[The factors of improving rice transformation efficiency].

The factors influencing the rice transformation frequency have been investigated by using 8 indica and japonica rice cultivars during the transformation procedures, such as induction of calli, selection, and regeneration. The following is the results: The immature embryos were pretreated in 4 degrees C for 4 or 7 days before inoculation, and the regeneration frequency of calli would be increased. High osmotic treatment may increase the possibility of transformation, especially for calli of 6 months or older. The transformation frequency was improved when selection pressure was removed during differentiation period. The V-type selection may be better than the normal selection. There is a difference of differentiation ability among the calli of three periods and it is found that the calli of two or three months are the best for transformation. Proline and DMSO can decrease browning rate of calli.

[Improvement of transformation frequency of rice mediated by Agrobacterium].

The factors influencing the frequency of rice transformation mediated by Agrobacterium have been investigated by using 16 commercially important indica and japonica rice cultivars or lines. The main results were as following: For most rice CC medium was the best for both callus initiation and subculture. With supplement of 2.5-5 mg/L ABA the quality of calli can be improved. The concentration of selective agent for Indica rice callus was lower than that for japonica rice callus. Agrobacterium tumefaciens strain EHA105 was more efficient than LBA4404 and AGL1 for rice transformation. The inhibitive effect of cefotaxime to Agrobacterium was better than that of carbenicillin. The partial desiccation treatment after co-cultivation was beneficial to inhibit the growth of Agrobacterium and increase transformation efficiency. A stable and efficient Agrobacterium-mediated transformation system has been established in ten different rice cultivars and fertile transgenic plants have been obtained.

[Stable inheritance of hpt gene in transgenic rice plants mediated by biolistic bombardment].

Japonica rice cv 77170 was transformed with pBI222 carrying hygromycin phosphotransferase gene (hpt) by using biolistic bombardment and fertile transgenic rice plants were obtained. In T1 and most T2 generation the performance of hygromycin resistance was normal. Only in T2 progeny of SG-15 some lines showed decreased resistance. Mendelian inheritance of hygromycin resistance was showed as single dominant locus and proved by Southern blotting analysis in T1 and T2 generation of all transgenic plants. Multiple copies of hpt integrated into rice genome. These copies linked closely and inherited stablly. Msp I digested Southern blotting showed that methylation of hpt existed commonly in transgenic plants and their progeny, therefore uncomplete silencing of hpt in some T2 progeny of SG-15 was not associated with hpt methylation probably.

Linomide (roquinimex) affects the balance between pro- and anti-inflammatory cytokines in vitro in multiple sclerosis.

OBJECTIVES: Multiple sclerosis (MS) is characterized by high levels of circulating mononuclear cells (MNC) that respond to myelin proteins like myelin basic protein (MBP) in vitro by expressing mRNA of both pro-inflammatory cytokines, e.g. interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT) that may make MS worse, and anti-inflammatory cytokines like IL-4, IL-10 and transforming growth factor-beta (TGF-beta) that may act beneficially. Substances that down-regulate cytokines such as TNF-alpha or promote IL-10 or TGF-beta can be anticipated to affect MS beneficially. MATERIAL AND METHODS: In situ hybridization to detect and enumerate IFN-gamma, TNF-alpha, LT, IL-4, IL-10 and TGF-beta mRNA expressing blood MNC after stimulation with myelin basic protein (MBP), control antigens and without antigen in presence and absence of Linomide (roquinimex, LS-2616) was employed. In parallel, ELISPOT assay to detect MBP- and PHA-reactive IFN-gamma secreting blood MNC+/-Linomide was used. RESULTS: Here we report that Linomide, a synthetic immunomodulator, at concentrations effective in vivo reduces the number of MBP-reactive TNF-alpha and increases MBP-reactive IL-10 and TGF-beta mRNA expressing MNC from MS patients' blood when analysed in vitro. Compared to dexamethasone, Linomide up-regulated levels of blood MNC expressing mRNA of TGF-beta after culture in presence of MBP. CONCLUSIONS: Changes of cytokine balance towards a production of anti-inflammatory cytokines could be a desirable effect to be evaluated in future drug studies of Linomide-like substances. At present, Linomide is not evaluable in MS clinical trials due to side-effects.

Neutralising and binding anti-interferon-beta-I b (IFN-beta-I b) antibodies during IFN-beta-I b treatment of multiple sclerosis.

Interferon-beta-I b (IFN-beta-I b) is an immunomodulatory therapy of multiple sclerosis (MS), reducing the numbers and severity of exacerbations and the total lesion load measured by magnetic resonance imaging of the brain. The benefits of IFN-beta-I b could be hampered by the development of neutralising antibodies against the compound. Our results confirmed earlier studies, showing that 42% of MS patients treated with IFN-beta-I b for more than 3 months had developed neutralising antibodies. The occurrence of binding anti-IFN-beta-I b antibodies, presently not believed to impede the clinical efficacy of IFN-beta-I b, were demonstrated by an immunoassay in some patients after 1 month of treatment and in 78% after 3 months. The development of binding antibodies seemed to be an early phenomenon, preceding the appearance of neutralising antibodies. Antibodies crossreacting with IFN-beta-I a and natural IFN-beta were also found in a majority of IFN-beta-I b treated patients with high titres of binding antibodies. Employing a solid-phase enzyme-linked immunospot (ELISPOT) assay, 68% of MS patients treated with IFN-beta-I b for 1-23 months had elevated numbers of anti-IFN-beta-I b-antibody secreting cells in blood, compared to 18% of untreated MS patients and 20% among patients with other neurological diseases. Thus, our findings confirm that IFN-beta-I b is immunogenic in MS patients. High levels of anti-IFN-beta-I b antibody secreting cells were, however, also found in two untreated control patients with inflammatory diseases, suggesting that anti-IFN-beta-I b antibodies might also occur spontaneously.