Affected and non-affected skin fibroblasts from systemic sclerosis patients share a gene expression profile deviated from the one observed in healthy individuals.

OBJECTIVES: To evaluate the gene expression profile of fibroblasts from affected and non-affected skin of systemic sclerosis (SSc) patients and from controls. MATERIALS AND METHODS: Labeled cDNA from fibroblast cultures from forearm (affected) and axillary (non-affected) skin from six diffuse SSc patients, from three normal controls, and from MOLT-4/HEp-2/normal fibroblasts (reference pool) was probed in microarrays generated with 4193 human cDNAs from the IMAGE Consortium. Microarray images were converted into numerical data and gene expression was calculated as the ratio between fibroblast cDNA (Cy5) and reference pool cDNA (Cy3) data and analyzed by R environment/Aroma, Cluster, Tree View, and SAM softwares. Differential expression was confirmed by real time PCR for a set of selected genes. RESULTS: Eighty-eight genes were up- and 241 genes down-regulated in SSc fibroblasts. Gene expression correlation was strong between affected and non-affected fibroblast samples from the same patient (r>0.8), moderate among fibroblasts from all patients (r=0.72) and among fibroblasts from all controls (r=0.70), and modest among fibroblasts from patients and controls (r=0.55). The differential expression was confirmed by real time PCR for all selected genes. CONCLUSIONS: Fibroblasts from affected and non-affected skin of SSc patients shared a similar abnormal gene expression profile, suggesting that the widespread molecular disturbance in SSc fibroblasts is more sensitive than histological and clinical alterations. Novel molecular elements potentially involved in SSc pathogenesis were identified.

Transcriptional changes in U343 MG-a glioblastoma cell line exposed to ionizing radiation.

Glioblastoma multiforme (GBM) is a highly invasive and radioresistant brain tumor. Aiming to study how glioma cells respond to gamma-rays in terms of biological processes involved in cellular responses, we performed experiments at cellular context and gene expression analysis in U343-MG-a GBM cells irradiated with 1 Gy and collected at 6 h post-irradiation. The survival rate was approximately 61% for 1 Gy and was completely reduced at 16 Gy. By performing the microarray technique, 859 cDNA clones were analyzed. The Significance Analysis of Microarray algorithm indicated 196 significant expressed genes (false discovery rate (FDR) = 0.42%): 67 down-regulated and 97 up-regulated genes, which belong to several classes: metabolism, adhesion/cytoskeleton, signal transduction, cell cycle/apoptosis, membrane transport, DNA repair/DNA damage signaling, transcription factor, intracellular signaling, and RNA processing. Differential expression patterns of five selected genes (HSPA9B, INPP5A, PIP5K1A, FANCG, and TPP2) observed by the microarray analysis were further confirmed by the quantitative real time RT-PCR method, which demonstrated an up-regulation status of those genes. These results indicate a broad spectrum of biological processes (which may reflect the radio-resistance of U343 cells) that were altered in irradiated glioma cells, so as to guarantee cell survival.

Immunoglobulin V-lambda transcription profiling of systemic lupus erythematosus patients reveals biased usage of genes located near the Jlambda-Clambda segments.

To evaluate the distribution of usage and to quantify the transcription levels of the immunoglobulin lambda variable (IGLV) genes in patients with systemic lupus erythematosus (SLE) and normal individuals (NIs), cDNA samples from peripheral blood lymphocytes were prepared and probed with IGLV-specific oligonucleotides. Because recombinations involving V-lambda pseudogenes are nonproductive, we analysed the IGLV productive repertoire, as cDNAs were copied from IGLV mRNA producing B lymphocytes. Increased expression of the IGLV8a gene in SLE led us to analyse the transcription levels of all IGLV genes. We developed an expression profiling approach to scan the entire V-lambda locus on chromosome 22q11.2. The transcription profiling showed that usually the V-lambda genes located near the Jlambda-Clambda cluster were preferentially expressed in both groups, i.e. patients and NIs, with the expression levels of SLE patients being significantly higher. However, genes displaying peaks of expression independent of Jlambda-Clambda cluster proximity were observed along the IGLV locus. Our data permit us to conclude that there are differences in V-lambda gene expression between SLE patients and NIs, and a preferential usage of genes located near the Jlambda-Clambda cluster. The data also demonstrate the occurrence of Vlambda-Jlambda-Clambda-productive recombinations independent of gene localization along the locus.

Onset of T-cell receptor Vbeta8.1 and Dbeta2.1 V(D)J recombination during thymus development of inbred mouse strains.

The assembling of T-cell receptor (TCR alpha/beta and gamma/delta) genes depends on the V(D)J recombination occurring in early thymocytes during thymus ontogeny. The V(D)J recombination reaction is directed by a recombinase complex from the RAG-1 and RAG-2 genes, and is modulated by several other gene products. Due to the essential role of the TCRbeta in thymocyte differentiation, it is important to define with precision the temporal emergence of the TCRbeta recombination in normal non-manipulated mouse strains. We analysed the onset of V(D)J recombination between TCRVbeta8.1 and Jbeta2.1 gene segments during fetal development of the thymus in three non-manipulated inbred strains of mice; BALB-c, C57BL/6 and CBA. We show that the emergence of the V(D)J recombination at the TCRbeta locus differs among strains, suggesting an in vivo role of the different genetic backgrounds in driving gene rearrangements.

Emergence of TCR alpha/beta V(D)J recombination and transcription during ontogeny of inbred mouse strains.

The maturation of T-cells depends on V(D)J recombination at the TCR alpha/beta and gamma/delta loci that occurs in the thymus during fetal development. Due to the essential role of the TCRbeta in thymocyte differentiation, it is important to define with precision the temporal emergence of the TCRbeta rearrangement and its expression in normal non-manipulated mouse strains. We studied the onset of the V(D)J recombination and transcription of the TCR Valpha3 and Vbeta11 genes during ontogeny in Balb-c, C57B1/6 and CBA inbred mouse strains. Our data show differences in the emergence of recombination in both TCR alpha and beta loci among strains. The transcriptions of these loci followed respective recombinations and we detected an early germline transcript before TCR beta locus recombination in the CBA strain.