglobulin lambda 8 (IGLV8S1) gene contributes to autoreactive B-cell repertoire of auto-immune patients. Accumulation of immune complexes of these anti-DNA autoantibodies causes severe systemic inflammation in SLE. The current treatment of lupus disease is based on immunosuppressive drugs, but the precise role for this therapy remains to be defined. To evaluate the in vivo effect of combined immunosuppressive treatment on B-lymphocytes repertoire of SLE patients, we have developed an approach using the IGLV8S1 gene as a marker. The transcription of this gene in treated SLE patients was increased. However, we observed a trend, in these patients, to conserve complementarity determining regions (CDRs) and framework regions (FRs) of Vlambda8 polypeptide light chain deduced sequence, from its germline counterpart. Sequencing IGLV8S1 cDNA of untreated SLE patients, taken as a control for treatment effect, displayed a decreased frequency of silent somatic mutations (consequently high frequency of replacement mutations) in the Vlambda8 polypeptide chain deduced sequence. These data suggest that the immunosuppressive drug treatment modulates the positive selection of somatically mutated Vlambda8 light chain.

Adult↗

PubMed2002

Chromosomal rearrangements involving telomeric DNA sequences in Balb/3T3 cells transfected with the Ha-ras oncogene.

Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism.

Animals↗