Glial Fibrillary Acidic Protein mRNA Research Articles

Novel approach for detecting prohibited species-specific central nervous system tissue contamination in meat by one-step real-time reverse transcriptase PCR.

The dissemination of prohibited species-specific central nervous system (CNS) tissue contamination in meat must be tracked to mitigate human health risk associated with bovine spongiform encephalopathy. The efficiency of compliance monitoring and risk control measures taken by concerned regulatory authorities at meat production facilities to avoid such contamination depends on the ability to detect CNS tissue with a reliable and adequately sensitive quantitative method. A rapid and convenient one-step real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was developed based on the absolute quantification of glial fibrillary acidic protein (GFAP) mRNA as a marker for CNS tissue contamination in meat. The GFAP RNA quantity corresponding to a percentage of CNS tissue in artificially spiked meat was determined using an appropriate in vitro transcribed target GFAP RNA as a calibration standard in the assay. The assay had a linear dynamic range of 10(2) to 10(9) copies of target RNA and was able to detect 0.01% CNS contamination in meat. Further evaluation consisted of an analysis of 272 random meat cuts from carcasses and 109 ground meat samples received from a federally inspected abattoir and two meat processing facilities, respectively, over a 5-month period. The analyzed samples were all negative for CNS tissue contamination at an arbitrarily set lower threshold of 0.025%. Overall, the newly developed one-step qRT-PCR may be useful as an objective quantitative compliance monitoring tool and for setting an acceptable low tolerance threshold for such contamination in meat.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200–250 g) were submitted to 1-hour ischemia and treated with NPCs (4 × 10⁶ cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-β and TNF-α expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1β, TNF-α and INF-γ). Our data suggested that NPC therapy improved renal function by influencing immunological responses.

Viability of estrogen and lithium‐treated primary brain cell cultures following glutamate insult and NR1 mRNA expression of pretreated cultures

Glutamate is an excitatory neurotransmitter that facilitates calcium influx, and excess calcium influx is detrimental to brain cells. Excitotoxicity is implicated in many neurological diseases and estrogen (E2) and lithium can influence excitotoxicity inversely: E2 facilitates calcium influx, while lithium impedes this influx. We harvested cortical and hippocampal cells of C57BL/6J fetal mice and pre‐treated for 48 hours with E2, lithium or combined E2/lithium. Cultures were then exposed to a toxic level of glutamate (100μM). Cell viability using fluorescein diacetate/propidium iodide assay revealed that only lithium pretreatment increased viability of cortical cultures and combined pretreatment did not. All pretreated hippocampal cultures reduced cell viability. Total RNA was also isolated from pre‐treated cultures and reverse transcribed. cDNA was then amplified via PCR using a glutamate receptor subunit (NR1), neuron and glia gene‐specific primers. Combined E2/lithium treatment reduced NR1 mRNA expression for both culture types. Additionally, these cultures expressed increased glial fibrillary acidic protein (GFAP) mRNA while neurofilament‐heavy chain (NF‐H) mRNA was low. Our data suggest lithium may protect against glutamate insult, but combined doses of E2 and lithium interfere with/prevent protection against glutamate excitotoxicity and NR1 mRNA expression among these cultures.

Poly-T oligonucleotides enhance TLR7/8 agonist induced responses in glial cells through a TLR7-dependent mechanism (135.11)

Abstract The brain does not contain a resident dendritic cell population so resident cells such as microglia and astrocytes play an important role in initiating innate immune responses in the central nervous system (CNS) following virus infection. However, the activation of these cells following stimulation of virus-recognizing toll-like receptors (TLRs) such as TLR7 and TLR8 remains unclear. Poly T oligonucleotides (pT-ODNs) were recently shown to enhance signaling through TLR8, including murine TLR8 which was previously thought to be non-functional. In the current study, we analyzed the ability of pT-ODNs to enhance TLR7/8 activation of murine astrocytes and microglia. We found that the TLR7/8 agonist CL075 (3M002) induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines. Addition of pT-ODNs enhanced the expression of proinflammatory cytokines and chemokines as well as the microglia/macrophage activation markers F4/80 and Iba1. Surprisingly, pT-ODNs enhancement of CL075-induced glial activation was abolished in TLR7 deficient mice, with the exception of Gfap (glial fibrillary acidic protein) mRNA upregulation. Thus in glial cells, pT-ODNs enhanced TLR7/8-induced proinflammatory responses through a TLR7-dependent pathway. This research was supported by the Intramural Research Program of the NIH.

Intermediate filament transcription in astrocytes is repressed by proteasome inhibition

Increased expression of the astrocytic intermediate filament protein glial fibrillary acidic protein (GFAP) is a characteristic of astrogliosis. This process occurs in the brain during aging and neurodegeneration and coincides with impairment of the ubiquitin proteasome system. Inhibition of the proteasome impairs protein degradation; therefore, we hypothesized that the increase in GFAP may be the result of impaired proteasomal activity in astrocytes. We investigated the effect of proteasome inhibitors on GFAP expression and other intermediate filament proteins in human astrocytoma cells and in a rat brain model for astrogliosis. Extensive quantitative RT-PCR, immunocytochemistry, and Western blot analysis resulted unexpectedly in a strong decrease of GFAP mRNA to <4% of control levels [Control (DMSO) 100+/-19.2%; proteasome inhibitor (epoxomicin) 3.5+/-1.3%, n=8; P < or = 0.001] and a loss of GFAP protein in astrocytes in vitro. We show that the proteasome alters GFAP promoter activity, possibly mediated by transcription factors as demonstrated by a GFAP promoter-luciferase assay and RT(2) Profiler PCR array for human transcription factors. Most important, we demonstrate that proteasome inhibitors also reduce GFAP and vimentin expression in a rat model for induced astrogliosis in vivo. Therefore, proteasome inhibitors could serve as a potential therapy to modulate astrogliosis associated with CNS injuries and disease.

Estradiol and lithium chloride specifically alter NMDA receptor subunit NR1 mRNA and excitotoxicity in primary cultures

Glutamate facilitates calcium influx via NMDAR, and excess calcium influx increases excitotoxicity — a pathological characteristic of neurological diseases. Both 17β-estradiol (E2) and lithium influence NMDAR expression/signaling and excitotoxicity. This led us to hypothesize that combined E2 and lithium will alter NMDAR expression and excitotoxicity. We tested this hypothesis using primary cell cultures from the cortex and hippocampus of C57BL/6J fetal mice pretreated with E2, lithium chloride (LiCl) and combined E2/LiCl for 12, 24 or 48 h. We examined cultures for brain cell type and changes in cell type caused by experimental procedures using glia and neuron gene specific primers. These cultures expressed increased glial fibrillary acidic protein (GFAP) mRNA with low neurofilament-heavy chain (NF-H) mRNA expression. Subsequent analysis of cortical cell cultures indicated that combined E2/LiCl decreased NR1 mRNA expression after a 12 and 48 h treatment period. Combined E2/LiCl also reduced NR1 mRNA expression in hippocampal cultures but only after a 48 h treatment period. LiCl-treated hippocampal cultures also reduced NR1 mRNA expression after a 24 and 48 h treatment. We next examined the response of 48 h pretreated cultures to a toxic level of glutamate. Excitotoxicity was measured using fluorescein diacetate/propidium iodide (FDA/PI) cell viability assay. Results from FDA/PI assay revealed that LiCl pretreatment increased viability for cortical cultures while E2 and combined E2/LiCl reduced viability. All pretreatments for hippocampal cultures failed to increase viability. Our results showed combined E2/LiCl reduced NR1 mRNA and prevented protection against glutamate excitotoxicity in glial primary cultures.

Synaptic mRNAs are modulated by learning

We have recently demonstrated that brain plastic events significantly modify synaptic protein synthesis measured by the incorporation of [(35)S]methionine in brain synaptosomal proteins. Notably, in rats learning a two-way active avoidance task, the local synthesis of two synaptic proteins was selectively enhanced. Because this effect may be attributed to transcriptional modulation, we used reverse transcriptase-polymerase chain reaction methods to determine the content of discrete synaptosomal mRNAs in rats exposed to the same training protocol. Correlative analyses between behavioral responses and synaptosomal mRNA content showed that GAT-1 mRNA (a prevalent presynaptic component) correlates with avoidances and escapes in rat cerebellum, while glial fibrillary acid protein mRNA (an astrocytic component) correlates with freezings in cerebellum and cerebral cortex. These observations support the hypothesis that synaptic protein synthesis may be transcriptionally regulated. The cellular origin of synaptic transcripts is briefly discussed, with special regard to those present at large distances from neuron somas.

Expression of myelin basic protein and glial fibrillary acidic protein genes in human glial tumors

Analysis of the expression of genes encoding myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) genes in human glial tumors was carried out for determination of the expression specificity of these genes according to tumor types and their malignancy. Low levels of MBP mRNA in astrocytoma specimens of malignancy grades II-IV and significantly higher levels in perifocal zone adjacent to them have been determined by Northern hybridization. Diffuse astrocytomas and anaplastic astrocytomas are characterized mostly by low level of MBP gene expression and high level of GFAP gene expression, but distinct subtypes of diffuse and anaplastic astrocytomas with high level of MBP gene and low level of GFAP gene expression can be also detected that may be the reflection of different oncogenic pathways. Very low levels or even absence of MBP mRNA were revealed in oligodendroglioma and all oligoastrocytomas. Thus, Northern hybridization data are correlated with Serial Analysis of Gene Expression (SAGE). Obtained results show that MBP is nonspecific marker for tumors of oligodendroglial origin, but determination of relative levels of MBP and GFAP mRNAs may be useful for glial tumors recognition. By such a way, these two genes together with previously found by us YKL-40 and TSC-22 can be included into the gene panel for the determination of so called "gene signatures" of brain tumors. However, severe requirements in relation to a clinical value of these "gene signatures" can not be formulated without their verification on plenty of clinical samples of tumors and valid control.

Limited remyelination in Theiler's murine encephalomyelitis due to insufficient oligodendroglial differentiation of nerve/glial antigen 2 (NG2)‐positive putative oligodendroglial progenitor cells

Limited remyelination is a key feature of demyelinating Theiler's murine encephalomyelitis (TME). It is hypothesized that a dysregulation of differentiation of oligodendroglial progenitor cells (OPCs) represents the main cause of insufficient regeneration in this model of multiple sclerosis. TME virus (TMEV)-infected SJL/J mice were evaluated by footprint analysis, light and electron microscopy, immunohistology, confocal immunofluorescence and RT-qPCR at multiple time points ranging from 1 h to 196 days post infection (dpi). Footprint analysis revealed a significantly decreased stride length at 147 and 196 dpi. Demyelination progressively increased from 14 towards 196 dpi. A mild amount of remyelination was detected at 147 and 196 dpi. Early onset axonal injury was detected from 14 dpi on. TMEV RNA was detectable throughout the observation period and markedly increased between 7 and 28 dpi. Intralesional nerve/glial antigen 2 (NG2)-positive OPCs were temporarily increased between 28 and 98 dpi. Similarly, a transient upregulation of NG2 and platelet-derived growth factor alpha-receptor mRNA was noticed. In contrast, intralesional 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-positive oligodendrocytes were decreased between 56 and 196 dpi. Although CNPase mRNA remained unchanged, myelin basic protein mRNA and especially its exon 2 containing splice variants were decreased. Glial fibrillary acidic protein (GFAP)-positive astrocytes and GFAP mRNA were increased in the late phase of TME. A mildly increased colocalization of both NG2/CNPase and NG2/GFAP was revealed at 196 dpi. Summarized, the present results indicated a dysregulation of OPC maturation as the main cause for the delayed and limited remyelination in TME. A shift of OPC differentiation from oligodendroglial towards astrocytic differentiation is postulated.

Detection of Bovine Central Nervous System Tissue as Bovine Spongiform Encephalopathy Risk Material by Real-Time Reverse Transcriptase-PCR in Raw and Cooked Beef Products

There is increasing evidence of the association of the new variant of Creutzfeldt-Jacob disease (nvCJD) in humans with bovine spongiform encephalopathy (BSE) in cattle. Many countries established legislation of banning central nervous system (CNS) tissues, which are regarded as BSE-specified risk materials (SRM), in human food supply because of the potential transmission of BSE to humans. A real-time reverse transcriptase-PCR assay using the bovine glial fibrillary acidic protein (GFAP) mRNA template for the detection of CNS tissues in raw and cooked beef products was developed in this study. The results showed that (1) this method can detect CNS tissues from bovine and ovine origins, but not from porcine and avian ones; (2) GFAP mRNA can only be detected from brain and spinal cords rather than other tissues; (3) the GFAP mRNA was detectable in CNS tissues even after dilution to 0.001%; and (4) the assay was unaffected by heat treatment at 100°C for 30 min or storage at room temperature for 4 days, and at 4°C for at least 15 days.

Blood–brain barrier impairment with enhanced SP, NK‐1R, GFAP and Claudin‐5 expressions in experimental cerebral toxocariasis

Infection by Toxocara canis in humans may cause cerebral toxocariasis (CT). Appreciable numbers of T. canis larvae cross the blood-brain barrier (BBB) to invade the brain thus causing CT. In the present studies, we evaluated the BBB permeability and BBB injury as assessed by the cerebral Evans blue (EB) concentration as well as by pathological changes and glial fibrillary acidic protein (GFAP) expression in T. canis -infected mice monitored from 3 days (dpi) to 8 weeks post-infection (wpi). The vasodilation neuropeptides, the expressions of substance P (SP) and its preferred binding neurokinin-1 receptor (NK-1R) as well as claudin-5 of tight-junction proteins associated with BBB impairment were also assessed by Western blotting and reverse-transcriptase polymerase chain reaction. Results revealed that BBB permeability increased as evidenced by a significantly elevated EB concentration in brains of infected mice. BBB injury appeared due to enhanced GFAP protein and mRNA expressions from 4 to 8 wpi. Leukocytes might have been unrelated to BBB impairment because there was no inflammatory cell infiltration despite T. canis larvae having invaded the brain; whereas markedly elevated SP protein and NK-1R mRNA expressions concomitant with enhanced claudin-5 expression seemed to be associated with persistent BBB impairment in this experimental CT model.

Neurobehavioral basis of the impaired nurturing in mice lacking the immediate early gene FosB

The transcription factor FosB is induced in neurons of the medial preoptic area (MPOA) during parenting, through activation of the extracellular signal-regulated kinase (ERK). FosB mutant (−/−) postpartum mice and virgin mice that are exposed to pups show defective nurturing behavior. The FosB (−/−) MPOA fails to fully up-regulate SPRY1 and Rad, the feedback regulators of ERK and calcium signaling, respectively. Here we studied FosB function by examining the gene expression profiles and the behavioral characteristics of FosB (−/−) mice. We found that FosB (−/|−) mice exhibited not only decreased parenting but also decreased infanticide compared with (+/) littermates. We then performed gene expression analysis in the MPOA of FosB (−/−) mice compared with the wild-type littermates. We found up-regulation of glial fibrillary acidic protein (GFAP), C4, and Ela1 mRNA in the MPOA of FosB (−/−) mice; all of these gene products were implicated in general neuropathological conditions. Immunohistochemical analysis showed that up-regulation of GFAP was not restricted to MPOA but extended throughout the forebrain, including the cerebral cortex and striatum. Such pervasive GFAP up-regulation suggested that FosB (−/−) mice might have other behavioral abnormalities than nurturing. Indeed, these mice showed a clear alteration in emotionality, detected by the acoustic startle, elevated plus maze, and passive avoidance tests. These results suggest that FosB (−/−) mice have broader neurobehavioral dysfunctions, with which the nurturing defect might share the common mechanism.

Protection against Human Immunodeficiency Virus Type 1 Tat Neurotoxicity by Ginkgo biloba Extract EGb 761 Involving Glial Fibrillary Acidic Protein

Human immunodeficiency virus (HIV)-1 Tat protein is an important pathogenic factor in HIV-associated neuropathogenesis. Despite recent progress, the molecular mechanisms underlying Tat neurotoxicity are still not completely understood. However, few therapeutics have been developed to specifically target HIV infection in the brain. Recent development of an inducible brain-specific Tat transgenic mouse model has made it possible to define the mechanisms of Tat neurotoxicity and evaluate anti-neuroAIDS therapeutic candidates in the context of a whole organism. Herein, we demonstrate that administration of EGb 761, a standardized formulation of Ginkgo biloba extract, markedly protected Tat transgenic mice from Tat-induced developmental retardation, inflammation, death, astrocytosis, and neuron loss. EGb 761 directly down-regulated glial fibrillary acidic protein (GFAP) expression at both protein and mRNA levels. This down-regulation was, at least in part, attributable to direct effects of EGb 761 on the interactions of the AP1 and NF-kappaB transcription factors with the GFAP promoter. Most strikingly, Tat-induced neuropathological phenotypes including macrophage/microglia activation, central nervous system infiltration of T lymphocytes, and oxidative stress were significantly alleviated in GFAP-null/Tat transgenic mice. Taken together, these results provide the first evidence to support the potential for clinical use of EGb 761 to treat HIV-associated neurological diseases. Moreover, these findings suggest for the first time that GFAP activation is directly involved in Tat neurotoxicity, supporting the notion that astrocyte activation or astrocytosis may directly contribute to HIV-associated neurological disorders.

Cannabidiol in vivo blunts β‐amyloid induced neuroinflammation by suppressing IL‐1β and iNOS expression

Pharmacological inhibition of beta-amyloid (Abeta) induced reactive gliosis may represent a novel rationale to develop drugs able to blunt neuronal damage and slow the course of Alzheimer's disease (AD). Cannabidiol (CBD), the main non-psychotropic natural cannabinoid, exerts in vitro a combination of neuroprotective effects in different models of Abeta neurotoxicity. The present study, performed in a mouse model of AD-related neuroinflammation, was aimed at confirming in vivo the previously reported antiinflammatory properties of CBD. Mice were inoculated with human Abeta (1-42) peptide into the right dorsal hippocampus, and treated daily with vehicle or CBD (2.5 or 10 mg kg(-1), i.p.) for 7 days. mRNA for glial fibrillary acidic protein (GFAP) was assessed by in situ hybridization. Protein expression of GFAP, inducible nitric oxide synthase (iNOS) and IL-1beta was determined by immunofluorescence analysis. In addition, ELISA assay of IL-1beta level and the measurement of NO were performed in dissected and homogenized ipsilateral hippocampi, derived from vehicle and Abeta inoculated mice, in the absence or presence of CBD. In contrast to vehicle, CBD dose-dependently and significantly inhibited GFAP mRNA and protein expression in Abeta injected animals. Moreover, under the same experimental conditions, CBD impaired iNOS and IL-1beta protein expression, and the related NO and IL-1beta release. The results of the present study confirm in vivo anti-inflammatory actions of CBD, emphasizing the importance of this compound as a novel promising pharmacological tool capable of attenuating Abeta evoked neuroinflammatory responses.

Future Contrast Agents for Molecular Imaging in Stroke

The objective of this article is to illustrate both the potential and the limitations of molecular imaging in stroke research. By molecular imaging we mean the visual representation of biological processes at the cellular and molecular level. The use of molecular imaging for stroke diagnosis is still at a very preliminary stage and many of these procedures have only been tested in animals. In rats, stroke therapy using stem cells can be monitored by magnetic resonance imaging (MRI), green fluorescent protein (GFP) or luciferase (LUC) imaging. The migration of macrophages, which take up intravenously administered iron-based contrast agents and then migrate to the area of infarction, can already be observed in stroke patients. With MRI, the new agent Gd-DTPA-sLexA that binds to E- and P-selectin can specifically visualize selectin-mediated early endothelial activation after transient focal ischemia "in vivo". Decreased glial fibrillary acidic protein (GFAP) gene expression can be imaged in vivo by scintigraphy 24 hours after cerebral ischemia using a peptide nucleic acid antisense conjugate labeled with 111In and that hybridizes to the rat GFAP mRNA. Technetium-99m hydrazine nicotinamide-labeled HYNIC-annexin V SPECT can not only detect sites of neuronal injury in stroke patients but also can monitor the effects of neuroprotective therapy with a monoclonal antibody raised against FasLigand (FasL) in rats. Finally, information about cell metabolism in the infarct region can be gained using certain intracellular tracers [e.g. 18F-fluoromisonidazole (FMISO)]. Imaging benzodiazepine receptors with 11C-flumazenil (FMZ) can distinguish between irreversibly damaged and viable penumbra tissue early after stroke.

Reactive nonproliferative gliosis predominates in a chronic mouse model of glaucoma

In many CNS diseases, proliferation becomes dysregulated; cells divide and participate in pathological processes. Gliosis is a fundamental CNS response to trauma or disease in which cell hypertrophy and proliferation play prominent roles. The DBA/2J mouse is a glaucoma model in which mice experience gliosis concomitant with raised intraocular pressure that leads to a slow and progressive retinal ganglion cell axonopathy. We sought to determine if glaucomatous changes in DBA/2 retina would alter the regulation of cell proliferation, specifically in relation to retinal glia. Astrocyte and Müller glia populations within DBA/2 retina upregulated glial fibrillary acidic protein mRNA and protein compared with C57Bl/6; microglial cell number increased twofold from 4 to 10 months. Various bromodeoxyuridine (BrdU) injection paradigms were used to label dividing cells in DBA/2 and C57Bl/6 retina at 4 and 10 months of age. Very modest cell division in the retina, primarily in ganglion cell and inner nuclear layers, was observed at all ages. Immunohistochemistry indicated cell turnover primarily of NG2+ pericytes and Iba1+ microglia; astrocytes and Müller glia did not proliferate. There were no significant differences in BrdU+ cell numbers in 4 and 10-month-old retina, though 4-month retina had generally fewer BrdU+ cells than 10-month. C57Bl/6 retinas had fewer BrdU+ cells than DBA/2 retinas at all ages. These data show that, in contrast to gliosis in other CNS trauma and neurodegenerative diseases, glaucomatous changes in retina do not include substantive cell proliferation. Retinal changes in a chronic model of glaucoma engender a reactive, not proliferative, gliosis response.

Detection of central nervous system tissues in meat products: Validation and standardization of a real-time PCR-based detection system

Several phenotypic as well as genotypic methods have been published describing the detection of central nervous system (CNS) tissues that are part of the bovine spongiform encephalopathy (BSE) risk material in food products. However, none of these methods is able to differentiate between CNS tissue of the banned ruminant species and tissues of other animal species. A quantitative and species-specific real-time RT-PCR method has been developed that enables the reliable identification of CNS tissues in meat and meat products. This method is based on a messenger (m)RNA assay that uses bovine, ovine and caprine glial fibrillary acidic protein (GFAP) encoding gene sequences as markers. The in-house validation studies evaluated the tissue specificity of up to 15 bovine tissues and the standardization of absolute as well as relative quantitative measurement. The specific amplification of spinal cord and brain tissue GFAP cDNA has been shown previously. In addition, two commercially available ELISA kits were used for the comparative analysis of artificially contaminated minced meat. Small quantities of bovine brain that had been stored over the recommended period of 14 days were examined. The real-time PCR method proved to be suitable for the detection of 0.1% CNS tissue. No false negative results were observed. The quantitative detection of GFAP mRNA using real-time RT-PCR seems a suitable tool in routine diagnostic testing that assesses the illegal use of CNS tissue in meat and meat products. The stability of the selected target region of the GFAP mRNA also allows the detection of CNS tissues after the meat has been processed.

Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

The expression of glial fibrillary acidic protein (GFAP) by perivascular cells of many mammalian organs suggests an as yet unknown function of this intermediate filament protein in the maintenance of homeostasis and vascular permeability at the blood-tissue interface. Although a similar situation may exist at the air-tissue interface, the cellular distribution of GFAP in skin tissue has never been demonstrated. To approach this issue, we have employed immunofluorescence and Western blotting techniques to detect GFAP in skin sections of young and adult humans, normal rodents, and two types of mutant mice, as well as in rat lung sections, and in cultured human keratinocytes and fibroblasts. Colocalization with antigens known to be associated with GFAP in other tissues was also tested. Epidermal and hair follicle keratinocytes and dermal fibroblasts showed distinct staining for GFAP as well as colocalization with alpha-actin, metallothionein, and antigens of the class-II major histocompatibility complex (MHC II). GFAP was also identified in rat alveolar fibroblasts which, in common with keratinocytes, form part of the air-tissue interface. GFAP was upregulated together with MHC II in nude mice but was barely detectable in the skin of non-obese diabetic severe combined immunodeficiency mice, suggesting a possible involvement in antigen-presenting functions. The intriguing distribution of a common set of antigens both in certain cells of the integumentary system and at the blood-tissue interfaces of internal organs suggests the involvement of these proteins in universal mechanisms controlling tissue homeostasis and protection.

Collaborative Trial for Validation of a Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay for Detection of Central Nervous System Tissues as Bovine Spongiform Encephalopathy RiskMaterial: Part 1

A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120 degrees C) and the medium heat-treated samples (sausages cooked at 80 degrees C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.

Aspirin-induced blockade of NF-kB activity restrains up-regulation of glial fibrillary acidic protein in human astroglial cells

The marked induction of glial fibrillary acidic protein (GFAP) has been observed in astrocytes during neuropathological processes accompanying reactive gliosis; however, the precise molecular mechanism(s) underlying this GFAP induction remains poorly resolved. Therefore, in this study, we examined whether the change of nuclear factor-kappa B (NF-κB) activity can influence GFAP expression levels. Aspirin, widely used to prevent NF-κB activity, reduced the levels of GFAP mRNA and protein in human astroglial cells including human glioblastoma A172 cells and primary human brain astrocyte cells (HBAs). Furthermore, aspirin inhibited the effects of hypoxic injury on the up-regulation of GFAP expression in HBAs. We confirmed the repressive effect of aspirin on GFAP transcription by GFAP promoter-driven reporter assay and found that one NF-κB binding site conserved in the mouse and human GFAP gene promoters is critical for this effect. To further delineate whether NF-κ Bi s directly involved in the regulation of GFAP gene expression, we transfected A172 cells with an expression vector encoding a super-repressor IκBα protein (IκBα-SR) to specifically inhibit NF-κB activity and found the marked reduction of GFAP protein levels in IκBα-SR-transfectant cells. Taken together, our results suggest that NF-κB may play pivotal roles in GFAP gene expression.