Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A.

Questions remain concerning the long-term efficacy, safety, and site(s) of transgene expression following adeno-associated vector (AAV) therapy. We report a long-term follow-up of 8 (male = 4, hemizygous, and female = 4, homozygous) dogs with severe hemophilia A treated with a single portal vein infusion of a B-domain-deleted (BDD)-canine FVIII (cFVIII) AAV vector (median dose = 1.25 × 1013 vg/kg, AAV2 = 4, AAV6 = 3, and AAV8 = 1). After a median follow-up of 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage = 12.7%, chromogenic = 7.2%) was seen in all responding dogs (n = 6), with improvement in annualized bleed rates (pre = 3.9 vs post = 0.3 event per year; P = .003). Anti-AAV capsid neutralizing antibodies (nAbs) toward the dosed capsid were detected throughout the study, with limited cross-reactivity to other capsids. nAb titers for all capsid serotypes declined with time, although they remained at levels precluding redosing with the same capsid. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median = 0.15 vg per diploid genome), with lower levels in the spleen in 4 dogs (median = 0.005 vg per diploid genome). Consistent with the liver-specific promoter, BDD-cFVIII mRNA was only detected in the liver. Postmortem examination demonstrated no evidence of chronic liver disease or liver malignancy. Persistent FVIII expression and an improved bleeding phenotype was seen for more than a decade after vector delivery. This is the longest follow-up reported in a preclinical model supporting long-term efficacy and safety of AAV-mediated gene therapy.

Fibroblast growth factor 13 regulates glioma cell invasion and is important for bevacizumab-induced glioma invasion.

Glioblastoma has the poorest prognosis, and is characterized by excessive invasion and angiogenesis. To determine the invasive mechanisms, we previously used two glioma cell lines (J3T-1 and J3T-2) with different invasive phenotypes. The J3T-1 showed abundant angiogenesis and tumor cell invasion around neovasculature, while J3T-2 showed diffuse cell infiltration into surrounding healthy parenchyma. Microarray analyses were used to identify invasion-related genes in J3T-2 cells, and the expressed genes and their intracellular and intratumoral distribution patterns were evaluated in J3T-2 cell lines, human glioma cell lines, human glioblastoma stem cells and human glioblastoma specimens. To determine the role of the invasion-related genes, invasive activities were evaluated in vitro and in vivo. Fibroblast growth factor 13 (FGF13) was overexpressed in J3T-2 cells compared to J3T-1 cells, and in human glioma cell lines, human glioblastoma stem cells and human glioblastoma specimens, when compared to that of normal human astrocytes. Immunohistochemical staining and the RNA-seq (sequencing) data from the IVY Glioblastoma Atlas Project showed FGF13 expression in glioma cells in the invasive edges of tumor specimens. Also, the intracellular distribution was mainly in the cytoplasm of tumor cells and colocalized with tubulin. Overexpression of FGF13 stabilized tubulin dynamics in vitro and knockdown of FGF13 decreased glioma invasion both in vitro and in vivo and prolonged overall survival of several xenograft models. FGF13 was negatively regulated by hypoxic condition. Silencing of FGF13 also decreased in vivo bevacizumab-induced glioma invasion. In conclusion, FGF13 regulated glioma cell invasion and bevacizumab-induced glioma invasion, and could be a novel target for glioma treatment.

Integrin antagonist augments the therapeutic effect of adenovirus-mediated REIC/Dkk-3 gene therapy for malignant glioma.

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) was identified as a gene whose expression is reduced in many human cancers. REIC/Dkk-3 expression is also downregulated in malignant glioma and regulates cell growth through caspase-dependent apoptosis. cRGD (EMD121974), an antagonist of integrins, has demonstrated preclinical efficacy against malignant glioma. In this study, we investigated the antiglioma effect of combination therapy using an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) and cRGD. Quantitative real-time reverse-transcription PCR revealed the reduction of REIC/Dkk-3 mRNA levels in malignant glioma cell lines. The reduction of REIC/Dkk-3 protein expression in malignant glioma cell lines was also confirmed with western blot analysis. After treatment with Ad-REIC and cRGD, the proliferative rate of malignant glioma cells was significantly reduced in a time-dependent manner. In vivo, there was a statistically significant increase in the survival of mice treated with Ad-REIC and cRGD combination therapy compared with Ad-REIC monotherapy. We identified an apoptotic effect following monotherapy with Ad-REIC. Moreover, cRGD augmented the antiglioma efficacy of Ad-REIC. These results may lead to a promising new approach for the treatment of malignant glioma.

The integrin inhibitor cilengitide enhances the anti-glioma efficacy of vasculostatin-expressing oncolytic virus.

Oncolytic viral (OV) therapy has been considered as a promising treatment modality for brain tumors. Vasculostatin, the fragment of brain-specific angiogenesis inhibitor-1, shows anti-angiogenic activity against malignant gliomas. Previously, a vasculostatin-expressing oncolytic herpes simplex virus-1, Rapid Antiangiogenesis Mediated By Oncolytic virus (RAMBO), was reported to have a potent antitumor effect. Here, we investigated the therapeutic efficacy of RAMBO and cilengitide, an integrin inhibitor, combination therapy for malignant glioma. In vitro, tube formation was significantly decreased in RAMBO and cilengitide combination treatment compared with RAMBO or cilengitide monotherapy. Moreover, combination treatment induced a synergistic suppressive effect on endothelial cell migration compared with the control virus. RAMBO, combined with cilengitide, induced synergistic cytotoxicity on glioma cells. In the caspase-8 and -9 assays, the relative absorption of U87ΔEGFR cell clusters treated with cilengitide and with RAMBO was significantly higher than that of those treated with control. In addition, the activity of caspase 3/7 was significantly increased with combination therapy. In vivo, there was a significant increase in the survival of mice treated with combination therapy compared with RAMBO or cilengitide monotherapy. These results indicate that cilengitide enhanced vasculostatin-expressing OV therapy for malignant glioma and provide a rationale for designing future clinical trials combining these two agents.

Appendiceal mucocele: sonographic findings.

BACKGROUND: Appendiceal mucocele (AM) is a relatively rare disease, and its sonograms (US) have not been sufficiently analyzed. METHODS: We studied the US findings of five patients with AM, with special attention to AM size, shape, internal echoes, and the mode of back echoes. RESULTS: All five cases showed an elongated mass in the lower right abdomen. Internal echoes were present in all cases and M-mode US confirmed the movement of those echoes. The echogenecity of the lesion changed according to the frequency of the transducer used. Only one case showed posterior echo enhancement, and no case showed lateral shadowing. CONCLUSION: AM appears as an elongated echo-poor mass without posterior echo enhancement. The cyst wall is less distinct than what one would expect for a cyst. When encountering such a mass in the lower right abdomen, one should strongly suspect an AM. In such cases, appropriate diagnostic and therapeutic strategies are especially necessary to prevent rupture that results in development of pseudomyxoma peritonei.

Dilatation of the cystic duct in patients with obstructive jaundice.

BACKGROUND: It is well known that obstructive jaundice causes biliary dilatation, but sonographic (US) findings of the cystic duct in patients with obstructive jaundice are rarely reported. METHODS: We reviewed US findings in 25 such patients. RESULTS: US findings of dilated cystic duct could be divided roughly into two patterns: tortuous and tubular. The former pattern was a tortuously dilated cystic duct arising from the gallbladder neck, running predominantly deeply, turning anteromedially, and then joining the posterior face of the common hepatic duct. The latter pattern was a straight dilated cystic duct running parallel with the common hepatic duct, giving the appearance of a septated bile duct. CONCLUSION: A better understanding of the US findings of dilated cystic duct translates into improved biliary US diagnosis.

Retroperitoneal cyst: sonographic findings.

BACKGROUND: Retroperitoneal cyst (RC) is a relatively rare disease, and its sonographic (US) findings have not been analyzed sufficiently. METHODS: We studied US findings of five patients with RC, with special attention to location, size, shape, internal echoes, the presence or absence of lateral shadowing, and the mode of back echoes. RESULTS: In all cases, the cyst was situated behind (four cases) or lateral to (one case) the pancreas: behind or lateral to the pancreatic head in two cases, behind the pancreatic body in one case, and behind the pancreatic tail in two cases. Four cases showed a round mass (three cases) or multiple round mass (one case). Internal echoes were present in those cases and showed a "pseudo-solid" pattern. In those cases, M-mode US confirmed the movement of those internal echoes. In the remaining case, the lesion was imaged as an irregularly shaped multilocular mass. No case showed posterior echo enhancement, and no case showed lateral shadowing. No blood flow signals from the lesion were seen. CONCLUSION: Unlike ordinary cysts, RC usually is imaged as a round mass behind the pancreas, with dense internal echoes without lateral shadowing or posterior echo enhancement, which presents a "pseudo-solid" pattern. A diagnosis of RC should be considered when encountering a mass with such US findings.

Accessory hepatic duct: sonographic findings.

Accessory hepatic ducts are relatively rare biliary anomalies. They have been reported only sporadically, and very few reports have described their sonographic (US) findings. Our experience with two such cases suggested that a diagnosis of an accessory hepatic duct should be considered when US shows a fine tubular structure or an anechoic or hypoechoic oval mass with acoustic shadowing at the level between the hepatic hilus and the common bile duct. A good knowledge of the US findings will determine the diagnostic and therapeutic strategies.

Abdominal lymphoma: differentiation from pancreatic carcinoma with Doppler US.

BACKGROUND: Marked lymphadenopathy around the pancreas due to lymphoma (abdominal lymphoma) occasionally can mimic a total pancreatic carcinoma on ultrasonography (US). We investigated whether US and color Doppler US allowed differentiation between those pathologies. METHODS: We analyzed the US and color Doppler results of 12 cases of abdominal peripancreatic lymphoma and 21 cases of total pancreatic carcinoma. RESULTS: With regard to shape, echogenicity of the lesion, and mode of vascular involvement, there was no difference between groups. With regard to maximal velocities and resistive indices of the involved vessels, there was no difference between groups. However, the presence of turbulent flows in the involved vessels was seen exclusively in the pancreatic carcinoma group. CONCLUSION: The presence or absence of turbulent flow in the involved vessels is a very important finding for differentiating abdominal lymphomas from total pancreatic carcinomas.

Mesenteric lipoma: report of a case with emphasis on US findings.

Mesenteric lipoma (ML) is a relatively rare disease that has been very infrequently reported. We present the clinical data and medical imaging results of an asymptomatic case with ML incidentally detected by sonography (US). On US the lesion was imaged as a well-demarcated hypoechoic mass with multiple linear echoes. The mass also changed location under probe compression. The lesion was imaged as a clearly demarcated non-enhanced homogeneous fat-density mass on CT. Color Doppler US and angiography confirmed the avascular nature of the mass. Knowledge of the US findings in this case is useful for the diagnosis of ML.

Emphysematous cholecystitis: sonographic findings.

BACKGROUND: Emphysematous cholecystitis (EC) is a rare but life-threatening complication of acute cholecystitis, and an early diagnosis is required to prevent delay in patient management. Because sonography (US) is the first choice for diagnosing gallbladder diseases, US findings of EC should be understood more precisely. METHODS: We reviewed US findings of 11 surgically proven cases of EC (with small amounts of gas in three cases and large amounts in eight cases) and compared those with patients' clinical data. RESULTS: (1) In cases with small amounts of gas, US showed an echogenic line with a distinct ring-down artifact or a "powder snow-like" speckled posterior echo. (2) In cases with large amounts of gas, US showed a wide spiculated echogenic band with a powder snow-like speckled posterior echo or a speckled acoustic shadowing. In all cases, the presence of gas prevented visualization of the gallbladder wall. (3) US did not differentiate gas localized to the gallbladder wall and gas extending to the surrounding hepatic tissue. (4) Two diabetic cases showed gas throughout the intrahepatic bile ducts. In those cases, the time from diagnosis to recovery was relatively long. CONCLUSION: Our series showed some characteristic US patterns of EC. A good understanding of its US findings and appropriate emergent management will reduce the serious morbidity and mortality rates caused by EC.

Segmental chronic cholecystitis: sonographic findings and clinical manifestations.

BACKGROUND: In chronic cholecystitis, the gallbladder (GB) wall is usually evenly involved, whereas marked segmental thickening of the GB wall (segmental cholecystitis) seldom is reported. We wanted to define its clinical manifestations and sonographic (US) findings. METHODS: We reviewed the clinical and US data of 13 cases and compared these results with those of 30 patients with chronic cholecystitis with evenly thickened GB walls (usual-cholecystitis group). RESULTS: (a) All cases of segmental cholecystitis showed the portion distal to the kinking to be markedly thickened. (b) The thickened portion corresponded to the fundus in three cases, the body and fundus in seven cases, and the fundus, body, and infundibulum in three cases; and the thickened distal portion contained many stones in 11 cases. (c) There was no difference in the maximal diameters of the GB walls between the segmental-cholecystitis group and the usual-cholecystitis group. However, there was a significant difference in the minimal diameters of the GB walls between groups. CONCLUSION: Knowledge of the US findings and clinical presentations of segmental cholecystitis can help in the development of appropriate diagnostic and therapeutic strategies.

Determination of serotonin in microdialysis samples from rat brain by microbore column liquid chromatography with post-column derivatization and fluorescence detection.

The present paper describes a new method for on-line determination of 5-HT in brain microdialysates from awake rats by microbore column liquid chromatography with post-column derivatization and fluorescence detection. The derivatization reagent contained 1 mM benzylamine and 0.5 mM potassium hexacyanoferrate (III), both dissolved in a mixture of acetonitrile and 25 mM borate buffer (pH 11.0) (1:1, v/v). The limit of detection (S/N=3) for 5-HT was 0.5 fmol/20 microl. The samples were injected every 20 min onto a microbore column packed with C18 silica gel. The method exhibits an excellent stability over the periods of at least 12-24 h. The basal levels of 5-HT from 25 awake rats were 7.10+/-1.06 fmol/20 microl in the dorsal hippocampus and 4.64+/-0.91 fmol/20 microl (mean+/-SD) in the striatum. The 5-HT release increased to about 1500% during the perfusion with 100 mM K(+) containing Ringer solution or it was reduced to 60 or 40% during the perfusion with 1 microM tetrodotoxin or calcium free Ringer, respectively. The new method can be used to monitor extracellular 5-HT following acute systemic drug administration.

Duodenal carcinoma: sonographic findings.

BACKGROUND: Duodenal carcinoma is a relatively rare tumor and its sonographic (US) findings have rarely been reported. METHODS: We reviewed the clinical data and US results of 15 patients with histologically proven duodenal carcinoma to pinpoint the contribution of US to the diagnosis. RESULTS: US was useful for not only detecting the duodenal carcinoma (13 of 15, 86.7%), which altered the endoscopic procedure in nine patients, but also for evaluating vascular involvement (marked portal invasion in four cases and inferior vena cava invasion in two cases). Four of the eight carcinomas arising from the second or third portion showed this vascular invasion. CONCLUSION: After reviewing the clinical data and US results of 15 cases of duodenal carcinoma, we found that US was useful for detecting duodenal carcinoma and determining vascular invasion. Thus, US contributed significantly to alter the endoscopic procedure and patient management.

Liver tumors in fatty liver: difficulty in ultrasonographic interpretation.

BACKGROUND: Fatty liver and liver tumors are very frequent diseases. Sonography (US) currently is the initial diagnostic tool for hepatic exploration. However, there is a marked paucity of US findings of tumors in fatty liver. METHODS: We studied the US findings of 41 lesions (31 patients) with this combination, with special attention paid to internal echoes, marginal echoes, and especially the mode of back echoes, and compared them with the US results of 64 lesions (38 patients) without fatty liver. RESULTS: Comparing the group having liver tumor without fatty liver with the group having liver tumor with fatty liver showed that (a) the number of hypoechoic lesions increased (29 of 41, 70.7%, vs. 16 of 64, 25.0.%), (2) the tumor margin appeared indistinct (32 of 41, 78.0%, vs. nine of 64, 14.1%), and (c) the lesions showing posterior echo enhancement increased (34 of 41, 82.9%, vs. eight of 64, 12.5%). CONCLUSION: Liver tumors in fatty liver are expected to show unusual patterns on US, so we should consider this difficulty when interpreting these US findings and we should not make a conclusion without including other imaging modalities.

Splenic lymphoma: differentiation from splenic cyst with ultrasonography.

BACKGROUND AND METHODS: Lymphoma can be nearly anechoic and mimic a cyst on ultrasonography (US). To investigate whether this phenomenon occurs at the level of the spleen, we analyzed the US findings of 38 cases of splenic lymphoma and 16 cases of splenic cyst. RESULTS: (1) With regard to shape, echogenicity of the lesion, and mode of posterior echo, there was no difference between splenic lymphomas and splenic cysts. However, the boundaries of the lesions were indistinct in splenic lymphomas and distinct in splenic cysts. (2) Blood flow signals and vascular penetration were seen exclusively in splenic lymphomas. CONCLUSION: The mode of boundary echo (distinct or indistinct) distinguishes splenic lymphomas from splenic cysts. Color Doppler US increases the diagnostic confidence of US.

Peripheral bone metastasis from hepatocellular carcinoma: sonographic findings.

BACKGROUND AND METHODS: We reviewed medical and radiologic records of five patients with bone metastasis from hepatocellular carcinoma (HCC) to evaluate the role of sonography (US) in the diagnosis of this bone metastasis. RESULTS: The metastatic lesions were clearly visualized by US as expansive, homogeneous, soft tissue masses with bony destruction, and color Doppler US showed fine vessels within the lesions. After treatment, US showed a decrease in tumor size with an increase in echogenicity and a decrease in blood flow. Hepatic US at the time of bone metastasis showed a portal tumor thrombus in all cases. CONCLUSION: When US detects a portal venous tumor thrombus in HCC patients, attention should be directed not only to the liver but also to bone to improve patient care. US is useful not only in detecting the metastatic lesion but also in evaluating the treatment effect.

Anti-AIDS agents. 46. Anti-HIV activity of harman, an anti-HIV principle from Symplocos setchuensis, and its derivatives.

Matairesinol (1) and harman (5), identified from Symplocos setchuensis, were found to inhibit HIV replication in H9 lymphocyte cells. Anti-HIV evaluation of 28 derivatives of 5 revealed that compound 19 showed potent activity with EC(50) and therapeutic index values of 0.037 microM and 210, respectively.

Choledochal cyst due to anomalous pancreatobiliary junction in the adult: sonographic findings.

BACKGROUND: Adult cases of choledochal cyst due to anomalous pancreaticobiliary duct junction have been rarely reported. At present, sonography (US) is the first tool for diagnosing biliary disorders. The aim of this study was to reevaluate the US findings of choledochal cysts due to anomalous pancreaticobiliary duct junction in adults. METHODS: We reviewed the clinical manifestations and US findings of 12 such adult cases confirmed by endoscopic retrograde cholangiopancreatic ductography (ERCP). Patients were assigned to three groups: (a) associated with biliary carcinoma (two cases), (b) associated with choledocholithiasis (one case), and (c) not associated with other abdominal diseases (nine cases). RESULTS: Patients in group c were asymptomatic, and the lesions were detected incidentally detected by US. In contrast, patients in group a sought medical care because of symptoms such as jaundice and those in group b sought medical attention because of abdominal pain. The diameter of a dilated bile duct on US was considerably less than that of ERCP (ERCP: 26-58 mm, mean = 37.6 mm; US: 13-32 mm, mean = 21.8 mm). Its diameter changed significantly under probe compression when the dilated bile duct took a purely cystic form but changed very little when it took a tubular form. CONCLUSION: Unlike cases in children, adult cases of choledochal cyst are generally asymptomatic. Careful US observation of the bile duct is thus expected to detect asymptomatic adult choledochal cysts cases. Cyst diameter can change significantly under probe compression, so it is important not to compress the bile duct during routine US examination.

Mycotic abdominal aneurysm: report of a case with emphasis on the presence of gas echoes.

Mycotic abdominal aneurysm is a relatively rare disease and its sonographic (US) findings have rarely been reported. We present a case of mycotic aneurysm of the common iliac artery in which the initial US showed a slightly wall-thickened abdominal aorta with gas echoes in the wall, a second US 6 days later showed a rapid increase in aortic size, and an emergent surgical intervention showed a mycotic aneurysm. This interesting observation suggested that the presence of gas echoes is a useful US finding indicating the beginning of a mycotic aneurysm and, when encountering this US finding, that emergent treatment should be considered to prevent delay in patient management.