14-3-3- and II/3-10-gene expression as molecular markers to address viability and growth activity of Echinococcus multilocularis metacestodes

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

Evolution of gene expression changes in newborn rats after mechanical ventilation with reversible intubation

Mechanical ventilation (MV) is life-saving but potentially harmful for lungs of premature infants. So far, animal models dealt with the acute impact of MV on immature lungs, but less with its delayed effects. We used a newborn rodent model including non-surgical and therefore reversible intubation with moderate ventilation and hypothesized that there might be distinct gene expression patterns after a ventilation-free recovery period compared to acute effects directly after MV. Newborn rat pups were subjected to 8 hr of MV with 60% oxygen (O(2)), 24 hr after injection of lipopolysaccharide (LPS), intended to create a low inflammatory background as often recognized in preterm infants. Animals were separated in controls (CTRL), LPS injection (LPS), or full intervention with LPS and MV with 60% O(2) (LPS + MV + O(2)). Lungs were recovered either directly following (T:0 hr) or 48 hr after MV (T:48 hr). Histologically, signs of ventilator-induced lung injury (VILI) were observed in LPS + MV + O(2) lungs at T:0 hr, while changes appeared similar to those known from patients with chronic lung disease (CLD) with fewer albeit larger gas exchange units, at T:48 hr. At T:0 hr, LPS + MV + O(2) increased gene expression of pro-inflammatory MIP-2. In parallel anti-inflammatory IL-1Ra gene expression was increased in LPS and LPS + MV + O(2) groups. At T:48 hr, pro- and anti-inflammatory genes had returned to their basal expression. MMP-2 gene expression was decreased in LPS and LPS + MV + O(2) groups at T:0 hr, but no longer at T:48 hr. MMP-9 gene expression levels were unchanged directly after MV. However, at T:48 hr, gene and protein expression increased in LPS + MV + O(2) group. In conclusion, this study demonstrates the feasibility of delayed outcome measurements after a ventilation-free period in newborn rats and may help to further understand the time-course of molecular changes following MV. The differences obtained from the two time points could be interpreted as an initial transitory increase of inflammation and a delayed impact of the intervention on structure-related genes.

Modulation of chemokine gene expression by Shiga-toxin producing Escherichia coli belonging to various origins and serotypes

Infection with Shiga-toxin producing Escherichia coli (STEC) may result in the development of the haemolytic-uremic syndrome (HUS), the main cause of acute renal failure in children. While O157:H7 STEC are associated with large outbreaks of HUS, it is difficult to predict whether a non-O157:H7 isolate can be pathogenic for humans. The mucosal innate immune response plays a central role in the pathogenesis of HUS; therefore, we compared the induction of IL-8 and CCL20 in human colon epithelial cells infected with strains belonging to different serotypes, isolated from cattle or from HUS patients. No correlation was observed between strain virulence and chemokine gene expression. Rather, the genetic background of the strains seems to determine the chemokine gene expression profile. Investigating the contribution of different bacterial factors in this process, we show that the type III secretion system of O157:H7 bacteria, but not the intimate adhesion, is required to stimulate the cells. In addition, H7, H10, and H21 flagellins are potent inducers of chemokine gene expression when synthesized in large amount.

Gene expression in cultured endometrium from women with different outcomes following IVF

Estradiol and progesterone are crucial for the acquisition of receptivity and the change in transcriptional activity of target genes in the implantation window. The aim of this study was to differentiate the regulation of genes in the endometrium of patients with recurrent implantation failure (IF) versus those who became pregnant after in vitro fertilization (IVF) treatment. Moreover, the effect of embryo-derived factors on endometrial transcriptional activity was studied. Nine women with known IVF outcome (IF, M, miscarriage, OP, ongoing pregnancy) and undergoing hysteroscopy with endometrial biopsy were enrolled. Biopsies were taken during the midluteal phase. After culture in the presence of embryo-conditioned IVF media, total RNA was extracted and submitted to reverse transcription, target cDNA synthesis, biotin labelling, fragmentation and hybridization using the Affymetrix Human Genome U133A 2.0 Chip. Differential expression of selected genes was re-analysed by quantitative PCR, in which the results were calculated as threshold cycle differences between the groups and normalized to Glyceraldehyde phosphate dehydrogenase and beta-actin. Differences were seen for several genes from endometrial tissue between the IF and the pregnancy groups, and when comparing OP with M, 1875 up- and 1807 down-regulated genes were returned. Real-time PCR analysis confirmed up-regulation for somatostatin, PLAP-2, mucin 4 and CD163, and down-regulation of glycodelin, IL-24, CD69, leukaemia inhibitory factor and prolactin receptor between Op and M. When the different embryo-conditioned media were compared, no significant differential regulation could be demonstrated. Although microarray profiling may currently not be sensitive enough for studying the effects of embryo-derived factors on the endometrium, the observed differences in gene expression between M and OP suggest that it will become an interesting tool for the identification of fertility-relevant markers produced by the endometrium.

Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts

The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

Stress-induced modulation of NF-κB activation, inflammation-associated gene expression, and cytokine levels in blood of healthy men

Acute psychosocial stress stimulates transient increases in circulating pro-inflammatory plasma cytokines, but little is known about stress effects on anti-inflammatory cytokines or underlying mechanisms. We investigated the stress kinetics and interrelations of pro- and anti-inflammatory measures on the transcriptional and protein level. Forty-five healthy men were randomly assigned to either a stress or control group. While the stress group underwent an acute psychosocial stress task, the second group participated in a non-stress control condition. We repeatedly measured before and up to 120min after stress DNA binding activity of the pro-inflammatory transcription factor NF-κB (NF-κB-BA) in peripheral blood mononuclear cells, whole-blood mRNA levels of NF-κB, its inhibitor IκBα, and of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6, and the anti-inflammatory cytokine IL-10. We also repeatedly measured plasma levels of IL-1ß, IL-6, and IL-10. Compared to non-stress, acute stress induced significant and rapid increases in NF-κB-BA and delayed increases in plasma IL-6 and mRNA of IL-1ß, IL-6, and IκBα (p's<.045). In the stress group, significant increases over time were also observed for NF-κB mRNA and plasma IL-1ß and IL-10 (p's<.055). NF-κB-BA correlated significantly with mRNA of IL-1β (r=.52, p=.002), NF-κB (r=.48, p=.004), and IκBα (r=.42, p=.013), and marginally with IL-6 mRNA (r=.31, p=.11). Plasma cytokines did not relate to NF-κB-BA or mRNA levels of the respective cytokines. Our data suggest that stress induces increases in NF-κB-BA that relate to subsequent mRNA expression of pro-inflammatory, but not anti-inflammatory cytokines, and of regulatory-cytoplasmic-proteins. The stress-induced increases in plasma cytokines do not seem to derive from de novo synthesis in circulating blood cells.

Molecular cloning, expression analysis and assignment of the porcine tumor necrosis factor superfamily member 10 gene (TNFSF10) to SSC13q34-->q36 by fluorescence in situ hybridization and radiation hybrid mapping

We have cloned the complete coding region of the porcine TNFSF10 gene. The porcine TNFSF10 cDNA has an ORF of 870 nucleotides and shares 85% identity with human TNFSF10, and 75% and 72% identity with rat and mouse Tnfsf10 coding sequences, respectively. The deduced porcine TNFSF10 protein consists of 289 amino acids with the calculated molecular mass of 33.5 kDa and a predicted pI of 8.15. The amino acid sequence similarities correspond to 86, 72 and 70% when compared with human, rat and mouse sequences, respectively. Northern blot analysis detected TNFSF10-specific transcripts (approximately 1.7 kb) in various organs of a 10-week-old pig, suggesting ubiquitous expression. Real-time RT-PCR studies of various organs from fetal (days 73 and 98) and postnatal stages (two weeks, eight months) demonstrated developmental and tissue-specific regulation of TNFSF10 mRNA abundance. The chromosomal location of the porcine TNFSF10 gene was determined by FISH of a specific BAC clone to metaphase chromosomes. This TNFSF10 BAC clone has been assigned to SSC13q34-->q36. Additionally, the localization of the TNFSF10 gene was verified by RH mapping on the porcine IMpRH panel.

Hepatic gene expression profile in mice perorally infected with Echinococcus multilocularis eggs

Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown.

Pheochromocytoma in rats with multiple endocrine neoplasia (MENX) shares gene expression patterns with human pheochromocytoma

Pheochromocytomas are rare neoplasias of neural crest origin arising from chromaffin cells of the adrenal medulla and sympathetic ganglia (extra-adrenal pheochromocytoma). Pheochromocytoma that develop in rats homozygous for a loss-of-function mutation in p27Kip1 (MENX syndrome) show a clear progression from hyperplasia to tumor, offering the possibility to gain insight into tumor pathobiology. We compared the gene-expression signatures of both adrenomedullary hyperplasia and pheochromocytoma with normal rat adrenal medulla. Hyperplasia and tumor show very similar transcriptome profiles, indicating early determination of the tumorigenic signature. Overrepresentation of developmentally regulated neural genes was a feature of the rat lesions. Quantitative RT-PCR validated the up-regulation of 11 genes, including some involved in neural development: Cdkn2a, Cdkn2c, Neurod1, Gal, Bmp7, and Phox2a. Overexpression of these genes precedes histological changes in affected adrenal glands. Their presence at early stages of tumorigenesis indicates they are not acquired during progression and may be a result of the lack of functional p27Kip1. Adrenal and extra-adrenal pheochromocytoma development clearly follows diverged molecular pathways in MENX rats. To correlate these findings to human pheochromocytoma, we studied nine genes overexpressed in the rat lesions in 46 sporadic and familial human pheochromocytomas. The expression of GAL, DGKH, BMP7, PHOX2A, L1CAM, TCTE1, EBF3, SOX4, and HASH1 was up-regulated, although with different frequencies. Immunohistochemical staining detected high L1CAM expression selectively in 27 human pheochromocytomas but not in 140 nonchromaffin neuroendocrine tumors. These studies reveal clues to the molecular pathways involved in rat and human pheochromocytoma and identify previously unexplored biomarkers for clinical use.

Gene expression profile of osseointegration of a hydrophilic compared with a hydrophobic microrough implant surface

OBJECTIVES: To compare the gene expression profile of osseointegration associated with a moderately rough and a chemically modified hydrophilic moderately rough surface in a human model. MATERIAL AND METHODS: Eighteen solid screw-type cylindrical titanium implants, 4 mm long and 2.8 mm wide, with either a moderately rough (SLA) or a chemically modified moderately rough (SLActive) surface were surgically inserted in the retromolar area of nine human volunteers. The devices were removed using a trephine following 4, 7 and 14 days of healing. The tissue surrounding the implant was harvested, total RNA was extracted and microarray analysis was carried out to identify the differences in the transcriptome between the SLA and SLActive surfaces at days 4, 7 and 14. RESULTS: There were no functionally relevant gene ontology categories that were over-represented in the list of genes that were differentially expressed at day 4. However, by day 7, osteogenesis- and angiogenesis-associated gene expression were up-regulated on the SLActive surface. Osteogenesis and angiogenesis appeared to be regulated by BMP and VEGF signalling, respectively. By day 14, VEGF signalling remains up-regulated on the SLActive surface, while BMP signalling was up-regulated on the SLA surface in what appeared to be a delayed compensatory response. Furthermore, neurogenesis was a prominent biological process within the list of differentially expressed genes, and it was influenced by both surfaces. CONCLUSIONS: Compared with SLA, SLActive exerts a pro-osteogenic and pro-angiogenic influence on gene expression at day 7 following implant insertion, which may be responsible for the superior osseointegrative properties of this surface.

Proliferation, differentiation and gene expression of osteoblasts in boron-containing associated with dexamethasone deliver from mesoporous bioactive glass scaffolds

Boron is one of the trace elements in the human body which plays an important role in bone growth. Porous mesopore bioactive glass (MBG) scaffolds are proposed as potential bone regeneration materials due to their excellent bioactivity and drug-delivery ability. The aims of the present study were to develop boron-containing MBG (B-MBG) scaffolds by sol-gel method and to evaluate the effect of boron on the physiochemistry of B-MBG scaffolds and the response of osteoblasts to these scaffolds. Furthermore, the effect of dexamethasone (DEX) delivery in B-MBG scaffold system was investigated on the proliferation, differentiation and bone-related gene expression of osteoblasts. The composition, microstructure and mesopore properties (specific surface area, nano-pore volume and nano-pore distribution) of B-MBG scaffolds have been characterized. The effect of boron contents and large-pore porosity on the loading and release of DEX in B-MBG scaffolds were also investigated. The results have shown that the incorporation of boron into MBG scaffolds slightly decreases the specific surface area and pore volume, but maintains well-ordered mesopore structure and high surface area and nano-pore volume compared to non-mesopore bioactive glass. Boron contents in MBG scaffolds did not influence the nano-pore size distribution or the loading and release of DEX. B-MBG scaffolds have the ability to maintain a sustained release of DEX in a long-term span. Incorporating boron into MBG glass scaffolds led to a controllable release of boron ions and significantly improved the proliferation and bone-related gene expression (Col I and Runx2) of osteoblasts. Furthermore, the sustained release of DEX from B-MBG scaffolds significantly enhanced alkaline phosphatase (ALP) activity and gene expressions (Col I, Runx2, ALP and BSP) of osteoblasts. These results suggest that boron plays an important role in enhancing osteoblast proliferation in B-MBG scaffold system and DEX-loaded B-MBG scaffolds show great potential as a release system to enhance osteogenic property for bone tissue engineering application.

Electric pulses augment reporter gene expression in the beating heart

Gene therapy of the heart has been attempted in a number of clinical trials with the injection of naked DNA, although quantitative information on myocellular transfection rates is not available. The present study aimed to quantify the efficacy of electropulsing protocols that differ in pulse duration and number to stimulate transfection of cardiomyocytes and to determine the impact on myocardial integrity.

Increased endometrial placenta growth factor (PLGF) gene expression in women with successful implantation

To analyze the vascularization of the endometrium via hysteroscopy and to assess its correlation with angiogenic factor gene expression and embryo implantation rate.

Life stage differences in mammary gland gene expression profile in non-human primates

Breast cancer (BC) is the most common malignancy of women in the developed world. To better understand its pathogenesis, knowledge of normal breast development is crucial, as BC is the result of disregulation of physiologic processes. The aim of this study was to investigate the impact of reproductive life stages on the transcriptional profile of the mammary gland in a primate model. Comparative transcriptomic analyses were carried out using breast tissues from 28 female cynomolgus macaques (Macaca fascicularis) at the following life stages: prepubertal (n = 5), adolescent (n = 4), adult luteal (n = 5), pregnant (n = 6), lactating (n = 3), and postmenopausal (n = 5). Mammary gland RNA was hybridized to Affymetrix GeneChip(®) Rhesus Macaque Genome Arrays. Differential gene expression was analyzed using ANOVA and cluster analysis. Hierarchical cluster analysis revealed distinct separation of life stage groups. More than 2,225 differentially expressed mRNAs were identified. Gene families or pathways that changed across life stages included those related to estrogen and androgen (ESR1, PGR, TFF1, GREB1, AR, 17HSDB2, 17HSDB7, STS, HSD11B1, AKR1C4), prolactin (PRLR, ELF5, STAT5, CSN1S1), insulin-like growth factor signaling (IGF1, IGFBP1, IGFBP5), extracellular matrix (POSTN, TGFB1, COL5A2, COL12A1, FOXC1, LAMC1, PDGFRA, TGFB2), and differentiation (CD24, CD29, CD44, CD61, ALDH1, BRCA1, FOXA1, POSTN, DICER1, LIG4, KLF4, NOTCH2, RIF1, BMPR1A, TGFB2). Pregnancy and lactation displayed distinct patterns of gene expression. ESR1 and IGF1 were significantly higher in the adolescent compared to the adult animals, whereas differentiation pathways were overrepresented in adult animals and pregnancy-associated life stages. Few individual genes were distinctly different in postmenopausal animals. Our data demonstrate characteristic patterns of gene expression during breast development. Several of the pathways activated during pubertal development have been implicated in cancer development and metastasis, supporting the idea that other developmental markers may have application as biomarkers for BC.