10-year trends in the treatment and outcomes of patients with first-episode schizophrenia.

OBJECTIVE: The first episode of schizophrenia is a critical period for illness course and outcomes. We aimed to investigate treatments and outcomes during the first year after the diagnosis of schizophrenia. METHOD: Pharmacoepidemiologic inception cohort study of all newly diagnosed patients with schizophrenia in Denmark (n = 13,600) 1996-2005. RESULTS: From 1996 to 2005, the mean age at first diagnosis decreased significantly (29.2-26.1 years), more patients received antipsychotics (67.2-80.7%, annual OR = 1.07, CI: 1.06-1.09, P < 0.001) and antipsychotic polypharmacy for >4 months (16.7-37.1%, OR = 1.14, CI: 1.12-1.57, P < 0.001). The antipsychotic defined daily dosage (DDD) doubled (150-332 DDD, P < 0.001), use of antidepressants (24.3-40.6%, P < 0.001). Bed days [89.9 days (CI: 81.8-98.8) to 71.8 days, CI: 63.7-80.8, P < 0.0001] decreased, whereas outpatient contacts [10.2 (CI: 9.5-11.0) to 21.4 (CI: 19.9-21.0), P < 0.0001] doubled. CONCLUSION: Between 1996 and 2005, there was an earlier recognition of schizophrenia, intensified outpatient treatment, increased use and dosing of antipsychotics and antidepressants, but also more antipsychotic polypharmacy.

[Mortality and causes of death in patients with eating disorders in Denmark, 1970-1993]. / Dødelighed og årsager til død hos patienter med spiseforstyrrelser i Danmark, 1970-1993.

INTRODUCTION: The aim of this study was to describe standardised mortality rates and causes of death in patients with eating disorders, who were admitted to Danish psychiatric and somatic hospitals during the period, 1970 to 1993. METHODS: By means of record-linkage, the study covered all patients admitted for and diagnosed as suffering from an eating disorder, according to the ICD-8 classification system, at any Danish psychiatric (since 1970) or somatic department (since 1977) during the period 1970-1993. The study comprised 2763 patients 237 of whom were males. The maximum follow-up time was 23 years and the mean follow-up time 10.3 years. RESULTS: The crude mortality at follow-up was 8.4%. A significant excessive mortality was demonstrated, as the standardised mortality ratio (SMR) of the total patient population was 6.69 (CI 5.68-7.83) and the highest rate ratio (RR) of 14.92 (CI 9.66-22.03) related to women aged 25-29. CONCLUSION: With its 2763 patients, this is the largest study of a population with eating disorders ever published. The study documents a significant excessive mortality among such patients in all age groups.

Anorexia nervosa and cancer risk.

OBJECTIVE: Energy restriction reduces the incidence of malignant tumors in experimental animals, but evidence for a similar effect in humans is lacking. To test the hypothesis in humans, we investigated cancer incidence among patients with anorexia nervosa, who have had an extremely low intake of calories for prolonged periods of their lives. METHODS: Patients with anorexia nervosa (2151 women and 186 men) during 1970-1993 were identified in the population-based Danish Psychiatric Case Register and the National Registry of Patients. The cohort was linked to the Danish Cancer Registry, and cancer incidence among cohort members was compared with that of the general population. RESULTS: The overall cancer incidence among women with anorexia nervosa was reduced by a factor of 0.80 (95% confidence interval 0.52-1.18) below that of the general population on the basis of 25 observed and 31.4 expected cases. Among men, two cases of cancer were observed, both confined to the brain, whereas 0.2 cases were expected. CONCLUSIONS: The finding of a slight reduction in cancer risk among women with anorexia nervosa may support the theory that a low-energy diet may decrease tumor development in humans. However, longer follow-up and control for confounding factors are needed to obtain more convincing evidence.

Mortality and causes of death in eating disorders in Denmark 1970-1993: a case register study.

OBJECTIVE: The aim of this study is to describe mortality rates and causes of death for patients with eating disorders. METHOD: By means of record-linkage, the study includes all patients admitted and diagnosed as suffering from an eating disorder according to the ICD-8 classification system during the period 1970-1993 at any Danish psychiatric (since 1970) or somatic department (since 1977). The study includes 2,763 cases, of which 237 are males. Maximum follow-up time is 23 years and mean follow-up time is 10.3 years. RESULTS: Crude mortality at follow-up is 8.4%. A significant excess mortality is demonstrated since the standardized mortality ratio (SMR) of the total patient population is 6.69 (CI 5.68-7.83) and the highest rate ratio (RR) of 14.92 (CI 9.66-22.03) relates to women aged 25-29. DISCUSSION: The study documents a significant excess mortality among eating-disordered patients.

[Neuroleptic malignant syndrome after treatment with olanzapine]. / Malignt neuroleptikumsyndrom efter behandling med olanzapin.

A case of neuroleptic malignant syndrome (NMS) in a 23 year old male patient is reported. The symptoms were hyperthermia, muscle rigidity, change in mental status, sinus tachycardia, creatinine phosphokinase elevation and myoglobinuria. The patient suffered from severe muscle pain and compromised respiratory function. Treatment was cessation of neuroleptic medication and institution of intensive medical care focusing on symptomatic treatment. One week after admission clinical status and laboratory findings were normalized and the patient was readmitted to a psychiatric hospital. The neuroleptic medication of the reported patient had been olanzapine during seven months at a dose of 25 mg daily. The day before onset of NMS the pharmacological treatment was supplemented by 100 mg of clozapine. The cause of onset of NMS in this case is discussed. Clozapine, an atypical neuroleptic, is known to have reduced potential to cause NMS and in such cases without extrapyramidal symptoms. Olanzapine, however, has not yet been reported to cause NMS. Alternatively the cause of onset of NMS in this patient could be explained by the combination treatment and possible synergistic effect of the two antipsychotic drugs. Further research in this field is needed.

Modeling the growth and proteinase A production in continuous cultures of recombinant Saccharomyces cerevisiae.

Overexpression of the homologous protein proteinase A (PrA) in Saccharomyces cerevisiae has been achieved by inserting the PrA gene (PEP4) with its own promoter on a 2mu multicopy plasmid. With this system the specific PrA production rate was found to be described well by a linear function of the oxidative glucose metabolism, the reductive glucose metabolism, and the oxidative ethanol metabolism, with a significant lower yield resulting from the reductive glucose metabolism compared with the oxidative glucose metabolism. To describe the experimental data, a simple mathematical model has been set up. The model is based on an assumption of a limited respiratory capacity as suggested by Sonnleitner and Käppeli but extended to describe production of an extracellular protein. The model predicts correctly the critical dilution rate to be between 0.15 and 0.16 h(-1), the decrease in the biomass yield above the critical dilution rate, and the production of proteinase A at different dilution rates. Both the experimental data and model simulations suggest that the optimum operating conditions for protein production is just at the critical dilution rate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 447-454, 1997.

Mathematical modeling of proteinase A overproduction by Saccharomyces cerevisiae.

A simple, structured model was developed to describe the growth and product formation behavior of two recombinant strains of Saccharomyces cerevisiae (JG176 and JG180), both overproducing extracellular proteinase A. The model parameters were estimated to data from continuous fermentations obtained at steady-state conditions. Model predictions show good agreement with experimental data obtained by batch fermentations. The two concerned organisms are distinguished from each other by the type of promoter on the plasmids controlling the proteinase A expression. The proteinase A transcription is controlled by the natural proteinase A promoter in JG176 and by a tpi promoter in JG180. By means of experiments and simulations, the extracellular product formation from the two strains with different promoter systems was compared in batch and continuous fermentations. The results showed that the proteinase A formation kinetic from JG176 was a combination of growth and nongrowth associated (production in the stationary growth phase), whereas the proteinase A formation from JG180 was truly growth associated (production in the exponential growth phase). In both batch and continuous cultivations JG176 gave the highest product concentrations and volumetric productivities.

The use of antibodies for characterization and quantification of a recombinant protein.

In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4). Polyclonal and monoclonal antibodies were raised to study the product heterogeneity. Characterization of proteinase A was performed by immunoelectrophoresis and immunoblotting techniques. None of the monoclonal antibodies raised against proteinase A was found to react with the glycosyl side chains; thus cross-reaction with other glycosylated proteins (e.g. carboxypeptidase Y) was very low. This study allowed us to develop an ELISA method for the quantification of proteinase A in culture supernatants as well as the evaluation of monoclonal antibodies for their use in immunoaffinity chromatography.

Production of proteinase A by Saccharomyces cerevisiae in a cell-recycling fermentation system: experiments and computer simulations.

Overproduction of proteinase A by recombinant Saccharomyces cerevisiae was investigated by cultivations in a cell-recycling bioreactor. Membrane filtration was used to separate cells from the broth. Recycling ratios and dilution rates were varied and the effect on enzyme production was studied both experimentally and by computer simulations. Experiments and simulations showed that cell mass and product concentration were enhanced by high ratios of recycling. Additional simulations showed that the proteinase A concentration decreased drastically at high dilution rates and the optimal volumetric productivities were at high dilution rates just below washout and at high ratios of recycling. Cell-recycling fermentation gave much higher volumetric productivities and stable product concentrations in contrast to simple continuous fermentation.

Extra- and intracellular amino acid concentrations in continuous Chinese hamster ovary cell culture.

A recombinant DNA Chinese hamster ovary (CHO) cell line that produces tissue-type plasminogen activator (tPA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day with three different asparagine concentrations in the feed (0.05, 2.55 and 7.55 mM). The up-shift in asparagine concentration caused an up-shift in asparagine consumption [15.7 and 31.4 nmol (10(6) cells)-1 h-1] and intracellular concentration (2.19 and 18.7 mM). The up-shift was accompanied by an increased production of ammonium, glycine and alanine, and a metabolic shift whereby the cells began to produce aspartate and glutamate, which were consumed before the shift. The tPA production was reduced in the up-shift culture. This might be explained by ammonium inhibition, but alternatively by a surprising down-shift in the intracellular concentration of many amino acids, a down-shift that was not observed in the extracellular concentrations or consumption rates. For efficient physiological engineering of mammalian cells it is necessary to include both extracellular and intracellular measurements and to consider the transport into and out of the cells.

Perfusion systems for hybridoma cells based on sedimentation in chambers and Erlenmeyer flasks.

An inclined sedimentation chamber and a modified 250-ml Erlenmeyer flask have been used as separation devices for perfusion fermentations with hybridoma cells. The maximum cell density is increased 2-16-fold compared to batch fermentations when the separation units are used. When the sedimentation chamber is used, IgG is continuously produced and the daily production is increased by a factor 3.7 compared to batch fermentation.

Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture.

A recombinant DNA Chinese hamster ovary (CHO) cell line which produces tissue-type plasminogen activator (t-PA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day-1. The cultivation consisted of four phases with four different ammonium chloride concentrations (0, 2.5, 5, and 7.5 mM) in the feed medium, causing a reactor ammonium concentration of up to 8 mM. Cell growth was not inhibited by these high ammonium concentrations, as cell densities of around 2.3 x 10(6) cells mL-1 were established. In contrast, the production of t-PA was reduced under high ammonium concentration. The decrease in specific t-PA production could be due to either a negative ammonium influence on productivity or a limitation of medium components, e.g., amino acids. Cell metabolism was changed under high ammonium concentrations, seen most clearly by a decrease in specific ammonium production by a factor of 8 and an increase in specific alanine production of 30%.

Observations on the influence of glutamine, asparagine and peptone on growth and t-PA production of Chinese hamster ovary (CHO) cells.

When a transfected CHO cell, that produces tissue-type Plasminogen Activator, t-PA, was transferred from a medium based on 5% Fetal Calf Serum, FCS, to a medium based on 0.8% casein peptone with variable glutamine and asparagine content, it was observed, that the growth of the cells changed from anchorage dependant to suspension culture giving more reproducible cultivations. In the FCS culture t-PA was unstable, observed as a decline in t-PA concentration after 250 h. This decline in t-PA concentration was not observed in the serum free culture, although there was a decline in productivity after 200 h. This change in production profile may be attributed to either no proteolytic attack from serum or by scavenging of proteolytic activities produced by the cells from the peptone peptides. Increasing amounts of glutamine/asparagine gave higher production of t-PA in synchrony with an increasing production of ammonia/ammonium ions. Ammonia inhibition does not seem to be a key factor for this cell line as seen with many others.

Enhanced antibody production associated with altered amino acid metabolism in a hybridoma high-density perfusion culture established by gravity separation.

A high density hybridoma perfusion culture was established by separating and recycling cells from the product stream to the reactor using a simple external sedimentation-based separator-an inclined modified Erlenmeyer flask. After 3 weeks, when the optimal perfusion rate of 1.0 day-1 had been reached, viable cell density stabilized at around 10 x 10(6) cells ml-1, a level five times that obtained by simple batch culture. The efficiency of the separator was enhanced by cell flocculation. Specific antibody productivity, which was initially 0.4 micrograms 1 x 10(6) cells-1 h-1, decreased to half that value while cell density was increasing, but recovered to the initial level when the culture finally stabilized at a high cell density. During the final phase, when viable cell density and specific antibody production were high, there was a marked shift in metabolism. Consumption of the two most important substrates for energy generation, glucose and glutamine, caused their broth concentrations to decrease to 1.5 mM and 1 mM, respectively, from input medium concentrations of 25 mM and 10 mM, respectively. At the same time there was an increase in the specific production of glycine and aspartate, their broth concentrations reaching 1.5 mM and 0.02 mM, respectively. We suggest that this shift in metabolism results in enhanced production of ATP from glutamine. The specific glucose consumption and lactate production also indicate that there is a shift to more energy efficient metabolism. The mechanism whereby this leads to enhanced specific antibody production remains to be elucidated. Nevertheless, the combination of high cell density and enhanced productivity obtained with the present perfusion culture resulted in a high monoclonal antibody production-100 mg 1-1 d-1.

Automatic containment sampling of recombinant escherichia coli fermentations.

A containment sampling system for shake flasks and fermentors has been developed from a blood collection system used in hospitals. The core of the system is a collection vial with a vacuum inside. When a needle connected to the fermentation fluid penetrates a rubber seal on the vial, a sample is withdrawn. The system has been developed in two versions, a manual method for shake flasks, and an automated version for fermentors including cool storage of samples. The sampling system offers the same safety for fermentation containment as the original system offers safety for patients and hospital staff.

A comparison of simple growth vessels and a specially designed bioreactor for the cultivation of hybridoma cells.

Three tank type bioreactors of very simple design were compared to a commercially available laboratory-scale bioreactor, designed especially for mammalian cell culture, for their ability to support hybridoma growth and antibody production under batch culture conditions. The comparison reveals quite similar numbers for maximum viable cell densities and IgG production, despite large differences in vessel and agitator geometry and aeration mode. Furthermore, some data indicate that the hydrodynamic stress level in the growth vessels may influence the specific production rate of the cells and thus the overall productivity of the reactors.

An easy-to-handle semi-automated method for media development using a colorimetric viability assay and fractional factorial designs.

A rapid and effective semi-automated screening method has been developed for the development of growth media for mammalian cell culture. The method has proven to be a powerful tool for preliminary evaluation and comparison of new media formulations, but has some inherent disadvantages, which are important to recognise in order to interpret the results.

Quantitative binding studies of a monoclonal antibody to immobilized protein-A.

Binding constants and column capacities are important factors for evaluating an affinity chromatography system. Scatchard plots based on classical equilibrium binding have been used to demonstrate how association constants and column capacities can be computed from simple binding experiments and a commercial computer program. The analysis has been demonstrated on a monoclonal antibody type IgG-1 Kappa against Serratia marcescens nuclease and a commercial protein-A column, Prosep-A. Additional analyses were performed with the same antibody and other protein-A affinity systems and the different binding constants and column capacities obtained confirmed the value of the analysis for evaluating an affinity system.

Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli.

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.