Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis.

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.

Initial treatment of acute graft-versus-host disease with a murine monoclonal antibody directed to the human alpha/beta T cell receptor.

A murine IgG2b monoclonal antibody directed to the constant part of the human alpha/beta T cell receptor (BMA031) was investigated in a pilot study as an initial treatment for acute graft-versus-host disease (aGvHD) after allogeneic bone marrow transplantation. The treatment protocol consisted of 5 mg BMA031 on 5 consecutive days with continuation of the prophylactic baseline immuno suppression using cyclosporin. Seven patients with grades II-III acute graft-versus-host disease were entered on the protocol and six patients completed the full treatment course. Mild to moderate acute adverse reactions to the first BMA031 infusion occurred in three patients. A nearly complete decline of circulating T lymphocytes was observed during BMA031 therapy, but the T cells returned to pretreatment values within 1 week after the last infusion. Serum pharmacokinetics of free antibody best fitted to a two-compartment open model with a mean initial half-life of 6 h and an estimated mean terminal half-life of 40 h. One patient developed antimurine antibodies of the IgM subclass. In five patients a complete and sustained resolution of all disease manifestations was attained, while in one patient a temporary response of skin involvement with aGvHD was noted. These results indicate that BMA031 can be safely administered as initial treatment of aGvHD. The therapeutic responses observed warrant its further clinical evaluation in this setting.