Two-dimensional patterning of thin coatings for the control of tissue outgrowth

Control of the precise location and extent of cellular attachment and proliferation, and of tissue outgrowth is important in a number of biomedical applications, including biomaterials and tissue engineered medical devices. Here we describe a method to control and direct the location and define boundaries of tissue growth on surfaces in two dimensions. The method relies on the generation of a spatially defined surface chemistry comprising protein adsorbing and non-adsorbing areas that allow control over the adsorption of cell-adhesive glycoproteins. Surface modification was carried out by deposition of thin acetaldehyde and allylamine plasma polymer coatings on silicon wafer and FEP substrates, followed by grafting of a protein resistant layer of poly(ethylene oxide). Spatially controlled patterning of the surface chemistry was achieved by masking during plasma polymerization. XPS and AFM were used to provide evidence of successful surface modifications. Adsorption of the extracellular matrix protein collagen I followed by tissue outgrowth experiments with bovine corneal epithelial tissue for up to 21 days showed that two-dimensional control over tissue outgrowth is achievable with our patterning method over extended time frames. The method promises to be an effective tool for use in a number of in vitro and in vivo applications.     

Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells

MRL/lpr mice develop a systemic autoimmune disease similar to systemic lupus erythematosus in humans. The mice show progressive lymphadenopathy due to the accumulation of an unusual population of CD4^?8^?(DN) B220⁺ αβ⁺ T cells. We bred MRL/lpr mice with mice lacking CD4⁺ or CD8⁺ T cells by gene targeting via homologous recombination in embryonal stem cells to determine the roles of these cells in the autoimmune disease. No difference in survival or autoantibody levels was noted between CD8-/- lpr and littermate controls. Interestingly, these CD8-/- lpr mice have a reduced level of B220⁺ DN T cells despite the fact that the degree of lymphadenopathy was unaltered. CD4-/- lpr mice had a diminished autoimmune disease with a reduction in autoantibody production and skin vasculitits, and increased survival compared to littermate controls. However, CD4-/- lpr mice had an enhanced splenomegaly that developed massively by 16–20 weeks of age (5 to 8 greater than lpr control mice) due to the accumulation of DN B220⁺ T cells. In addition, there were no differences in peripheral lymph node enlargement, although the proportion of DN B220⁺ T cells was about twofold higher in the CD4-/- lpr mice. These cells were phenotypically identical to the DN population in control lpr mice, indicating that the accumulating DN T cells can be dissociated from the autoimmune disease in these mice. Collectively, our results reveal that the autoimmune disease is dependent on CD4⁺, but not CD8⁺ T cells, and that many of the B220⁺ DN T cells traverse a CD8 developmental pathway.     

The human T cell receptor alpha-delta locus: a physical map of the variable, joining and constant region genes

In this study a physical macro-restriction map of the entire human alpha locus that spans about 1000 kilobase pairs and includes the V alpha, J alpha and C alpha genes is presented. Evidence is provided that gene duplications were involved in the increase of genomic diversity of V alpha genes. In addition, we show a detailed map of a 40-kb region located approximately 100 kg upstream of the human C alpha gene. Direct evidence is provided to support that the human alpha chain locus, like the murine, also contains another T cell constant region gene in the alpha chain locus, the human delta chain gene. In addition, two J segments and one D segment have been identified. Using these genomic probes, we show that several T cell lines, including those known to express the surface gamma/delta heterodimer, have rearranged this region. The design of two separate centers of rearrangement within one locus that are involved in rearrangement events at different times, and the presence of high number of J segments in this region, may render the locus highly vulnerable to chromosomal translocation during T cell development.     

Clonal rearrangements of T-cell receptor and immunoglobulin genes and immunophenotypic antigen expression in different subclasses of Hodgkin's disease

Twenty-two cases of Hodgkin's disease (HD), representing the 4 different subclasses, were studied by immunophenotypic and immunogenotypic analysis. Quantitative immunophenotypic analysis of HD infiltrates showed a predominance of CD3-positive T cells in all subtypes except the lymphocytic depletion (HDLD) subtype. Only 5 samples of HD [2 of lymphocytic predominance (HDLP), 2 of mixed cellularity (HDMC), and one of nodular sclerosis type (HDNS)] were found to have both their Ig and T-cell antigen receptor (TcR) genes in the germ-line configuration. The remaining patients with HDLP (3 cases), HDNS (5 cases), and HDMC (4 cases), all exhibited rearrangements of either TcR gamma or TCR gamma and TcR beta genes, while all 5 cases of HDLD had either TCR gamma or immunoglobulin heavy-chain gene rearrangement. These results substantiate the view that Hodgkin's lymphomas contain clonal lymphocyte populations and that different rearrangement patterns may be associated with different subclasses of HD.     

Matrix-assisted laser desorption ionization mass spectrometry detection of proteins adsorbed in vivo onto contact lenses

Identification of the biomolecules that form the first adsorbed monolayer, which thus effect "interface conversion", in competitive adsorption from multicomponent biological solutions can be challenging because of limitations in mass resolution and sensitivity of established techniques. In this study matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry is developed and applied as a novel surface analytical method to enable analysis of adsorbed multicomponent biomolecule layers directly on the biomaterial surfaces. We show that proteins adsorbed in vivo (on human eyes) on contact lenses can be detected rapidly and conveniently by the diagnostic highly resolved mass signals recorded by MALDI mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers at levels substantially below monolayer coverage. Identification is done by comparison with molecular masses of known proteins. Specifically, it is shown that in addition to lysozyme, other low molecular weight proteins adsorb from human tear fluid onto contact lenses; these proteins had not been detected in earlier studies using other techniques.     

Surface-MALDI mass spectrometry in biomaterials research

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new surface analysis method with unique capabilities that complement established biomaterial surface analysis methods such as XPS and ToF-SSIMS. These new MALDI variant methods, which we shall collectively summarize as Surface-MALDI-MS, are capable of desorbing adsorbed macromolecules from biomaterial surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established biochemical techniques such as SDS-PAGE, and can in some circumstances be used for the quantitative analysis of adsorbed protein amounts. At this early stage of development, however, limitations exist: in some cases proteins are not detectable, which appears to be related to tight surface binding. This review summarizes ways in which Surface-MALDI-MS methods have been applied to the study of a range of issues in biomaterials surfaces research.     

Über die Hemmung der antidiuretischen Wirkung von Morphin und Dromoran durch (-)-3-Hydroxy-N-Allyl-Morphinan (Laevallorphan) beim Menschen

Zusammenfassung Nach orientierenden Vorversuchen über die Höhe der sicher wirksamen Dosen wurde an 20 gesunden Versuchspersonen der antidiuretische Effekt von Morphin und Dromoran mit Hilfe des Wasser- und Konzentrationsversuches untersucht. Die Antidiurese konnte durch gleichzeitige Verabfolgung von (-)-3-Hydroxy-N-allyl-Morphinan bei Morphin deutlich gehemmt und bei Dromoran, entsprechend seiner geringeren antidiuretischen Wirkung, fast völlig aufgehoben werden. Ein antidiuretischer Effekt von Laevallorphan selbst wurde nicht beobachtet.Versuchspräparat RO 1-7700 der Fa. Hoffmann-La Roche, der für die Überlassung der Versuchsmengen hier bestens gedankt wird.     

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