[The association between frontotemporal connecting fibers and seizure severity in patients with temporal lobe epilepsy].

The patients with temporal lobe epilepsy (TLE) admitted in the Department of Neurology, Zhongshan Hospital, Fudan University from June 2009 to February 2012 were prospectively enrolled. The diffusion tensor imaing was performed on the patients at the time of enrollment and 3 years later. The fractional anisotropy (FA) values of the white matter connecting fibers(bilateral hooked, arcuate, cingulate, and superior longitudinal tracts), the connecting fibers of both hemispheres(anterior union, anterior callosal forceps, posterior forceps, and bilateral fornix), and fibers of perirhinal cortices system(bilateral radiating crown and anterior limb of the internal capsule) were measured by the region of interest method. The severity of epilepsy was evaluated using the Veterans Administration Seizure Type and Frequency Rating Scale(VA-2) and National Hospital Seizure Severity Scale (NHS3). A total of 51 patients with TLE were screened, with 27 patients completing the 3-year follow-up. There were 13 males and 14 females with an age of (32±11) years and a follow-up duration of (39.1±1.1) months. During the follow-up, 6 patients had increased/unchanged NHS3 or VA-2 scores, while 21 patients had decreased scores. Three years later, the FA values of the bilateral arcuate fasciculus, the right superior longitudinal fasciculus, the right radial coronal and corpus callosum anterior forceps in TLE patients decreased compared to baseline(P<0.05). However, compared to the patients with decreased VA-2 scores during the follow-up, the degree of increase in FA values (ΔFA, follow-up FA value-baseline FA value) of the ipsilateral hook bundle caused by epilepsy was more significant in the group with increased/unchanged VA-2 scores (decreased score group vs increased/unchanged score group:-0.032±0.063 vs 0.018±0.043, t=2.305, P=0.035). The value of ΔFA in epileptic patients with increased/unchanged NHS3 scores (0.075±0.113) was higher compared to those with decreased scores (-0.079±0.099, t=2.804, P=0.010). Correlation analysis also showed the changes in FA values of epileptic lateral fasciculus (r=0.503, P=0.009) and arcuate fasciculus (r=0.602, P=0.001)were positively correlated with the changes in VA-2 and HNS3 scores, respectively. The seizure severity in patients with TLE was closely associated with the microstructure changes in the frontal and temporal white matter, especially the arcuate and uncinate tracts, on the same side that caused seizures, which may indicate the white matter remodeling and abnormal network reformation associated with seizures.

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.

[Pharmacokinetics of contragestazol (DL-111-IT), a new non-steroid antifertility agent in monkeys].

The pharmacokinetics of contragestazol, an early pregnancy temperating agent [3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole, DL-111-IT] was studied in Rhesus monkey. The blood concentration of DL-111-IT was determined by coupled column system HPLC method. Using an aqueous vehicle (20% cremophor EL in saline) DL-111-IT was injected intravenously to monkeys at doses of 25, 12.5 and 6.3 mg.kg-1. Blood drug concentration were measured. Using a programmable calculator the calculated pharmacokinetic parameters were as follows: alpha 1.83 h-1, 4.71 h-1 and 3.61 h-1, beta 0.15 h-1, 0.08 h-1 and 0.09 h-1; T1/2 beta 6.63 h, 10.2 h and 10.1 h; AUC 9.54 micrograms.h-1.ml-1, 3.94 micrograms.h-1.ml-1 and 3.75 micrograms.h-1.ml-1. An oil solution of DL-111-IT was injected intramuscularly in monkeys at doses of 50, 25 and 12.5 mg.kg-1. Its blood concentrations were determined at 0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 8, 12 and 24 h after administration. From the time vs concentration curve, the pharmacokinetic parameters obtained were as follows: Ka 0.98 h-1, 1.03 h-1 and 1.45 h-1; Ke 0.42 h-1, 0.37 h-1 and 0.60 h-1; T1/2Ke 1.66 h, 1.90 h and 1.16 h; T (peak) 1.52 h, 1.57 h and 1.09 h; AUC 4.86 micrograms.h-1.ml-1, 5.61 micrograms.h-1.ml-1 and 1.74 micrograms.h-1.ml-1.

[Studies on quantitative determination of total flavonoid in qingpi, zhiqiao and zhishi by TLC-densitometric methods].

A method for the determination of total flavonoid contents in Pericarpium Citri Reticulatae Viride (Qingpi), Fructus Aurantii (Zhiqiao) and Fructus Aurantii Immaturus (Zhishi) was established by the use of TLC-densitometry. The results show that the total flavonoid contents in Qingpi and Zhishi are higher than in Zhiqiao. The content in Zhiqiao is 9.81%-15.85%.