Powerful and controllable angiogenesis by using gene-modified cells expressing human hepatocyte growth factor and thymidine kinase

OBJECTIVES: This study investigated the possibility of achieving angiogenesis by using gene-modified cells as a vector. BACKGROUND: Although gene therapy for peripheral circulation disorders has been studied intensively, the plasmid or viral vectors have been associated with several disadvantages, including unreliable transfection and uncontrollable gene expression. METHODS: Human hepatocyte growth factor (hHGF) and thymidine kinase (TK) expression plasmids were serially transfected into NIH3T3 cells, and permanent transfectants were selected (NIH3T3 + hHGF + TK). Unilateral hindlimb ischemia was surgically induced in BALB/c nude mice, and cells were transplanted into the thigh muscles. All effects were assessed at four weeks. RESULTS: The messenger ribonucleic acid expression and protein production of hHGF were confirmed. Assay of growth inhibition by ganciclovir revealed that the 50% (median) inhibitory concentration of NIH3T3 + hHGF + TK was 1000 times lower than that of NIH3T3 + hHGF. The NIH3T3 + hHGF + TK group had a higher laser Doppler blood perfusion index, higher microvessel density, wider microvessel diameter, and lower rate of hindlimb necrosis, as compared with the plasmid- and adenovirus-mediated hHGF transfection groups or the NIH3T3 group. The newly developed microvessels were accompanied by smooth muscle cells, as well as endothelial cells, indicating that they were on the arteriolar or venular level. Laser Doppler monitoring showed that the rate of blood perfusion could be controlled by oral administration of ganciclovir. The transplanted cells completely disappeared in response to ganciclovir administration for four weeks. CONCLUSIONS: Gene-modified cell transplantation therapy induced strong angiogenesis and collateral vessel formation that could be controlled externally with ganciclovir.     

Monitoring of hepatocellular carcinoma, following proton radiotherapy, with contrast-enhanced color Doppler ultrasonography

Background We have reported that proton radiotherapy for hepatocellular carcinoma (HCC) is a safe and effective therapeutic option. However, it is difficult to evaluate its effect in certain cases. Recently, it has been reported that the usage of contrast-enhanced color Doppler ultrasonography (CECDU) can improve diagnostic accuracy, both in terms of the presence of hepatic tumor and in the evaluation of treatment. The aim of this study was to determine the usefulness of CECDU in assessing the therapeutic response of HCC treated with proton radiotherapy.Methods Twenty-two patients treated with the proton radiotherapy were studied. We inspected HCC lesions by CECDU, before and after the irradiation, over time. The magnitude of blood flow in the HCC was quantified on still images by CECDU. The ratio of the number of color pixels against that of the total number of pixels in the tumor area was defined as the tumor blood flow ratio (TBFR).Results Immediately after the proton treatment, a transient increase of blood flow in the tumor was recognized in more than half of the patients, while the TBFR was unchanged or decreased in the remaining patients. At longer periods after irradiation, the TBFR in all HCCs gradually decreased, and this reduction of TBFR was statistically significant from 9 months after irradiation. These findings are consistent with those obtained previously by computed tomography (CT) as well as magnetic resonance imaging (MRI).Conclusions We propose CECDU as a useful diagnostic option for the evaluation of HCC treated with proton radiotherapy.Presented in part at the annual meeting of the American Gastroenterological Association in May 2001.     

Appropriate Heart Rate in a Patient with Repaired Tetralogy of Fallot

Appropriate heart rate in a failing pulmonary ventricle remains unknown, particularly in congenital heart disease with unique hemodynamics. A 71-year-old male with repaired tetralogy of Fallot and a pacemaker for a sinus node dysfunction suffered from heart failure symptoms with preserved left ventricular function. Simply changing the pacemaker’s lower rate from 60 to 75 bpm, New York Heart Association classification improved from III to II, and hemodynamic parameters drastically improved. We regarded this case as informative. Appropriate heart rate could be higher in congenital patients with failing right and non-failing left ventricles than in adults with malfunctioning LV.     

Paradoxical regional myocardial uptake between 201Thallium and 123I-BMIPP SPECT in patients with cardiomyopathy

 The clinical significance of the paradoxical mismatched phenomenon between ²⁰¹Tl and ¹²³I-BMIPP is still unknown. We report two cases that revealed paradoxical regional myocardial uptake between two tracers in patients with cardiomyopathy. There may be abnormal myocardium in these patients where active transportation of ²⁰¹Tl is disturbed and passive transportation of ¹²³I-BMIPP is not disordered. Received: July 31, 2002 / Accepted: September 21, 2002 Correspondence to K. Ueshima     

A patient of hypogonadotropic hypogonadism accompanied by growth hormone deficiency and decreased bone mineral density who attained normal growth

We present here a rare case of hypopituitarism accompanied by growth hormone (GH) deficiency and hypogonadotropic hypogonadism, in which the patient attained normal height but was of eunuchoid appearance. A 23-year-old man who had not reached puberty was referred to Saitama Medical School for hormonal evaluation. Basal hormonal data and hormone-stimulating tests revealed impaired secretion of GH, gonadotropins and adrenocorticotropic hormone (ACTH). Serum levels of testosterone, estrone, estradiol and estriol were all below the detectable ranges. The patient's plasma ACTH responded to corticotropin releasing hormone, but not to insulin-induced hypoglycemia. Serum GH showed a minimal response to GH-releasing hormone, but was unresponsive to insulin-induced hypoglycemia. Serum luteinizing hormone and follicle stimulating hormone did not respond to luteinizing hormone-releasing hormone. The results were compatible with a diagnosis of hypothalamic hypopituitarism. Magnetic resonance images of the brain showed a small anterior pituitary, an ectopic posterior lobe and transection of the pituitary stalk. Although the patient showed signs of hypopituitarism, he finally attained normal height, possibly because of failed epiphyseal maturation. His bone mineral density was markedly reduced to 0.647 g/cm2 in the lumbar spine; this level was 61.7% of the average level of healthy young males. Our findings were compatible with a recently advocated view that estrogen is important in promoting epiphyseal fusion and in determining bone density in males as well as females.     

Inflammation and insulin resistance are independently related to all-cause of death and cardiovascular events in Japanese patients with type 2 diabetes mellitus

Insulin resistance (IR)/hyperinsulinemia and low-grade inflammation (high-sensitivity C-reactive protein [hs-CRP]) can predict cardiovascular disease. However, because IR and inflammation (IF) have not been evaluated simultaneously, it is not known whether IR and IF are independently related to cardiovascular disease. Furthermore, the combined effect of IR and IF on the prediction of cardiovascular disease is presently unknown. Thus, we measured insulin sensitivity (K index of the insulin tolerance test; KITT) and hs-CRP in 350 Japanese patients with type 2 diabetes, and followed them for 1-7 years (mean, 4.5 years). During the follow-up, 33 patients died and 53 patients developed non-fatal coronary artery disease or stroke (endpoint). Age, systolic blood pressure, current smoking, past history of cardiovascular disease, KITT, and hs-CRP independently and significantly correlated with endpoint. One-S.D. difference was associated with a significant increase of relative risk in KITT (1.45; 95% CI 1.09-1.91) and hs-CRP (1.30; 1.04-1.67). When patients were subdivided to tertile, the relative risk in the highest tertile of KITT was 1.76 (95% CI 1.01-3.11) and hs-CRP was 2.00 (1.03-3.85) compared with the patients with lowest tertile. The relative risk in the highest tertile of both KITT and hs-CRP was 5.32 (1.18-24.0) compared with the lowest tertile of both values. In conclusion, low-grade IF and IR are independently related to all-cause of death and cardiovascular disease in Japanese patients with type 2 diabetes. Coexistence of low-grade IF and IR amplify this effect.     

Gore-Tex jump graft for portal vein thrombosis following living donor liver transplantation

Portal vein thrombosis is a rare surgical complication following liver transplantation, which remains a cause of graft loss and death. We describe here the treatment of portal vein thrombosis following living donor liver transplantation using an extended left lobe graft. The patient was treated with a Gore-Tex vascular jump graft extra-anatomically interposed between the recipient superior mesenteric vein and the donor umbilical vein. This technique allowed the hepatic hilum to be left untouched and supplied suitable blood flow to the hepatic allograft. Our experience suggests that this innovative technical solution can be helpful in the effort to rescue cases of hepatic allograft with vascular complications.     

A case of late onset mitochondrial neuromyopathy

A 68-year-old woman was admitted with dysesthesia on both soles, hands and buttocks. She first noticed the dysesthesia in the left first toe two years before admission. One year before admission, serum creatine kinase and lactate dehydrogenase levels were elevated but she had no clinical symptoms suggesting myopathy. Nerve conduction study showed no apparent abnormalities, and an electromyogram showed mild myogenic change. Computed tomography of the muscle showed fatty degeneration of the trunk, major gluteus muscle, and biceps femoris muscle and atrophic change of the biceps femoris muscle. Muscle biopsy revealed a slight variation in fiber size, the presence of cytochrome c oxidase (CCO)-negative fibers by CCO staining, ragged-red fibers by Gomori trichrome staining, and mild denervation fiber by neuron specific enolase (NSE) staining. Analysis of her mitochondrial DNA (mtDNA) revealed a large deletion of mtDNA (approximately 8.5 Kb), and mitochondrial neuromyophy was diagnosed. The frequency of mtDNA deletion increases with aging. Although her mtDNA abnormality was compatible with chronic progressive external ophthalmoplegia (CPEO), we speculate that the aging process may be contributed to the mtDNA abnormality, which would be related to the late onset of her symptoms. As the phenotype of this mitochondrial disorder shows a wide variation and mtDNA abnormality is more frequent among elderly people, we should consider the possibility of mitochondrial disorders even in elderly people.     

Hepatic mitochondrial dysfunction in senescence-accelerated mice: correction by long-term,orally administered physiological levels of melatonin

Mitochondrial oxidative damage from free radicals may be a factor underlying aging. We investigated whether long-term administration of physiological levels of melatonin, a direct free radical scavenger and indirect antioxidant, influences mitochondrial respiratory activity in liver of senescence-accelerated mice (SAM). Liver was obtained in the middle of dark period of the daily light:dark cycle from SAMP8, a strain of mice prone to accelerated senescence, and from SAMR1, a senescence-resistant strain, at 6 and 12 months of age. Respiratory control index (RCI), adenosine-5-diphosphate (ADP)/O ratio, State 3 respiration and dinitophenol (DNP)-dependent uncoupled respiration exhibited significant age-associated decreases in SAMP8. SAMP8 also showed significant age-associated reductions in respiratory chain complex I and IV activities. No age-related effects were found in these parameters in SAMR1. Daily oral melatonin administration (2 microg/mL of drinking fluid) beginning at 7 months of age significantly increased RCI, State 3 respiration, DNP-dependent uncoupled respiration, and complex I and IV activities in both mouse strains when they were 12 months old. These results reveal age-related reductions in mitochondrial function in SAM mice which are modified by melatonin; the most likely explanation for the corrective actions of melatonin relate to its antioxidative actions in mitochondria and other portions of the cell. The implication of the findings is that melatonin may be beneficial during aging as it reduces the deteriorative oxidative changes in mitochondria and other portions of the cell associated with advanced age.     

Induction of alpha/beta interferon and gamma interferon in mice infected with Listeria monocytogenes during pregnancy.

Alpha/beta interferon (IFN-alpha/beta) was induced in the bloodstream of mice 48 h after intravenous infection with Listeria monocytogenes, whereas IFN-gamma was induced in the bloodstream 6 h after stimulation with specific antigen on day 5 of infection in virgin mice. In contrast, no IFN-alpha/beta or IFN-gamma was produced in the bloodstream of pregnant mice after L. monocytogenes infection. However, unusual acid-labile IFN-alpha/beta instead of IFN-gamma was produced in some of the pregnant mice in response to specific antigen. The bacterial growth in the organs of pregnant mice in the early stage of infection was normal, but resulted in the delay of T-cell-dependent elimination of bacteria from the organs of pregnant animals in the late stage, and numerous bacteria were detected in both the placenta and the fetus. The significance of the IFN system induced by L. monocytogenes infection in pregnant mice is discussed.