Effects of SV40 T antigen transduction on human corneal endothelial cell wound healing in vitro.

OBJECTIVE: The aim of this study was to investigate the endothelial wound healing effects of SV40 large T and small t antigen transduction on cultured human corneal endothelial cells (HCEC). METHODS: Human corneal endothelial cells were infected with either mock solution, Ad green fluorescent protein (GFP), or Ad SV40 T/t antigen/GFP, then mechanically wounded 48 h later. The endothelial wound healing rate was quantified by an analysis of the photographs taken every 12 h after wounding. The characteristics of Ad SV40 T/t Ag/GFP-infected human corneal endothelial cells were evaluated with cell morphology, cell density, contact inhibition, and cytoskeletal features using rhodamine phalloidin to stain F-actin. DNA synthesis was assessed using 5-Bromo-2'-deoxy-uridine (BrdU) labeling. RESULTS: Wound healing rates in the first 12 and 24 h after wounding were significantly faster in the Ad SV40 T/t antigen/GFP-infected group than the other two groups. In all three groups, the morphology, cell density, and cytoskeletal features of cells at confluency was similar and contact inhibition retained. There were no differences in the pattern of F-actin and endothelial cell density 4 d after wound closure. However, during the process of wound healing, prominent stress fibers in migrating cells near the wound edge were noted in normal cells at 36 h after wounding, whereas the Ad SV40 T/t Ag/GFP-infected cells showed similar changes as early as 12 h after wounding. BrdU staining results revealed that the Ad SV40 T/t antigen/GFP-infected group had labeled cells showing DNA synthesis in the wound area at 12 h after wounding, while no labeled cells were found in the other two groups. CONCLUSIONS: In an in vitro model, transduction of human corneal endothelial cells using a recombinant adenoviral vector expressing SV40 T/t antigen enhanced both the wound healing rate and proliferative capacity, especially in the first 12 h after wounding, and the characteristic morphologic features of the infected cells were maintained.     

Homocysteine, folate, vitamin B-12 and vitamin B-6 in patients receiving antiepileptic drug monotherapy

We hypothesized that elevated plasma homocysteine concentrations (hyperhomocysteinemia) exist in patients receiving antiepileptic drugs (AED), and a long-term administration of AED may result in an increased risk of occlusive vascular disease in these patients. A total of 62 patients who received AED monotherapy (phenytoin, lamotrigine, carbamazepine or valproate) participated in this study. Blood concentrations of homocysteine, folate, vitamin B-12 and pyridoxal-5'-phosphate (PLP, a coenzyme form of vitamin B-6) were measured, and thermolabile genotypes of 5, 10-methylenetetrahydrofolate reductase (MTHFR) were also determined. Of 62 patients, only seven (11.4%) had hyperhomocysteinemia. Of 20 patients who received phenytoin, three (15.0%) had hyperhomocysteinemia, whereas 85% of these had plasma folate concentrations below the normal range. However, erythrocyte folate concentrations were abnormally low in only 25% of the patients who received phenytoin. Valproate administration increased serum vitamin B-12 concentrations. Over 55% of the entire patients had PLP concentrations below the normal range, although the reason is unknown. Only three patients had the homozygous thermolabile genotype of MTHFR; therefore, meaningful statistical analysis was not possible in this study. However, one patient with homozygous genotype who received phenytoin therapy had hyperhomocysteinemia with poor folate nutritional status, and the other two had normal homocysteine concentrations with normal folate status. Our data suggest that hyperhomocysteinemia is not a serious clinical concern in epileptic patients when folate nutriture is adequate.     

An Experimental Study of Atrial Natriuretic Peptide Levels and the Effect of Inhaled Nitric Oxide After Pneumonectomy

(Received for publication on Feb. 24, 1999; accepted on Nov. 11, 1999)     

Tuberculous peritonitis defying diagnosis: Report of a case

(Received for publication on Feb. 10, 1999; accepted on Nov. 11, 1999)     

Ameliorated glomerular injury in mice overexpressing brain natriuretic peptide with renal ablation

Brain natriuretic peptide (BNP) is a cardiac hormone produced by the ventricle, and its secretion is markedly increased in heart failure, hypertension, and renal failure. Transgenic mice that overexpress BNP in the liver (BNP-Tg) were recently generated, resulting in low BP. To elucidate the role of BNP in renal pathophysiology, the effect of chronic excess of BNP in transgenic mice on glomerular injury after subtotal nephrectomy induced by resection of the renal poles was examined. After nephrectomy, glomerular cross-sectional areas in control nontransgenic mice markedly increased as compared with those in sham-operated mice (+81 +/- 7%), whereas there was only a modest increase in BNP-Tg (+10 +/- 6%). Expansion of the mesangial area and increase in the intraglomerular cell number were also inhibited in BNP-Tg. Glomerular expressions of transforming growth factor-beta and fibronectin were increased with hypertrophy and were significantly suppressed in BNP-Tg. Furthermore, increases in the urinary albumin excretion and BP were significantly ameliorated in BNP-Tg. Chronic hydralazine treatment in nephrectomized nontransgenic mice failed to inhibit glomerular hypertrophy. These findings indicate that the chronic excess of BNP in mice ameliorates glomerular hypertrophy and mesangial expansion after renal ablation. The results also suggest that the observed effects of natriuretic peptides under reduced renal mass are not due merely to systemic BP reduction and may be therapeutically applicable in various renal diseases.     

Effect of wortmannin on the suppression of thermotolerance in tsAF8 cells.

Phosphatidylinositol 3 kinases (PI-3 kinases) play a crucial role in the inositol phospholipid signaling pathway. The effect of wortmannin, an inhibitor of PI-3 kinases, on the suppression of thermotolerance development was investigated in the temperature-sensitive cell line tsAF8. When wortmannin was added immediately after the preheating, the thermotolerance was suppressed, with this suppression being dose-dependent from 0.01 to 0.1 microM, but not dose-dependent from 0.1 to 20 microM. Our results suggest that PI-3 kinases might play an important role in thermotolerance development in tsAF8 cells.     

The influence of dendritic cell infiltration and vascular endothelial growth factor expression on the prognosis of non-small cell lung cancer.

Vascular endothelial growth factor (VEGF) is well known to be produced by many human tumors, and it also plays an important role in tumor neovasculature formation. In addition to angiogenesis promotion, recent basic research has shown that VEGF has another function that allows it to inhibit dendritic cell (DC) maturation. However, very little is known about VEGF-dependent DC inhibition in a clinical setting. In this study, we analyzed the immunohistochemical expression of VEGF, microvessel density (MVD), and intratumoral DC infiltration in 132 surgically resected lung cancer specimens. We also evaluated the influence of these factors on their survival by a multivariate statistical analysis. VEGF expression was positively related to MVD (P = 0.0003) and negatively related to the degree of DC infiltration (P = 0.0232). A multivariate analysis also showed the VEGF expression, MVD, and DC infiltration to be independent prognostic factors. Moreover, we also accurately analyzed patient prognoses using the double stratification method for determining VEGF expression and DC infiltration. The patient group with a high VEGF expression/low DC infiltration showed a worse prognosis (P < 0.0001), whereas the group with a low VEGF expression/high DC infiltration had a better prognosis (P = 0.0001).     

Phase III study of concurrent versus sequential thoracic radiotherapy in combination with cisplatin and etoposide for limited-stage small-cell lung cancer: results of the Japan Clinical Oncology Group Study 9104.

PURPOSE: To evaluate the optimal timing for thoracic radiotherapy (TRT) in limited-stage small-cell lung cancer (LS-SCLC), the Lung Cancer Study Group of the Japan Clinical Oncology Group conducted a phase III study in which patients were randomized to sequential TRT or concurrent TRT. PATIENTS AND METHODS: We treated 231 patients with LS-SCLC. TRT consisted of 45 Gy over 3 weeks (1.5 Gy twice daily), and the patients were randomly assigned to receive either sequential or concurrent TRT. All patients received four cycles of cisplatin plus etoposide every 3 weeks (sequential arm) or 4 weeks (concurrent arm). TRT was begun on day 2 of the first cycle of chemotherapy in the concurrent arm and after the fourth cycle in the sequential arm. RESULTS: Concurrent radiotherapy yielded better survival than sequential radiotherapy (P =.097 by log-rank test). The median survival time was 19.7 months in the sequential arm versus 27.2 months in the concurrent arm. The 2-, 3-, and 5-year survival rates for patients who received sequential radiotherapy were 35.1%, 20.2%, and 18.3%, respectively, as opposed to 54.4%, 29.8% and 23.7%, respectively, for the patients who received concurrent radiotherapy. Hematologic toxicity was more severe in the concurrent arm. However, severe esophagitis was infrequent in both arms, occurring in 9% of the patients in the concurrent arm and 4% in the sequential arm. CONCLUSION: This study strongly suggests that cisplatin plus etoposide and concurrent radiotherapy is more effective for the treatment of LS-SCLC than cisplatin plus etoposide and sequential radiotherapy.