Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment.

SUMMARYThe plasma membrane H + -ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H + -ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H + -ATPase subfamilies having different properties.

Bone marrow samples of 16 patients (two adults and 14 children) with a B lineage acute lymphoblastic leukaemia (ALL), and in whom Ig heavy chain gene rearrangements were detectable at diagnosis using polymerase chain reaction (PCR), were studied during evolution using PCR. The VDJ junctional fragment of the Ig heavy chain rearranged gene was amplified at diagnosis. After length reduction by restriction digestion, the amplified fragment was recovered by chromatography, labelled using a specific hexamer as a primer and directly used as a clonospecific probe. The sensitivity of the PCR ranged from 1:10(4) to 1:10(5) cells, depending on the patient's rearrangement. Residual disease (MRD) was detected in most of the patients achieving a complete remission after induction therapy, regardless of the long-term outcome of treatment. However, in patients remaining in complete remission, the level of MRD showed a tendency to decrease and ultimately become undetectable for variable periods of time, while in patients eventually relapsing there was a trend for MRD to persist at stable levels and even to increase before relapse was clinically evident. We conclude that the use of a simplified methodology for obtaining a clonospecific probe from the Ig heavy chain gene, though less sensitive than the sequencing methodology, is a valuable and readily available tool to monitor MRD in a high proportion of B lineage ALL.

Immunosuppressive drugs significantly affect proliferation and viability of MSCs, but neither TAC nor SIR had a detrimental impact on MSCs' immunomodulatory properties. In this large-animal model, addition of short course of immunosuppression seems to overcome the immune response to intracardiac allogeneic MSCs, which was recently demonstrated to occur in the absence of immunosuppression.

We describe here the potent specific immunosuppression obtained in vitro by LO-CD2a, a rat mAb directed against the human CD2 molecule. Addition of low dose LO-CD2a (40 ng/ml) at the time of mixed lymphocyte culture (MLC) initiation inhibits 80% of the proliferation and, more impressive, addition of the mAb 4 days after culture initiation at a similar concentration still suppresses 50% of the MLC. When responder T cells previously treated with LO-CD2a are challenged a second time by the same donor or third party allogeneic cells, hyporesponsiveness occurs in both cases, although reactivity to T cell mitogenic stimulation persists. Finally, the low production of cytokines such as tumor necrosis factor-alpha and IFN-gamma after incubation of human T cells with LO-CD2a suggests the absence of T cell activation. These results demonstrate that LO-CD2a mAb has a significant immunosuppressive effect and induces hyporesponsiveness in vitro, thereby suggesting potential efficacy in vivo for the treatment of acute rejection and for the induction of tolerance in allotransplantation.