Search for the optimal genotoxicity assay for routine testing of chemicals: Sensitivity and specificity of conventional and new test systems.

Many conventional in vitro tests that are currently widely used for routine screening of chemicals have a sensitivity/specificity in the range between 60 % and 80 % for the detection of carcinogens. Most procedures were developed 30-40 years ago. In the last decades several assays became available which are based on the use of metabolically competent cell lines, improvement of the cultivation conditions and development of new endpoints. Validation studies indicate that some of these models may be more reliable for the detection of genotoxicants (i.e. many of them have sensitivity and specificity values between 80 % and 95 %). Therefore, they could replace conventional tests in the future. The bone marrow micronucleus (MN) assay with rodents is at present the most widely used in vivo test. The majority of studies indicate that it detects only 5-6 out of 10 carcinogens while experiments with transgenic rodents and comet assays seem to have a higher predictive value and detect genotoxic carcinogens that are negative in MN experiments. Alternatives to rodent experiments could be MN experiments with hen eggs or their replacement by combinations of new in vitro tests. Examples for promising candidates are ToxTracker, TGx-DDI, multiplex flow cytometry, γH2AX experiments, measurement of p53 activation and MN experiments with metabolically competent human derived liver cells. However, the realization of multicentric collaborative validation studies is mandatory to identify the most reliable tests.

Atlantic bluefin tuna Thunnus thynnus feeding ecology in the northern Gulf of Mexico: a preliminary description of diet from the western Atlantic spawning grounds.

A combination of stomach contents, nitrogen stable-isotope and tissue C:N values are presented to demonstrate feeding activity of Atlantic bluefin tuna Thunnus thynnus on the Gulf of Mexico (GOMEX) spawning grounds. Diets include teleosts, cephalopods, crustaceans and a pelagic tunicate (Pyrosoma atlanticum). Results reveal the need to classify the GOMEX as a T. thynnus feeding ground.

The novel immunotoxin HM1.24-ETA' induces apoptosis in multiple myeloma cells.

Despite new treatment modalities, the clinical outcome in a substantial number of patients with multiple myeloma (MM) has yet to be improved. Antibody-based targeted therapies for myeloma patients could make use of the HM1.24 antigen (CD317), a surface molecule overexpressed on malignant plasma cells and efficiently internalized. Here, a novel immunotoxin, HM1.24-ETA', is described. HM1.24-ETA' was generated by genetic fusion of a CD317-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA'). HM1.24-ETA' inhibited growth of interleukin 6 (IL-6)-dependent and -independent myeloma cell lines. Half-maximal growth inhibition was observed at concentrations as low as 0.3 nM. Target cell killing occurred via induction of apoptosis and was unaffected in co-culture experiments with bone marrow stromal cells. HM1.24-ETA' efficiently triggered apoptosis of freshly isolated/cryopreserved cells of patients with plasma cell leukemia and MM and was active in a preclinical severe combined immunodeficiency (SCID) mouse xenograft model. Importantly, HM1.24-ETA' was not cytotoxic against CD317-positive cells from healthy tissue (monocytes, human umbilical vein endothelial cells). These results indicate that CD317 may represent a promising target structure for specific and efficient immunotoxin therapy for patients with plasma cell tumors.

The novel tribody [(CD20)(2)xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells.

Bispecific antibodies (bsab) offer a promising approach for optimizing antibody-based therapies. In the present study, [(CD20)(2)xCD16], a recombinant CD20- and CD16-directed bsab in the tribody format, was designed to optimize recruitment of FcγRIII (CD16)-positive effector cells. [(CD20)(2)xCD16] retained the antigen specificities of the parental monoclonal antibodies and binding to FcγRIIIa was not compromised by the F/V polymorphism at amino-acid position 158. [(CD20)(2)xCD16] mediated potent lysis of lymphoma cell lines and freshly isolated tumor cells from patients, even at low picomolar concentrations (∼10 pM). Irrespective of the CD16a allotype, potency as well as efficacy of lysis obtained with the tribody was significantly higher than lysis triggered by rituximab. Tumor cell killing also occurred when autologous NK cells were used as effector cells. Compared with rituximab, the tribody demonstrated depletion of autologous B cells in ex vivo whole blood assays at 100-fold lower antibody concentration. In mice with a reconstituted humanized hematopoietic system, established by transplantation of human CD34-positive cord blood cells, this novel tribody significantly depleted autologous human B cells. Thus, tribodies such as [(CD20)(2)xCD16], recruiting CD16-positive effector cells, may represent promising candidates for clinical development.

Human kappa light chain targeted Pseudomonas exotoxin A--identifying human antibodies and Fab fragments with favorable characteristics for antibody-drug conjugate development.

Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.

Novel sampling methods for atmospheric semi-volatile organic compounds (SOCs) in a high altitude alpine environment.

High- and low-volume active air samplers as well as bulk deposition samplers were developed to sample atmospheric SOCs under the adverse conditions of a mountain environment. Active sampling employed separate filters for different European source regions. Filters were switched depending on daily trajectory forecasts, whose accuracy was evaluated post hoc. The sampling continued on three alpine summits over five periods of four months. The prevailing trajectories varied stronger between sampling periods than between stations. The sampling equipment (active and bulk deposition) proved dependable for operation in a mountain environment, with idle times being mainly due to non-routine manipulations and connectivity.

Electroporation of isolated higher-plant mitochondria: transcripts of an introduced cox2 gene, but not an atp6 gene, are edited in organello.

To facilitate the analysis of RNA processing in plant mitochondria, a method was established for introducing foreign DNA into mitochondria isolated from maize and sorghum. This method permits the uptake of DNA of up to 11 kb into the mitochondrial matrix. In vitro incubation of maize mitochondria in a specific buffer system was found to permit splicing and editing of newly synthesized RNAs for a period of at least 7 h. This was shown both for transcripts of endogenous mitochondrial genes (atp6, cox2) and for transcripts derived from an introduced Arabidopsis thaliana cox2 gene. In contrast, when a Sorghum bicolor atp6 gene was introduced into isolated maize mitochondria, the gene was transcribed, but the RNA was not edited, although all the editing sites in maize and sorghum atp6 RNA are identical. This may indicate the presence of transcript-specific cis -acting regions in the up- or downstream untranslated sequences of the mRNA. The system described here should allow further dissection of the mechanism of RNA editing in plant mitochondria.

Serum 25-hydroxyvitamin D and parathyroid hormone in patients with ankylosing spondylitis before and after a three-week rehabilitation treatment at high altitude during winter and spring.

Does a sojourn at high altitude during the winter and spring improve vitamin D status (and possibly suppress parathyroid hormone [PTH]) in patients with ankylosing spondylitis (AS)? In 73 patients with AS, serum concentrations of 25-hydroxy-vitamin D [25(OH)D] and PTH were determined before and after a three-week rehabilitation treatment at Bad Gastein (1000 m above sea level). At the first examination, serum 25(OH)D was median (25th, 75th percentile) 15.5 ng mL-1 (10.0 ng mL-1, 20.6 ng mL-1). Thirteen patients (18%) had a 25(OH)D concentration below 8 ng mL-1. In 53 patients (73%) the level was below 20 ng mL-1. After the sojourn, 25(OH)D significantly (p = 0.02) increased to 19.7 (11.3, 24.6) ng mL-1. PTH did not change significantly, being 32 (22.4, 43.9) pg mL-1 before and 30.3 (24.1, 39.9) pg mL-1 after the sojourn. Analysing different periods of sojourn, a significant (p < 0.001) increase in 25(OH)D was found in April but not in the other months. Patients with ankylosing spondylitis may have extremely low levels of 25(OH)D. The results of the present study suggest that a sojourn at high altitude in early spring is liable to reduce vitamin D deficiency.

Analysis of acid esterase activity and polymorphism as an aid for the classification of human low-grade malignant non-Hodgkin's lymphomas.

Investigating a link between enzyme histochemical and recent immunohistochemical results, the authors studied the activity and the polymorphism of acid esterase (EC 3.1.1.6) in well defined human B-cell lymphomas. TWelve cases of chronic B-lymphocytic leukemia, 18 cases of centroblastic/centrocytic follicular lymphoma, and 17 cases of lymphoplasmacytic/lymphoplasmacytoid lymphoma, as diagnosed according to Kiel classification, were subjected to enzyme assay and isoelectric focusing of acid esterase. Enzyme values revealed no characteristic distribution among the lymphoma entities. The isoenzyme pattern, specific to normal human B-lymphocytes, were regularly detectable in all lymphoma entities. The results document the B-cell origin of the analyzed subsets of B-cell malignancies, although distinctive acid esterase patterns were lacking. The prevalence of three anodal isoenzymes in cases of chronic B-lymphocytic leukemia, hardly detectable in other lymphoma entities, were interpreted as the expression of a clonal proliferation, arrested at a certain differentiation stage of B-cells common for the majority of the tumor cells. Hence, further evidence is provided supporting the view that the studied entities represent B-cell neoplasias at different stages of differentiation expressing a variety of different markers.