Development of the hybrid Sleeping Beauty: baculovirus vector for sustained gene expression and cancer therapy.

Antiangiogenesis is an appealing anticancer approach but requires continued presence of the antiangiogenic agents, which can be remedied by gene therapy. Baculovirus is an emerging gene delivery vector but only mediates transient expression (<7 days); thus, this study primarily aimed to develop a hybrid baculovirus for sustained antiangiogenic gene expression and cancer therapy. We first constructed plasmids featuring adeno-associated virus inverted terminal repeats (AAV ITRs), oriP/Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1) or Sleeping Beauty (SB) transposon and compared their efficacies in terms of persistent expression. In human embryonic kidney (HEK293) cells, AAV ITR failed to prolong the expression while oriP/EBNA1 moderately extended the expression to 35 days. In contrast, the SB system led to stable expression beyond 77 days even without antibiotic selection. Given this finding, we constructed a hybrid SB baculovirus expressing the SB transposase and harboring the transgene cassette flanked by inverted repeat/direct-repeat (IR/DR) elements recognizable by SB. The hybrid SB baculovirus efficiently transduced mammalian cells and mediated an expression duration longer than that by conventional baculoviruses, thanks to the transgene persistence and integration. The SB baculovirus (Bac-SB-T2hEA/w) expressing the antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) also enabled prolonged hEA expression. With sustained hEA expression, Bac-SB-T2hEA/w repressed the angiogenesis in vivo, hindered the growth of two different tumors (prostate tumor allografts and human ovarian tumor xenografts) in mice and extended the life span of animals. These data altogether implicated the potential of the hybrid SB-baculovirus vector for prolonged hEA expression and for the treatment of multiple types of angiogenesis-dependent tumors.

Baculovirus vectors for antiangiogenesis-based cancer gene therapy.

Baculovirus is an insect virus that is non-pathogenic to humans and has emerged as a promising gene therapy vector. Since solid tumor growth/metastasis critically relies on angiogenesis and hEA, a fusion protein comprising human endostatin and angiostatin, exhibits potent antiangiogenic and antitumor efficacy in mouse models; this study aimed to evaluate the feasibility of baculovirus for hEA expression and antiangiogenesis-based cancer gene therapy. Toward this end, we constructed Bac-hEA that mediated transient hEA expression and Bac-ITR-hEA that exploited the adeno-associated virus inverted terminal repeats (ITRs) for prolonged hEA expression. Western blot and ELISA analyses showed that both Bac-hEA and Bac-ITR-hEA expressed hEA in transduced mammalian cells, yet Bac-ITR-hEA only marginally prolonged the hEA expression. In comparison with Bac-hEA, nonetheless, Bac-ITR-hEA significantly enhanced the hEA expression level that concurred with augmented antiangiogenic properties, as demonstrated by cell proliferation, migration and tubule network formation assays. Importantly, intratumoral injection of Bac-ITR-hEA into prostate cancer mouse models, when compared with Bac-hEA, exerted stronger antiangiogenic effects in vivo, more potently inhibited tumor growth and significantly prolonged mouse survival. This study collectively supported the notion that hEA is an effective antiangiogenic protein and proved the potential of baculovirus as a vector for antiangiogenesis-based cancer therapy, which may be combined with chemotherapy, radiotherapy or gene therapies using other vectors.

RUNX1 mutations are frequent in chronic myelomonocytic leukemia and mutations at the C-terminal region might predict acute myeloid leukemia transformation.

Runt-related transcription factor 1 (RUNX1) is essential for normal hematopoiesis. RUNX1 mutations have rarely been reported in chronic myelomonocytic leukemia (CMML). We examined RUNX1 mutations in 81 patients with CMML at initial diagnosis. Mutational analysis was performed on bone marrow samples by direct sequencing of all reverse transcription PCR products amplified with three primer pairs that cover the entire coding sequences of RUNX1b. Thirty-two RUNX1 mutations were detected in 30 patients (37%); 23 mutants were located in the N-terminal part and 9 in the C-terminal region. The mutations consisted of 9 missense, 1 silent, 7 nonsense and 15 frameshift mutations. Two patients had biallelic heterozygous mutations. There was no difference in overall survival between patients with and without RUNX1 mutations, but a trend of higher risk of acute myeloid leukemia (AML) progression was observed in mutation-positive patients (16/30 vs 17/51, P=0.102), especially in patients with C-terminal mutations (P=0.023). The median time to AML progression was 6.8 months in patients with C-terminal mutations compared with 28.3 months in those without mutations (P=0.022). This study showed for the first time a high frequency of RUNX1 mutations in CMML. C-terminal mutations might be associated with a more frequent and rapid AML transformation.

Functional recovery of chronic complete idiopathic transverse myelitis after administration of neurotrophic factors.

STUDY DESIGN: Case report. OBJECTIVE: To evaluate the functional recovery of chronic complete idiopathic transverse myelitis (ITM) after administration of acidic fibroblast growth factor (aFGF). METHODS: A 28-year-old woman presented with a 4-year history of spastic paralysis, sensory level at T10, urinary retention and constipation due to ITM. In all, 20 microg aFGF bolus injection was applied via intradural lumbar puncture, which was repeated every 5 months for 15 months. RESULTS: At 3 weeks after first injection, the patient experienced vague sensation at approximately T12-L1 dermatomes. At 2 months after the second injection, muscle activities and gait pattern were recorded in bilateral gluteus and hip abductors as she ambulated with long leg brace and axillary crutches. Increased walking speeds, reduced pelvic tilting and reduced compensatory trunk rotation during the swing phase were also demonstrated as compared to the initial gait analysis. At 18 months after injection, motor evoked potentials were obtained in hip abductors of both legs. CONCLUSIONS: aFGF may increase the efficacy of spinal reactivation/regeneration and is a potential remedy for chronic transverse myelitis.

Integrating a health IC card system into a hospital information system.

As an experiment, a health IC card has been linked into the hospital information system of the National Cheng Kung University (NCKU) Hospital. The NCKU Hospital Information System (NCKU-HIS) services all the procedures for patient registration, billing, prescriptions, and the processing and storing of test results. The health IC card system provides good quality information to the doctors, the hospital, the patients, and the insurance organizations. For a fully comprehensive system, it is essential to integrate the health IC card system into the operational NCKU-HIS, the communication protocols, and the national insurance procedures. The experiment was designed to see if the development of such a system was feasible, and the results have been impressive. It has proved possible to operate the health IC card system, linking into different machines under different computing environments. A single computer system to store all the data of all the patients and to process all the nation's healthcare information would require almost unlimited mainframe processing power and unlimited storage. It would be very difficult to construct such a large computerized information system. All the hospitals would be required to use the same data format to process their information, and a large national healthcare network would have to be built, making the collection and storage of personal medical information very difficult and probably inefficient and unsatisfactory. Even if it was possible, it would be a very costly and time-consuming task. An IC card is cheap and easy to use, and offers an alternative solution to the problem of handling the nation's healthcare information. The original healthcare IC card experiment (started in 1990) was limited to the NCKU Hospital and the Taiwan Government Employees' Clinic. The experimental system was designed to be fully integrated into the NCKU-HIS and has been widely accepted by the users, especially the IC card-holding patients. It is also well-suited to the management of healthcare insurance. An expanded healthcare IC card system was implemented from the end of 1993 and covers six clinics and six hospitals in Penghu County. The operation of the registration and billing subsystems are supported by PC workstations in a client/server network as these subsystems are used very frequently. Some of the other computerized functions in the NCKU-HIS are supported by the mainframe TANDEM computer system. The interfaces with the communication protocols and application software provide for the effective two-way transfer of data and programs. For future use, consideration is being given to the employment of the Health Level Seven (HL/7) protocol for electronic data interchange. National standards such as Chinese information processing, the use of standard codes, the authorization of the content of the IC card, and communication protocols are already incorporated in the system.

Activation of epileptogenic activities by etomidate in electrocorticoencephalography (ECoG) during operation for epilepsy.

Epileptic surgery is a radical and ablative treatment for medically refractory epilepsy. Electrocorticoencephalography (ECoG) obtained by subdural strip electrodes should always be used during operation for precise localization of epileptic focus and mapping the extent of its involvement. But difficulties and ambiguities exist when the patient's ECoG is suppressed, either owing to the anticonvulsant used, being at the stage of posterictal period, or the effects of anesthetics during operation. Then, intentional activation of epileptogenic activity should be performed to locate the pathological focus in order to accomplish a successful surgical treatment. Etomidate has been considered as an abnormal electroencephalogram (EEG) activator and its use is not recommended in patients with epilepsy. But lesser dose of etomidate as an activator for ECoG has not been investigated. The study reported here established that etomidate as an hypnotic has not only anesthetic properties but, paradoxically, also activates epileptogenic activity. With the latter pharmacologic characteristic, etomidate can be used to deliberately activate the spikes of the potentially epileptogenic tissue, the delineation and localization of which may help the surgeon determine to what extent the pathological cortex be resected in surgical treatment of a refractory epilepsy. The current recommended dosage of etomidate at veterans General Hospital-Taipei for ECoG in epileptic surgery is 0.1-0.15 mg/kg/iv.

Quantitative structure-activity relationships and possible mechanisms of action of bispyridinium oximes as antidotes against pinacolyl methylphosphonofluoridate.

The antidotal efficacy of bispyridinium oximes against the poisoning by pinacolyl methylphosphonofluoridate can be correlated well with their physicochemical parameters. Good correlation was observed between the efficacy of antagonism against pinacolyl methylphosphonofluoridate and antinicotinic action of these oximes. X-ray structural analysis showed that these oximes possessed structural similarity to nicotine and acetylcholine of nicotinic conformation. A new model of antidotal action, other than reactivation, against the poisoning by pinacolyl methylphosphonofluoridate was proposed for these bispyridinium oximes through nicotinic receptor binding. The antagonistic efficacy of these antidotes against pinacolyl methylphosphonofluoridate may be attributed to their direct antagonism at nicotinic receptor as well as reactivation of inhibited acetylcholinesterase.

Heterogeneous platinum-catalyzed hydrogenation of dialkyl(diolefin)platinum(II) complexes: A new route to platinum surface alkyls.

Platinum metal catalyzes the reduction of dialkyl(diolefin)platinum(II) complexes by dihydrogen to alkanes and platinum(0). The reaction involves adsorption of the platinum(II) complex on the platinum(0) catalyst surface with conversion of the alkyl moieties to platinum surface alkyls; these appear as alkane products. The platinum atom originally present in the soluble organoplatinum species becomes part of the platinum(0) surface.