Pathophysiological analysis of uninephrectomized db/db mice as a model of severe diabetic kidney disease.

Diabetic nephropathy, included in diabetic kidney disease (DKD), is the primary disease leading to end-stage renal disease (ESRD) or dialysis treatment, accounting for more than 40% of all patients with ESRD or receiving dialysis. Developing new therapeutics to prevent the transition to ESRD or dialysis treatment requires an understanding of the pathophysiology of DKD and an appropriate animal model for drug efficacy studies. In this study, we investigated the pathophysiology of diabetic kidney disease with type 2 diabetes in uninephrectomized db/db mice. In addition, the nephrectomized db /db mice from 10 weeks to 42 weeks were used to assess the efficacy of long-term administration of the angiotensin-II-receptor antagonist losartan. The blood and urinary biochemical parameters, main pharmacological endpoint of the losartan therapy, were periodically measured. And at the end, histopathological analysis was performed. Uninephrectomized db/db mice clearly developed obesity and hyperglycemia from young age. Furthermore, they showed renal pathophysiological changes, such as increased urinary albumin-creatinine ratio (UACR) (the peak value 3104 ± 986 in 40-week-old mice), glomerular hypertrophy and increased fibrotic areas in the tubulointerstitial tubules. The blood pressure in the losartan group was significantly low compared to the normotensive Vehicle group. However, as expected, Losartan suppressed the increase in UACR (829±500) indicating the medication was sufficient, but the histopathological abnormalities including tubular interstitial fibrosis did not improve. These results suggest that the uninephrectomized db/db mice are useful as an animal model of the severe DKD indicated by the comparison of the efficacy of losartan in this model with the efficacy of losartan in clinical practice.

Effect of vitamin D supplementation on pegylated interferon/ribavirin therapy for chronic hepatitis C genotype 1b: a randomized controlled trial.

Chronic HCV-infected patients tend to have vitamin D deficiency, suggesting that vitamin D supplementation may enhance the efficacy of treatment with pegylated interferon (PEG-IFN) and ribavirin (RBV). We therefore assessed the effects of vitamin D supplementation on viral response to PEG-IFN/RBV. Eighty-four patients with HCV genotype 1b were randomized, 42 to oral vitamin D supplementation (1000 IU/day) and 42 to nonsupplementation (control), from week 8 to the end of PEG-IFN/RBV therapy. The primary end point was undetectable HCV RNA at week 24 (viral response [VR]). VR rate at week 24 was significantly higher in the vitamin D than in the control group (78.6% vs 54.8% P = 0.037). Adverse events were similar in both groups. When patients were subdivided by IL28B SNP rs8099917 genotype, those with the TT genotype group showed a significantly higher VR rate at week 24 with than without vitamin D supplementation (86.2% vs 63.3% vs P = 0.044). Although patients with the TG/GG genotype, who were relatively resistant to PEG-IFN treatment, had similar VR rates at week 24 with and without vitamin D supplementation, the decline in viral load from week 8 to week 24 was significantly greater with than without vitamin D supplementation. Multivariate analysis showed that rs8099917 genotype and vitamin D supplementation contributed significantly to VR at week 24. SVR rates were similar in the vitamin D and control groups [64.3% (27/42) vs 50% (21/42), P = 0.19]. Vitamin D supplementation may enhance the effects of PEG-IFN/RBV in HCV genotype 1b-infected patients.

Impact of aging on the development of hepatocellular carcinoma in patients with hepatitis C virus infection in Japan.

BACKGROUND: It is difficult to study the long-term outcome of hepatitis C virus (HCV) infection because chronic infection is often asymptomatic and duration of the disease is prolonged. The clinical outcome of HCV infection remains unclear in patients of advanced age. METHODS: Among 575 patients consecutively diagnosed with hepatocellular carcinoma (HCC) from 1988 to 1999 at Hiroshima University, we examined 430 with HCV. We studied the differences between males and females in the following characteristics: age at first diagnosis of HCC, Child grade, various tumour factors, history of blood transfusion, duration to development of HCC, and history of alcohol intake. RESULTS: The incidence of HCC patients with HCV increased in elderly persons, including female patients. Background liver function was significantly better for female patients (P < 0.001). In both genders, the duration between blood transfusion and diagnosis of HCC was significantly shorter when the patients received blood transfusion at an older age (P < 0.001). In habitual drinkers, the average age at first diagnosis of HCC was significantly younger (P < 0.001), and duration to development of HCC significantly shorter (P < 0.05). The percentage of atomic bomb survivors among HCV-positive HCC patients was significantly higher than that among HCV-negative HCC patients (P < 0.05). CONCLUSIONS: Patients with HCV might exhibit slow disease progression and develop HCC finally with aging regardless of gender. Patients of advanced age with HCV, even female patients, should therefore be closely followed.

Long-term prognosis of patients undergoing transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma: comparison of cisplatin lipiodol suspension and doxorubicin hydrochloride emulsion.

PURPOSE: To evaluate long-term prognosis of transcatheter arterial chemoembolization (TACE) with use of cisplatin (CDDP) lipiodol (LPD) suspension (CDDP/LPD) compared with that with use of doxorubicin hydrochloride (ADM) LPD emulsion (ADM/LPD) in patients with unresectable hepatocellular carcinoma (HCC). MATERIALS AND METHODS: One hundred eight patients were treated with use of CDDP/LPD and 26 were treated with use of ADM/LPD. Survival rates and frequency of side effects and complications in the CDDP/LPD group were compared with those in the ADM/LPD group. RESULTS: CDDP/LPD was given at a dose of 15-70 mg (mean dose, 41 mg), whereas ADM/LPD was given at a dose of 20-100 mg (mean dose, 57 mg) throughout the study period. The survival rates in the CDDP/LPD group were 81% at 1 year, 41% at 3 years, 19% at 5 years, and 13% at 7 years, whereas those in the ADM/LPD group were 67% at 1 year, 18% at 3 years, and 0% at 5 years. The CDDP/LPD group showed significantly better survival than the ADM/LPD group (P <.05). In the CDDP/LPD group, there was a significant prolongation of survival in patients with monofocal HCC (P <.05) and patients with HCC assessed as an almost complete LPD accumulation (P <.05). There were no significant differences in survival rates in the ADM/LPD group according to tumor size and number of tumors. Hepatic failure was observed in 8% of all procedures and was not different between the two therapeutic groups. Renal dysfunction was observed in 2% of all treatments involving CDDP/LPD, and it resolved spontaneously with appropriate medications. CONCLUSIONS: TACE with use of low-dose CDDP was efficacious for unresectable HCC and had few complications. TACE with use of CDDP may contribute to prolongation of the life span of patients with HCC versus TACE with use of ADM.

Three-dimensional portography using multislice helical CT is clinically useful for management of gastric fundic varices.

OBJECTIVE: This study seeks to evaluate three-dimensional (3D) helical CT portography as a tool for examining patients with gastric fundic varices. SUBJECTS AND METHODS: We compared 3D helical CT portography and conventional angiographic portography in 30 consecutive patients with gastric fundic varices. We assessed whether 3D helical CT portography is useful in selecting patients and in evaluating the results of balloon-occluded retrograde transvenous obliteration. RESULTS: Three-dimensional helical CT portography simultaneously depicted second or third branches of the intrahepatic portal vein and provided images of entire portosystemic collaterals. On 3D helical CT portography, gastric fundic varices were seen in 30 patients (100%), left gastric veins in 19 (63%), posterior gastric veins or short gastric veins in 28 (93%), gastrorenal shunts in 27 (90%), paraumbilical veins in three (10%), and inferior phrenic veins in two patients (7%). Findings of 3D helical CT portography and conventional angiographic portography were in close agreement. However, in four patients, posterior gastric veins or short gastric veins were not seen on conventional angiographic portography images of the spleen, but they were clearly revealed on 3D helical CT portography. Treatment was successful in all patients except one. Three-dimensional helical CT portography could easily evaluate therapeutic results. CONCLUSION: Three-dimensional helical CT portography proved so effective that it can be considered a less invasive alternative than conventional angiographic portography in assessing portosystemic collaterals. CT portography is useful in selecting candidates from patients with gastric fundic varices for retrograde transvenous obliteration and also in evaluating therapeutic results.

Immuno-histochemical detection of human telomerase reverse transcriptase in human liver tissues.

Although telomerase activity in hepatocellular carcinoma (HCC) increases in accordance with degree of histological undifferentiation, it is unknown whether the level of telomerase activity in HCC reflects of the degree of activity in individual cells or the frequency of telomerase-positive HCC cells. Non-cancerous liver tissues exhibit low but significant levels of telomerase activity, but the nature of telomerase-positive cells in these tissues is unclear. In this study, we performed immunohistochemical staining using specific antibody against telomerase reverse transcriptase (hTERT) protein in 15 HCC samples and 13 adjacent non-cancerous liver tissues. There were hTERT-positive hepatocytes, though very low frequency, in non-cancerous liver tissues. The frequencies in hTERT positive hepatocytes were very well correlated with clinicopathological parameters and telomerase activity levels: the average frequencies of chronic hepatitis was 0.2%, liver cirrhosis 0.2%, well-differentiated HCC 3.0%, moderately differentiated HCC 28%, and poorly differentiated HCC 95%. The intensity of staining varied among cells within a given specimen, and correlation with degree of histological undifferentiation was less obvious. Portions of migrating lymphocytes and biliary epithelial cells were also hTERT-positive. These findings indicate that the upregulation of telomerase activity with degree of undifferentiation of HCC is mainly due to the increase in frequency of hTERT positive HCC cells.

Telomere reduction in human liver tissues with age and chronic inflammation.

Telomere shortening in human liver with aging and chronic inflammation was examined by hybridization protection assay using telomere and Alu probes. The reduction rate of telomere repeats in normal liver (23 samples from patients 17-81 years old) was 120 bp per year, which is in good agreement with the reported reduction rate in fibroblasts of 50-150 bp at each cell division and replacement rate of human liver cells, once a year. Mean telomere repeat length shortened to about 10 kbp in normal livers from 80-year-old individuals. The number of telomere repeats in chronic hepatitis (26 samples) and liver cirrhosis (11 samples) was significantly lower than that in normal liver of the same age (P < 0. 01). Telomere length in all these chronic liver disease samples, other than two exceptions, was not reduced shorter than 5 kbp, which was assumed to give a limit of proliferation (Hayflick's limit) to untransformed cells.

Expression of telomerase component genes in hepatocellular carcinomas.

The aim of the study was to clarify the role of telomerase component genes in hepatocarcinogenesis and to examine both the relationship between the expression of telomerase component genes and histological differentiation in hepatocellular carcinoma (HCC) and the relationship between expression levels of telomerase component genes and telomerase activity in HCCs. Telomerase is a ribonucleoprotein enzyme composed of a template RNA and several proteins. Recently, three such telomerase component genes have been identified: human telomerase reverse transcriptase (hTERT); human telomerase RNA component (hTERC); and telomerase-associated protein 1 (TEP1). The expression of these components was evaluated in 34 HCCs and 24 non-cancerous liver tissues by reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of hTERT mRNA was detected in most HCCs, but not in the non-cancerous tissues (P<0.01). Expression of hTERC was detected in both HCCs and non-cancerous tissues, but the expression level in HCCs was higher than that in non-cancerous tissues (P<0.01) and tended to increase as histological differentiation became less marked. The expression level of hTERT mRNA correlated with relative telomerase activity (P<0.01). These results suggest that telomerase reactivation during hepatocarcinogenesis might be regulated by only hTERT and an increase in telomerase activity level in tumour progression might be regulated by both hTERT and hTERC.

Precancerous hepatic nodules had significant levels of telomerase activity determined by sensitive quantitation using a hybridization protection assay.

BACKGROUND: Telomeric repeat amplification protocol using internal telomerase assay standard (ITAS) (conventional TRAP) has detected telomerase activity in various malignant tumors. With conventional TRAP, it is difficult to differentiate quantitatively low levels of telomerase activity between well-differentiated hepatocellular carcinomas (HCCs) and dysplastic nodules because of quantitative limitation. To apply a telomerase assay for differential diagnosis, we used a hybridization protection assay combined with TRAP (TRAP/HPA). This combination had better sensitivity and wider linearity than conventional TRAP. METHODS: TRAP/HPA was applied for quantitative measurement of telomerase activity in various hepatic tissues. Telomerase activity was evaluated in 10 precancerous hepatic nodules, 17 well-differentiated HCCs, 19 moderately differentiated HCCs, 5 poorly differentiated HCCs, 22 nontumorous chronic hepatic disease samples, and 2 normal liver tissues. RESULTS: Telomerase activity in HCCs tended to increase according to the malignant transformation. The average relative telomerase activity in 0.6 microg protein, which was expressed as cell equivalent activity of MKN-1, a gastric carcinoma cell line, was 8.5 in precancerous hepatic nodules, 87 in well-differentiated HCCs, 265 in moderately differentiated HCCs, 447 in poorly differentiated HCCs, and 0.4 in nontumorous hepatic tissues, including chronic liver diseases. CONCLUSIONS: TRAP/HPA was sensitive enough to distinguish the telomerase activity in precancerous hepatic nodules from that in other lesions. Telomerase activity in precancerous hepatic nodules was higher than that in nontumorous hepatic tissues. However, the activity in precancerous hepatic nodules was lower than that in well-differentiated HCCs, although statistically not significant. The authors suggest that precancerous hepatic nodules with telomerase activity above the diagnostic cutoff level (twice the highest activity in nontumorous hepatic tissues, or the 2 cell equivalent activity of MKN-1) should be treated as malignancy.

Brain metastasis from hepatocellular carcinoma after radical hepatectomy.

Brain metastasis from hepatocellular carcinoma (HCC) is a rare, yet perplexing problem in patients with cancer. We report on 5 patients with metastasis of HCC to the brain after radical hepatectomy. Intrahepatic recurrence occurred in 3 patients, and distant metastasis to sites other than the brain was observed in 3 patients (lung, 2; bone, 1). The symptoms for brain metastasis included headache, hemiparesis, and vomiting. Hemorrhage was found in 4 of 5 patients. All patients had a single nodular lesion in the brain. The alpha-fetoprotein levels were more than 10,000 ng/ml in 4 patients. Two patients underwent surgical resection, 1 received cranial irradiation, and 2 were administered corticosteroids. The interval between diagnosis of the primary cancer and detection of brain metastasis ranged from 2 to 54 months. The mean survival period was only 3 months after diagnosis of brain metastasis. All 5 patients died of neurologic causes. Because no effective treatment for brain metastasis from HCC is available, further study is needed.

Hepatic resection with tumor thrombectomy for hepatocellular carcinoma with tumor thrombi in the major vasculatures.

BACKGROUND/AIMS: It is still controversial whether surgical or non-surgical treatments should be adopted for hepatocellular carcinomas (HCC) with tumor thrombi (TT) in the major vasculatures. We evaluate the effectiveness of and the indications for hepatic resection with tumor thrombectomy for such patients. METHODOLOGY: Seventeen patients with TT in the major vasculatures caused by HCC were enrolled. Eleven patients had Vp3 TT, 5 patients had Vv3 TT, and 1 patient had Vp3 and Vv3 TT, concurrently. Out of the 17 patients, 13 underwent hepatic resections with tumor thrombectomies and the remaining 4 received only hepatic resections without tumor thrombectomies. RESULTS: In patients with Vp3 TT, median and mean survival times were 7.8 and 18.5 months, respectively, and 1- and 5-year survival rates were 36.4% and 18.2%, respectively. In patients with Vv3 TT, median and mean survival times were 9.9 and 8.4 months, respectively. Patients who underwent hepatic resections with tumor thrombectomies had significantly better prognoses than those who did not receive tumor thrombectomies (p=0.0039). CONCLUSIONS: The prognosis of HCC patients with TT in the major vasculatures, who have relatively small primary tumors, good hepatic functional reserves and no distant metastases should be good, if hepatic resections with tumor thrombectomies are performed.

Adjuvant hepatic arterial infusion chemotherapy after radical hepatectomy for hepatocellular carcinoma--results of long-term follow-up.

BACKGROUND/AIMS: This clinical study aimed to clarify the effectiveness and indication of adjuvant hepatic arterial infusion chemotherapy (HAIC) that is performed to prevent recurrence after radical hepatectomy for hepatocellular carcinoma (HCC). METHODOLOGY: From January 1986 to December 1992, 135 HCC patients, who tolerated curative hepatic resection in which all of the macroscopic HCC was removed, were included in this study. They were divided into two groups. One group was comprised of 68 patients who received HAIC after radical hepatectomy (HAIC (+) group), and the other group was comprised of 67 patients who received radical hepatectomy alone (HAIC (-) group). In the HAIC (+) group, an emulsion of doxorubicin (30-50 mg) and lipiodol (3-5 ml) was injected from a reservoir every 2 or 3 months for 1 year. RESULTS: The cumulative survival rates in the HAIC (+) group (79.1%, 54.5% and 39.9% at 3, 5, and 7 years after hepatectomy, respectively) were better than those in the HAIC (-) group (69.2%, 38.1% and 26.8%, respectively) (p = 0.086). The disease-free survival rates in the HAIC (+) group (50.8%, 31.7% and 25.6% at 3, 5, and 7 years after hepatectomy, respectively) were significantly better than those in the HAIC (-) group (25.7%, 20.6% and 6.4%, respectively) (p = 0.006). This improvement was evident for 3 years after hepatectomy. The adjuvant HAIC was effective especially in patients with good liver function, whose tumor size ranged between 2.1 cm and 5 cm in diameter, and who received a minor hepatic resection. CONCLUSIONS: Adjuvant HAIC was effective in preventing recurrence after radical hepatectomy for HCC. This treatment is especially indicated for patients with good liver function, whose tumor size ranges between 2.1 cm and 5 cm in diameter, and who have received a minor hepatic resection.

Factors affecting postoperative prognosis in the solitary-nodule type of hepatocellular carcinoma: experience of 132 cases in our institute.

A retrospective analysis of clinical and pathological factors was performed on 132 surgical cases with solitary-nodule type HCC in our hospital. The overall cancer-free survival rates after 1, 3 and 5 years were 82.2%, 42.3% and 26.5%, respectively. With univariate analysis, the significant prognostic factors for survival were tumor size, cancer cell infiltration of the fibrous capsule of the tumor (fc-inf), invasion into portal vein (vp), and intrahepatic metastasis (im), while significant prognostic factors for non-recurrence were tumor size, fc-inf, vp, im, Edmondson-Steiner's classification and perioperative blood transfusion. With multivariate analysis for recurrence, significant factors were vp, clinical stage (CS), and perioperative blood transfusion. Therefore, prognostic factors for long-term survival in surgical cases of HCC are thought to be good hepatic function, absence of portal invasion, and avoidance of perioperative blood transfusion if possible.

Indications for palliative reduction surgery for hepatocellular carcinoma with multiple intrahepatic metastases.

We evaluated the efficacy of and the indications for palliative reduction surgery as a procedure to improve the prognosis of hepatocellular carcinoma (HCC) patients with multiple intrahepatic metastases. From January 1986 to October 1997, 25 HCC patients with multiple intrahepatic metastases who underwent necessary palliative reduction surgery due to advanced disease, were participated in the study. The 1-, 3-, 5-year survival rates of 25 patients with reduction surgery were 54.7%, 29.9%, 22.4%, respectively. Moreover, the 1-, 3-, 5-year survival rates of patients with some postoperative supplemental treatment were 68.8%, 41.2% and 30.9%, respectively. All 6 patients who had been alive for more than 3 years had had primary tumors with diametric sizes of approximately 5 cm and had undergone, at most, one segmentectomy. These patients received postoperative supplemental treatments. The indication for palliative reduction surgery for HCC patients with multiple intrahepatic metastases was patients with a relatively small primary tumor (around 5 cm) which could be removed by one segmentectomy or less.

Thoracoscopic microwave coagulation therapy for hepatocellular carcinoma.

Thoracoscopic microwave coagulation therapy (MCT) is a new therapeutic approach for hepatocellular carcinoma (HCC) in segments VII and VIII, which allows minimal access to the tumor and complete tumor ablation. In this study, four patients with HCC in segments VII and VIII underwent thoracoscopic MCT as a less invasive therapeutic option due to advanced liver cirrhosis and/or severe complications. Tumor sizes ranged from 15 to 30 mm in diameter and the tumors were well differentiated in 2 patients, moderately in one and poorly in one patient. Microwave irradiation was performed at an 80 W output with a 60-sec duration via a thoracoscopic route and the total duration ranged from 4 to 24 min (mean: 17 min). Patients recovered rapidly to preoperative conditions and no mortality was occurred. Complications were observed in one patient, including pleural effusion and fever elevation, but were cured conservatively. Postoperative computed tomography (CT) showed complete tumor ablation with a cancer-free margin, which is thought to be equivalent to a limited hepatic resection. This preliminary study suggests that thoracoscopic MCT might be a new, less invasive option providing a cure for HCC in segments VII and VIII in patients with advanced liver cirrhosis and severe complications.

[Telomere length and telomerase activity in hepatocellular carcinoma].

Telomerase activity and terminal restriction fragment (TRF) length were examined in hepatocellular carcinoma (HCC). Telomerase activity was assayed by telomeric repeat amplification protocol (TRAP) connected with an internal telomerase assay standard (ITAS). The incidence of strong telomerase activity (highly variable level compared with the activity of non-cancerous liver tissue) was 79% in well, 84% in moderately, and 100% in poorly differentiated HCC, while 0% in non-cancerous liver tissues. The incidence of TRF length alteration (reduction or elongation) was 53% in HCC. The incidence of TRF alteration was significantly higher in HCC exceeding 3 cm in diameter, moderately or poorly differentiated in histology. Telomerase activity was not associated with TRF length alteration in HCC. In conclusion, strong telomerase activity and TRF length alteration increased with HCC tumor progressions.