Bidirectional regulation of emotional memory by 5-HT1B receptors involves hippocampal p11.

Cognitive impairments are common in depression and involve dysfunctional serotonin neurotransmission. The 5-HT1B receptor (5-HT(1B)R) regulates serotonin transmission, via presynaptic receptors, but can also affect transmitter release at heterosynaptic sites. This study aimed at investigating the roles of the 5-HT(1B)R, and its adapter protein p11, in emotional memory and object recognition memory processes by the use of p11 knockout (p11KO) mice, a genetic model for aspects of depression-related states. 5-HT(1B)R agonist treatment induced an impairing effect on emotional memory in wild type (WT) mice. In comparison, p11KO mice displayed reduced long-term emotional memory performance. Unexpectedly, 5-HT(1B)R agonist stimulation enhanced memory in p11KO mice, and this atypical switch was reversed after hippocampal adeno-associated virus mediated gene transfer of p11. Notably, 5-HT(1B)R stimulation increased glutamatergic neurotransmission in the hippocampus in p11KO mice, but not in WT mice, as measured by both pre- and postsynaptic criteria. Magnetic resonance spectroscopy demonstrated global hippocampal reductions of inhibitory GABA, which may contribute to the memory enhancement and potentiation of pre- and post-synaptic measures of glutamate transmission by a 5-HT(1B)R agonist in p11KO mice. It is concluded that the level of hippocampal p11 determines the directionality of 5-HT(1B)R action on emotional memory processing and modulates hippocampal functionality. These results emphasize the importance of using relevant disease models when evaluating the role of serotonin neurotransmission in cognitive deficits related to psychiatric disorders.

Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.

Adeno-associated viral (AAV) vectors are non-pathogenic, integrating DNA vectors in which all viral genes are removed and helper virus is completely eliminated. To evaluate this system in the post-mitotic cells of the brain, we found that an AAV vector containing the lacZ gene (AAVlac) resulted in expression of beta-galactosidase up to three months post-injection in vivo. A second vector expressing human tyrosine hydroxylase (AAVth) was injected into the denervated striatum of unilateral 6-hydroxydopamine-lesioned rats. Tyrosine hydroxylase (TH) immunoreactivity was detectable in striatal neurons and glia for up to four months and we also found significant behavioural recovery in lesioned rats treated with AAVth versus AAVlac controls. Safe and stable TH gene transfer into the denervated striatum may have potential for the genetic therapy of Parkinson's disease.

Mapping and targeted viral activation of pancreatic nerves in mice reveal their roles in the regulation of glucose metabolism.

A lack of comprehensive mapping of ganglionic inputs into the pancreas and of technology for the modulation of the activity of specific pancreatic nerves has hindered the study of how they regulate metabolic processes. Here we show that the pancreas-innervating neurons in sympathetic, parasympathetic and sensory ganglia can be mapped in detail by using tissue clearing and retrograde tracing (the tracing of neural connections from the synapse to the cell body), and that genetic payloads can be delivered via intrapancreatic injection to target sites in efferent pancreatic nerves in live mice through optimized adeno-associated viruses and neural-tissue-specific promoters. We also show that, in male mice, the targeted activation of parasympathetic cholinergic intrapancreatic ganglia and neurons doubled plasma-insulin levels and improved glucose tolerance, and that tolerance was impaired by stimulating pancreas-projecting sympathetic neurons. The ability to map the peripheral ganglia innervating the pancreas and to deliver transgenes to specific pancreas-projecting neurons will facilitate the examination of ganglionic inputs and the study of the roles of pancreatic efferent innervation in glucose metabolism.

Peroral gene therapy of lactose intolerance using an adeno-associated virus vector.

Gene therapy is usually reserved for severe and medically refractory disorders because of the toxicity, potential long-term risks and invasiveness of most gene transfer protocols. Here we show that an orally administered adeno-associated viral vector leads to persistent expression of a beta-galactosidase transgene in both gut epithelial and lamina propria cells, and that this approach results in long-term phenotypic recovery in an animal model of lactose intolerance. A gene 'pill' associated with highly efficient and stable gene expression might be a practical and cost-effective strategy for even relatively mild disorders, such as lactase deficiency.

Continuous renewal of the axonal pathway sensor apparatus by insertion of new sensor molecules into the growth cone membrane.

BACKGROUND: Growth cones at the tips of growing axons move along predetermined pathways to establish synaptic connections between neurons and their distant targets. To establish their orientation, growth cones continuously sample for, and respond to, guidance information provided by cell surfaces and the extracellular matrix. To identify specific guidance cues, growth cones have sensor molecules on their surface, which are expressed differentially during the temporospatial progress of axon outgrowth, at levels that depend on the pattern of neural activity. However, it has not been elucidated whether a change in gene expression can indeed change the molecular composition and, hence, the function of the sensor apparatus of growth cones. RESULTS: We have constructed adenoviral gene transfer vectors of the chicken growth cone sensor molecules axonin-1 and Ng-CAM. Using these vectors, we initiated the expression of axonin-1 and Ng-CAM in rat dorsal root ganglia explants during ongoing neurite outgrowth. Using specific surface immunodetection at varying time points after infection, we found that axonin-1 and Ng-CAM are transported directly to the growth cone and inserted exclusively in the growth cone membrane and not in the axolemma of the axon shaft. Furthermore, we found that axonin-1 and Ng-CAM do not diffuse retrogradely, suggesting that the sensor molecules are integrated into multimolecular complexes in the growth cone. CONCLUSIONS: During axon outgrowth, the pathway sensor apparatus of the growth cone is continuously updated by newly synthesized sensor molecules that originate directly from the transcription/translation machinery. Changes in the expression of sensor molecules may have a direct impact, therefore, on the exploratory function of the growth cone.

Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation.

This gene transfer experiment is the first Parkinson's Disease (PD) protocol to be submitted to the Recombinant DNA Advisory Committee. The principal investigators have uniquely focused their careers on both pre-clinical work on gene transfer in the brain and clinical expertise in management and surgical treatment of patients with PD. They have extensively used rodent models of PD for proof-of-principle experiments on the utility of different vector systems. PD is an excellent target for gene therapy, because it is a complex acquired disease of unknown etiology (apart from some rare familial cases) yet it is characterized by a specific neuroanatomical pathology, the degeneration of dopamine neurons of the substantia nigra (SN) with loss of dopamine input to the striatum. This pathology results in focal changes in the function of several deep brain nuclei, which have been well-characterized in humans and animal models and which account for many of the motor symptoms of PD. Our original approaches, largely to validate in vivo gene transfer in the brain, were designed to facilitate dopamine transmission in the striatum using an AAV vector expressing dopamine-synthetic enzymes. Although these confirmed the safety and potential efficacy of AAV, complex patient responses to dopamine augmenting medication as well as poor results and complications of human transplant studies suggested that this would be a difficult and potentially dangerous clinical strategy using current approaches. Subsequently, we and others investigated the use of growth factors, including GDNF. These showed some encouraging effects on dopamine neuron survival and regeneration in both rodent and primate models; however, uncertain consequences of long-term growth factor expression and question regarding timing of therapy in the disease course must be resolved before any clinical study can be contemplated. We now propose to infuse into the subthalamic nucleus (STN) recombinant AAV vectors expressing the two isoforms of the enzyme glutamic acid decarboxylase (GAD-65 and GAD-67), which synthesizes the major inhibitory neurotransmitter in the brain, GABA. The STN is a very small nucleus (140 cubic mm or 0.02% of the total brain volume, consisting of approximately 300,000 neurons) which is disinhibited in PD, leading to pathological excitation of its targets, the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNpr). Increased GPi/SNpr outflow is believed responsible for many of the cardinal symptoms of PD, i.e., tremor, rigidity, bradykinesia, and gait disturbance. A large amount of data based on lesioning, electrical stimulation, and local drug infusion studies with GABA-agonists in human PD patients have reinforced this circuit model of PD and the central role of the STN. Moreover, the closest conventional surgical intervention to our proposal, deep brain stimulation (DBS) of the STN, has shown remarkable efficacy in even late stage PD, unlike the early failures associated with recombinant GDNF infusion or cell transplantation approaches in PD. We believe that our gene transfer strategy will not only palliate symptoms by inhibiting STN activity, as with DBS, but we also have evidence that the vector converts excitatory STN projections to inhibitory projections. This additional dampening of outflow GPi/SNpr outflow may provide an additional advantage over DBS. Moreover, of perhaps the greatest interest, our preclinical data suggests that this strategy may also be neuroprotective, so this therapy may slow the degeneration of dopaminergic neurons. We will use both GAD isoforms since both are typically expressed in inhibitory neurons in the brain, and our data suggest that the combination of both isoforms is likely to be most beneficial. Our preclinical data includes three model systems: (1) old, chronically lesioned parkinsonian rats in which intraSTN GAD gene transfer results not only in improvement in both drug-induced asymmetrical behavior (apomorphine symmetrical rotations), but also in spontaneous behaviors. In our second model, GAD gene transfer precedes the generation of a dopamine lesion. Here GAD gene transfer showed remarkable neuroprotection. Finally, we carried out a study where GAD-65 and GAD-67 were used separately in monkeys that were resistant to MPTP lesioning and hence showed minimal symptomatology. Nevertheless GAD gene transfer showed no adverse effects and small improvements in both Parkinson rating scales and activity measures were obtained. In the proposed clinical trial, all patients will have met criteria for and will have given consent for STN DBS elective surgery. Twenty patients will all receive DBS electrodes, but in addition they will be randomized into two groups, to receive either a solution containing rAAV-GAD, or a solution which consists just of the vector vehicle, physiological saline. Patients, care providers, and physicians will be blind as to which solution any one patient receives. All patients, regardless of group, will agree to not have the DBS activated until the completion and unblinding of the study. Patients will be assessed with a core clinical assessment program modeled on the CAPSIT, and in addition will also undergo a preop and several postop PET scans. At the conclusion of the study, if any patient with sufficient symptomatic improvement will be offered DBS removal if they so desire. Any patients with no benefit will simply have their stimulators activated, which would normally be appropriate therapy for them and which requires no additional operations. If any unforeseen symptoms occur from STN production of GABA, this might be controlled by blocking STN GABA release with DBS, or STN lesioning could be performed using the DBS electrode. Again, this treatment would not subject the patient to additional invasive brain surgery. The trial described here reflects an evolution in our thinking about the best strategy to make a positive impact in Parkinson Disease by minimizing risk and maximizing potential benefit. To our knowledge, this proposal represents the first truly blinded, completely controlled gene or cell therapy study in the brain, which still provides the patient with the same surgical procedure which they would normally receive and should not subject the patient to additional surgical procedures regardless of the success or failure of the study. This study first and foremost aims to maximally serve the safety interests of the individual patient while simultaneously serving the public interest in rigorously determining in a scientific fashion if gene therapy can be effective to any degree in treating Parkinson's disease.

Estrogen increases HIP-70/PLC-alpha messenger ribonucleic acid in the rat uterus and hypothalamus.

In the ventromedial hypothalamus (VMH) of female rats, estrogen induces a protein isoform, HIP-70, whose sequence is identical to a protein reported to be phosphoinositol-specific phospholipase-C-alpha (PLC-alpha). Since previous studies explored induction only at the protein level, we examined both the distribution of HIP-70/PLC-alpha mRNA in various tissues and the effects of estrogen on HIP-70/PLC-alpha mRNA. Using slot blot analysis, we found that HIP-70/PLC-alpha mRNA is most abundant in pituitary, uterus, and VMH of female rats compared with other brain regions and tissues. Since these are target tissues for estrogen action, we examined the effects of estrogen on the abundance of HIP-70/PLC-alpha mRNA in these areas. Levels of HIP-70/PLC-alpha mRNA increased greater than 3-fold in the uterus 18 h after estrogen treatment. HIP-70/PLC-alpha mRNA in the VMH also increased about 35% 3 h after estrogen treatment. In situ hybridization corroborated the induction in the ventrolateral ventromedial hypothalamus. No effect of estrogen was observed on pituitary PLC-alpha mRNA. These results indicate that estrogen does increase HIP-70/PLC-alpha mRNA levels in certain tissues. Since the induction of HIP-70/PLC-alpha mRNA in VMH is relatively modest compared to the much larger induction of the HIP-70 protein isoform, regulation of HIP-70/PLC-alpha may entail both pre- and posttranslational mechanisms. Because members of the PLC family catalyze the hydrolysis of phosphatidyl inositol, potentially activating several secondary mediators (intracellular Ca2+, protein kinase-C, and eicanosoids), this second messenger pathway may mediate some effects of estrogen.

Transfection of human lactotroph adenoma cells with an adenovirus vector expressing tyrosine hydroxylase decreases prolactin release.

Pituitary adenomas are common intracranial neoplasms, for which surgery and radiation are usually not curative. In attempting to develop gene therapy as a better approach to treating pituitary adenomas, we chose lactotroph adenomas as a model. The rationale for the use of this model is based on the observation that dopamine agonists decrease prolactin secretion by lactotroph adenomas, and also decrease their size. We transfected primary cultures of human lactotroph adenoma cells with an adenovirus vector containing a cDNA which encodes a human tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. Transfection induced expression of tyrosine hydroxylase and increased production of dopamine, resulting in the predicted biologic effect of decreased prolactin secretion. These results demonstrate the potential for gene therapy of lactotroph adenomas and perhaps other pituitary adenomas, which are less amenable to pharmacologic treatment than lactotroph adenomas.

Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-gamma.

We investigated the feasibility of local treatment or tumor vaccination with a herpes simplex virus (HSV) type 1-defective vector. The vector was engineered to express murine interferon-gamma (IFN-gamma) for experimental gene therapy against mouse glioma Rous sarcoma virus (RSV). The murine IFN-gamma gene was driven by the cytomegalovirus promoter. The helper virus (tsk) was thermosensitive; consequently, this vector could only proliferate at 31 degrees C. A high level of murine IFN-gamma expression was confirmed in vitro and in vivo by immunohistochemistry using anti-mouse IFN-gamma monoclonal antibody. This engineered vector (dvHSV/MulFN-gamma) inhibited the proliferation of mouse glioma RSV cells in vitro, and an intratumoral (i.t.) local injection of the vector caused i.t. necrosis in vivo. The immunological effect of dvHSV/MulFN-gamma was also examined in a mouse glioma RSV cell implantation model. A subcutaneous (s.c.) implant of 1 x 10(6) mouse glioma RSV cells after treatment with dvHSV/MulFN-gamma was rejected. However, the implant after treatment with an engineered HSV-defective vector containing an antisense nucleotide sequence of the murine IFN-gamma gene was not rejected. In addition, in another group of mice in which RSV cells treated with dvHSV/MulFN-gamma were implanted into a femoral (s.c.) region and nontreated RSV cells were implanted into a contralateral femoral (s.c.) region, the implanted RSV cells were rejected. The rejection of the implanted mouse glioma RSV was blocked by anti-asialo GM1, which was known to inhibit natural killer cell activity. These results revealed that the HSV-defective vector could realize a high efficiency of transfection to glioma cells through short-time treatment, and that the IFN-gamma gene transferred to the cells had the effect of tumor vaccination, which was suggested be related to natural killer cells. In conclusion, dvHSV/MulFN-gamma may be useful for the gene therapy of malignant glioma through either i.t. local injection or a practical tumor vaccination with ex vivo gene transfer.

Gene transfer of wild-type p53 results in restoration of tumor-suppressor function in a medulloblastoma cell line.

The replacement of functional genes into cells that lack genes or have mutant genes is the basis of gene therapy. In cancer, where cells often have multiple genetic defects, the replacement of critical genes may suffice to suppress cell growth or induce cell death. The high frequency of mutations of the p53 tumor-suppressor gene in human cancers, including primary brain tumors, suggests that p53 plays a critical role in carcinogenesis and tumor progression. We report the successful transfer of the wild-type p53 gene using a defective herpes simplex viral vector into a human medulloblastoma cell line containing a mutant copy of p53. Upon gene transfer, we detected novel expression of wild-type p53 protein in the cells. In addition, the p53 protein was functionally active, since gene transfer resulted in increased levels of mdm2 proteins and induced cell cycle arrest of the majority of transduced cells. To our knowledge, this is the first report of the use of this vector system to carry wild-type p53. We conclude that defective herpes simplex viral vectors can transfer and express p53 in human primary brain tumor cells in vitro, restoring wild-type p53 tumor-suppressor functions.

Detection of messenger RNA and low-abundance heteronuclear RNA with single-stranded DNA probes produced by amplified primer extension labeling.

We describe a procedure for detection of low-abundance cellular RNAs by in situ hybridization histochemistry, using single-stranded DNA probes produced by amplified primer extension labeling with Taq polymerase. We have used this approach to detect a number of high- and low-abundance RNA species and have found it to be a simple and reproducible method of obtaining sensitive probes for in situ hybridization studies. For example, DNA probes generated by amplified primer extension labeling can detect low-abundance heteronuclear RNAs in individual neurons. Since this procedure does not involve recombinant DNA technology or microbiological facilities, it should prove useful to a wide variety of investigators studying the regulation of gene expression at the cellular level.

Linear dimeric interleukin-2 obtained by the use of a defective herpes simplex viral vector: conformation-activity relationship.

An interleukin-2 dimer, produced enzymatically by a nerve-derived transglutaminase in vitro, is cytotoxic to oligodendrocytes, unlike the immune-derived monomeric interleukin-2. The object of this study was to establish a way to produce a dimer of interleukin-2 in quantities, by means of genetic engineering, and to confirm that the structure of the resulting molecule is critical for its function. A defective herpes simplex virus vector was utilized for overproduction of a dimeric interleukin-2. The resulting linear dimer, which is a translational product, differs from the enzymatically produced dimer, which is a posttranslational modification of interleukin-2. The linear dimer, while retaining the known interleukin-2 activity of monomeric interleukin-2 with respect to mitogenicity on T cells, was not cytotoxic to oligodendrocytes. This finding suggests that the lack of cytotoxicity of the linear dimeric interleukin-2 is not caused by a loss of activity during its preparation but is related to its conformational structure, which evidently does not meet the requirements for cytotoxicity. This study opens the way to the design at the transcriptional level of modified proteins and their efficient production, provided that the new transcript encodes for the desired modification in the protein at the appropriate sites.

Expression of the growth-associated protein B-50/GAP43 via a defective herpes-simplex virus vector results in profound morphological changes in non-neuronal cells.

This study describes the creation and application of a defective herpes simplex viral (HSV) vector for B-50/GAP-43, a neural growth-associated phosphoprotein. We demonstrate abundant expression of B-50/GAP-43 in cultured non-neuronal cells (African green monkey kidney cells [vero cells] and Rabbit skin cells) via this HSV vector. When B-50/GAP-43 was expressed in non-neuronal cells major morphological changes occurred that included extensive membrane ruffling, the formation of filopodia and long thin extensions reminiscent of neurites. These extensions often terminated in growth cone-like structures. Quantitation of these morphological changes at different times following infection demonstrates that the surface area of the B-50/GAP-43-expressing cells started to increase between 6 and 10 h post-infection. At 72 h, B-50/GAP-43-positive cells were 3.0 times larger in size and one third of the cells expressed long processes with a mean length of 165 +/- 14.5 microns. Ultrastructural studies of cells 48 h after infection revealed that B-50/GAP-43 is predominantly localized at the plasma membrane of the elaborated processes. Some immunoreactivity was associated with vesicular structures that appear to be in-transit in the processes. These observations suggest that B-50/GAP-43 acts at the plasmamembrane to induce a neuron-like morphology in non-neuronal cells persisting for several days in culture. In the future the defective viral vector will enable gene transfer to express B-50/GAP-43 in neurons in vivo in order to study its involvement in regenerative sprouting and neuroplasticity.

Efficient adenoviral vector-directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium.

Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that Ad-LacZ, a prototype first generation adenoviral vector containing an expression cassette for the reporter gene LacZ, directs transgene expression to mature and immature olfactory neurons and to sustentacular cells. The technique to apply the vector to the nasal cavity and the amount of viral vector per mouse are important variables that determine the success of viral vector-mediated gene transfer to the mouse olfactory neuroepithelium. Slow infusion of the viral vector solution in fully anaesthetized mice yields the best result in terms of the number of epithelial cells transduced. Infection of the olfactory neuroepithelium with a moderate amount of viral vector (10(9) plaque-forming units (PFU)) results in transgene expression in many cells throughout the epithelium for 8-12 days, followed by a decline in transduced cells at 25 days postinstillation of the virus This decrement in transgene expression is consistent with the natural turnover process that occurs in the epithelium throughout adulthood. At high viral loads (1.3 x 10(10) PFU) extinction of transgene expression occurs as early as 8 days postinjection and is accompanied by epithelial degeneration indicating that the vector dose used should be carefully chosen. Taken together, the current observations demonstrate that adenoviral vectors are effective tools to genetically modify the adult mouse olfactory neuroepithelium in vivo.

Defective viral vectors as agents for gene transfer in the nervous system.

Viral vectors have attracted great interest as vehicles for gene therapy. Due to concerns regarding continued viral gene expression in several systems, new approaches have been sought for gene transfer in the nervous system. This article reviews the general concepts and basic biology of defective viral vectors. These are vectors which can package into a viral coat but contain no viral genes, thereby allowing efficient gene transfer in the absence of viral gene expression in target cells. The defective herpes simplex virus (HSV) vector has been applied to numerous interesting questions in neurobiology. The inability to completely eliminate helper viruses has raised concern regarding the application of this vector to human disease. The adeno-associated virus (AAV) vector has recently been introduced into the nervous system. This vector harbors no viral genes, however helper viruses can also be completely eliminated from the system. Although the smaller size may limit the range of applications for this vector, it has received great interest as a potential agent for gene therapy in the nervous system. Potential future directions are discussed as well.

Tension pneumocephalus resulting from iatrogenic subarachnoid-pleural fistulae: report of three cases.

BACKGROUND: Symptomatic pneumocephalus may result from a cerebrospinal fluid leak communicating with extradural air. However, it is a rare event after thoracic surgical procedures, and its management and physiology are not widely recognized. METHODS: During the past 2 years, we have identified 3 patients who developed pneumocephalus after thoracotomy for tumor resection. Only 1 patient had a discernible spinal fluid leak identified intraoperatively. Two patients experienced delayed spinal fluid drainage from their chest tubes and subsequently developed profound lethargy, confusion, and focal neurologic signs. The third patient was readmitted to the hospital with a delayed pneumothorax and altered mental status. Radiographic imaging in all patients showed significant pneumocephalus of the basilar cisterns and ventricles. RESULTS: The first 2 patients were managed by discontinuation of the chest tube suction and bedrest. The third patient underwent surgical reexploration and nerve root ligation. All 3 patients had resolution of their symptoms within 72 hours. CONCLUSIONS: Pneumocephalus is a rare, but serious, complication of thoracotomy. Previous patients reported in the literature have been managed with reoperation to ligate the nerve roots. However, the condition resolved nonoperatively in 2 of our patients. Discontinuation of chest tube suction may be definitive treatment and is always the important initial management to decrease cerebrospinal fluid extravasation into the pleural space and allow normalization of neurologic symptoms.

Long-term gene transfer in porcine myocardium after coronary infusion of an adeno-associated virus vector.

BACKGROUND: Viral vector-mediated gene transfer into the heart represents a potentially powerful tool for studying both cardiac physiology as well as gene therapy of cardiac disease. We report here the use of a defective viral vector, which expresses no viral gene products, for gene transfer into the mammalian heart. Previous studies have used recombinant viral vectors, which retained viral genes and yielded mostly short-term expression, often with significant inflammation. METHODS: An adeno-associated virus vector was used that contains no viral genes and is completely free of contaminating helper viruses. The adeno-associated virus vector was applied to rat hearts by direct intramuscular injection; adeno-associated virus was also infused into pig hearts in vivo via percutaneous intraarterial infusion into the coronary vasculature using routine catheterization techniques. RESULTS: Gene transfer into rat heart yielded no apparent inflammation, and expression was observed for at least 2 months after injection. Infusion into pig circumflex coronary arteries resulted in successful transfer and expression of the reporter gene in cardiac myocytes without apparent toxicity or inflammation; gene expression was observed for at least 6 months after infusion. CONCLUSIONS: We report the use of adeno-associated virus vectors in the cardiovascular system as well as successful myocardial gene transfer after percutaneous coronary artery infusion of viral vectors in a large, clinically relevant mammalian model. These results suggest that safe and stable gene transfer can be achieved in the heart using standard outpatient cardiac catheterization techniques.

In situ PCR for in vivo detection of foreign genes transferred into rat brain.

Here we describe the use of in situ PCR to detect a viral transgene in rat brain. Previously, we have reported in vivo gene transfer by using a defective herpes simplex viral vector in mammalian brain (Kaplitt, M.G., Pfaus, J.G., Kleopoulos, S.P., Hanlon, B.A., Rabkin, S.D., Pfaff, D.W., Mol. Cell. Neurosci. 2 (1991) 320-330). For detection of the LacZ transgene, we have used histochemical staining for the protein product, beta-galactosidase, and in situ hybridization for its mRNA, but the DNA itself cannot be reliably detected with conventional methods. Therefore we have adapted the technique of in situ PCR, so that we may detect minute quantities of transgenic vector DNA following in vivo gene. The brain sections, prefixed, were treated with PBS-detergent before PCR amplification to increase permeability for peptides and oligonucleotides across cellular barriers in brain tissue. Pretreatment with detergent retained better brain morphology than the more widely used proteinase treatment. The PCR mixture containing dNTPs, primers, digoxigenin-dUTP (Dig-dUTP) and buffer was loaded onto each brain section. Slides containing brain sections were placed in an aluminum boat and then on the block of the thermal cycler. Temperature was brought to 82 degrees C before adding Taq polymerase ('hot start' method). Dig-labeled PCR amplified fragments were then detected by alkaline-phosphatase-linked anti-digoxigenin-antibody. Positive signals were seen within the nucleus of transduced neurons, indicating presence of viral DNA. Enhanced specificity was observed with the use of Dig-labeled primers which eliminates the possibility of non-specific viral DNA detection through primer-independent reactions. Overall, this technique can serve not only as an internal control for transgene presence during comparisons of experimental groups of animals, but may also have clinical applications including the detection of viral infection in human brain such as HIV in pathology specimens.

Assessment of disease severity in late infantile neuronal ceroid lipofuscinosis using multiparametric MR imaging.

BACKGROUND AND PURPOSE: LINCL is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene that encodes for tripeptidyl peptidase 1, a lysosomal enzyme necessary for the degradation of products of cellular metabolism. With the goal of developing quantitative noninvasive imaging biomarkers sensitive to disease progression, we evaluated a 5-component MR imaging metric and tested its correlation with a clinically derived disease-severity score. MATERIALS AND METHODS: MR imaging parameters were measured across the brain, including quantitative measures of the ADC, FA, nuclear spin-spin relaxation times (T2), volume percentage of CSF (%CSF), and NAA/Cr ratios. Thirty MR imaging datasets were prospectively acquired from 23 subjects with LINCL (2.5-8.4 years of age; 8 male/15 female). Whole-brain histograms were created, and the mode and mean values of the histograms were used to characterize disease severity. RESULTS: Correlation of single MR imaging parameters against the clinical disease-severity scale yielded linear regressions with R2 ranging from 0.25 to 0.70. Combinations of the 5 biomarkers were evaluated by using PCA. The best combination included ADC, %CSF, and NAA/Cr (R2=0.76, P<.001). CONCLUSIONS: The multiparametric disease-severity score obtained from the combination of ADC, %CSF, and NAA/Cr whole-brain MR imaging techniques provided a robust measure of disease severity, which may be useful in clinical therapeutic trials of LINCL in which an objective assessment of therapeutic response is desired.