Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components

Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.     

Comparison of the effects of cilnidipine and amlodipine on ambulatory blood pressure.

Cilnidipine is a novel and unique 1,4-dydropyridine derivative calcium antagonist that exerts potent inhibitory actions not only on L-type but also on N-type voltage-dependent calcium channels. Blockade of the neural N-type calcium channel inhibits the secretion of norepinephrine from peripheral neural terminals and depresses sympathetic nervous system activity. The purpose of this study was to assess the effect of cilnidipine and amlodipine on ambulatory blood pressure (BP) levels. We performed 24-h ambulatory BP monitoring before and after once-daily use of cilnidipine (n=55) and amlodipine (n=55) in 110 hypertensive patients. Both drugs significantly reduced clinic and 24-h systolic BP (SBP) and diastolic BP (DBP) (p < 0.005). However, the reductions of 24-h (-1.19+/-6.78 vs. 1.55+/-6.13 bpm, p=0.03), daytime (-1.58+/-6.72 vs. 1.68+/-7.34 bpm, p=0.02) and nighttime (-1.19+/-5.72 vs. 1.89+/-6.56 bpm, p=0.01) pulse rate (PR) were significantly greater in the cilnidipine group than the amlodipine group. There was no correlation between the degree of daytime SBP change and that of daytime PR change after amlodipine treatment (r=-0.08, n.s.), but there was a significant negative correlation between the degree of daytime SBP change and that of day-time PR change after cilnidipine treatment (r=-0.27, p<0.05). N-type calcium channel blockade by cilnidipine may not cause reflex tachycardia, and may be useful for hypertensive treatment.     

Left ventricular oozing rupture following acute myocardial infarction

We describe the case of an 85-year-old woman in whom pericardiocentesis, prolonged bed rest and blood pressure control were performed without surgery to successfully treat an oozing-type myocardial rupture due to myocardial infarction.     

A case of intracoronary protruded thrombus caused after bailout stenting for side branch occlusion

A 69-year-old woman underwent percutaneous coronary intervention for a severe stenotic lesion in the bifurcation of the mid-left anterior descending artery and first diagonal branch. A single stent was implanted into the left anterior descending artery. After the stent strut was dilated by balloon inflation in the diagonal branch, dissection occurred at the ostium of the diagonal branch and resulted in side branch occlusion due to hematoma. Bailout stenting was performed in the diagonal branch, but thrombus projection occurred in the left anterior descending artery. Aspiration, balloon inflation and thrombolytic therapy were performed, but distal embolism developed. This case illustrates that thrombus projection caused by stenting in a side branch may occur as a rare complication in percutaneous coronary intervention.     

Correlation of anatomic and hemodynamic features with aortic valve leaflet deformity in doubly committed subarterial ventricular septal defect

Summary The records of 153 patients with doubly committed subarterial ventricular septal defect (DCVSD) who underwent intracardiac repair were analyzed to evaluate factors responsible for aortic valve leaflet deformity. The patients were divided into two groups according to their echocardiographic and angiographic features as well as anatomic findings at operation: DCVSD without (17/153, 11.1%) and with arterial valve offsetting (136/153, 88.9%). Aortic regurgitation (AR) was much more prevalent in the patients with (50.0%) than in those without leaflet deformity (2.2%,P < 0.01). Arterial valve offsetting is one of the major contributing factors to the development of leaflet deformity, accounting for 5.9% in the patients without offsetting and 46.3% in those with offsetting (P < 0.01). Among the patients with arterial valve offsetting, the pulmonary-to-systemic pressure ratio was significantly higher (P < 0.01) in the patients without (0.76 ± 0.14) than in those with leaflet deformity (0.36 ± 0.12), suggesting that pulmonary hypertension might prevent the aortic valve leaflet from prolapsing in DCVSD. In addition, increased severity of aortic valve leaflet deformity and subsequent AR were observed with increasing age. These results suggest that aging and the presence of arterial valve offsetting as well as the absence of pulmonary hypertension might be factors responsible for aortic valve leaflet deformity and subsequent AR in DCVSD. The anatomic and hemodynamic features in DCVSD have a great impact on the development of aortic valve leaflet deformity and subsequent AR.     

Identification of quantitative trait loci for cardiac hypertrophy in two different strains of the spontaneously hypertensive rat.

Cardiac hypertrophy and left ventricular hypertrophy are known to be substantially controlled by genetic factors. As an experimental model, we undertook genome-wide screens for cardiac mass in F2 populations bred from the stroke-prone spontaneously hypertensive rats (SHRSP) and normal spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) of a Japanese colony. Two F2 cohorts were independently produced: F2(SHRSP x WKY) (110 male and 110 female rats) and F2(SHR x WKY) (151 male rats). The ratio of heart weight to body weight (Hw/Bw) was evaluated at 12 months of age in F2(SHRSP x WKY) after salt-loading for 7 months, and at around 15 weeks of age in F2(SHR x WKY) who had been fed a normal rat chow diet. Subsequent to an initial screen with 251 markers in F2(SHRSP x WKY) male progeny, 170 and 161 markers were selected and characterized in F2(SHRSP x WKY) female progeny and F2(SHR x WKY) male progeny, respectively. Markers from four chromosomal regions showed suggestive or significant linkage to Hw/Bw. The strongest and the most consistent linkage was found in the vicinity of D3Mgh16 on rat chromosome (RNO) 3 (a maximal log of the odds score reached 4.0 to 6.6 across the F2 populations studied). In the other three regions on RNO6, RNO10 and RNO13, the degree of linkage was more prominent in either males or females. These data provide solid evidence for a "principal" RNO3 quantitative trait loci regulating Hw/Bw in SHRSP and SHR, and also suggest the possible presence of sexual dimorphism in regard to genetic susceptibility for cardiac hypertrophy.     

Intravascular brachytherapy for native coronary ostial in-stent restenotic lesions

OBJECTIVES: We analyzed the effects of vascular brachytherapy (VBT) on ostial in-stent restenosis (ISR). BACKGROUND: In-stent restenosis has a high recurrence rate after percutaneous reintervention. The recurrence rate of ostial ISR lesions and the impact of VBT remain unknown. METHODS: We evaluated 133 patients with native coronary ostial ISR from a pooled database of 990 patients enrolled in randomized VBT trials. Independent quantitative angiography was performed at baseline and follow-up in 45 gamma, 27 beta, and 61 placebo patients. RESULTS: Binary restenosis was significantly higher in placebo than radiated patients (75.4% vs. 17.8% in gamma vs. 22.2% in beta, p < 0.0001). The treatment effect of both gamma (odds ratio [OR] 0.06; 95% confidence interval [CI] 0.02 to 0.17) and beta VBT (OR 0.10; 95% CI 0.03 to 0.31) was maintained after controlling for differences in baseline lesion length. Proximal and distal radiation edge restenosis rates were similar among the groups. Vascular brachytherapy of true aorto-ostial lesions (n = 34) was similarly beneficial: restenosis rates of placebo versus gamma or beta patients of 83.3% versus 6.7% versus 28.6%, p = 0.0002. CONCLUSIONS: Conventional treatment of ostial ISR is associated with a recurrence rate of over 75%. Vascular brachytherapy with either gamma or beta sources results in significant and similar reductions in restenosis compared with placebo. Similar benefits after VBT prevail in true aorto-ostial lesions.