SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti‐inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic ...contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen‐specific or non‐specific stimulation. It downregulates the production of Th1 interleukin (IL)‐2, interferon‐γ and Th2 (IL‐4, IL‐10) type cytokines after antigen‐specific stimulation of a human T‐helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin‐stimulated transcription of a reporter gene coupled to the human IL‐2 promoter in the human T‐cell line Jurkat and the IgE/antigen‐mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)‐α promoter in the murine mast‐cell line CPII. It does not, however, affect the human TNF‐α promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcγRIII receptor. SDZ ASM 981 also prevents the release of preformed pro‐inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro‐inflammatory cytokines from T cells and mast cells in vitro.
We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A ...(CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.
Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC50 = 6.9 ± 0.9 nM) but is structurally ...different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the C26C27 exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity of 2 for cyclophilin (IC50 = 29 ± 2.1 nM) is essentially identical to SFA, which indicates that the interaction between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This observation was confirmed by the X-ray crystal structure resolved at 2.1 Å of cyclophilin A complexed to macrolide 16, a close analogue of 2. The X-ray crystal structure showed that macrolide 16 binds to the same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure−activity relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or (2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide unit, which together provide an extremely strong affinity for cyclophilin A.
Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type‐specific gene expression patterns that constitute cellular identities. To test whether the ...developmental potential of fetal brain‐derived cells that form floating sphere colonies (neurospheres) can be modified by destabilizing their epigenotype, neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived neurosphere cells treated with a combination of trichostatin A (TSA) plus 5‐aza‐2′‐deoxycytidine (AzaC) (TSA/AzaC neurosphere cells) yielded long‐term, multilineage and transplantable neurosphere‐derived haematopoietic repopulation. Untreated neurosphere cells exhibited no haematopoietic repopulation activity. The neurosphere‐derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of neurosphere cells followed by transplantation enables the generation of neurosphere‐derived haematopoietic cells.
We have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations ...polylysine or protamine, we have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle Wagner, E., Zenke, M., Cotten, M., Beug, H. \& Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414. We demonstrate here that this procedure is useful for a human leukemic cell line. We enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell-permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, we show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks "transferrinfection," as does incubation of the cells at 18⚬C.
A large membrane proteinase 3 (mPR3)-positive neutrophil subset (mPR3high) is a risk for Wegener's granulomatosis (WG). The relationship between mPR3 expression and clinical manifestations was ...investigated in 81 WG patients and mPR3 expression was studied in CD34+ stem cell-derived human neutrophils. The mPR3high neutrophil percentage correlated with renal function, anemia, and albumin at the time of presentation. The mPR3high neutrophil percentage and renal failure severity correlated directly after 5 yr. For elucidating mechanisms that govern mPR3 expression, studies were conducted to determine whether the genetic information that governs mPR3 expression resides within the neutrophils, even without stimuli possibly related to disease. CD34+ hematopoietic stem cells were differentiated to neutrophils, and their mPR3 expression was determined. A two-step amplification/differentiation protocol was used to differentiate human CD34+ hematopoietic stem cells into neutrophils with G-CSF. The cells progressively expressed the neutrophil surface markers CD66b, CD35, and CD11b. The ferricytochrome C assay demonstrated a strong respiratory burst at day 14 in response to PMA but none at day 0. Intracellular PR3 was detectable from day 4 by Western blotting. An increasing percentage of a mPR3-positive neutrophil subset became detectable by flow cytometry, whereas a second subset remained negative, consistent with a bimodal expression. Finally, human PR3-anti-neutrophil cytoplasmic autoantibodies induced a stronger respiratory burst, compared with human control IgG in stem cell-derived neutrophils. Taken together, these studies underscore the clinical importance of the WG mPR3 phenotype. The surface mPR3 on resting cells is probably genetically determined rather than being dictated by external factors.
Einleitung:
Das humane Pankreaskarzinom (PDAC) gehört zu den Krebserkrankungen mit der höchsten Letalität. Dabei handelt es sich um eine komplexe Tumorerkrankung mit durchschnittlich 60 genetischen ...Veränderungen, die ca. 12 verschiedene Signalwege betreffen. Mit wachsendem biologischen Verständnis rücken zunehmend Netzwerke von Transkriptionsfaktoren in den Focus. Der Transkriptionsfaktor TBX3 spielt dabei eine wichtige Rolle in der Regulation von embryonischen Stammzellen, es konnte jedoch darüber hinaus in einem Kollektiv von resezierten PDACs bereits eine Verschlechterung der Prognose bei TBX3 Expression gezeigt werden.
Ziele:
Untersuchung der Rolle von TBX3 im humanen PDAC.
Methodik:
Grundlage
in vitro
Experimente: Lentivirus-mediierte Überexpression von TBX3 in etablierten humanen Pankreaskarzinom- und Primärzelllinien. Grundlage
in vivo
Experimente: Chick Chorioallantoic Membrane (CAM) Assay. Korrelation der TBX3 Expression mittels humanem Tissue-Micro-Array und Tumorgewebe.
Ergebnis:
Zunächst konnte in humanen PDAC Proben eine Überexpression von TBX3, gegenüber gesundem Pankreasgewebe, nachgewiesen werden. Ein Einfluss von TBX3 auf die Proliferationsrate konnte nicht nachgewiesen werden, jedoch zeigt sich nach Überexpression ein signifikant gesteigertes Invasions- und Migrationsverhalten der Tumorzellen,
in vitro
und
in vivo
. Im CAM Assay konnte zusätzlich ein deutlicher Größenunterschied der Tumoren zu Gunsten der TBX3 Überexpressionsgruppe gezeigt werden. Im humanen PDAC geht die Überexpression von Tbx3 mit einer vermehrten Akquise von Stammzelleigenschaften einher und fördert dabei signifikant die Bildung von Spheres. Es konnte abschließend eine TBX3-abhängige Krebsstammzell-Population separiert werden, welche sich durch eine autokrine TBX3-ACTIVIN/NODAL Signalschleife aktiviert.
Schlussfolgerung:
Es konnte gezeigt warden das TBX3 ein weiteres Pluripotenz-assoziiertes Gen ist das eine Rolle in der Karzinogenese des humanen Pankreaskarzinoms spielt.
The absorption and disposition of pimecrolimus, a calcineurin inhibitor developed for the treatment of inflammatory skin diseases, was investigated in four healthy volunteers after a single oral dose ...of 15 mg of (3)Hpimecrolimus. Supplementary information was obtained from in vitro experiments. Pimecrolimus was rapidly absorbed. After t(max) (1-3 h), its blood concentrations fell quickly to 3% of C(max) at 24 h, followed by a slow terminal elimination phase (average t(1/2) 62 h). Radioactivity in blood decreased more slowly (8% of C(max) at 24 h). The tissue and blood cell distribution of pimecrolimus was high. The metabolism of pimecrolimus in vivo, which could be well reproduced in vitro (human liver microsomes), was highly complex and involved multiple oxidative O-demethylations and hydroxylations. In blood, pimecrolimus was the major radiolabeled component up to 24 h (49% of radioactivity area under the concentration-time curve(0-24) h), accompanied by a large number of minor metabolites. The average fecal excretion of radioactivity between 0 and 240 h amounted to 78% of dose and represented predominantly a complex mixture of metabolites. In urine, 0 to 240 h, only about 2.5% of the dose and no parent drug was excreted. Hence, pimecrolimus was eliminated almost exclusively by oxidative metabolism. The biotransformation of pimecrolimus was largely catalyzed by CYP3A4/5. Metabolite pools generated in vitro showed low activity in a calcineurin-dependent T-cell activation assay. Hence, metabolites do not seem to contribute significantly to the pharmacological activity of pimecrolimus.
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