An organoselenium compound inhibits Staphylococcus aureus biofilms on hemodialysis catheters in vivo.

Colonization of central venous catheters (CVCs) by pathogenic bacteria leads to catheter-related bloodstream infections (CRBSIs). These colonizing bacteria form highly antibiotic-resistant biofilms. Staphylococcus aureus is one of the most frequently isolated pathogens in CRBSIs. Impregnating CVC surfaces with antimicrobial agents has various degrees of effectiveness in reducing the incidence of CRBSIs. We recently showed that organoselenium covalently attached to disks as an antibiofilm agent inhibited the development of S. aureus biofilms. In this study, we investigated the ability of an organoselenium coating on hemodialysis catheters (HDCs) to inhibit S. aureus biofilms in vitro and in vivo. S. aureus failed to develop biofilms on HDCs coated with selenocyanatodiacetic acid (SCAA) in either static or flowthrough continuous-culture systems. The SCAA coating also inhibited the development of S. aureus biofilms on HDCs in vivo for 3 days. The SCAA coating was stable and nontoxic to cell culture or animals. This new method for coating the internal and external surfaces of HDCs with SCAA has the potential to prevent catheter-related infections due to S. aureus.

Serum inhibits P. aeruginosa biofilm formation on plastic surfaces and intravenous catheters.

INTRODUCTION: Biofilm formation on medical devices such as intravenous catheters is a serious manifestation of Pseudomonas aeruginosa infections. Serum has bactericidal activity, a function of multiple serum components. In this study, we determined the effect of serum and serum components on the formation of P. aeruginosa biofilm. MATERIALS AND METHODS: We examined the effect of adult bovine serum (ABS) or bovine serum albumin (BSA) on biofilm development on plastic coverslips. This was done using both static and continuous flow-through culture systems and P. aeruginosa strain PAO1. Biofilms were quantified using crystal violet assays and visualized using confocal scanning laser microscopy and scanning electron microscopy. We examined the effect of ABS on PAO1 swimming and twitching motilities (both contribute to P. aeruginosa biofilm development). We also analyzed the inhibitory effect of adult human serum (AHS) and plasma (AHP) on PAO1 biofilm development on plastic coverslips and intravenous catheters. RESULTS: Compared with M9 minimal medium (M9), 10% ABS-supplemented medium (M9/ABS-10) caused a significant decrease in biofilm development. Coverslips precoated with M9/ABS-10 failed to develop biofilm when placed in M9. In addition to reduced biofilm formation, adding ABS to M9 reduced an already-developed PAO1 biofilm. Compared with M9, M9/ABS-10 enhanced PAO1 twitching motility considerably, but did not affect swimming motility. Similar to ABS, BSA blocked biofilm formation but did not affect PAO1 twitching motility. Both AHS and AHP blocked PAO1 biofilm formation on plastic coverslips and intravenous catheters. CONCLUSIONS: These results suggest that as part of the host innate resistance, serum inhibits P. aeruginosa biofilm formation on plastic surfaces, including intravenous catheters. Two possible scenarios for this inhibition include blocking the direct interaction between P. aeruginosa and the substrates, and the enhancing P. aeruginosa twitching motility.

Cerebellar granule cells cultured from adolescent rats express functional NMDA receptors: an in vitro model for studying the developing cerebellum.

In the developing rat cerebellum functional NMDA receptors (NMDARs) expressing the NR2C subunit have been identified on or after postnatal day 19. We obtained primary cultured cells from 19- to 35-day-old rat cerebellum that expressed few oligodendrocytes or astrocytes. Cultured cells were immunoreactive for neuron-specific proteins thus indicating a neuronal population. The primary neuron present was the granule cell as indicated by immunofluorescence for the GABA(A) alpha 6 subunit. Whole-cell patch-clamp experiments indicated that functional NMDARs were present. Functional characteristics of NMDARs expressed in cerebellar granule cells (CGCs) obtained from adolescent animals were similar to those previously reported for NMDARs expressed in CGCs obtained from neonatal rats. Cultured CGCs obtained from older animals contained NMDARs that were inhibited by EtOH and were less sensitive to the NR2B subunit-specific antagonist Ro 25-6981. Furthermore, NMDA-induced currents were smaller than those observed in CGCs. Western blot analysis indicated the presence of the NMDA NR2A and NR2C subunits, but not the NR2B in cultures obtained from the adolescent rats. CGCs obtained from adolescent rats express functional NMDARs consistent with a developmental profile observed in vivo.

Bergmann glia utilize active caspase-3 for differentiation.

Recently, functions associated with caspase have been modified from their well-established role in apoptosis. Although caspases are still regarded as mediators of apoptosis, some of the pro-apoptotic caspases, namely caspase-8, -14 and -3 also regulate differentiation in certain cell types, namely myelomonocytic cells, osteoblasts, skeletal muscle cells, keratinocytes, and T lymphocytes. In the central nervous system, non-apoptotic active caspase-3 expression has been located in proliferating and differentiating neuronal cells of the ventricular zone and external granular layer of the developing cerebellar cortex. We previously demonstrated that active caspase-3 expression was not limited to neuronal cells but also was located in the Bergmann glia of the postnatal cerebellum. In that study, active caspase-3 immunolabeling did not markedly colocalize with Ki67, a proliferation marker, but was present in differentiating Bergmann glia that expressed brain lipid binding protein (BLBP) and thus, by its localization, suggested a role in the differentiation of Bergmann glia. The current study addresses the function of caspase-3 in Bergmann glia development by utilizing a Bergmann glial culture preparation. Inhibition of caspase-3 activity by the peptide inhibitor, DMQD-FMK, increased the number of proliferating precursor glial cells and decreased the number of differentiating Bergmann glia, without significantly altering the non-glial active caspase-3 negative population. The transformation in the developmental state of Bergmann glia occurring after suppression of caspase-3 activity strongly suggests an involvement of this enzyme in promoting differentiation of Bergmann glia.

The Peridural Membrane of the Human Spine is Well Innervated.

There is evidence that low back pain may originate from a peridural membrane (PDM) at the inferior and medial aspect of neural foramen of the lumbar spine. The objective of this investigation was to determine if this membrane contains neural elements suggestive of sensory innervation with nociceptive function. Spines of four embalmed and three non-embalmed human cadavers were dissected using a sagittal approach to the neural foramen. Seventeen samples of the peridural membrane overlying the neural foramen were collected for immunohistochemistry (IHC) examination by light microscopy and transmission electron microscopy (TEM). Chromagin tagged antibodies to protein gene product 9.5 (PGP9.5) and S-100, and fluorescent antibodies to substance P and calcitonin gene related peptide (CGRP) were used to label neural structures in tissue sections cut from paraffin embedded blocks. This approach allows good visualization of all neural elements, small sensory, and nociceptive nerve fibers in particular. Neural elements were found in all samples. Marked presence of small nerve fibers was observed in 12 of 15 samples. IHC and TEM evaluation revealed myelinated as well as unmyelinated fibers in the peridural membrane. CGRP and substance P immunoreactive fibers indicative of nociceptive function were abundant. These findings confirm and expand evidence that the peridural membrane in human is well innervated and contains sensory nociceptive nerve fibers suggestive of a nociceptive function of the membrane.

In vitro chondrocyte behavior on porous biodegradable poly(e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation.

In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.

Active caspase-3 expression during postnatal development of rat cerebellum is not systematically or consistently associated with apoptosis.

Development is a dynamic process that includes an intricate balance between an increase in cell mass and an elimination of excess or defective cells. Although caspases have been intimately linked to apoptotic events, there are a few reports suggesting that these cysteine proteases can influence the differentiation and proliferation of cells. Specifically, the active form of caspase-3, which has been classified as an executor of apoptosis, recently has been implicated in a nonapoptotic role in the regulation of the cell cycle, cell proliferation, and cell differentiation. This study investigated the nonapoptotic function and phenotypic expression of active caspase-3-positive cells in the external granule cell layer (EGL) of the postnatal rat cerebellum by using biochemical and immunohistochemical analyses, respectively. Evidence that negates an apoptotic function for the caspase-3-positive EGL cells includes a failure to exhibit chromatin condensation (assessed with TOPRO), phosphatidyl serine externalization (Annexin V labeling), or DNA fragmentation (TUNEL labeling). Proliferative (Ki67-positive) and differentiated (TUJ1-positive) cells within the EGL exhibited a cytosolic expression of caspase-3, whereas terminally differentiated granule cells (NeuN-positive) in the internal granular layer and the migrating granule cells did not express active caspase-3. Thus, this study supports a nonapoptotic role for active caspase-3 in cells residing in the EGL and suggests a possible involvement in EGL proliferation and differentiation.

Differential sensitivity of prefrontal cortex and hippocampus to alcohol-induced toxicity.

The prefrontal cortex (PFC) is a brain region responsible for executive functions including working memory, impulse control and decision making. The loss of these functions may ultimately lead to addiction. Using histological analysis combined with stereological technique, we demonstrated that the PFC is more vulnerable to chronic alcohol-induced oxidative stress and neuronal cell death than the hippocampus. This increased vulnerability is evidenced by elevated oxidative stress-induced DNA damage and enhanced expression of apoptotic markers in PFC neurons. We also found that one-carbon metabolism (OCM) impairment plays a significant role in alcohol toxicity to the PFC seen from the difference in the effects of acute and chronic alcohol exposure on DNA repair and from exaggeration of the damaging effects upon additional OCM impairment in mice deficient in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR). Given that damage to the PFC leads to loss of executive function and addiction, our study may shed light on the mechanism of alcohol addiction.

Helicase-like transcription factor (Hltf) regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac transcription networks, and collagen biogenesis.

HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum) brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05) - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf's regulation of the G2/M transition (p=9.726E(-15)) of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2) and lysyl (Plod2) collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3) for collagen trimerization, and lysyl oxidase (Loxl2) for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps) was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and fragmented. Thus, silencing Hltf during heart organogenesis compromised DNA double-strand break repair, and caused aberrant collagen biogenesis altering the structural network that transmits cardiomyocyte force into muscle contraction.

Role of helicase-like transcription factor (hltf) in the G2/m transition and apoptosis in brain.

HLTF participates in transcription, chromatin remodeling, DNA damage repair, and tumor suppression. Aside from being expressed in mouse brain during embryonic and postnatal development, little is known about Hltf's functional importance. Splice variant quantification of wild-type neonatal (6-8 hour postpartum) brain gave a ratio of 5:1 for Hltf isoform 1 (exons 1-25) to isoform 2 (exons 1-21 with exon 21 extended via a partial intron retention event). Western analysis showed a close correlation between mRNA and protein expression. Complete loss of Hltf caused encephalomalacia with increased apoptosis, and reduced viability. Sixty-four percent of Hltf null mice died, 48% within 12-24 hours of birth. An RNA-Seq snapshot of the neonatal brain transcriptome showed 341 of 20,000 transcripts were altered (p < 0.05) - 95 up regulated and 246 down regulated. MetaCore™ enrichment pathway analysis revealed Hltf regulates cell cycle, cell adhesion, and TGF-beta receptor signaling. Hltf's most important role is in the G2/M transition of the cell cycle (p  =  4.672e-7) with an emphasis on transcript availability of major components in chromosome cohesion and condensation. Hltf null brains have reduced transcript levels for Rad21/Scc1, histone H3.3, Cap-E/Smc2, Cap-G/G2, and Aurora B kinase. The loss of Hltf in its yeast Rad5-like role in DNA damage repair is accompanied by down regulation of Cflar, a critical inhibitor of TNFRSF6-mediated apoptosis, and increased (p<0.0001) active caspase-3, an indicator of intrinsic triggering of apoptosis in null brains. Hltf also regulates Smad7/Bambi/Tgf-beta/Bmp5/Wnt10b signaling in brain. ChIP confirmed Hltf binding to consensus sequences in predicted (promoter Scgb3a1 gene) and previously unidentified (P-element on chromosome 7) targets. This study is the first to provide a comprehensive view of Hltf targets in brain. Moreover, it reveals how silencing Hltf disrupts cell cycle progression, and attenuates DNA damage repair.

Garlic ointment inhibits biofilm formation by bacterial pathogens from burn wounds.

When thermal injury damages the skin, the physical barrier protecting underlying tissues from invading micro-organisms is compromised and the host's immune system becomes supressed, facilitating colonization and infection of burn wounds with micro-organisms. Within the wound, bacteria often develop biofilms, which protect the bacteria from the immune response and enhance their resistance to antibiotics. As the prophylactic use of conventional antibiotics drives selection of drug-resistant strains, the use of novel agents to prevent biofilm formation by wound pathogens is essential. In the present study, we utilized our recently developed in vitro wound biofilm model to examine the antibiofilm activity of garlic (Allium sativum). Wound pathogens were inoculated on sterile cellulose discs, exposed to formulated garlic ointment (GarO) or ointment base, and incubated to allow biofilm development. Biofilms were quantified and visualized microscopically. GarO prevented biofilm development by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, and caused a 2-5 log reduction of the bioburden within Enterococcus faecalis biofilms. Additionally, GarO disrupted partially developed biofilms produced by S. aureus, S. epidermidis and A. baumannii. The antistaphylococcal activity of GarO was stable for over 3 months at room temperature. Thus, GarO could be used as a prophylactic therapy to prevent wound biofilms caused by both Gram-negative and Gram-positive bacteria from forming, and may be a potential therapy for disrupting established staphylococcal biofilms.

An in vitro model for studying the effects of continuous ethanol exposure on N-methyl-D-aspartate receptor function.

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl-d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post-ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I(NMDA)) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I(NMDA) from CEE-treated cells were significantly larger than I(NMDA) from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I(NMDA) were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial-neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial-neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.

Prolactin induces Jak2 phosphorylation of RUSHY195.

Jak2/RUSH-mediated prolactin signaling culminates in RUSH-1α-DNA-binding. Heretofore, Jak2-specific phosphorylation residues in RUSH were unknown. Genpathway's discovery approaches correlated RUSH-DNA binding (-126/-121) in uteroglobin's proximal promoter with recruitment of the transcriptional machinery. NetPhos 2.0 server found a single tyrosine phosphorylation site in RUSH's minimal DNA-binding domain. Y195 had identical context and prediction scores (0.52) for rabbit and human (HLTF) orthologs. The mouse ortholog (Hltf) had a higher prediction score (0.897). Affinity purified RUSHY195ph antibodies recognized native tyrosine phosphorylated RUSH protein immunoprecipitated from nuclear extracts. When R5020-treated HRE-H9 cells±the Jak2 inhibitor, Tyrene CR4, were stimulated with prolactin, confocal immunofluorescence images provided conclusive evidence that Jak2 mediated the availability of phosphorylated RUSHY195 in nucleus and cytoplasm. Catalytically active Jak2 is ipso facto a RUSH site-specific tyrosine kinase. Immunoprecipitation/Western blotting revealed both phosphorylation at Y195 and the physical interaction between p-Jak2/RUSH/HLTF/Hltf are evolutionarily conserved across three mammalian (rabbit, human, mouse) orthologs.

An in vitro biofilm model to examine the effect of antibiotic ointments on biofilms produced by burn wound bacterial isolates.

PURPOSE: Topical treatment of burn wounds is essential as reduced blood supply in the burned tissues restricts the effect of systemic antibiotics. On the burn surface, microorganisms exist within a complex structure termed a biofilm, which enhances bacterial resistance to antimicrobial agents significantly. Since bacteria differ in their ability to develop biofilms, the susceptibility of these biofilms to topically applied antibiotics varies, making it essential to identify which topical antibiotics efficiently disrupt or prevent biofilms produced by these pathogens. Yet, a simple in vitro assay to compare the susceptibility of biofilms produced by burn wound isolates to different topical antibiotics has not been reported. METHODS: Biofilms were developed by inoculating cellulose disks on agar plates with burn wound isolates and incubating for 24h. The biofilms were then covered for 24h with untreated gauze or gauze coated with antibiotic ointment and remaining microorganisms were quantified and visualized microscopically. RESULTS: Mupirocin and triple antibiotic ointments significantly reduced biofilms produced by the Staphylococcus aureus and Pseudomonas aeruginosa burn wound isolates tested, as did gentamicin ointment, with the exception of one P. aeruginosa clinical isolate. CONCLUSIONS: The described assay is a practical and reproducible approach to identify topical antibiotics most effective in eliminating biofilms produced by burn wound isolates.

Prolactin-induced Jak2 phosphorylation of RUSH: a key element in Jak/RUSH signaling.

Jak2/Stat-mediated prolactin signaling culminates in Stat5a-DNA-binding. However, not all Jak2-dependent genes have Stat5 sites. Western analysis with inhibitors showed Jak2 is a proximal intermediate in prolactin-induced RUSH phosphorylation. Transfection assays with HRE-H9 cells showed the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcription of the RUSH gene. Jak2 inhibitors or targeted RUSH-site mutation blocked the prolactin effect. RUSH co-immunoprecipitated with phospho-Jak2 from nuclear extracts. Jak2 inhibitors abolished the nuclear pool of phospho-RUSH not the nuclear content of RUSH in HRE-H9 cells. Nucleolar-affiliated partners, e.g. nucleolin, were identified by microLC/MS/MS analysis of nuclear proteins that co-immunoprecipitated with RUSH/GST-RING. RUSH did not exclusively co-localize with fibrillarin to the nucleolus. MG-132 (proteasomal inhibitor) failed to block Tyrene CR4-mediated decrease in phospho-RUSH, and did not promote RUSH accumulation in the nucleolus. These studies authenticate prolactin-dependent Jak2 phosphorylation of RUSH, and provide functional implications on the RUSH network of nuclear interactions.

Actin depolymerization contributes to ethanol inhibition of NMDA receptors in primary cultured cerebellar granule cells.

We have previously reported that a 30s ethanol (10 and 100mM) pre-exposure significantly enhanced EtOH inhibition of N-methyl-d-aspartate (NMDA-induced currents)-induced peak currents in primary cultured cerebellar granule cells (CGCs). The purpose of this study was to determine if intracellular factors play a role in ethanol pre-exposure-enhanced inhibition of NMDA-induced currents and if so, to identify the intracellular target(s) mediating this effect. Ethanol pre-exposure-enhanced inhibition was reduced when ethanol was present intracellularly prior to the initiation of the pretreatment protocol. Similar to results acquired with the whole-cell configuration, ethanol pre-exposure-enhanced inhibition of NMDA-induced currents was also observed in the perforated patch-clamp mode. Collectively, these results suggest an intracellular target not easily dialyzed from the cell. Perturbation of the actin cytoskeleton was responsible for the ethanol pre-exposure-enhanced inhibition of NMDA-induced currents was supported by the observation that the intracellular presence of the actin stabilizer phalloidin prevented ethanol pre-exposure-enhanced inhibition. Similar to the effects of ethanol, the depolymerizing agent latrunculin A inhibited NMDA-induced currents after a 30s pretreatment exposure with full recovery of receptor function after washout of the drug. Furthermore, latrunculin A occluded the enhanced inhibition of NMDA-induced currents by ethanol pre-exposure for both 10 and 100mM ethanol. The microtubule depolymerizing agent taxol had no affect on ethanol pretreatment-enhanced inhibition of NMDA-induced currents. Confocal microscopy with phalloidin-FITC indicated that F-actin filaments in neurites were depolymerized after a 30s treatment of either latrunculin A or 100mM ethanol. Our observations indicate that ethanol inhibition of NMDAR function may involve perturbation of the actin cytoskeleton.

Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells.

BACKGROUND: Glial cells are critical in the functioning of the central nervous system (CNS), including responsiveness to injury and immunocompetence. The immune and inflammatory response involves the inducible form of nitric-oxide synthase (iNOS), and subsequent nitric oxide (NO) production. Previously, we have demonstrated that ethanol inhibits cytokine-induced iNOS expression and activity in rat glial cells. Evidence of ethanol-induced effects on iNOS in human glial cells is nonexistent. Herein, the conditions necessary for significant iNOS induction in human A172 astrocytoma cells have been characterized, and subsequently, the effects of ethanol on iNOS expression have been investigated. METHODS: A172 cells were analyzed immunohistochemically for the astrocyte markers, glial fibrillary acidic protein (GFAP) and S-100beta. The ability of A172 cells to express iNOS was assessed by stimulating cells with interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), bacterial lipopolysaccharide (LPS), L-arginine, and tetrahydrobiopterin (BH4) in various combinations. Following stimulation, iNOS induction was monitored via measurement of nitrite production and in vitro iNOS enzyme activity. Time-course (6-24 hr) studies assessed the effects of ethanol (50-200 mM) on iNOS induction. RESULTS: Immunohistochemistry analysis confirmed that A172 cells were phenotypically, astrocytic. Induction of nitrite production by a cytomix [IFNgamma (100 ng/ml) + TNFalpha (30 ng/ml) + IL-1beta (5 ng/ml)] was differentially enhanced by exposure to supplemental factors including LPS, L-arginine, and BH4. Nitrite production was greatest over the initial 24 hr of stimulation with iNOS enzyme activity peaking at 12 hr. Acute (6-24 hr) exposure of activated cells to 50 mM ethanol enhanced iNOS activity recovered from the cytosol, whereas 200 mM ethanol decreased it. Ethanol had no direct effect on the catalytic activity of the enzyme. CONCLUSIONS: The present study is the first published report of ethanol-induced modulation of iNOS expression in human glial cells. The data suggest that ethanol is influencing iNOS enzyme levels most profoundly. Altered astrocyte function may be a point of ethanol-induced perturbation in CNS immune function. These findings should lend insight into the role of ethanol on human CNS immunity and brain injury.