Platelet-derived endothelial cell growth factor gene therapy for limb ischemia

OBJECTIVES: Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to thymidine phosphorylase (TP), and it can induce angiogenesis, including arteriogenesis, in chronically ischemic canine myocardium. Because its effect on peripheral arterial disease has not been elucidated, we investigated whether overexpression of PD-ECGF/TP could ameliorate chronic limb ischemia in rabbits. METHODS: Left femoral arteries were resected from 24 male rabbits. After 10 days, a plasmid vector containing human PD-ECGF/TP complimentary DNA was injected into 10 sites in the adductor muscles. Control groups received either the LacZ plasmid vector or saline vehicle only (n = 8 per group). Blood pressure was measured in the calf before surgery, at the onset of ischemia, 10 days later, and 20 and 30 days after gene transfer. Collateral vessel development and limb perfusion were assessed by angiography, and resected tissues underwent molecular and histologic examination. RESULTS: In the PD-ECGF/TP group, human PD-ECGF/TP messenger RNA and protein were still detected at 30 days after treatment. Calf blood pressure decreased significantly after femoral artery resection in all three groups. It subsequently showed a greater increase in the PD-ECGF/TP group than in either control group, and the difference was significant at 20 days after treatment (PD-ECGF/TP, 97.4 +/- 7.4; LacZ, 58.6 +/- 6.9; saline, 41.3 +/- 3.6). Immunohistochemical staining demonstrated an increased ratio of capillaries and arterioles to muscle fibers in the PD-ECGF/TP group (2.14 +/- 0.13 and 1.51 +/- 0.06), but not in the LacZ group (1.39 +/- 0.04 and 0.71 +/- 0.05) or the saline group (1.34 +/- 0.05 and 0.71 +/- 0.04, P < .01). The angiographic score was higher in the PD-ECGF/TP group (0.96 +/- 0.08) than in the LacZ group (0.50 +/- 0.02) or saline group (0.51 +/- 0.03) at 30 days after gene transfer (P < .01). CONCLUSIONS: This study demonstrated that PD-ECGF/TP gene transfer induced angiogenesis and decreased ischemia in a rabbit hindlimb model by promoting arteriogenesis, suggesting that targeting this gene may be a promising therapeutic strategy for peripheral vascular disease.     

Adrenal androgen levels as predictors of outcome in castration‐resistant prostate cancer patients treated with combined androgen blockade using flutamide as a second‐line anti‐androgen

Objectives: To analyze the clinical effects of flutamide as a second‐line anti‐androgen for combined androgen blockade in patients with castration‐resistant prostate cancer (CRPC) initially treated with bicalutamide as a first‐line anti‐androgen. Methods: Our study population consisted of 16 patients with CRPC who were treated with flutamide (375 mg daily) as second‐line hormonal therapy. Dehydroepiandrosterone (DHEA), androstenedione, androstenediol, testosterone and dihydrotestosterone were measured to investigate the relationship between plasma androgens and outcome following treatment. Furthermore, adrenal androgen levels in a medium of adrenal cancer cell line were also measured. Results: Second‐line hormonal therapy using flutamide resulted in a reduction of the prostate‐specific antigen (PSA) level in 14 (87.5%) of 16 patients. A PSA decline greater than 50% was observed in 8 (50%) of the 16 patients. The duration of median responsiveness was 6.25 months. PSA elevation of baseline androstenediol level was a predictive factor of PSA responsiveness. The lower DHEA group improved the duration of responsiveness to flutamide. In vitro, 3 µmol/L flutamide suppressed DHEA, androstenedione and androstenediol synthesis compared with bicalutamide in a medium of adrenal cancer cell line. Conclusions: Our data show that flutamide suppresses the adrenal androgens in comparison with bicalutamide. The responsiveness and response duration of flutamide can be predicted in patients with a higher baseline androstenediol level and a lower DHEA level. Metabolites from adrenal androgens contribute to the progression of prostate cancer.     

Viability of liver grafts from fasted donor rats: relationship to sinusoidal endothelial cell apoptosis

Previous studies have shown that livers from fasted donors appear to tolerate long-term preservation better than livers from fed donors, but the mechanism is not clear. Some studies have shown that the apoptosis of sinusoidal endothelial cells (SEC) appeared to be a pivotal mechanism of ischemia/reperfusion injury in liver transplantation. The purpose of the present investigation was to evaluate the relation of SEC apoptosis to liver viability in rats after liver transplantation, comparing findings for fasted and fed donors. Wistar rats were used as donors and recipients. The fed group had access to solid feed and water ad libitum. The fasted group was allowed access only to water for 4 days prior to liver harvest. All rat livers were preserved with University of Wisconsin (UW) solution at 2 °C for 24 h. After preservation, the livers were orthotopically transplanted, and survival time was measured. Apoptosis was determined by in-situ staining for apoptotic cells, using a TdT-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay and electron microscope (EM) examination separately. The 14-day survival rates after 24-h preservation were 0% (0/11) for recipients of livers from fed donors and 91% (10/11) for recipients of livers from fasted donors. There was no significant difference in the numbers of TUNEL-positive SEC after 24-h preservation between the two groups. However, at 6 h after transplantation, the number of TUNEL-positive SEC was significantly higher in the fed group than in the fasted group. These results suggest that donor fasting decreases SEC apoptosis after reperfusion alone, and that this may be related to the protection of the liver graft from reperfusion injury. Received: December 22, 2000 / Accepted: February 15, 2001     

Nailfold capillary abnormality and pulmonary hypertension in systemic sclerosis

Background Patients with systemic sclerosis (SSC) show a capillary abnormality of nailfolds with controversial correlation with organ involvement. Our purpose was to study the correlation between this nailfold capillary abnormality and pulmonary hypertension in patients with SSC. Methods We studied the nailfold capillaries, using capillary microscopy, and the pulmonary arterial pressure, using right-heart catheterlzation, in 44 patients with SSC. Canonical discriminant analysis was used to define the capillary abnormality in patients with SSC, which was then compared with that of 40 normal controls. The correlations between the patterns of nailfold capillaries and the cardiopulmonary findings. Including the pulmonary arterial pressure, were examined using Fisher's test. Results Thirty-two of 44 patients with SSC could be differentiated from normal controls by our definition of the SSC pattern. The SSC pattern correlated significantly with elevated pulmonary vascular resistance, as well as with pulmonary fibrosis, eiectrocardiographic abnormalities, decreased vital capacity, and decreased diffusing capacity for carbon monoxide. All SSC patients with pulmonary hypertension showed this SSC pattern. In patients with elevated pulmonary arterial pressure, capillary microscopy and diffusion capacity for carbon monoxide (DCCM) showed the highest rate of abnormalities. A limited-type SSC significantly correlated with DCCM and with anticentromere antibody, and the diffuse-type SSC with pulmonary fibrosis and anti-sci-70 antibody. Conclusion Our data suggest that in patients with SSC, nailfold capillary abnormalities correlate with pulmonary arterial hypertension as well as with clinical and laboratory findings indicating pulmonary hypertension.     

REP-1 gene mutations in Japanese patients with choroideremia

· Background: Choroideremia (CHM) is an X-linked progressive dystrophy of the choroid, retinal pigment epithelium, and retina. Recently, the REP-1 gene was isolated and the causative mutations in the gene were detected in patients with CHM. In a previous study, we described a Japanese family with CHM who had a mutation in the REP-1 gene. In the present study, we performed extensive analysis of the REP-1 gene in patients with CHM from several institutions in Japan. · Methods: Twenty-six patients with CHM and 5 unaffected females from 22 independently ascertained families were examined. Exons 1–15 of the REP-1 gene were screened by single-strand conformation polymorphism. The DNA fragments suspected of any variations were directly sequenced. · Results: Fifteen different mutations, including one previously reported mutation, were detected in 18 families. In addition, carrier status was proven in four unaffected females found to be heterozygous for the mutant allele. · Conclusions: Fifteen different mutations of the REP-1 gene were detected in 18 Japanese families. There were no hot spots for the mutations and no missense mutations. The results show that REP-1 gene defects cause CHM in Japanese patients, and the mutations in these Japanese patients differed from the mutations reported for CHM patients in Europe, Canada, and America except for R267X and 1313delTC. These findings suggest that the mutations occurred independently in the Japanese patients. Received: 13 August 1998 Revised version received: 16 November 1998 Accepted: 9 December 1998     

Quantitative analysis of α1(I) and α1(III) procollagen mRNA expression in systemic sclerosis skin tissue – an in situ hybridization study

Abstract Human α1(I) and α1(III) procollagen mRNA expression in skin tissue from 15 systemic sclerosis (SSc) patients and from 7 normal control subjects was quantitatively analyzed using in situ hybridization. The grains accumulating in each area, representing procollagen mRNA expression per cell, were counted. To normalize the results from each subject, the number of cells and the number of grains per cell were divided by the area of the skin specimen (in square millimeters). The number of cells per square millimeter expressing α<1(I) and α1(III) procollagen mRNA in SSc skin was significantly elevated compared with normal control skin (both P < 0.01). The number of grains per cell per square millimeter expressing α1(III) procollagen mRNA in SSc skin was also significantly elevated compared with normal control skin (P < 0.01). The relationship between procollagen mRNA expression and the histological findings in SSc was also studied. The numbers of cells and grains per cell per square millimeter expressing α1(I) procollagen mRNA in fibrotic zone SSc skin were significantly elevated compared with normal control skin (both P < 0.01). The numbers of cells and grains per cell per square millimeter expressing α1(III) procollagen mRNA in SSc skin were significantly elevated compared with normal control skin (both P < 0.01) and with border zone SSc skin (number of cells P < 0.01, number of grains P < 0.05). These results indicate an increase in the number of cells showing elevated expression of α1(I) and α1(III) procollagen mRNA, and a close relationship between α1(I) and α1(III) procollagen mRNA expression and the histological findings in SSc. Received: 24 February 1999 / Received after revision: 20 May 1999 / Accepted: 16 August 1999     

Recent decline in hospital mortality among patients with acute myocardial infarction.

BACKGROUND: Many patients with acute myocardial infarction will still die after admission. Recent trends in hospital mortality were analyzed to identify aspects that need improvement. METHODS AND RESULTS: A total of 1,247 patients admitted to Kinki University School of Medicine within 24 h of the onset of infarction were analyzed between 1975 and 2001. The percentage of patients discharged with 100% occlusion decreased gradually from 31.3% during 1975-1982 to 2.1% during 1998-2001, while those with 50% stenosis or less gradually increased from 12.5% to 82.5% during the same period (trends: p < 0.01). The cardiac death rate was 17.1% in 1975-1982, and 7.7% in 1998-2001, showing a significant decrease with time (p < 0.01). This decrease was particularly marked among those admitted within 6 h of the onset of infarction. Death due to cardiac rupture decreased significantly with time (p < 0.001). In contrast, the non-cardiac death rate, amounting to 2.2% on average, did not decline. CONCLUSIONS: Cardiac deaths due to acute myocardial infarction have decreased markedly of late. However, patients must be admitted within 6 h of the onset of infarction to benefit from this improvement. More effort should be made to improve the general care of patients in order to reduce the incidence of non-cardiac death.     

Next-Generation Sequencing-Based Molecular Diagnosis of Choroideremia

We screened patients with choroideremia using next-generation sequencing (NGS) and identified a novel mutation and a known mutation in the CHM gene. One patient presented an atypical fundus appearance for choroideremia. Another patient presented macular hole retinal detachment in the left eye. The present case series shows the utility of NGS-based screening in patients with choroideremia. In addition, the presence of macular hole in 1 of the 2 patients, together with a previous report, indicated the susceptibility of patients with choroideremia to macular hole.Key Words: Choroideremia, Next-generation sequencing, Diagnosis, Utility