Overexpression of Ad5 precursor terminal protein accelerates recombinant adenovirus packaging and amplification in HEK-293 packaging cells.

Recombinant adenoviruses are one of the most common vehicles for efficient in vitro and in vivo gene deliveries. Here, we investigate whether exogenous precursor terminal protein (pTP) expression in 293 cells improves the efficiency of adenovirus packaging and amplification. We used a piggyBac transposon-based vector and engineered a stable 293 line that expresses high level of Ad5 pTP, designated as 293pTP. Using the AdBMP6-GLuc that expresses green fluorescent protein (GFP), BMP6 and Gaussia luciferase, we found that the infectivity of AdBMP6-GLuc viral samples packaged in 293pTP cells was titrated up to 19.3 times higher than that packaged in parental 293 cells. AdBMP6-GLuc viral samples packaged in 293pTP cells exhibited significantly higher transduction efficiency in 143B and immortalized mouse embryonic fibroblast (iMEF) cells, as assessed by fluorescence-activated cell sorting analysis of GFP-positive cells, the luciferase activity assay and BMP6-induced osteogenic marker alkaline phosphatase activities in iMEFs. When adenovirus amplification efficiency was analyzed, we found that 293pTP cells infected with AdBMP6-GLuc yielded up to 12.6 times higher titer than that in parental 293 cells, especially at lower multiplicities of infection. These results strongly suggest that exogenous pTP expression may accelerate the packaging and amplification of recombinant adenoviruses. Thus, the engineered 293pTP cells should be a superior packaging line for efficient adenovirus production.

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma.

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery.

Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.

Fluorescence-based functional assay for Wnt/beta-catenin signaling activity.

Aberrant activation of beta-catenin signaling has been implicated in the development of human cancers. As a Wnt signal transducer, beta-catenin forms a complex with the lymphocyte enhancer-binding factor/T cell factor transcription factor and activates downstream targets that promote cell proliferation. Here we developed a Wnt-dependent beta-catenin-mediated heterologous transactivation system, which consisted of a chimeric transcription factor constructed by fusing the GAL4 DNA-binding domain with the full-length beta-catenin, and a GAL4-responsive reporter expressing GFP. The chimeric transcription factor was highly unstable and exerted no detectable transactivating effect on the GAL4-responsive reporter. However, lithium and Wnt1 significantly stabilized this chimeric transactivator, indicating that this transactivation system is regulated by beta-catenin in a Wnt-responsive fashion. Thus, this transactivation system could be used as a functional reporter to identify potential upstream factors that deregulate beta-catenin signaling during tumorigenesis, as well as to screen for potential anti-cancer agents that specifically inhibit beta-catenin signaling in human tumors.

Tetracycline-regulated gene expression mediated by a novel chimeric repressor that recruits histone deacetylases in mammalian cells.

Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be developed. Histone deacetylases have recently been shown to play an important role in regulating gene expression. Here we investigated whether a more tightly controlled expression could be achieved by using a novel chimeric repressor that recruits histone deacetylases to a tetracycline-responsive promoter. This chimeric repressor was engineered by fusing the tetracycline repressor (TetR) with an mSin3-interacting domain of human Mad1 and was shown to bind the tetO(2) element with high affinity, and its binding was efficiently abrogated by doxycycline. The chimeric repressor was shown to directly interact with mSin3 of the histone deacetylase complex. This inducible system was further simplified by using a single vector that contained both a chimeric repressor expression cassette and a tetracycline-responsive promoter. When transiently introduced into mammalian cells, the chimeric repressor system exhibited a significantly lower basal level of luciferase activity (up to 25-fold) than that of the TetR control. When stably transfected into HEK 293 cells, the chimeric repressor system was shown to exert a tight control of green fluorescent protein expression in a doxycycline dose- and time-dependent fashion. Therefore, this novel chimeric repressor provides an effective means for more tightly regulated gene expression, and the simplified inducible system may be used for a broad range of basic and clinical studies.

High-dose intra-arterial cisplatin boost with hyperfractionated radiation therapy for advanced squamous cell carcinoma of the head and neck.

PURPOSE: To evaluate the tolerance and efficacy of intra-arterial (IA) cisplatin boost with hyperfractionated radiation therapy (HFX-RT) in patients with advanced squamous cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: Forty-two patients with locally advanced primary SCCHN were treated on consecutive phase I/II studies of HFX-RT (receiving a total of 76.8 to 81.6 Gy, given at 1.2 Gy bid) and IA cisplatin (150 mg/m(2) received at the start of and during RT boost treatment). RESULTS: Acute grade 3 to 4 toxicities were as follows: grade 4 and grade 3 mucosal toxicity occurred in three (7%) and 31 patients (69%), respectively, and grade 3 hematologic, infectious, and skin events occurred in one patient each. Eight of 24 patients (33%) were unable to receive a second planned dose of IA cisplatin because of general anxiety (n = 5), nausea and/or emesis (n = 2), or asymptomatic occlusion of an external carotid artery (n = 1). Thirty-seven patients (88%) experienced complete response (CR) at primary site. Twenty-nine (85%) of 34 patients presenting with nodal disease experienced CR. The actuarial 2-year rates of locoregional control and disease-specific and overall survival are 73%, 63%, and 57%, respectively, with a median active follow-up of 30 months. CONCLUSION: In this highly unfavorable subset of patients, these results seem superior to previously reported chemoradiation regimens in more favorable patients. Use of a second dose of IA cisplatin boost was associated with increased toxicity without obvious therapeutic gain. This novel strategy allows for an incremental increase in the treatment intensity of the HFX-RT regimen recently established as superior to once-a-day RT.

The relationship between dysphonia and sinusitis: a pilot study.

Though some clinicians believe chronic sinusitis is associated with dysphonia, studies confirming such a relationship are not available. This pilot study investigated vocal characteristics of chronic sinusitis patients compared to those of nonsinusitis patients. Nineteen male patients were evaluated in a single-blind, nonrandomized, prospective study at a Department of Veterans Affairs Medical Center. After candidates for the experimental and control groups were thoroughly screened [nasal and laryngology examinations, sinus computed tomography (CT) scan, history], 10 chronic sinusitis patients were assigned to the experimental group, nine patients without chronic sinusitis were assigned to the control group. Visi-Pitch analysis of their speech evaluated fundamental frequency, intensity, and perturbation. The evaluator described voice quality and vowel prolongation. Videostroboscopic examination evaluated glottic closure, supraglottic activity, vocal fold edge, amplitude, mucosal wave, and nonvibrating portion. Visi-Pitch analysis and vowel prolongation for each group was compared using an independent t-test. The average fundamental frequency mean was 113.43+/-32.75 Hz for the control group and 112.29+/-19.71 for the experimental group. This study established methods and parameters to determine if a relationship between chronic sinusitis and dysphonia actually exists. It did not establish a relationship between these entities, possibly due to the small sample size enrolled in the study, but determined that 126 subjects would be necessary to achieve significant results.

Auricular endochondral pseudocysts: diagnosis and management.

The auricle can be the site of a variety of cystic lesions, many of which involve either potential spaces between the auricular cartilage and the perichondrium or spaces within the skin and subcutaneous tissues. An auricular endochondral pseudocyst is a fluid collection located within the cartilaginous structure of the auricle. The auricular hematoma and the auricular pseudocyst may represent opposing ends of a continuum of damage and repair of traumatic insults. Whereas the hematoma represents a significant acute traumatic event resulting in cleavage between the perichondrium and the cartilage, the pseudocyst could be the outcome of chronic lower grade trauma. In this instance, the perichondrium not only separates from the cartilage but may be induced to regenerate an outer cartilaginous wall exacting the contour of the separated perichondrium, thus completing a cartilaginous auricular pseudocyst. This firm, cartilaginous outer cyst wall accounts for the increased difficulty in treating pseudocysts and clearly requires a different surgical treatment than for the hematoma. Having reviewed a substantial number of case reports, we recommend incision and drainage of the cavity followed by obliteration of the cavity by curettage, sclerosing agent, and pressure dressing. More invasive techniques should be reserved for an uncommon recurrence after adequate initial treatment.

The reliability of six faculty members in identifying important OSCE items.

PURPOSE: To measure the agreement among faculty members about the importance of items on a checklist used to grade an objective structured clinical examination (OSCE) station. METHODS: Six faculty members rated the importance of 47 items for an OSCE station in which students took the history of a patient with sore throat and hoarseness. RESULTS: Of the 47 items, the raters independently identified 15 items as very important. The reliability of each individual rater was fair (averaged value of alpha = .63.) The reliability of the mean rating of the six raters was high (alpha = .91). CONCLUSIONS: The results strongly suggest that when a group of faculty members cooperatively identifies the important items to be included in an OSCE checklist, the reliability of the checklist is superior to one created by a single author.

Prophylactic antibiotic drops after tympanostomy tube placement.

OBJECTIVE: To evaluate the effectiveness of prophylactic polymyxin B sulfate-neomycin sulfate-hydrocortisone drops in decreasing the incidence of posttympanostomy otorrhea. DESIGN: Prospective randomized controlled study. SETTING: University referral center. PATIENTS: Three hundred patients undergoing tympanostomy tube placement (including those undergoing tonsillectomy, adenoidectomy, or both) were randomized into three groups. INTERVENTION: The use of polymyxin B-neomycin-hydrocortisone drops. Patients in group 1 received no antibiotic drops; group 2, a single dose intraoperatively, and group 3, an intraoperative dose followed by a 5-day course. MAIN OUTCOME MEASURE: Posttympanostomy otorrhea. RESULTS: A statistically significant decrease was observed in the incidence of posttympanostomy otorrhea between the control (16.4%) and treatment groups (group 2, 8.3%; group 3, 8.1%) (P = .011). A single dose of antibiotics was effective when patients' middle ears were dry or had serous effusions. A 5-day course was indicated for those whose ears had mucoid or purulent contents. CONCLUSIONS: Antibiotic ear drops are indicated in all patients. A single dose is as effective as a 5-day course, but our data support a longer course in certain subgroups.

Anomalies of the branchial apparatus: a case report and review of embryology, anatomy and development.

The majority of congenital anomalies occurring in the neck are related to the development of the branchial apparatus. This area is responsible for the development of multiple structures in the head and neck and thus its anatomy and embryology are complex. Although cysts, sinuses, and fistulas of the branchial region are not common occurrences, they do present in a variety of settings and are generally easily managed if diagnosed early. A fundamental understanding of their anatomy, embryology, and presentation is needed to appropriately diagnose and manage them. These fundamentals are reviewed and a case report is used to illustrate one of the more common presentations.

Use of standardized patients to identify deficits in student performance and curriculum effectiveness.

Much literature supports the value of performance-based tests for obtaining an overall assessment of clinical skills; however, these tests can also identify specific deficits in medical curriculum and student learning. This study, conducted over a period of 2.5 years, used standardized patients to identify deficiencies in medical students' evaluation of a patient with "hoarseness and cough." Each of 230 students performed a physical examination and took a medical history, and each was evaluated on 29 items according to predetermined criteria. We conclude the following: (1) When examining a patient complaining of hoarseness and cough, students frequently did not screen for hemoptysis, thyroid problems, and otalgia; (2) Overall test performance correlated positively with the time of year of the rotation, but screening for certain items (including hemoptysis, thyroid problems, and otalgia) appeared not to depend on rotation; (3) Several important items significantly differentiated the more knowledgeable student from the less knowledgeable student.

Intracranial pressure changes during bilateral radical neck dissections.

The management of the contralateral neck in patients with head and neck cancer who have undergone a radical neck dissection (RND) is controversial. A number of these patients will require a second RND. Sacrifice of both internal jugular veins (IJV) has been felt to lead to increased intracranial pressure (ICP) with subsequent neurologic sequelae. From 1987 to 1991 four patients had staged bilateral RNDs at the West Virginia University. In these patients a subarachnoid bolt was placed to directly monitor ICP. Jugular bulb, mean arterial, pulmonary artery, and central venous pressures were monitored. Electroencephalographic (EEG) monitoring was also performed. All patients demonstrated elevations in ICP immediately on head rotation. Further marked elevations were noted immediately after IJV ligation with a maximum peak at 30 minutes. Pressure levels of greater than 40 mm Hg were observed in three of four patients. Systemic hypertension was observed in response to elevated ICP (Cushing's reflex). All patients studied recovered from surgery without significant sequelae. Within 24 hours the ICP had returned to normal in all patients. Three patients required intraoperative intervention to lower their ICP. We demonstrate that even in a staged second RND there are significant rises in ICP. These are to a level that suggests emergency medical intervention is required. We feel that when the second IJV is sacrificed an increase in ICP should be anticipated, monitored, and treated accordingly.

Audiometric thresholds in osteomyelitis patients treated with gentamicin-impregnated methylmethacrylate beads (Septopal).

Use of gentamicin-impregnated methylmethacrylate beads is a relatively new and effective method of treating osteomyelitis. Although previous studies have concluded that such therapy is associated with low systemic toxicity, no data are available relating the use of this treatment to ototoxicity and hearing loss. Audiometric thresholds from 28 cases of osteomyelitis treated by surgical debridement and long-term systemic antibiotics alone (14 subjects) or gentamicin-impregnated methylmethacrylate beads either alone or combined with systemic antibiotics (14 subjects) were analyzed. Results showed one permanent threshold shift in the gentamicin-impregnated methylmethacrylate beads group (8%) versus four permanent threshold shifts in the systemic antibiotics alone group (29%). Osteomyelitis patients treated with gentamicin-impregnated methylmethacrylate beads are at no more risk and are probably at less risk of experiencing ototoxicity than patients treated with conventional long-term systemic antibiotics.

Cutaneous squamous carcinoma and related lesions.

The epidemiologic, etiologic, and prognostic factors in cutaneous squamous carcinoma depend heavily upon the status of the immune systems of the skin and body. Because UV radiation is both mutagenic and immunosuppressive, exposure to it will ultimately set the stage for the development of cutaneous carcinoma. If other immunobiologic-altering factors are added to UV radiation exposure, then both a susceptibility to invasion by oncogenic viruses and the development of squamous cell disorders, including squamous cell carcinoma, will exist. Although these factors are not totally controllable, a better understanding of them will allow the physician to more effectively tailor preventive care, treatment decisions, and counseling advice.

Comparison of complications following frontal sinus fractures managed with exploration with or without obliteration over 10 years.

Two hundred twelve patients were treated for facial or skull trauma at the West Virginia University Hospital between the years 1977 and 1987. Sixty-six of these patients had frontal sinus or nasofrontal duct trauma. Follow-up information was obtained on 64 of these patients through clinic visits, chart review, questionnaires to patients and physicians, and telephone calls to the patients. Follow-up greater than 1 year was obtained on 52 patients. Sixty-four patients were managed either with a frontal sinus obliteration or with an open exploratory procedure. The incidence of complications occurring in the past 10 years after each of these procedures is compared. Because the indications for each procedure vary somewhat, data is presented on fracture etiology, associated injuries, specific fracture location, fracture displacement, severity of injury, and associated cerebrospinal fluid leaks.

Nontraumatic subluxation of the atlantoaxial joint: Grisel's syndrome.

Grisel's syndrome is defined as subluxation of the atlantoaxial joint not associated with trauma or bone disease. Primarily an affliction of children, the disorder may occur in association with any condition that results in hyperemia and pathologic relaxation of the transverse ligament of the atlantoaxial joint, including several common otolaryngic entities. Grisel's syndrome has been noted infrequently in the otolaryngologic literature; this paper reports a case from West Virginia University Hospital of a 6-year-old boy with radiographic evidence of retropharyngeal cellulitis following meningitis progressing to atlantoaxial subluxation. Discussed are the pathophysiologic aspects of Grisel's syndrome with an anatomic explanation recently described to account for the syndrome.