Luiz F. L. Reis

Critical role of a common transcription factor, IRF-1, in the regulation of IFN-beta and IFN-inducible genes.

Interferon regulatory factor 1 (IRF‐1) is a protein that binds to cis‐elements within the promoter of interferon (IFN)‐beta and some IFN‐inducible genes. We used a human fibroblast line, GM‐637, to generate stable transfectants constitutively expressing IRF‐1 mRNA in either the sense or antisense orientation. Upon induction with poly‐(I).poly(C) or Newcastle disease virus, cells expressing sense IRF‐1 mRNA produced significantly higher levels of IFN‐beta mRNA and protein than control cells, whereas cells expressing antisense IRF‐1 mRNA produced little or no IFN‐beta mRNA and protein. Furthermore, clear differences were seen among the transfectants in the level of expression of two IFN‐induced genes (2′‐5′‐oligoadenylate synthetase and class I HLA). Our data show that IRF‐1 is essential for the induced expression of the IFN‐beta gene. The results also indicate an important role of IRF‐1 in the expression of IFN‐inducible genes and suggest a role for IRF‐1 in many other cytokine actions.

Increased Mortality and Inflammation in Tumor Necrosis Factor-Stimulated Gene-14 Transgenic Mice after Ischemia and Reperfusion Injury

TSG-14/PTX3 is a gene inducible by tumor necrosis factor (TNF)-alpha, interleukin-1 beta, and lipopolysaccharide in fibroblasts, macrophages, and endothelial cells. It encodes a 42-kd secreted glycoprotein that belongs to the pentraxin family of acute-phase proteins. Recently, we demonstrated that TSG-14 transgenic mice (TSG-14tg) overexpressing the murine TSG-14 gene under control of its own promoter are more resistant to lipopolysaccharide-induced shock and to polymicrobial sepsis caused by cecal ligation and puncture. Here we show that after ischemia and reperfusion (I/R) injury, TSG-14tg mice have an impaired survival rate, which appeared secondary to a markedly increased inflammatory response, as assessed by the local (duodenum and ileum) and remote (lung) enhancement in vascular permeability, hemorrhage, and neutrophil accumulation. Moreover, tissue concentrations of TNF-alpha, interleukin-1 beta, KC, and MCP-1 were higher in TSG-14tg as compared to wild-type mice after I/R injury. Of note, elevated TNF-alpha concentrations in serum were only observed in TSG-14tg mice and blockage of TNF-alpha action prevented lethality of TSG-14tg mice. These results demonstrate that transgenic expression of TSG-14 induces an enhanced local and systemic injury and TNF-alpha-dependent lethality after I/R. Taken together, our data point to a critical role of TSG-14 in controlling acute inflammatory response in part via the modulation of TNF-alpha expression.

Gene Expression Profile Associated with Response to Doxorubicin-Based Therapy in Breast Cancer

Purpose: This study was designed to identify genes that could predict response to doxorubicin-based primary chemotherapy in breast cancer patients. Experimental Design: Biopsy samples were obtained before primary treatment with doxorubicin and cyclophosphamide. RNA was extracted and amplified and gene expression was analyzed using cDNA microarrays. Results: Response to chemotherapy was evaluated in 51 patients, and based on Response Evaluation Criteria in Solid Tumors guidelines, 42 patients, who presented at least a partial response (≥30% reduction in tumor dimension), were classified as responsive. Gene profile of samples, divided into training set (n = 38) and independent validation set (n = 13), were at first analyzed against a cDNA microarray platform containing 692 genes. Unsupervised clustering could not separate responders from nonresponders. A classifier was identified comprising EMILIN1, FAM14B, and PBEF, which however could not correctly classify samples included in the validation set. Our next step was to analyze gene profile in a more comprehensive cDNA microarray platform, containing 4,608 open reading frame expressed sequence tags. Seven samples of the initial training set (all responder patients) could not be analyzed. Unsupervised clustering could correctly group all the resistant samples as well as at least 85% of the sensitive samples. Additionally, a classifier, including PRSS11, MTSS1, and CLPTM1, could correctly distinguish 95.4% of the 44 samples analyzed, with only two misclassifications, one sensitive sample and one resistant tumor. The robustness of this classifier is 2.5 greater than the first one. Conclusion: A trio of genes might potentially distinguish doxorubicin-responsive from nonresponsive tumors, but further validation by a larger number of samples is still needed.

Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and kynurenine formation during in vivo infection with Toxoplasma gondii: induction by endogenous gamma interferon and requirement of interferon regulatory factor 1.

ABSTRACT The induction of indoleamine 2,3-dioxygenase (INDO) expression and the tryptophan (Trp)-kynurenine (Kyn) metabolic pathway during in vivo infection with Toxoplasma gondii was investigated. Decreased levels of Trp and increased formation of Kyn were observed in the lungs, brain, and serum from mice infected with T. gondii. Maximal INDO mRNA expression and enzyme activity were detected in the lungs at 10 to 20 days postinfection. Further, the induction of INDO mRNA expression, Trp degradation and Kyn formation were completely absent in tissues from mice deficient in IFN-γ (IFN-γ−/−) or IFN regulatory factor -1 (IRF-1−/−). These findings indicate the important role of endogenous IFN-γ and IRF-1 in the in vivo induction of the Trp-Kyn metabolic pathway during acute infection with T. gondii. In contrast, expression of INDO mRNA and its activity was preserved in the tissues of TNF-receptor p55- or inducible nitric oxide synthase-deficient mice infected with T. gondii. Together with the results showing the extreme susceptibility of the IFN-γ−/− and the IRF-1−/− mice to infection with T. gondii, our results indicate a possible involvement of INDO and Trp degradation in host resistance to early infection with this parasite.

cDNA microarray analysis of genes associated with ERBB2 (HER2/ neu ) overexpression in human mammary luminal epithelial cells

To investigate changes in gene expression associated with ERBB2, expression profiling of immortalized human mammary luminal epithelial cells and variants expressing a moderate and high level of ERBB2 has been carried out using cDNA microarrays corresponding to approximately 6000 unique genes/ESTs. A total of 61 significantly up- or downregulated (2.0-fold) genes were identified and further validated by RT–PCR analysis as well as microarray comparisons with a spontaneously ERBB2- overexpressing breast cancer cell line and ERBB2-positive primary breast tumors. The expression and clinical relevance of proteins predicted to be associated with ERBB2 overexpression in breast cancers were analysed together with their clinical relevance by antibody screening using a tissue array. Differentially regulated genes include those involved in cell–matrix interactions including proline 4-hydroxylase (P4HA2), galectin 1 (LGALS1) and galectin 3 (LGALS3), fibronectin 1 (FN1) and p-cadherin (CDH3), and cell proliferation (CRIP1, IGFBP3) and transformation (S100P, S100A4). A number of genes associated with MYC signalling were also differentially expressed, including NDRG1, USF2 and the epithelial membrane proteins 1 and 3 (EMP1, EMP3). These data represent profiles of the transcriptional changes associated with ERBB2-related pathways in the breast, and identify novel and potentially useful targets for prognosis and therapy.

TSG-14 transgenic mice have improved survival to endotoxemia and to CLP-induced sepsis

Tumor necrosis factor‐stimulated gene 14 (TSG‐14)/PTX3 was identifiedoriginally as a TNF‐α and IL‐1β‐stimulated gene from normal, humanforeskin fibroblasts and vascular endothelial cells, respectively. TSG‐14 gene encodes a 42‐kDa‐secreted glycoprotein with acarboxy‐terminal half that shares homology with the entire sequence of C‐reactive protein (CRP) and serum amyloid P component (SAP),acute‐phase proteins of the pentraxin family. Some experimentalevidence suggests that TSG‐14 plays a role in inflammation, yet itsfunction and mechanism of action remain unclear. We have generatedtransgenic mice that overexpress the murine TSG‐14 gene under thecontrol of its own promoter. From eight transgenic founders, twolineages were derived and better characterized: Tg2 and Tg4, carryingtwo and four copies of the transgene, respectively. TSG‐14 transgenicmice were found to be more resistant to the endotoxic shock induced byLPS and to the polymicrobial sepsis caused by cecal ligation andpuncture (CLP). Moreover, macrophages derived from the transgenic miceproduced higher amounts of nitric oxide in response to IFN‐γ,TNF‐α, and LPS as compared with macrophages from wild‐type animals, and the augmented response appears to be the consequence of a higherresponsiveness of transgenic macrophages to IFN‐γ. The data shownhere are the first in vivo evidence of the involvement of TSG‐14 in the inflammatory process and suggest a role for TSG‐14 in thedefense against bacterial infections.

Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.

Macrophage signaling by glycosylphosphatidylinositol-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes

Activation of cells from the innate immune system has an important role in host resistance to early infection with the intracellular protozoan parasite, Trypanosoma cruzi. Here we review the studies that have identified and structurally characterized the glycosylphosphatidylinositol (GPI) anchors, as parasite molecules responsible for the activation of cells from the macrophage lineage. We also cover the studies that have identified the receptor, signaling pathways as well as the array of genes expressed in macrophages that are activated by these glycoconjugates. We discuss the possible implications of such response on the host resistance to T. cruzi infection and the pathogenesis of Chagas disease.

Molecular classifiers for gastric cancer and nonmalignant diseases of the gastric mucosa

High incidence of gastric cancer-related death is mainly due to diagnosis at an advanced stage in addition to the lack of adequate neoadjuvant therapy. Hence, new tools aimed at early diagnosis would have a positive impact in the outcome of the disease. Using cDNA arrays having 376 genes either identified previously as altered in gastric tumors or known to be altered in human cancer, we determined expression signature of 99 tissue fragments representing normal gastric mucosa, gastritis, intestinal metaplasia, and adenocarcinomas. We first validated the array by identifying molecular markers that are associated with intestinal metaplasia, considered as a transition stage of gastric adenocarcinomas of the intestinal type as well as markers that are associated with diffuse type of gastric adenocarcinomas. Next, we applied Fisher’s linear discriminant analysis in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Many classifiers could distinguish between normal and tumor samples, whereas, for the distinction of gastritis from tumor and for metaplasia from tumor, fewer classifiers were identified. Statistical validations showed that trios that discriminate between normal and tumor samples are powerful classifiers to distinguish between tumor and nontumor samples. More relevant, it was possible to identify samples of intestinal metaplasia that have expression signature resembling that of an adenocarcinoma and can now be used for follow-up of patients to determine their potential as a prognostic test for malignant transformation.

Differential regulation of TSG-14 expression in murine fibroblasts and peritoneal macrophages.

Tumor necrosis factor (TNF)‐stimulated gene 14 (TSG‐14, also termed PTX3) encodes a secreted glycoprotein whose carboxyterminal half shares sequence similarity with the pentraxin family of acute phase proteins (C‐reactive protein and serum amyloid P component). We compared TSG‐14 mRNA expression in cultures of murine BALB/c 3T3 fibroblasts and thioglycollateelicited peritoneal macrophages. TNF and interleukin‐1 (IL‐1) potently induced TSG‐14 expression in 3T3 fibroblasts but not in peritoneal macrophages. Lipopolysaccharide (LPS) elicited TSG‐14 expression in both cell types, but induction in 3T3 cells and macrophages showed several distinct characteristics. Whereas in 3T3 fibroblasts TSG‐14 mRNA was rapidly up‐regulated by LPS, expression in macrophages was substantially delayed. Furthermore, cycloheximide greatly reduced LPS‐induced TSG‐14 mRNA up‐regulation in macrophages but not in 3T3 cells. Finally, interferon‐γ (IFN‐γ; but not IFN‐α/β) inhibited LPS‐induced TSG‐14 expression in macrophages and not in 3T3 fibroblasts. The antioxidant pyrrolidine dithiocarbamate inhibited LPS‐induced nuclear factor‐κB (NF‐κB) activation and TSG‐14 expression in macrophages. In contrast, IFN‐γ did not inhibit NF‐κB function as measured by IκB‐α and IκB‐β degradation, IκB‐α resynthesis, or electrophoretic mobility shift analysis. Inhibition of LPS‐induced TSG‐14 mRNA expression by IFN‐γ in macrophages was also observed in the presence of cycloheximide and in cells from STAT1 null mice, suggesting that IFN‐γ inhibits TSG‐14 expression through an unconventional mechanism. J. Leukoc. Biol. 67: 387–395; 2000.