Dendritic cells in bone marrow at diagnosis and after chemotherapy in adult patients with acute myeloid leukaemia.

Dendritic cells (DCs) develop in the bone marrow from haematopoietic progenitor cells. Two subsets, plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), have been identified. Little is known regarding DC levels in bone marrow of patients with acute myeloid leukaemia (AML) before and after chemotherapy. We investigated relative pDC and mDC levels in bone marrow from 37 hospital controls and 60 patients with AML [at diagnosis, complete remission (CR) and follow-up] using four-colour flow cytometry. The pDC immunophenotype was characterized as lin-/HLA-DR+/CD123 +  and mDC as lin-/HLA-DR+/CD11c+. In 69% of patients with AML, no DCs were detected at diagnosis. At CR, mDC levels were the same in patients with AML and hospital controls while pDC levels were slightly lower. There was no association between minimal residual disease or survival rates and DC levels. Patients with low mDC levels at CR were more likely to suffer from complicated infections, although the difference was not statistically significant. Altogether, there was a profound decrease in DC levels in patients with AML at diagnosis. DC levels increased at CR and were higher than in hospital controls after post-remission therapy, suggesting that DCs recover after repeated chemotherapy. There may be an association between mDC levels and infectious complications.

Growth, carcass characteristics, and profitability of organic versus conventional dairy beef steers.

Bull calves (n=49), born at the University of Minnesota West Central Research and Outreach Center (Morris) between March and May 2011, were used to compare growth measurements and profitability of conventional and organic dairy steers. Calves were assigned to 1 of 3 replicated groups at birth: conventional (CONV; n=16), organic (pasture and concentrate; ORG; n=16), or organic grass only (GRS; n=17), and analysis of variables was on a pen basis. Breed groups of calves were Holstein (HO; n=9); Holsteins (n=11) maintained at 1964 breed average level; crossbreds (n=19) including combinations of HO, Montbéliarde, and Swedish Red; and crossbreds (n=10) including combinations of HO, Jersey, Swedish Red, and Normande. The CONV steers were fed a diet of 80% concentrate and 20% forage. The ORG steers were fed a diet of organic corn, organic corn silage, and at least 30% of their diet consisted of organic pasture during the grazing season. The GRS steers grazed pasture during the grazing season and were fed high-quality hay or hay silage during the nongrazing season. Intakes of a total mixed ration were recorded daily with herd management software. A profit function was defined to include revenues and expenses for beef value, feed intake, pasture intake, health cost, and yardage. The GRS (358.6 kg) steers had lesser total gains from birth to slaughter than ORG (429.6 kg) and CONV (534.5 kg) steers. Furthermore, the GRS (0.61 kg/d) steers had lesser average daily gain from birth compared with ORG (0.81 kg/d) and CONV (1.1 kg/d) steers. The GRS and ORG steers had smaller rib eye area (49.5 and 65.8 cm(2), respectively) compared with CONV (75.4 cm(2)) steers. For profitability, GRS steers had 43% greater profit than CONV steers due to organic beef price premiums and lower feed costs. On the other hand, ORG steers had substantially less profit than CONV steers. The higher cost of production for the ORG steers is due to the extreme high value of organic corn. The results of the current study illustrate the economic potential of alternative strategies for growing and marketing male offspring of organic dairy cattle in the Midwest.

Fatty acid profiles, meat quality, and sensory attributes of organic versus conventional dairy beef steers.

Meat from Holstein and crossbred organic and conventional dairy steers were evaluated and compared for fatty acid profiles, meat quality, sensory attributes, and consumer acceptance. Bull calves (n=49) were randomly assigned to 1 of 3 replicated groups: conventional (CONV), organic (ORG, pasture + concentrate), or grass-fed organic (GRS) and were born at the University of Minnesota West Central Research and Outreach Center (Morris, MN) between March and May 2011. The CONV steers (n=16) were fed a diet that contained 80% concentrate and 20% forage, and ORG steers (n=16) were fed a diet of organic corn, organic corn silage, and organic protein supplement. Furthermore, ORG steers consumed at least 30% of diet dry matter of high-quality organic pasture during the grazing season. The GRS steers (n=17) consumed 100% forage from pasture during the grazing season and high-quality hay or hay silage during the nongrazing season. The ORG steers had fat that was greater in oleic acid (C18:1) than the GRS and CONV steers (47.1, 36.1, and 39.9%, respectively). The GRS steers (21.9%) were lower for monounsaturated fat than the ORG (42.1%) and CONV (40.4%) steers. Furthermore, the GRS steers tended to have greater n-3 fat and had lower n-6 fat than the ORG and CONV steers. Consequently, the GRS (1.4%) steers had a lower n-6-to-n-3 fat ratio than the ORG (12.9%) and CONV (10.0%) steers. The GRS (2.6 kg) steers had steaks that were not different for Warner-Bratzler shear force than ORG (2.3 kg) steaks; however, the GRS steaks tended to have greater shear force than the CONV (2.0 kg) steaks. The 3 steer group had steaks that were not different for color brightness (L*; 0 = black and 100 = white) and yellowness/blueness (b*; positive values = yellow and negative values = blue) values; however, the GRS (10.5) steaks had lower redness/greenness (a*; positive values = red and negative values = green) values than CONV (14.5) steaks. For sensory attributes (0- to 120-point scale), no differences were observed for ORG (71.3) and CONV (69.2) steers for overall consumer liking of the beef; however, the GRS (56.3) steers had the lowest overall liking among beef consumers. The ORG (73.3) steers had greater flavor liking than the GRS (56.8) and CONV (69.2) steers. Conversely, the GRS (6.3) steers had the highest scores for off-flavor (0- to 20-point scale) compared with the ORG (3.9) and CONV (4.1) steers. The results of the current study suggest that a potential market may exist for organic grass-fed dairy steers in the United States, but quality and consistency of the beef needs to be improved.

Whole-milk feeding duration, calf growth, and profitability of group-fed calves in an organic production system.

The objective of this study was to evaluate the effect of early-life feeding duration on growth and economics of group-fed organic dairy calves. Heifer calves born during the spring of 2011 (n = 67) and the spring of 2012 (n = 57) were used to evaluate the effect of weaning age, growth, and profitability of group-fed calves fed once per day in an organic dairy production system. Calves were assigned to replicate feeding groups of 10 in super hutches by birth order, and were born at the University of Minnesota West Central Research and Outreach Center, Morris organic dairy. Breed groups were Holsteins (n = 15) selected for high production, Holsteins (n = 23) maintained at 1964 breed-average level, crossbreds (n = 54) including combinations of Holstein, Montbéliarde, and Swedish Red, and crossbreds (n = 32) including combinations of Holstein, New Zealand Friesian, Jersey, and Swedish Red. Groups of calves were weaned at 30 (EW, early weaning), 60 (MW, mid weaning), or 90 (LW, late weaning) d of age, and groups were fed 1.5% of birth weight of 13% total solids organic whole milk once daily and weaned when the group of 10 calves consumed an average of 0.91 kg of organic calf starter per calf per day for 4 consecutive days. Body measurements were recorded at birth, weekly during the preweaning period, at weaning, and monthly thereafter. Profitability was estimated as a function of the total cost for organic milk and organic calf starter for weaning groups to weaning and to the first 90 d of age. Preweaning group performance was weaning age, EW: 47.6d, MW: 64.5d, LW: 93.7d; weaning weight, EW: 61.8 kg, MW: 79.2 kg, LW: 108.1 kg; and gain per day, EW: 0.51 kg/d, MW: 0.63 kg/d, LW: 0.75 kg/d. Body weight (BW) did not differ among weaning groups at 90 d of age; however, MW calves had lower 120-d BW than did LW calves. The EW calves did not differ from either MW or LW calves for 120-d BW. Total feed costs to weaning for groups were $1,092.97 for EW calves, $1,871.24 for MW calves, and $2,956.64 for LW calves. The cost per kilogram of gain was higher for the EW ($5.54) group of calves than for the MW ($4.60) or LW ($4.14) groups during the preweaning period. Total costs and cost per kilogram of gain for the first 90 d of age were lowest for EW ($1,595.59, $3.02) calves and highest for LW ($2,956.64, $4.13) calves, respectively.

Inhibition of monoacylglycerol lipase by troglitazone, N-arachidonoyl dopamine and the irreversible inhibitor JZL184: comparison of two different assays.

BACKGROUND AND PURPOSE: Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays. EXPERIMENTAL APPROACH: MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated. KEY RESULTS: Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC(50) values of 1.1, 4.9, 0.78 and 3.1µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ(12,14) -prostaglandin J(2) ≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC(50) 41µM) than hydrolysis of NPA by the human MGL lysates. CONCLUSIONS AND IMPLICATIONS: 'Hits' in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators.

Development of an analytical method to determine avermectins in water, sediments and soils using liquid chromatography-tandem mass spectrometry.

A comprehensive analytical multi-residue method has been developed for the determination of seven avermectins (abamectin, doramectin, ivermectin, emamectin benzoate, eprinomectin, moxidectin and selamectin) in surface water, sediment and soil samples. Solid samples were extracted applying pressurised liquid extraction followed by a solid-phase extraction (SPE) clean-up step. For aqueous samples, extraction was done utilising only SPE. All compounds were measured using liquid chromatography coupled to tandem mass spectrometry using atmospheric pressure chemical ionisation. The recoveries were 38-67% (relative standard deviation: 9-26%), 63-88% (16-23%) and 63-80% (9-15%) for spiked Rhine water, spiked sediment and spiked soil samples, respectively, and limit of quantifications were 2.5-14 ngl(-1) in water and 0.5-2.5 ngg(-1) in soil and sediment.

Dendritic cell regeneration in the bone marrow of children treated for acute lymphoblastic leukaemia.

Dendritic cells (DC) play a pivotal role in coordinating functions of the immune system. Little is known about DC levels in the bone marrow (BM) of patients receiving cytostatic treatment. We investigated DC levels by flow cytometry in BM at diagnosis, during and post-treatment in 76 children with acute lymphoblastic leukaemia (ALL). The levels of both plasmacytoid DC (pDC) and myeloid DC (mDC) were profoundly reduced at diagnosis. However, the levels of pDC and mDC were significantly higher in T-precursor ALL patients when compared with B-precursor ALL patient group (P = 0.044 and 0.041 respectively). Both subsets normalized in both standard-risk (SR) and high-risk patients after the end of induction at day 50. Patients with minimal residual disease (MRD) at day 50 had significantly higher pDC levels than MRD-negative patients (P = 0.021). In B-precursor SR ALL patients, mDC levels but not pDC levels decreased during prolonged maintenance treatment, remaining reduced at the end of treatment (P = 0.032) and at 6 months post-treatment (P = 0.028). In conclusion, levels of DC in BM normalize quickly in children treated for ALL. Long-term treatment may more profoundly affect mDC subset, which shows reduced levels several months after treatment.

Discriminant function analysis as decision support system for the diagnosis of acute leukemia with a minimal four color screening panel and multiparameter flow cytometry immunophenotyping.

Despite several recommendations for standardization of multiparameter flow cytometry (MFC) the number, specificity and combinations of reagents used by diagnostic laboratories for the diagnosis and classification of acute leukemias (AL) are still very diverse. Furthermore, the current diagnostic interpretation of flow cytometry readouts is influenced arbitrarily by individual experience and knowledge. We determined the potential value of a minimal four-color combination panel of 13 monoclonal antibodies (mAbs) with a CD45/sideward light scatter-gating strategy for a standardized MFC immunophenotyping of the clinically most relevant subgroups of AL. Bone marrow samples from 155 patients with acute myeloid leukemia (AML, n=79), B-cell precursor acute lymphoblastic leukemia (BCP-ALL, n=29), T-cell precursor acute lymphoblastic leukemia (T-ALL, n=12) and normal bone marrow donors (NBMD, n=35) were analyzed. A knowledge-based learning algorithm was generated by comparing the results of the minimal panel with the actual diagnosis, using discriminative function analysis. Correct classification of the test sample according to lineage, that is, BCP-ALL, T-ALL, AML and differentiation of NBMD was achieved in 97.2% of all cases with only six of the originally applied 13 mAbs of the panel. This provides evidence that discriminant function analysis can be utilized as a decision support system for interpretation of flow cytometry readouts.

Shape-selective extraction of PCBs and dioxins from fish and fish oil using in-cell carbon fractionation pressurized liquid extraction.

This paper describes a new shape-selective, pressurized liquid extraction (PLE) procedure for extracting polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and PCBs from food and feed samples with an integrated carbon fractionation step. Initially this was done using specially designed inserts for 34-mL cells, but subsequently, large solid cells (66 mL) were machined to increase the capacity and robustness of the system. Depending on the carbon load and extraction solvent strength, the non-ortho PCBs were recovered either with the bulk of the PCBs or with the PCDD/Fs. The former is preferable if PCDD/Fs are the targets. In most cases, however, data are required for all indicator PCBs, WHO-PCBs, and PCDD/Fs. Therefore, further efforts focused on developing, optimizing, and validating a cost- and time-efficient PLE procedure that can extract these targets, separate non-ortho PCBs and PCDD/Fs from the bulk of the PCBs, allow gravimetric fat determinations, and requires a minimum of postextraction cleanup. The performance of the resulting procedure was assessed in experiments with a fish tissue reference material. The trueness of the WHO-PCB-TEQ, PCDD/F-TEQ, and total-TEQ data were -8, -5, and -7%, respectively, and the corresponding CVs were 1.5, 0.5, and 1.3%; within the limits set by the European community for gas chromatography-high-resolution mass spectrometry methods for food and feed control.

Quality assurance of the donation process.

The quality of the organs harvested from a deceased donor is of critical importance for the outcome of the transplantations. During 2005, a quality assurance project was initiated to evaluate the donor management, harvest operation, and flow of information during the donation process. Three kinds of questionnaires were used in each donation. They were completed by the transplant coordinator, the harvesting surgeon, and the surgeons performing the liver and kidney transplantations. Feedback is given to the harvesting teams within 2 weeks after the procedures. The most important findings related to missed information concerning organ abnormalities or organ damage from the procurement operation. Procurement of organs from a deceased donor involves a complex chain of events. Based on our experiences in this 1-year project, we believe that standardized registration of the various parts of the process and structured feedback to the staff give possibilities to improve performance. After minor modifications, this method for quality assurance has been introduced as a permanent part of our donation procedure. We believe that this strategy can help to detect weaknesses and improve transplant outcomes.

Changing donation pattern in central Sweden.

Since 1990, the Organisation for Organ donation in Central Sweden has registered the numbers of donations at the various hospitals in the area. During this period, a significant decrease in donation rate was observed in the large hospitals, while there was an increase in donation rate in the smaller hospitals. Taken together, the small hospitals are now at least as important as the large hospitals. Possible reasons for the observed change in donation pattern are discussed.

Admission N-terminal pro-brain natriuretic peptide and its interaction with admission troponin T and ST segment resolution for early risk stratification in ST elevation myocardial infarction.

OBJECTIVE: To assess the long term prognostic value of N-terminal pro-brain natriuretic peptide (NT-proBNP) on admission and its prognostic interaction with both admission troponin T (TnT) concentrations and resolution of ST segment elevation in fibrinolytic treated ST elevation myocardial infarction (STEMI). DESIGN AND SETTING: Substudy of the ASSENT (assessment of the safety and efficacy of a new thrombolytic) -2 and ASSENT-PLUS trials. PATIENTS: NT-proBNP and TnT concentrations were determined on admission in 782 patients. According to NT-proBNP concentrations, patients were divided into three groups: normal concentration (for patients < or = 65 years, < or = 184 ng/l and < or = 268 ng/l and for those > 65 years, < or = 269 ng/l and < or = 391 ng/l in men and women, respectively); higher than normal but less than the median concentration (742 ng/l); and above the median concentration. For TnT, a cut off of 0.1 microg/l was used. Of the 782 patients, 456 had ST segment resolution (< 50% or > or = 50%) at 60 minutes calculated from ST monitoring. MAIN OUTCOME MEASURES: All cause one year mortality. RESULTS: One year mortality increased stepwise according to increasing concentrations of NT-proBNP (3.4%, 6.5%, and 23.5%, respectively, p < 0.001). In receiver operating characteristic analysis, NT-proBNP strongly trended to be associated more with mortality than TnT and time to 50% ST resolution (area under the curve 0.81, 95% confidence interval (CI) 0.72 to 0.9, 0.67, 95% CI 0.56 to 0.79, and 0.66, 95% CI 0.56 to 0.77, respectively). In a multivariable analysis adjusted for baseline risk factors and TnT, both raised NT-proBNP and ST resolution < 50% were independently associated with higher one year mortality, whereas raised TnT contributed independently only before information on ST resolution was added to the model. CONCLUSION: Admission NT-proBNP is a strong independent predictor of mortality and gives, together with 50% ST resolution at 60 minutes, important prognostic information even after adjustment for TnT and baseline characteristics in STEMI.

Analysis of minimal residual disease in childhood acute lymphoblastic leukemia: comparison between RQ-PCR analysis of Ig/TcR gene rearrangements and multicolor flow cytometric immunophenotyping.

Detection of minimal residual disease (MRD) in follow-up samples from patients with ALL is essential for evaluation of treatment response. We applied multicolor flow cytometry and real-time quantitative PCR (RQ-PCR) to compare MRD results in 71 follow-up samples from 22 children treated for ALL. When results obtained by flow cytometry and RQ-PCR were grouped into positive-negative categories, a significant level of agreement was found in 72% of samples (P<0.001). However, if a cutoff level of 0.01% was applied, the concordance was 89%. MRD could be quantified in 19 samples by both methods, showing a strong correlation (P<0.01). Nevertheless, MRD levels differed more than five-fold between both methods in 4/19 samples. In 20 (28%) samples, the two techniques showed discordant results. Most discordant results (17/20) were due to the limited sensitivity of flow cytometry analysis within the range 0.01-0.001%; remaining discordant results were due to the instable or subclonal IG/TCR gene rearrangements or a limited quantitative range of the applied RQ-PCR targets. Although concordant results could be obtained by flow cytometry and RQ-PCR analysis, MRD levels may differ. Therefore, MRD data obtained by these two techniques are not yet easily exchangeable.

Flow cytometric follow-up of minimal residual disease in bone marrow gives prognostic information in children with acute lymphoblastic leukemia.

Using flow cytometry (FC) and live gate (LG) analysis we have followed levels of minimal residual disease (MRD) in the bone marrow (BM) of 70 consecutive patients with childhood acute lymphoblastic leukemia (59 B precursor ALL and 11 T-ALL) treated according to the Nordic (NOPHO-92) protocols. Thorough studies of B and T cell antigen expression patterns in normal BM performed during BIOMED 1 Concerted Action on MRD, made it possible to tailor individual protocols of marker combinations for follow-up in 97% of patients. In 12% of LG analyses, the numbers of cells exceeded 10(6) and in 82% exceeded 10(5), giving the sensitivity level of MRD detection 10(-5) and 10(-4), respectively. The median follow-up time was 53 months. Patients with MRD levels > or = 0.01% at follow-up time-points during and after first induction, and at the end of treatment had significantly lower disease-free survival by comparison to patients with MRD values <0.01%. Seven of nine patient with recurrence in the BM showed under treatment persisting MRD levels > or = 0.01% of BM cells. This was also observed in another two patients with infant leukemia who relapsed. In conclusion, the investigation of levels and the dynamics of MRD by sensitive and quantitative FC can provide a basis for further clinical studies for at least upgrading of therapy.

Alternative extraction methods for Zearalenone: Microwave Assisted Extraction and Accelerated Solvent Extraction.

Zearalenone (ZON) was extracted from wheat and corn by using Microwave Assisted Extraction and Accelerated Solvent Extraction. A factorial design approach was applied to evaluate the influence of the most important extraction parameters such as temperature, time and solvent extraction mixture on fortified cereals. ZON was determined by LC-MS without performing any clean-up after the extraction to better evaluate the extraction efficiency. The selected extraction conditions were tested on samples which had been previously used in an international proficiency test. Applying the selected extraction conditions both alternative extraction procedure provided satisfactory results comparable to the most commonly used method.

Comparison of fat retainers in accelerated solvent extraction for the selective extraction of PCBs from fat-containing samples.

Five types of fat retainers were investigated for the lipid-free extraction of PCBs from fat-containing matrixes using accelerated solvent extraction: florisil, basic alumina, neutral alumina, acidic alumina, and sulfuric acid-impregnated silica. All of the fat retainers generated fat-free extracts when the fat/fat retainer ratio was 1:40. Sulfuric acid-impregnated silica and florisil were the only retainers that gave completely clear extracts, and the former was the only not to show any reaction when treated with sulfuric acid after the extraction. Using sulfuric acid-impregnated silica as fat retainer, on-line cleanup of fat-containing matrixes was possible, as demonstrated for naturally contaminated fish meal as well as certified cod liver oil (CRM 349).

pH-dependent spectral properties of HpIX, TPPS2a, mTHPP and mTHPC.

Lower extracellular pH in tumors as compared to normal tissues has been proposed to be a factor contributing to the tumor selective uptake of several photosensitizers. Therefore, the pH dependence of absorption and fluorescence spectral properties of four different drugs relevant for photodynamic therapy (hematoporphyrin IX [HpIX], disulfonated meso-tetraphenylporphine [TPPS2a], meso-tetra(3-hydroxyphenyl)porphine [mTHPP] and meso-tetra(3-hydroxyphenyl)chlorin [mTHPC]) has been examined. Spectral analysis of the dyes dissolved in phosphate buffered saline (PBS) indicates pH-dependent modification in the physiologically important region (6.0-8.0) only in the case of HpIX. This modification is probably related to the protonation of carboxylic groups. Spectral changes of HpIX in PBS observed at acidic pH values < 5, as well as those of the rest of the drugs (inflection points of titration curves occurred at about 5.1, 3.8 and 2.4 for TPPS2a, mTHPP and mTHPC, respectively), are likely to be due to the protonation of imino nitrogens. The tumor localizing properties of mTHPP and mTHPC reported in the literature appear to be due to factors other than pH-dependent changes in the lipophilicity of the drugs.

On-line clean-up of pressurized liquid extracts for the determination of polychlorinated biphenyls in feedingstuffs and food matrices using gas chromatography-mass spectrometry.

This paper describes a fast and simple pressurized liquid extraction method for the determination of polychlorinated biphenyls (PCBs) in feedingstuffs and food matrices. The method is based on a simultaneous extraction/clean-up step requiring a minimum of sample handling. The final analysis was performed with gas chromatography-mass spectrometry. Seven PCBs (28, 52, 101, 118, 138, 153 and 180) were analyzed, all of which are indicator congeners that, according to European legislation should be included in the analytical monitoring program. The extracted matrices were spiked feed for poultry and two certified reference materials naturally contaminated with PCBs (cod-liver oil and milk powder), which showed excellent conformity with certified data.