Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy

CD8(+) cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8(+) T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 x 10(6) cells following 5 weeks of culture. Expanded cells contained primarily CD3(+) T-cells, of which CD8(+) T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro (51)Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8(+) T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. (c) 1996 John Wiley & Sons, Inc.     

A metabolic model for biological phosphorus removal by denitrifying organisms

A metabolic model for biological phosphorus removal under denitrifying conditions has been established. The model is based on previous work with aerobic phosphorus removal. The form of the kinetic equations used is the same as for the aerobic model. The main difference is the value of P/NADH(2) ratio in the electron transport phosphorylation with nitrate (delta(N)). This value was determined independently from batch tests with an enriched culture of denitrifying phosphorus-removing bacteria. The measured delta(N) was approximately 1.0 mol ATP/mol NADH(2). This indicates that the energy production efficiency with nitrate compared to oxygen is approximately 40% lower. These batch tests were also used to identify a proper set of kinetic parameters. The obtained model was subsequently applied for the simulation of cyclic behavior in an anaerobic-anoxic sequencing batch reactor at different biomass retention times. The simulation results showed that the metabolic model can be used successfully for the denitrifying dephosphatation process. The obtained kinetic parameters for denitrifying enrichment cultures, however, deviated from those obtained for the aerobic enrichment cultures. (c) 1996 John Wiley & Sons, Inc.     

Sediment quality assessment in the delta of rivers Rhine and Meuse based on field observations,bioassays and food chain implications

The quality of sediment was assessed in 46 sites in the delta of the rivers Rhine and Meuse (The Netherlands) by means of physical-chemical analysis, field observations on the macrobenthic community structure, accumulation studies and bioassays using Chironomus riparius, Daphnia magna and Photobacterium phosphoreum. The results of chemical analyses were classified using national criteria for sediment quality. Results of field studies and bioassays were classified using criteria derived from research in reference areas or based on data from literature. Risk assessment was carried out according to the sediment quality triad and by means of multi criteria analysis (MCA). The Triad approach was used to demonstrate causal relations between effects on the macrozoobenthos community structure, effects demonstrated in bioassays and sediment pollution. This was done by making a comparison of sediment contamination levels with toxicity data from literature for the test organisms of the bioassays. Using the MCA method, for each site a numerical value was derived for total environmental risk in the present situation, based on observed effects. In this way, a relative risk ranking of all sites was realized. The MCA values for the present situation were also compared with MCA scores based on estimated risks after remediation in 1995, in order to set priorities for sites where remediation is expected to cause a significant reduction in environmental risk. In most of the 46 sites studied so far, the macrofauna community was poorly developed, judged by a low number of benthic species, low abundances and a high dominance of species regarded as relatively tolerant to chemical pollution. In bioassays high sediment toxicity was demonstrated for 24 sites. Using the sediment quality triad approach, 25 sites were identified as areas where pollution can be held responsible for the effects observed in the field. From a comparison of contaminant concentrations in different types of food with maximum tolerable risk levels, and the application of a bioaccumulation model it was concluded that the sediment pollution also implies high risks for plant-, benthos- or fish-eating birds (secondary poisoning of top predators). In the Nieuwe Merwede highest MCA risk scores were found for shallow parts where highly polluted sediments are found. It is concluded that the sediment quality triad and the MCA provide additional information which can be used to establish priorities for remedial action. Based on an ecotoxicological evaluation of the improved quality of new sediments that will be deposited after removal of the polluted sediments in the Nieuwe Merwede, it is concluded that in this upstream part of the Rhine delta remedial action will be effective.     

Responses of restored grassland ditch vegetation to hydrological changes, 1989–1992

The impact of hydrological manipulation of an unfertilised, Dutch peat grassland area on plant species composition on ditch banks and in ditchwater was studied. The hydrological manipulation involved raising the groundwater level by admitting nutrient-poor, Ca-rich groundwater in one compartment, and by retaining precipitation in another compartment. A third compartment served as control.The plant species composition showed significant correlations with the following hydrochemical parameters: in bank vegetation with K⁺ concentration, and winter and summer groundwater levels, water depth and elevation; in aquatic vegetation with pH, the concentrations of Cl^- organic-C and NH₄ ⁺ water temperature and elevation.The number of terrestrial plant species increased after compartmentation from 97 to 122; 16 submerged and floating species were found. Most new terrestrial species probably emerged in response to cessation of fertilization and biomass removal, since they showed no preference for any compartment. Five species showed preference for the groundwater compartment and two for the rainwater compartment. Of the new terrestrial plants, seven were relatively rare: Carex panicea, C. oederi, C. pallescens, C. vesicaria, Galium uliginosum, Juncus acutiflorus and Stellaria uliginosa. Of the aquatic plants, eight were relatively rare: Chara globularis, two Callitriche spp., four potamogetonaceae and Ranunculus circinatus.This study indicates that hydrological manipulation of grassland systems in which fertilisation has ceased has profound effects on the vegetation in the ditches of these systems. Bank vegetation responds more slowly and to other hydrochemical factors than aquatic vegetation. Short-term responses in terms of increase in diversity of vegetation pattern and species richness are promising. Long-term responses are not yet known.     

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring

AFLP^TM is a new technique to generate large numbers of molecular markers for genetic mapping. The method involves the selective amplification of a limited number of DNA restriction fragments out of complex plant genomic DNA digests using PCR. With six primer combinations 264 segregating AFLP amplification products were identified in a diploid backcross population from non-inbred potato parents. The identity of an AFLP marker was specified by the primer combination of the amplification product and its size estimated in bases. The segregating AFLP amplification products were mapped by using a mapping population with 217 already known RFLP, isozyme and morphological trait loci. In general, the AFLP markers were randomly distributed over the genome, although a few clusters were observed. No indications were found that AFLP markers are present in other parts of the genome than those already covered by RFLP markers. Locus specificity of AFLP markers was demonstrated because equally sized amplification products segregating from both parental clones generally mapped to indistinguishable maternal and paternal map positions. Locus specificity of AFLP amplification products will allow to establish the chromosomal identity of linkage groups in future mapping studies.Since AFLP technology is a multi-locus detection system, it was not possible to identify the AFLP alleles which belong to a single AFLP locus. The consequences of a genetic analysis based on single alleles, rather than on loci with two or more alleles on mapping studies using progenies of non-inbred parents are discussed.     

Inheritance and genetic mapping of resistance to Alternaria alternata f. sp. lycopersici in Lycopersicon pennellii

The fungal pathogen Alternaria alternata f. sp. lycopersici produces AAL-toxins that function as chemical determinants of the Alternaria stem canker disease in the tomato (Lycopersicon esculentum). In resistant cultivars, the disease is controlled by the Asc locus on chromosome 3. Our aim was to characterize novel sources of resistance to the fungus and of insensitivity to the host-selective AAL-toxins. To that end, the degree of sensitivity of wild tomato species to AAL-toxins was analyzed. Of all members of the genus Lycopersicon, only L. cheesmanii was revealed to be sensitive to AAL-toxins and susceptible to fungal infection. Besides moderately insensitive responses from some species, L. pennellii and L. peruvianum were shown to be highly insensitive to AAL-toxins as well as resistant to the pathogen. Genetic analyses showed that high insensitivity to AAL-toxins from L. pennellii is inherited in tomato as a single complete dominant locus. This is in contrast to the incomplete dominance of insensitivity to AAL-toxins of L. esculentum. Subsequent classical genetics, RFLP mapping and allelic testing indicated that high insensitivity to AAL-toxins from L. pennellii is conferred by a new allele of the Asc locus.     

Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11

At the nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot (Daucus carota L.) cell variant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type cells. The causative component in the conditioned medium has been identified as an acidic 32 kD endochitinase. An antiserum raised against the 32 kD chitinase detected this protein in culture medium from ts11 embryo cultures grown at the permissive temperature as well as at the nonpermissive temperature. No difference in biochemical characteristics or in effect on ts11 embryo development could be detected between the 32 kD chitinase purified from wild-type cultures and the chitinase from ts11 cultures grown at the permissive or at the nonpermissive temperature. Compared to the amount present in a ts11 embryo culture at the permissive temperature, a reduction in the amount of 32 kD chitinase was observed during the temperature-sensitive period at the nonpermissive temperature. These results imply that the arrested embryo phenotype of ts11 is not the result of a structural difference in its 32 kD chitinase, but is the result of a transient decrease in the amount of 32 kD chitinase present. Morphological observations indicate that the ts11 phenotype is pleiotropic and also affects the cell wall of nonembryogenic cells. © 1995 Wiley-Liss, Inc.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).