Richard Kowalski

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.

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.

BACKGROUND: Long-term use of immunosuppressants is associated with significant morbidity and mortality in transplant recipients. A simple whole blood assay that has U.S. Food and Drug Administration clearance directly assesses the net state of immune function of allograft recipients for better individualization of therapy. A meta-analysis of 504 solid organ transplant recipients (heart, kidney, kidney-pancreas, liver and small bowel) from 10 U.S. centers was performed using the Cylex ImmuKnow assay. METHODS: Blood samples were taken from recipients at various times posttransplant and compared with clinical course (stable, rejection, infection). In this analysis, 39 biopsy-proven cellular rejections and 66 diagnosed infections occurred. Odds ratios of infection or rejection were calculated based on measured immune response values. RESULTS: A recipient with an immune response value of 25 ng/ml adenosine triphosphate (ATP) was 12 times (95% confidence of 4 to 36) more likely to develop an infection than a recipient with a stronger immune response. Similarly, a recipient with an immune response of 700 ng/ml ATP was 30 times (95% confidence of 8 to 112) more likely to develop a cellular rejection than a recipient with a lower immune response value. Of note is the intersection of odds ratio curves for infection and rejection in the moderate immune response zone (280 ng/ml ATP). This intersection of risk curves provides an immunological target of immune function for solid organ recipients. CONCLUSION: 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.