Tumor necrosis factor induces distinct patterns of caspase activation in WEHI-164 cells associated with apoptosis or necrosis depending on cell cycle stage.

TNF is unusual among the death receptor ligands in being able to induce either apoptotic or necrotic cell death. We have observed that in WEHI 164 fibrosarcoma, cells the mode of TNF-induced cell death is dependent on the stage of the cell cycle. Cells arrested in G(0)/G(1) undergo necrosis, while those progressing through the cell cycle undergo apoptosis. TNF induces caspase activity in both settings, and the broad spectrum caspase inhibitor zVAD-fmk inhibits this activity and blocks both TNF-induced apoptosis and necrosis. Inhibition of oxygen radical accumulation does not block cytotoxicity. The presence and activation of specific caspases were examined by Western blotting. The procaspase-8a isoform was down-regulated in proliferating cells. Procaspases-8b and -7 were cleaved during TNF-induced apoptosis but not necrosis. Thus, a different pattern of caspase expression and activation occurs dependent on the cell cycle and which may determine the mode of cell death.     

Enhanced sensitivity to IL-2 signaling regulates the clinical responsiveness of IL-12-primed CD8(+) T cells in a melanoma model

The optimal expansion, trafficking, and function of adoptively transferred CD8(+) T cells are parameters that currently limit the effectiveness of antitumor immunity to established tumors. In this study, we addressed the mechanisms by which priming of self tumor-associated Ag-specific CD8(+) T cells influenced antitumor functionality in the presence of the inflammatory cytokine IL-12. In vitro priming of mouse tumor-specific CD8(+) T cells in the presence of IL-12 induced a diverse and rapid antitumor effector activity while still promoting the generation of memory cells. Importantly, IL-12-primed effector T cells dramatically reduced the growth of well-established s.c. tumors and significantly increased survival to highly immune resistant, established intracranial tumors. Control of tumor growth by CD8(+) T cells was dependent on IL-12-mediated upregulation of the high-affinity IL-2R (CD25) and a subsequent increase in the sensitivity to IL-2 stimulation. Finally, IL-12-primed human PBMCs generated tumor-specific T cells both phenotypically and functionally similar to IL-12-primed mouse tumor-specific T cells. These results highlight the ability of IL-12 to obviate the strict requirement for administering high levels of IL-2 during adoptive cell transfer-mediated antitumor responses. Furthermore, acquisition of a potent effector phenotype independent of cytokine support suggests that IL-12 could be added to adoptive cell transfer clinical strategies in cancer patients.     

Below-ground competition between trees and grasses may overwhelm the facilitative effects of hydraulic lift

Under large East African Acacia trees, which were known to show hydraulic lift, we experimentally tested whether tree roots facilitate grass production or compete with grasses for below‐ground resources. Prevention of tree–grass interactions through root trenching led to increased soil water content indicating that trees took up more water from the topsoil than they exuded via hydraulic lift. Biomass was higher in trenched plots compared to controls probably because of reduced competition for water. Stable isotope analyses of plant and source water showed that grasses which competed with trees used a greater proportion of deep water compared with grasses in trenched plots. Grasses therefore used hydraulically lifted water provided by trees, or took up deep soil water directly by growing deeper roots when competition with trees occurred. We conclude that any facilitative effect of hydraulic lift for neighbouring species may easily be overwhelmed by water competition in (semi‐) arid regions.     

Pooling local expert opinions for estimating mammal densities in tropical rainforests

Methods currently used for assessing wildlife density in rainforests are time and money consuming. The precision of the most commonly used methods is disputed, but accepted because more exact methods are not available. In this study a new method of wildlife density estimation is explained. The new method is less time and money consuming, but yields comparable results with classical methods. The method was tested in the field in Cameroon and compared with transect surveys in the area and with relevant literature. The Pooled Local Expert Opinion (PLEO) method is based on the knowledge of local experts. A number of hunters were asked to estimate wildlife abundance in a specified area, after which the density/km² was calculated for 33 wildlife species. These estimates were pooled and extrapolated for the whole study area. Elephant (Loxodonta africana) density outside the National Park was estimated to be 0.06 animals/km², and 0.3 inside. Buffalo (Syncerus caffer) density for the study area was estimated at 0.2 animals/km² and gorilla (Gorilla gorilla) density at 1.05 per km². Transect surveys carried out at the same time for considerably more money, taking far more time, produced too few data to calculate densities. The evaluation of the PLEO method was favourable and the method offers a substitute for conventional methods of estimating wildlife density in rainforests. The methodology is simple and it can be incorporated in many tropical biodiversity and conservation projects. It can also be used for long-term monitoring of wildlife status in a given area. In contrast with classical methods, the PLEO method is low in cost and assures local ownership of the results.     

An assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling

A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (32Pi). Intra- and extracellular 32PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added 32Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated.     

Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors.     

Detecting femur–insert collisions to improve precision of fluoroscopic knee arthroplasty analysis

Fluoroscopic analysis is an important tool for assessing in vivo kinematics of knee prostheses. Most commonly, a single-plane fluoroscopic setup is used to capture the motion of prostheses during a particular task. Unfortunately, single-plane fluoroscopic analysis is imprecise in the out-of-plane direction. This can result in reconstructing physically impossible poses, in which—for example—the femoral component intersects with the insert, as the normal pose estimation process does not take into account the relation between the components. In the proposed method, the poses of both components are estimated simultaneously, while preventing femur–insert collisions. In a phantom study, the accuracy and precision of the new method in estimating the relative pose of the femoral component were compared to those of the original method. With reverse engineered models, the errors in estimating the out-of-plane position decreased from 2.0±0.7 to 0.1±0.1 mm, without effects on the errors in rotations and the in-plane positions. With CAD models, the errors in estimating the out-of-plane position decreased from 5.3±0.7 mm (mean±SD) to 0.0±0.4 mm, at the expense of a decreased precision for the other position or orientation parameters. In conclusion, collision detection can prevent reconstructing impossible poses and it improves the position and motion estimation in the out-of-plane direction.     

De Visuele Associatietest (VAT) als instrument voor de ouderenpsycholoog

The Visual Association Test (VAT) is a brief learning task that consists of six line drawings of pairs of interacting objects (association cards). Subjects are asked to name or identify each object and later are presented with one object from the pair (the cue) and asked to name the other (the target). The VAT was administered in a consecutive sample of 174 psychogeriatric day care participants with mild to major neurocognitive disorder. Comparison of test performance with normative data from non-demented subjects revealed that 69% scored within the range of a major deficit (0–8 over two recall trials), 14% a minor, and 17% no deficit (9–10, and ≥10 respectively).VAT-scores correlated with another test of memory function, the Cognitive Screening Test (CST), based on the Short Portable Mental Status Questionnaire (r = 0.53). Tests of executive functioning (Expanded Mental Control Test, Category Fluency, Clock Drawing) did not add significantly to the explanation of variance in VAT-scores.Fifty-five participants (31.6%) were faced with initial problems in naming or identifying one or more objects on the cue cards or association cards. If necessary, naming was aided by the investigator. Initial difficulties in identifying cue objects were associated with lower VAT-scores, but this did not hold for difficulties in identifying target objects.A hierarchical multiple regression analysis was used to examine whether linear or quadratic trends best fitted VAT performance across the range of CST scores. The regression model revealed a linear but not a quadratic trend. The best fitting linear model implied that VAT scores differentiated between CST scores in the lower, as well as in the upper range, indicating the absence of floor and ceiling effects, respectively. Moreover, the VAT compares favourably to word list-learning tasks being more attractive in its presentation of interacting visual objects and cued recall based on incidental learning of the association between cues and targets.For practical purposes and based on documented sensitivity and specificity, Bayesian probability tables give predictive power of age-specific VAT cutoff scores for the presence or absence of a major neurocognitive disorder across a range of a priori probabilities or base rates.     

Withdrawal of long-term diuretic medication in elderly patients: a double blind randomised trial.

OBJECTIVES: About 20% of elderly people use long-term diuretic medication, but there is doubt whether prolonged diuretic medication on such a large scale is necessary. We performed a study to assess what proportion may successfully be withdrawn from diuretic therapy. DESIGN: Double blind randomised controlled trial with six month follow up. SETTING: General practice. SUBJECTS: 202 patients taking long-term diuretics without manifest heart failure or hypertension. INTERVENTIONS: Patients were allocated to either placebo (withdrawal group, n = 102) or continuation of diuretic treatment (control group, n = 100). MAIN OUTCOME MEASURE: Occurrence of clinical conditions requiring diuretic therapy based on fixed criteria. RESULTS: During follow up diuretic therapy was required in 50 patients in the withdrawal group and 13 in the control group (risk difference 36%; 95% confidence interval 22% to 50%). Heart failure was the most frequent cause of prescribing diuretic therapy (n = 25). Cessation of diuretic therapy caused a mean increase in systolic blood pressure of 13.5 (9.2 to 17.8) mm Hg and in diastolic pressure of 4.6 (1.9 to 7.3) mm Hg. CONCLUSION: Withdrawal of long-term diuretic treatment in elderly patients leads to symptoms of heart failure or increase in blood pressure to hypertensive values in most cases. Any attempt to withdraw diuretic therapy requires careful monitoring conditions, notably during the initial four weeks.