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Biomedical subjects

U Kühn

Publications and source records attributed to U Kühn.

At least 19 recordsLinked to original sources
PubMed2005

Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth.

The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. It is believed that this tissue contains stem cells and lineage committed progenitor cells or precursor cells (PCs) for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the isolation of PCs derived from dental follicle of human third molar teeth. These fibroblast-like, colony forming and plastic adherent cells expressed putative stem cell markers Notch-1 and Nestin. We compared gene expressions of PCs, human mesenchymal stem cells (hMSCs), periodontal ligament cells (PDL-cells) and osteoblasts (MG63) for delimitation of PCs. Interestingly, PCs expressed higher amounts of insulin-like growth factor-2 (IGF-2) transcripts than hMSCs. Differentiation capacity was demonstrated under in vitro conditions for PCs. Long-term cultures with dexamethasone produced compact calcified nodules or appeared as plain membrane structures of different dimensions consisting of a connective tissue like matrix encapsulated by a mesothelium-like cellular structure. PCs differentially express osteocalcin (OCN) and bone sialoprotein (BS) after transplantation in immunocompromised mice but without any sign of cementum or bone formation. Therefore, our results demonstrate that cultured PCs are unique undifferentiated lineage committed cells residing in the periodontium prior or during tooth eruption.

Adolescent↗
PubMed2003

[Rare tumors of the parotid gland. Lymphadenoma of a sebaceous gland and extracranial metastasis from glioblastoma].

Tumors of the parotid gland can be benign or malignant lesions.The pathophysiology of rare tumors of the parotid gland is often not sufficiently explored in this paper two rare histological entities are described. In one patient a benign sebaceous lymph-adenoma was histologically diagnosed. This type of tumor accounts for only 0.196 of all adenomas. The international literature is not conclusive about whether the tumor originates in the parotid gland itself or in the parotid lymph nodes. The second patient presented with a metastasis from a glioblastoma in the parotid gland. Again, the mechanism for metastasis of an intra-cerebral tumor to the parotid gland remains unknown. Iatrogenic seeding during a neurosurgical intervention is a probable explanation. The actual clinical course of his patient and the cases described in literature render surgical removal of such a metastasis questionable.

Adenoma↗
PubMed2001

The hSkn-1a POU transcription factor enhances epidermal stratification by promoting keratinocyte proliferation.

Skn-1a is a POU transcription factor that is primarily expressed in the epidermis and is known to modulate the expression of several genes associated with keratinocyte differentiation. However, the formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation, and a role for Skn-1a in this process has not been previously demonstrated. Here, our results show, surprisingly, that human Skn-1a contributes to epidermal stratification by primarily promoting keratinocyte proliferation and secondarily by enhancing the subsequent keratinocyte differentiation. In organotypic raft cultures of both primary human keratinocytes and immortalized HaCaT keratinocytes, human Skn-1a expression is associated with increased keratinocyte proliferation and re-epithelialization of the dermal substrates, resulting in increased numbers of keratinocytes available for the differentiation process. In these same raft cultures, human Skn-1a expression enhances the phenotypic changes of keratinocyte differentiation and the upregulated expression of keratinocyte differentiation genes. Conversely, expression of a dominant negative human Skn-1a transcription factor lacking the C-terminal transactivation domain blocks keratinocytes from proliferating and stratifying. Keratinocyte stratification is dependent on a precise balance between keratinocyte proliferation and differentiation, and our results suggest that human Skn-1a has an important role in maintaining epidermal homeostasis by promoting keratinocyte proliferation.

Cell Differentiation↗
PubMed2000

Nuclear inclusions in oculopharyngeal muscular dystrophy consist of poly(A) binding protein 2 aggregates which sequester poly(A) RNA.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of the disease is caused by short (GCG)(8-13) expansions in the PABP2 gene. This gene encodes the poly(A) binding protein 2 (PABP2), an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we report that PABP2 is detected in filamentous nuclear inclusions, which are the pathological hallmark of OPMD. Using both immunoelectron microscopy and fluorescence confocal microscopy, the OPMD-specific nuclear inclusions appeared decorated by anti-PABP2 antibodies. In addition, the inclusions were labeled with antibodies directed against ubiquitin and the subunits of the proteasome and contained a form of PABP2 that was more resistant to salt extraction than the protein dispersed in the nucleoplasm. This suggests that the polyalanine expansions in PABP2 induce a misfolding and aggregation of the protein into insoluble inclusions, similarly to events in neurodegenerative diseases caused by CAG/polyglutamine expansions. No significant differences were observed in the steady-state poly(A) tail length in OPMD and normal myoblasts. However, the nuclear inclusions were shown to sequester poly(A) RNA. This raises the possibility that in OPMD the polyalanine expansions in the PABP2 protein may interfere with the cellular traffic of poly(A) RNA.

Cell Nucleus↗
PubMed2000

Polarized secretion of the regulated secretory protein chromogranin A.

Bovine chromogranin A (CgA), together with secreted alkaline phosphatase (SEAP) as an external control for apical secretion were expressed in MDCK cells to test if CgA contains sorting signals for polarized secretion. CgA, SEAP, and the endogenous apical marker GP80 were secreted 75-80% apically. Basolateral secretion of SEAP was inhibited 40% by ammonium chloride. Sulfate labeling and digestion with chondroitinase ABC revealed a 120 kDa proteoglycan-CgA and 75 kDa CgA. Inhibition of proteoglycan synthesis did not affect apical secretion of CgA. As CgA is not N-glycosylated, we used tunicamycin to test if cellular N-glycosylation is required for apical sorting. Tunicamycin reversed the polarity of secretion of CgA to the basolateral side. These results suggest that CgA contains dominant apical and recessive basolateral sorting information.

Animals↗
PubMed2000

The nuclear poly(A) binding protein, PABP2, forms an oligomeric particle covering the length of the poly(A) tail.

The mammalian nuclear poly(A) binding protein, PABP2, controls the length of the newly synthesized poly(A) tail on messenger RNAs. To gain a better understanding of the mechanism of length control, we have investigated the structure of the PABP2.poly(A) complex. Electron microscopy and scanning force microscopy studies reveal that PABP2, when bound to poly(A), forms both linear filaments and discrete-sized, compact, oligomeric particles. The maximum diameter of the filament is 7 nm; the maximum diameter of the particle is 21(+/-2) nm. Maximum particle size is realized when the PABP2. poly(A) complex is formed with poly(A) molecules 200-300 nt long, which corresponds to the average length of the newly synthesized poly(A) tail in vitro and in vivo. The equilibrium between filaments and particles is highly sensitive to ionic strength; filaments are favored at low ionic strength, while particles predominate at moderate to high ionic strength. Nitrocellulose filter binding and gel mobility shift assays indicate that the PABP2.poly(A) particle formed on A(300) is not significantly more stable than complexes formed with smaller species of poly(A). These results are discussed in the context of the proposed functions for PABP2.

Allosteric Regulation↗
PubMed2000

Deciphering the cellular pathway for transport of poly(A)-binding protein II.

Poly(A)-binding protein II (PABP2) is an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In the cytoplasm, a distinct protein (PABP1) binds to poly(A) tails and participates in mRNA translation and stability. How cytoplasmic PABP1 substitutes for nuclear PABP2 is still unknown. Here we report that PABP2 shuttles back and forth between nucleus and cytoplasm by a carrier-mediated mechanism. A potential novel type of nuclear localization signal exists at the C-terminus of the protein, a domain that is highly enriched in methylated arginines. PABP2 binds directly to transportin in a RanGTP-sensitive manner, suggesting an involvement of this transport receptor in mediating import of the protein into the nucleus. Although PABP2 is small enough to diffuse passively through the nuclear pores, protein fusion experiments reveal the existence of a facilitated export pathway. Accordingly, no transport of PABP2 to the cytoplasm occurs at 4 degrees C. In contrast, export of PABP2 continues in the absence of transcription, indicating that transport to the cytoplasm is independent of mRNA traffic. Thus, rather than leaving the nucleus as a passive passenger of mRNAs, the data suggest that PABP2 interacts with the nuclear export machinery and may therefore contribute to mRNA transport.

3T3 Cells↗
PubMed1999

The Drosophila poly(A)-binding protein II is ubiquitous throughout Drosophila development and has the same function in mRNA polyadenylation as its bovine homolog in vitro.

The poly(A)-binding protein II (PABP2) is one of the polyadenylation factors required for proper 3'-end formation of mammalian mRNAs. We have cloned Pabp2, the gene encoding the Drosophila homolog of mammalian PABP2, by using a molecular screen to identify new Drosophila proteins with RNP-type RNA-binding domains. Sequence comparison of PABP2 from Drosophila and mammals indicates that the most conserved domains are the RNA-binding domain and a coiled-coil like domain which could be involved in protein-protein interactions. Pabp2 produces four mRNAs which result from utilization of alternative poly(A) sites and encode the same protein. Using an antibody raised against Drosophila PABP2, we show that the protein accumulates in nuclei of all transcriptionally active cells throughout Drosophila development. This is consistent with a general role of PABP2 in mRNA polyadenylation. Analysis of Drosophila PABP2 function in a reconstituted mammalian polyadenylation system shows that the protein has the same functions as its bovine homolog in vitro : it stimulates poly(A) polymerase and is able to control poly(A) tail length.

Amino Acid Sequence↗
PubMed1998

Induction of tyrosine phosphorylation in human MHC class II-positive antigen-presenting cells by stimulation with contact sensitizers.

To investigate the intracellular signaling mechanisms involved in the activation of APC by contact sensitizers, we studied the induction of tyrosine phosphorylation by these agents. Selective analysis of phosphotyrosine (p-tyr) in human Langerhans cells and different mononuclear cell types was achieved using a multicolor flow-cytometric technique. Stimulation with contact sensitizers revealed a distinct increase in p-tyr exclusively for MHC class II-positive cells. For different haptens, irritants, as well as activators of distinct signal transduction pathways, it was demonstrated that only strong sensitizers or the protein tyrosine phosphatase inhibitor sodium orthovanadate or cross-linking of MHC class II molecules were able to induce formation of p-tyr in human blood-derived dendritic cells serving as model for the dendritic cell family. This event required physiologic cell culture conditions and was blocked by specific inhibitors of protein tyrosine kinases. No evidence for the inhibition of protein tyrosine phosphatases by haptens was found. Western blot analysis of monocyte-enriched populations revealed an augmented phosphorylation of distinct proteins after hapten stimulation partly resembling the pattern noticed after cross-linking of HLA-DR molecules. In dendritic cells generated from mononuclear progenitors, the protein tyrosine kinase inhibitor genistein was able to block tyrosine phosphorylation as well as production of IL-1beta mRNA transcripts. Our data underline the unique capacity of haptens to activate APC and the important role of tyrosine phosphorylation for this process.

Antigen-Presenting Cells↗
PubMed1997

Flow-cytometric screening for the modulation of receptor-mediated endocytosis in human dendritic cells: implications for the development of an in vitro technique for predictive testing of contact sensitizers.

The aim of this study was to explore the usefulness of human blood dendritic cells (DC) in the development of an in vitro model for predictive testing of contact sensitizers. A method was established to monitor the influence of chemicals on the intracellular targeting of antibody-crosslinked MHC class II molecules after their uptake by human DC. Using a three-colour flow-cytometric technique, freshly prepared DC were distinguished from other MHC class II-bearing cell types such as B-cells and monocytes in unseparated mononuclear cell suspensions of healthy volunteers. The assay is based on the pH-sensitivity of internalized fluorescein-coupled MHC class II specific antibodies. Quenching of fluorescence intensity due to internalization into acidic intracellular compartments was observed with untreated DC whereas internalization into less acidic structures following stimulation with strong contact sensitizers ensured that the fluorescence intensity was conserved. The usefulness of this approach for predictive testing of the preservatives MI/MCI, imidazolidinyl urea, methyl-4-hydroxy-benzoate and 2-phenoxyethanol in comparison to the strong allergen DNFB and the irritants sodium lauryl sulphate and dithranol was explored. Whereas low concentrations of MI/MCI resembled the strong allergen DNFB, high concentrations of imidazolidinyl urea were required for a moderate response. Methyl-4-hydroxy-benzoate and 2-phenoxyethanol as well as the irritants SLS and dithranol failed to induce a significant effect in this assay. The non-responsiveness to the latter compounds reflected their minor or absent capacity to induce contact hypersensitivity in humans, whereas DNFB, MI/MCI and imidazolidinyl urea are well established contact sensitizers. These data suggest that the capacity of a chemical to modulate endocytotic mechanisms in dendritic cells in vitro seems to reflect the probability of that substance acting as a hapten in vivo.

Antibodies, Monoclonal↗
PubMed1997

Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions.

Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-alpha/IL-1/IL-6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)-gamma and no IL-4 or IL-10. Production of IFN-gamma was significantly induced by addition of PGE2, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN-gamma(low), IL-4(neg) cells, TNF-alpha/IL-1/IL-6 promoted growth of IFN-gamma(intermediate), IL-4(neg) CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN-gamma production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-alpha/IL-1/IL-6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF-alpha/IL-1, IL-6 + PGE2-cultured DC seem to be optimal for generation of IFN-gamma-producing CD4/CD8+ T cells.

Cell Differentiation↗
PubMed1997

Immunostimulatory effects of platinum compounds: correlation between sensitizing properties in vivo and modulation of receptor-mediated endocytosis in vitro.

The sensitizing properties of different complex salts of platinum were defined in vivo by means of the popliteal lymph node (PLN) assay in mice. Hexa- and tetrachloroplatinates were confirmed to be highly immunogenic, inducing vigorous primary immune responses in the draining PLN following single subcutaneous injections. Flow-cytometric analysis revealed a dramatic increase in the total number of cells expressing proliferating cell nuclear antigen. The majority of these cells were of the T helper phenotype (CD4+) reflecting the T-cell dependence of the PLN response induced by Pt salts such as Na2[PtCl6] or Na2[PtCl4]. In contrast, [Pt(NH3)4]Cl2 failed to elicit a significant increase in PLN cell proliferation when compared with saline-treated controls. The differential immunogenicity of the Pt compounds found in vivo directly correlated with their capacity to modulate mechanisms of receptor-mediated endocytosis in murine Langerhans cells in vitro. The reactivity of Na2[PtCl6] or Na2[PtCl4] resembled that of potent contact sensitizers in this endocytosis assay whereas [Pt(NH3)4]Cl2 proved to be mert. These results suggest that [Pt(NH3)4]Cl2 might be less harmful to humans than hexa- or tetrachloroplatinates. As demonstrated with Pt compounds, monitoring of direct effects of low-molecular-weight chemicals on antigen-presenting dendritic cells in vitro is able to predict their sensitizing potential in vivo.

Adjuvants, Immunologic↗
PubMed1996

Xenopus poly(A) binding protein: functional domains in RNA binding and protein-protein interaction.

Subsets of the four RNA binding domains (RBD 1 to 4) in the Xenopus poly-adenylate binding protein (PABP) have distinct affinities and specificities for RNA. RBDs 1 plus 2 exhibit RNA affinity and selectivity equal to the wild-type (WT) protein. RBDs 3 plus 4 have distinct selectivity and about ten-fold reduced affinity for A23, and the isolated RBDs 2 or 3 or 4 exhibit about 100-fold reduced affinity for A23 in comparison to WT. For the full-length protein, independent RNA contacts have been mapped by UV crosslinking with RBDs 1/2 and RBDs 3/4. The carboxy-terminal, non-RBD portion of the protein does not contribute to RNA affinity or selectivity, but confers homodimerization activity on PABP. RBDs 3 and 4 cooperate with the C terminus to gain poly(A) organizing activity, i.e. the ability to form an RNP with multiple, regularly spaced copies of PABP on a poly(A) substrate.

Animals↗
PubMed1996

An approach to predictive testing of contact sensitizers in vitro by monitoring their influence on endocytotic mechanisms.

Endocytotic activation of epidermal Langerhans cells (LC) by immunogenic haptens is an early event during development of allergic contact dermatitis. In this work a fast and objective flow-cytometric assay for predictive in vitro testing of contact sensitizers by monitoring their influence on endocytotic mechanisms in murine LC was developed. Epidermal cell suspensions were labelled with a monoclonal antibody directed to MHC class II molecules and pH-sensitive fluorochrome-coupled second-step reagents. For untreated LC a significant quenching of fluorescence intensity by internalization of the MHC-antibody complexes into acidic compartments was noticed. Similar results were obtained in the presence of irritants, the lectin concanavalin A and the phorbol ester phorbol 12-myristate 13-acetate. In contrast stimulation with several well-defined sensitizing compounds resulted in partial conservation of the fluorescence intensity due to the internalization of the labelled complexes into less acidic compartments. Monitoring this modulation of endocytosis is an effective in vitro method to test for properties typical for moderate and strong contact sensitizers. It will help to assess the risk of unknown chemicals to act as haptens and should be useful for restriction of animal experimentation in this field.

Animals↗
PubMed1995

A new method for phenotyping proliferating cell nuclear antigen positive cells using flow cytometry: implications for analysis of the immune response in vivo.

The incorporation of radioactive nucleotides into newly synthesized DNA has been established as a standard method for the detection of proliferation in eucaryotic cells. Unfortunately the use of this method makes it harder to obtain information on the phenotype of proliferating cells in mixed cell populations. For this reason we established a flow-cytometric approach employing a monoclonal antibody specific for murine as well as human proliferating cell nuclear antigen (PCNA) and a double labeling technique for detection of cell membrane-expressed phenotypic markers. The efficiency of this immunostaining procedure was confirmed by simultaneous and highly specific detection of PCNA in nuclear structures as well as cell membrane-expressed antigens using cytological techniques. In vitro experiments with mitogen- and alloantigen-stimulated murine lymph node cells (LNC) and human peripheral blood mononuclear leukocytes (PBML) revealed a good correlation of total [3H]thymidine incorporation into DNA and expression of PCNA. For the analysis of proliferating cells activated in vivo the method was employed to evaluate the local lymph node assay which assesses the allergenicity of small chemicals. LNC prepared from the cervical lymph nodes of mice treated on 4 consecutive days with sensitizing concentrations of the contact allergens oxazolone, TNCB and DNFB as well as the irritants benzoic acid and SLS in comparison to the solvent control showed a dramatic increase in the total amount of proliferating cells for contact allergen-treated animals in comparison to the solvent control and irritant-treated mice. In addition a detailed phenotyping of the proliferating cell populations was possible. This approach offers an easy to perform, non-radioactive method for the assessment of proliferation of murine as well as human leukocytes in vitro and especially in vivo and will be of great advantage for situations where the phenotype of proliferating cellular subsets in heterogeneous populations is of interest.

Allergens↗