In-depth neuropharmacokinetic analysis of antipsychotics based on a novel approach to estimate unbound target-site concentration in CNS regions: link to spatial receptor occupancy.

The current study provides a novel in-depth assessment of the extent of antipsychotic drugs transport across the blood-brain barrier (BBB) into various brain regions, as well as across the blood-spinal cord barrier (BSCB) and the blood-cerebrospinal fluid barrier (BCSFB). This is combined with an estimation of cellular barrier transport and a systematic evaluation of nonspecific brain tissue binding. The study is based on the new Combinatory Mapping Approach (CMA), here further developed for the assessment of unbound drug neuropharmacokinetics in regions of interest (ROI), referred as CMA-ROI. We show that differences exist between regions in both BBB transport and in brain tissue binding. The most dramatic spatial differences in BBB transport were found for the P-glycoprotein substrates risperidone (5.4-fold) and paliperidone (4-fold). A higher level of transporter-mediated protection was observed in the cerebellum compared with other brain regions with a more pronounced efflux for quetiapine, risperidone and paliperidone. The highest BBB penetration was documented in the frontal cortex, striatum and hippocampus (haloperidol, olanzapine), indicating potential influx mechanisms. BSCB transport was in general characterized by more efficient efflux compared with the brain regions. Regional tissue binding was significantly different for haloperidol, clozapine, risperidone and quetiapine (maximally 1.9-fold). Spatial differences in local unbound concentrations were found to significantly influence cortical 5-HT2A receptor occupancy for risperidone and olanzapine. In conclusion, the observed regional differences in BBB penetration may potentially be important factors contributing to variations in therapeutic effect and side effect profiles among antipsychotic drugs.

Evaluation of a community-based drowning prevention programme in northern Islamic Republic of Iran.

This study evaluated the feasibility of a drowning intervention package in northern Islamic Republic of Iran. A quasi-experimental design used pre- and post-observations among residents and tourists in water-recreation beach areas of intervention and control regions by the Caspian Sea and in residents near the Caspian Sea coastline. The fatal drowning rate in the studied resident population in the provinces fell from 4.24 per 100 000 residents at baseline to 3.04 per 100,000 residents at endline. The risk of death from drowning in the intervention areas in the water-recreation area was greater during the pre-intervention (OR = 1.15, 95% CI: 0.66-2.01) than the implementation period (OR = 0.24, 95% CI: 0.15-0.37). The risk of drowning can be reduced by implementing increased supervision and raising community awareness.

Ecology. The value of nature and the nature of value.

Ecosystems are capital assets: When properly managed, they yield a flow of vital goods and services. Relative to other forms of capital, however, ecosystems are poorly understood, scarcely monitored, and--in many important cases--undergoing rapid degradation. The process of economic valuation could greatly improve stewardship. This potential is now being realized with innovative financial instruments and institutional arrangements.

A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia.

Development of antibody drugs against novel targets and pathways offers great opportunities to improve current cancer treatment. We here describe a phenotypic discovery platform enabling efficient identification of therapeutic antibody-target combinations. The platform utilizes primary patient cells throughout the discovery process and includes methods for differential phage display cell panning, high-throughput cell-based specificity screening, phenotypic in vitro screening, target deconvolution, and confirmatory in vivo screening. In this study the platform was applied on cancer cells from patients with Chronic Lymphocytic Leukemia resulting in discovery of antibodies with improved cytotoxicity in vitro compared to the standard of care, the CD20-specific monoclonal antibody rituximab. Isolated antibodies were found to target six different receptors on Chronic Lymphocytic Leukemia cells; CD21, CD23, CD32, CD72, CD200, and HLA-DR of which CD32, CD200, and HLA-DR appeared as the most potent targets for antibody-based cytotoxicity treatment. Enhanced antibody efficacy was confirmed in vivo using a patient-derived xenograft model.

Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries.

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Cellular discriminants for a biological classification of human colon carcinoma.

We categorized established human colon carcinoma cell lines into three biological groups. Our studies were performed on six colorectal cancer lines representing the proposed three groups. Group I consisted of two lines designated LoVo and SW 48; Group II comprised two lines called SW 480 and SW 620; and Group III was represented by lines SW 403 and SW 1116. Group I consisted of the most differentiated cells. This differentiation encompassed morphological markers, gland and signet ring formation, and ciliary development. The outstanding morphological characteristic of Group III was the development of numerous multinucleated giant cells. The range and modal chromosome number increased from Group I to Group III, a change reflected by the higher DNA content of these cells as measured by flow cytometry. Carcinoembryonic antigen synthesis was maximal for Group III and virtually absent for Group II. The number of clonogenic cells decreased from Group I to Group III, while the proportion of nonproliferating cells calculated both by experiments using continuous labeling with tritiated thymidine, and by the primer-available alpha-DNA polymerase index, increased from Group I through Group III. Another important cytokinetic difference was that Group I had an exponential cell cycle stage distribution not seen for the other groups. Cells in Group I were easily propagated in athymic (nude) rats by s.c. injection; cells in Group II injected s.c. grew for about 30 days and then regressed spontaneously. Cells in Group III could only be grown when inoculated intracerebrally. Thus, our studies have now confirmed and extended the hypothesis that cultured human colorectal carcinomas can be separated into at least three groups on the basis of morphological differentiation, chromatin distribution, carcinoembryonic antigen production, cytokinetic properties, and xenograft propagation. Perhaps this classification is just the tip of the iceberg, and future studies will determine the existence of additional groups or subgroups on the basis of other markers. However, at present it appears established that malignant cells with a common histological origin in the gut express their phenotypic potential in a sufficiently discrete manner as to permit their classification into distinct biological groups. Thus, the stage is set for extrapolating this in vitro classification for an in vivo segregation of human colorectal tumors into categories with specific properties and diverse prognosis.

The interaction between different domains of staphylococcal protein A and human polyclonal IgG, IgA, IgM and F(ab')2: separation of affinity from specificity.

Binding properties of staphylococcal protein A (SpA) to different human immunoglobulins have been investigated. In this analysis, intact SpA as well as SpA-derived fragments containing one to five IgG-binding domains of different compositions, were used. The affinity binding constants of the different proteins to human polyclonal IgG, IgA, IgM and F(ab')2-fragments as well as their binding capacity to the immunoglobulin molecules were determined. The results show that although all the proteins bound to IgG, regardless of size or composition, the binding strength differed significantly. Proteins containing five domains have a stronger affinity for IgG than those containing one or two. There were no marked differences in binding strength between different domains. However, the binding ability to IgA and IgM showed a marked difference between the various SpA-derived proteins of different compositions. This discrepancy was correlated to differences in their relative binding properties to isolated F(ab')2-fragments of IgG. Hence, we conclude that the binding affinity is mainly affected by the number of domains, whereas the binding specificity is to a large extent determined by which domains are selected.

HIV transmission by blood transfusions in Stockholm 1979-1985: nearly uniform transmission from infected donors.

Thirty-five out of approximately 800 known HIV-seropositive people in Stockholm by mid-1986 were blood donors during the period 1979-1986. Almost all, i.e. 349 recipients of their blood components (red blood cells, platelets, plasma) could be traced. One hundred and eighty were still alive and 112 of these on further analysis, were suspected of being infected. They were contacted and all but one agreed to be tested for HIV antibodies. Fifty recipients were found to be seropositive. They had been transfused with blood components from 14 of the 35 donors. The earliest observed transmission occurred in June 1982. The patterns of HIV transmission showed, with only one exception, that each donor who had transmitted HIV to one recipient had also transmitted it to all later recipients. Appropriate preserved sera and clinical records from five of the donors who had not transmitted the virus were found and analysed. The result indicated that these donors had acquired their HIV infection after their last blood donation. In conclusion, our study indicates that every antibody-positive donor transmits HIV to almost every recipient.

A gene fusion system for generating antibodies against short peptides.

A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZ8 and pEZZ18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57-70] of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by affinity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The results suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.

Placebo-controlled study of mesulergine in Parkinson's disease.

We used a new D2 dopamine agonist, mesulergine (8-alpha-amino-ergoline, CU 32-085), to treat 20 patients (12 men and 8 women), mean age 62.6 (SEM = 1.7) and mean duration of illness 5.9 (SEM = 1.0) years. Wearing-off effect was the principal indication for new therapy in 15 patients, and the others had inadequate response to levodopa. All continued on levodopa therapy, and 10 patients were studied in a double-blind controlled test. The mean motor disability decreased from 2.8 (SEM = 0.12) to 1.6 (SEM = 0.18) with mesulergine (p less than 0.0001) and increased to 1.9 (SEM = 0.20) with placebo (p less than 0.001). Tremor improved most, followed by rigidity, bradykinesia, gait, and postural instability. Side effects included dyskinesia, light-headedness, hallucinations, nausea, vomiting, drowsiness, and ankle edema, but, in general, mesulergine was tolerated well.

Tanning habits and sunburn in a Swedish population age 13-50 years.

As part of a skin cancer control programme, we studied the occurrence of self-reported outdoor tanning, sunbed use and ultraviolet (UV)-induced erythema in an urban area. A cross-sectional questionnaire study of 6000 adolescents aged 13-19 years, and 4000 adults aged 20-50 years was applied. Non-response was analysed for outdoor tanning and sunbed use. Results, in general, did not differ between responders and non-responders. Females aged 17-29 years tanned outdoors most frequently. Sunbed use and related erythema was twice as common in young females. In males, outdoor tanning was not age-related. In the past 12 months, 55% reported sunburn and/or burn from sunbed use, one-third were burned in Sweden, one-quarter on sunny resorts abroad. Sunburn occurs frequently. Compliance with recommendations for sunbed use is poor, especially among adolescents and young adults. To reduce the occurrence of erythema, the influence of risk settings upon behaviours is a critical issue for exploration.

Inhibition of antibody binding to viable cells by liposomes containing purified rat spleen glycoprotein.

The capacity of liposomes with inserted RT-1 histocompatibility antigen to bind anti-RT-1 antibodies varies depending on their lipid composition and mode of preparation. The binding capacity of liposomes prepared by dialysis is different from that of liposomes prepared by gel filtration or of small membrane protein micelles. A new assay for analysis of membrane antigens has been developed. Liposomes are used to compete for antibody binding with in vitro cultured cells while binding of antibody to the same adherent liposomes is simultaneously assayed.

A biotin-labelled antigen radioimmunoassay (BILA) for antibodies to membrane antigens useful for monoclonal antibody screening.

Viable cells or protein extracts were labelled with N-hydroxysuccinimidobiotin and used as target antigens in a biotin-labelled antigen radioimmunoassay (BILA). The binding of the biotinylated antigens to capture antibodies coated on the bottoms of 96-well plastic plates were measured using 125I-labelled streptavidin as the detection step. The assay circumvents some of the problems associated with solid-phase RIA and permits screening of antibodies to undefined protein antigens present in very small amounts in complex protein solutions.

Magnetic carbohydrate nanoparticles for affinity cell separation.

Magnetically responsive nanoparticles were prepared from enzymatically hydrolysed starch and magnetite. Two different monoclonal antibodies were covalently coupled to the particles. The antibody-coupled particles were in the size range of 100-300 nm and had an iron content of about 60%. Using 100 micrograms of magnetic particles (coupled with monoclonal mouse anti-rat Ig kappa light chain antibody) a very high depletion of surface Ig positive cells (mostly B-cells) from one million rat peripheral blood mononuclear cells could be achieved. The separation efficiency was evaluated by flow cytofluorometric analysis. This technique permits the detection of a small number of surface Ig positive cells among 10,000 negative cells.

Use of a monoclonal rat anti-mouse Ig light chain (RAMOL-1) antibody reduces background binding in immunohistochemical and fluorescent antibody analysis.

Rat monoclonal antibodies (MAb) directed to mouse Ig heavy and light chain determinants were produced. A rat anti-mouse light chain MAb (RAMOL-1) which bound to all (24/24) mouse Ig of the kappa light chain type and with varying strength to 4/4 lambda light chain-bearing Ig was evaluated as a general secondary reagent, together with two MAb that bound to the heavy chain of mouse IgG. They were conjugated with biotin or FITC and used in immunohistochemical and immunofluorescence assays to detect mouse monoclonal antibodies binding to antigens expressed in rat and human tissues and cells. As compared to commercially available polyclonal reagents, RAMOL-1 gave higher staining contrast by showing lower background staining and equal or higher staining of the primary MAb tested. This was a result of two main effects. First, crossreactivity with endogenous Ig and tissue type-specific determinants was eliminated. With polyclonal anti-mouse Ig reagents, binding to endogenous Ig was noted in vascular spaces and on Ig-bearing cells, and to rat gastric mucosa and epithelial tumor tissue in frozen tissue sections, even when diluted in high concentrations of serum homologous to the tissue. Second, binding of the secondary reagent was reduced to cells and tissues prone to have high nonspecific binding capability, such as monocytes/macrophages and formalin-fixed, paraffin-embedded tissue. Owing to unlimited and reproducible access to this homogeneous reagent, RAMOL-1 is used as second antibody to standardize the procedure used for immunohistochemical grading of human malignant tumors by determination of blood group antigen expression detected with mouse MAb.

Inhibitory effects of selenium on 1,2-dimethylhydrazine and methylazoxymethanol acetate induction of colon tumors.

Sprague-Dawley rats were injected weekly with either 1,2-dimethylhydrazine (DMH) or methylazoxymethanol acetate (MAM). Addition of 4 ppm selenium (sodium selenite) in the drinking water reduced the number of rats developing DMH-induced colon tumors from 13 to 6 groups of 15 each. The total number of tumors observed in these two groups was 39 in the DMH treated and 11 in the DMH plus selenium. The incidence of MAM-induced tumors was 93% (14/15) with the selenium additive and 100% (14/14) when MAM was administered without the selenium supplement. However, selenium decreased the total number of colon tumors induced by MAM to 42 tumors as compared to a total of 73 tumors in rats receiving only MAM. Both carcinogens induced tumors with a higher frequency in the transverse colon as compared to either the proximal or distal colon. Selenium at this level did not affect the weight gain of the animals.

Treatment of alcoholism.

Special problems in the treatment of alcoholism are discussed. Risk groups, for instance children of alcoholics, should be identified as early as possible so that special attention can be paid to them. Treatment motivation plays a very central role, and motivating a patient for long-term treatment often is the most difficult part of the work. For alcohol dependent alcoholics, total abstinence should be the goal of treatment. To achieve this, psychotherapy consisting of supportive, behavioral and dynamic elements is recommended. To achieve effective therapy it should be carried out for a considerable length of time.

National adaptations of the ICD rules for classification--a problem in the evaluation of cause-of-death trends.

In 1981, the registration routines of the Swedish cause-of-death register were adjusted. The aim of this study was to assess what influence these changes in registration practice might have had on the cause-of-death trends after 1981. The Eighth Revision of the International Classification of Diseases (ICD-8) was used throughout the study period (1976-1985). Significant changes in the registered number of cases were found in 13 of the 18 diagnostic groups scrutinized. Four main types of outcomes were observed: (a) the number of underlying causes increased while the number of contributing causes decreased or vice versa; (b) the number of both underlying and contributory causes changed in the same direction, due to the transfer of a diagnostic group from one ICD category to another; (c) the number of both underlying and contributory causes changed in the same direction, but not due to the transfer of a diagnostic group; or (d) the number of either underlying or contributory causes changed, but not both. In general, the altered registration practice led to more conditions that are often considered as terminal complications to other diseases being registered as the underlying cause of death. While most of the 1981 instructions meant a more literal application of the ICD-8, those concerning cardiac valvular diseases deviated substantially from it. We conclude that (a) important changes in registration practice may occur at any point in time, and not only in connection with the implementation of a new version of the ICD; and (b) national adaptations of the ICD coding instructions may amount to a reversal of the instructions included in the ICD manuals. These findings must be considered when comparing cause-of-death statistics from different countries, and both underlying and contributing cause-of-death statistics should be considered in such analyses of cause-of-death trends.

Correlative fine specificity of several Thomsen-Friedenreich disaccharide-binding proteins with an effect on tumor cell proliferation.

Epithelial cancer cells show increased cell surface expression of mucin antigens with aberrant O-glycosylation, notably type I core (Galbeta1-3GalNAcalpha), termed Thomsen-Friedenreich disaccharide (TFD), a chemically well-defined carbohydrate antigen with a proven link to malignancy. Several TFD-binding proteins influence the proliferation of cells to which they bind. We studied the fine specificity of TFD-binding proteins and its relationship with epithelial tumor cell proliferation. Competitive binding assays against asialoglycophorin showed that Agaricus bisporus lectin (ABL) and human anti-TFD monoclonal antibody (mAb) TF1 were inhibited only by TFD and its alpha-derivatives. Peanut agglutinin (PNA), mAb TF2, and mAb TF5 were also inhibited by other carbohydrates such as lacto-N-biose (Galbeta1-3GlcNAc), lactose, and (Mealpha or beta) Gal, indicating lower recognition of the axial C-4 hydroxyl group position of GalNAc from TFD, and the major relevance of the terminal Gal on interaction of these three TFD-binding proteins. In the direct glycolipid-binding assay, ABL bound mostly to alpha-anomeric TFD-bearing glycolipids, whereas PNA interacted mainly with beta-linked TFD. Of the three anti-TFD mAbs analyzed, all bound N5b (terminal beta-TFD), but only TF2 interacted with N6 (terminal alpha-TFD). These findings indicate that TFD-binding proteins that stimulate the proliferation of epithelial tumor cell lines recognize mainly a terminal beta-Gal region of beta-linked TFD, whereas ABL, which inhibits the proliferation of these tumor cells, binds mainly to subterminal GalNAc of alpha-anomeric TFD.