Lentiviral-mediated fetal gene therapy for monogenic disorders: development of an in vitro rabbit model.

OBJECTIVE: Fetal gene replacement is a novel, potential therapy for monogenic disorders which are diagnosed prenatally. The purpose of this study was to develop in vitro, respiratory-epithelium targeted, lentiviral (LV)-mediated gene transfer in fetal rabbit tracheas. METHODS: Via triple plasmid transfection, vesicular stomatitis virus-G (VSV-G)-pseudotyped LV vector containing green fluorescent protein (GFP) marker gene, under the control of a cytomegalovirus promoter was constructed. LV bioavailability in rabbit amniotic fluid (AF) was evaluated by infectivity assays of 293T cell monolayers in variable concentrations of AF. Fetal tracheas from time-mated rabbits (term gestation, G = 31 days) were collected on G 23-25 days, and placed in tissue culture (substrate-enriched DMEM, 37 degrees C, 5% CO(2)/room air). The tracheal cultures were transfected with 1 x 10(5) LV particles, and analyzed daily for: reporter gene by polymerase chain reaction, and reporter gene product (GFP) by whole-mount fluoroscopy and immunohistochemistry. RESULTS: 293T cell infectivity assays confirmed bioavailability of LV in rabbit AF. Following in vitro transfection, GFP DNA and GFP were detectable in fetal rabbit tracheas by 4 and 5 days, respectively. Immunocytochemistry localized GFP to the luminal aspect of tracheal epithelium. CONCLUSIONS: In vitro, LV-mediated GFP gene transfer to fetal rabbit tracheas occurs within 4 days, and gene expression is evident by 5 days post-transfection. This observation, and the bioavailability of LV through AF, suggests the appropriateness of this model for the future evaluation of in vivo, transamniotic gene delivery strategies.

An audit of cancer diagnosis in a Canadian children's hospital: Quality, timing and efficiency.

BACKGROUND AND OBJECTIVE: The diagnosis of paediatric cancer requires multidisciplinary cooperation to achieve both a timely diagnosis and efficient resource use. The authors undertook a 12-month audit of paediatric cancer cases to assess BC's Children's Hospital's (Vancouver, British Columbia) diagnostic process from the perspective of quality (timing and accuracy of diagnosis) and procedural efficiency, with an emphasis on the impact on resource use in the departments of radiology, pathology, anesthesia and surgery. METHODS: Malignancies (excluding brain and cortical bone primary tumours, for which the preoperative diagnostic workup is often completed before admission) diagnosed between January 1 to December 31, 2003, were reviewed. Data collected included total outpatient versus inpatient procedures, number and timing of diagnostic procedures, general anesthesia (GA) requirements, and lag times from admission to biopsy to diagnosis during the initial hospitalization. RESULTS: Fifty-four patients were identified. Only 10 patients (19%) had an outpatient diagnostic procedure. One hundred seventeen inpatient diagnostic procedures were performed, with only 50% occurring within regular working hours. Thirty-one per cent of patients required two or more procedural GAs during their initial hospital admission. The mean lag time to biopsy was 2.6 days and to a pathological diagnosis was 1.2 days. CONCLUSIONS: Despite timeliness, the process of cancer diagnosis at BC Children's Hospital requires hospital admission and a significant consumption of resources outside of regular working hours. Opportunities for improvement include maximizing outpatient workup, allocating oncology operating room time to increase the percentage of weekday procedures and improving interdisciplinary procedural coordination to reduce the GA requirements per patient.

Lentiviral vector-mediated, in vivo gene transfer to the tracheobronchial tree in fetal rabbits.

BACKGROUND/PURPOSE: Fetal gene replacement is a novel, potential therapy for antenatally diagnosed monogenic disorders. The purpose of this study was to evaluate in vivo techniques of lentiviral (LV) vector-mediated gene transfer to the tracheobronchial tree in a rabbit model of fetal gene therapy. METHODS: Via triple plasmid transfection, vesicular stomatitis virus-G-pseudotyped LV vector containing green fluorescent protein (GFP) reporter gene under the control of a cytomegalovirus promoter was constructed. In vivo gene transfer of 5 x 10(6) LV particles to fetuses of time-mated NZW rabbits (term = 31 days) was attempted using 2 techniques: (1) direct amniotic injection (gestation = 24-26 days) and (2) direct tracheal injection (gestation = 26 days). Injected fetuses and saline-injected littermate controls were delivered and killed on gestational day 30. Fetal and maternal tissues were analyzed. RESULTS: Both in vivo techniques produced gene transfer to fetal tissues (trachea, lung, liver, intestine), including those of some controls. In one prep, GFP DNA was identified in maternal lung. CONCLUSIONS: Lentiviral vector-mediated GFP gene transfer to fetal rabbit tracheobronchial epithelium occurs within 4 days of transfection by both amniotic injection or direct fetal tracheal injection. This in vivo model confirms bioavailability of vector through amniotic fluid with some cross-infection of adjacent fetuses. Vector access to fetal tissues appears to be by both luminal and hematogenous routes. Transplacental gene transfer from fetus to mother may occur in this model.

Can we predict the failure of thoracostomy tube drainage in the treatment of pediatric parapneumonic collections?

BACKGROUND/PURPOSE: Tube thoracostomy is a standard method of treating pediatric parapneumonic collections. Despite recent work denoting thoracoscopy as a superior method of treatment, few studies have looked at factors predictive of tube thoracostomy failure. We reviewed parapneumonic collections initially treated with tube thoracostomy to identify such factors. METHODS: Nontuberculous parapneumonic collections treated initially with tube thoracostomy over a 10-year period were reviewed. A "failed primary tube thoracostomy" was defined as the presence of worsening clinicoradiological signs requiring a further chest procedure (ie, thoracoscopy, thoracotomy, or second thoracostomy). RESULTS: Fifty-eight patients were identified. Forty-three percent failed primary tube thoracostomy. Within group F (failure group), 32% of patients had a concomitant medical condition (P < .001). Sixty percent of group F patients had duration of symptoms for more than 1 week compared with only 24% of group S (successful group) (P < .001). CONCLUSIONS: Our results suggest that primary treatment of parapneumonic collections with tube thoracostomy is likely to be unsuccessful in patients who are symptomatic for more than a week or who have a concomitant medical condition. A more aggressive primary surgical intervention is suggested for this group.