Measured versus estimated glomerular filtration rate in the Calvert equation: influence on carboplatin dosing.

PURPOSE: Carboplatin is frequently dosed to achieve a desired area under the plasma concentration-time curve (AUC) by using the Calvert or Chatelut equations to estimate carboplatin clearance. Accurate determination of glomerular filtration rate (GFR) is necessary to correctly calculate carboplatin clearance using the Calvert equation. In clinical practice, the Cockcroft-Gault formula is frequently used to estimate GFR, but this practice has been reported to under- and overestimate carboplatin clearance. The purpose of this trial was to compare determinations of carboplatin clearance using the Chatelut equation and four separate GFR determinations, including 99mTc-DTPA, the Cockcroft-Gault formula, a 24-h urine collection and a 2-h urine collection. METHODS: Carboplatin clearance was estimated in 21 previously untreated extensive-stage small-cell lung cancer patients. GFR was determined using 99mTc-DTPA, the Cockcroft-Gault formula, 24-h urine collection and 2-h urine collection. Serum and urine creatinine concentrations were measured using enzymatic assays. The carboplatin clearance was then calculated by individually adding 25 to the four GFR determinations based on the Calvert equation, which states that carboplatin clearance equals GFR + 25 (nonrenal clearance). The carboplatin clearance was also estimated using the Chatelut equation. The five determinations of carboplatin clearance were compared using Friedman's test and post-hoc Wilcoxon signed rank tests. Precision and bias for each carboplatin clearance determination were calculated assuming that 99mTc-DTPA provided the most accurate measure of GFR. RESULTS: A statistically significant difference was found between the five methods of estimating carboplatin clearance (P < 0.001). No difference was found between carboplatin clearance calculated using 99mTc-DTPA and the Chatelut equation, the Cockcroft-Gault formula or the 2-h urine collection. The Chatelut equation provided more precision and less bias than the 2-h urine collection (median precision 20% and 30%, median bias -1% and -18%, respectively). CONCLUSION: Compared to 99mTc-DTPA, the Chatelut equation more accurately estimates carboplatin clearance than the Cockcroft-Gault formula, the 2-h urine collection and the 24-h urine collection. The greater negative bias found for the latter three estimates of carboplatin clearance could result in underdosing of carboplatin.

Biomechanics of bone fusion.

Bone fusion can be achieved by one or more of three methods: in situ, onlay, and interbody fusion. Interbody implants provide the spine with the ability to bear an axial load. They function optimally when placed along the neutral axis and produce little, if any, significant bending moment. Interbody implants may be comprised of bone, non-bone materials such as acrylic, or a combination of both such as in interbody cages. In this report the authors' goal is to provide some insight into the theoretical, as well as practical, biomechanical factors that influence bone fusion, focusing on interbody implants. They review the concept of stress shielding and its impact on fusion. With the attendant biomechanical nuances of the different regions of the spine, they discuss region-specific strategies involved in successful fusion. Finally, they review intraoperative techniques that will improve the chance of achieving a successful arthrodesis.

Benefits of a nurse directed over the counter medication dispensing system in an automotive plant. A preliminary study.

Information is limited about the effects of the availability of over the counter medicines (OTCs) at a worksite on workers' ability to remain at work and the effect on health care utilization. The purpose of this preliminary study was to assess workers' perceptions related to the benefits of having a nurse directed over the counter medication dispensing system (OTCMS) at an automotive manufacturing plant. Fifty-six percent of 257 randomly selected workers who participated in semistructured interviews indicated they used OTCs as the first intervention when at home, and 88% had obtained OTCs from the plant's medical department. The workers were overwhelmingly positive about having an OTCMS available at their worksite. Eighty-nine percent indicated that having OTCs available from the occupational health nurse made it possible for them to stay at work and complete their shift. Not only did the workers find the OTCMS to be an asset in their worksite, but they also highly recommended having an OTCMS available in work settings that do not presently have one in place.

Recovery of functional memory T cells in lung transplant recipients following induction therapy with alemtuzumab.

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 CMV-specific T-cell function was assessed longitudinally in 23 lung transplant (LTx) recipients using T-cell assays (ImmuKnow and T Cell Memory, 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.

Establishing pediatric immune response zones using the Cylex ImmuKnow assay.

For all transplant patients, the transplant physician must balance the risk of rejection caused by under-immunosuppression against the risk of drug toxicity, secondary infections and post-transplant lymphoproliferative disorder with over-immunosuppression. A Food and Drug Administration (FDA)-approved in vitro assay, the Cylex ImmuKnow assay, provides a global assessment of cellular immune function to help monitor the immune status of immunosuppressed patients. This assay uses the plant lectin phytohemagglutinin to stimulate lymphocytes; an ATP assay is then used to measure the degree of activation of CD4 T cells. However, the normal values for this assay were developed with healthy adult patients. In this study, we determined the normal ranges for the ImmuKnow assay in healthy children and compared those values to levels obtained in healthy adults and in stable pediatric renal transplant patients. We found that healthy children 12 yr of age and older showed immune function levels indistinguishable from adults, while healthy children under 12 had significantly lower immune function levels than adults. For adults, the ImmuKnow assay zones (in ng/mL ATP) of strong, moderate and low immune function correspond to >525, 225 to 525, and <225. In children under 12, we found the corresponding zones to be >395, 175-395 and <175 ng/mL. The median value for normal adults is 415, whereas it is only 295 for children <12 yr of age and this value decreases to 165 in stable renal transplant patients <12 yr of age (compared with 258 for stable adult renal transplant patients). Thus, this study provides critical information necessary to utilize the ImmuKnow assay with pediatric patients. In adults, the degree of immune function as assessed by the ImmuKnow assay helps to predict patients at risk for infection or rejection. If further studies in pediatric patients document the same and is true for children, then the ImmuKnow assay will provide a useful adjunct tool to prevent over- or under-immunosuppression as newly developed drugs are utilized or drug treatment is altered because of drug side effects, toxicity, concurrent illnesses or rejection.

Unambiguous classification of microtubule-ends in vitro: dynamic properties of the plus- and minus-ends.

To understand the mechanism of dynamic instability of microtubule growth and shortening, one needs a means of reliably determining the polarity of the microtubules under investigation. Sea urchin sperm-tail axonemal fragments nucleate the growth of both plus-ended and minus-ended microtubules, but their polarity is not apparent by video-enhanced DIC microscopy. The polarity of a microtubule is usually assessed by observing differences between the rates and lengths of growth and shortening excursions of the two ends. In practice, though, a significant fraction of the population of microtubules displays characteristics intermediate between the average characteristics of either end, thereby escaping classification. Excluding these "intermediate" microtubules from the measured populations introduces bias into the understanding of microtubule dynamic instability. We circumvent this problem by making use of the plus-end directed movement of the microtubule-dependent molecular motor kinesin to determine the polarity of any given microtubule unambiguously. Carboxylated-microspheres coated with kinesin, which are clearly visible by DIC microscopy, were used to determine the polarity of a microtubule. The dynamics were then observed. Kinesin was found to have no marked effect on dynamic instability. By this technique, we show that the distributions of properties that describe microtubule dynamic instability (rates and lengths of growth and shortening as well as frequencies of interconversion between these phases) of plus-ends overlap to a significant extent with those of minus-ends. It is this overlap that obscures the usual classification of the ends. Therefore, models describing microtubule dynamic instability need to incorporate the broad and overlapping range of properties of the two ends.

Microtubule-associated protein 2 alters the dynamic properties of microtubule assembly and disassembly.

The influence of microtubule-associated protein 2 (MAP2) on the dynamics of microtubule assembly and disassembly from axonemal fragments was characterized in vitro in solutions of pure tubulin and varying concentrations of MAP2. A mechanistic description of interactions between MAP2 and individual microtubules was developed from analysis of recorded images obtained by video-enhanced differential-interference-contrast light microscopy. MAP2 decreased the rates and lengths of shortening events and decreased the frequency of transitions between growth and shortening over a wide range of concentrations, thereby producing the increases in net microtubule growth previously described by light-scattering techniques. Increases in rates and lengths of elongation phases, as well as rescue frequencies (i.e. transition from shortening to growth), were observed under conditions in which microtubules are expected to be saturated with MAP2. During early stages of nucleated assembly, MAP2 greatly increased the number of microtubules growing from a given axoneme and caused elongation of "curved" structures which may be sheet-like microtubules.

Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel (Taxol(R)).

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.

The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant cells.

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.

Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than taxol.

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.