Adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 suppresses seizure activity in vivo.

Neuropeptide Y (NPY) is a 36-amino-acid peptide that attenuates seizure activity following direct infusion or adeno-associated virus (AAV)-mediated expression in the central nervous system. However, NPY activates all NPY receptor subtypes, potentially causing unwanted side effects. NPY13-36 is a C-terminal peptide fragment of NPY that primarily activates the NPY Y2 receptor, thought to mediate the antiseizure activity. Therefore, we investigated if recombinant adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 could alter limbic seizure sensitivity. Rats received bilateral piriform cortex infusions of AAV vectors that express and constitutively secrete full-length NPY (AAV-FIB-NPY) or NPY13-36 (AAV-FIB-NPY13-36). Control rats received no infusion, as we have previously shown that vectors expressing and secreting reporter genes like GFP (AAV-FIB-EGFP), as well as vectors expressing peptides that lack secretion sequences (AAV-GAL) have no effect on seizures. One week later, all animals received kainic acid (10 mg kg(-1), intraperitoneally), and the latencies to wet dog shakes and limbic seizure behaviors were determined. Although both control and vector-treated rats developed wet dog shake behaviors with similar latencies, the latencies to class III and class IV limbic seizures were significantly prolonged in both NPY- and NPY13-36-treated groups. Thus, AAV-mediated expression and constitutive secretion of NPY and NPY13-36 is effective in attenuating limbic seizures, and provides a platform for delivering therapeutic peptide fragments with increased receptor selectivity.

GABAB receptor antagonist SGS742 improves spatial memory and reduces protein binding to the cAMP response element (CRE) in the hippocampus.

Memory storage in the brain requires protein synthesis initiated through signaling pathways that control transcription. Such mechanisms are under active investigation for therapies in disorders involving cognitive dysfunction. Long-term memory can be improved by inhibiting activation or reducing expression of transcription factors such as ATF4/CREB2 and some C/EBP family members which appear to serve as memory suppressors. Here, we provide evidence that GABAB receptor antagonists may enhance cognition, at least in part, by this mechanism. We tested a GABAB receptor antagonist, SGS742 (CGP36742), on hippocampal-dependent memory and hippocampal nuclear CRE-binding activity in rats. As a result, acute in vivo administration of SGS742 both improved memory and reduced total hippocampal CRE-binding activity of which a large proportion in the basal state could be immunoneutralized with CREB2 antibodies. Consistent with its activity on information storage mechanisms, acute SGS742 effectively improved long-term memory in retrograde protocols, in which drug was given at times when memory formation can be interrupted by blocking new protein production. In conclusion, GABAB antagonists may provide a pharmacological therapy for cognitive impairment, sharing mechanistic features with genetic approaches to reduce CREB2 activity and to augment long-term memory.

Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element.

Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604-1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the "off" state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other cis-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in the A/D elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged.

Inducible long-term gene expression in brain with adeno-associated virus gene transfer.

Recombinant adeno-associated virus (rAAV) vectors hold promise for treating a number of neurological disorders due to the ability to deliver long-term gene expression without toxicity or immune response. Critical to these endeavors will be controlled expression of the therapeutic gene in target cells. We have constructed and tested a dual cassette rAAV vector carrying a reporter gene under the control of the tetracycline-responsive system and the tetracycline transactivator. Transduction in vitro resulted in stable expression from the vector that can be suppressed 20-fold by tetracycline treatment. In vivo experiments, carried out to 6 weeks, demonstrated that vector-transduced expression is sustained until doxycycline administration upon which reporter gene expression is reduced. Moreover, the suppression of vector-driven expression can be reversed by removal of the drug. These studies demonstrate long-term regulated gene expression from rAAV vectors. This system will provide a valuable approach for controlling vector gene expression both in vitro and in vivo.

Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

The proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5' end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis.

Evidence for a reexcitability gap in man after treatment with type I antiarrhythmic drugs.

The intention of this study was to determine whether type IA antiarrhythmic drugs cause a disparity between refractoriness and repolarization, and if so, its magnitude. We simultaneously measured monophasic action potential duration (MAPD) and effective refractory period (ERP) at a right ventricular site in 11 patients without overt right ventricular disease. The pacing protocol, which included sinus rhythm and a 600 and 400 msec cycle length of ventricular drive, was performed at baseline and after the intravenous administration of 15 mg/kg of procainamide in nine patients, and of 10 mg/kg of quinidine in two patients. Despite trivial changes in sinus rates, drug therapy caused a 10% to 17% increase in MAPD and ERP that shortened with decreasing drive cycle lengths. At baseline there was a small gap (10 to 13 msec) between MAPD and ERP in sinus rhythm and with a 600 or 400 msec drive. However, the type IA drug caused a significant widening of this gap that was most profound in sinus rhythm (45 msec) and that shortened but was not abolished with a 600 and 400 msec drive (28 and 29 msec, respectively). The disparity was caused in one third of cases by postrepolarization refractoriness. Type I drugs increase the difference between repolarization and refractoriness, and this effect is partially reversed with increases in heart rate. The clinical implications of these findings are discussed.

Laryngeal adenocarcinoma, not otherwise specified, treated with carbon dioxide laser excision and postoperative radiotherapy.

Glandular carcinomas of the larynx are aggressive neoplasms that comprise less than 1% of all laryngeal malignancies. Adenocarcinoma, not otherwise specified, is the most common histologic type. The rarity of these lesions has prohibited clarification of definitive therapy. Traditionally, radical surgery is performed because of the usually high stage at presentation and the relative insensitivity to radiotherapy. A case of glottic T1 adenocarcinoma, not otherwise specified, is reported. The tumor presented clinically as a vocal fold granuloma. The treatment modalities included endoscopic excision with the carbon dioxide laser and postoperative radiotherapy. The patient has been followed up for 39 months without evidence of recurrence or metastases. The current literature is reviewed in regard to treatment of laryngeal glandular carcinomas.

Angle-tipped Baron suction tubes.

Modified Baron no. 3 suctions angled at the tips have been found to be valuable during otologic surgery. The 3-mm length has been found most helpful and its use is recommended.

Is outpatient suction cautery tonsillectomy safe in a community hospital setting?

Tonsillectomy and adenotonsillectomy are frequently performed operations. They are typically done as a day-of-surgery admission with discharge on the first postoperative day. Five hundred consecutive tonsillectomies and adenotonsillectomies performed by the authors were retrospectively reviewed to determine if these procedures could safely be performed on an outpatient basis. Primary postoperative hemorrhage was found to be rare using the suction cautery technique. Secondary hemorrhage occurred most commonly on the sixth postoperative day and the overall postoperative bleed rate was 7%. Our conclusion was that suction cautery tonsillectomy and adenotonsillectomy were safe to perform on an outpatient basis.

The Haberman suction elevator.

A suction elevator for nasal surgery has been developed. Surgical trial has demonstrated effectiveness and I recommend consideration of its use.

False-positive MRI and CT findings of an acoustic neuroma.

CT and MRI are excellent imaging modalities available in the work-up of a patient with suspected acoustic neuroma. A case is presented where both CT and MRI suggested the presence of a small acoustic neuroma. Exploration of the cerebellopontine angle and internal auditory canal revealed no evidence of a tumor. A vascular loop around the inferior and superior vestibular nerves was identified and decompression completely eliminated the symptoms. Even with today's sophisticated diagnosis tools available to evaluate a suspected acoustic neuroma, the definitive diagnosis and subsequent treatment may change at surgery.

Serum reverse T3 and amiodarone efficacy.

The use of serum reverse T3 (rT3) levels in the assessment of amiodarone efficacy is controversial. We prospectively studied 10 patients with frequent ventricular ectopy and symptomatic ventricular tachycardia (VT) treated with amiodarone. Serial 24-h Holter monitor, thyroid function studies including serum rT3, and 12-lead electrocardiogram were performed on each patient at baseline, and at 1, 4, 12, and 24 weeks of oral amiodarone therapy. Serial Holter monitors on therapy were analyzed for 100% suppression of VT, 90% suppression of couplets, and 85% suppression of ventricular ectopic beats (VEBs) compared with baseline Holter, defining patient groups VT-, Co-, and VEB-, respectively. Lack of arrhythmia suppression to this degree defined groups as VT+, Co+, and VEB+. There were no statistically significant differences in rT3 levels between VT+ and VT- groups, Co+ and Co- groups, or the VEB+ and VEB- groups. VT suppression could not be predicted at any rT3 level. We conclude that serum rT3 is an insensitive means of assessing amiodarone efficacy.

Modification of horizontal jaws of alligator forceps.

Modification of horizontal opening alligator forceps has been developed. In my experience, this instrument has made certain maneuvers in otologic surgery easier.

Candida epiglottitis.

Acute epiglottitis is most commonly of bacterial origin. A case of Candida epiglottis in a healthy, nondebilitated patient is reported. A literature search did not produce a previously reported case.

Recurrent acquired atresia of the external auditory canal and associated canal cholesteatoma.

Acquired atresia of the external auditory canal associated with medial canal cholesteatoma is a rare finding. Recurrence of this pathologic picture redevelops three times. Various speculations regarding possible etiologic factors, as well as audiometry and treatment are discussed.