Improved therapeutic effect on malignant glioma with adenoviral suicide gene therapy combined with temozolomide.

Malignant gliomas (MGs) are cancers with poor prognosis and limited therapeutic options. Herpes Simplex virus-1 thymidine kinase expressed from adenoviruses with prodrug ganciclovir (TK/GCV) is the best-characterized suicide gene therapy, whereas temozolomide (TMZ) is the first-line chemotherapy for MG. However, the potential of their combination has not been studied thoroughly. The aim of this study was to evaluate the therapeutic response of this combination and to study whether addition of valproic acid (VPA) could benefit the treatment outcome. Efficacies of different treatments were first studied in vitro in BT4C rat MG cells. Therapeutic assessment in vivo was done in an immunocompetent rat MG model for treatment efficacy and toxicity. In vitro, VPA was able to significantly enhance cytotoxicity and increase adenovirus-mediated transduction efficiency up to sevenfold. In vivo, rats receiving TK/GCV+TMZ had notably smaller tumors and enhanced survival (P<0.001) in comparison with control rats. However, VPA was not able to further enhance the treatment response in vivo. Leukocytopenia and thrombocytopenia were the major side effects. We conclude that careful optimization of the treatment schedules and doses of individual therapies are necessary to achieve an optimal therapeutic effect with TK/GCV+TMZ combination. No further in vivo benefit with VPA was observed.

Increased invasion of malignant gliomas after 15-LO-1 and HSV-tk/ganciclovir combination gene therapy.

Recent clinical trials for malignant glioma have drawn attention to the potential therapeutic efficacy of herpes simplex virus-thymidine kinase (HSV-tk) suicide gene therapy. Nevertheless, because of the nature of these tumors, it is believed that no single treatment alone is able to combat this fatal disease. Combination therapies may provide a solution to further improve therapies against malignant gliomas. We have recently demonstrated that 15-lipoxygenase-1 (15-LO-1) is able to inhibit tumor angiogenesis as well as enhance apoptosis in tumors. As a result, we studied the potential additive/synergistic effects of 15-LO-1 gene therapy when combined with HSV-tk gene therapy for the treatment of malignant gliomas. For that, BT4C malignant glioma cells were implanted into BDIX male rats. Fourteen days after tumor cell implantation, animals were transduced using adenoviral vectors either with HSV-tk alone or in combination with 15-LO-1. The results show that the combination gene therapy neither improved inhibition of tumor growth nor did it show any benefit on survival. Instead, a profound effect on the migratory properties of the tumor cells was found, resulting in decreased survival. Similar to conventional therapies, the combination of two therapeutic genes may result in unexpected side effects, not seen when given alone.

Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors.

Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.

(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

Gene therapy: the first approved gene-based medicines, molecular mechanisms and clinical indications.

As gene therapy has matured from clinical trials to the first commercial products, understanding of the mechanisms of gene delivery has increased tremendously. This has also been reflected in viral vector development, creating a number of new approaches to tackle issues in transduction efficiency, biodistribution and viral safety. This review will highlight the most important issues and advancements in vector development, administration, surface modification, integration to host genome and safety. The gene therapy products currently available or near market approval, based on p53 expression (Gendicine and Advexin), conditionally replicative adenoviruses (Oncorine) and thymidine kinase + ganciclovir therapy (Cerepro), are introduced with emphasis on the molecular mechanisms of action.

Fatal poisonings in young drug addicts in the Nordic countries: a comparison between 1984-1985 and 1991.

Fatal poisonings among young drug addicts (15-34 years) in the five Nordic countries, Denmark, Finland, Iceland, Norway and Sweden in 1991 were investigated and compared to a similar investigation for 1984-1985 (Sweden for 1984 only). A common definition of 'drug addict' has been applied by the participating countries. In both investigations, the greatest number of drug addict deaths was seen in Denmark calculated per 10(5) inhabitants, followed in descending order by Norway, Sweden, Finland and Iceland. An increased number of deaths was observed from 1984-1985 to 1991 in all five countries. The increase in Denmark and Sweden was small while the number of deaths was more than doubled in Norway and Finland. The increased number of cases in Norway and Sweden in 1991 is mainly due to a greater number of deaths in the age group 25-34 years. In Finland, the increased number was seen mainly in the age group 15-24 years. In the two investigations heroin/morphine caused most of the fatal poisonings in Norway and Sweden. In Denmark, heroin/morphine caused about half of the fatal poisonings only, and strong analgesics other than heroin/morphine caused about one third of the deaths. In 1984-1985 it was methadone, propoxyphene and ketobemidone and in 1991 mostly methadone. The number of heroin/morphine related deaths in Finland increased from 1984-1985 to 1991, but other drugs and poisons caused a much higher proportion of the deaths. Pentobarbital caused the only fatal poisoning in Iceland in 1991.

Drug addict deaths in the Nordic countries: a study based on medicolegally examined cases in the five Nordic countries in 1991.

The study includes medicolegally examined deaths among drug addicts in 1991 in the five Nordic countries: Denmark, Finland, Iceland, Norway and Sweden. A common definition of 'drug addict' was applied by the participating countries. The greatest number of drug addict deaths per 10(5) inhabitants was observed in Denmark followed, in descending order by Norway, Sweden, Finland and finally Iceland with only four deaths. The main difference between the countries was found in the number of fatal poisonings. The distribution according to geographical regions showed that about half of all drug addict deaths occurred in the metropolitan areas. Of the capitals, the greatest number of fatal poisonings per 10(5) inhabitants was seen in Oslo, followed by Copenhagen with a similar number, Stockholm with only the half, and Helsinki with a quarter. Heroin/morphine dominated as cause of death in fatal poisonings in Norway and Sweden. In Denmark, heroin/morphine caused about half of the fatal poisonings only, and nearly one third of the fatal poisonings was caused by methadone. Except for two cases in Sweden, methadone deaths were not seen in the other Nordic countries. Amphetamine caused one tenth of the fatal poisonings in Sweden. In Finland only one tenth of the deaths were caused by heroin/morphine and more by codeine, ethylmorphine and different drugs and poisons not classified in Single Convention on Narcotic Drugs 1961 or the International Convention on Psychotropic Substances 1971. A widespread use of alcohol, cannabis and benzodiazepines, diazepam especially, was seen in all the countries.

Drugs usage of drivers suspected of driving under the influence of alcohol and/or drugs. A study of one week's samples in 1979 and 1993 in Finland.

The extent of drug use among drivers suspected of driving under the influence of alcohol and/or drugs in Finland was studied. All blood samples submitted to the laboratory during 1 week in two study periods, in 1979 (n = 298) and 1993 (n = 332), were analyzed for alcohol and psychotropic drugs. Drugs classified as hazardous to traffic safety were detected in 7.0% of the samples in 1979 and 26.8% in 1993. Benzodiazepines were the most frequently found drugs in both years: 6.0% of the cases in 1979 and 22.9% in 1993. Illegal drugs were found in 4% of the cases in 1993. Of the samples tested, 296 in 1979 and 317 in 1993 were from drivers suspected of driving under the influence of alcohol only. In 1979 every fourteenth and in 1993 every fourth of these suspected drunken drivers had drugs in their blood. Drugs, other than alcohol, were found six times more often than expected by the police. The results indicate that the trend of drug use, multidrug use and drug abuse is increasing among cases suspected of driving under the influence of alcohol/drugs.

Objectively measured tobacco exposure during pregnancy: neonatal effects and relation to maternal smoking.

OBJECTIVES: To measure quantitatively and objectively the maternal and fetal tobacco exposure during pregnancy and its neonatal effects. DESIGN: Tobacco exposure was assessed from maternal serum samples, obtained during the first half of pregnancy and from umbilical serum samples obtained at delivery, by measuring the concentration of nicotine metabolite, cotinine. Data on the respective pregnancies and neonates were collected from the Finnish Medical Birth Registry. SETTING: Finland. SUBJECTS: One thousand two hundred and thirty-seven pregnancies and newborns, representing all pregnancies resulting in a liveborn infant during one week in one country. MAIN OUTCOME MEASURES: Gestational age, birthweight and crown-heel length of newborns. RESULTS: Cotinine (> 6 micrograms/l) was detected in either maternal or umbilical serum in 300 pregnancies, and these mothers and newborns were classified as exposed. Important differences occurred between measured exposure and reported smoking behaviour. Of the exposed mothers, 38% were nonsmokers and 3.4% of the nonexposed mothers were smokers. Tobacco exposure was associated with shorter gestational age, reduced birthweight and shorter crown-heel length of the newborns. After correction for parity, gender, and gestational age, the exposed newborns were on average 188 g (95% confidence interval (CI) 123-253 g) lighter and 10 mm (95% CI 7-13 mm) shorter than the nonexposed newborns. One micrograms/ml of cotinine in maternal serum resulted in a mean decrease of 1.29 g (95% CI 0.55-2.02 g) in birthweight and in a mean decrease of 0.059 mm (95% CI 0.035-0.083 mm) in birth length. Maternal cotinine concentrations better explained the neonatal findings than the reported smoking habits. CONCLUSIONS: There is a quantitative dose and effect relation between tobacco exposure and a decrease in the gestational age at birth and size of the neonate. The smoking habit reported by mothers themselves is not an accurate measure of fetal tobacco exposure.

Seasonal variation in high density lipoprotein cholesterol.

We investigated the seasonal variation in high density lipoprotein cholesterol (HDL) in 142 dyslipidemic (non-HDL-cholesterol > or = 5.2 mmol/l) middle-aged men in the placebo group of the Helsinki Heart Study over the 5-year trial period. A seasonal pattern was found in HDL fluctuation, with a 4.5% drop during mid-winter (5-year mean 1.192 +/- 0.265 mmol/l) compared with a stable level (5-year mean 1.248 +/- 0.281 mmol/l) during the rest of the year (P < 0.001). A less pronounced seasonal variation in HDL was observed in 85 subjects receiving gemfibrozil. Although affecting pretrial HDL level in cross-sectional analyses, age, alcohol consumption, dietary adherence, physical activity and serum triglycerides had no influence on the seasonality of HDL variation. Smoking had a slight attenuating effect on the variation pattern. Pretrial HDL was influenced by relative weight, but there was also an inverse relationship between HDL and body weight variations, i.e. the annual drop in HDL coincided with the annual peak in body weight. However, seasonal HDL variation was not directly reflected in the annual variation in CHD incidence.

Conventional laboratory tests as indicators of heavy drinking in young university students.

Conventional laboratory tests: gamma glutamyltransferase (GGT), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), mean corpuscular volume (MCV), cholesterol, and high-density lipoprotein cholesterol (HDL-cholesterol) were studied as possible markers of heavy drinking in a sample of 1,619 first-year university students in Helsinki. Alcohol consumption was measured on a frequency-volume scale. No significant (p < 0.001) correlation between the laboratory tests and reported drinking was found. However, significant differences between different drinking groups were found in GGT, MCV, and HDL-cholesterol in both female and male students and in cholesterol in male students. We conclude that, even if alcohol drinking has some impact on GGT, MCV, HDL-cholesterol, and cholesterol (males), these tests are neither specific nor sensitive enough to be used as screening tests for heavy drinking in young students.

[Occasion and risk for detecting driving in alcoholic intoxication]. / Anlass und Risiko der Entdeckung des Fahrens in alkoholisiertem Zustand.

Detection of offenders driving while intoxicated (DW) (mandatory BAC limit at 0.50%) in Finland was investigated. Almost every fifth offender was detected at road blocks and every tenth of them because of abnormal driving. Compared to the local police the national traffic police had arrested much more offenders for traffic violation (38% vs. 11%) but much less because of accidents (12% vs. 23%). The reason of detection was in a significant way dependent on the following variables: BAC, stage of inebriation, vehicle, day of week and hour of day. The risk of detection is very low, i.e. about 1 to 300. However, the risk has doubled from 1979 to 1984, but thereafter slightly declined. The study provides information for the strategy of the police in traffic supervision.

[Accidents with fatal outcome in Finnish leisure boating 1986-1988]. / Unfälle mit tödlichem Ausgang im finnischen Wasserverkehr während der Freizeit 1986-1988.

All 291 fatal accidents (510 persons on board, 318 drowned) in water traffic in Finland in 1986-1988 were investigated by specific teams. Only some data of this extensive investigation are presented in this study. Staggering and falling in boat because of drunkenness, falling over and sinking of boat were the main causes of getting into water of the people aboard. Only 3.5% of the drowned had used life jackets and 9.7% of them could not swim. The reduced ability to swim because of alcohol and the exhaustion were in about half of the drowned the actual cause and the cold water in one third the background factor for drowning. The results indicate that fatal accidents in water traffic are a major problem of males (95.9%) and give important information for countermeasures.

Alcohol screening with the Alcoscan test strip in forensic praxis.

The Alcoscan test strip was applied as an assay for the screening of alcohol in vitreous humor and urine samples in autopsy cases and in saliva from drunken drivers. The method gives instant and reliable semi-quantitative information on the presence of alcohol and is valuable when considering the necessity of chemical sampling especially during autopsy.

Isolation of silica-dependent protein from rat lung with special reference to development of fibrosis.

Silicosis was produced experimentally in rats by single intratracheal injections of various doses of SiO2 dust. The weight of the lungs as well as the contents of total nitrogen, collagen, nucleic acids (especially RNA), and lipids increased in accordance with the dose and the time interval. Fibrogenic stimulation in vitro was shown by the supernatant of the homogenized lung in the incorporation of proline into incubated granulation tissue or lung fibroblasts. The fibrogenic factor-activity depended more on the time interval after the injection than on the SiO2 dose. Electrophoresis of the soluble proteins in the silicotic rat lungs showed a protein of 16,000 Da, which was dependent on the time interval following SiO2 administration as well as on the dose itself, and which originated from macrophages. This protein was purified by repeated gel-filtration chromatography. It stimulated collagen synthesis in granulation-tissue cells at a concentration of about 10(-10) M in a dose-dependent way. It was acidic by amino acid composition but differed from calmodulin which also increased collagen synthesis in granulation-tissue cells in vitro. The ability of non-fractionated macrophage preparations to stimulate the incorporation of proline into collagen correlated inversely with the gross alkaline RNase activity.

Fatal intoxications in the Nordic countries. A forensic toxicological study with special reference to young drug addicts.

Fatal intoxications in the 15-34 age group in the five Nordic countries during the years 1984 and 1985 (Sweden only in 1984) were investigated. The known drug addicts were studied separately. The highest incidence of intoxications, calculated per 10(5) population, was found in Finland (11.3), followed by Denmark (10.3), Sweden (8.5), Iceland (7.2) and Norway (6.6). The percentage of intoxications caused by drugs was 92 in Denmark, 71 in Norway, 66 in Sweden, 50 in Finland and 17 in Iceland. Ethanol intoxications were seen 5-7 and 2-3 times as frequently in Finland and in Iceland, respectively, than in the other three countries. Carbon monoxide intoxications accounted for two-thirds of all fatal intoxications in Iceland. Drug addicts accounted for 62% of all fatal intoxications in the Danish material. The corresponding figures were 33% in the Norwegian, 16% in the Swedish and 5% in the Finnish material. No deaths in drug addicts were found in Iceland. Most drug addicts in Denmark, Norway and Sweden died of hard drugs and most in Norway and Sweden, from heroin or morphine, whereas in Denmark other strong analgesics, such as methadone, dextropropoxyphene and ketobemidone, accounted for 40% of all hard-drug-related fatal intoxications. To a certain extent the results reflect differences in the legal autopsy routines in the various Nordic countries. However, the ascertainment of drug addicts is assumed to be near-complete in each country.

[Unexpected natural death in street traffic]. / Der unerwartete natürliche Tod im Strassenverkehr.

All natural deaths of motor vehicle drivers and bicyclists in traffic were retrospectively studied from the police and forensic autopsy protocols in the province Uusimaa in 1982-1985. Of the cases 57 were males and 3 females. The annual prevalence of natural deaths at wheel greatly increased with the age and was 260 and 231 per 1 million drivers and bicyclists aged 60-69 years, respectively. The most common underlying cause of death was chronic ischaemic heart disease, often known before (42%), and often combined with acute myocardial infarction (62%). In about half of the cases there were injuries. However, only in 5 cases the injuries had contributed so death. The presence of alcohol and drugs did not play any role. These deaths differed significantly by the event and demography of the parties from those killed in traffic accidents. The results suggest that sudden deaths at wheel are not very common in Finland. However, in all kinds of traffic deaths the police investigation should be completed by the forensic autopsy.

Aetiology of congenital hypothyroidism in Finland.

In Finland a nationwide screening programme for congenital hypothyroidism (CHT) has operated since 1980 with complete coverage. Among the total of 307,000 newborns screened, the incidence per 100,000 was 24.6 for thyroid dysgenesis and 4.0 for dyshormonogenesis. We conclude that, when screening is based on cord serum TSH, the false-positive results are caused mainly by difficult delivery. The most important factors associated with dyshormonogenesis were CHT in the family, origin from a geographic risk area, and origin of both mother and father from the same community. These reflect the autosomal recessive inheritance. The risk factors for dysgenesis were female gender, CHT in the family, birth in a geographic risk area, and birth during a risk period of the year.

[Fatal poisoning in Scandinavia--narcotic addicts are a high-risk group]. / Dödliga förgiftningar i Norden--narkomaner en utsatt grupp.

This study on fatal intoxications in the Nordic countries indicates an over-representation of drug addicts in Denmark, Norway and Sweden. All cases of fatal intoxications in persons 15-34 years of age submitted to medicolegal autopsy and subsequent toxicological examination in the Nordic countries during 1984-1985 included in the study. A comparison between the Nordic countries is possible because the same definition of a drug addict and classification of drugs are used. The numbers of fatal intoxications among drug addicts per 10(5) population were 6.3 in Denmark, 2.1 in Norway, 1.3 in Sweden, 0.6 in Finland and none in Iceland. About 70 per cent of the drug addicts in Norway and Sweden died from heroin/morphine intoxication. In Denmark only 55 per cent of the drug addicts died from heroin/morphine and 36 per cent died from overdoses of the legal drugs methadone, dextropropoxyphene and ketobemidone.

Increased serum acetate as a marker of problem drinking among drunken drivers.

727 consecutive drunken drivers were studied for laboratory markers of excessive alcohol consumption. Serum gamma-glutamyltransferase and alanine aminotransferase showed no differences and aspartate aminotransferase and blood alcohol concentration only small differences between groups of first and repeating drunk driving offenders. The best laboratory test to differentiate the repeating offenders with probably more serious alcohol problems from the first offenders was in our material serum acetate, the mean serum acetate level of the repeating offenders being highly significantly (P less than 0.001) higher than that of the first offenders or nonalcoholic controls. Serum acetate also differentiated first offenders from nonalcoholic controls (P less than 0.001). Our results suggest that serum acetate could be used for the screening of problem drinking among drunken drivers.