Differenzierung und Entwicklung / Differentiation and Development
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Quantitative Real Time-PCR gene expression analysis was used to determine the halfmaximal inhibition of the cardiomyocyte gene expression IC50 Exp caused by the test compound. To predict embryotoxic effects in vivo from the determined in vitro data, these parameters were used for the classification of the test chemicals as non, weak or strong embryotoxic via a mathematical prediction model PM.
In the majority of cases comparable halfmaximal inhibiting concentrations were calculated in the conventional and molecular EST that resulted in the identical classification of the tested chemicals concerning their embryotoxic potential. These predictions were consistent with in vivo findings that testifies the stability and the usefulness of the parameters used in the conventional and molecular EST. MAMac, which is known as a developmental neurotoxin in vivo, represented the single exception.
Genetische, cytohistologische und biochemische Grundlagen der Entwicklung. — Genetical, cytohistological and biochemical foundations of development. Differenzierung und Entwicklung / Differentiation and Development.
Its misclassification as compared to in vivo data may originate from the limitations of the model system that is based on mesodermal differentiation. Thus, specific effects on neuronal developmental processes obviously cannot be detected completely.
Regulation der Genexpression bei der Erythroiden Differenzierung
Gene expression analysis showed that test substance concentrations which were proved to be inhibiting on the morphological differentiation of cardiomyocytes caused a repression of cardiac-specific marker gene expression as well. Thereby, IC50 Exp-values proved to be just as sensitive as the conventional parameters and can provide valuable and supportive data. They allowed a prediction of the embryotoxic potential of the chemicals in vivo already at day 5 and day 7 of in vitro differentiation.
Moreover, gene expression analysis of appropriate differentiation specific genes could be used to investigate mechanisms that are responsible for embryotoxic properties of the test compounds. Thus, the EST is considered to represent a new, predictive screening test especially in the pharmaceutical industry to detect the embryotoxic potential of chemical compounds early in the process of compound development. Once subtype-specific epitopes that are conserved among a particular subtype have been identified, monolclonal antibodies mabs will be generated against such peptides.
If regions representing continuous stretches of suitable epitopes can be found, such polypeptides will be expressed in eukaryotic or procaryotic expression systems. The main short-term goal is the development of a universally applicable indirect, or blocking format to allow host-independent antibody detection ELISA for the subtype-specific detection of selected influenzavirus A antibodies, e. Ultimately, a complete set of 16 HA and 9 NA antigens, as well as mabs against inter-subtype-specific, intra-type-conserved epitopes will be available, allowing the development of a highly configurable multi-purpose ELISA array for the rapid characterization of antibodies against any influenza A virus.
Molekulare Mechanismen in Zelldifferenzierung und Zellteilung
To overcome the current problems and limitations involved in influenza virus antibody detection and subtyping, we have decided to address the issue of rapid typing of anti-influenza antibodies by a comprehensive approach, which focuses on the expression of recombinant HA and NA proteins to be used as antigens, determination of inter- and intra-subtype-specific epitopes, and generation of appropriate mabs that can be used in competitive ELISAs.
The ultimate goal of this work is an ELISA-based differentiation of antibodies agains all 16 HA and all 9 NA influenza virus subtypes, which works equally well for an unlimited number of bird and mammalian host species.
Since this is quite an ambitious and comprehensive project, we will focus on certain HA and NA subtypes and develop the respective tools in a prioritized way. As a minimal output after the 4 year project duration we anticipate at least to have a fully validated ELISA available that allows to differentiate H5 and H7 antibodies from other HA subtypes, as well as N1 from other NA subtypes. Since both an indirect and a blocking-format ELISA will be developed, the test should work with sera from any bird or mammalian host species. We also expect to gain important information about the inter- and itra-subtype specificity of immunogenic epitopes on the HA and the NA protein.
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This will be the basis for a next gereration of diagnostic tools with increased specificity and sensitivity; and once appropriate mabs against these epitopes have been generated, they can also be used for rapid typing of new influenza virus strains in an antigen-trapping ELISA, but also in immunohistochemistry, not only for diagnostic purposes, but also for influenza pathogenesis studies, e.
A highly configurable ELISA for the subtype-specific detection and characterization of anti-influenza virus antibodies will be available. Independent of the test format indirect or blocking and the type of antigens being employed, the ELISA will be available in a highly flexible and configurable format, allowing a tailor-made setup to be used depending on the intended use: i In its simplest format, the ELISA will only contain one HA subtype, and, if simultaneous NA subtyping is required, one NA subtype.
In this format replicates of three columns of a well plate will be coated with negative antigen or left blank, if HA and NA antigens are not used as fusion proteins , HA and NA antigens, respectively. This format would be suitable for screening purposes, allowing the simultaneous testing of 28 single wells or 14 duplicate wells sera per plate. Based on the current worldwide spread of H5N1 highly desirable uses of this test format would e. In this case, a maximum number of 92 sera could be tested on one plate, if single wells and a blocking ELISA format are used which does not require the inclusion of negative antigen wells.
H5N1-infected birds on the other hand will be identified based on their positive reactivity with the N1 antigen irrespective whether they had also been vaccinated with H5N2 vaccine. This would allow to type 5 sera per plate one series of 16 wells reserved for control reactions with 16 HA reference sera.
To this end, the most sophisticated array format would be the combination of the 16 HA with the 9 NA antigens in a two-dimensional array format, where all the different combinations of HA with NA antigen would be coated by mixing one HA and one NA, respectively, in one well of on the plate. However, such a format would require the use of well plates in order to include all the necessary controls.
Detection and especially differentiation of antibodies against avian influenza virus in sera from birds and mammals is still largely done using the hemagglutination inhibition HI test. They allowed a prediction of the embryotoxic potential of the chemicals in vivo already at day 5 and day 7 of in vitro differentiation. Particularly preferred are heart muscle cells cardiomyocytes. The phloem-localized thiamine transporter, polyamine uptake transporter 3 PUT3 , mediates thiamine translocation from the shoots to the roots. According to the invention, the promoters are selected from cell-specific promoters, and development-specific promoters.
Furthermore, such an array format would require a considerable amount of test serum unless the sera can be used sufficiently diluted , which is not feasable for sera from small bird species. The use of highly influenza virus subtype-specific antigens, eventually in combination with subtype-specific polyclonal antisera or mabs will result in the availability of a highly customizable, highly configurable ELISA, depending on the questions to be addressed. Taken together, a test system for influenza virus laboratory diagnosis will be available for a broad range of very different types of studies to be performed, such as i rapid typing of antibodies, ii use of the ELISA as a DIVA diagnostic tool, iii epidemiological tracing, iv for surveillance projects e.
The materials obtained in the present project can be used as reagents for additional purposes.
Social Differentiation
The HA and NA subtype-specific antigens and antibodies polyclonal sera, mabs produced in the present work will not only serve as tools in an antibody ELISA, but will also be available for several additional purposes. However, these additional applications are beyond the time frame and personal capacity of this project and would require additional manpower, or they could be used as starting materials in future projects: i A new generation of antigen ELISA could be developed in the form of a sandwich ELISA, where e.
The immobilized influenza virus would then be detected by a PO-labeled or unlabeled mab being specific for the respective homologous subtype. The availability of a complete set of recombinant specific antigens and mabs against all relevant subtypes would allow to very quickly develop such tests for any given influenza virus subtype.
This could be important if new influenza virus strains are detected for which immediate typing is required, e. For instance, these reagents could be used for subtype-sprecific high-throughput testing of swab samples for virus detection or screening of sera for antibody detection using fluorescence polarization technique Jolley and Nasir, A second appealing application is the use of these reagents in biosensor technology, where either antigens or antibodies are coupled to nanosensors, allowing an extremely sensitive detection of either pathogens or antibodies.
Among many available techniques, cantilever sensors will be considered as the most promising technology, since this technique has already been described for the detection of microbial pathogens for review, see Hansen and Thundat, No special biosafety requirements are necessary for production and use of this test system. The HA and NA typing of antibodies or at least the production of the necessary reagents, i. In contrast, the production of the reagents described here do not require special biosafety measures related to the protection of the personnel or the environment against influenza virus, since all the materials used in the ELISA are non-infectious.
No matter what ELISA format is being used to detect antibodies, the test can be performed in any diagnostic laboratory.