Assessing combinations of cytotoxic agents using leukemia cell lines

The mainstay of clinical anti-neoplastic chemotherapy is multi-agent combinations, most of which were developed empirically. To speed research and decrease costs, there is increasing interest in moving new drugs into clinical trials in potentially active combinations based upon pre-clinical testing data. Because testing drug combinations in animals is expensive and complex, defining drug combinations initially in cell culture assays is essential. For in vitro testing we employ a panel of well-characterized cell lines and DIMSCAN, a semi-automatic fluorescence-based digital image microscopy system that quantifies relative total (using a DNA stain) or viable [using fluorescein diacetate (FDA)] cell numbers in tissue culture multi-well-plates ranging from 6 to 384 wells per plate. DIMSCAN is a rapid and efficient tool for conducting in vitro cytotoxicity assays across a 4 log dynamic range. The specificity of detecting viable cells with FDA is achieved by use of digital image processing and chemical quenching of fluorescence in non-viable cells with eosin Y. Cytotoxicity measured by DIMSCAN was found to be comparable to manual trypan blue dye exclusion counts or colony formation in soft agar, but with a significantly wider dynamic range, that enables drug combination studies used to detect synergistic or antagonistic interactions in cell lines from both solid tumors and leukemias. While different mathematical models have been proposed for evaluating drug interactions, which can be classified as synergistic (combinations demonstrating greater than the additive activity expected from each agent alone), additive, or antagonistic (drugs showing less activity in combination than expected from the sum of each agent alone), we generally find the Combination Index method (as developed by Chou, et al.) to be the most suitable for evaluating of drug interactions in cell culture assays.     

Multiple OXPHOS deficiency in the liver of a patient with CblA methylmalonic aciduria sensitive to vitamin B12

Summary  An adult patient with methylmalonic aciduria due to defective cobalamin synthesis (CblA) responsive to vitamin B₁₂ presented suddenly with severe visual impairment ascribed to optic atrophy followed by a fatal multiorgan failure and lactic acidosis but low methylmalonic acid in plasma and urine. Multiple deficiency of oxidative phosphorylation was found in the patient’s liver. We suggest that patients with B₁₂-sensitive methylmalonic aciduria who have a milder clinical course should be carefully monitored for long-term complications. Competing interests: None declared References to electronic databases: Methylmalonic aciduria (MMA): OMIM 251000, 277400, 251100 (CblA), 277410 (CblD), 251110 (CblB), 277380, 606169. Adenosylcobalamin: EC 2.7.7.62. Methylmalonyl-coenzyme A mutase (MUT): EC 5.4.99.2. Citrate synthase: EC 2.3.3.1. Succinate-CoA ligase (GDP-forming): EC 6.2.1.4. Succinate-CoA ligase (ADP-forming): EC 6.2.1.5. Succinate dehydrogenase (ubiquinone): EC 1.3.5.1. Fumarate hydratase: EC 4.2.1.2. ATP citrate synthetase: EC 2.3.3.8. Pyruvate dehydrogenase: EC 1.2.4.1. Pyruvate carboxylase: EC 6.4.1.1. Nucleoside-diphosphate kinase: EC 2.7.4.6. Presented at the Annual Symposium of the SSIEM, Lisbon, Portugal, 2–5 September 2008.     

Pharmacologic rationale for early G-CSF prophylaxis in cancer patients and role of pharmacogenetics in treatment optimization

The use of recombinant human granulocyte colony stimulating factors (G-CSF) has become an integral part of supportive care during cytotoxic chemotherapy. Current guidelines recommend the use of G-CSF in patients with substantial risk of febrile neutropenia. However, little consensus exists about optimal timing and tailoring of this therapy. Based on the known effects of chemotherapy and G-CSF on bone marrow compartments, we propose a model that supports the prophylactic rather than therapeutic use of G-CSF therapy. In addition, several genetic alterations in G-CSF signalling pathway have been described. These genetic variants may predict the risk of febrile neutropenia and response to G-CSF. Thus, future pharmacogenetic/omics studies in this field are warranted. Through the identification of patients at risk and the knowledge of biological basis for optimal timing, hopefully we should soon be able to improve the application of the existing guidelines for G-CSF therapy and patient's prognosis.     

An angiographic evaluation of restenosis rate at a six-month follow-up of patients with ST-elevation myocardial infarction submitted to primary percutaneous coronary intervention

BACKGROUND: Percutaneous coronary intervention (PCI) is considered to be the optimal type of revascularization in patients with ST-segment elevation myocardial infarction (STEMI). However, the long-term effectiveness of this procedure can be reduced by restenosis. This investigation was aimed at a prospective evaluation, in a group of STEMI patients of "the real world" (not involved in randomised trials), of the angiographic restenosis rate at a 6-month follow-up, and at identifying the relationship between restenosis and the patients' characteristics. MATERIALS AND METHODS: Our study population consisted of 123 patients with STEMI submitted to primary PCI to then undergo stress echocardiography 3 months after PCI and an angiographic evaluation at a 6-month follow-up. RESULTS: a) In real life the restenosis rate is quite high (42.3%); b) no correlation was found between patients' clinical characteristics and restenosis; c) restenosis rate was higher in patients with bare metal stents than in those with drug-eluting stents (55.8% vs. 11.1%; p<0.001); in patients with longer stents (21.6+/-8.62 vs 18.1+/-6.34 mm, p=0.015) and when more than one stent was implanted. Moreover, a consistent number of patients showed restenosis though asymptomatic. CONCLUSIONS: Our data suggest that primary PCI is associated with a high incidence of angiographic restenosis. No correlation was found between patients' clinical characteristics and restenosis. The length and the number of implanted stents seem to be associated with a more probable restenosis at six-month angiographic evaluation. Drug-eluting stent implantation seems to be associated with a lower incidence of restenosis even in STEMI patients.     

Did Duty Hour Reform Lead to Better Outcomes Among the Highest Risk Patients?

Background

Earlier work demonstrated that ACGME duty hour reform did not adversely affect mortality, with slight improvement noted among specific subgroups.

Objective

To determine whether resident duty hour reform differentially affected the mortality risk of high severity patients or patients who experienced post-operative complications (failure-to-rescue).

Design

Observational study using interrupted time series analysis with data from July 1, 2000 - June 30, 2005. Fixed effects logistic regression was used to examine the change in the odds of mortality or failure-to-rescue (FTR) in more versus less teaching-intensive hospitals before and after duty hour reform.

Participants

All unique Medicare patients (n = 8,529,595) admitted to short-term acute care non-federal hospitals and all unique VA patients (n = 318,636 patients) with principal diagnoses of acute myocardial infarction, congestive heart failure, gastrointestinal bleeding, stroke or a DRG classification of general, orthopedic or vascular surgery.

Measurements and Main Results

We measured mortality within 30 days of hospital admission and FTR, measured by death among patients who experienced a surgical complication. The odds of mortality and FTR generally changed at similar rates for higher and lower risk patients in more vs. less teaching intensive hospitals. For example, comparing the mortality risk for the 10% of Medicare patients with highest risk to the other 90% of patients in post-reform year 1 for combined medical an OR of 1.01 [95% CI 0.90, 1.13], for combined surgical an OR of 0.91 [95% CI 0.80, 1.04], and for FTR an OR of 0.94 [95% CI 0.80, 1.09]. Findings were similar in year 2 for both Medicare and VA. The two exceptions were a relative increase in mortality for the highest risk medical (OR 1.63 [95% CI 1.08, 2.46]) and a relative decrease in the high risk surgical patients within VA in post-reform year 1 (OR 0.52 [95% CI 0.29, 0.96]).

Conclusions

ACGME duty hour reform was not associated with any consistent improvements or worsening in mortality or failure-to-rescue rates for high risk medical or surgical patients.KEY WORDS: medical errors internship and residency, education, medical, graduate, personnel staffing and scheduling, continuity of patient care     

Markers of inflammation, thrombosis and endothelial activation correlate with carotid IMT regression in stable coronary disease after atorvastatin treatment

Background and aimsMIAMI is a prospective multicenter clinical study designed to investigate the relationship between changes in carotid intima-media thickness (C-IMT) and changes in circulating markers of inflammation, thrombosis and endothelial activation in stable coronary patients treated for 20 ± 3.7 months with 20 mg/day atorvastatin.Methods and resultsEighty-five subjects had their C-IMT, blood lipids and soluble markers measured at baseline, at the 12th month and at the end of the study. Almost all soluble markers decreased upon treatment except for high-sensitivity C-reactive protein (hs-CRP), interleukin-18 (IL-18), tissue factor pathway inhibitor-free (TFPI-free) and soluble vascular cell adhesion molecules-1 (sVCAM-1) which did not change significantly, and interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and soluble CD40 ligand (sCD40L) which increased. sCD40L, fibrinogen, tissue factor pathway inhibitor-total (TFPI-total), soluble intercellular adhesion molecules-1 (sICAM-1), sE-selectin, interleukin-8 (IL-8) and von Willebrand factor (vWF) changed significantly even after application of the Bonferroni correction for multiple comparisons. Changes in lipids did not correlate with C-IMT regression either when considered singly or when combined in a lipid score. Changes in soluble markers correlated poorly with C-IMT regression when analyzed singly, but strongly when combined in relevant composite scores (inflammation/coagulation score, endothelial activation score, soluble markers score and total score).ConclusionIn patients with stable coronary artery disease treated with moderate doses of atorvastatin, carotid IMT regression correlated with changes of inflammation, thrombosis and endothelial activation profiles.     

Aminoindazole PDK1 Inhibitors: A Case Study in Fragment-Based Drug Discovery

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Intrahippocampal LSD accelerates learning and desensitizes the 5-HT2A receptor in the rabbit, Romano et al.

Rationale  

Parenteral injections of d-lysergic acid diethylamide (LSD), a serotonin 5-HT_2A receptor agonist, enhance eyeblink conditioning. Another hallucinogen, (±)-1(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), was shown to elicit a 5-HT_2A-mediated behavior (head bobs) after injection into the hippocampus, a structure known to mediate trace eyeblink conditioning.     

Mycobacterium tuberculosis Beijing Lineage Favors the Spread of Multidrug-Resistant Tuberculosis in the Republic of Georgia

High rates and transmission of multidrug-resistant (MDR) tuberculosis (TB) have been associated with the Mycobacterium tuberculosis complex (MTBC) Beijing lineage, pointing to the importance of pathogen genetic factors for the modulation of infection outcome and epidemiology. We present here an in-depth analysis of the population structure of MTBC strains from the Republic of Georgia, a high-incidence setting at the Black Sea Coast. Phylogenetic lineages were identified based on 24-locus MIRU-VNTR (for mycobacterial interspersed repetitive unit-variable number tandem repeat) and spoligotyping analysis. Clusters of strains with identical genotyping profiles were determined as an indicator for the rate of recent transmission. Among the 183 M. tuberculosis isolates investigated, the most prominent lineage found was Beijing (26%), followed by the LAM (18%), Ural (12%), and Haarlem (5%) strains. A closely related previously undefined phylogenetic group (62 strains) showed a genotyping pattern similar to laboratory strain H37RV and was denominated as “Georgia-H37RV-like.” Although isoniazid resistance was found among strains of different lineages, MDR TB was nearly completely restricted to Beijing strains (P < 0.0001). Approximately 50% of the isolates were grouped in clusters, indicating a high rate of recent transmission. Our data indicate that, in addition to the confirmation of the importance of Beijing genotype strains for the TB epidemiology in former Soviet Union countries, a high-population diversity with strains of the LAM, Ural, Haarlem, and a previously undefined lineage represents nearly two-thirds of the strains found in Georgia. Higher rates among previously treated and MDR TB patients point to a higher potential of lineage Beijing to escape therapy and develop MDR TB.Drug-resistant Mycobacterium tuberculosis complex (MTBC) strains have emerged worldwide as a serious threat for tuberculosis (TB) control. Rates of multidrug-resistant (MDR) strains (i.e., resistance at least to isoniazid [INH] and rifampin [RIF]) have reached levels of up to 14% among patients never treated and up to 40% among previously treated patients in several MDR TB “hot spots” such as such as Karakalpakstan (Uzbekistan) and Kazakhstan in Eastern Europe (10, 37). Every year an estimated 489,000 cases of MDR TB arise globally (36). MDR TB is associated with much poorer treatment outcomes than for drug-susceptible TB, with a much higher risk of developing further resistances (2, 7). Prolonged periods of infectivity result in enhanced transmission of drug-resistant strains, further accelerating the rates of drug resistance (3). Even more worrisome is the emergence of a nearly untreatable form of TB, namely, extensively drug-resistant TB (XDR TB), which is defined as MDR plus additional resistance to any fluoroquinolone and at least one of three injectable drugs (i.e., amikacin, kanamycin, or capreomycin). A recent survey confirmed the worldwide presence of XDR strains, with rates of up 15% of MDR TB cases (30).Considering the difficulties and problems associated with the treatment of resistant TB, high levels of MDR and XDR TB have the clear potential to jeopardize TB control on a local or national level. In addition to various measures for strengthening TB control such as rapid case detection, proper treatment, and rapid detection of drug resistance, the long-term effect of the emergence of drug-resistant strains on the worldwide TB epidemic also depends on the relative fitness of the MDR and XDR strains compared to susceptible strains (3).In clinical MTBC strains, drug resistance results from chromosomal mutations in particular genes that confer resistance, which might also have an effect on bacterial fitness (5, 38). Although initial experiments indicated a lower fitness of e.g., INH-resistant strains, recent results confirm that the fitness of resistant strains depends on the kind of mutations, as well on the strain''s genetic background (13). Furthermore, the initial adverse effects on bacterial fitness might be reversed by compensatory mutations occurring during long-term infection and ongoing transmission. In fact, MDR variants have been described to have even an enhanced fitness compared to susceptible progenitor strains (13). If these figures are used in models for prediction of the MDR TB epidemic, it turns out that even in the case of a well-functioning TB control system, small subpopulations of comparatively fit MDR clones might outcompete susceptible and less-fit resistant strains and become the dominant clones in future with dramatic consequences for TB treatment and control (5).The most striking association between a mycobacterial genetic background and drug resistance documented thus far has been described for strains of the so-called Beijing lineage. These strains have been found to be involved in outbreaks and the transmission of MDR TB in several areas of the world (15). In Eastern Europe, a rising number of studies report a clear association between Beijing genotype infection and drug resistance (8, 11, 15, 28). Furthermore, large clusters of dominant clones have been determined that might indicate the development of “highly transmissible” MDR Beijing strains circulating in the community (28). Similar observations have been recently reported from South Africa, where a rapidly spreading highly resistant clone represents nearly half of all cases in the George subdistrict (35).However, the overall picture of the correlation between bacterial genotype disease characteristics is incomplete. The majority of studies focused on particular strain types such as the Beijing genotype only, basically, because they are easy to recognize by applying genotyping techniques such as IS6110 DNA fingerprint and spoligotyping (34). Based on these markers, a variety of strains were not classifiable into phylogenetic lineages or clonal complexes since the genotyping information was not informative, e.g., due to homoplasy (4, 6). Therefore, the presence of particular genotypes might simply be overlooked and, consequently, the association with clinical characteristics could not be investigated or false associations have been obtained. Since this question is of scientific and public health relevance, further studies addressing the population structure of the MTBC applying more appropriate genetic markers are urgently needed. More recently, a new genotyping techniques based on mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing was developed that allows the simultaneous high-resolution discrimination of clinical isolates for epidemiological studies and a valid phylogenetic strain classification (26, 31).In the present study, we used MIRU-VNTR typing and spoligotyping to investigate the population structure of strains obtained from patients living in the Republic of Georgia, where high rates of MDR TB have been recently reported (14, 20). We specifically analyzed the association between Beijing genotype and drug resistance. Furthermore, the data have been used to classify determine the whole variety of strains circulating in Georgia and to describe new clonal complexes and/or phylogenetic lineages. Phylogenetic strain classifications have been correlated with clinical characteristics.