Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced motor paralysis.

Nonsense-mediated mRNA decay (NMD) is an RNA surveillance mechanism that requires upframeshift protein 1 (UPF1). This study demonstrates that human UPF1 exerts protective effects in a rat paralysis model based on the amyotrophic lateral sclerosis (ALS)-associated protein, TDP-43 (transactive response DNA-binding protein 43 kDa). An adeno-associated virus vector (AAV9) was used to express TDP-43 throughout the spinal cord of rats, inducing reproducible limb paralysis, to recapitulate the paralysis in ALS. We selected UPF1 for therapeutic testing based on a genetic screen in yeast. The expression of human TDP-43 or human UPF1 in the spinal cord was titrated to less than twofold over the respective endogenous level. AAV9 human mycUPF1 clearly improved overall motor scores in rats also expressing TDP-43. The gene therapy effect of mycUPF1 was specific and reproducible compared with groups receiving either empty vector or green fluorescent protein vector controls. The gene therapy maintained forelimb motor function in rats that would otherwise become quadriplegic. This work helps validate UPF1 as a novel therapeutic for ALS and other TDP-43-related diseases and may implicate UPF1 and NMD involvement in the underlying disease mechanisms.

AAV9-mediated erythropoietin gene delivery into the brain protects nigral dopaminergic neurons in a rat model of Parkinson's disease.

We have recently shown that intrastriatal injection of recombinant human erythropoietin (EPO) protects dopaminergic (DA) neurons in the substantia nigra (SN) from 6-hydroxydopamine (6-OHDA) toxicity in a rat model of Parkinson's disease. However, systemic administration of EPO did not protect nigral DA neurons, suggesting that the blood-brain barrier limits the passage of EPO protein into the brain. In the present study, we used an adeno-associated viral (AAV) serotype 9 (AAV9) vector to deliver the human EPO gene into the brain of 6-OHDA-lesioned rats. We observed that expression of the human EPO gene was robust and stable in the striatum and the SN for up to 10 weeks. EPO-immunoreactive (IR) cells were widespread throughout the injected striatum, and EPO-IR neurons and fibers were also found in the ipsilateral SN. Enzyme-linked immunosorbent assay and western blot analyses exhibited dramatic levels of EPO protein in the injected striatum. As a result, nigral DA neurons were protected against 6-OHDA-induced toxicity. Amphetamine-induced rotational asymmetry and spontaneous forelimb use asymmetry were both attenuated. Interestingly, we also observed that intrastriatal injection of AAV9-EPO vectors led to increased numbers of red blood cells in peripheral blood. This highlights the importance of using an inducible gene delivery system for EPO gene delivery.

Nuclear magnetic resonance-determined lipoprotein subclass profile in the DCCT/EDIC cohort: associations with carotid intima-media thickness.

AIMS: To relate nuclear magnetic resonance lipoprotein subclass profiles (NMR-LSP) and other lipoprotein-related factors with carotid intima-media thickness (IMT) in Type 1 diabetes. METHODS: Lipoprotein-related factors were determined in sera (obtained in 1997-1999) from 428 female [age 39 +/- 7 years (mean +/- SD)] and 540 male (age 40 +/- 7 years) Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) participants. NMR quantifies chylomicrons, three very low-density lipoprotein (VLDL) subclasses, intermediate density lipoprotein (IDL), three low-density lipoprotein (LDL) subclasses, two high-density lipoprotein (HDL) subclasses, mean VLDL, LDL and HDL size, and LDL particle concentration. Conventional lipids, ApoA1, ApoB and Lp(a) and in vitro LDL oxidizibility were also measured. IMT was determined (in 1994-1995) using high-resolution B-mode ultrasound. Relationships between IMT and lipoproteins were analysed by multiple linear regression, controlling for age, diabetes-related factors, and cardiovascular disease (CVD) risk factors. RESULTS: IMT associations with lipoproteins were stronger for the internal than the common carotid artery, predominantly involving LDL. Internal carotid IMT was positively (P < 0.05) associated with NMR-based LDL subclasses and particle concentration, and with conventional LDL-cholesterol and ApoB in both genders. Common carotid IMT was associated, in men only, with large VLDL, IDL, conventional LDL cholesterol and ApoB. CONCLUSIONS: NMR-LSP reveals significant associations with carotid IMT in Type 1 diabetic patients, even 4 years after IMT measurement. NMR-LSP may aid early identification of high-risk diabetic patients and facilitate monitoring of interventions. Longer DCCT/EDIC cohort follow-up will yield CVD events and IMT progression, permitting more accurate assessment of pre-morbid lipoprotein profiles as determinants of cardiovascular risk in Type 1 diabetes.

Lipoproteins and diabetic microvascular complications.

Risk factors for the microvascular complications (nephropathy and retinopathy) of Type 1 and Type 2 diabetes mellitus and the associated accelerated atherosclerosis include: age, diabetes duration, genetic factors, hyperglycaemia, hypertension, smoking, inflammation, glycation and oxidative stress and dyslipoproteinaemia. Hypertriglyceridaemia, low HDL and small dense LDL are common features of Type 2 diabetes and Type 1 diabetes with poor glycaemic control or renal complications. With the expansion of knowledge and of clinical and research laboratory tools, a broader definition of 'lipid' abnormalities in diabetes is appropriate. Dyslipoproteinaemia encompasses alterations in lipid levels, lipoprotein subclass distribution, composition (including modifications such as non-enzymatic glycation and oxidative damage), lipoprotein-related enzymes, and receptor interactions and subsequent cell signaling. Alterations occur in all lipoprotein classes; chylomicrons, VLDL, LDL, HDL, and Lp(a). There is also emerging evidence implicating lipoprotein related genotypes in the development of diabetic nephropathy and retinopathy. Lipoprotein related mechanisms associated with damage to the cardiovascular system may also be relevant to damage to the renal and ocular microvasculature. Adverse tissue effects are mediated by both alterations in lipoprotein function and adverse cellular responses. Recognition and treatment of lipoprotein-related risk factors, supported by an increasing array of assays and therapeutic agents, may facilitate early recognition and treatment of high complication risk diabetic patients. Further clinical and basic research, including intervention trials, is warranted to guide clinical practice. Optimal lipoprotein management, as part of a multi-faceted approach to diabetes care, may reduce the excessive personal and economic burden of microvascular complications and the related accelerated atherosclerosis.

Induction of uncoupling protein 1 by central interleukin-6 gene delivery is dependent on sympathetic innervation of brown adipose tissue and underlies one mechanism of body weight reduction in rats.

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.

Burn wound itch control using H1 and H2 antagonists.

This study investigated the use of a combination of H1 and H2 antagonists and topical medications to control burn wound itch. Graeco-Latin square assignment provided an oral combination of 1) cetirizine and cimetidine or 2) diphenhydramine and placebo in four divided doses. The study protocol lasted 16 days divided into 4-day intervals, scoring itch before the initial dose of medication and at 1-hour, 6-hour, and 12-hour intervals after the first medication. A significant difference between mean itch scores across the four times was observed (Wilks' Lambda F = 26.52, df = 3, P <.0005). A three-way nested repeated measures interaction effect (Wilks' Lambda F = 9.85, df = 9, P <.0005) was observed representing a significantly different pattern on days 1 to 4 of the study compared with the remaining days. Controlling for the effect of topical medications, the cetirizine/cimetidine combination demonstrated a dramatic improvement at 1 and 6 hours, and a moderate improvement at 12 hours after initial medication for the day when compared with the diphenhydramine/placebo combination.

Effect of inflammatory cytokines on the metabolism of low-density lipoproteins by human vascular endothelial cells.

Cytokines have been shown to activate multiple, varied metabolic pathways in endothelial cells. Little information is available concerning the effects of inflammatory cytokines on lipoprotein metabolism by vascular endothelial cells. Human umbilical vein endothelial cells (HuECs) and bovine aortic endothelial cells (BAECs) were incubated with the inflammatory cytokines recombinant human interleukin-1beta (IL-1), tumor necrosis factor alpha (TNF), interferon gamma (gamma-IF), and interferon beta (beta-IF) at increasing concentrations (0.1 to 1,000 U/mL), for increasing periods (6 to 72 hours). After the incubation period, the media were removed and replaced with serum-free media containing radiolabeled native or acetylated low-density lipoprotein (Ac-LDL) and the rates of degradation and accumulation of radiolabeled LDL were determined. The degradation and accumulation of 125I-LDL were significantly increased (P < .02) in HuECs preincubated with IL-1 (100 U/mL) compared with control incubations without the cytokine or incubations containing gamma-IF, beta-IF, or TNF. This resulted from a 38% increase in LDL receptor protein in cells incubated with IL-1. The increased rate of LDL catabolism by HuECs incubated with IL-1 was accompanied by a significant increase (P < .05) in the rate of cholesteryl ester synthesis in the cells. Cholesteryl ester synthesis rates in HuECs preincubated with gamma-IF, beta-IF, or TNF did not differ significantly from the rates in control incubations. The effect of preincubation with cytokine on the activity of the scavenger receptor was also determined. There were no significant differences in the rate of degradation or accumulation of radiolabeled Ac-LDL in control incubations compared with cultures preincubated with IL-1, gamma-IF, beta-IF, or TNF. There also were no significant differences in the rate of catabolism of native LDL or Ac-LDL in BAECs preincubated with cytokines. Although cytokines have been shown previously to alter the binding of monocytes to endothelial cells, there was no significant increase in the binding of monocytes to cultures incubated with IL-1 plus LDL compared with IL-1 alone. In summary, we now demonstrate that cytokines, specifically IL-1, may alter LDL metabolism by human vascular endothelial cells and alter endothelial cell cholesterol metabolism. These changes in endothelial cell metabolism provide additional evidence supporting the critical role of cytokines in atherogenesis.

Native and modified LDL activate extracellular signal-regulated kinases in mesangial cells.

Glycation and/or oxidation of LDL may promote diabetic nephropathy. The mitogen-activated protein kinase (MAPK) cascade, which includes extracellular signal-regulated protein kinases (ERKs), modulates cell function. Therefore, we examined the effects of LDL on ERK phosphorylation in cultured rat mesangial cells. In cells exposed to 100 microg/ml native LDL or LDL modified by glycation, and/or mild or marked (copper-mediated) oxidation, ERK activation peaked at 5 min. Five minutes of exposure to 10-100 microg/ml native or modified LDL produced a concentration-dependent (up to sevenfold) increase in ERK activity. Also, 10 microg/ml native LDL and mildly modified LDL (glycated and/or mildly oxidized) produced significantly greater ERK activation than that induced by copper-oxidized LDL +/- glycation (P < 0.05). Pretreatment of cells with Src kinase and MAPK kinase inhibitors blocked ERK activation by 50-80% (P < 0.05). Native and mildly modified LDL, which are recognized by the native LDL receptor, induced a transient spike of intracellular calcium. Copper-oxidized (+/- glycation) LDL, recognized by the scavenger receptor, induced a sustained rise in intracellular calcium. The intracellular calcium chelator (EGTA/AM) further increased ERK activation by native and mildly modified LDL (P < 0.05). These findings demonstrate that native and modified LDL activate ERKs 1 and 2, an early mitogenic signal, in mesangial cells and provide evidence for a potential link between modified LDL and the development of glomerular injury in diabetes.

NGF gene transfer to intrinsic basal forebrain neurons increases cholinergic cell size and protects from age-related, spatial memory deficits in middle-aged rats.

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.

Adeno-associated virus vectors: activity and applications in the CNS.

Transgenic strategies are useful for functional studies and they may also lead to novel therapies. Controlling transgene expression in defined cell populations over time is increasingly important for both functional and gene therapy experiments. The adeno-associated virus (AAV) vector may provide sufficient spatio-temporal control of gene expression for these purposes. This paper reviews in vivo somatic gene transfer methodology using AAV. Advantageous features of this system include neuronal gene expression that is: (1) efficient; (2) long-lived; and (3) non-toxic. Thus, AAV-mediated gene transfer is a good method for functional genomic research. From characterizing vector activity in the brain using different combinations of promoters and transgenes in the mid to late 1990s, researchers continue to discover novel uses of AAV for both basic and clinical neuroscience.

Anti-modified LDL antibodies, LDL-containing immune complexes, and susceptibility of LDL to in vitro oxidation in patients with type 2 diabetes.

We investigated the hypothesis that modified lipoproteins trigger an immune response leading to the production of autoantibodies and subsequently to the formation of atherogenic immune complexes (IC). We recruited 20 type 2 diabetic patients with macrovascular disease, 14 nondiabetic patients with coronary artery disease (CAD), and 34 healthy control subjects matched for age, sex, and race. Serum antibodies to oxidized and glycated LDL did not differ significantly among the 3 groups. Serum IC contained variable, but not statistically different, amounts of IgG, IgM, and IgA. In contrast, the content of cholesterol in IC isolated from diabetic patients was significantly higher than that in IC isolated from control subjects, and the content of apolipoprotein (apo)-B was significantly higher than that in IC isolated from control subjects and patients with CAD. Cholesteryl ester accumulation in human monocyte-derived macrophages incubated with IC, a measure of the atherogenic potential of IC, was significantly higher in macrophages incubated with red blood cell-adsorbed IC isolated from diabetic patients compared with IC isolated from control subjects (P < 0.03) or from patients with CAD (P < 0.04) and was strongly correlated with the content of apoB (r = 0.68, P < 0.001) and cholesterol (r = 0.61, P < 0.001) in IC. LDL from diabetic patients was more susceptible to oxidation in vitro, was significantly smaller, and contained significantly less alpha-tocopherol than LDL isolated from subjects in the other groups. In addition, the n-3 polyunsaturated fatty acid content of phospholipids and cholesteryl esters in LDL isolated from diabetic patients was significantly increased (P < 0.05) compared with that from patients with CAD or from control subjects. We postulate that LDL size, susceptibility to oxidation, and lipid fatty acid composition may play a critical role in the production of antibodies to oxidized LDL and consequently in the formation of LDL-containing IC in patients with type 2 diabetes.

Prevention of 6-hydroxydopamine-induced rotational behavior by BDNF somatic gene transfer.

Brain-derived neurotrophic factor (BDNF) was expressed via injection of viral vector into the substantia nigra pars compacta (SNc) to investigate its influence on nigrostriatal dopaminergic activity and locomotor behavior. The recombinant adeno-associated virus (rAAV) vector, pTR-BDNFmyc, incorporated the neuron-specific enolase (NSE) promoter and the internal ribosome entry site (IRES) element driving expression of both epitope-tagged BDNF and green fluorescent protein (GFP) bicistronically. The control vector, pTR-UF4, incorporated NSE promoter-driven GFP expression only. Transgene expression persisted in both vector groups throughout the 9 month course of the study. Partial 6-hydroxydopamine (6-OHDA) lesions were conducted in the SNc ipsilateral to, and 6 months after, transduction with either the pTR-BDNFmyc or the pTR-UF4. Transgenic BDNFmyc had no effect on the number of tyrosine hydroxylase (TH)-labeled neurons in the SNc after 6-OHDA-lesions, but did block the amphetamine-induced, ipsiversive, turning-behavior caused by the lesion in the pTR-UF4 group. The BDNFmyc-transduced group also demonstrated more locomotor activity and rotational activity contralateral to the lesioned side than did the pTR-UF4-transduced group. Long-term, stable expression of BDNF can therefore modulate locomotor activity without significantly affecting nigrostriatal dopaminergic survival.

Pre-enrichment of modified low density lipoproteins with alpha-tocopherol mitigates adverse effects on cultured retinal capillary cells.

PURPOSE: We determined whether pre-enrichment of low density lipoproteins (LDL) with alpha-tocopherol mitigates their adverse effects, following in vitro glycation, oxidation or glycoxidation, towards cultured bovine retinal capillary endothelial cells (RCEC) and pericytes. METHODS: LDL, while still in plasma obtained and pooled from non-diabetic humans, was supplemented in vitro with alpha-tocopherol. It was then isolated and modified in vitro by glycation, minimal oxidation, and glycoxidation. Bovine RCEC and pericytes were exposed to LDL (100mg protein/ ml) for three days. Cell count was determined by cell counting, supernatant levels of plasminogen activator inhibitor-1 (PAI-1) and endothelin-1 (ET-1) by ELISA, and nitrite levels by spectroscopic colorimetric assay. RESULTS: While pre-enrichment of LDL with alpha-tocopherol did not reduce the measured extent of lipoprotein modification, it abolished the reduction in cell count observed with glycated, oxidized and glycoxidized LDL v. normal LDL. Pre-enrichment of LDL with alpha-tocopherol also reduced RCEC supernatant PAI-1 and ET-1 (corrected for cell counts) and increased RCEC and pericyte-associated supernatant nitrite levels: such effects of alpha-tocopherol may inhibit clot formation and favor vasodilatation. CONCLUSIONS: Enrichment of LDL with alpha-tocopherol abolishes adverse effects of glycated, mildly oxidized, and glycoxidized LDL on cultured retinal cell count, and mitigates adverse effects on modulators of fibrinolysis and vascular tone. Direct evidence is required before Vitamin E supplementation is recommended for people with diabetes.

Generation of aberrant sprouting in the adult rat brain by GAP-43 somatic gene transfer.

The expression of GAP-43 was modulated genetically in the adult rat nigrostriatal or septohippocampal pathway using recombinant adeno-associated virus (rAAV) vectors incorporating the neuron specific enolase (NSE) promoter and either a rat GAP-43 cDNA or the corresponding antisense sequence. Bicistronic expression of green fluorescent protein (GFP) enabled us to evaluate transduced neurons selectively. Single injections of rAAV into the substantia nigra pars compacta (SNc) transduced both dopaminergic and non-dopaminergic neurons stably for the 3-month duration of the study. Transduction with the GAP-43 vector in this region: (1) increased GAP-43 mRNA levels 2-fold compared to controls; (2) led to GAP-43 immunoreactivity in neuronal perikarya, axons, and dendrites that was not observed otherwise; and (3) resulted in GAP-43/ GFP-positive axons that were traced to the striatum where they formed clusters of aberrant nets. The GAP-43 antisense vector, in contrast, decreased neuropil GAP-43 immunoreactivity compared to controls in the SNc. In septum, injections of the GAP-43 expressing vector also caused aberrant clusters of GAP-43 labelled fibers in terminal fields, i.e., fornix and hippocampus, that were not observed in control tissues. It therefore appears that rAAV vectors provide a novel approach for modulating intraneuronal GAP-43 expression in the adult brain.

Long-term actions of vector-derived nerve growth factor or brain-derived neurotrophic factor on choline acetyltransferase and Trk receptor levels in the adult rat basal forebrain.

Trophic factor gene therapy may provide a rational treatment strategy for neurodegenerative disease. Recombinant adeno-associated virus vectors, incorporating a neuron-specific promoter driving bicistronic expression of green fluorescent protein and either nerve growth factor or brain-derived neurotrophic factor, transduced 10,000-15,000 neurons in the medial septum for periods of at least six months. Both cholinergic and non-cholinergic neurons expressed green fluorescent protein. Nerve growth factor and brain-derived neurotrophic factor vectors produced up to 50% increases in immunohistochemical detection of the acetylcholine-synthesizing enzyme in septal neurons ipsilateral to the injection. Increased levels of this enzyme, choline acetyltransferase, persisted for six months with the brain-derived neurotrophic factor vector. The nerve growth factor vector increased Trk receptor immunoreactivity in a volume of brain exceeding that of the transduced cells. Counterstaining for the neuronal marker, NeuN, or Nissl substance did not reveal any vector toxicity at any time-point. It therefore appears that the lasting effects of vector-mediated trophic factor gene transfer will offer a new approach for modulating septal cholinergic transmission and Trk receptor activity.

Transmission of two novel mutations in a pedigree with familial lecithin:cholesterol acyltransferase deficiency: structure-function relationships and studies in a compound heterozygous proband.

Two novel mutations were identified in a compound heterozygous male with lecithin:cholesterol acyltransferase (LCAT) deficiency. Exon sequence determination of the LCAT gene of the proband revealed two novel heterozygous mutations in exons one (C110T) and six (C991T) that predict non-conservative amino acid substitutions (Thr13Met and Pro307Ser, respectively). To assess the distinct functional impact of the separate mutant alleles, studies were conducted in the proband's 3-generation pedigree. The compound heterozygous proband had negligible HDL and severely reduced apolipoprotein A-I, LCAT mass, LCAT activity, and cholesterol esterification rate (CER). The proband's mother and two sisters were heterozygous for the Pro307Ser mutation and had low HDL, markedly reduced LCAT activity and CER, and the propensity for significant reductions in LCAT protein mass. The proband's father and two daughters were heterozygous for the Thr13Met mutation and also displayed low HDL, reduced LCAT activity and CER, and more modest decrements in LCAT mass. Mean LCAT specific activity was severely impaired in the compound heterozygous proband and was reduced by 50% in individuals heterozygous for either mutation, compared to wild type family members. It is also shown that the two mutations impair both catalytic activity and expression of the circulating protein.

Suicide by burning: a retrospective review of the Akron Regional Burn Center.

At the Akron Regional Burn Center from January 1978 through March 1995, 36 cases of attempted suicide by burning were identified in 34 patients. The overall incidence rate was 1 per cent of all burn center admissions to this institution. Of these patients, 21 of 34 were male and 13 of 34 were female. There were 10 of 34 lethal cases. A high incidence of prior psychiatric illness was identified. In all, 22 of 34 patients had a prior psychiatric diagnosis. Depression was the most common psychiatric diagnosis by history. The method most commonly used was a flame with the addition of a flammable liquid. These results are presented, discussed and compared to a review of the literature on the topic.

Neuron-specific transduction in the rat septohippocampal or nigrostriatal pathway by recombinant adeno-associated virus vectors.

Viral vector-mediated gene transfer in brain can provide a means for gene therapy and functional studies. However, robust and persistent transgene expression in specific populations of the adult brain has been difficult to achieve. In an attempt to produce localized and persistent transduction in rat brain, we compared recombinant adeno-associated virus (rAAV) vectors incorporating either the immediate early cytomegalovirus (CMV) promoter or the neuron-specific enolase (NSE) promoter. Transduction in hippocampus resulting from the NSE promoter-containing construct was more efficient and persistent than that resulting from the CMV promoter-containing construct. Most hippocampal cells transduced with the NSE promoter had multipolar neuron morphology. Neurons with glutamatergic morphology were transduced weakly. In order to produce a local supply of neurotrophic factor to cells that degenerate under certain disease and experimental conditions, the NSE promoter was utilized to drive expression of brain-derived neurotrophic factor (BDNF) in medial septum or substantia nigra. In this construct, the NSE promoter drives dicistronic expression of BDNF and an enhanced version of green fluorescent protein (GFP). We estimated 3000-15,000 GFP-positive cells per injection of rAAV into septum or substantia nigra, a transduction ratio of 5-20 infectious virus particles per transduced cell. This frequency may be sufficient for trophic factor gene therapy as well as for investigating specific protein function in "topical (i.e., localized) transgenic" animals produced by rAAV.

Ventricular cholecystic shunts in children.

Hydrocephalus is a prevalent pediatric problem, and ventricular peritoneal shunting is the preferred procedure for surgical treatment. A system may become dysfunctional if the distal end of the catheter fails to drain because of intraabdominal adhesions, cerebral spinal fluid cysts, or peritonitis. Then additional revisions become necessary. Pediatric surgeons can come to the aid of their neurosurgical colleagues by placing the distal end of the catheter into the pleural space, cardiac atrium, ureter, stomach, or fallopian tube. These have all met with limited success. The ventricular cholecystic shunt has been used in some difficult-to-manage cases of hydrocephalus, and our experience with the procedure is described. In the complicated patient, we have had success placing the distal catheters in the gallbladder. Between 1986 and 1995 sixteen patients have had ventriculocholecystic shunts placed. The mean age was 10.8 years, ranging from 0.9 to 23 years. Prior shunting procedures averaged 4.1 (range 0 to 11). The follow-up period ranged from 4 weeks to 7.5 years, with a median follow-up of 3 years. No shunts were removed or revised specifically because of gallbladder complications such as infected bile, stones, or distal catheter occlusion. Seven patients experienced other shunt-related problems that were not caused by distal catheters, ie, staphylococcal infections and intracerebral problems. Eleven of the original shunts are still functioning. Two patients died of causes not related to the ventriculoperitoneal shunt. Because of their simplicity of placement, low incidence of complication, and similarity to ventriculoperitoneal shunts, the ventricular cholecystic shunt should be considered when other sites are unattainable.