Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay.

The methyl-thiazol-tetrazolium (MTT) assay is a drug resistance assay which cannot discriminate between malignant and non-malignant cells. We previously reported that samples with > or = 80% leukaemic cells at the start of culture give similar results in the MTT assay and the differential staining cytotoxicity assay, in which a discrimination between malignant and non-malignant cells can be made. However, the percentage of leukaemic cells may change during culture, which might affect the results of the MTT assay. We studied 106 untreated childhood acute lymphoblastic leukemia (ALL) samples with > or = 80% leukaemic cells at the start of culture. This percentage decreased below 80% in 28%, and below 70% in 13%, of the samples after 4 days of culture. A decrease below 70% occurred more often in case of 80-89% leukaemic cells (9/29) than in case of > or = 90% leukaemic cells at the start of culture (5/77, P = 0.0009). Samples with < 70% leukaemic cells after culture were significantly more resistant to 6 out of 13 drugs, and showed a trend towards being more resistant to two more drugs, than samples with > or = 80% leukaemic cells. No such differences were seen between samples with 70-79% and samples with > or = 80% leukaemic cells after culture. We next studied in another 30 ALL samples whether contaminating mononuclear cells could be removed by using immunoamagnetic beads. Using a beads to target cell ratio of 10:1, the percentage of leukaemic cells increased from mean 72% (s.d. 9.3%) to mean 87% (s.d. 6.7%), with an absolute increase of 2-35%. The recovery of leukaemic cells was mean 82.1% (range 56-100%, s.d. 14.0%). The procedure itself did not influence the results of the MTT assay in three samples containing only leukaemic cells. We conclude that it is important to determine the percentage of leukaemic cells at the start and at the end of the MTT assay and similar drug resistance assays. Contaminating mononuclear cells can be successfully removed from ALL samples using immunomagnetic beads. This approach may increase the number of leukaemic samples which can be evaluated for cellular drug resistance with the MTT assay or a similar cell culture drug resistance assay

Orientational order in dipolar fluids consisting of nonspherical hard particles

We investigate fluids of dipolar hard particles by a certain variant of density-functional theory. The proper treatment of the long range of the dipolar interactions yields a contribution to the free energy which favors ferromagnetic order. This corrects previous theoretical analyses. We determine phase diagrams for dipolar ellipsoids and spherocylinders as a function of the aspect ratio of the particles and their dipole moment. In the nonpolar limit the results for the phase boundary between the isotropic and nematic phase agree well with simulation data. Adding a longitudinal dipole moment favors the nematic phase. For oblate or slightly elongated particles we find a ferromagnetic liquid phase, which has also been detected in computer simulations of fluids consisting of spherical dipolar particles. The detailed structure of the phase diagram and its evolution upon changing the aspect ratio are discussed in detail.Comment: 35 pages LaTeX with epsf style, 11 figures in eps format, submitted to Phys. Rev.

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: A systematic analysis for the Global Burden of Disease Study 2015

Background: The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods: We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors—the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings: Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57·8% (95% CI 56·6–58·8) of global deaths and 41·2% (39·8–42·8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211·8 million [192·7 million to 231·1 million] global DALYs), smoking (148·6 million [134·2 million to 163·1 million]), high fasting plasma glucose (143·1 million [125·1 million to 163·5 million]), high BMI (120·1 million [83·8 million to 158·4 million]), childhood undernutrition (113·3 million [103·9 million to 123·4 million]), ambient particulate matter (103·1 million [90·8 million to 115·1 million]), high total cholesterol (88·7 million [74·6 million to 105·7 million]), household air pollution (85·6 million [66·7 million to 106·1 million]), alcohol use (85·0 million [77·2 million to 93·0 million]), and diets high in sodium (83·0 million [49·3 million to 127·5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation: Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Funding: Bill & Melinda Gates Foundation

Cellular drug resistance in childhood leukemia

The response to chemotherapy is determined essentially by two factors: first, pharmacokinetic factors, determining which concentration of drug reaches the malignant cells, and second, cellular drug resistance of these cells, determining how many of them will be killed by that concentration of drug. The study of cellular drug resistance has been stimulated by the development of short-term 'total cell kill' assays, such as the MTT assay, for use on patient samples. The drug resistance profiles differed markedly between ALL and ANLL, between immunophenotypic and karyotypic subgroups within ALL, and between initial and relapsed ALL. The results of the MTT assay showed a significant relation between the antileukemic activity of prednisolone in vitro and the clinical response to systemic monotherapy with that drug. At multivariate analysis including several well-known prognostic factors (WBC, age, immunophenotype) only the in vitro resistance to prednisolone, dexamethasone, l-asparaginase, and daunorubicin was significantly related to clinical outcome. At multiple regression analysis, combination of the results for prednisolone, l-asparaginase, and vincristine made it possible to distinguish between three patient groups with increasing levels of drug resistance and markedly different probabilities of 2-year disease-free survival: 100%, 83%, and 60%. These results show that in vitro drug resistance testing can give a correct prediction of prognosis, superior to that of currently used prognostic factors. Stratification of prognostic groups based on the results of drug resistance testing is feasible and should be introduced into new clinical trials. Many questions now remaining could be answered within carefully designed preclinical and clinical studies

Glucocorticoid resistance in childhood leukemia

Glucocorticoids (GC) are being used in the treatment of childhood leukemia for several decades, most successfully in newly diagnosed acute lymphoblastic leukemia (ALL). However, GC resistance is seen in 10-30% of untreated ALL patients, and is much more frequent in relapsed ALL and in acute nonlymphoblastic leukemia (ANLL). Sensitivity or resistance to GC can be measured using a cell culture drug resistance assay. For this purpose, we use the colorimetric methyl-thiazol-tetrazolium (MTT) assay. We have shown that GC resistance in childhood leukemia is related to clinical and cell biological features, and to the clinical outcome after multi-drug chemotherapy. These results are summarized in this review. In addition, we describe the apoptotic 'cell-lysis pathway' by which GC exert their antileukemic activity. This description provides a model to discuss the mechanisms of GC resistance, and to summarize the relevant literature. Possible levels of resistance relate to the diffusion of GC through the cell membrane, binding to the GC receptor (GCR), activation of the GC-GCR complex, translocation of the complex into the nucleus, binding to DNA, endonuclease-mediated DNA fragmentation, and DNA repair. A low number of GCR has been shown to be the cause of resistance in some children with ALL. However, GC resistance is likely to be caused at the post-receptor level in most leukemias. Unfortunately, there is still a lack of knowledge relating to the clinical relevance of mechanisms of GC resistance at the post-receptor level. Studies on the mechanisms of GC resistance other than those directly related to the GCR should be initiated, especially if patient material is used, as the results might indicate ways to circumvent or modulate GC resistance. A further increase in our knowledge regarding the relation between GC resistance and patient and cell biological features, the clinical relevance of GC resistance, and the mechanisms of GC resistance in leukemia patients, may contribute to further improvement in the results of GC therapy in leukemia

Clinical and cell biological features related to cellular drug resistance of childhood acute lymphoblastic leukemia cells

Several clinical and cell biological features, such as sex, age, leukemic cell burden, morphologic FAB type, and immunophenotype, have prognostic value in childhood acute lymphoblastic leukemia (ALL). The explanation for their prognostic significance is unclear, but might be related to cellular drug resistance. We prospectively studied the relation between the above mentioned features with resistance to 13 drugs in 144 childhood ALL samples obtained at initial diagnosis. The MTT assay was used for drug resistance testing. The interindividual differences in drug resistance were very large and exceeded those between the several subgroups. There was generally no significant relation between sex, leukemic cell burden, and FAB type with drug resistance. However, subgroups with a worse prognosis as defined by age ( 120 months at diagnosis) or immunophenotype (pro-B ALL and T-ALL) did show relatively resistant drug resistance profiles as compared to the subgroups with a better prognosis (age 18-120 months, common and pre-B ALL). Within the group of common and pre-B ALL and compared to the intermediate age-group, samples of the younger children were significantly more resistant to daunorubicin, mitoxantrone and teniposide, and samples of the older children were significantly more resistant to prednisolone and mercaptopurine. Pro-B ALL samples were significantly more resistant to I-asparaginase and thioguanine, and T-ALL samples were significantly more resistant to prednisolone, dexamethasone, I-asparaginase, vincristine, vindesine, daunorubicin, doxorubicin, teniposide, and ifosfamide, than the group of common and pre-B ALL cases. We conclude that the prognostic significance of age and immunophenotype in particular may be explained, at least partly, by its relation with resistance to certain drugs. The results of this study may be useful for future rational improvements of chemotherapeutic regimens in childhood ALL

Favorable prognosis of hyperdiploid common acute lymphoblastic leukemia may be explained by sensitivity to antimetabolites and other drugs: Results of an in vitro study

DNA hyperdiploidy is a favorable prognostic factor in childhood acute lymphoblastic leukemia (ALL). The explanation for this prognostic significance is largely unknown. We have studied whether DNA ploidy was related to cellular resistance to 12 drugs, assessed with the methyl-thiazol- tetrazolium assay, in samples of 74 children with common (CD10+ precursor B- cell) ALL. Sixteen patients had hyperdiploid ALL cells and 58 patients had nonhyperdiploid ALL cells. Hyperdiploid ALL cells were more sensitive to mercaptopurine (median, 9.0-fold; P = .000003), to thioguanine (1.4-fold; P = .023), to cytarabine (1.8-fold; P = .016), and to I-asparaginase (19.5-fold; P = .022) than were nonhyperdiploid ALL cells. In contrast, these two ploidy groups did not differ significantly in resistance to prednisolone, dexamethasone, vincristine, vindesine, daunorubicin, doxorubicin, mitoxantrone, and teniposide. The percentage of S-phase cells was higher (P = .05) in the hyperdiploid ALL samples (median, 8.5%) than in the nonhyperdiploid ALL samples (median, 5.7%). However, the percentage of cells in S-phase was not significantly related to in vitro drug resistance. We conclude that the favorable prognosis associated with DNA hyperdiploidy in childhood common ALL may be explained by a relative sensitivity of hyperdiploid common ALL cells to antimetabolites, especially to mercaptopurine and to I-asparaginase