A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome.

Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.

The effects of FR167653 on pulmonary ischemia-reperfusion injury in dogs.

BACKGROUND: Ischemia-reperfusion injury may result in the local release of proinflammatory cytokines. A newly synthesized organic compound, FR167653, has been characterized as a potent suppressant of interleukin-1 beta and tumor necrosis factor-alpha. We investigated the effects of FR167653 on ischemia-reperfusion injury of the lung by using an in situ warm lung ischemia model in dogs. METHODS: Thirteen dogs were divided into two groups; seven dogs were assigned to the control group, and six were assigned to the FR167653-treated group. The latter group was administered FR167653 (1 mg/kg/hr) continuously beginning 30 minutes before induced ischemia and ending 2 hours after reperfusion. Warm ischemia was induced for 3 hours by clamping the pulmonary artery and veins. The left main bronchus was bisected and anastomosed 3 hours later. Arterial oxygen saturation, left pulmonary vascular resistance, and cardiac output were measured. Blood was collected to measure interleukin-1 beta level, and the lung specimen was harvested for histologic study. RESULTS: Arterial oxygen saturation levels after 30 minutes and 2 hours of reperfusion were significantly (p < 0.05) higher in the FR167653-treated group than in the control group. After 30 minutes of reperfusion, cardiac output deterioration was significantly (p < 0.05) greater in the control group than in the FR167653-treated group, and left pulmonary vascular resistance was significantly (p < 0.05) lower in the FR167653-treated group than in the control group. The 3-day survival rate was 67% in the FR167653-treated group and 14% in the control group. There were statistically significant differences (p < 0.05) between the survival rates of the two groups. Alveolar damage with interstitial edema and hyaline membranes localized along the alveolar duct were observed in the control group; whereas reduced interstitial edema was observed in the FR167653-treated group. Blood levels of IL-1 beta were lower in the FR167653-treated group than in the control group. CONCLUSION: FR167653 seems to generate a protective effect relative to ischemia-reperfusion injury of the lung in the early stage of tissue damage.

Effect of lazaroid U-74389G on pulmonary ischemia-reperfusion injury in dogs.

Lipid peroxidation induced by oxygen free radicals is a contributing factor in ischemia-reperfusion injury. Lazaroid U-74389G (LAZ-G) is a group of new synthetic 21-aminosteroids and inhibits irondependent lipid peroxidation. We investigated the effects of LAZ-G on pulmonary ischemia-reperfusion injury in dogs. Twenty dogs were divided into three groups. In the LAZ-G group (n = 6), LAZ-G was administered 15 min before ischemia. In the St group (n = 5), methylprednisolone was injected 15 min before ischemia and 15 min before reperfusion. In the control group (n = 9), the vehicle of Lazaroid was injected 15 min before ischemia. Warm ischemia was induced for 3 h by clamping the pulmonary artery and veins. Arterial oxygen saturation (SaO2), cardiac output (CO), left pulmonary vascular resistance (L-PVR), and blood levels of interleukin-1beta mRNA were measured. The lung specimen was harvested for histologic study and polymorphonuclear neutrophils (PMNs) counting. SaO2 levels at 30 min and 2 h after reperfusion were significantly higher in the LAZ-G group than in the control group. After 30 min of reperfusion, CO was significantly better in the LAZ-G group than in the St and control groups, and the L-PVR level was significantly lower in the LAZ-G group than in the control group. Survival rates were significantly better in the LAZ-G group than in the control group. Histological damages and PMNs infiltration were more severe in the control group than in the LAZ-G group. Interleukin-1beta mRNA levels were lower in the LAZ-G group than in the control group. Lazaroid U-74389G appears to generate a protective effect against ischemia-reperfusion injury of the lung.

Gallbladder carcinoma with choledochoduodenal fistula: a case report with surgical treatment.

A 79 year-old man was admitted to our hospital because of upper abdominal pain and nausea. A mobile tumor was palpable in the right upper abdomen. Abdominal ultrasonography, computed tomography and celiac angiography revealed a gallbladder tumor. Endoscopic retrograde cholangiopancreatography revealed a fistula 1.5 cm oral to the orifice of the papilla of Vater, dilatation of the common bile duct, and a filling defect in the gallbladder. Pancreatoduodenectomy associated with reconstruction using Imanaga's method was performed under a pre-operative diagnosis of gallbladder carcinoma with choledochoduodenal fistula. The gallbladder contained a tumor and two bilirubin stones impacted in the orifice of the duodenal papilla. Histological studies confirmed that the gallbladder tumor was a mucinous adenocarcinoma and had not infiltrated the bile duct. We speculated that choledochoduodenal fistula stimulated the development of cancer due to chronic irritation from pancreatic juice reflux.

[Trend of bacterial meningitis in children over a 14 year period (1981 through 1994) in Japan--an analysis based on studies in 27 institutions].

We observed 266 children with purulent meningitis in 27 institutions in Japan during the 14 years from 1981 on dividing these years into 3 periods, 1981-1985, 1986-1990 and 1991-1994, and studied the trend of causative organisms identified in 254 among the 266 patients. Their ages were less than 3 months after birth in 50 children and 3 months or older in 216: there were 141 boys and 125 girls. The causative organisms were H. influenzae in 134 patients and S. pneumoniae in 50, most of them being aged 3 months or older. Next to the above bacteria ranked S. agalactiae in 29 and E. coli in 12, many of the patients were aged less than 3 months. Staphylococcus spp. was found in 7 patients and about 70% of them were aged 3 months or older. L. monocytogenes was found in 4 patients and N. meningitidis in 3 and they were aged 3 months or older in both patient groups. S. pyogenes, Enterococcus spp., Peptostreptococcus spp., P. Mirabilis and Enterobacter spp. were detected each in 1 patient. The causative organism was unknown in 21 patients and there was no double infection. H. influenzae were detected in 18 patients in 1981-1985 period (36.7%), in 56 in 1986-1990 (54.9%) and in 60 in 1991-1994 (63.8%) showing an increasing tendency, but S. pneumoniae exhibited neither an increasing nor decreasing tendency. There was a decreasing tendency with S. agalactiae and E. coli, but the details were not clear because there were few patients aged less than 3 months. Although the period of coexistence of 4 main bacterial species was not made clear in this study. Listeria is considered to develop mainly in the early childhood, and we believe that the conventional way of using a cephem preparation and ampicillin combined for patients under 6 years need not be altered. However, panipenem (phonetic) is likely to be effective for insensible S. pneumoniae for the time being.

[Influence of dexamethasone on the clinical course of bacterial meningitis in children. Especially on secondary fever. Experiences in 27 institutions].

Of pediatric patients with purulent meningitis seen at the institutions listed in the title page of this paper between 1986 and 1994, 93 patients treated with antibiotics and dexamethasone (DXM) were compared with 91 patients treated with antibiotics alone. The patients receiving antibiotics with dexamethasone achieved overall improvement in inflammatory symptoms and signs and cerebrospinal fluid findings and became afebrile significantly earlier than those receiving antibiotics alone. However, some of the patients became febrile again. The secondary fever rate for the DXM group was much higher than that for the antibiotic alone group (p < 0.0001). In most of the rebounded cases, the body temperature rose above 38 degrees C and remained elevated for 2-4 days. Cerebrospinal fluid (CSF) was cultured daily in 54 and 32 patients receiving antibiotics with and without DXM, respectively. Although this study was not a controlled study in a strict sense, these patients compared. In both groups, the CSF became mostly culture-negative within 48 hours. In a few patients receiving DXM, however, it became culture-negative after 72 hours or longer. DXM caused an adverse effect in a patient with meningitis caused by Streptococcus pneumoniae. The adverse effect was mild gastrointestinal bleeding, which recovered spontaneously. From the findings described above, the use of DXM combined with antibiotic therapy was considered to accelerate the relief from fever and improvement of inflammatory symptoms and signs and CSF findings. The body temperature rose again in more than half of the patients receiving DXM, but fell to normal spontaneously without treatment. The elevation doubtlessly could not be distinguished from recurrence of the meningitis itself or complications. It seems to be likely that no treatment but careful observation is required even if the fever recurs as far as the CSF findings showed favorable progress with excelluent general conditions. When DXM is given, it is essential that CSF tests and culture are repeated during the early stages and the progress is monitored carefully.

[Unresectable gastric cancer followed by remarkably effective tumor disappearance and good quality of life for 10 months after CDDP/5'-DFUR combination chemotherapy--a case report].

A 40-year-old woman was admitted to the hospital because of dysphagia and severe anemia (Hb 4.5 g/dl). She was diagnosed as having an advanced gastric cancer, which was unresectable because of liver metastasis, esophageal invasion and paraaortic lymph node metastasis. Combination chemotherapy with CDDP/5'-DFUR was started. CDDP of 80 mg/m2 was administered twice every 4 weeks by a 24-hour drip infusion method, and oral 5'-DFUR of 1,400 mg/m2 was administered for 4 days prior to the first administration of CDDP. Then, 5'-DFUR of 500 mg/m2 was given every day except for 7 days after the first administration of CDDP. Her performance status before the chemotherapy was 3, and improved to 1 a month after the first administration of CDDP. The patient was discharged very much improved on the 45th day after the first administration of CDDP. The side effect was nausea but tolerable. Six months after the first administration, her cancer disappeared on X-ray films, endoscopic and CT examinations. Her PS improved to 0, and she has remained alive with a good QOL for 10 months after the second administration of CDDP.

[Effect of combination chemotherapy with multiple drugs (FLMP therapy) based on the circadian rhythms of the human body in advanced recurrent gastric cancer].

Combination chemotherapy with multiple drugs (FLMP therapy), in which the drugs were determined based on biochemical modulation and the dosing schedule was established in accordance with the circadian rhythms of the human body, was performed in cases of advanced recurrent gastric cancer. The drugs were administered according to the following schedule: 500 mg of 5-FU (continuous) on days 1-5 (the dose was increased during the night), 20 mg of LV on days 1-5 (at 6 PM), 2 mg of MMC on day 5 (at 9 AM) and 60-80 mg of CDDP on day 5 (at 6 PM). A five-day course was administered by intravenous drip or hepatic arterial infusion at intervals of 4 weeks. Of 14 patients treated, the effect was estimated to be CR in 3, PR in 6, NC in 3, and PD in 2. The effectiveness rate was 62.3% overall, and the rate by administration route was 6/10 (60.0%) for i.v. and 3/4 (75.0%) for i.a. The side effects were slight. Those of grade 3 or more included anorexia in 5%, nausea and vomiting in 1%, stomatitis in 1% and leukopenia in 1%. This therapy, administered in accordance with the theory of chronotherapy, caused few side effects, and thus is considered a promising treatment for gastric cancer.

[The effect of combination chemotherapy to adapted to chronotherapy with 5-fluorouracil, leucovorin, mitomycin C and cisplatin in patients with gastric or colorectal cancer].

We performed combination chemotherapy adapted to chronotherapy with 5-fluorouracil, leucovorin, mitomycin C and cisplatin in 11 patients with gastric cancer and 7 with colorectal cancer. Treatment consisted of a 5-day course of continuous arterial or intravenous infusion of 5-FU (500 mg/body/day), arterial or intravenous infusion of leucovorin (20 mg/body/day) at 6:00 p.m. on days 1-5, arterial or intravenous infusion of mitomycin C (2 mg/body) at 9:00 a. m. on day 5, and arterial or intravenous infusion of cisplatin (20-80 mg/body) at 6:00 p.m. on day 5. The effective rate against gastric cancer was 73%; however, the effective rate against colorectal cancer was 29%. During and after this therapy, there was only a little appetite loss, nausea and stomatitis.

Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA: implication for involvement of the highly ordered structure and cell type-specific transacting factors.

Hepatitis C virus (HCV) carries an internal ribosomal entry site (IRES) within the 5' portion of the RNA. To identify structures that influence efficiency of the translation initiation, relative activities of modified IRESs were examined by using engineered bicistronic mRNAs, between the two cistrons of which various mutant IRESs were inserted. An IRES derived from genotype 2b is at least two times more efficient than one from genotype 1b in cultured cells. Activity ratios of genotype 2b IRES to 1b IRES differ in magnification among cultured cells, suggesting the difference in assortment of IRES-related host factors among individual cell types. Recombinant IRESs between the genotypes show similar or higher activities compared with 2b IRES in cell-free systems and show intermediate activities in cultured cells. Patterns of relative activities of those IRESs indicate that the IRES activity is not regulated by defined structure(s), although a cluster of different nucleotides is observed in the genome region of nucleotides 176-224 between the two alleles. The results suggest that a highly ordered structure formed by the entire 5' portion of the RNA is important for the IRES activity. The 5' border of HCV IRES was examined by using a series of deletion RNAs in various systems. The results strongly suggest that the border resides between nucleotide positions 28 and 45. Patterns of relative activities of the deletion IRESs differ in translation systems or cell types. These results imply that interactions of HCV RNA with the related transacting factor(s) may differ in the translation systems or cell types.

The effective dose of FR167653 in pulmonary ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion injury may result in the local release of proinflammatory cytokines. FR167653 is a potent suppressant of interleukin-1 and tumor necrosis factor-alpha. In a previous study, we reported the efficacy of FR167653 in canine ischemia-reperfusion models. In this report we investigated the dose of FR167653 effective in pulmonary ischemia-reperfusion injury in a canine model. METHODS: Adult mongrel dogs, weighing 9 to 13 kg, were allocated into five groups. FR167653 was continuously infused (FR-A (n = 7): 1 mg/kg/hr; FR-B (n = 6): 0.5 mg/kg/hr; FR-C (n = 6): 0.1 mg/kg/hr; FR-D (n = 5): 0.05 mg/kg/hr) from 30 minutes prior to ischemia to 2 hours after reperfusion. In the control group (n = 7), a vehicle was given continuously. Warm ischemia was induced for 3 hours. Arterial oxygen saturation (SaO2), left pulmonary vascular resistance (L-PVR), and cardiac output (CO) were measured. The lung was harvested for histologic study. RESULTS: SaO2 levels after 2 hours of reperfusion were significantly (p < 0.05) higher in groups FR-A, B, C, and D than in the control group. Just after reperfusion, CO deterioration was significantly (p < 0.05) greater in the control group than in the FR-treated groups, and the L-PVR level was significantly (p < 0.05) lower in groups FR-A, B, and C than in the control group. There were statistically significant differences (p < 0.05) in the survival rates of groups FR-A and B and the control group. Histological damage was more severe in the control group than in the FR-treated groups. CONCLUSION: FR 167653 seems to ameliorate ischemia-reperfusion injury of the lung dose-dependently.

Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3' untranslated region of RNA.

The genome of hepatitis C virus (HCV) is a single-stranded RNA of positive polarity that has a poly(U/C) tract followed by a highly conserved 98-nt sequence, termed the X region, in the 3' untranslated region (UTR). To investigate the effect of the 3'UTR on the HCV translation that depends on the internal ribosomal entry site (IRES), we prepared a deletion HCV RNA, MA delta, that lacked the RNA region from nt 1286 to 8785. A series of MA delta RNAs that differ in the primary structure of their 3'UTR, were generated and examined for their translation efficiencies in reticulocyte lysates. Deletion of the poly(U/C) tract and/or stem-loop structure (SL) 3 region of 3'X resulted in enhancement of the translation efficiency. Translation of MA delta RNA was inhibited by the addition of recombinant polypyrimidine tract-binding protein (PTB). A similar inhibition by PTB, however, was observed when an RNA lacking the poly(U/C) tract or SL3 region was used. The inhibitory effect by PTB was not obvious for MA delta (1041) RNA composed of nt 1 to 1041 but MA delta (8928) RNA composed of nt 1 to 1285 and nt 8786 to 8928. These results suggest that the observed down-regulation of HCV translation by the 3'UTR is mediated by some host factor(s) other than PTB, and that a PTB site for inhibition resides in the coding sequence of nt 1042 to 8928 of MA delta RNA.