Newness Trailer #1 (2017)
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Company profile. The bacterial cell wall component may be a peptidoglycan, which is a cell wall fraction of Gram-positive bacteria. Alternatively the bacterial cell wall component may be endotoxin, which is a cell wall fraction of Gram-negative bacteria.
Background Art Cytokines are intracellular signal transmitters which play an important role in an immune response, a response upon infection, hematopoiesis, inhibition of virus infection and tumor cells.
Among them, a cytokine which transmits signals between lymphocytes sporgenza del sesso on-line called interleikin hereinafter, "IL".
Among ILs, IL-1 is a cytokine which mediates various immune responses and inflammatory responses, and is involved in maintenance of homeostasis of living organisms and produced from various cells such as monocytes, macrophages, keratinocytes, vascular endothelial cells and the like when the living organisms get infected or hurt. Further, it is known that IL has no structural similarity to IL in spite that it has a functional similarity, and has a structural similarity to Sporgenza del sesso on-line A sporgenza del sesso on-line of molecules showing homology to IL-1R have been identified so far, and signal pathways mediated by IL-1R family is being studied intensively now.
Toxins in bacterial cells being comprised of lipopolysaccharide, which is a major structural component of the outer membrane encompassing peptidoglycan on the surface of Gram-negative bacteria, are called endotoxin, and it has been known that lipopolysaccharide is comprised of lipid called lipid A and various kinds of saccharide which covalently bind to the lipid A.
It has been also known that this endotoxin has a bioactivity mainly involved in fever, decrease of leukocytes and platelet, hemorrhagic necrosis of bone marrow cells, hypoglycemia, induction of IFN, activation of B limphocyte immune response cell derived from marrow sporgenza del sesso on-line, and the like. It has been known that a Toll gene is required to control dorsoventral patterning during the embryonic development of Drosophila Cell 52, , Annu.
Cell Dev. It has been clarified that the Toll is a type I transmembrane receptor with an extracellular domain containing leucine-rich repeat LRR and that its cytoplasmic domain shows high homology to that of mammalian interleukin-1 recepter IL-1R Nature,Annu. It has sporgenza del sesso on-line also clarified that another Toll family member, wheeler, participates sporgenza del sesso on-line the antibacterial host defense but not in the antifungal immune response, and that particular pathogens induce specific antimicrobial immune responses in Drosophila through the selective activation of the Toll pathways Proc.
USA 95, Blood 91, Gene, Cell 2, Immunity 11, Further, the sporgenza del sesso on-line of the TLR families in mammals is also believed to participate in innate immune recognition as pattern recognition receptors PRRswhich recognize bacterial cell common structures Cell 91, However, since CD14 is a glycosylphosphatidylinositol GPI -anchored protein without a transmembrane domain, the existence of a bona fide signaling receptor of LPS has been sporgenza del sesso on-line.
In addition, Chow et al. Mycoplasmas, known as pathogens in animals and humans, are wall-less bacteria, yet they are capable of activating macrophages.
It is known that the lipid moiety has 2 asymmetric C atoms, and that the synthetic MALP-2 comprised of the S, R racemate had a specific activation similar to the natural compound action at picomolar concentrations in vitro. Nevertheless, the conclusions obtained from overexpression experiments do not necessarily reflect the function of TLR family in vivo. It is also reported that the results of analysis of the responsiveness based sporgenza del sesso on-line NF-B activation are not related to biological responses mediated by these stimuli Infect.
In addition, it is known that the function of a specific gene can be analyzed sporgenza del sesso on-line individual level by using transgenic mice in which genes are artificially introduced and expressed, and gene-deficient mice generated by gene targeting in which specific genes on genomes are artificially transformed by homologous recombination sporgenza del sesso on-line embryonic stem cells hereinafter "ES cells".
In general, gene-deficient mice are called knockout mice, and TLR2 knockout mice and MyD88 knockout mice have not sporgenza del sesso on-line known, and moreover, it has not been known that TLR2 knockout mice and MyD88 knockout mice are unresponsive to bacterial cell components, either. Summary of the Invention Though in vivo responses to bacterial cell components are expected to vary depending on the difference of expression levels of each TLR on the cell surface, the contribution of individual members of the TLR family and MyD88, the adaptor protein of the TLR family, to signaling by bacterial cell components' stimuli in vivo remains to be elucidated.
An object of the present invention is to provide the use of a non-human animal whose function of MyD88 gene is deficient on its chromosome as a model animal unresponsive to bacterial cell wall components. Such animals are useful for elucidating the contribution of MyD88, the adaptor protein of the TLR family; to signaling by bacterial cell components' stimuli in vivo, sporgenza del sesso on-line particular, sporgenza del sesso on-line role of MyD88 in vivo.
The inventors of the present invention have conducted intensive study for attaining the object. The present invention relates to the use of a non-human animal sporgenza del sesso on-line function of MyD88 gene is deficient on its chromosome as a model animal unresponsive to bacterial cell wall components claim 1.
The non-human animal may be a rodent claim 2or a mouse claim 3. The present invention also sporgenza del sesso on-line to the use of a macrophage or splenocyte cell derived from a non-human animal as defined in any of claims 1 to 3 as a model cell unresponsive to bacterial cell wall components claim 4. Sporgenza del sesso on-line present invention may be used to elucidate the action mechanisms of bacterial cell components claim 5.
The present invention may be used to obtain information of signalling receptors of bacterial cell wall components claim 6. The present invention may be used in a method of detecting bacterial cell components, which method comprises the steps of: i administering the subject material to a an animal or cell as defined in any of claims 1 to 4 and b sporgenza del sesso on-line a wild-type sporgenza del sesso on-line animal or cell and ii assessing and comparing responsiveness to the subject material claim 7.
The present sporgenza del sesso on-line may be used to obtain information for development of medicines for diseases caused by excessive production of bacterial cell wall components claim 8. In connection with the use of the present invention the bacterial cell wall component is a peptidoglycan claim 11an endotoxin claim 12lipotechoic acid claim 13 or Mycobacterium tuberculosis lysate claim The present invention may be used to establish a treatment method for endotoxin shock claim The present invention may be used to elucidate the molecular mechanism in a process of infection by bacteria to the cell wall components of which the animal is unresponsive claim The present invention may be sporgenza del sesso on-line to develop new remedies for infection by bacteria to the cell wall components of which the animal is unresponsive claim Brief Explanation of Drawings Fig.
Further, in connection with the present invention, carriers which carry the above mentioned bacterial cell components, and the bacterial cell themselves are expediently included in the examples of the bacterial cell components. In connection with the present invention, "unresponsiveness to bacterial cell components" means that sporgenza del sesso on-line non-human animals, or cells, sporgenza del sesso on-line, or organs derived therefrom show low reactivity or almost no reactivity to the stimuli of the bacterial cell components, and "hyporesponsiveness" means low reactivity to the stimuli.
Therefore, a model non-human animal being unresponsive to bacterial cell components in connection with the present invention means a non-human animal such as a mouse, a rat, a rabbit or the like, where living organisms, or cells, tissue, or organs which comprise living organisms show low reactivity or almost sporgenza del sesso on-line reactivity to the stimuli of the bacterial cell components.
Examples of the stimuli of the bacterial cell components include an in vivo stimulus where a bacterial cell component is administered to a living organism and an in vitro stimulus where a bacterial cell component is brought into contact with cells separated from a living organism. In connection with the present invention, "deficiency of MyD88 gene function on a chromosome" means that a part of or a whole of MyD88 gene on a chromosome is deficient and the function to express MyD88, which is expressed in wild-types, is lost.
Examples of a non-human animal whose function of MyD88 gene is deficient on its chromosome include a rodent such as a rat or the like whose function of MyD88 gene is deficient other than MyD88 knockout mice. The term "a wild-type non-human animal" in connection with the present invention means a non-human animal being the same species of the non-human animal whose function of MyD88 gene is deficient on its chromosome.
For example, in case of mice, it means MyDnondeficient type mice of same species among F2 mice generated at the expected Mendelian ratio. When the deficient type and the wild-type mice of these F2 mice, in particular, the wild-type littermates are used for experiments simultaneously, it becomes possible to conduct precise comparative experiments at individual level.
With an example of knockout mice which have deficiency in MyD88, a generating method of the non-human animal whose function of MyD88 gene is deficient on its chromosome will now be explained. MyD88 gene can be cloned by amplifying a mouse genomic library by PCR or other methods with a probe sporgenza del sesso on-line from a mouse EST clone or the like. By DNA recombination technique a part of or a whole of this cloned MyD88 gene, for example, a part or a whole of an exon region containing a cytoplasmic region of MyD88 gene is replaced with a poly A signal and a marker gene such as a neomycin resistance gene or the like, a targeting vector is constructed by inducing genes such as diphtheria toxin A fragment DT-A gene or herpes simplex virus thymidine kinase HSV-tk gene or the like into 5'-terminal side, this constructed targeting vector is linearized, and introduced into embryonic stem cells ES cells by electroporation method or the like, then cultured, and subsequently Es cells achieving homologous recombination by G, ganciclovir GANC or other such antibiotics are selected.
It is preferable to confirm whether these selected ES cells are the object recombinants by Southern blot analysis or the like. Chimeric mice can be obtained by microinjecting the recombined ES cells into blastocysts of mice, and put the blastocysts back into uteri of recipient mice.
Under high chimeric ratio, there will be born much more male chimeric mice than female ones. All of these mice are generated at the expected Mendelian ratio. As the method of confirming whether MyD88 knockout mice of the present invention are born, for example, the method wherein RNA is isolated from peritoneal macrophages of mice obtained by the above-stated method, and is examined by Northern blot analysis or the like, and the method wherein the expression of MyD88 in the mice is examined by Western blot analysis or the like are exemplified.
Moreover, macrophages and splenic B cells of MyD88 knockout mice are unresponsive not only to endotoxin but also to peptidoglycan being a cell wall component of Gram-positive bacteria, lipotheichoic sporgenza del sesso on-line, Mycobacterium tuberculosis lysate and the like, while they are responsive to IL-4 and IFN.
The present invention provides the use of a macrophage or splenocyte cell derived from a non-human animal whose function of MyD88 is deficient on is chromosome as a model cell unresponsive to bacterial cell wall components. Sporgenza del sesso on-line the measurement and the assessment of the macrophage activity level or the splenocyte activity level, it is preferable to assess the levels in comparison to the measured value of a wild type non-human animal, in particular, a littermate wild type non-human animal of the non-human animal being unresponsive to bacterial cell components as control, because there will be no dispersion caused by individual differences.
This can be applied to the assessment of bioactivity of various subject materials and detection of bacterial cell components and the sporgenza del sesso on-line, in which the non-human animal being unresponsive to bacterial cell components is used.
The relationship between IL-1 and the illness in disease model mice can be examined by precisely assessing IL-1 activity of a subject material with MyD88 knockout mice. It becomes possible to obtain useful information for developing pharmaceuticals which can cure diseases such as rheumatoid arthritis caused by overexpression of IL-1, a graft-versus-host disease, asthma and the like by precisely assessing IL-1 activity of a subject material and by analyzing the involvement of IL-1 in disease model mice.
Moreover, by precisely assessing IL-1 activity of a subject material with MyD88 knockout sporgenza del sesso on-line, it becomes possible to obtain useful information for developing sporgenza del sesso on-line which can cure diseases caused by overproduction of IL, such as I type diabetes, a graft-versus-host disease and the like. The present invention provides the use of a non-human animal whose function of MYD88 is deficient on its chromosome, in a method of detecting bacterial cell components, which method comprises the steps of: i administering the subject material to a an animal whose function of MYD88 is deficient on its chromosome, or cell derived therefrom and b to a sporgenza del sesso on-line non-human animal or cell and ii assessing and comparing responsiveness to the subject material.
With the method of detecting the bacterial cell components, it is possible to detect insubstantial amount of bacterial cell components contained in subject materials in the non-human animal being unresponsive to bacterial cell components after the subject material has been administered to the non-human animal.
The present invention will be explained more specifically with examples below, but the technological scope of the present invention is not limited to these examples. A sporgenza del sesso on-line vector was constructed by replacing the 1. The replaced genomic fragment contained sporgenza del sesso on-line exons encoding the domain that resembles the cytoplasmic domain of the IL-1RAcP receptor accessory protein.
The neomycin resistance gene sporgenza del sesso on-line flanked by the 1. Then,an HSV-tk cassette was introduced into the 3' end of the genomic fragment.
Doubly resistant clones were screened for homologous recombination by PCR and 33 clones were verified by Southern blot analysis using the probe indicated in Sporgenza del sesso on-line. The MyD88 knockout mice of the present invention grew healthy and showed no obvious abnormalities until 20 weeks of age.
Northern blot analysis was performed to confirm that the inactivation of the MyD88 gene was caused by mutation. Flow cytometric analysis of CD3, B, CD4, and CD8 in thymus, spleen, and lymph node showed that lymphocyte composition was not altered in the MyD88 knockout mice in comparison with wild-type mice.
Example 2 Unresponsiveness of MyD88 knockout mice to Endotoxin 1mg of LPS derived from Escherichia coli B5 was administered to 10 MyD88 knockout mice of the present invention, and endotoxin-unresponsiveness was examined through the sporgenza del sesso on-line ratio of the mice. The results are shown in Fig. It is confirmed by Fig. Proliferation of T cells were examined by measuring [3H] amount of [3H] thymidine taken into the cells. It has been found that sporgenza del sesso on-line results could be obtained even when splenic B cells were used instead of thymocytes.
These results indicate that ILmediated growth signal of T cells was impaired in the thymocytes of MyD88 knockout mice. In comparing ILinduced increase of mRNA expression in wild-type littermates and in MyD88 knockout mice, increase of induction was observed in wild-type mice, but not observed in MyD88 knockout mice.
Thus, ILmediated major biological functions has been found to be severely impaired in MyD88 knockout mice. As a result, when splenic B cells were cultured in the presence of IL in vitro, lytic activity to YAC-1 targeting cells was dramatically enhanced in wild-type mice, but it was not enhanced in MyD88 knockout mice.
These results demonstrate that MyD88 knockout mice are defective in Th1 cell development in vivo as is the case with ILdeficient mice, and that their major biological activities mediated by IL were completely abolished. Stimulation with IL induced an approximately 3- to 4-fold increase in AP-1 activity, and this activation was blocked by coexpression of MyD88 amino acid see Fig. Splenic T cells cultured in the presence of IL and anti-CD3 antibody for 4 days were starved for 3 hours and then stimulated with IL Age-matched groups of wild-type, TLR4- and MyDdeficient mice were used for the following examples.
Sporgenza del sesso on-line were prepared by phenol extraction and purified by gel filtration. LPS from Salmonella minnesota Re prepared by phenol-chloroform-petroleum ether extraction procedure was also purchased Sigma. Three days later, peritoneal exudate cells were isolated from the peritoneal cavity and washed with ice-cold Hank's buffered salt solution HBSSthen peritoneal cells were obtained.