Expression of tlr4, md2 and cd14 in equine blood leukocytes during endotoxin infusion and in intestinal tissues from healthy horses

The expression of tlr4, md2 and cd14 was studied in equine blood leukocytes and in intestinal samples using real time PCR. The stability of three commonly used reference genes, glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxantine ribosyltransferase (HPRT) and succinate dehydrogenase complex subunit A (SDHA), was evaluated using qbase(PLUS). The equine peripheral blood mononuclear cells (eqPBMC) examined were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynuclotide 2216 (CpG-ODN 2216) or obtained from horses before, during and after infusion of endotoxin. Intestinal tissue from healthy horses was sampled at ileum, right dorsal colon and rectum. Ranking of the three reference genes used for normalisation identified the combination HPRT/SDHA as most suitable both when determined ex vivo in leukocytes obtained from experimentally induced endotoxaemia and in eqPBMC activated in vitro while HPRT/GAPDH were most appropriate for the intestinal samples. The relative amounts of mRNA for TLR4 and MD-2 increased threefold during in vitro activation of the cells with CpG-ODN 2216 but was decreased in cultures stimulated with PMA/ionomycin. A transient elevation in the transcription of tlr4 and md2 was also evident for equine blood leukocytes following endotoxaemia. The levels of mRNA for CD14 on the other hard remained unaffected both during the induction of endotoxaemia and in the in vitro stimulated PBMCs. A low steady expression of TLR4, MD-2 and CD14 mRNA was demonstrated for the intestinal samples with no variation between the intestinal segments analysed. Thus, the foundation for real time PCR based levels of analysis of mRNA for all three components in the equine LPS receptor complex in different intestinal segments was set, making it possible to carry out future expression studies on clinical material. (C) 2012 Elsevier B.V. All rights reserved.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Amygdala gene expression of NMDA and GABAA receptors in patients with mesial temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and affects 40% of the patients. Seizures arising from the mesial temporal lobe structures (i.e., amygdala and hippocampus) are common, whereas neocortical seizures are rare. In recent years, many studies aimed to identify the pattern of gene expression of neurotransmitters involved in molecular mechanisms of epilepsy. We used real-time PCR to quantify the expression of GABAA (subunits a1, beta 1, beta 2) and NMDA (subunits NR1, NR2A, and NR2B) receptor genes in amygdalae of 27 patients with TLE and 14 amygdalae from autopsy controls. The NR1 subunit was increased in patients with epilepsy when compared with controls. No differences were found in expression of NMDA subunits NR2A and NR2B or in a1, beta 1, and beta 2 subunits of GABAA receptors. Our results suggest that the NR1 subunit of NMDA receptors is involved in the amygdala hyperexcitability in some of the patients with TLE. (C) 2010 Wiley Periodicals, Inc., Inc.

Expression of Mitochondrial Non-coding RNAs (ncRNAs) Is Modulated by High Risk Human Papillomavirus (HPV) Oncogenes

The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.

Expression of angiogenic factors in placenta of stressed rats

The aim of the present study was to analyse the influence of stress on pregnant rats, particularly in terms of maternal, placental and fetal weight, placental morphology and placental gene expression of the angiogenic factors Vegfa and Pgf and their receptors. The parameters were evaluated on gestation Day 20. Maternal, fetal and placental weights were statistically lower in stressed animals than controls, suggesting abnormalities in gestational physiology. Morphologically the placentas of rats subjected to stress were reduced in size and weight, with few glycogen cells and a significant increase in the number of apoptotic cells. Stress caused an increase in placental gene expression of Vegfa (P < 0.05) and a reduction in Pgf, Flt1 and Kdr expression (P < 0.05). It has been suggested that increased VEGF is associated with vasodilatation and hypotension, but in this model persistent hypertension was present. This study suggests that the limited hypotensive Vegfa response to stress-induced hypertension could result from reduced expression of Flt1/Kdr disrupting specific VEGF pathways. These findings may elucidate one of the multiple possible factors underlying how stress modulates placental physiology, and could aid the understanding of stress-induced gestational disorders.

Expression profile of apoptosis-related genes in childhood adrenocortical tumors: low level of expression of BCL2 and TNF genes suggests a poor prognosis

Background: Impaired apoptosis has been implicated in the development of childhood adrenocortical tumors (ACT), although the expression of apoptosis-related gene expression in such tumors has not been reported. Methods: The mRNA expression levels of the genes CASP3, CASP8, CASP9, FAS, TNF, NFKB, and BCL2 were analyzed by quantitative real-time PCR in consecutive tumor samples obtained at diagnosis from 60 children with a diagnosis of ACT and in 11 non-neoplastic adrenal samples. BCL2 and TNF protein expression was analyzed by immunohistochemistry. Results: A significant association was observed between tumor size >= 100 g and lower expression levels of the BCL2 (P=0.03) and TNF (P=0.05) genes; between stage IV and lower expression levels of CASP3 (P=0.008), CASP9 (P=0.02), BCL2 (P=0.002), TNF (P=0.05), and NFKB (P=0.03); Weiss score >= 3 and lower expression of TNF (P=0.01); unfavorable event and higher expression values of CASP9 (P=0.01) and lower values of TNF (P=0.02); and death and lower expression of BCL2 (P=0.04). Underexpression of TNF was associated with lower event-free survival in uni- and multivariate analyses (P<0.01). Similar results were observed when patients with Weiss score <3 were excluded. Conclusion: This study supports the participation of apoptosis-related genes in the biology and prognosis of childhood ACT and suggests the complex role of these genes in the pathogenesis of this tumor.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Gene expression of peripheral blood lymphocytes may discriminate patients with schizophrenia from controls

To identify a classifier in schizophrenia, blood gene expression profiling was applied to patients with schizophrenia under different treatments and to controls. Expression of six genes discriminated patients with sensitivity of 89.3% and specificity of 90%, supporting the use of peripheral blood as biological material for diagnosis in schizophrenia. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Intestinal Lymph-Borne Factors Induce Lung Release of Inflammatory Mediators and Expression of Adhesion Molecules After an Intestinal Ischemic Insult

Background. Intestinal ischemia and reperfusion (I/R) is a documented cause of acute lung injury (ALI) and systemic inflammation. We previously reported that obstruction of thoracic lymphatic flow during intestinal I/R blunts pulmonary neutrophil recruitment and microvascular injury and decreases the systemic levels of tumor necrosis factor. Here, we consider the existence of a gut-lung axis promoting the induction of systemic inflammation, whereby drained intestinal lymph stimulates lung expression of adhesion molecules and matrix components and generation of inflammatory mediators. Material and Methods. Upon administration of anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min, followed by 2 h of intestinal reperfusion (I/R); groups of rats were subjected to I/R with or without thoracic lymphatic duct ligation immediately before the procedure. The non-manipulated rats were used to investigate basal parameters. Results. Obstruction of thoracic lymphatic flow before intestinal I/R decreased the ability of cultured lung tissue explants to release IL-1 beta, IL-10, and VEGF. In contrast, lymphatic obstruction normalized the elevated lung expression of PECAM-1 caused by intestinal I/R. On the other hand, lung E-selectin expression was significantly reduced, whereas fibronectin expression and collagen synthesis were not affected. Lymph levels of LTB4 and TXB2 were found to be significantly increased. Conclusions. These data suggest that lymph factors drained from the intestine during ischemic trauma stimulate the lung to generate inflammatory mediators and alter the expression of adhesion molecules. Disturbances in lung homeostasis mediated by lymph might contribute to the spread of inflammatory processes, thereby accounting for the systemic inflammation induced by intestinal I/R. (C) 2012 Elsevier Inc. All rights reserved.

Expression of RANTES, eotaxin-2, ICAM-1, LFA-1 and CCR-3 in chronic rhinosinusitis patients with nasal polyposis

PURPOSE: To compare gene expression of the chemokines RANTES and eotaxin-2, its receptor, CCR-3, adhesion molecule ICAM-1 and its receptor LFA-1 in eosinophilic polyps and in control normal nasal mucosa. METHODS: Gene expression was quantified by Real Time PCR in polyps (n=35) and in healthy nasal mucosa (n=15). RESULTS: Eosinophilic polyps showed a higher expression of eotaxin-2 and RANTES, but not of CCR-3, ICAM-1 or LFA-1 compared to control nasal mucosa. CONCLUSION: Eosinophilic polyps present greater expression of eotaxin-2 and RANTES, but not of CCR-3, ICAM-1 or LFA-1 compared to control nasal mucosa.

Intrauterine food restriction alters the expression of uncoupling proteins in brown adipose tissue of rat newborns

A previous study from our laboratory showed that maternal food restriction (MFR) delays thermoregulation in newborn rats. In neonates brown adipose tissue (BAT) is essential for thermogenesis due to the presence of uncoupling proteins (UCPs). The aim of this study was to evaluate the influence of MFR on the UCPs mRNA and protein expression in BAT and skeletal muscle (SM) of the newborn rat. Female Wistar EPM-1 control rats (CON) received chow ad libitum during pregnancy, whereas food-restricted dams (RES) received 50% of the amount ingested by CON. Fifteen hours after birth, the litters were weighed and sacrificed. Blood was collected for hormonal analysis. BAT and SM were used for determination of UCPs mRNA and protein expression, and Ca2+-ATPase sarcoplasmic reticulum (SERCA1). RES pups showed a significant reduction in body weight and fat content at birth. MFR caused a significant increase in the expression of UCP1 and UCP2 in BAT, without changes in UCP3 and SERCA1 expression in BAT and SM. No differences between groups were found for leptin, T4 and glucose levels. RES pups showed increased insulin and decreased T3 levels. The delay in development of thermoregulation previously described in RES animals appears not to result from impairment in thermogenesis, but from an increase in heat loss, since MFR caused low birth weight in pups, leading to greater surface/volume ratio. The higher expression of UCP1 and UCP2 in BAT suggests a compensatory mechanism to increased thermogenesis. (C) 2011 Elsevier Ltd. All rights reserved.

Ethanol consumption increases the expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and metalloproteinases in the rat kidney

Objectives The effects of longterm ethanol consumption on the levels of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and metalloproteinase-2 (MMP-2) were studied in rat kidney. Methods Male Wistar rats were treated with 20% ethanol (v/v) for 6 weeks. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of eNOS and iNOS were assessed by immunohistochemistry and quantitative real-time polymerase chain reaction, respectively. MMP-2 activity was determined by gelatin zymography. Histopathological changes in kidneys and indices of renal function (creatinine and urea) and tissue injury (mitochondrial respiration) were also investigated. Results Chronic ethanol consumption did not alter malondialdehyde levels in the kidney. Ethanol consumption induced a significant increase in renal nitrite and nitrate levels. Treatment with ethanol increased mRNA expression of both eNOS and iNOS. Immunohistochemical assays showed increased immunostaining for eNOS and iNOS after treatment with ethanol. Kidneys from ethanol-treated rats showed increased activity of MMP-2. Histopathological investigation of kidneys from ethanol-treated animals revealed tubular necrosis. Indices of renal function and tissue injury were not altered in ethanol-treated rats. Conclusions Ethanol consumption increased renal metalloproteinase expression/activity, which was accompanied by histopathological changes in the kidney and elevated NO generation. Since iNOS-derived NO and MMPs contribute to progressive renal injury, the increased levels of NO and MMPs observed in ethanol-treated rats might contribute to progressive renal damage.

Analysis of the mRNA expression of the S100 beta protein in adipocytes of patients with diabetes mellitus, type 2

Objective: This study aims to explore the possible relationship between the expression level of S100 beta protein mRNA with diabetes mellitus type 2 in adipocytes from patients with this disease in comparison with normoglycemic individuals. Materials and methods: Samples of adipose tissue of eight patients from the coronary section of the Institute Dante Pazzanese of Cardiology (IDPC), four in Group Diabetes and four of Normoglycemic group, were evaluated by RT-PCR real time. Results: An increase around 15 times values, between the threshold cycle (Delta Ct), of mRNA expression of S100 beta protein in adipocytes of the diabetes group was observed in comparison to the control group (p = 0.015). Conclusion: Our results indicate, for the first time, that there is coexistence of increased expression of the S100 beta and the type 2 diabetes mellitus gene. Arq Bras Endocrinol Metab. 2012;56(7):435-40

Forced Expression of Nanog in Human Bone Marrow-Derived Endothelial Cells Activates Other Six Pluripotent Genes

Human endothelial cells (ECs) have the ability to make up the lining of blood vessels. These cells are also capable of neovascularization and revascularization and have been applied in various clinical situations. With the aim of understanding the effect of NANOG superexpression on ECs, we transduced the Nanog gene into the ECs. Nanog is highly expressed in embryonic stem cells (ESCs) and is essential for pluripotency and self-renewal. However, Nanog can also be expressed in somatic stem cells, and this gene is related to great expansion capacity in vitro. We found that ECs expressing Nanog showed expression of other stemness genes, such as Sox2, FoxD3, Oct4, Klf4, c-myc, and beta-catenin, that are not normally expressed or are expressed at very low levels in ECs. Nanog is one of the stemness genes that can activate other stemness genes, and the upregulation of the Nanog gene seems to be critical for reprogramming cells. In this study, the introduction of Nanog was sufficient to alter the expression of key genes of the pluripotent pathway. The functional importance of Nanog for altering the cell expression profile and morphology was clearly demonstrated by our results.