Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas: A pilot case-control analysis

Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern of CTCF binding sites 5 and 6 using methylationsensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic methylation pattern was maintained in CTCF binding site 5 in 22 heterozygous out of 91 samples analyzed. Nevertheless, a biallelic methylation pattern was detected in CTCF binding site 6 in a subgroup of HNSCC patients as a somatic acquired feature of tumor cells. An atypical biallelic methylation was also observed in both tumor and lymphocyte DNA from two patients, and at a high frequency in the control group (29 out of 64 informative controls). Additionally, we found that the C/T transition detected by HhaI RFLP suppressed one dinucleotide CpG in critical CTCF binding site 6, of a mutation showing polymorphic frequencies. Although a heterogeneous methylation pattern was observed after DNA sequencing modified by sodium bisulfite, the biallelic methylation pattern was confirmed in 9 out of 10 HNSCCs. These findings are likely to be relevant in the epigenetic regulation of the DMR, especially in pathological conditions in which the imprinting of IGF2 and H19 genes is disrupted.

Gene expression analysis reveals novel altered genes in nasal polyps

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

Annexin 1 mimetic peptide protects against renal ischemia/reperfusion injury in rats

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

Structural and ultrastructural evidence for telocytes in prostate stroma

The prostate comprises a glandular epithelium embedded within a fibromuscular stroma. The stroma is a complex arrangement of cells and extracellular matrix (ECM) components in addition to growth factors, regulatory molecules, remodelling enzymes, blood vessels, nerves and immune cells. The principal sources of ECM components are fibroblasts and smooth muscle cells (SMC), which synthesize the structural and regulatory components of the ECM. Telocytes (TCs) were recently described as a novel stromal cell type that exhibited characteristic features. The aim of this study was to confirm the presence of TCs in prostate stromal tissue of gerbils, as the stromal compartment of this gland is a dynamic microenvironment. We used transmission electron microscopy (TEM), light microscopy and immunohistochemistry methods to provide morphological evidence for the presence of TCs. Cells that resembled TCs were observed in gerbil prostatic stroma. These cells had small cellular bodies with very thin and extremely long cellular processes. They were found primarily in the subepithelial area and also at the periphery of SMC layers. TCs also exhibited moniliform processes, caveolae and nuclei surrounded by small amounts of cytoplasm. Close contacts between TC podomers were evident, particularly in the adjacent epithelial compartment. This morphological evidence supported the presence of TCs in the gerbil prostatic stroma, which we report for the first time. © 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

DNA methylation patterns of steroid receptor genes ESR1, ESR2 and PGR in deep endometriosis compromising the rectum

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

β-Globin polymorphisms in amerindian populations from the brazilian amazon

Objectives: This investigation was performed to examine genetic variation at the beta-globin locus in a sample of 30 healthy individuals from native populations in South America. The patterns of haplotypic variation were compared with those of previous studies including samples for various worldwide populations in an attempt to make inferences about the occupation of the Americas from a deeper temporal perspective than is typically available with haploid markers. Methods: A 2.67-kb segment containing the beta-globin gene and its flanking regions was examined for genetic variation in a sample of 60 chromosomes from native populations in South America. The fragment was PCR-amplified and directly sequenced. To determine linkage relationships in compound heterozygotes, we used the amplification refractory mutation system. In addition, we assessed genetic variability and differentiation among populations, and we performed tests of selective neutrality. These analyses were performed for Brazilian Amerindian group and other worldwide populations previously studied. Results: Eleven polymorphic sites were found in the studied fragment, which distinguished eight different haplotypes, three recombinants haplotypes (present as single copies) and five previously described haplotypes, including some of those most highly differentiated. Genetic variation found in the pooled sample is substantial. Conclusions: Although only five known haplotypes are observed in Amazonia, some of these are highly divergent, resulting in patterns of molecular polymorphism equal to or higher than those from other world regions. Am. J. Hum. Biol. 2012. (C) 2012 Wiley Periodicals, Inc.

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha and RC-3095 (10 ng/mL), Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis. GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-kappa B and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-alpha. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00083

Abnormal function of monoamine oxidase-A in comorbid major depressive disorder and cardiovascular disease: Pathophysiological and therapeutic implications (Review)

The association between major depressive disorder (MDD) and cardiovascular disease (CVD) is among the best described medical comorbidities. The presence of MDD increases the risk of cardiac admissions and mortality and increases healthcare costs in patients with CVD, and similarly, CVD affects the course and outcome of MDD. The potential shared biological mechanisms involved in these comorbid conditions are not well known. However, the enzyme monoamine oxidase-A (MAO-A), which has a key role in the degradation of catecholamines, has been associated with the pathophysiology and therapeutics of both MDD and CVD. Increased MAO-A activity results in the dysregulation of downstream targets of this enzyme and thus affects the pathophysiology of the two diseases. These deleterious effects include altered noradrenaline turnover, with a direct elevation in oxidative stress parameters, as well as increased platelet activity and cytokine levels. These effects were shown to be reversed by MAO inhibitors. Here, a model describing a key role for the MAO-A in comorbid MDD and CVD is proposed, with focus on the shared pathophysiological mechanisms and the potential therapeutic relevance of agents targeting this enzyme.

MyD88 Signaling Pathway Is Involved in Renal Fibrosis by Favoring a T(H)2 Immune Response and Activating Alternative M2 Macrophages

Inflammation contributes to the pathogenesis of chronic kidney disease (CKD). Molecules released by the inflamed injured tissue can activate toll-like receptors (TLRs), thereby modulating macrophage and CD4+ T-cell activity. We propose that in renal fibrogenesis, M2 macrophages are recruited and activated in a T helper subset 2 cell (TH2)-prone inflammatory milieu in a MyD88- dependent manner. Mice submitted to unilateral ureteral ligation (UUO) demonstrated an increase in macrophage infiltration with collagen deposition after 7 d. Conversely, TLR2, TLR4 and MyD88 knockout (KO) mice had an improved renal function together with diminished TH2 cytokine production and decreased fibrosis formation. Moreover, TLR2, TLR4 and MyD88 KO animals exhibited less M2 macrophage infiltration, namely interleukin (IL)-10+ and CD206+ CD11bhigh cells, at 7 d after surgery. We evaluated the role of a TH2 cytokine in this context, and observed that the absence of IL-4 was associated with better renal function, decreased IL-13 and TGF- β levels, reduced arginase activity and a decrease in fibrosis formation when compared with IL-12 KO and wild-type (WT) animals. Indeed, the better renal outcomes and the decreased fibrosis formation were restricted to the deficiency of IL-4 in the hematopoietic compartment. Finally, macrophage depletion, rather than the absence of T cells, led to reduced lesions of the glomerular filtration barrier and decreased collagen deposition. These results provide evidence that future therapeutic strategies against renal fibrosis should be accompanied by the modulation of the M1:M2 and TH1:TH2 balance, as TH2 and M2 cells are predictive of fibrosis toward mechanisms that are sensed by innate immune response and triggered in a MyD88-dependent pathway.