Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole-organ intervertebral disc model

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted up-regulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was down-regulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity.

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.

A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone

BACKGROUND AND AIMS: Heterotopic ossification (HO) is a pathological bone formation process in which ectopic bone is formed in soft tissue. The formation of bone depends on the expression of the osteoblast phenotype. Earlier studies have shown conflicting results on the expression of phenotype markers of cells originating from HO and normal bone. The hypothesis of the present study is that cells from HO show an altered expression of osteoblast-specific phenotype markers compared to normal osteoblasts. The aims of the study were to further characterize the expression of osteoblast phenotypemarkers and to provide a comparison with other study results. PATIENTS AND METHODS: Using an in vitro technique, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and immunohistochemistry, we compared the phenotype gene expression (type I collagen, alkaline phosphatase, Cbfa-1, osteocalcin) of osteoblasts from resected HO and normal bone (iliac crest). RESULTS: Cells from HO expressed the osteoblast phenotype (type I collagen, alkaline phosphatase) but were characterized by a depleted osteocalcin expression. The expression of Cbfa-1 (osteocalcin transcription gene) showed a large variety in our study. Preoperative radiotherapy had no effect on phenotype expression in cells from HO. CONCLUSION: Our results provide a characterization of cells originating from HO and support the thesis of an impaired osteoblast differentiation underlying the formation of HO. The transcription axis from Cbfa-1 to osteocalcin could be involved in the pathogenesis of HO.

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

Differential expression of CD45 on canine microglial cells

CD45, also called leucocyte common antigen is a transmembrane protein tyrosine phosphatase on the surface of nearly all white blood cells and has a functional role in signal transduction. In the brain, the expression of CD45 can be used to distinguish microglial cells with a characteristic phenotype of CD11b/c+ and CD45(low) from other central nervous system (CNS) macrophages which show an expression of CD11b/c+ and CD45(high). In the course of pathological changes in the CNS, microglia in rodents is known to readily upregulate expression of various surface molecules, such as CD45. Understanding the mechanisms that regulate expression of surface molecules is essential to study the pathogenesis of CNS diseases. In the present study, the expression of CD45 on microglia of 42 dogs was examined ex vivo by means of flow cytometry. The dogs were classified in two groups according to the histopathological diagnosis in the CNS. All dogs without changes in the CNS (group I; n = 22) only showed low percentages of CD45+ microglial cells. In group II consisting of 20 dogs with different intracranial diseases varying results were obtained. Thirteen dogs showed a low percentage of CD45+ microglial cells whereas seven dogs exhibited high percentages of microglial cells expressing CD45. Evaluation of expression intensity in these seven dogs revealed two subpopulations of CD45+ microglial cells: a large subpopulation with CD45(low) and a small subpopulation with CD45(high). The expression intensity of CD45(high) was comparable with that of canine monocytes. It was attempted to correlate these findings to age of the animals, underlying disease, duration of clinical signs, medical treatment, occurrence of seizure activity and the expression of other surface molecules. It appeared that dogs with high percentages of CD45+ suffered from long-lasting CNS disease with seizures. In future studies, the reason and consequences for upregulated CD45 in long-lasting CNS diseases has to be further evaluated.

Adenoviral expression of IKs contributes to wavebreak and fibrillatory conduction in neonatal rat ventricular cardiomyocyte monolayers

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K(+) current (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of I(Ks)) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in I(Ks)-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the I(Ks) group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the I(Ks) reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.

Expression of zebra fish aromatase cyp19a and cyp19b genes in response to the ligands of estrogen receptor and aryl hydrocarbon receptor

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17beta-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERalpha, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligands.

Placental ABCA1 expression is reduced in primary antiphospholipid syndrome compared to pre-eclampsia and controls

The ATP binding cassette transporter A1 (ABCA1) mediates cellular cholesterol and phospholipid efflux, and is implicated in phosphatidylserine translocation and apoptosis. Loss of functional ABCA1 in null mice results in severe placental malformation. This study aimed to establish the placental localisation of ABCA1 and to investigate whether ABCA1 expression is altered in placentas from pregnancies complicated by pre-eclampsia and antiphospholipid syndrome. ABCA1 mRNA and protein localisation studies were carried out using in situ hybridization and immunohistochemistry. Comparisons of gene expression were performed using real-time PCR and immunoblotting. ABCA1 mRNA and protein was localised to the apical syncytium of placental villi and endothelia of fetal blood vessels within the villi. ABCA1 mRNA expression was reduced in placentas from women with APS when compared to controls (p<0.001), and this was paralleled by reductions in ABCA1 protein expression. There were no differences in ABCA1 expression between placentas from pre-eclamptic pregnancies and controls. The localisation of ABCA1 in human placenta is consistent with a role in cholesterol and phospholipid transport. The decrease in ABCA1 protein in APS may reflect reduced cholesterol transport to the fetus affecting the formation of cell membranes and decreasing the level of substrate available for steroidogenesis.

Stimulation-specific contribution of p38 and JNK to IFN-beta gene expression in human macrophages

Induction of interferon-beta (IFN-beta) gene expression is a tightly regulated process, and a plethora of studies identified the signal transduction pathway TANK-binding kinase-1 (TBK-1)/IFN regulatory factor-3 (IRF-3) as essential to the induction of IFN-beta gene expression. Data regarding the role of p38 and JNK are rare, however. We investigated the contribution of these kinases to IFN-beta expression in human macrophages treated with poly(I:C), lipopolysaccharide (LPS), Sendai virus, or vesicular stomatitis virus (VSV). We found that all the stimuli induced IFN-beta mRNA, albeit to a different extent. Whereas LPS and VSV induced the phosphorylation of p38 and JNK, neither poly(I:C) nor Sendai virus led to the detection of phosphospecific signals. When inhibiting p38, a VSV-triggered IFN-beta mRNA response was inhibited, whereas inhibiting JNK suppressed an LPS-triggered response, but only when macrophages were primed with IFN-gamma. Neither poly(I:C)-induced nor Sendai virus-induced IFN-beta mRNA expression was affected when p38 and JNK were inhibited. Collectively, the data show that the contribution of p38 and JNK to the expression of IFN-beta occurs in a stimulation-specific manner in human macrophages.

Expression of TCL1 in hematologic disorders

OBJECTIVE: TCL1, MTCP1 and TCL1b are three members of a new family of oncogenes that are expressed in T cell leukemias of ataxia telangiectasia patients (T-PLL, T-CLL). TCL1 is located at 14q32.1 and activated by juxtaposition to the alpha/delta-locus at 14q11 or beta-locus at 7q35 of the T cell receptor during the reciprocal translocations t(14;14)(q11;q32), t(7;14)(q35;q32), or inversion inv(14)(q11;q32). TCL1 encodes a predominantly cytoplasmic protein of 114 aa (14 kD) of unknown function. Recent studies suggest that TCL1 promotes cell survival rather than stimulating cell proliferation, as previously proposed. METHODS: In an attempt to clarify the contexts in which TCL1 is expressed, we investigated TCL1 expression in 114 lymphoma and leukemia patients by Northern blot, RT-PCR and immunohistochemistry. RESULTS: TCL1 expression is restricted to lymphoid cells, and is found in neoplastic (T and B cell neoplasms, and Hodgkin's disease) and nonneoplastic proliferations (reactive lesions). Out of 114 cases, 18 neoplasms of myeloid and 4 cases of epithelial origin were TCL1-negative. In lesions of the lymphoid system, both low- and high-grade lymphomas were found to express TCL1. CONCLUSIONS: We propose that TCL1 expression especially in high-grade B cell non-Hodgkin's lymphomas might interfere with B cell differentiation and promote the transition from low- to high-grade lymphoma.

Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores

BACKGROUND: Diagnosis and prognosis in breast cancer are mainly based on histology and immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) material. Recently, gene expression analysis was shown to elucidate the biological variance between tumors and molecular markers were identified that led to new classification systems that provided better prognostic and predictive parameters. Archived FFPE samples represent an ideal source of tissue for translational research, as millions of tissue blocks exist from routine diagnostics and from clinical studies. These should be exploited to provide clinicians with more accurate prognostic and predictive information. Unfortunately, RNA derived from FFPE material is partially degraded and chemically modified and reliable gene expression measurement has only become successful after implementing novel and optimized procedures for RNA isolation, demodification and detection. METHODS: In this study we used tissue cylinders as known from the construction of tissue microarrays. RNA was isolated with a robust protocol recently developed for RNA derived from FFPE material. Gene expression was measured by quantitative reverse transcription PCR. RESULTS: Sixteen tissue blocks from 7 patients diagnosed with multiple histological subtypes of breast cancer were available for this study. After verification of appropriate localization, sufficient RNA yield and quality, 30 tissue cores were available for gene expression measurement on TaqMan(R) Low Density Arrays (16 invasive ductal carcinoma (IDC), 8 ductal carcinoma in situ (DCIS) and 6 normal tissue), and 14 tissue cores were lost. Gene expression values were used to calculate scores representing the proliferation status (PRO), the estrogen receptor status and the HER2 status. The PRO scores measured from entire sections were similar to PRO scores determined from IDC tissue cores. Scores determined from normal tissue cores consistently revealed lower PRO scores than cores derived from IDC or DCIS of the same block or from different blocks of the same patient. CONCLUSION: We have developed optimized protocols for RNA isolation from histologically distinct areas. RNA prepared from FFPE tissue cores is suitable for gene expression measurement by quantitative PCR. Distinct molecular scores could be determined from different cores of the same tumor specimen.

TLR expression on neutrophils at the pulmonary site of infection: TLR1/TLR2-mediated up-regulation of TLR5 expression in cystic fibrosis lung disease

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Expression profiling with RNA from formalin-fixed, paraffin-embeeded material

Background Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge. Results We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97. Conclusion We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.

Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.

Expression of adenosine A2a receptors gene in the olfactory bulb and spinal cord of rat and mouse

The expression of adenosine A2a receptors (A2aR) in the mammalian striatum is well known. In contrast the exact distribution of A2aR in other regions of the central nervous system remains unclear. The aim of this study was to investigate the A2aR gene expression in the rat olfactory bulb and spinal cord, two regions which are seldom included in mapping studies. Secondly, we compared the A2aR expression in the rat and in the mouse brain. Hybridization histochemistry was performed with an S35-labelled radioactive oligonucleotide probe. The results show strong expression of A2aR in the mouse and rat striatum in accordance with previous reports. In the olfactory bulb a weak but specific expression of A2aR was found in the granular cell layer in both species. In contrast, no significant expression of the A2aR gene was observed in other parts of the brain or the rat spinal cord. The presence of the A2aR in the mammalian olfactory bulb suggests a functional role for this receptor in olfaction.