Transplantation of CD6-depleted peripheral blood stem cells after DLA-haploidentical bone marrow transplantation contributes to engraftment and tolerance in a preclinical model of stem cell transplantation

Human leukocyte antigen (HLA)-haploidentical stem cell transplantation is an opportunity for nearly all patients lacking an HLA matched stem cell donor. However, graft rejection and graft-versus-host disease (GvHD) as well as infectious complications still result in high treatment-related mortality. Here, we used the dog as a preclinical model for the study of tolerance induction with the aim to optimize and to improve a clinical protocol of haploidentical stem cell transplantation. For this purpose CD6-depleted peripheral blood stem cells (PBSCs) were transfused 6d after transplantation of unmodified bone marrow from dog leukocyte antigen (DLA)-haploidentical littermate donors in order to induce immune tolerance. Besides hematopoietic stem cells CD6-depleted PBSC contain, NK cells and a minority of suppressive CD8-positive cells that may suppress activated T lymphocytes. Recipients were conditioned with, cyclophosphamide and antithymocyte globulin (ATG) preceded by a transfusion of donor buffy coat and either 1, 2 or 3 × 3.3 Gy total body irradiation (TBI). Postgrafting immunosuppression was limited to 30 d of cyclosporine and methotrexate. The additional administration of CD6-depleted PBSCs after unmodified marrow could not prevent GvHD, but it may improve engraftment and chimerism after conditioning with 2 × 3.3 Gy TBI. Reasons for incomplete suppression and possible improvements for clinical applications are discussed.     

The impact of extraction methodologies on the toxicity of sediments in the zebrafish (Danio rerio) embryo test

Purpose

Traditionally, methods for sediment extractions are characterised using chemical analyses. However, in order to evaluate sediment extracts with regard to biological effects and, thus, bioaccessibility, extraction methods have to be compared to effect data obtained from experiments with in situ exposure scenarios, i.e., sediment contact tests. This study compares four extraction methods and sediment contact test data from a previous project with respect to predictive power in the fish embryo test with zebrafish (Danio rerio).

Materials and methods

A natural and an artificial sediment spiked with a mixture of six organic pollutants (2,4-dinitrophenol, diuron, fluoranthene, nonylphenol, parathion and pentachlorophenol) were extracted using (a) membrane dialysis extraction (MDE), (b) a Soxhlet procedure, (c) hydroxypropyl-??-cyclodextrin (HPCD) or (d) Tenax^®-TA. Whereas the former two are regarded being exhaustive with respect to non-covalently bound contaminants, the latter two are considered to predict bioaccessibility. Resulting extracts were tested in the fish embryo assay with D. rerio for embryotoxic and teratogenic potential.

Results and discussion

Mortalities caused by organic extracts from Soxhlet extraction and MDE were high. However, HPCD extracts turned out to be at least as effective as extracts obtained with these two methods. One possible reason might be short ageing time of the spiked sediments. Only Tenax^®-TA extracts gave results comparable to the sediment contact assay for natural sediment, but revealed low reproducibility. Significant differences between natural and artificial sediment were found for extracts obtained with techniques using native (i.e., non-freeze-dried) sediments, i.e., HPCD and Tenax^®-TA. In contrast, MDE and Soxhlet extracts used freeze-dried sediment and did not differentiate between natural and artificial sediment. Therefore, freeze-drying has likely altered and equalised sediment properties that influence accessibility, such as composition of bacterial communities and organic matter quality.

Conclusions

Four extraction methods were successfully characterised with respect to their stringency and predictiveness for bioaccessibility. MDE was confirmed as an alternative to Soxhlet extraction. High mortalities induced by HPCD extracts underline the need to include ageing into consideration when assessing sediments. Although Tenax^®-TA may basically be used to predict bioaccessibility in the fish embryo test, the high variability observed warrants further investigation of the relation between effect and extractability. Apparently, freeze-drying can severely affect sediment properties, potentially eliminating individual properties of natural sediments.     

Schwermetallgehalte in landwirtschaftlich relevanten stoffen

Die Gehalte für die sechs Schwermetalle Cd, Cr, Cu, Ni, Pb und Zn in landwirtschaftlich relevanten Stoffgruppen (mineralische und organische Düngestoffe, Kulturpflanzen und tierische Produkte) wurden aus Literaturdaten für die beiden statistischen Maßzahlen Mittelwert und Spannweite der Mittelwerte in Übersichtstabellen zusammengefasst.     

Yield and tuber characteristics of 4x progeny from 2x×2x crosses

Summary Unrelated, unselected 2x haploid-species hybrids that produce 2n eggs, by second division restitution and 2n pollen due to first division restitution were intercrossed to generate 4x progeny. The 19, 4x families were compared with seven cultivars for tuber yield, tuber appearance and tuber set. The mean yield of the 4x families was 30% higher than the mean yield of the cultivars. The best five 4x families outyielded the best five cultivars by 57% and 69% at two locations. Cultivars were superior for tuber appearance and tuber set. The high yields of the 4x families are due to genetic diversity of the 2x parents and their ability to transmit that diversity to the 4x progeny utilizing 2n gametes. The potential of the 2x×2x breeding method is dependent on developing 2x hybrid clones that form 2n eggs and possess desirable tuber types; clones producing 2n pollen and good tuber quality are available.