Peroxidase Labeling Research Articles

Proteomics Approaches for Biomarker and Drug Target Discovery in ALS and FTD.

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are increasing in prevalence but lack targeted therapeutics. Although the pathological mechanisms behind these diseases remain unclear, both ALS and FTD are characterized pathologically by aberrant protein aggregation and inclusion formation within neurons, which correlates with neurodegeneration. Notably, aggregation of several key proteins, including TAR DNA binding protein of 43 kDa (TDP-43), superoxide dismutase 1 (SOD1), and tau, have been implicated in these diseases. Proteomics methods are being increasingly applied to better understand disease-related mechanisms and to identify biomarkers of disease, using model systems as well as human samples. Proteomics-based approaches offer unbiased, high-throughput, and quantitative results with numerous applications for investigating proteins of interest. Here, we review recent advances in the understanding of ALS and FTD pathophysiology obtained using proteomics approaches, and we assess technical and experimental limitations. We compare findings from various mass spectrometry (MS) approaches including quantitative proteomics methods such as stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tagging (TMT) to approaches such as label-free quantitation (LFQ) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) in studies of ALS and FTD. Similarly, we describe disease-related protein-protein interaction (PPI) studies using approaches including immunoprecipitation mass spectrometry (IP-MS) and proximity-dependent biotin identification (BioID) and discuss future application of new techniques including proximity-dependent ascorbic acid peroxidase labeling (APEX), and biotinylation by antibody recognition (BAR). Furthermore, we explore the use of MS to detect post-translational modifications (PTMs), such as ubiquitination and phosphorylation, of disease-relevant proteins in ALS and FTD. We also discuss upstream technologies that enable enrichment of proteins of interest, highlighting the contributions of new techniques to isolate disease-relevant protein inclusions including flow cytometric analysis of inclusions and trafficking (FloIT). These recently developed approaches, as well as related advances yet to be applied to studies of these neurodegenerative diseases, offer numerous opportunities for discovery of potential therapeutic targets and biomarkers for ALS and FTD.

Aptamer-Structure Switch Coupled with Horseradish Peroxidase Labeling on a Microplate for the Sensitive Detection of Small Molecules.

Detection of small molecules with good sensitivity, high throughput, simplicity, and generality using aptamers is desired but still remains challenging. We described an aptamer-structure-switch assay coupled with horseradish peroxidase (HRP) labeling on microplates for sensitive absorbance and chemiluminescence detection of small molecules. This assay relies on competition for affinity binding to a limited HRP-labeled aptamer between small-molecule targets and immobilized short DNA strands complementary to the aptamer (cDNA) on a microplate. In the absence of targets, the HRP-labeled aptamer hybridizes with the cDNA on the microplate, and HRP catalyzes substrate into product, generating absorbance or chemiluminescence signals. The binding of small-molecule targets to aptamers causes displacement of HRP-labeled aptamers from the cDNA and signal decrease. In chemiluminescence-analysis mode, the assay achieved detection of aflatoxin B1 (AFB1), ochratoxin A (OTA), and adenosine triphosphate (ATP) with detection limits of 10 pM, 20 pM, and 20 nM, respectively. This assay does not require enzyme-labeled small molecules or the conjugation of small molecules on solid phase. HRP, as an enzyme label, here allows for easily obtainable and highly active signal amplification. This microplate assay is rapid and promising for high-throughput analysis. It shows potential for wide applications in the detection of small molecules.

Immunohistochemical method of simultaneous detection of neurons and astrocytes in the rat brain

This study aimed to develop a method of simultaneous immunohistochemical detection of neurons and astrocytes using peroxidase labeling with subsequent analysis by light microscopy. A pair of antigens, each of which specifically identifies either neurons or astrocytes, was chosen (as markers neuronal nuclear antigen and glial fibrillary acidic protein were used, respectively). This method not only identifies the studied cells, but it also assesses their functional state under normal and pathological conditions. The proposed staining protocol allows significant simplification and optimization to simultaneously detect two antigens and to improve the quality of the slices obtained.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Ultrasensitive magnetic ELISA of zearalenone with pre-concentration and chemiluminescent detection

In this study, an enzyme-linked immunosorbent assay (ELISA) of mycotoxin zearalenone (ZEN) based on magnetic iron nanoparticles (MNPs) was developed. Highly dispersed MNPs used as carriers to immobilize anti-ZEN antibodies allowed the immunoassay to be carried out in a pseudo-homogeneous mode through the rapid formation of immune complexes in the reaction media, their separation by a magnetic field, and the heterogeneous detection of the bound peroxidase label. The traditional colorimetric substrate of a peroxidase label was changed to a highly sensitive chemiluminescent one for the detection of specific complexes’ formation. The developed analysis consisted of two main stages: first, the interaction of MNP-based immunosorbent with free ZEN in a large-volume sample and its transfer to a smaller volume (preliminary concentration of the analyte) and then binding with ZEN–peroxidase complexes. A proposed novel combination of a 50-fold pre-concentration of ZEN with chemiluminescence detection allows for the determination of zearalenone with the detection limit of 4 pg/mL. Therefore, more than an order of magnitude increase of the assay sensitivity was achieved compared with the same assay format with colorimetric detection (0.06 ng/mL) or chemiluminescent MNP–ELISA without a pre-concentration of the analyte (0.1 ng/mL). The analysis was validated for the determination of zearalenone in wine. An assay based on magnetic concentration and chemiluminescent detection can be proposed as a universal analytical tool for the rapid assessment of food contamination by mycotoxins and other hazardous substances.

DNA biosensors based on gold nanoparticles-modified graphene oxide for the detection of breast cancer biomarkers for early diagnosis

Two different DNA (ERBB2c and CD24c) modified gold nanoparticles and graphene oxide loaded on glassy carbon electrodes were prepared for early detection of breast cancer markers by electrochemical detection of HER2. Comparative study of ERBB2c and CD24c for the detection was carried out. A “sandwich-type” detection strategy was employed in this electrochemical DNA biosensor and its response was measured by amperometric detection. The electrochemical signal enhancement achieved via gold nanoparticles and grapheme oxide system allowed for sensitive detection of the breast cancer biomarker ERBB2 and the control marker CD24. The modified graphene oxide was characterised using Raman spectroscopy, UV–visible spectroscopy, Fourier transform infrared spectroscopy transmission electron microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The various steps involved in the modification of a glassy carbon electrode with graphene oxide, gold nanoparticles and DNA probes, target and reporter probe were electrochemically characterised using cyclic voltammetry and electrochemical impedance spectroscopy. Using amperometric detection of a horse radish peroxidase label, detection limits of 0.16nM and 0.23nM were obtained with sensitivity 378nA/nM and 219nA/nM for ERBB2 andCD24 respectively.

Analysis of morphological and functional changes of kidney endothelium in systemic rheumatic diseases

Introduction. The nature of the lesion and the pathogenesis of kidney vessels damage in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Henoch-Schönlein purpura (HSP) and microscopic polyangiitis (MPA) remains not enough studied, although the severity of such vasculopathy determines the prognosis of systemic autoimmune rheumatic diseases.The aim and purpose of the work was to evaluate the nature of morphological changes in the endothelium of the capillaries and arterioles of the kidneys in SLE, RA, VHS and MPA, connection with clinical and laboratory factors of the diseases' course and the influence of systemic vascular endothelial dysfunction.Materials and methods. The kidney biopsy results and data of laboratory analysis of vascular endothelial function were analysed in 94 patients, among which 41 persons suffered from SLE, 17 – RA, 24 - HSP and 12 – MPA. The ratio of men and women in these groups was respectively 1:7, 1:2, 2:1 and 1:3, the average age of the patients - 38, 50, 28 and 41 years, duration of disease from its manifestation - 12, 12, 10 and 4 years, the distribution of patients I, II and III degree of activity of the pathological process - as a 1:2:3, 1:2:2, 2:1:1, 1:2:9. Kidney biopsy is performed only in patients with the presence of proteinuria (excluded patients with AA amyloidosis). Carried out echocardiography, doppler ultrasound of the vessels and biomicroscopy of the conjunctiva vessels; by immune-enzyme analysis were studied indicators of vascular endothelial growth factor, endothelin-1, thromboxane-A2, homocysteine, prostacyclin, cyclic guanosine monophosphate, E- and P-selectin. Histological sections of the kidneys were stained with hematoxylin-eosin, alcian blue and van Gieson, becoming the PAS-reaction. In addition, the immune-enzyme (with peroxidase label) and fluorescence immunoassay methods of research of kidney tissue were performed.Results. The proliferation of capillary endothelium was peculiar with a greater extent to SLE and MPA, and arteriolar endothelium - only to MPA, whereas these changes were not peculiar to RA. Deposits of immunoglobulin (Ig) A in the blood vessels were the most typical for HSP and C1q-complement component - for SLE and MPA. The lesions of the endothelium of the capillaries and arterioles of the kidneys depend on the severity of the skin syndrome, peripheral neuropathy, leukocytoclastic enantemy and hands and feet capillaritis. Vascular endothelial dysfunction as vasodilators and vasoconstrictors imbalance occurs in 35 % of RA patients with renal disease, in 39 % SLE, 52 % HSP and 100 % MPA. Morphological lesions of the renal vascular endothelium are closely related to the severity of instrumental signs of extrarenal systemic angiopathy. Plasma levels of vascular endothelial growth factor determine proliferation of glomerular endothelium in patients with MPA, and in the development of renal immune endothelial deposits (IgA, IgG, IgM, C3, C1q) in SLE participate endothelin-1 and E-selectin, in RA - P-selectin, VSH - homocysteine.Conclusions. All patients with SLE, RA, HSP and MPA peculiar changes of the capillaries and arterioles endothelium of the kidneys glomeruli, in which genesis the disorders of systemic endothelial dysfunction of vessels participate.

Optimized dendrimer-encapsulated gold nanoparticles and enhanced carbon nanotube nanoprobes for amplified electrochemical immunoassay of E. coli in dairy product based on enzymatically induced deposition of polyaniline

A highly sensitive immunosensor was reported for Escherichia coli assay in dairy product based on electrochemical measurement of polyaniline (PAn) that was catalytically deposited by horseradish peroxidase (HRP) labels. Herein, the immunosensor was developed by using poly(amidoamine) dendrimer-encapsulated gold nanoparticles (PAMAM(Au)) as sensing platform. Importantly, the optimal HAuCl4/PAMAM ratio was investigated to design the efficient PAMAM(Au) nanocomposites. The nanocomposites were proven to not only increase the amount of immobilized capture antibody (cAb), but also accelerate the electron transfer process. Moreover, the {dAb-CNT-HRP} nanoprobes were prepared by exploiting the amplification effect of multiwalled carbon nanotubes (CNTs) for loading detection antibody (dAb) and enormous HRP labels. After a sandwich immunoreaction, the quantitatively captured nanoprobes could catalyze oxidation aniline to produce electroactive PAn for electrochemical measurement. On the basis of signal amplification of the PAMAM(Au)-based immunosensor and the {dAb-CNT-HRP} nanoprobes, the proposed strategy exhibited a linear relationship between the peak current of PAn and the logarithmic value of E. coli concentration ranging from 1.0×102 to 1.0×106cfumL−1 with a detection limit of 50cfumL−1 (S/N=3), and the electrochemical detection of E. coli could be achieved in 3h. The electrochemical immunosensor was also used to determine E. coli in dairy product (pure fresh milk, infant milk powder, yogurt in shelf-life and expired yogurt), and the recoveries of standard additions were in the range of 96.8–108.7%. Overall, this method gave a useful protocol for E. coli assay with high sensitivity, acceptable accuracy and satisfying stability, and thus provided a powerful tool to estimate the quality of dairy product.

Doing more with less: fluorescence in situ hybridization and gene sequencing assays can be reliably performed on archival stained tumor tissue sections.

Little is known about molecular testing on tumor tissue retrieved from stained sections, for which there may be a clinical need. We retrospectively analyzed 112 sections from 56 tumor patients using either fluorescence in situ hybridization (FISH) with different probes (19 sections from 17 patients) or Sanger or targeted next generation sequencing for detection of BRAF, EGFR, KRAS, C-KIT, and TP53 mutations (93 sections from 39 patients). Tumor tissue sections had been stained by hematoxylin and eosin (H&E) (42 sections) or by immunohistochemistry for cytoplasmic or nuclear/nuclear-cytoplasmic markers (70 sections) with a peroxidase (P-IHC, with 3,3'-diaminobenzidine as chromogen) or alkaline phosphatase label (AP-IHC, with Warp Red™ as chromogen). For FISH analysis, the concordance rate between the original diagnosis and that obtained on H&E- or P-IHC-stained tissue sections (AP-IHC was not on record for this set of patients) was 95% (18 out of 19 tumor sections). Only one tumor sample, diffusely positive for MLH1, did not yield any nuclear hybridization signal. For sequencing analysis, the concordance rate was 100% on negative P-IHC and positive AP-IHC-stained sections, regardless of the subcellular localization of the reaction product. Mutations were detected in only 52% of cases expressing nuclear/nuclear-cytoplasmic markers, regardless of the sequencing technology used (p = 0.0002). In conclusion, stained sections may be a valuable resource for FISH or sequencing analysis, but on cases expressing nuclear markers sequencing results need to be interpreted cautiously.

Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH)

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with rRNA-targeted oligonucleotide probes has significantly improved the identification of microorganisms in various environmental samples. However, one of the major constraints of CARD-FISH is the low probe penetration due to the high molecular weight of the horseradish peroxidase (HRP) label. Recently, this limitation has been overcome by a novel signal amplification approach termed in situ DNA-hybridization chain reaction (in situ DNA-HCR). In this study, we present an improved and accelerated in situ DNA-HCR protocol (quickHCR-FISH) with increased signal intensity, which was approximately 2 times higher than that of standard in situ DNA-HCR. In addition, the amplification time was only 15min for the extension of amplifier probes from the initiator probe compared to 2h in the original protocol. The quickHCR-FISH was successfully tested for the quantification of marine bacteria with low rRNA contents in both seawater and sediment samples. It was possible to detect the same number of marine bacteria with quickHCR-FISH compared to CARD-FISH within only 3h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.

Improvements in neuroelectrophysiological and rear limb functions in rats with spinal cord injury after Schwann cell transplantation in combination with a C5a receptor antagonist.

We measured the effect of Schwann cell transplantation and complement factor 5a (C5a) receptor antagonist on nerve function recovery in rats with spinal cord injury. Experimental spinal cord injury was induced in eighty Wistar rats and these were randomly divided into four treatment groups: culture medium and saline injection (control group), Schwann cell injection (cell transplantation group), C5a receptor antagonist injection (C5a receptor antagonist group), and both Schwann cell and C5a receptor antagonist injections (combination group). Rear limb functional recovery was assessed 1, 2, 4, 6, and 8 weeks after the spinal cord injury with the tilt table test and the Basso, Beattie, Bresnahan scale. Sex-determining region Y (SRY) gene expression was measured at week 4 and horseradish peroxidase (HRP) labeling was used at week 8 to further assess the recovery of neuroelectrophysiological functions. The rear limb functional assessment showed that the combination group had better outcomes than the cell transplantation and C5a receptor antagonist groups. All treatment groups had better outcomes than control. Only the cell transplantation and combination groups showed SRY expression. The number of HRP-positive nerve fibers in the different groups ranked as follows: combination group > cell transplantation and C5a receptor antagonist > control. The refractory period and amplitude of the induced potential in the combination group were significantly greater than in the other three groups. These results suggest that the combination of Schwann cell transplantation and the C5a receptor antagonist enhances the regeneration of injured synapses and improves limb function and electrophysiology.

Electrical signaling of enzyme-linked immunosorbent assays with an ion-sensitive field-effect transistor

Optical laboratory-based immunoassays, such as enzyme-linked immunosorbent assay (ELISA) give a high sensitivity and specificity of various fatal diseases. However, these assays are no longer efficient in on-spot diagnostics of wide-spreading and contagious infections. At this point in time, portable and handhold devices play a pivotal role in infectious diseases with quick diagnostics at or near the site of the disease propagation. In this paper, we demonstrated a novel electrical immunoassay of ELISA that was not based on optical signaling but on electrical signaling. This was done by combining an ion-sensitive field-effect transistor (ISFET) with ELISA. By harnessing the catalytic reaction of alkaline phosphatase that precipitated silver particles, we effectively overcame the chronic Debye screening length issue of the ISFET. Ultimately, small signal ranging from 1pg/mL to 10ng/mL was immensely amplified with the ALP label, regardless of buffer conditions. The sensor platform herein surpassed a sensing capability of conventional ELISA that is considered to have a LOD on the order of ~1ng/mL. The results were compared with those of horseradish peroxidase label, which is generally used for optical analyses in ELISA. Our newly developed ISFET-based portable sensor holds a large potential for point-of-care tools in a variety of diseases, without being limited by the need for expensive equipment such as spectrophotometers.

Covalent functionalization of gold nanoparticles as electronic bridges and signal amplifiers towards an electrochemical immunosensor for botulinum neurotoxin type A

This work introduced an efficient approach for modification of AuNPs with multicomponents by diazonium salt couplings. The multifunctionalized AuNPs with protruding functional groups that allow simple bioconjugation to large amounts of biomolecules have been successfully used as electronic bridges and signal amplifiers for an electrochemical immunosensor towards the detection of BoNT/A. The one-step anchoring AuNPs strategy has greatly increased the efficiency for attachment of biomolecules and subsequently increased the sensitivity. Sensitivity was further amplified by preparation of bioconjugates particles containing horseradish peroxidase (HRP) labels along with detection antibodies (AbL) attached to AuNPs. The immunosensor can be used for the detection of BoNT/A over the range of 4–35pgmL−1 with the lowest detection limit of 1pgmL−1 and assay time of 10min. The herein sensing strategy is rapid, robust, selective, sensitive, and is promising for future fabrication of point-of-care devices.

Human eosinophil adhesion and receptor expression.

Eosinophils migrate from the bone marrow in response to cytokines and chemokines which induce the expression and activation of adhesion receptors. In understanding the recruitment of eosinophils, protocols to identify eosinophil adhesion and receptor expression have been identified. In this summary, the eosinophil peroxidase and fluorescent labeling assays are described as measurements of indirect and direct eosinophil adhesion, respectively. Additional protocols that identify eosinophil receptor expression via immunofluorescent microscopy and flow cytometry are also described.

Application of BrdU in studying postnatal neurogenesis

PURPOSES: We applied the thymidine analog, 5-bromo-2'-deoxyuridine (BrdU) that labels DNA to study neurogenic events in many brain regions of the postnatal mammalian brain and to evaluate its paradigms, pitfalls and limitations. MATERIAL AND METHODS: Different BrdU paradigms and various time points between postnatal day P0 and P51 were applied to Wistar rats. Paraffin sections for single or double BrdU immunohistochemistry combined with neuronal markers nestin, Dcx, MAP(2) and NeuN by peroxidase or fluorescence labeling were examined. Sections were analyzed using standard or confocal microscopy and stereology. RESULTS: Peak neurogenesis was found out in the dentate gyrus during the first postnatal week of life with a progressive decline after P9. Areas with substantial neurogenesis such as subventricular zone of the lateral ventricles, subgranular zone of the hippocampal dentate gyrus, and cerebellum were observed. By P14 to P18, cell proliferation was reduced in most brain areas with exception of the dentate gyrus. Numerical densities of BrdU(+) cells in all evaluated brain regions were higher in the younger animals and decreased with age. Most newly-generated cells became nonneuronal cells (endothelial and glial cells). BrdU colocalization with neuronal markers was rarely found out mainly within the dentate gyrus and cerebellum. CONCLUSION: In the developing rat brain, BrdU labeling showed regional distribution. It was dose-, age- and survival time-dependent. Our findings provided useful information on BrdU application in the study of postnatal neurogenesis. Scripta Scientifica Medica 2013; 45(1): 24-28.

Enzyme-based ultrasensitive electrochemical biosensors

Signal amplification in conventional enzyme-based biosensors is not high enough to achieve the ultrasensitive detection of biomolecules. In recent years, signal amplification has been improved by combining enzymatic reactions with redox cycling or employing multienzyme labels per detection probe. Electrochemical-chemical redox cycling and electrochemical-chemical-chemical redox cycling allow ultrasensitive detection simply by including one or two more chemicals in a solution without the use of an additional enzyme and/or electrode. Multiple horseradish peroxidase labels on magnetic bead carriers provide high signal enhancement along with a multiplex detection possibility. In both cases, the detection procedures are the same as those in conventional enzyme-based electrochemical sensors.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

ELCR: electrochemical detection of single DNA base changes via Ligase Chain Reaction

Simple, inexpensive and wide-scaled analysis of single DNA base changes ('point mutations' which include SNP's, somatic mutations and epigenetic changes) is one of the holy grail's of point-of-care diagnostics. Herein, we present eLCR: a simple methodology that fuses Ligase Chain Reaction (LCR) with electrochemical detection based on DNA-mediated charge transport. LCR generates long and short "knife" motifs representing the presence or absence of single DNA base changes, which are then detected electrochemically by either methylene blue intercalation or horseradish peroxidase labelling.

Fabrication of immunosensor microwell arrays from gold compact discs for detection of cancer biomarker proteins.

A simple method is reported to fabricate gold arrays featuring microwells surrounding 8-electrodes from gold compact discs (CDs) for less than $0.2 per chip. Integration of these disposable gold CD array chips with microfluidics provided inexpensive immunoarrays that were used to measure a cancer biomarker protein quickly at high sensitivity. The gold CD sensor arrays were fabricated using thermal transfer of laserjet toner from a computer-printed pattern followed by selective chemical etching. Sensor elements had an electrochemically addressable surface area of 0.42 mm(2) with RSD <2%. For a proof-of-concept application, the arrays were integrated into a simple microfluidic device for electrochemical detection of cancer biomarker interleukin-6 (IL-6) in diluted serum. Capture antibodies of IL-6 were chemically linked onto the electrode arrays and a sandwich immunoassay protocol was developed. A biotinylated detection antibody with polymerized horseradish peroxidase labels was used for signal amplification. The detection limit of IL-6 in diluted serum was remarkably low at 10 fg mL(-1) (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL(-1). These easily fabricated, ultrasensitive, microfluidic immunosensors should be readily adapted for sensitive detection of multiple biomarkers for cancer diagnostics.

Acidic Fibroblast Growth Factor (FGF) Potentiates Glial-mediated Neurotoxicity by Activating FGFR2 IIIb Protein

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.