Gene therapy for malignant mesothelioma: current prospects and challenges.

Malignant mesothelioma, developed in the thoracic cavity, is resistant to current treatments. Suppression of the local tumor growth is beneficial to the patients since mesothelioma infrequently metastasizes to extrapleural organs. A majority of the tumors have a homologous genetic deletion at the INK4A/ARF locus that includes the p14ARF and the p16INK4A genes, and the genetic defect results in an inactivation of the p53-mediated pathways and in progression of cell cycle through pRb phosphorylation. Preclinical studies targeting the genetic abnormality with adenoviruses showed that restoration of the p53 pathways induced pRb dephosphorylation and subsequently produced anti-tumor effects. A number of preclinical studies with different genes and vector systems demonstrated the therapeutic efficacy and raised the possibility of gene therapy in clinical settings. An intrapleural administration of vectors has several advantages in transducing pleural mesothelioma but activates rapid antibody production which impedes further gene expression. There have been several clinical studies conducted for mesothelioma and these trials showed the feasibility of intrapleural administrations of adenovirus vectors. In this review we summarize major preclinical and clinical gene therapy for mesothelioma, and discuss the advantages of gene therapy in the context of stimulating host immune systems. Accumulating clinical data suggest that an intrapleural administration of viral vectors has distinct aspects which are not observed in other administration routes.

Neuroimaging of histamine H1-receptor occupancy in human brain by positron emission tomography (PET): a comparative study of ebastine, a second-generation antihistamine, and (+)-chlorpheniramine, a classical antihistamine.

AIMS: Sedation induced by antihistamines is widely recognized to be caused by their penetration through the blood-brain-barrier and the consequent occupation of brain histamine H1-receptors. We previously studied the mechanism of sedation caused by antihistamines using positron emission tomography (PET). Recently, we revealed the nonsedative characteristic of ebastine, a second-generation antihistamine, with cognitive performance tests. In the present study, H1-receptor occupation by ebastine was examined in the human brain using PET. METHODS: Ebastine 10 mg and (+)-chlorpheniramine 2 or 6 mg were orally given to healthy male volunteers. PET scans with [11C]-doxepin, a potent H1-receptor antagonist, were conducted near tmax of respective drugs. Other volunteers in the control group also received PET scans. The binding potential of doxepin (BP = Bmax/Kd) for available brain H1-receptors was imaged on a voxel-by-voxel basis through graphical analysis. By setting regions of interest, the H1-receptor occupancy of drugs was calculated in several H1-receptor rich regions. RESULTS: Brain distribution of radioactivity after ebastine treatment was similar to that without any drugs. However, after the oral administration of 2 mg (+)-chlorpheniramine, the level was lower than after ebastine and nondrug treatments. Graphical analysis followed by statistical parametric mapping (SPM96) revealed that H1-receptor rich regions such as cortices, cingulate gyrus and thalamus were regions where the BPs after ebastine were significantly higher than after (+)-chlorpheniramine (2 mg). H1-receptor occupancies in cortex were approximately 10% by ebastine and > or = 50% by either dose of (+)-chlorpheniramine (95% confidence interval for difference in the mean receptor occupancies: 27%, 54% for 2 mg and 35%, 62% for 6 mg vs ebastine, respectively). Receptor occupancies increased with increasing plasma concentration of (+)-chlorpheniramine, but not with concentration of carebastine, an active metabolite of ebastine. CONCLUSIONS: Ebastine (10 mg orally) causes brain histamine H1-receptor occupation of approximately 10%, consistent with its lower incidence of sedative effect, whereas (+)-chlorpheniramine occupied about 50% of brain H1-receptors even at a low but sedative dose of 2 mg; occupancy of (+)-chlorpheniramine was correlated with plasma (+)-chlorpheniramine concentration.

Inappropriate discharges by fourth generation implantable cardioverter defibrillators in patients with ventricular arrhythmias.

The study prospectively investigated the incidence, cause and efficient management of inappropriate discharge by the fourth generation implantable cardioverter-defibrillator (ICD) system in 45 patients (mean age, 57+/-16 years). During the follow-up period of 27+/-17 months, 18 patients (40%) experienced one or more inappropriate therapies: sinus and supraventricular tachycardia (15 patients) and T wave oversensing (3 patients). In the 15 patients, re-programming of the tachycardia detection interval and/or additional treatment with beta-blocking agents were effective. In the 3 patients with T wave oversensing, the arrythmia was associated with an increase in T wave amplitude, change in T wave morphology and decreased R wave amplitude, and re-programming of the sensitivity of the local electrogram or changing the number of intervals to detect ventricular tachycardia decreased the number of inappropriate discharges in all 3 patients. In conclusion, inappropriate therapies are common problems in patients treated with the fourth generation ICD system, but most of them can be resolved using the dual-chamber ICD system. However, in patients with T-wave oversensing, it is difficult to avoid inappropriate discharge completely, even if the dual-chamber ICD system is implanted.

Absolute configuration of (6S,9S)-roseoside from Polygonum hydropiper.

From Polygonum hydropiper L., a C13-norisoprenoid glucoside was isolated and its absolute configuration was established to be (6S,9S)-roseoside (1) by spectroscopic evidence and X-ray crystallographic analysis of its acetate derivative (2). In addition, the stereostructure of roseoside from Canthium subcordatum was revised to the (6S,9S) configuration.

Correlation between the effective refractory period and activation-recovery interval calculated from the intracardiac unipolar electrogram of humans with and without dl-sotalol treatment.

In experimental studies and/or human body surface mapping, the activation-recovery interval (ARI) is used as a parameter to estimate local repolarization. However, it has not been clarified whether the ARI calculated from the intracardiac unipolar electrogram of humans reasonably represents the local effective refractory period (ERP). Measurement of ARIs at multiple ventricular sites can be helpful in assessing the dispersion of ventricular refractoriness of humans, so we examined the relationship between ERP and ARI in the control state and under treatment with dl-sotalol during clinical electrophysiologic studies (EPS). Of 19 patients, an EPS was performed in the control state in 12 and during treatment with dl-sotalol in the other 7. Quadripolar electrode catheters with an interelectrode distance of 5 mm were placed at the right atrium and in the right ventricle. Using atrial pacing, the heart rate was increased incrementally by 10 beats/min, and ERP and ARI were measured for each pacing rate. The ERP at the right ventricle was measured by single extrastimulation between the first and third distal electrodes of the catheter in the right ventricle, and the ARI was calculated from the second distal unipolar electrode of the same catheter as the interval between the minimum derivative of the intrinsic deflection and the maximum derivative of the T wave. In all patients, the unipolar electrogram was stable during the entire EPS, and 83 data points in the control group and 50 in the dl-sotalol group were analyzed. At each pacing rate, the beat-to-beat difference of ARI was less than 10 ms. As the atrial pacing rate increased, the ERP and ARI were progressively shortened, and linear regression analysis revealed an excellent correlation between ERP and ARI. At the same pacing rate, the ERP and ARI in the dl-sotalol group were longer than those in the control group, but no difference was observed in the slope (close to 1.0) and in the intercept of the regression lines between ERP and ARI. In the human ventricle, the ARI calculated from the intracardiac unipolar electrogram represents the local ERP both in the control state and under treatment with dl-sotalol. The ARI can be used as a parameter of local refractoriness and used to study the distribution of refractoriness in the human ventricle.

Molecular cloning of a cDNA coding for feline liver xanthine dehydrogenase.

A cDNA coding for feline liver xanthine dehydrogenase (XDH, EC 1.1.204) was amplified by RT-PCR and cloned for determining the sequence. The clones contained an open reading frame of 4002 base pairs encoding 1333 amino acid residues. The calculated molecular weight and isoelectric point were approximately 146 kDa and 7.0. Comparison of the deduced amino acid sequences indicated remarkable high homology, i.e., the amino acid residues of feline XDH shared approximately 90%, 87%, 87% and 86% identity with those of human, bovine, rat and mouse, respectively. The anino acid sequences of two putative iron-sulfur centers, one NAD binding site and one molybdenum binding site were well conserved among mammalian animals.

Partial cloning of the hormone-binding domain of the cortisol receptor in tilapia, Oreochromis mossambicus, and changes in the mRNA level during embryonic development.

Cortisol is one of the central hormones in osmoregulation in fish, especially in seawater adaptation. A cDNA of 453 bp was cloned from liver mRNA of freshwater-reared tilapia (Oreochromis mossambicus), by reverse transcription polymerase chain reaction (RT-PCR) with primers designed for the hormone-binding domain of glucocorticoid receptors (GRs) in mammals and rainbow trout. The sequence of PCR product has 83% homology to the trout GR at the nucleotide level and 92% at the amino acid level. The PCR product of tilapia showed highest homology (74% at the amino acid level) to GR among human steroid hormone receptors, including mineralocorticoid receptor. The length of the receptor mRNA of tilapia was about 6.5 kb as determined by Northern blot hybridization. The mRNA concentration in the gills was relatively higher among various organs, the highest concentration being observed in blood cells. Signal intensity of the receptor message in the gills was stronger in fish reared in freshwater than in those reared in seawater or in concentrated (160%) seawater. During early development of tilapia, the highest concentration of receptor mRNA in the total RNA extracted from the whole egg was found just after fertilization, and its concentration decreased steadily toward hatching. The absolute amount of receptor mRNA per egg increased gradually before the initiation of cortisol production by the embryo. When embryos were transferred from fresh water to seawater 2 days before hatching, no difference was observed in the signal intensity of the receptor mRNA among embryos after 1, 2 (the day of hatching), 4, and 7 days.

Nucleotide sequence of prothrombin gene in abnormal prothrombin-producing hepatocellular carcinoma cell lines.

A protein induced by vitamin K absence or antagonist II, PIVKA-II is synthesized in the liver and possesses a structure similar to prothrombin except that ten glutamic acid residues in amino-terminal Gla domain are not completely gamma-carboxylated and are functionally inactive. This protein can be detected in the plasma of patients with hepatocellular carcinoma (HCC) and used as a new tumor marker. To analyze the mechanism of PIVKA-II production in HCC tissue, the prothrombin gene of PIVKA-II-secreting HCC cell lines was sequenced to detect the mutation in the Gla domain and carboxylase recognition site of leader sequence located on exons I and II that may cause the inhibition of carboxylation. Exons I and II and donor and acceptor site of intron I of the prothrombin gene in two HCC cell lines, PLC/PRF/5 and huH-2, were analyzed by polymerase chain reaction (PCR), and the product was sequenced directly. In addition, RNA samples of these cell lines were used for complementary DNA synthesis, followed by PCR and sequencing. The nucleotide sequences of the Gla domain in both HCC cell lines were conserved. One nucleotide change was detected at nt.554 (adenine to guanine), but this did not influence the amino acid sequence. Splicing sites between exons I and II, the leader sequence of the precursor prothrombin, and protease target sites also were conserved as the reported prothrombin gene, and mutations reported for other des-gamma-carboxy coagulation factors were not detected. These results also were confirmed by DNA analysis of seven human fresh-frozen samples (three PIVKA-II-positive HCC samples and four control specimens). The mechanism of PIVKA-II production in HCC is still unclear, but it is not caused by mutation in the prothrombin gene.

Expression of novel DNA-binding protein with zinc finger structure in various tumor cells.

We have isolated from B16 mouse melanoma cells a complementary DNA (Mel-18), whose deduced amino acid sequence possesses a characteristic zinc finger structure. Immunostaining with antibodies raised against partial Mel-18 peptide sequences demonstrated nuclear localization of the gene product. We have also demonstrated that this protein has DNA-binding capacity, and the zinc finger is responsible for the DNA binding. At the transcriptional level the Mel-18 mRNA was detected in all tumor cells examined as well as melanoma cells (ontogenically of neural origin) but was scarcely present in normal tissues except neural organs. The transcript is developmentally regulated. These data suggest that Mel-18 may play a role in transcriptional regulation and also in control of cell proliferation and/or neural cell development.

5-epideoxyloganic acid from Nepeta cataria.

On the basis of exhaustive (1)H- and (13)C-NMR spectral studies and chemical transformations, the structure of nepetolglucosylester isolated from Nepeta cataria L. was revised as (5R, 8S, 9R)-7-deoxyloganic acid, which was renamed 5-epideoxyloganic acid.

8-Epi-iridodial glucoside from Boschniakia rossica.

Boschnaside, 8-epi-iridodial glucoside a new iridoid glucoside was isolated from Boschniakia rossica Hult., and its absolute structure was determined by chemical correlation of boschnaloside tetraacetate with boschnaside tetraacetate.