Biomedical subjects

M Nakamuta

Publications and source records attributed to M Nakamuta.

At least 19 recordsLinked to original sources

PubMed2006

Interventional navigation for abdominal therapy based on simultaneous use of MRI and ultrasound.

An interventional navigation system designed for percutaneous abdominal therapies was proposed, and a pilot study was carried out to assess the proposed system. Integration of US to MRI-based segmentation and 3D display of tumours can help physicians deal with instabilities such as respiratory motion and soft tissue shift that are inherent in abdominal interventions. In addition to the 3D display of the needle and tumours, we adapted the system for the abdominal applications and incorporated a process to correct the mismatch in needle path between MRI and US. The preliminary results of phantom and animal experiments indicated that the proposed method could combine the advantages of both MRI and US. The time required to determine the optimal needle insertion path by using this system was significantly less than that required when either US or MRI guidance alone was employed. The developed system was applied in two patients who underwent PEIT therapy, and its clinical feasibility was partially confirmed.

Abdomen↗

PubMed2005

Cyclosporine suppresses cell growth and collagen production in hepatic stellate cells.

BACKGROUND: In HCV-related graft hepatitis, immunosuppression has been implicated in rapid progression to cirrhosis, a serious clinical issue. We investigated the effects of cyclosporine or tacrolimus on cell growth and collagen production by hepatic stellate cells (HSC), which play a role in hepatic fibrosis. MATERIALS AND METHODS: Cultured rat HSCs and human HSC-derived TWNT-4 cells were evaluated for proliferation, type I collagen, phosphorylation states of mitogen-activated protein kinases extracellular signal-regulated kinase 1/2; [MAPKs Erk1/2], c-Jun N-terminal kinase (JNK, p38), as well as the expression of collagen, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) genes. RESULTS: Cyclosporine suppressed cell growth and collagen production in a concentration-dependent manner. At clinically relevant concentrations of 0.125 micromol (150 ng/mL) cyclosporine significantly reduced collagen production per cell by more than 50%. Similarly, tacrolimus also reduced both collagen concentration and cell number; however, tacrolimus at a clinically relevant concentration of 12.5 nmol (10 ng/mL) did not significantly reduce collagen production. Treatment with cyclosporine reduced type I collagen and TIMP-1 expression and enhanced MMP-1 expression. Cyclosporine also inhibited phosphorylation strongly for JNK and p38, and weakly inhibited for Erk1/2. CONCLUSION: These findings demonstrated that cyclosporine suppresses cell growth and collagen production, suggesting that it may have an antifibrogenic effect.

Animals↗

PubMed2004

Liver lesions of visceral larva migrans due to Ascaris suum infection: CT findings.

The purpose of this study was to analyze computed tomographic (CT) findings of hepatic lesions due to Ascaris suum infection. CT of the liver in three patients, all of whom had immunoserologically confirmed A. suum infection, were retrospectively reviewed. Twenty-five lesions were identified in total. Two radiologists analyzed CT findings in a consensus fashion, with particular interest in the margin, shape, and location of the lesions. Hepatic lesions were ill-defined (22 of 25), small (3-35 mm; average, 11 mm), and nodular (18 of 25) or wedge (three of 25) in shape. Most were located in periportal (16 of 25) or subcapsular (six of 25) regions. Hepatic nodules due to visceral larva migrans of A. suum were located mainly in periportal or subcapsular regions, which may represent periportal eosinophilic granuloma, its pathologic feature. The results were considered to represent the pathophysiology of this entity.

Adult↗

PubMed2004

Movement of a novel serum tumour marker, RCAS1, in patients with biliary diseases.

BACKGROUND: We have demonstrated immunohistochemically that RCAS 1 antigen is expressed in biliary neoplasms. Serum RCAS 1 levels are also elevated in a high percentage of patients with intra-hepatic cholangiocarcinoma. AIM: The study was designed to determine whether serum levels of RCAS1 are of clinical significance as a tumour marker for biliary tract tumour, in comparison to CA19-9. PATIENTS AND METHODS: In 38 patients with biliary carcinoma (gallbladder carcinoma, extra-hepatic and intra-hepatic cholangiocarcinoma and ampullary carcinoma), we measured serum RCAS1 and CA19-9 levels. For control, serum samples from patients with benign biliary disease and healthy volunteers were also examined. RESULTS: We established a threshold value for RCAS1 of 17.5 U/ml, which permitted discrimination between malignant and non-malignant biliary diseases. In comparison to CA 19-9, serum RCAS1 was more sensitive and specific for malignancy, and was not influenced by cholestasis. RCAS1 levels varied with respect to the disease course and the effect of clinical treatment. CONCLUSIONS: Serum RCAS1 appears to be valuable as a diagnostic index for biliary carcinomas, as well as for evaluating the progression of cancers during therapy. We speculate that RCAS1 is a clinically more significant serum marker for biliary neoplasms than CA19-9.

Antigens, Neoplasm↗

PubMed2003

A novel tumour marker RCAS1 in a case of extramammary Paget's disease.

A 66-year-old male, whose primary skin lesion in extramammary Paget's disease had been surgically resected 4 years previously, was hospitalized with liver metastases. Hepatic arterial infusion chemotherapy was carried out and the tumours clearly reduced in size. Serum levels of some common tumour markers were not elevated, even prior to therapy. We measured serum levels of a novel tumour-associated antigen, RCAS1, because its expression was detected in the tumour cells. The patient's serum RCAS1 level was elevated (22.0 U/mL) before therapy and fell during (10.5 U/mL) and after (5.0 U/mL) therapy. Therefore, serum RCAS1 levels may be valuable as a potential biomarker for monitoring therapeutic efficacy against Paget's disease.

Aged↗

PubMed2001

A DnaJ protein, apobec-1-binding protein-2, modulates apolipoprotein B mRNA editing.

Mammalian homologues of DnaJ proteins, also known as Hsp40 proteins, are co-chaperonins that complement Hsp70 chaperone function. Using the yeast two-hybrid system, we cloned an apolipoprotein (apo) B mRNA editing complementation protein, called apobec-1-binding protein-2 (ABBP-2), and found that it is a Class II DnaJ homologue. ABBP-2 binds to apobec-1, the mammalian apoB mRNA editase, via its J domain and neighboring G/F domain. It is a ubiquitously expressed protein, and, by transfection analysis of GFP-ABBP-2, we found that the protein is located in both the nucleus and cytosol of transfected cells, with predominance in the nucleus. Down-regulation of ABBP-2 expression in cultured cells inhibits endogenous apobec-1-mediated apoB mRNA editing. Like other Hsp40 proteins, ABBP-2 binds to Hsp70 and has ATPase-stimulating activity. Apobec-1-mediated apoB mRNA editing activity of in vitro tissue extracts requires the presence of Hsp70/ABBP-2. Although exogenously added ATP is not required for editing activity, removal of the endogenous ATP present in these extracts, which disrupts ABBP-2-Hsp70 interaction, completely inhibits editing. ABBP-2 differs from previously described auxiliary proteins (ABBP-1, ACF, and GRY-RBP) in that it does not contain any RNA recognition motifs. Not only is ABBP-2 required for efficient apoB mRNA editing, this newly discovered apobec-1-binding protein may help determine the subcellular distribution and trafficking of apobec-1 via its interaction with the chaperonin Hsp70.

Adenosine Triphosphate↗

PubMed2001

Expression of a tumor-associated antigen RCAS1 in hepatocellular carcinoma.

RCAS1 has been reported as a tumor-associated antigen in uterine and ovarian carcinomas. In vitro studies on RCAS1 indicated that it might function as an apoptosis-inducing factor since binding between RCAS1 and its receptor induced apoptosis in receptor-expressing cells. In this study, 68 surgically resected samples of hepatocellular carcinoma (HCC) were prepared and RCAS1 expression was examined immunohistochemically, because RCAS1 was also positive in all HCC cell lines tested. Clinical and pathological parameters were then compared between RCAS1-positive and -negative HCC cases. As a result, RCAS1 is expressed in 26.5% of HCC cases and vascular invasion is observed at a much higher rate in the RCAS1-positive cases (72.2%) than in RCAS1-negative cases (24.0%). RCAS1 is not an antigen specific for gynecological cancers. In HCC cases, the RCAS1-positive percentage is not high, however, RCAS1-positive HCCs exhibited a trend towards invasive character.

Animals↗

PubMed2001

Serum levels of HCV RNA and core protein before and after incubation at 37 degrees C for 24 h.

The kinetics of HCV during interferon (IFN) therapy have recently been described and the estimated virion half-life is an average of 2.7 h, suggesting that HCV infection is highly dynamic. The aim of this study was to evaluate serum levels of HCV-RNA and HCV core protein (HCV-Ag) before and after incubation at 37 degrees C for 24 h. We also evaluated the viral kinetics during IFN treatment by determining their serum levels at 0, 24 and 48 h, and day 8 after the start of treatment. The decay slope was calculated as the logarithm of the ratio of HCV-RNA levels at 0 and 24 h of incubation: log(virus load) 24 h-log(virus load) 0 h and the estimated half-life was also calculated. The decay slope was -1.66+/-0.75 (-4.12 to -0.18) (mean+/-S.D. (range)) and the estimated virion half-life was 6.2+/-6.9 h (1.8-39.3). The HCV-RNA level was rapidly decreased to 6.8+/-13.1% of the initial load after incubation independently of the serotype. In contrast, the HCV-Ag level after incubation for 24 h was 98.7+/-12.2% of the initial level. The synthesized naked HCV-RNA (equivalent to 10(7) copy/ml) was not detected after 1-min incubation. These data suggested that HCV virions are very unstable and collapsed rapidly and that HCV-RNA, existing outside of virions, is immediately degraded in serum, whereas HCV-Ag remains stable. IFN treatment caused a rapid decrease in the levels of both HCV-RNA and HCV-Ag. The HCV-RNA decay slope was -1.95+/-0.96 (range: -3.48 to -0.50) and was similar to that seen in the incubation study. Our result suggested the significance of measuring HCV-Ag during clinical management independently of HCV-RNA, especially because of its high stability.

Journal Article↗

PubMed2001

Telomerase activity is repressed during differentiation along the hepatocytic and biliary epithelial lineages: verification on immortal cell lines from the same origin.

Recent investigations indicate that telomerase activity regulates the life span of cells by compensating for telomere shortening during DNA replication. In addition, as differentiation progresses, telomerase activity is reduced in several different cell lineages. These findings lend support to the theory that more immature cells have greater remaining proliferative capacity and longer life span. However, it has not been directly demonstrated that the differentiation along a hepatocytic or a bile ductal lineage is accompanied by reduction of telomerase activity. In this study, we present direct evidence that telomerase activity is reduced during hepatocytic and biliary epithelial differentiation by using our unique cell lines including a stem-like cell line, ETK-1. When hepatocytic differentiation was induced in ETK-1 by 5-azacytidine, telomerase activity decreased significantly. Similarly, when we compared the telomerase activity on SSP-25 and RBE cell lines from the same origin but representing different maturation stages of cholangiocarcinoma, more mature cells were found to possess significantly lower activity. These results indicate that the generally accepted relationship between telomerase activity and differentiation stage also applies in the hepatocytic and biliary epithelial lineages.

Azacitidine↗

PubMed2001

Decline of hepatitis C virus load in serum during the first 24 h after administration of interferon-beta as a predictor of the efficacy of therapy.

BACKGROUND/AIMS: Hepatitis C virus (HCV) kinetics during interferon (IFN)-alpha treatment have been evaluated recently, however, little is known about the resultant viral kinetics in IFN-beta treatment. In this study, we evaluated HCV kinetics during the first 24 h of IFN-beta treatment, and also assessed their relationship to therapeutic outcomes. METHODS: We measured HCV RNA levels at 0 and 24 h after the initiation of IFN-beta treatment, and we calculated the decay slope, viral half-life, and viral production and clearance. Then we analyzed these factors as they related to therapeutic responses with IFN-beta as well as to clinical variables, i.e. genotype, diversity of hyper variable region, and histological findings. RESULTS: Patients with sustained responses (SR) displayed steeper decay slopes of the viral load than those without SR (2.87 +/- 1.41 vs. 1.82 +/- 1.66, P = 0.031). On the other hand, the decay slope was not affected by the clinical variables. The values of viral half-life and viral production and clearance showed no significant correlation to the response and the clinical variables. CONCLUSION: This study demonstrated that the decay slope of the viral load during the first 24 h is related to the virological response to IFN-beta treatment.

Adolescent↗

PubMed2001

A selective ROCK inhibitor, Y27632, prevents dimethylnitrosamine-induced hepatic fibrosis in rats.

BACKGROUND: p160ROCK is a direct Rho target which mediates Rho-induced assembly of focal adhesions and stress fibers. We previously reported that Rho signaling pathways are involved in the activation of hepatic stellate cells (HSC) in vitro. The aim of the present study was to test the hypothesis that an inhibitor specific for p160ROCK (Y27632) could prevent experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. METHODS: Y27632 was given orally at 30 mg/kg daily for 4 weeks after the first injection of DMN. The degree of fibrosis was evaluated by image analysis and also by measurements of collagen and hydroxyproline content in the liver. The expression of alpha-smooth muscle actin (alpha-SMA) in the liver and in the primary cultured HSC was also evaluated. Semi-quantitative RT-PCR was performed to evaluate the expression of type I collagen mRNA in the liver. RESULTS: Y27632 treatment significantly decreased the occurrence of DMN-induced hepatic fibrosis and reduced the collagen and hydroxyproline content and alpha-SMA expression in the liver. The expression of alpha-SMA in HSC was also suppressed in vitro. CONCLUSIONS: These findings indicate that inhibitors of the Rho-ROCK pathway might be useful therapeutically in hepatic fibrosis.

Actins↗

PubMed2001

Pirfenidone inhibits dimethylnitrosamine-induced hepatic fibrosis in rats.

1. In the present study, we investigated the preventive effects of pirfenidone (PFD), an antifibrotic agent, on experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. 2. Treatment with DMN caused a significant decrease in bodyweight and liver weight. Oral PFD (500 mg/kg daily for 4 weeks) essentially prevented this DMN-induced loss in bodyweight and tended to suppress the loss in liver weight. There were no significant differences in liver weight and serum L-alanine aminotransferase levels between PFD-treated and -untreated groups. Pirfenidone has no major side effects in vivo. 3. Pirfenidone suppressed the induction of hepatic fibrosis determined by histological evaluation and reduced hepatic hydroxyproline levels. Expression of mRNA for type I collagen and transforming growth factor-beta in the liver was also suppressed by PFD treatment. 4. Because hepatic stellate cells (HSC) are the major cellular source of extracellular matrix in hepatic fibrosis, we examined the effects of PFD on type I collagen production in vitro using rat primary HSC cultures. Pirfenidone inhibited collagen production in HSC culture in a dose-dependent manner. 5. These results demonstrate that the inhibitory effects of PFD against hepatic fibrosis may be due, at least in part, to blockade of collagen production by HSC and suggest that PFD may be potentially useful in the prevention of the development of hepatic fibrosis.

Animals↗

PubMed2001

Kinetics of the hepatitis C virus during interferon therapy as a marker of therapeutic response.

BACKGROUND: The viral load and subtype of hepatitis C virus (HCV) are predictors of the efficacy of interferon (IFN) therapy. The kinetics of HCV during IFN therapy have been described recently, suggesting that HCV infection is highly dynamic. These observations have raised the issue as to whether early monitoring of the viral load can help guide IFN therapy. METHODS: We measured HCV-RNA levels at 0, 24 and 48 h after the start of IFN-alpha treatment (10 MU daily for 2 weeks and then three times weekly for 22 weeks) or IFN-beta treatment (6 MU daily for 6 weeks). Then we analyzed the relationship between HCV kinetics and therapeutic response using stepwise multivariate logistic regression analysis. RESULTS: The exponential decay slope of the viral load during the first 24 h, not the first 48 h or the next 24 h, was a predictor of viral eradication at 6 months after completion of the treatment (sustained response; P = 0.0023). This decay slope was not affected by the HCV serotype or the type of IFN used. Initial viral load and HCV serotype were also predictors, as reported previously (P < 0.0001 and P = 0.0347, respectively). We also proposed a model using a prognostic index that predicted a sustained response with more than 80% sensitivity, specificity and efficacy in an independent and external group of patients. CONCLUSION: This study demonstrated that the exponential decay slope of the viral load during the first 24 h was an important predictor of the response to IFN therapy as well as the initial viral load and HCV serotype. The model may also be useful for the clinical management of IFN therapy.

Antiviral Agents↗

PubMed2001

Prevalence of hepatitis B or C virus infections in patients with non-Hodgkin's lymphoma.

BACKGROUND: Hepatitis C virus (HCV) and hepatitis B virus (HBV) are not only hepatotropic, but also lymphotropic viruses. Recently, some reports suggested that these viruses may participate in the development of malignant lymphoproliferative disorders. METHODS: We investigated the prevalence of HCV or HBV infection in 348 patients with non-Hodgkin's lymphoma (NHL). We also compared these prevalences with those in blood donors as a control group representing the general population in our area (n= 1,513,358). Next, we evaluated the clinical and pathologic characteristics of HCV- or HBV-infected NHL cases. Non-Hodgkin's lymphoma was classified according to the Working Formulation classification. RESULTS: Thirty-seven cases (14.9%) were found to be infected with HCV or HBV; of these, 20 (8.1%) were infected with HCV, and 17 (6.9%) with HBV. In male NHL patients, the rate of HCV infection was significantly higher than in an age- and sex-matched population in the same area (P < 0.001, Mantel-Haenszel test). The rate of HBV infection also tended to be higher in the population (P = 0.0551). In contrast, in female NHL patients, the rate of HCV or HBV infection was not higher than in the general population. In HCV-infected cases, 15 cases (75%) had B-cell NHL and 16 cases (80%) were classified as being in the intermediate grade; B-cell NHL comprised 83% of all NHL cases. In HBV-infected NHL cases, 11 (65%) were of B-cell type and 10 (58%) were classified as being in the intermediate grade. CONCLUSIONS: The high prevalence of HCV or HBV infections in our study population provides epidemiologic evidence suggesting that HCV and HBV infections may be involved in the development of a subgroup of NHL in males. Our investigation also revealed that both HCV- and HBV-infected NHL patients showed certain similarities in clinical and pathologic manifestations.

Adult↗

PubMed2001

Activation of retinoic X receptor and peroxisome proliferator-activated receptor-gamma inhibits nitric oxide and tumor necrosis factor-alpha production in rat Kupffer cells.

Activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), which forms a heterodimer with retinoic X receptor (RXR), inhibit the production of certain inflammatory mediators. To clarify the role of the PPAR gamma:RXR signaling pathway in Kupffer cells, we studied the effect of an RXR agonist and PPARgamma agonist on LPS-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production. An RXR-specific agonist, Ro47-5944, and a PPAR gamma-specific agonist, AD4833 (pioglitazone hydrochloride), each inhibited LPS-induced NO and TNF-alpha production. The combined treatment of Ro47-5944 and AD4833 resulted in enhanced inhibition, and suppressed the mRNA levels of NO and TNF-alpha. PPAR gamma:RXR activation did not affect the level of LPS-induced phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase. PPAR gamma:RXR activation also did not affect nuclear factor kappa B (NF-kappa B) nuclear translocation nor NF-kappa B and activator protein 1 (AP-1) activation in the electrophoretic mobility-shift assay. Finally, PPAR gamma:RXR activation suppressed the LPS-induced promoter activity of the NF-kappa B-luciferase reporter gene in RAW 264.7 cells. These data imply that PPARgamma:RXR activation suppresses LPS-induced NO and TNF-alpha production in Kupffer cells, and that this inhibition occurred at the transcriptional level. Although no consensus PPAR gamma:RXR-responsive element in the promoter regions of the inducible isoform of nitric oxide synthase (iNOS) and TNF-alpha genes was found, PPAR gamma:RXR may interfere with NF-kappa B and AP-1 transcriptional activity. Our data also suggest a potential therapeutic approach for moderating hepatic injury such as endotoxin shock in which Kupffer cell activation has been implicated.

Animals↗

PubMed2001

The selenoorganic compound ebselen suppresses liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats.

Ebselen (2-phenyl-1,2-benzoisoselenazol-3[2H]-one) is a selenoorganic compound containing selenium that has various pharmacological effects, including anti-inflammatory and antioxidant activity. Kupffer cells, residual hepatic macrophages, play an important role in the development of liver injury by producing free radicals and cytokines. The aim of this study is to evaluate whether ebselen suppresses macrophage-associated liver injury in rats. In vivo, we examined the effects of ebselen on liver injury, induced by Propionibacterium acnes and lipopolysaccharide (P. acnes-LPS), in rats where hepatic macrophages are considered to be primarily involved in injury development. Ebselen administration reduced the incidence of death following hepatic failure by P. acnes-LPS (82% vs. 20%, p<0.05). Serum levels of alanine aminotransferase, at 5 h after LPS administration, were significantly lower in the ebselen-treated group than in the control group (202.4+/-100.3 IU/l vs. 558.4+/-146.4 IU/l, p<0.05). Histological evidence of injury, such as necrosis, hemorrhage, and degeneration, was also suppressed by ebselen. Further, to assess the mechanisms involved, we investigated the production of cytokines and superoxide anions produced by activated hepatic macrophages in vivo. Serum levels of TNF alpha, interleukin-18 (IL-18)/IFN gamma-inducing factor (IGIF), and interferon gamma (IFN gamma) at 1 h after LPS administration were significantly lower in the ebselen-treated group. Formazan depositions, which were generated by the perfusion of the liver with nitroblue tetrazolium, were also observed less frequently in the ebselen treated group, suggesting a suppression in the release of superoxide anion from activated hepatic macrophages. In addition, we examined the effects of ebselen on cytokine production and mRNA expression, in vitro, using rat primary Kupffer cell culture. Ebselen also inhibited TNF alpha production and mRNA expression in vitro. These data imply that ebselen suppresses liver injury by inhibiting the production and/or release of proinflammatory cytokines and superoxide from activated hepatic macrophages. These data also suggest that ebselen is potent in the prevention of hepatic injury, such as endotoxemia, where hepatic macrophage activation has been implicated.

Alanine Transaminase↗

PubMed2001

Dimethyl sulfoxide inhibits dimethylnitrosamine-induced hepatic fibrosis in rats.

We studied the preventive effects of dimethyl sulfoxide (DMSO) on experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. Treatment with DMN caused a significant decrease in body and liver weight. Oral DMSO (2 ml/kg daily for 4 weeks) essentially prevented this DMN-induced body and liver weight loss with no major side effects. DMSO suppressed the induction of hepatic fibrosis, as determined by histological evaluation, and reduced hepatic hydroxyproline. It also suppressed the expression of mRNA for type I collagen in the liver. Because hepatic stellate cells (HSC) are the major cellular source of the collagen in hepatic fibrosis, we examined the effects of DMSO on collagen production in vitro using rat primary HSC culture. However, it was found that DMSO did not inhibit the collagen production in vitro. We next evaluated the effects of DMSO on tumor necrosis factor alpha (TNFalpha) and nitric oxide (NO) production by Kupffer cells, because these factors represent major activator of HSC, and because monocyte-macrophage infiltration has been implicated as being pathogenetically important for hepatic fibrosis induced by DMN. DMSO inhibited lipopolysaccharide (LPS)-induced TNFalpha and NO production, and reduced TNFalpha mRNA levels. DMSO also suppressed the LPS-induced nuclear factor kappa B activation in a murine macrophage-like cell line. These results suggest that the inhibitory effects of DMSO on hepatic fibrosis may be primarily exerted via blocking of DMN-induced inflammation. These results also implied that DMSO may be potentially useful for preventing the development of hepatic fibrosis.

Animals↗

PubMed2000

Hammerhead ribozyme as a therapeutic agent for hyperlipidemia: production of truncated apolipoprotein B and hypolipidemic effects in a dyslipidemia murine model.

In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we used adenovirus-mediated vector to target hammerhead ribozyme at GUA(6679) downward arrow of apoB mRNA (designated AvRB15) in the liver of a dyslipidemic mouse model that is deficient in apoB mRNA editing enzyme and overexpresses human apoB100. In this study, we delivered approximately 4 x 10(11) virus particles of AvRB15 (active ribozyme) or AvRB15-mutant (inactive ribozyme) to the animals. Using Southern blot analysis, we readily detected RB15 DNA in the mouse liver as long as day 35 after injection. This result was correlated with the RNA expression of RB15 by RNase protection assay. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site was confirmed by sequencing. Importantly, the levels of human and mouse apoB mRNA decreased approximately 80% after AvRB15 transduction. There was a marked decrease in plasma cholesterol, triglyceride, and human apoB of 42, 51, and 62%, respectively, when compared with the inactive ribozyme-treated group. Moreover, ribozyme cleavage of apoB mRNA generated a truncated protein of the expected size (apoB48.1), which was associated with lipoprotein particles in the very low density, low density, and high density lipoprotein fractions. Taken together, these results indicate that apoB mRNA-specific hammerhead ribozyme can be used as a potential therapeutic agent to modulate apoB gene expression and to treat hyperlipidemia.

Animals↗