Search

Introduction: Large-sample epidemiological studies of tobacco cigarette smoking routinely assess so-called "lifetime prevalence" of tobacco dependence. This work delves into the earliest stages of smoking involvement, focusing on newly incident tobacco cigarette smokers in the very recent past, and examines hypothesized subgroup variation in count processes that become engaged once smoking starts. Here, the term "count process" has two components: (a) whether smoking will be persistent and (b) the rate of smoking, conditional upon membership in a latent class of smokers who will persist, as estimated under the zero-inflated Poisson (ZIP) model for complex survey data., Methods: We estimate these ZIP parameters for nationally representative samples of newly incident smokers in the United States (all with smoking initiation within 24 months of assessment). Data are from the 2004-2007 National Surveys on Drug Use and Health., Results: Once cigarette smoking started, roughly 40%-45% persisted, and the estimated median rate was five smoking days/30 days, conditional on membership in the latent class of persistent smokers. Among non-Hispanic recent-onset cigarette smokers, Whites, Black/African Americans, Asians, and Native American/Alaskan Natives did not differ, but recent-onset smokers of Hispanic origin and those of Pacific Islander background had comparatively less cigarette involvement., Discussion: Tobacco prevention and control initiatives may require elaboration in the form of brief interventions, including interpersonal and social transactions that might constrain a mounting frequency of days of smoking before daily smoking starts, and until conventional smoking cessation medication aids become indicated. These very-early stage interventions (VESI) might be mounted within family or peer groups or in the primary care or school settings, but randomized trials to evaluate VESI interventions will be required.

Few case series studies have addressed the issue of treatment response in patients with obsessive-compulsive disorder (OCD) and comorbid post-traumatic stress disorder (PTSD), and there are no prospective studies addressing response to conventional treatment in OCD patients with a history of trauma (HT). The present study aimed to investigate, prospectively, the impact of HT or PTSD on two systematic, first-line treatments for OCD. Two hundred and nineteen non-treatment-resistant OCD outpatients were treated with either group cognitive-behavioral therapy (GCBT n = 147) or monotherapy with a selective serotonin reuptake inhibitor (SSRI n = 72). Presence of HT and PTSD were assessed at intake, as part of a broader clinical and demographical baseline characterization of the sample. Severity and types of OCD symptoms were assessed with the Yale-Brown Obsessive-Compulsive Scale (YBOCS) and the Dimensional YBOCS (DYBOCS), respectively. Depression and anxiety symptoms were measured with the Beck Depression Inventory (BDI) and the Beck Anxiety Inventory (BAI). Both treatments had 12-week duration. Treatment response was considered as a categorical [35% or greater reduction in baseline YBOCS scores plus a Clinical Global Impression-Improvement rating of better (2) or much better (1)] and continuous variable (absolute number reduction in baseline YBOCS scores). Treatment response was compared between the OCD + HT group versus the OCD without HT group and between the OCD + PTSD group versus the OCD without PTSD group. Parametric and non-parametric tests were used when indicated. Data on HT and PTSD were available for 215 subjects. Thirty-eight subjects (17.67% of the whole sample) had a positive HT (OCD + HT group) and 22 subjects (57.89% of the OCD + HT group and 10.23% of the whole sample) met full DSM-IV criteria for PTSD. The OCD + HT and OCD without HT groups presented similar response to GCBT (60% of responders in the first group and 63% of responders in the second group, p = 1.00). Regarding SSRI treatment, the difference between the response of the OCD + HT (47.4%) and OCD without HT (22.2%) groups was marginally significant (p = 0.07). In addition, the OCD + PTSD group presented a greater treatment response than the OCD without PTSD group when treatment response was considered as a continuous variable (p = 0.01). The age when the first trauma occurred had no impact on treatment response. In terms of specific OCD symptom dimensions, as measured by the DYBOCS, OCD treatment fostered greater reductions for the OCD + PTSD group than for the OCD without PTSD group in the scores of contamination obsessions and cleaning compulsions, collecting and hoarding and miscellaneous obsessions and related compulsions (including illness concerns and mental rituals, among others). The OCD + PTSD group also presented a greater reduction in anxiety scores than the OCD without PTSD group (p = 0.003). The presence of HT or PTSD was not related to a poorer treatment response in this sample of non-treatment-resistant OCD patients. Unexpectedly, OCD patients with PTSD presented a greater magnitude of response when compared with OCD without PTSD patients in specific OCD symptom dimensions. Future studies are needed to clarify if trauma and PTSD have a more significant impact on the onset and clinical expression of OCD than on the conventional treatment for this condition, and whether OCD stemming from trauma would constitute a subtype of OCD with a distinct response to conventional treatment.

The in vivo progenitor of culture-expanded mesenchymal-like adipose-derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro. Immunohistostaining of intact adipose tissue was used to identify three markers (CD31, CD34, and CD146), which together unambiguously discriminate histologically distinct inner and outer rings of vessel-associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter flow cytometry in conjunction with stem/progenitor markers (CD90 and CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two endothelial populations were isolated by high speed flow cytometric sorting, expanded in short term culture, and tested for adipogenesis. The inner layer of stromal cells in contact with small vessel endothelium (pericytes) was CD146+/alpha-SMA+/CD90+/-/CD34-/CD31-; the outer adventitial stromal ring (designated supra adventitial-adipose stromal cells, SA-ASC) was CD146-/alpha-SMA-/CD90+/CD34+/CD31-. Capillary endothelial cells were CD31+/CD34+/CD90+ (endothelial progenitor), whereas small vessel endothelium was CD31+/CD34-/CD90- (endothelial mature). Flow cytometry confirmed these expression patterns and revealed a CD146+/CD90+/CD34+/CD31- pericyte subset that may be transitional between pericytes and SA-ASC. Pericytes had the most potent adipogenic potential, followed by the more numerous SA-ASC. Endothelial populations had significantly reduced adipogenic potential compared with unsorted expanded SVF cells. In adipose tissue, perivascular stromal cells are organized in two discrete layers, the innermost consisting of CD146+/CD34- pericytes, and the outermost of CD146-/CD34+ SA-ASC, both of which have adipogenic potential in culture. A CD146+/CD34+ subset detected by flow cytometry at low frequency suggests a population transitional between pericytes and SA-ASC.

Background: Neuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury., Results: After unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states., Conclusion: Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.

Multiple drug resistance, mediated by the expression and activity of ABC-transporters, is a major obstacle to antineoplastic therapy. Normal tissue stem cells and their malignant counterparts share MDR transporter activity as a major mechanism of self-protection. Although MDR activity is upregulated in response to substrate chemotherapeutic agents, it is also constitutively expressed on both normal tissue stem cells and a subset of tumor cells prior to the initiation of therapy, representing a built-in obstacle to therapeutic ratio. Constitutive and induced MDR activity can be detected in cellular subsets of disaggregated tissues, using the fluorescent substrates Rhodamine 123 and Hoechst 33342 for ABCB1 (also known as P-gp and MDR1) and ABCG2 (BCRP1). In this chapter, we will describe the complete procedure for the detection of MDR activity, including: (1) Preparing single-cell suspensions from tumor and normal tissue specimens; (2) An efficient method to perform cell surface marker staining on large numbers of cells; (3) Flow cytometer setup and controls; (4) Simultaneous measurement of Hoechst 33342 and Rhodamine123 transport; and (5) Data acquisition and analysis.

Sporothrix, one of the anamorph genera of Ophiostoma, includes the important human pathogen S. schenckii and various fungi associated with insects and sap stain of wood. A survey of fungi from wood utility poles in South Africa yielded two distinct groups of Sporothrix isolates from different geographical areas. DNA sequence and morphological data derived in this study showed that isolates in these groups represent two novel species in the S. schenckii-O. stenoceras species complex. A new species isolated from pine poles and rosebush wood and phylogenetically closely related to S. pallida is described here as Sporothrix stylites. Phylogenetic analyses also confirmed the synonymy of S. albicans and S. nivea with S. pallida. Sporothrix stylites and S. pallida also are related closely to the isolates from soil, previously treated as "environmental" isolates of S. schenckii. Soil isolates are clearly distinct from human isolates of S. schenckii. We describe the former here as Sporothrix humicola. The isolates from eucalypt poles group peripheral to most other species in the S. schenckii-O. stenoceras complex and are newly described as Sporothrix lignivora. Phylogenetic analyses of sequences of isolates from soil and wood together with those of clinical isolates showed that the human-pathogenic strains form an aggregate of several cryptic species.

One approach to treatment of negative cognitive effects associated with Alzheimer's disease and schizophrenia may involve activation of neuronal alpha7 nicotinic acetylcholine receptors (nAChRs). We used the alpha7-selective partial agonist 3-(4-hydroxy, 2-methoxy-benzylidene)anabaseine (4OH-GTS-21), the alpha7 modulator 5-hydroxyindole (5-HI), and recombinant adeno-associated virus (rAAV)-mediated alpha7 gene transfer in order to test the hypothesis whether combining these strategies would significantly increase indirect measures of alpha7 nAChR function, including measures of spontaneous synaptic events in CA1 pyramidal cells. 5-HI (1 mM), and 5-HI (1 mM)+4OH-GTS-21 (5 microM) increased the frequency of APV- and NBQX-sensitive currents, while 5-HI+4OH-GTS-21 increased the frequency and amplitude of bicuculline-sensitive currents. Effects on EPSCs were blocked with tetrodotoxin (TTX) (1 microM), but not by methyllycaconitine (MLA) (50 nM). Neither TTX nor MLA reduced the potentiation of IPSC frequencies. However, TTX blocked, and in some cases MLA reduced, the potentiation of IPSC amplitudes. These data suggest that effects of 5-HI+4OH-GTS-21 on EPSC frequency were associated with action potential-dependent transmitter release produced by 5HI, and that potentiation of IPSC amplitudes resulted at least in part, from activation of alpha7 nAChRs. Finally, rAAV-mediated alpha7 gene transfer did not alter the magnitude of effects produced by 5-HI or 5-HI+4OH-GTS-21. Thus, although we previously showed that direct measures of alpha7 nAChR function were enhanced by alpha7 gene transfer, indirect measures of alpha7 nAChRs function were not significantly enhanced by combining alpha7 gene transfer with either agonist activation or positive allosteric modulation of alpha7 nAChRs.

Human immunodeficiency virus type 1 (HIV-1) causes neuronal degeneration and, at a late stage, creates HIV-associated dementia (HAD) and other neurological abnormalities. Therefore, the need for neuroprotective agents is great. However, therapeutic agents that reduce HIV neurotoxicity are difficult to characterize and develop because rodents are not infected by HIV. This study was undertaken to develop an animal model of HIV neurotoxicity by using the HIV-1 envelope glycoprotein 120 (gp120). Vehicle or gp120 was injected acutely in the striatum of adult rats. gp120 produced loss of nigrostriatal neurons, as shown both by histochemical analysis of brain sections for apoptosis and biochemical determination of dopamine. The neurotrophin brain-derived neurotrophic factor (BDNF) delivered by a recombinant adeno-associated viral vector prevented gp120 toxicity. This study's results support the notion that gp120 produces a widespread neurotoxicity similar to that observed in HIV-positive individuals and that BDNF may be a suitable neuroprotective agent for HAD.

The accumulation and deposition of the 40-42-amino acid peptide amyloid beta (Abeta) is thought to be a critical event in the pathology of Alzheimer's disease (AD). Both passive and active immunizations against Abeta in amyloid-depositing transgenic mice have reduced Abeta pathology and improved memory-related behavior. Peripheral treatments with other amyloid-binding agents have also reduced Abeta pathology. The present study demonstrates that peripheral delivery of plasmid DNA coding for the amyloid-binding protein plasma gelsolin reduces brain Abeta in two separate amyloid-depositing transgenic mouse models of AD when inter-litter variability is accounted for. The reduction in Abeta pathology observed is accompanied by an apparent increase in activated and reactive microglia and soluble oligomeric forms of amyloid. These findings demonstrate that peripheral expression of plasma gelsolin may be a suitable gene-therapeutic approach for the prevention or treatment of AD.

While activation of alpha7 nicotinic receptors protects neurons from a variety of apoptotic insults in vitro, little is known about this neuroprotective action in vivo, especially under amyloidogenic conditions that mimic Alzheimer's disease. We therefore investigated the effects of 4OH-GTS-21, a selective partial agonist for these receptors, on septohippocampal cholinergic and GABAergic neuron survival following fimbria fornix (FFX) lesions in three strains of mice: C57BL/6J wild type mice; human presenilin-1 mutant M146L (PS1) transgenic mice; and mice expressing both mutant PS1 and Swedish mutant K670N/M671L amyloid precursor protein (APP). Initial studies to demonstrated that 4OH-GTS-21 is likely brain permeant based on its ability to improve passive avoidance and Morris water task behaviors in nucleus basalis-lesioned rats. In FFX-lesioned mice, twice per day i.p. injections of 1 mg/kg of 4OH-GTS-21 for 2 weeks promoted the survival and prevented the atrophy of septal cholinergic neurons. Septal parvalbumin-staining GABAergic neurons were not protected by this treatment, although they also express alpha7 nicotinic receptors, suggesting an indirect, nerve growth factor (NGF)-mediated mechanism. No protection of cholinergic neurons was observed in similarly treated PS1 or APP/PS1 transgenic mice. 4OH-GTS-21 treatment actually reduced cholinergic neuronal size in APP/PS1 mice. Hippocampal amyloid deposition was not affected by FFX lesions or treatment with this alpha7 nicotinic receptor agonist in APP/PS1 mice under these conditions. These results indicate that brain alpha7 nicotinic receptors are potential targets for protecting at-risk brain neurons in Alzheimer's disease, perhaps via their effects on NGF receptors; however, this protection may be sensitive under some conditions to environmental factors such as inhibitory amyloid-peptides.

Glycoprotein 120 (gp120) from the T-tropic strain of the human immunodeficiency virus type 1 has been shown to cause neuronal apoptosis through activation of the chemokine receptor CXCR4. Therefore, reducing CXCR4 expression may prevent gp120-mediated apoptosis. Brain-derived neurotrophic factor (BDNF) is known to reduce both gp120 neurotoxicity and CXCR4 expression in vitro. The scope of this work is to establish whether BDNF is neuroprotective against gp120 in vivo and, if so, whether this effect correlates with its ability to down-regulate CXCR4. Serotype 2 adeno-associated viral vector encoding for BDNF (rAAV-BDNF) or control vector was microinjected into the striata of adult rats. Two weeks later gp120 was injected into the same striatum, and apoptosis determined. Pretreatment with rAAV-BDNF prior to gp120 microinjection prevented caspase-3 activation as well as in situ terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling in the striatum and substantia nigra. In addition, rAAV-BDNF reversed the loss of tyrosine hydroxylase immunoreactivity induced by gp120 in both areas. CXCR4 expression was then determined by immunohistochemistry and RT-PCR, and found to be decreased in striata of rAAV-BDNF-treated rats. Conversely, BDNF heterozygous mice exhibited an increase in CXCR4 mRNA levels compared to wild-type littermates. Our data suggest that down-regulation of CXCR4 expression may contribute to the neuroprotective activity of BDNF against gp120 toxicity in the basal ganglia.

Brain alpha7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal alpha7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)-derived vectors. In order to determine whether alpha7 receptor-mediated cytotoxicity was dependent on receptor density, rat alpha7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [3H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an alpha7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat alpha7 nicotinic receptors increased [3H]MLA binding in this region in wild type and alpha7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no alpha7 receptor response did so following alpha7 receptor gene delivery in wild type mice. Recovery of alpha7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat alpha7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether alpha7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of alpha7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

For well over a decade, researchers in Porto Alegre, Brazil, have been documenting the extent of the AIDS epidemic in the region, with a specific focus on the linkages between drug use and HIV seropositivity. Virtually all of the studies conducted during those years found injection drug use (IDU) to be the major vector for HIV seropositivity in this population. However, recent research found that the number of IDUs had declined significantly. Qualitative interviews and focus groups suggested many reasons for this decline: (1) many had died, because they had never heard of AIDS or HIV, and were unaware of how HIV is transmitted. As a result, they had become infected through the sharing of injection paraphernalia. (2) The quality of street cocaine had declined, making injection difficult. (3) Because of a fear of AIDS, some shifted to the smoking of crack, which had become a newly availability commodity in the street culture. Within this context, this article describes the qualitative data describing the decline of cocaine injecting and the corresponding emergence of crack use in Porto Alegre, Brazil, and related HIV risks.

The gene encoding for the Swedish double mutation (K595N/M596L) of amyloid precursor protein (APP695Swe) was expressed bilaterally in adult rat hippocampus to determine its long-term effects on memory-related behavior as well as amyloid deposition. Recombinant adeno-associated viral serotype 2 (rAAV2) vectors were injected that contained either non-expressing DNA or cDNA encoding for APP695Swe under control of a chicken beta actin/cytomegalovirus promoter/enhancer. Immunolabeling human APP with the antibody 6E10 was observed throughout the cytoplasm of aspiny and, to a lesser extent, spine-bearing hippocampal neurons 6 and 12 months post-injection of the APP695Swe but not control vector. Abeta1-42 immunolabeling was identified in unusual immunoreactive objects within the hilus of the dentate gyrus and in the granule cell layer, proximal to the injection site. At 12 months post-transduction, rats that received the APP695Swe gene also demonstrated significant deficits in the acquisition and probe components of the spatial-memory-related Morris water task compared to control animals. These behavioral deficits occurred in the absence of any amyloid plaques, gliosis, or FluoroJade labeling of dying neurons. In conclusion, prolonged and localized APP695Swe expression in hippocampal neurons is sufficient to produce memory deficits without plaque formation or neuronal loss.

The medial septum-diagonal band (MS/DB) contains primarily cholinergic and GABAergic neurons that project to the hippocampus, and are important for learning and memory. Whole-cell patch clamp methods with brain slices from p11--p20 rats were used to measure MS/DB cell responses to focal somatic application of 1mM acetylcholine (ACh) and a series of current pulses was applied in order to assess firing frequencies and the presence of hyperpolarization-activated currents (Ih). We identified three types of cells: (1) cells with fast inward currents blocked by methyllycaconitine (MLA) with slow firing rates (3--12 Hz), accommodating action potentials, and no Ih (n=20); (2) cells with currents that had both fast (MLA-sensitive) and slow components that were blocked with mecamylamine (MEC) that showed fast firing (up to 60 Hz) and slow firing (up to 3 Hz), with accommodating and non-accommodating action potentials (n=46), 33% of which had Ih; and (3) cells not responsive to ACh with moderate firing rates (10--42 Hz), some with accommodating action potentials and some without (n=19), of which 92% had Ih. These results are among the first to demonstrate functional nicotinic receptors in the MS/DB. The data suggest that these receptors include alpha 7 and non-alpha 7 subtypes and that the expression of each is correlated with firing frequency and the presence of Ih. Responses to ACh were not affected by tetrodotoxin (TTX) and CdC l(2) but were blocked by MLA or MLA and MEC, suggesting that these currents involve direct activation of nicotinic receptors.

The alpha7-type nicotinic acetylcholine receptor (nAChR) has been recognized as a potential therapeutic target for the treatment of a variety of pathologic conditions, including schizophrenia, Alzheimer's disease, and peripheral inflammation. A unique feature of alpha7 nAChRs that tends to complicate functional assays intended to identify selective drugs for these receptors is the strong concentration-dependent desensitization of their agonist-evoked responses. At low agonist concentrations, voltage-clamp responses are small but tend to closely follow the solution exchange profile, whereas higher agonist concentrations produce responses that peak and then decay very rapidly, usually before the full drug concentration has been achieved. In this article, we report that an alpha7 T245S mutant, which has a point mutation at the sixth position in the alpha7 second transmembrane domain (T6'S), demonstrates a significant gain of function, sustaining current when exposed to relatively high agonist concentrations when expressed in Xenopus laevis oocytes and larger peak currents when expressed in mammalian GH4C1 cells. At the single-channel level, the T6'S mutant has a unitary conductance of 61.7 +/- 5.8 pS, similar to that reported for wild-type alpha7, but a vastly longer average open duration. In addition, channel burst activity indicates a greater than 40% probability of channel re-opening in the sustained presence of 30 muM acetylcholine, consistent with a greater overall open probability relative to wild-type alpha7. Unlike the alpha7 L248T gain-of-function mutant, the T6'S mutant exhibits a pharmacological profile that is remarkably similar to the wild-type alpha7 receptor, implicating it as a potentially useful tool for identifying therapeutic agents.

Nerve growth factor (NGF) therapy has been proposed to treat cognitive impairments in aged patients including those with Alzheimer's disease. Various viral vectors, including adeno-associated virus serotype 2 (AAV2), have been investigated for their ability to deliver NGF in brain. In this study, hybrid vectors (AAV2/5) consisting of the genome of recombinant AAV2 and the capsid of AAV serotype 5 were evaluated for their ability to deliver NGF and green fluorescent protein (GFP) genes into brain. Compared to AAV2, AAV2/5 consistently led to more septal neurons being transduced with GFP over a wider range of distribution. However, both types of vector provided similar levels of long-term (17 weeks) protection of septal cholinergic neurons from axotomy and led to similar levels of NGF accumulation in this region. These results demonstrate that rAAV-mediated NGF gene delivery is neuroprotective for an extended period of time, but that factors other than transduction efficiency appear to determine transgenic NGF expression in septum.

The hippocampus receives substantial input from the medial septum/diagonal band of broca (MS/DB) via the fibria-fornix (FF). Projections from the MS/DB innervate hippocampal interneurons that express alpha7 nicotinic receptors and regulate excitation in principal cell populations. In the present report we used stereotaxic surgery, whole-cell patch clamping, and immunohistochemical techniques to evaluate the effects of FF and MS/DB lesions on alpha7 nicotinic receptors in stratum radiatum interneurons. Focal somatic application of ACh (1 mM) evoked methyllycaconitine (MLA)-sensitive currents that were markedly reduced following aspirative lesions of the FF. Reductions in current amplitudes were prevented or restored to levels not significantly different from controls following in vivo treatment with the alpha7-selective agonist GTS-21, and GTS-21 treatment did not change current amplitudes measured in tissue from unlesioned animals. MS/DB injections of the selective cholinergic neurotoxin 192 IgG-saporin did not affect alpha7 receptor currents, although MS/DB ChAT and hippocampal AChE immunolabeling were significantly reduced. In contrast, kainic acid lesions of the MS/DB, potentially more selective for GABAergic projection neurons, produced significant reductions in current amplitudes. These findings are the first to show functional changes in alpha7 receptors following hippocampal denervation and suggest that MS/DB hippocampal innervation regulates functional aspects of hippocampal alpha7 receptors. The results confirm hippocampal alpha7 nicotinic receptors as viable therapeutic targets in diseases that involve degradation of the septohippocampal pathway and may indicate that GABAergic MS/DB hippocampal input plays a more substantial role in the regulation of alpha7 nicotinic receptor function than MS/DB hippocampal cholinergic input.

alpha7 Nicotinic receptors are calcium permeant and provide neuroprotection against many insults. We investigated the roles of intracellular calcium ions and downstream calcium channels in this protection. The alpha7 agonist GTS-21 prevented pheochromocytoma cell death induced by nerve growth factor + serum deprivation over a 3-day interval. This effect was blocked by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in a manner that did not appear to involve changes in receptor density. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked GTS-21-induced protein kinase C activation, a necessary process for protection. The insositol triphosphate calcium-channel blocker xestospongin C and the phospholipases C inhibitor U-73122 blocked protection, ryanodine partially attenuated protection, but the L-type channel antagonist nifedipine had no effect. ERK1/2 but not JNK and p38 were activated by GTS-21, and the ERK phosphorylation inhibitors PD98059 and U0126 blocked protection.

This study characterized the presynaptic dopaminergic properties of neuronally differentiated mouse embryonic stem (ES) cells. Approximately 30% of the ES cells expressed tyrosine hydroxylase (TH) immunoreactivity when co-cultured with PA6 cells. These cultures expressed high affinity, sodium-dependent dopamine uptake as well as depolarization-induced and calcium-dependent dopamine release of this transmitter. These and other important dopaminergic genes found expressed in these cultures by RT-PCR included Nurr1, vesicular monoamine transporter 2 (VMAT2), TH, dopamine transporter (DAT), and glial cell line-derived neurotrophic factor (GDNF) receptors c-Ret and GFRalpha1. These results demonstrate that differentiated ES cells have the presynaptic functions for maintaining dopaminergic homeostasis, which may be essential for their long-term use in restoring CNS levels of this transmitter.

Purpose: Brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) hold much promise for the protection of retinal ganglion cells against excitotoxic cell death. We tested the possibility of delivering these growth factors to retinal ganglion cells via an adeno-associated viral (AAV) vector and tested their efficacy in two models of excitotoxicity., Methods: Rat retinas were infected with AAV vectors encoding bFGF or BDNF. A control vector containing green fluorescent protein (GFP) was injected in the contralateral eye. Eyes were subjected to either an intravitreal injection of N-methyl-D-aspartate (NMDA) or optic nerve crush, and ganglion cell survival was evaluated., Results: AAV.CMV.bFGF and AAV.CBA.BDNF were neuroprotective against NMDA injection 1 month post-treatment. Additionally, AAV.CMV.bFGF was protective against optic nerve crush., Conclusion: AAV-mediated delivery of bFGF and BDNF can promote retinal cell survival following excitotoxic insult.

Recombinant adeno-associated virus-2 (rAAV2) under control of the chicken beta actin promoter/truncated CMV enhancer (CBA) was investigated for its ability to transduce primary cultures of rat brain neurons, microglia and astrocytes. This vector was highly effective in all three cell types in heparin-sensitive manners (astrocytes, microglia and neurons transduced by >98%, 75%, and 95%, respectively). However, astrocytes co-cultured with neurons were not transduced. rAAV2/CBA is an important new method for genetic manipulation of brain cells, though this may be modulated by interactions among cell types.

Background: Myoclonus-dystonia (M-D) is a movement disorder with autosomal dominant inheritance and reduced penetrance but may also occur sporadically. Recently, mutations in the epsilon-sarcoglycan gene (SGCE) were shown to cause M-D. Furthermore, single variants in the dopamine D2 receptor (DRD2) and DYT1 genes were found in combination with SGCE mutations in two M-D families, and another M-D locus was recently mapped to chromosome 18p11 in one family., Methods: The authors clinically and genetically characterised ten consecutive cases with myoclonus-dystonia; seven familial and three sporadic. Twenty nine M-D patients and 40 unaffected family members underwent a standardised clinical examination by a movement disorder specialist. Index cases were screened for mutations in the SGCE, DYT1, and DRD2 genes and for deletions of the SGCE gene. Suitable mutation negative families were tested for linkage to the SGCE region and to chromosome 18p11., Results: Two SGCE mutations were detected among the seven familial but no mutation in the sporadic cases. Haplotype analysis at the new M-D locus was compatible with linkage in two families and excluded in another family, suggesting at least one additional M-D gene. There were no obvious clinical differences between M-D families with and without detected mutations., Conclusion: M-D is genetically heterogeneous with SGCE mutations accounting for the disease in only part of the clinically typical cases.

Although the muscle-type and homomeric alpha7-type nicotinic acetylcholine receptors (nAChRs) share many structural features and bind alpha-bungarotoxin with high affinity, several important functional and pharmacological properties distinguish these two major nAChR subtypes. We have shown previously that amino acid sequence in the second transmembrane (TM) domain of the beta subunit is critical for pharmacological distinction between muscle type and heteromeric neuronal (e.g., ganglionic) nAChRs. We tested the hypothesis that homologous substitution of amino acid sequence from the muscle beta1 subunit into the alpha7 subunit would confer specific properties of muscle-type receptors to mutant alpha7 nAChRs. In this study, we show that a single amino acid substitution at the alpha7 TM2 6' position makes both biophysical and pharmacological properties of the mutant receptors resemble those of wild-type muscle nAChR. This mutation produces significant changes in acetylcholine potency and response kinetics, eliminating the characteristic fast desensitization of alpha7 and dramatically reducing divalent ion permeability relative to wild-type alpha7. The TM2 T6'F mutation also produces a profound increase in activation by succinylcholine compared with either wild-type alpha7 or neuronal beta-subunit-containing receptors and the loss of potentiation by 5-hydroxyindole. Thus, the alpha7 TM2 T6'F mutant displays several features that are similar to the muscle nAChR, some of which are not typically thought to be regulated by the pore-lining domain of the receptor.

Anabaseine is a marine worm toxin that is a relatively non-selective nicotinic agonist, activating both muscle-type and neuronal nicotinic acetylcholine receptors (nAChR) with varying efficacy. While anabaseine has significant activity with muscle-type and neuronal alpha 3 beta 4 and alpha 4 beta 2 receptors, benzylidene anabaseine (BA) derivatives have high selectivity for the alpha 7 receptor subtype. Two BA compounds with substituents at the 2 and 4 positions of the benzylidene ring, GTS-21 and 4OH-GTS-21, may have therapeutic potential for treating neuropathological disorders such as Alzheimer's disease due to their alpha 7 selectivity. In this study, we specifically investigated the influence of the benzylidene attachment to anabaseine on alpha 7 nicotinic receptor selectivity, as well as the effects of specific substituents at the 4- position of the benzylidene moiety. We demonstrate that benzylidene-attachment alone is sufficient to confer alpha 7 selectivity to anabaseine. Increased potency and receptor binding affinity was obtained with a 4-hydroxyl substitution. Two other 4-substituted benzylidene anabaseines, 3-(4'-methylthiobenzylidene)anabaseine (4-MeS-BA) and 3-(4-trifluoromethylbenzylidene) anabaseine (4-CF(3)-BA), offered very little agonist activity for any nicotinic receptors and instead were antagonists for both alpha 7 and neuronal alpha 3 beta 4 and alpha 4 beta 2 receptors. Since the relative amounts of agonist and antagonist activities for specific BA compounds vary with the specific drug/receptor combinations, benzylidene anabaseines provide valuable tools for nAChR drug-receptor structure-function relationships.

Polyethylenimine (PEI) is an effective vehicle for in vivo gene delivery in many tissues including brain. PEI mediates transgene expression in brain neurons and glia. To investigate whether PEI-mediated nerve growth factor (NGF) gene transfer protected axotomized septal cholinergic neurons, we injected linear PEI (in vivo jetPEI, Qbiogene) complexed with a plasmid encoding for mouse NGF (PEI/pNGF-W) into the rat septum. PEI complexed with a plasmid encoding for green fluorescent protein (PEI/pGFP) was used as the control. PEI-mediated gene expression was predominantly neuronal. Fimbria-fornix transections (FFTs), conducted 1 day after rats were injected with control vector, resulted in a 70% loss of septal cholinergic neurons. In contrast, PEI/pNGF-W injection prior to FFTs attenuated the loss of septal cholinergic neurons. This is the first study, to our knowledge, that shows the neuroprotective effects induced by PEI-mediated trophic factor gene transfer in brain., (Copyright 2004 Elsevier B.V.)

Nerve growth factor (NGF) therapy has been proposed to treat patients with age-related cognitive deficits, including those with Alzheimer's disease. One promising approach to delivering this protein into brain involves viral vectors. However, little is known about the effects of aging on gene transfer in brain generally and in particular its effect on transgenic NGF expression. To examine the transgene expression and biological effects of NGF gene transfer in adult and aged rats, we delivered mouse NGF with C-terminal myc-tag, using a recombinant adeno-associated virus serotype 2 (rAAV2) vector, into the septum of 6- and 21-month-old Fischer 344/Brown Norway hybrid rats. Other animals received a control vector encoding green fluorescent protein. As expected, this strain of rat demonstrated very few age-related deficits in spatial memory-related behavior in the Morris water task either before gene transfer (6 vs 21 months) or afterward (up to 11 vs 26 months). We found that rAAV2 vectors drove transgene expression in aged rats up to 5 months, although the level of transgene expression was lower than that of adult animals. We also showed that NGF gene transfer into the septum of aged animals induced local trophic effects by increasing the number and soma area of septal cholinergic neurons and improved distal synaptic activity by increasing the level of depolarization-induced acetylcholine (ACh) release from hippocampal synaptic terminals. Interestingly, NGF gene transfer suppressed depolarization-induced ACh release in adult rats. These findings show for the first time, to our knowledge, that septal NGF gene transfer modulates hippocampal nerve terminal function. These results are relevant for the potential clinical application of NGF gene therapy.

Neurofibrillary pathology was produced in the brains of adult rats after localized gene transfer of human tau carrying the P301L mutation, which is associated with frontotemporal dementia with parkinsonism. Within 1 month of in situ transfection of the basal forebrain region of normal rats, tau-immunoreactive and argyrophilic neuronal lesions formed. The fibrillar lesions had features of neurofibrillary tangles and tau immunoreactivity at light and electron microscopic levels. In addition to neurofibrillary tangles, other tau pathology, including pretangles and neuropil threads, was abundant and widespread. Tau gene transfer to the hippocampal region of amyloid-depositing transgenic mice produced pretangles and threads, as well as intensely tau-immunoreactive neurites in amyloid plaques. The ability to produce neurofibrillary pathology in adult rodents makes this a useful method to study tau-related neurodegeneration.

Although NGF gene therapy has been proposed to treat age- or disease-related brain cholinergic decline, little is known about the ectopic expression or function of this trophic factor after transduction in the brain especially over long intervals. The neuron-targeting, recombinant adeno-associated virus serotype 2 (rAAV2) vector was used to express mouse NGF with C-terminal myc-tag in septum using a full-length preproNGF sequence. While the predominant form of endogenous NGF immunoreactivity in septum was 31 kd of proNGF, almost all of the ectopic NGF-immunoreactivity attributable to the rAAV2-mediated transduction in this region was recovered as mature NGF. Transgene expression was found in both cholinergic and GABAergic neurons, with the number of transduced neurons dependent on vector dose. To determine the long-term effects of this NGF-expression on neuron function, fimbria-fornix (FF) lesions were conducted 6 months after NGF gene transfer. NGF gene transfer attenuated the lesion-induced loss of septal cholinergic but not GABAergic neurons, indicating that long-term expression did not eliminate this response, which has been noted over short intervals. The effects and dose dependency of NGF gene delivery on neuroprotection and neurotrophism were also examined. NGF transduction increased cholinergic cell size in the septum, but required a higher vector dose than neuroprotection. These results reveal potential long-term benefits as well as concerns for genetically modifying septal NGF gene expression to preserve neuronal viability and function.

A unique feature of alpha7 nicotinic acetylcholine receptor physiology is that, under normal physiological conditions, alpha7 receptors are constantly perfused with their natural selective agonist, choline. Studying neurons of hypothalamic tuberomammillary (TM) nucleus, we show that choline and the selective alpha7 receptor agonist 4OH-GTS-21 can regulate neuronal functions directly, via activation of the native alpha7 receptors, and indirectly, via desensitizing those receptors or transferring them into a state "primed" for desensitization. The direct action produces depolarization and thereby increases the TM neuron spontaneous firing (SF) rate. The regulation of the spontaneous firing rate is robust in a nonphysiological range of choline concentrations >200 microM. However, modest effects persist at concentrations of choline that are likely to be attained perineuronally under some conditions (20-100 microM). At high physiological concentration levels, the indirect choline action reduces or even eliminates the responsiveness of alpha7 receptors and their availability to other strong cholinergic inputs. Similarly to choline, 4OH-GTS-21 increases the TM neuron spontaneous firing rate via activation of alpha7 receptors, and this regulation is robust in the range of clinically relevant concentrations of 4OH-GTS-21. We conclude that factors that regulate choline accumulation in the brain and in experimental slices such as choline uptake, hydrolysis of ACh, membrane phosphatidylcholine catabolism, and solution perfusion rate influence alpha7 nAChR neuronal and synaptic functions, especially under pathological conditions such as stroke, seizures, Alzheimer's disease, and head trauma, when the choline concentration in the CSF is expected to rise.

Nerve growth factor (NGF) is known to promote both the survival of cholinergic neurons after injury and the regeneration of damaged cholinergic axons. Recent evidence has implicated NGF in the regulation of cholinergic axonal sprouting by intact neurons projecting to the hippocampus of rats, sustaining a lesion of the entorhinal cortex. We explored the possibility that NGF may regulate this lesion-induced cholinergic sprouting by injecting recombinant adeno-associated virus (rAAV) vector expressing NGF and green fluorescent protein (GFP) into the dentate gyrus of rats that were subsequently given unilateral entorhinal lesions. Sprague Dawley rats were unilaterally injected with (1) rAAV vector expressing NGF and GFP or (2) rAAV vector expressing GFP. Fourteen days after injection, the animals received lesions of the entorhinal area ipsilateral to the virus injection. Four days after lesion, GFP expression and the septodentate sprouting response in the dentate gyrus were assessed. Optical densitometric analyses revealed a significant increase in acetylcholinesterase label (a marker for cholinergic septodentate sprouting) in the ipsilateral outer molecular layer of the dentate gyrus in rats injected with rAAV vector expressing NGF. Thus, NGF-expressing rAAV vector enhanced the sprouting response of intact cholinergic neurons after unilateral entorhinal lesions in rats.

Assessing the efficacy of in vivo gene transfer often requires a quantitative determination of the number, size, shape, or histological visualization characteristics of biological objects. The optical fractionator has become a choice stereological method for estimating the number of objects, such as neurons, in a structure, such as a brain subregion. Digital image processing and analytic methods can increase detection sensitivity and quantify structural and/or spectral features located in histological specimens. We describe a hardware and software system that we have developed for conducting the optical fractionator process. A microscope equipped with a video camera and motorized stage and focus controls is interfaced with a desktop computer. The computer contains a combination live video/computer graphics adapter with a video frame grabber and controls the stage, focus, and video via a commercial imaging software package. Specialized macro programs have been constructed with this software to execute command sequences requisite to the optical fractionator method: defining regions of interest, positioning specimens in a systematic uniform random manner, and stepping through known volumes of tissue for interactive object identification (optical dissectors). The system affords the flexibility to work with count regions that exceed the microscope image field size at low magnifications and to adjust the parameters of the fractionator sampling to best match the demands of particular specimens and object types. Digital image processing can be used to facilitate object detection and identification, and objects that meet criteria for counting can be analyzed for a variety of morphometric and optical properties., (Copyright 2002 Elsevier Science (USA))

Demonstrating consistently reliable levels of expression is a critical part of any gene transfer study in order to assess variability and determine effective gene dosages. This article highlights some of the key methods for studying the expression levels of green fluorescent protein and neurotrophic factors after injections of adeno-associated virus (AAV) vectors into the brain. The data demonstrate greater spread and higher levels of expression using the cytomegalovirus/chicken beta-actin (CBA) promoter coupled with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), compared to earlier AAV serotype 2 vectors. Injections of either CBA-nerve growth factor (NGF)-WPRE or CBA-glial cell line-derived neutrotrophic factor-WPRE AAV vectors into the nucleus basalis of the basal forebrain led to clear and consistent elevation of the respective trophic factor as measured by enzyme-linked immunosorbent assay, with NGF vectors affecting the size and number of cholinergic neurons. AAV serotype may also be important for the spread of expression, since injecting an AAV-5 vector into the hippocampus led to higher-frequency transfection of dentate gyrus granule neurons, suggesting altered tropism relative to AAV-2., (Copyright 2002 Elsevier Science (USA))

Tuberomammillary histamine neurons (TM) of the posterior hypothalamus exclusively express alpha-bungarotoxin (alphaBgt) sensitive nicotinic receptors, providing a unique model system for studying physiological properties of native alpha7-like receptors. Here the properties of alphaBgt-sensitive receptors were investigated using the patch-clamp technique and rapid application of acetylcholine (ACh) or the alpha7-selective agonists, 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21), and choline. Alpha-Bgt-sensitive receptor responses to rapid application of high agonist concentrations were characterized by a transient current which rapidly decayed in a voltage-independent concentration-dependent manner to a relatively sustained slow current. Upon agonist removal, current persisted for several milliseconds (or longer) and increased above the level of the slow current (rebound). Lower agonist concentrations did not produce a rebound. Our analysis suggests that the current rebound represents a recovery phase from a low potency inhibition. This inhibition was voltage-dependent for ACh and choline but voltage-independent for 4OH-GTS-21. A slow form of desensitization was present which was relatively agonist-independent and was faster than the rate of 4OH-GTS-21 unbinding. Kinetic analysis revealed that the concentration dependence of the transient response amplitudes was compromised by solution exchange; net charge measurements over the late response phases were chosen as an alternative measure of concentration/response function. Our data suggest that low agonist concentrations can evoke a prolonged or tonic-like receptor activation. Functioning in this modality, receptors would regulate calcium homeostasis over a narrow, but therapeutically important, range of intracellular calcium concentrations. This could then provide the basis for cytoprotective effects of 4OH-GTS-21 and other nicotinic agonists, mediating trophic and neuromodulatory functions., (Copyright 2002 Elsevier Science B.V.)

Previous studies demonstrated that the rat neuron-specific enolase (NSE) promoter is effective for transgene expression in the brain in a variety of adeno-associated virus-2 vectors. This study evaluated the dose response and longer time course of this promoter and compared it to two cytomegalovirus/chicken beta-actin hybrid (CBA) promoter-based systems. NSE promoter-driven green fluorescent protein (GFP)-expressing neurons were found at doses as low as 10(7) particles, with expression increasing in a dose-dependent manner over a 3.3-log range. Bicistronic expression of GFP via an internal ribosome entry site coupled to the NSE promoter was also dose dependent, although the potency was decreased by 3.4-fold. The number of GFP-expressing neurons was stable for at least 25 months. The CBA promoter increased the numbers of GFP-expressing cells versus the NSE promoter, although the expression pattern remained neuronal and persisted for at least 18 months. The CBA promoter permitted detection of cells distal to the injection site that had retrogradely transported the vector from their terminal areas. Incorporating the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) into a CBA promoter vector induced greater expression levels in the hippocampus, as measured by stereological estimates of cell numbers and by Western blots, which demonstrated an 11-fold increase. Incorporation of the WPRE also improved transgene expression in primary neuronal cultures. The increased efficiency obtained with vector elements such as the CBA promoter and the WPRE may enhance the ability to genetically modify larger portions of the brain while requiring smaller doses and volumes.

Primary hippocampal neuronal cultures exhibited a concentration- and time-dependent loss of cells when exposed to ethanol (EtOH). EtOH-induced neurotoxicity was attenuated by 2,4-dimethoxybenzilidene anabaseine (DMXB) which selectively activates alpha7 nicotinic receptors in a concentration-dependent manner. We further investigated the mechanisms of the protective effect of DMXB on EtOH- induced neurotoxicity. We found that EtOH decreased the mitochondrial membrane potential and released cytochrome c from mitochondria at neurotoxic concentrations. DMXB (3 microm) attenuated both of these actions in a manner that was in turn blocked with the nicotinic antagonist methyllyconitine (MLA) 100 nm. Neither DMXB nor MLA alone affected these parameters. These results suggest that the neuroprotection conferred by alpha7 nicotinic receptor activation may be mediated, at least in part, through preventing the decrease in the mitochondrial membrane potential and the increase in the release of cytochrome c caused by EtOH.

Valproic acid (VPA) is a broad-spectrum anticonvulsant with well-documented teratogenic effects, but whose mechanism of action is largely unknown. In the present study we have examined the effects of VPA on the expression of two prominent substrates for protein kinase C (PKC) in the brain, MARCKS and GAP-43, which have been implicated in actin-membrane plasticity and neurite outgrowth during neuronal differentiation, respectively, and are essential to normal brain development. Immortalized hippocampal HN33 cells exposed to VPA exhibited reduced MARCKS protein expression and demonstrated increased GAP-43 protein expression, with concomitant alterations in cellular morphology, including an increase in the number and length of neurites and accompanied by a reduction in cell growth rate. The effects of VPA were observed at clinically relevant concentrations following chronic (>1 day) VPA exposure. We also present evidence for a VPA-induced alteration in PKC activity, as well as temporal changes in individual PKC isozyme expression. Inhibition of PKC with the PKC-selective inhibitor, LY333531, prevented the VPA-induced down-regulation of membrane-associated MARCKS, but had no effect on the cytosolic MARCKS reduction or the GAP-43 up-regulation. Inhibition of PKC by LY333531 enhanced the differentiating effects of VPA; additionally, LY333531 alone induced greater neurite outgrowth in this cell line. Collectively, these data indicate that VPA induces neuronal differentiation, associated with a reduction in MARCKS expression and an increase in GAP-43 expression, consistent with the hypothesis that a reduction in MARCKS at the membrane may be permissive for cytoskeletal plasticity during neurite outgrowth.

Recent studies have demonstrated that transplanted bone marrow cells can turn into unexpected lineages including myocytes, hepatocytes, neurons and many others. A potential problem, however, is that reports discussing such 'transdifferentiation' in vivo tend to conclude donor origin of transdifferentiated cells on the basis of the existence of donor-specific genes such as Y-chromosome markers. Here we demonstrate that mouse bone marrow cells can fuse spontaneously with embryonic stem cells in culture in vitro that contains interleukin-3. Moreover, spontaneously fused bone marrow cells can subsequently adopt the phenotype of the recipient cells, which, without detailed genetic analysis, might be interpreted as 'dedifferentiation' or transdifferentiation.

Mouse embryonic stem (ES) cells can proliferate indefinitely in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), or differentiate into all three germ layers upon removal of this factor. To determine cellular factors associated with self-renewal of undifferentiated ES cells, we used polymerase chain reaction-assisted cDNA subtraction to screen genes that are expressed in undifferentiated ES cells and down-regulated after incubating these cells in a differentiation medium without LIF for 48 h. The mRNA expression of a tetraspanin transmembrane protein, CD9, was high in undifferentiated ES cells and decreased shortly after cell differentiation. An immunohistochemical analysis confirmed that plasma membrane-associated CD9 was expressed in undifferentiated ES cells but low in the differentiated cells. Addition of LIF to differentiating ES cells reinduced mRNA expression of CD9, and CD9 expression was accompanied with a reappearance of undifferentiated ES cells. Furthermore, activation of STAT3 induced the expression of CD9, indicating the LIF/STAT3 pathway is critical for maintaining CD9 expression. Finally, addition of anti-CD9 antibody blocked ES cell colony formation and reduced cell viability. These results indicate that CD9 may play a role in LIF-mediated maintenance of undifferentiated ES cells.

Neurodegenerative diseases as a class do not have effective pharmacotherapies. This is due in part to a poor understanding of the pathologies of the disease processes, and the lack of effective medications. Gene delivery is an attractive possibility for treating these diseases. For the paradigm to be effective, efficient, safe and versatile vectors are required. In this study we evaluated three plasmid delivery systems for transgene expression in the rat hippocampus. Two of these systems were designed to have enhanced intracellular biodegradability. It was hypothesized that this system would be less toxic and could increase the free (non-vector) associated plasmids within the cell, leading to increased transgene activity. Polyethylenimine (PEI) and r-AAV-2 (recombinant adeno associated virus-2) were used as positive, non-viral and viral controls respectively, in the in vivo experiments. The results from the studies indicate there is a distinct difference between the various vectors in terms of total cells transfected, type of cell transfected, and toxicity. Non-viral systems were effective at transfecting both neurons and glia cells within the hippocampus, while the r-AAV-2 transfected mainly neurons. In summary, plasmid-mediated systems are effective for transgene expression within the brain and deserve further study.

Somatic cell gene transfer was used to express a mutant form of alpha-synuclein (alpha-syn) that is associated with Parkinson's disease (PD) in the rat substantia nigra (SN), a brain region that, in humans, degenerates during PD. DNA encoding the A30P mutant of human alpha-syn linked to familial PD was incorporated into an adeno-associated virus vector, which was injected into the adult rat midbrain. The cytomegalovirus/chicken beta-actin promoter was used to drive transgene expression. Over a 1-year time course, this treatment produced three significant features relevant to PD: (1) accumulation of alpha-syn in SN neuron perikarya, (2) Lewy-like dystrophic neurites in the SN and the striatum, and (3) a 53% loss of SN dopamine neurons. However, motor dysfunction was not found in either rotational or rotating rod testing. The lack of behavioral deficits, despite the significant cell loss, may reflect pathogenesis similar to that of PD, where greater than 50% losses occur before motor behavior is affected.

Interleukin-6 (IL-6) is a multifunctional cytokine that may have a role in energy regulation. Using a recombinant adeno-associated viral vector expressing murine interleukin-6 (rAAV-IL-6), we examined the chronic effects of centrally expressed IL-6 on food intake, body weight and adiposity in male Sprague-Dawley rats, and investigated the underlying mechanisms. Direct delivery of rAAV-IL-6 into rat hypothalamus suppressed weight gain and visceral adiposity without affecting food intake over a 5-week period. rAAV-IL-6 enhanced uncoupling protein 1 (UCP1) protein levels in interscapular brown adipose tissue (BAT). To investigate if the induction of UCP1 and the reduction in body weight are dependent on sympathetic innervation of BAT, we administered rAAV-IL-6 or a control vector into the hypothalamus of rats in which the interscapular BAT was unilaterally denervated. Over 21 days, there was no difference in food consumption or body weight between rAAV-IL-6- and control vector-treated rats. rAAV-IL-6 delivery increased UCP1 mRNA and protein levels in innervated BAT pads but not denervated BAT pads. Hypothalamic IL-6 signal transduction, indicated by phosphorylated signal transducer and activator of transcription 3 (P-STAT3) levels, was elevated by 2.6-fold at day 21, but returned to control levels by day 35. However, the suppressor of cytokine signaling-3 mRNA level was significantly elevated both at day 21 and day 35. These data demonstrate that chronic elevation of IL-6 in the CNS reduces body weight gain and visceral adiposity without affecting food intake. The mechanism involves sympathetic induction of UCP1 in BAT and, presumably, enhanced thermogenesis in BAT. Furthermore, chronic central IL-6 stimulation desensitizes IL-6 signal transduction characterized by reversal of elevated P-STAT3 levels.

The ability of a cationic lipid to deliver plasmid DNA (pDNA) in presence of the neurotoxic fragment of amyloid beta-peptide was evaluated. Pre-treatment of cells with betaAP (25-35) peptide resulted in a modest increase in transgene expression. When betaAP (25-35) peptide was mixed with the pDNA/liposome complex and used, the complexes lost their ability to transfect. However, the reverse sequenced betaAP (35-25) peptide demonstrated no significant differences in transgene expression in pre-treated cells, and in cells where betaAP (35-25) peptide was mixed with pDNA/liposome complexes and transfected. The amount of pDNA delivered to the cells was decreased in presence of betaAP (25-35) as measured with flow cytometry using fluorescently labeled liposomes. The decreased endocytosis may be due to their rod-like structure formation as demonstrated by electron microscopy and atomic force microscopy (AFM). These results demonstrate that betaAP (25-35) peptide may interfere with gene delivery with cationic systems.

Background: Major histocompatibility complex (MHC)-peptide tetrameric complexes (tetramers) are valuable tools for detecting and characterizing peptide-specific T cells. Because the frequency of these cells is generally very low, it may be difficult to discriminate between nonspecific and specific tetramer binding., Methods: A four-color flow cytometric assay that simultaneously measures tetramer, CD3, CD8, and CD14 was used to investigate the sensitivity and specificity of MHC class I tetramer staining. This was accomplished by using the influenza virus matrix protein peptide, GILGFVFTL (FLU), as a model recall antigen and the human immunodeficiency virus (HIV) reverse transcriptase peptide, ILKEPVHGV (HIV), as a model novel antigen. Peripheral blood mononuclear cells (PBMC) from 31 HLA-A2.1(+) and 10 HLA-A2.1(-) healthy individuals were stained with the tetramers., Results: The lower limit of detection was established at approximately 1/8,000. In HLA-A2(+) PMBC, frequencies of tetramer-positive CD8(+) T cells were log normally distributed and were high for FLU (1/910) but low for HIV (1/6,067). A novel competition assay, in which tetramer binding was shown to diminish subsequent staining with anti-CD3 antibody, was used to confirm the specificity of tetramer binding to the T-cell receptor (TCR) complex. The competition assay was validated by evaluating several anti-CD3 antibodies and showing that in PBMC from HLA-A2(-) subjects, spurious tetramer-positive events (1/20,000) failed to compete with CD3 binding. For the "recall" FLU tetramer, the degree of competition was proportional to the frequency, suggesting a selection of high avidity cells. Although CD3 competition was also highly correlated with the intensity of tetramer staining, competition allowed the identification of false positive cases with relatively high tetramer staining intensity., Conclusion: The data indicate that competition of CD3 binding allows confirmation of the specificity of tetramer binding to the TCR, extending the usefulness of tetramers in the frequency analysis of peptide-specific T lymphocytes., (Copyright 2000 Wiley-Liss, Inc.)

Nicotine treatment increases intracellular free Ca(2+) concentration [Ca(2+)](i), stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists alpha-bungarotoxin (alphaBTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca(2+)](i) and prevented the second prolonged elevation of [Ca(2+)](i), suggesting the involvement of alpha7 nAChRs. Two specific alpha7 nicotinic agonists, 3-(2,4-dimethoxybenzilidene)anabaseine (DMXB) and E, E-3-(cinnamylidene)anabaseine (3-CA), were found to elicit a small, delayed increase in [Ca(2+)](i) with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3-CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two- to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca(2+)](i) for up to 6 h. The increase in [Ca(2+)](i) or the elevation in TH mRNA by 3-CA was completely inhibited by alphaBTX. This study, for the first time, implicates stimulation of alpha7 nAChRs in the activation of TH and DBH gene expression.

Nicotinic receptors have been found to play a role in modulating pain transmission in the CNS. Activation of cholinergic pathways by nicotine and nicotinic agonists has been shown to elicit antinociceptive effects in a variety of species and pain tests. The involvement of alpha(7) nicotinic receptors in nicotinic analgesia was assessed after spinal (i.t.) and intraventricular (i.c.v.) administration in mice. Dose-dependent antinociceptive effects were seen with the alpha(7) agonist choline after spinal and supraspinal injection using the tail-flick test. Furthermore, alpha(7) antagonists MLA and alpha-BGTX significantly blocked the effects of choline. Dihydro-beta-erythroidine and mecamylamine failed to block choline-induced antinociception. These results strongly support the involvement of alpha(7) subunits in choline's antinociceptive effects. DMXB and 4-OH-DMXB, partial alpha(7) agonists, failed to elicit a significant antinociceptive effect. However, they blocked choline-induced antinociception in a dose-dependent manner following i.t. injection. This antagonism is probably related to their partial agonistic properties of the alpha(7) receptors. These studies suggest that activation of alpha(7) receptors in the CNS elicits antinociceptive effects in an acute thermal pain model.

Administration of nerve growth factor (NGF) by intracerebroventricular infusion or transplantation of NGF-secreting cells to the basal forebrain improves spatial memory in aged animals. Using the adeno-associated virus (AAV) vector system, basal forebrain neurons were transduced to produce NGF ectopically for long intervals (at least 9 months). Rats received intraseptal injections of either the control vector, pTR-UF4, or the pTR-NGFmyc at 3 months of age, prior to testing their performance in the Morris water task. An age-related decrease in the acquisition of the hidden platform location was found at 12 months of age in the pTR-UF4 control group, but not in the pTR-NGFmyc group. Further, when compared to 3 month old untreated animals, the control group, but not the pTR-NGFmyc group, was impaired at 12 months of age. Concomitant to preventing age-related memory deficits, the NGF gene transfer increased cholinergic neuron size by 34% in the medial septum. This approach may therefore represent a viable therapy for age-related dementia involving dysfunction in cholinergic activity and memory, such as Alzheimer's disease.

Background: WBC-reduced platelet components may be prepared by filtration or apheresis processing. Both methods have previously been shown to result in a residual total WBC content <5 x 10(6) per component. However, there may be differences in the efficacy of these techniques for removing certain WBC subsets., Study Design and Methods: Two multiparameter flow cytometric assays were developed and validated to perform WBC analysis on WBC-reduced platelets collected with two apheresis instruments (Amicus and COBE Spectra) and on 6 units of filtered pooled random-donor platelet concentrates., Results: All components contained <1 x 10(5) WBCs. The COBE Spectra and Amicus apheresis platelet components contained more WBCs than did filtered pooled platelets (p<0.05). Lymphocytes (T and B), monocytes, and granulocytes were identified in all components. Granulocyte content was lowest in the Amicus components and filtered pools. Monocytes were lowest in filtered pools. Amicus platelet components had fewer granulocytes and monocytes than the COBE Spectra platelets. Amicus and COBE Spectra components contained more lymphocytes than the filtered pools., Conclusion: Multiparameter flow cytometry can be used to quantify and characterize WBCs in WBC-reduced platelet components. WBC reduction by filtration or apheresis was highly effective. WBCs from each subset were identified in all components. Although filtered pools had the lowest numbers of WBCs, the very low numbers observed in all components suggests that the absolute quantitative differences in WBC subset content are of questionable clinical significance.

This study evaluated the effects of a novel, pH-sensitive surfactant, dodecyl 2-(1'-imidazolyl) propionate (DIP), on cationic lipid mediated transfection in primary rat brain neuronal and glial cultures. The cationic lipid complex DOTAP/DOPE (1, 2-dioleoyl-3-trimethylammonium propionate and dioleoyl phosphatidylethanolamine, respectively) was added over a range of concentrations (0-120 microg/ml) with DNA concentration kept constant (1.6 microg/ml). The neuron-specific enolase (NSE) and cytomegalovirus (CMV) promoters were found to drive green fluorescent protein (GFP) expression in neuron-enriched and glial cultures, respectively, using adeno-associated virus (AAV) derived constructs. NSE-driven GFP expression was not observed in glial cultures. Addition of DOTAP/DOPE increased transfection efficiency over a wide range of lipid concentrations (5-50 microg/ml) keeping DNA concentration constant (1.6 microg/ml). Addition of DIP to the lipid/DNA complex increased maximum transfection efficiencies in glial and neuronal cultures 2-3-fold. Transfection efficiencies were at their maximum with a similar total lipid concentration (50 microg/ml) in both cell-types in the presence of DIP. Neuronal cultures were more sensitive than glia to the toxic actions of DOTAP/DOPE, with or without DIP. These results indicate that AAV-mediated gene-transfer to neurons and glia can be facilitated by addition of a pH-sensitive surfactant to cationic liposome/DNA complexes and that endosomal escape could be a limiting factor in transgene expression.