Epothilones A and B, natural products with minimal structural analogy to taxoids, have effects similar to those of paclitaxel (Taxol(R)) in cultured cells and on microtubule protein, but differ from paclitaxel in retaining activity in multidrug-resistant cells. We examined interactions of the epothilones with purified tubulin and additional cell lines, including a paclitaxel-resistant ovarian carcinoma line with an altered beta-tubulin. The epothilones, like paclitaxel, induced tubulin to form microtubules at low temperatures and without GTP and/or microtubule-associated proteins. The epothilones are competitive inhibitors of the binding of [3H]paclitaxel to tubulin polymers. The apparent Ki values for epothilones A and B were 1.4 and 0.7 microM by Hanes analysis and 0.6 and 0.4 microM by Dixon analysis. In the paclitaxel-sensitive human cell lines we examined, epothilone B had greater antiproliferative activity than epothilone A or paclitaxel, while epothilone A was usually less active than paclitaxel. A multidrug-resistant colon carcinoma line and the paclitaxel-resistant ovarian line retained sensitivity to the epothilones. With Potorous tridactylis kidney epithelial (PtK2) cells examined by indirect immunofluorescence, microtubule bundles appeared more rapidly following epothilone B treatment, and there were different proportions of various mitotic aberrations following treatment with different drugs.
These data show that the Cylex ImmuKnow assay has a high negative predictive value and provides a target immunological response zone for minimizing risk and managing patients to stability.
Each year, 55 000 organ transplants are performed worldwide. Cumulatively, the number of living organ recipients is now estimated to be over 300 000. Most of these transplant recipients will remain on immunosuppressive drugs for the remainder of their lives to prevent rejection episodes. Controlled doses of these drugs are required to prevent over-medication, which may leave the patient susceptible to opportunistic infection and drug toxicity effects, or under-dosing, which may lead to shortened graft survival because of rejection episodes. This paper describes the result of a multicenter study conducted at the Universities of Pittsburgh, Alabama and Maryland to evaluate an in vitro assay (CylexTM Immune Cell Function Assay) for the measurement of global immune response in transplant patients receiving immunosuppressive therapy. The assay uses a whole blood sample to maintain the presence of the drug during incubation. Following overnight incubation of blood with phytohemagglutinin (PHA), CD4 cells are selected using paramagnetic particles coated with a monoclonal antibody to the CD4 epitope. The CD4-positive cells are targeted as major immunosuppressive drugs are designed to specifically inhibit T-cell activation which has been implicated in rejection. The data generated at these three sites were submitted in support of an Food and Drug Association (FDA) application for the use of this assay in the detection of cell-mediated immunity in an immunosuppressed population. The assay was cleared by the FDA on April 2, 2002. This cross-sectional study was designed to establish ranges for reactivity of this bioassay in the assessment of functional immunity for an individual solid organ recipient at any point in time.
Computer-assisted structure analysis indicated (+)-discodermolide, a polyhydroxylated alkatetraene lactone marine natural product, was an antimitotic compound, and we confirmed this prediction. Previous work had shown an accumulation of discodermolide-treated cells in the G2/M portion of the cell cycle, and we have now found that discodermolide arrests Burkitt lymphoma cells in mitosis. Discodermolide-treated breast carcinoma cells displayed spectacular rearrangement of the microtubule cytoskeleton, including extensive microtubule bundling. Microtubule rearrangement that occurred with 10 nM discodermolide required 1 microM taxol. Discodermolide had equally impressive effects on tubulin assembly in vitro. Near-total polymerization occurred at 0 degree C with tubulin plus microtubule-associated proteins (MAPs) under conditions in which taxol at an identical concentration was inactive. Without MAPs and/or without GTP, tubulin assembly was also more vigorous with discodermolide than with taxol under every reaction condition examined. Discodermolide-induced polymer differed from taxol-induced polymer in that it was completely stable at 0 degree C in the presence of high concentrations of Ca2+. In a quantitative assay designed to select for agents more effective than taxol in inducing assembly, discodermolide had an EC50 value of 3.2 microM versus 23 microM for taxol.
The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium. These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent. We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions. Furthermore, we demonstrated that discodermolide is a competitive inhibitor with [3H]paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM. Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines). Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide. The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times. Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment. A variety of spindle aberrations were observed after treatment with both drugs. The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.
Tissue regeneration strategies invoke cell-based therapies for effective tissue formation. Current assessment of mesenchymal stem cell (MSC) directed bone regeneration during in vivo assays is dependent on histologic determination of bone formation. It was the aim of this study to determine the relationship between bone sialoprotein (BSP) expression and osteocalcin expression with subsequent osteogenesis occurring in MSC-based implants. RT-PCR assessment of human actin, collagen type I, BSP, and osteocalcin indicated that undifferentiated cells did not express BSP or osteocalcin. Three weeks following implantation, human BSP could be identified in RNAs isolated from the retrieved implants. For every implant from which human BSP cDNA was amplified, parallel implants harvested at 6 weeks demonstrated bone formation at the histologic level. This study confirms that, in the context of the severe combined immunodeficiency disease (SCID) mouse model, culture-expanded, cryopreserved human MSCs have osteogenic potential and demonstrates that implanted cell gene expression can reveal the early onset of bone formation.
Profound T-cell depletion with the monoclonal antibody alemtuzumab facilitates reduced maintenance immunosuppression in abdominal and lung transplantation. While the phenotype of the post-depletional T cells has been characterized, little is known about their function. In the present study, global and CMVspecific T-cell function was assessed longitudinally in 23 lung transplant (LTx) recipients using T-cell assays (ImmuKnow ® and T Cell Memory TM , Cylex, Columbia, MD) during the first year posttransplant after induction therapy. Recovery of mitogen responses were seen at 2 weeks posttransplantation (65%PHA; 58% Con A), despite the low number of circulating T cells (<2%). These responses declined at 4-5 months (24%PHA; 54% Con A) and were partially reconstituted by 9 months (46% PHA; 73% Con A). CMV-specific responses recovered in 80% of R+ patients as early as 2 weeks posttransplant (n = 5) and 72% of patients had a memory response by 3 months (n = 11). In contrast, only 2 of 5 patients who did not exhibit memory responses pre-transplant (R-) developed transient CMV-specific T-cell responses. Our results show that profound depletion of T cells induced by alemtuzumab spares the functional subset of CMV-specific memory T cells. Conversely, CMV R-patients predepletion may require a prolonged period of prophylaxis.
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