Tracking the progeny of adoptively transferred virus-specific T cells in patients posttransplant using TCR sequencing.

Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirus-specific T-cell products to protect recipients of T-cell-depleted allogeneic stem cell grafts against viral reactivation. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem cell graft-derived T cells that survived T-cell depletion during conditioning or stem cell graft manipulation. Using messenger RNA sequencing of the T-cell receptor ß-chains of the individual virus-specific T-cell populations within these T-cell products, we were able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed in vivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific T cells that could also be detected before the T-cell product infusion. In addition, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivation. This study demonstrates that virus-specific T cells from prophylactically infused multiantigen-specific T-cell products can expand in response to antigen encounter in vivo and even persist in the absence of early viral reactivation.

HA-1H T-Cell Receptor Gene Transfer to Redirect Virus-Specific T Cells for Treatment of Hematological Malignancies After Allogeneic Stem Cell Transplantation: A Phase 1 Clinical Study.

Graft-vs.-leukemia (GVL) reactivity after HLA-matched allogeneic stem cell transplantation (alloSCT) is mainly mediated by donor T cells recognizing minor histocompatibility antigens (MiHA). If MiHA are targeted that are exclusively expressed on hematopoietic cells of recipient origin, selective GVL reactivity without severe graft-vs.-host-disease (GVHD) may occur. In this phase I study we explored HA-1H TCR gene transfer into T cells harvested from the HA-1H negative stem-cell donor to treat HA-1H positive HLA-A*02:01 positive patients with high-risk leukemia after alloSCT. HA-1H is a hematopoiesis-restricted MiHA presented in HLA-A*02:01. Since we previously demonstrated that donor-derived virus-specific T-cell infusions did not result in GVHD, we used donor-derived EBV and/or CMV-specific T-cells to be redirected by HA-1H TCR. EBV and/or CMV-specific T-cells were purified, retrovirally transduced with HA-1H TCR, and expanded. Validation experiments illustrated dual recognition of viral antigens and HA-1H by HA-1H TCR-engineered virus-specific T-cells. Release criteria included products containing more than 60% antigen-specific T-cells. Patients with high risk leukemia following T-cell depleted alloSCT in complete or partial remission were eligible. HA-1H TCR T-cells were infused 8 and 14 weeks after alloSCT without additional pre-conditioning chemotherapy. For 4/9 included patients no appropriate products could be made. Their donors were all CMV-negative, thereby restricting the production process to EBV-specific T-cells. For 5 patients a total of 10 products could be made meeting the release criteria containing 3-280 × 106 virus and/or HA-1H TCR T-cells. No infusion-related toxicity, delayed toxicity or GVHD occurred. One patient with relapsed AML at time of infusions died due to rapidly progressing disease. Four patients were in remission at time of infusion. Two patients died of infections during follow-up, not likely related to the infusion. Two patients are alive and well without GVHD. In 2 patients persistence of HA-1H TCR T-cells could be illustrated correlating with viral reactivation, but no overt in-vivo expansion of infused T-cells was observed. In conclusion, HA-1H TCR-redirected virus-specific T-cells could be made and safely infused in 5 patients with high-risk AML, but overall feasibility and efficacy was too low to warrant further clinical development using this strategy. New strategies will be explored using patient-derived donor T-cells isolated after transplantation transduced with HA-1H-specific TCR to be infused following immune conditioning.

CMV seronegative donors: Effect on clinical severity of CMV infection and reconstitution of CMV-specific immunity.

BACKGROUND: Cytomegalovirus (CMV)-specific T-cells are crucial to prevent CMV disease. CMV seropositive recipients transplanted with stem cells from a CMV seronegative allogeneic donor (R+D-) may be at risk for CMV disease due to absence of donor CMV-specific memory T-cells in the graft. METHODS: We analyzed the duration of CMV reactivations and the incidence of CMV disease in R+D- and R+D+ patients after alemtuzumab-based T-cell depleted allogeneic stem cell transplantation (TCD alloSCT). To determine the presence of donor-derived primary CMV-specific T-cell responses we analyzed the origin of CMV-specific T-cells in R+D- patients. RESULTS: The duration of CMV reactivations (54 versus 38 days, respectively, p = 0.048) and the incidence of CMV disease (0.14 versus 0.02, p = 0.003 at 1 year after alloSCT) were higher in R+D- patients compared to R+D+ patients. In R+D- patients, CMV-specific CD4+ and CD8+ T-cells were mainly of recipient origin. However, in 53% of R+D- patients donor-derived CMV-specific T-cells were detected within the first year. CONCLUSIONS: In R+D- patients, immunity against CMV was predominantly mediated by recipient T-cells. Nevertheless, donor CMV serostatus significantly influenced the clinical severity of CMV reactivations indicating the role of CMV-specific memory T-cells transferred with the graft, despite the ultimate formation of primary donor-derived CMV-specific T-cell responses in R+D- patients.

High Mutation Frequency of the PIGA Gene in T Cells Results in Reconstitution of GPI Anchor-/CD52- T Cells That Can Give Early Immune Protection after Alemtuzumab-Based T Cell-Depleted Allogeneic Stem Cell Transplantation.

Alemtuzumab (ALM) is used for T cell depletion in the context of allogeneic hematopoietic stem cell transplantation (alloSCT) to prevent acute graft-versus-host disease and graft rejection. Following ALM-based T cell-depleted alloSCT, relatively rapid recovery of circulating T cells has been described, including T cells that lack membrane expression of the GPI-anchored ALM target Ag CD52. We show, in a cohort of 89 human recipients of an ALM-based T cell-depleted alloSCT graft, that early lymphocyte reconstitution always coincided with the presence of large populations of T cells lacking CD52 membrane expression. In contrast, loss of CD52 expression was not overt within B cells or NK cells. We show that loss of CD52 expression from the T cell membrane resulted from loss of GPI anchor expression caused by a highly polyclonal mutational landscape in the PIGA gene. This polyclonal mutational landscape in the PIGA gene was also found in CD52- T cells present at a low frequency in peripheral blood of healthy donors. Finally, we demonstrate that the GPI-/CD52- T cell populations that arise after ALM-based T cell-depleted alloSCT contain functional T cells directed against multiple viral targets that can play an important role in immune protection early after ALM-based T cell-depleted transplantation.

Complement-dependent cytotoxicity induced by therapeutic antibodies in B-cell acute lymphoblastic leukemia is dictated by target antigen expression levels and augmented by loss of membrane-bound complement inhibitors.

To optimally utilize therapeutic monoclonal antibodies in the treatment of B-cell acute lymphoblastic leukemia (B-ALL) understanding their mechanisms of action and the factors influencing these mechanisms is required. We show strong correlations between target antigen expression levels and sensitivity to complement-dependent cytotoxicity (CDC) induced by rituximab, ofatumumab, or alemtuzumab in a panel of cell lines derived from primary B-ALL cells and in primary B-ALL samples. Simultaneous loss of expression of membrane-bound complement regulatory proteins (mCRP) CD55 and CD59 due to glycophosphatidylinositol-anchor deficiency, significantly increased sensitivity to CDC. Accordingly, induced increase in CD55 or CD59 expression protected cells against CDC. The extent of protection co-depended on antigen expression and antibody concentration. In contrast, natural variation in mCRP expression could not be used as a single factor to predict sensitivity to CDC. In conclusion, sensitivity of B-ALL cells to CDC was predominantly determined by antibody concentration and target antigen expression.

Carry-over of veterinary drugs from medicated to non-medicated feeds in commercial feed manufacturing plants.

Different compound feeds have to be manufactured in the same production line. As a consequence, traces of the first produced feed may remain in the production and get mixed with the next feed batches. This "carry-over" is unavoidable, and so non-medicated feed can be contaminated with veterinary drugs like antibiotics added to the previous batch of medicated feed. To monitor the carry-over of antibiotics in the Netherlands, 21 feed mills were visited and 140 samples of flushing feeds were collected and analysed for containing residues of antibiotics. Results show that 87% of all samples contain concentrations of antibiotics in the range of 0.1-154 mg/kg. It is expected that these levels - which are in the same range as previously found for the nowadays banned antimicrobial growth promoters (AMGPs) - have an effect on the occurrence of microbial resistance. Analysis of a second set of samples collected at four different feed mills directly after the production of oxytetracycline-medicated feed demonstrated that the first part of a flushing feed has much higher contamination than the last part of the batch. Furthermore, it was demonstrated that the carry-over percentage shows no correlation with the carry-over determined by one of the standard GMP+ procedures. These observations, unavoidable carry-over, inhomogeneous batches of feed with antibiotics and difficulties to predict the carry-over levels, together with the awareness of the increasing problem of microbial resistance, motivated the NEVEDI, association of Dutch Feed Producers, to announce that they will voluntarily stop the production of medicated feed in 2011. The alternatives for medicated feed are for example water or milk medication or the use of top-dressings at the farm. The consequences and possible new risks of carry-over at the farm are not completely clear yet.

Validation and transferability study of a method based on near-infrared hyperspectral imaging for the detection and quantification of ergot bodies in cereals.

In recent years, near-infrared (NIR) hyperspectral imaging has proved its suitability for quality and safety control in the cereal sector by allowing spectroscopic images to be collected at single-kernel level, which is of great interest to cereal control laboratories. Contaminants in cereals include, inter alia, impurities such as straw, grains from other crops, and insects, as well as undesirable substances such as ergot (sclerotium of Claviceps purpurea). For the cereal sector, the presence of ergot creates a high toxicity risk for animals and humans because of its alkaloid content. A study was undertaken, in which a complete procedure for detecting ergot bodies in cereals was developed, based on their NIR spectral characteristics. These were used to build relevant decision rules based on chemometric tools and on the morphological information obtained from the NIR images. The study sought to transfer this procedure from a pilot online NIR hyperspectral imaging system at laboratory level to a NIR hyperspectral imaging system at industrial level and to validate the latter. All the analyses performed showed that the results obtained using both NIR hyperspectral imaging cameras were quite stable and repeatable. In addition, a correlation higher than 0.94 was obtained between the predicted values obtained by NIR hyperspectral imaging and those supplied by the stereo-microscopic method which is the reference method. The validation of the transferred protocol on blind samples showed that the method could identify and quantify ergot contamination, demonstrating the transferability of the method. These results were obtained on samples with an ergot concentration of 0.02% which is less than the EC limit for cereals (intervention grains) destined for humans fixed at 0.05%.

Effective treatment of refractory CMV reactivation after allogeneic stem cell transplantation with in vitro-generated CMV pp65-specific CD8+ T-cell lines.

To treat patients with refractory cytomegalovirus (CMV) reactivation after allogeneic stem cell transplantation, a phase I/II clinical study on adoptive transfer of in vitro-generated donor-derived or patient-derived CMV pp65-specific CD8* T-cell lines was performed. Peripheral blood mononuclear cells from CMV seropositive donors or patients were stimulated with HLA-A*0201-restricted and/or HLA-B*0702-restricted CMV pp65 peptides (NLV/TPR) and 1 day after stimulation interferon-γ)-producing cells were enriched using the CliniMACS Cytokine Capture System (interferon-γ), and cultured with autologous feeders and low-dose interluekin-2. After 7-14 days of culture, quality controls were performed and the CMV-specific T-cell lines were administered or cryopreserved. The T-cell lines generated contained 0.6-17 × 10(6) cells, comprising 54%-96% CMV pp65-specific CD8 T cells, and showed CMV-specific lysis of target cells. Fifteen CMV-specific T-cell lines were generated of which 8 were administered to patients with refractory CMV reactivation. After administration, no acute adverse events and no graft versus host disease were observed and CMV load disappeared. In several patients, a direct relation between administration of the T-cell line and the in vivo appearance of CMV pp65-specific T cells could be documented. In conclusion, administration of CMV pp65-specific CD8* T-cell lines was found to be feasible and safe, and enduring efficacy of administered CMV pp65-specific CD8* T-cell lines could be demonstrated.

Monitoring phytoplankton and marine biotoxins in production waters of the Netherlands: results after one decade.

Shellfish products may be contaminated with marine biotoxins which, after consumption, may lead to human illness. The Netherlands has a regular monitoring programme for marine biotoxins and the possible toxic phytoplankton in shellfish production waters. The aim of the current study was to evaluate the presence of potential toxic phytoplankton species and marine biotoxins in Dutch production waters over the last decade, and to analyse the relationship between toxin levels and abundance of possible causative phytoplankton species. The results of the monitoring programme of the period 1999-2009 were used. The presence of Alexandrium spp. were negligible, but Pseudo-nitzschia spp. and phytoplankton causing diarrhetic shellfish poisoning (DSP toxin-producing phytoplankton) were present in nearly all three main production areas and years. The main DSP toxin-producing species was Dinophysis acuminata followed by D. rotundata and Prorocentrum lima. Toxins causing paralytic shellfish poisoning (PSP) and amnesic shellfish poisoning (ASP) were present in only a few individual shellfish samples, all at low levels. At the end of 2002, an episode of DSP toxicity was recorded, based on the rat bioassay results. Of the samples that were chemically analysed for DSP toxins in 2007 and 2008, about half of the samples in 2007 contained these toxins, although levels were low and no positive results were obtained using the rat bioassay. There was a slight positive correlation between concentrations of DSP toxin-producing phytoplankton and levels of DSP toxins in 2007. Increased DSP toxin levels were found up to 5 weeks after the peak in DSP toxin-producing phytoplankton. This positive, but weak, relationship needs to be confirmed in future research using more samples and chemical methods to quantify the presence of DSP toxins. If this relationship is further substantiated and quantified, it could be used within the current monitoring programme in the Netherlands to predict the risk areas regarding DSP toxicity in shellfish.

Quantitative determination of marine lipophilic toxins in mussels, oysters and cockles using liquid chromatography-mass spectrometry: inter-laboratory validation study.

Thirteen laboratories participated in an inter-laboratory study to evaluate the method performance characteristics of a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for marine lipophilic shellfish toxins. Method performance characteristics were evaluated for mussel (Mytilus edulis), oyster (Crassostrea gigas) and cockle (Cerastoderma edule) matrices. The specific toxin analogues tested included okadaic acid (OA), dinophysistoxins-1 and -2 (DTX1, -2), azaspiracids-1, -2 and -3 (AZA1, -2, -3), pectenotoxin-2 (PTX2), yessotoxin (YTX), and 45-OH-yessotoxin (45-OH-YTX). The instrumental technique was developed as an alternative to the still widely applied biological methods (mouse or rat bioassay). Validation was conducted according to the AOAC-harmonised protocol for the design, conduct and interpretation of method-performance studies. Eight different test materials were sent as blind duplicates to the participating laboratories. Twelve laboratories returned results that were accepted to be included in the statistical evaluation. The method precision was expressed as HORRATs. For the individual toxins (except for 45-OH-YTX) HORRATs were found to be ≤1.8 (median HORRAT=0.8) in all tested materials. The recoveries of OA-, AZA- and YTX-group toxins were within the range of 80-108% and PTX2 was within the range of 62-93%. Based on the acceptable values for precision and recovery, it was concluded that the method is suitable for official control purposes to quantitatively determine OA/DTXs, AZAs, YTXs and PTX2 in shellfish.

Tailored microarray platform for the detection of marine toxins.

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave clear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.

Carry-over of pyrrolizidine alkaloids from feed to milk in dairy cows.

Pyrrolizidine alkaloids are toxins present in many plants belonging to the families of Asteraceae, Boraginaceae and Fabaceae. Particularly notorious are pyrrolizidine alkaloids present in ragwort species (Senecio), which are held responsible for hepatic disease in horses and cows and may lead to the death of the affected animals. In addition, these compounds may be transferred to edible products of animal origin and as such be a threat for the health of consumers. To investigate the possible transfer of pyrrolizidine alkaloids from contaminated feed to milk, cows were put on a ration for 3 weeks with increasing amounts (50-200 g day(-1)) of dried ragwort. Milk was collected and sampled twice a day; faeces and urine twice a week. For milk, a dose-related appearance of pyrrolizidine alkaloids was found. Jacoline was the major component in milk despite being a minor component in the ragwort material. Practically no N-oxides were observed in milk, notwithstanding the fact that they constituted over 80% of the pyrrolizidine alkaloids in ragwort. The overall carry-over of the pyrrolizidine alkaloids was estimated to be only around 0.1%, but for jacoline 4%. Notwithstanding the low overall carry-over, this may be relevant for consumer health considering the genotoxic and carcinogenic properties demonstrated for some of these compounds. Analysis of the faeces and urine samples indicated that substantial metabolism of pyrrolizidine alkaloids is taking place. The toxicity and potential transfer of metabolites to milk is unknown and remains to be investigated.

Validation of a database on acrylamide for use in epidemiological studies.

BACKGROUND/OBJECTIVES: Acrylamide, a probable human carcinogen, was detected in various heat-treated foods such as French fries and potato crisps. Recently, positive associations have been found between dietary acrylamide intakes, as estimated with a food frequency questionnaire using an acrylamide database, and cancer risk in some epidemiological studies. As acrylamide levels vary considerably within the same type of foods, a validation study was performed to investigate whether use of an acrylamide food database containing calculated mean acrylamide content, based on extensive sampling and chemical analysis of Dutch foods (several samples per food), can classify subjects with respect to true acrylamide intake. SUBJECTS/METHODS: We used the data from a 24-h duplicate diet study. The acrylamide content of 39 Dutch 24-h duplicate diets collected in 2004 was estimated using the mean acrylamide levels of foods available from the database and the menu list, on which the participants of the duplicate diet study had listed the amounts of individual foods and drinks in household units. Next, the acrylamide content of the total duplicate diets was analytically measured and correlated to the estimated acrylamide contents. RESULTS: The Spearman's correlation coefficient between chemically determined acrylamide content and the calculated acrylamide content of the duplicate diets was 0.82 (P<0.001). CONCLUSIONS: This study indicates that it is possible to classify subjects with respect to acrylamide intake if mean instead of actual content of each food is applied. The database can therefore be applied in epidemiological studies on acrylamide intake and cancer risk, such as the Netherlands Cohort Study on Diet and Cancer.

Functional analysis of killer Ig-like receptor-expressing cytomegalovirus-specific CD8+ T cells.

Killer Ig-like receptors (KIR) are expressed by human NK cells and T cells. Although Ag-specific cytolytic activity and cytokine production of KIR(+) T cells can be inhibited by KIR ligation, the effect of KIR on proliferation is unclear. KIR(+) T cells have been reported to have a general proliferative defect. To investigate whether KIR(+) T cells represent end-stage dysfunctional T cells, we characterized KIR(+) CMV-specific T cells in allogeneic stem cell transplantation patients and healthy donors. In both patients and healthy donors, a significant percentage KIR(+) T cells was detected at various time points. All stem cell transplantation patients studied showed KIR expression on CMV-specific T cells, while not all donors had KIR-expressing CMV-specific T cells. From two of the patients and one donor KIR(+) CMV-specific T clones were isolated and analyzed functionally. T cells were detected that expressed KIR that could not encounter their corresponding KIR ligands in vivo, illustrating that KIR expression by these T cells was not based on functional selection but a random process. Our data demonstrate that KIR(+) T cells are fully functional T cells that are only restricted in effector functions and proliferation upon KIR ligation. The level of KIR-mediated inhibition of the effector functions and proliferation depended on the strength of TCR stimulation. We observed no diminished general proliferative capacity and therefore we conclude that these T cells do not represent end-stage dysfunctional T cells.

Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application.

BACKGROUND: Donor lymphocyte infusion is an effective form of adoptive immunotherapy for hematologic malignancies after allogeneic stem cell transplantation. Graft-versus-host disease, however, often develops due to recognition of ubiquitously-expressed minor histocompatibility antigens. Transfer of T-cell receptors recognizing hematopoiesis-restricted minor histocompatibility antigens to virus-specific T cells may be a powerful anti-tumor therapy with a low risk of graft-versus-host disease. The purpose of this study was to develop an optimal T-cell receptors-encoding multi-cistronic retroviral vector and an efficient method for generating T-cell receptors-engineered virus-specific T cells. DESIGN AND METHODS: Retroviral vectors encoding the T-cell receptors for the hematopoiesis-restricted minor histocompatibility antigen HA-2 with and without selection markers were compared for T-cell receptors surface expression and HA-2-specific lysis. In addition, two different methods, i.e. peptide stimulation of CD8(+) cells and Pro5 MHC pentamer-based isolation of antigen-specific T cells, were investigated for their efficiency to generate T-cell receptors-transduced virus-specific T cells. RESULTS: Bi-cistronic vectors without selection markers most efficiently mediated T-cell receptors surface expression and HA-2-specific lysis. Furthermore, both methods were useful for generating gene-modified cells, but the purity of virus-specific T cells was higher after pentamer isolation. Finally, the capacity of gene-modified cells to express the transgenic T-cell receptors at the cell surface markedly differed between virus-specific T cells and was correlated with lysis of relevant target cells. CONCLUSIONS: Our data support T-cell receptors gene transfer to pentamer-isolated virus-specific T cells using bi-cistronic retroviral vectors and illustrate the relevance of selection of gene-modified T cells with appropriate transgenic T-cell receptors surface expression for clinical gene therapy.

Collaborative study of a microbiological screening method (three-plate) for the banned antimicrobial growth promotors tylosin, virginiamycin, spiramycin, zinc bacitracin and avoparcin in animal feed.

A microbiological screening method (three-plate) for the detection of the antimicrobial growth promoters tylosin, spiramycin, virginiamycin, zinc bacitracin, and avoparcin in animal feed has been developed and validated successfully. A collaborative study involving 18 laboratories receiving 172 samples was carried out to verify the performance characteristics. The detection level for tylosin/virginiamycin/spiramycin, expressed in microbiological activity, was 1 mg kg(-1) (false-positives, 2%; false-negatives, 3, 0, and 6%, respectively). Avoparcin could be detected at 1 mg kg(-1) in feed in general (false-positives, 2%; false-negatives, 0%). However, in calf feed the sensitivity was lower. The percentages of false-negatives were found to be 12%, 7%, and 0% at 1, 3, and 5 mg kg(-1), respectively (false-positives, 4%). The limit of detection for zinc bacitracin was 3-5 mg kg(-1) (false-positives, 5-10%; false-negatives, 77% at 1 mg kg(-1), 45% at 2 mg kg(-1), 12% at 3 mg kg(-1), and 4% at 5 mg kg(-1)). The method allowed for a distinction to be made between the groups of antibiotics: avoparcin/zinc bacitracin versus tylosin/virginiamycin/spiramycin. This definitely gives added value to the method in the framework of a follow-up of positive screening results by post-screening and confirmatory analysis.

Improved microbial screening assay for the detection of quinolone residues in poultry and eggs.

An improved microbiological screening assay is reported for the detection of quinolone residues in poultry muscle and eggs. The method was validated using fortified tissue samples and is the first microbial assay to effectively detect enrofloxacin, difloxacin, danofloxacin, as well as flumequine and oxolinic acid, at or below their EU maximum residue limits (MRL). The accuracy of the assay was shown by analysing incurred tissue samples containing residue levels around the MRL. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of the quinolone concentration in these samples showed that the test plate can be used semi-quantitatively, allowing the definition of an "action level" as an inhibition zone above which a sample can be considered "suspect". The presented assay is a useful improvement or addition to existing screening systems.

Effects of Fusarium toxin-contaminated wheat and feed intake level on the biotransformation and carry-over of deoxynivalenol in dairy cows.

An experiment was carried out to examine the effects of feeding Fusarium toxin-contaminated wheat (8.21 mg deoxynivalenol (DON) and 0.09 mg zearalenone (ZON) per kg dry matter) at different feed intake levels on the biotransformation and carry-over of DON in dairy cows. For this purpose, 14 ruminal and duodenal fistulated dairy cows were fed a diet containing 60% concentrate with a wheat portion of 55% (Fusarium toxin-contaminated wheat (mycotoxin period) or control wheat (control period)) and the ration was completed with maize- and grass silage (50 : 50) on a dry matter basis. Daily DON intakes ranged from 16.6 to 75.6 mg in the mycotoxin period at dry matter intakes of 5.6-20.5 kg. DON was almost completely biotransformed to de-epoxy DON (94-99%) independent of the DON/feed intake, and the flow of DON and de-epoxy DON at the duodenum related to DON intake ranged from 12 to 77% when the Fusarium toxin-contaminated wheat was fed. In the serum samples, de-epoxy DON was detected in the range of 4-28 ng ml-1 in the mycotoxin period, while concentrations of DON were all below the detection limit. The daily excretion of DON and de-epoxy DON in the milk of cows fed the contaminated wheat varied between 1 and 10 microg and between 14 and 104 microg, respectively. The total carry-over rates as the ratio between the daily excretion of DON and de-epoxy DON into milk and DON intake were in the ranges of 0.0001-0.0002 and 0.0004-0.0024, respectively. Total carry-over rates of DON as DON and de-epoxy DON into the milk increased significantly with increasing milk yield. In the urine samples, de-epoxy DON was the predominant substance as compared with DON with a portion of the total DON plus de-epoxy DON concentration to 96% when the Fusarium toxin-contaminated wheat was fed, whereas the total residues of DON plus de-epoxy DON in faeces ranged between 2 and 18% of DON intake in the mycotoxin period. The degree of glucuronidation of de-epoxy DON was found to be approximately 100% in serum. From 33 to 80% of DON and from 73 to 92% of de-epoxy DON, and from 21 to 92% of DON and from 86 to 100% of de-epoxy DON were glucuronidated in the milk and urine, respectively. It is concluded that DON is very rapidly biotransformed to de-epoxy DON in the rumen and only negligible amounts of DON and de-epoxy DON were transmitted into the milk within the range of 5.6-20.5 kg day-1 dry matter intake and milk yields (fat corrected milk) between 10 and 42 kg day-1.

Aflatoxin M1 in milk powders: processing, homogeneity and stability testing of certified reference materials.

As part of the certification campaign of three candidate reference materials for the determination of aflatoxin M1 (AfM1) in whole milk powders, homogeneity, short- and long-term stability tests of naturally contaminated milk powders have been performed. The homogeneity of two AfM1-contaminated milk powders was studied by taking samples at regular intervals of the filling sequences and analysing in triplicate for their AfM1 contents by liquid chromatography with fluorescence detection (LC-FLD) using random stratified sampling schemes. The homogeneity testing of an AfM1 'blank' milk powder material was performed by determining the nitrogen content because AfM1 levels were below the limit of detection of the most sensitive determination method. The short-term stability of AfM1-contaminated milk powders was evaluated at three different storage temperatures (4, 18 and 40 degrees C). After storage times of 0, 1, 2 and 4 weeks, samples were investigated using LC-FLD. The long-term stability study comprised of measurements after 0, 6, 12 and 18 months after storage at -20 and 4 degrees C. Analyses were done by LC-FLD. Based on the homogeneity tests, the materials were sufficiently homogenous to serve as certified reference materials. Corresponding uncertainty contributions of 0.23-0.89% were calculated for the homogeneity. The stability measurements showed no significant trends for both short- and long-term stability studies. The long-term stability uncertainties of the AfM1-contaminated milk powders were 7.4 and 6.3%, respectively, for a shelf-life of 6 years and storage at -20 degrees C. Supplementary stability monitoring schemes over a long period of several years are currently ongoing.

Up-regulated expression in nonhematopoietic tissues of the BCL2A1-derived minor histocompatibility antigens in response to inflammatory cytokines: relevance for allogeneic immunotherapy of leukemia.

T cells directed against hematopoietic-restricted minor histocompatibility antigens (mHags) may mediate graft-versus-leukemia (GVL) reactivity without graft-versus-host disease (GVHD). Recently, the HLA-A24-restricted mHag ACC-1 and the HLA-B44-restricted mHag ACC-2 encoded by separate polymorphisms within the BCL2A1 gene were characterized. Hematopoietic-restricted expression was suggested for these mHags. We demonstrate BCL2-related protein A1 (BCL2A1) mRNA expression in mesenchymal stromal cells (MSCs) that was up-regulated by the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and/or interferon gamma (IFN-gamma). Analysis of cytotoxicity and IFN-gamma production illustrated that ACC-2-specific T cells did not recognize untreated MSCs or IFN-gamma-treated MSCs but showed specific recognition and killing of MSCs treated with TNF-alpha plus IFN-gamma. We hypothesize that under steady-state circumstances BCL2A1-specific T cells may exhibit relative specificity for hematopoietic tissue, but reactivity against nonhematopoietic cells may occur when inflammatory infiltrates are present. Thus, the role of BCL2A1-specific T cells in differential induction of GVL reactivity and GVHD may depend on the presence of inflammatory responses that may occur during GVHD.