Doença enxerto-versus hospedeiro: determinação da expressão de genes e alterações de vias associadas ao sistema imunológico usando metodos da biofísica

O transplante de medula óssea (TMO) é um procedimento terapêutico importante em casos relacionados à pacientes com leucemia ou linfoma. Em decorrência desse processo, uma reação conhecida como doença enxerto-versus-hospedeiro (GVHD) pode ocorrer em pacientes susceptíveis como conseqüência da presença de células imunocompetentes do doador. Entretanto, não existe um modelo para descrever completamente as ações relacionadas ao mecanismo imunológico da GVHD desde a fase que inicializa a doença até a fase efetora. O Objetivo geral deste estudo é a investigação da resposta imunológica considerando-se o sistema HLA (antígenos leucocitários humano) em pacientes que desenvolveram a GVHD em decorrência do TMO. O National Cancer Institute (NCI) – Pathway interaction Database e Reactome foram usados como bases de dados com o objetivo de se estudar a expressão de genes e vias relacionados às Classes I e II do sistema HLA (antígenos leucocitários humano). O estudo considerou a mudança de expressão de genes relacionados às 17 vias do sistema imunológico com potencialidade para se expressar em pacientes que desenvolveram a GVHD associada à TMO. Dados referentes aos transcriptomas foram obtidos utilizando-se a plataforma GPL570 Affymetrix Genoma Humano U133 Plus. A atividade relativa foi usada para determinar as alterações das vias em amostras de GVHD em relação ao controle. As análises foram realizadas utilizando-se o software Via Complex e Bioconductor. Observou-se aumento significativo da expressão de genes ralacionados às vias do sistema imune adaptativo, antígenos associados às Classe I e II do HLA, fosforilação de CD3 e CD247, sinalização dos receptores de células T em CD4+ nativas e ativação de NF-kapa β nas células B. Também observou-se alterações significativas na mudança de expressão dos genes associados às vias relacionadas à super família de moléculas B7:CD28\CTLA-4 quando comparadas ao controle. Isso pode indicar a necessidade de geração de um segundo sinal co-estimulador em GVHD, acionado pelas moléculas dessa super família. O aumento da expressão do gene CD69 nas amostras experimentais caracteriza a ativação celular e, portanto, a sinalização de estímulos em GVHD. Os achados obtidos neste estudo contribuem para melhor elucidar o mecanismo imunopatogênico associado à GVHD. P

Effects of nemorosone, isolated from the plant Clusia rosea, on the cell cycle and gene expression in MCF-7 BUS breast cancer cell lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Up-regulation of fas and fasL pro-apoptotic genes expression in type 1 diabetes patients after autologous haematopoietic stem cell transplantation

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell-mediated destruction of pancreatic beta cells, resulting in insulin deficiency and hyperglycaemia. Recent studies have described that apoptosis impairment during central and peripheral tolerance is involved in T1D pathogenesis. In this study, the apoptosis-related gene expression in T1D patients was evaluated before and after treatment with high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT). We also correlated gene expression results with clinical response to HDI-AHSCT. We observed a decreased expression of bad, bax and fasL pro-apoptotic genes and an increased expression of a1, bcl-xL and cIAP-2 anti-apoptotic genes in patients' peripheral blood mononuclear cells (PBMCs) compared to controls. After HDI-AHSCT, we found an up-regulation of fas and fasL and a down-regulation of anti-apoptotic bcl-xL genes expression in post-HDI-AHSCT periods compared to pre-transplantation. Additionally, the levels of bad, bax, bok, fasL, bcl-xL and cIAP-1 genes expression were found similar to controls 2 years after HDI-AHSCT. Furthermore, over-expression of pro-apoptotic noxa at 540 days post-HDI-AHSCT correlated positively with insulin-free patients and conversely with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients' PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy.

Genes and gene expression in the brain of the alcoholic

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABA(A) receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate and gamma-aminobutyric acid (GABA) receptors to influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy from alcoholics without alcohol-related disease, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABB2, EAAT2, and 5HTT genotypes did not divide alcoholic cases and controls on N-methyl-D-aspartate (NMDA) receptor parameters. In contrast, alcohol dehydrogenase (ADH)3 genotype interacted significantly with NMDA receptor efficacy and affinity in a region-specific manner. EAAT2 genotype interacted significantly with local GABAA receptor subunit mRNA expression, and GABB2 and DRD2B genotypes with p subunit isoform protein expression. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence. (C) 2004 Elsevier Ltd. All rights reserved

Genes and gene expression in the brains of human alcoholics

Chronic alcohol misuse by human subjects leads to neuronal loss in regions such as the superior frontal cortex (SFC). Propensity to alcoholism is associated with several genes. γ-Aminobutyric acid (GABA)A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the regional presentation of GABAA and glutamate-NMDA (N-methyl-d-aspartate) receptors in SFC. Autopsy tissue was obtained from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and matched controls. ADH1C, DRD2B, EAAT2, and APOE genotypes modulated GABAA-β subunit protein expression in SFC toward a less-effective form of the receptor. Most genotypes did not divide alcoholics and controls on glutamate-NMDA receptor pharmacology, although gender and cirrhosis did. Genotype may affect amino acid transmission locally to influence neuronal vulnerability.

Genes and gene expression in human alcoholic brain

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABA-A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate N-methyl-D-aspartate (NMDA) and GABA-A receptors to influence the severity of alcohol-induced brain damage. Cerebral cortex tissue was obtained at autopsy from alcoholics without disease comorbid with alcoholics, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABRB2, SLC1A2, and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters. In contrast, a specific alcohol dehydrogenase (ADHIC) genotype interacted significantly with NMDA efficacy and affinity in a region-specific manner SLC1A2 (glutamate transporter-2) genotype interacted significantly with local GABAA receptor b subunit mRNA expression, and ADHIC, DRD2B, SLC1A2, and APOE genotypes with b subunit isoform protein expression. In the latter instance, possession of the alcoholism- associated allele altered b isoform protein expression patterns toward a less-efficacious form of the GABA-A receptor in the pathologically vulnerable region. GABRB2 and GRIN2B (NMDA receptor 2B subunit} Genotypes were associated with significant regional difference in the pattern of b subunit protein isoform expression, but this was not influenced by alcoholism status. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence.

Integration of global SNP-based mapping and expression arrays reveals key regions, mechanisms, and genes important in the pathogenesis of multiple myeloma.

Multiple myeloma is characterized by genomic alterations frequently involving gains and losses of chromosomes. Single nucleotide polymorphism (SNP)-based mapping arrays allow the identification of copy number changes at the sub-megabase level and the identification of loss of heterozygosity (LOH) due to monosomy and uniparental disomy (UPD). We have found that SNP-based mapping array data and fluorescence in situ hybridization (FISH) copy number data correlated well, making the technique robust as a tool to investigate myeloma genomics. The most frequently identified alterations are located at 1p, 1q, 6q, 8p, 13, and 16q. LOH is found in these large regions and also in smaller regions throughout the genome with a median size of 1 Mb. We have identified that UPD is prevalent in myeloma and occurs through a number of mechanisms including mitotic nondisjunction and mitotic recombination. For the first time in myeloma, integration of mapping and expression data has allowed us to reduce the complexity of standard gene expression data and identify candidate genes important in both the transition from normal to monoclonal gammopathy of unknown significance (MGUS) to myeloma and in different subgroups within myeloma. We have documented these genes, providing a focus for further studies to identify and characterize those that are key in the pathogenesis of myeloma.

Exposure To Bisphenol-a During Pregnancy Partially Mimics The Effects Of A High-fat Diet Altering Glucose Homeostasis And Gene Expression In Adult Male Mice.

Bisphenol-A (BPA) is one of the most widespread EDCs used as a base compound in the manufacture of polycarbonate plastics. The aim of our research has been to study how the exposure to BPA during pregnancy affects weight, glucose homeostasis, pancreatic β-cell function and gene expression in the major peripheral organs that control energy flux: white adipose tissue (WAT), the liver and skeletal muscle, in male offspring 17 and 28 weeks old. Pregnant mice were treated with a subcutaneous injection of 10 µg/kg/day of BPA or a vehicle from day 9 to 16 of pregnancy. One month old offspring were divided into four different groups: vehicle treated mice that ate a normal chow diet (Control group); BPA treated mice that also ate a normal chow diet (BPA); vehicle treated animals that had a high fat diet (HFD) and BPA treated animals that were fed HFD (HFD-BPA). The BPA group started to gain weight at 18 weeks old and caught up to the HFD group before week 28. The BPA group as well as the HFD and HFD-BPA ones presented fasting hyperglycemia, glucose intolerance and high levels of non-esterified fatty acids (NEFA) in plasma compared with the Control one. Glucose stimulated insulin release was disrupted, particularly in the HFD-BPA group. In WAT, the mRNA expression of the genes involved in fatty acid metabolism, Srebpc1, Pparα and Cpt1β was decreased by BPA to the same extent as with the HFD treatment. BPA treatment upregulated Pparγ and Prkaa1 genes in the liver; yet it diminished the expression of Cd36. Hepatic triglyceride levels were increased in all groups compared to control. In conclusion, male offspring from BPA-treated mothers presented symptoms of diabesity. This term refers to a form of diabetes which typically develops in later life and is associated with obesity.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. one hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p < 0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p < 0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p = 0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4 +/- 12.4%) and apolipoprotein B (apoB) (17.0 +/- 31.3%) when compared with the higher quartile (>0.085: LDL-c = 40.3 +/- 14.3%; apoB = 32.5 +/- 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism. and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin. (C) 2008 Elsevier Inc. All rights reserved.

Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.

Hematopoietic stem cells (HSC) are probably the best understood somatic stem cells and often serve as a paradigm for other stem cells. Nevertheless, most current techniques to genetically manipulate them in vivo are either constitutive and/or induced in settings of hematopoietic stress such as after irradiation. Here, we present a conditional expression system that allows for externally controllable transgenesis and knockdown in resident HSCs, based on a lentiviral vector containing a tet-O sequence and a transgenic mouse line expressing a doxycyclin-regulated tTR-KRAB repressor protein. HSCs harvested from tTR-KRAB mice are transduced with the lentiviral vector containing a cDNA (i.e., Green Fluorescent Protein (GFP)) and/or shRNA (i.e., p53) of interest and then transplanted into lethally irradiated recipients. While the vector is effectively repressed by tTR-KRAB during homing and engraftment, robust GFP/shp53 expression is induced on doxycyclin treatment in HSCs and their progeny. Doxycylin-controllable transcription is maintained on serial transplantation, indicating that repopulating HSCs are stably modified by this approach. In summary, this easy to implement conditional system provides inducible and reversible overexpression or knock down of genes in resident HSCs in vivo using a drug devoid of toxic or activating effects.

Genomic organization and gene expression in a chromosomal region of Leishmania major.

Little is known about the relation between the genome organization and gene expression in Leishmania. Bioinformatic analysis can be used to predict genes and find homologies with known proteins. A model was proposed, in which genes are organized into large clusters and transcribed from only one strand, in the form of large polycistronic primary transcripts. To verify the validity of this model, we studied gene expression at the transcriptional, post-transcriptional and translational levels in a unique locus of 34kb located on chr27 and represented by cosmid L979. Sequence analysis revealed 115 ORFs on either DNA strand. Using computer programs developed for Leishmania genes, only nine of these ORFs, localized on the same strand, were predicted to code for proteins, some of which show homologies with known proteins. Additionally, one pseudogene, was identified. We verified the biological relevance of these predictions. mRNAs from nine predicted genes and proteins from seven were detected. Nuclear run-on analyses confirmed that the top strand is transcribed by RNA polymerase II and suggested that there is no polymerase entry site. Low levels of transcription were detected in regions of the bottom strand and stable transcripts were identified for four ORFs on this strand not predicted to be protein-coding. In conclusion, the transcriptional organization of the Leishmania genome is complex, raising the possibility that computer predictions may not be comprehensive.

Interleukins (IL)-1 and IL-2 control IL-2 receptor alpha and beta expression in immature thymocytes.

Functional high-affinity interleukin-2 receptors (IL-2R) contain three transmembrane proteins, IL-2R alpha, beta and gamma. We have investigated the expression of IL-2R alpha and beta genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL-2R alpha on their surface. Stimulation of IL-2R alpha+ and IL-2R alpha- immature thymocytes with phorbol 12-myristate 13-acetate and calcium ionophore induces synthesis of IL-2R alpha and IL-2R beta mRNA. Most of this response depends on autocrine stimulation by IL-2. IL-1 synergizes with IL-2 to induce a 120-fold increase in IL-2R alpha mRNA and a 14-fold increase in IL-2R beta mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL-2 plays a major role in the regulation of IL-2R expression in normal immature thymocyte. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.

Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant variant of human glial tumors. A prominent feature of this tumor is the occurrence of necrosis and vascular proliferation. The regulation of glial neovascularization is still poorly understood and the characterization of factors involved in this process is of major clinical interest. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine released by leukocytes and by a variety of cells outside of the immune system. Recent work has shown that MIF may function to regulate cellular differentiation and proliferation in normal and tumor-derived cell lines, and may also contribute to the neovascularization of tumors. Our immunohistological analysis of MIF distribution in GBM tissues revealed the strong MIF protein accumulation in close association with necrotic areas and in tumor cells surrounding blood vessels. In addition, MIF expression was frequently associated with the presence of the tumor-suppressor gene p53. To substantiate the concept that MIF might be involved in the regulation of angiogenesis in GBM, we analyzed the MIF gene and protein expression under hypoxic and hypoglycemic stress conditions in vitro. Northern blot analysis showed a clear increase of MIF mRNA after hypoxia and hypoglycemia. We could also demonstrate that the increase of MIF transcripts on hypoxic stress can be explained by a profound transcriptional activation of the MIF gene. In parallel to the increase of MIF transcripts, we observed a significant rise in extracellular MIF protein on angiogenic stimulation. The data of our preliminary study suggest that the up-regulation of MIF expression during hypoxic and hypoglycemic stress might play a critical role for the neovascularization of glial tumors.