The current gene transfer technology for single chain (scFv)-based chimeric immune receptor (CIR) has relied on retrovirus and lentivirus vectors which require a long time to obtain sufficient number of transduced cells and stably incorporate into genome. To ameliorate these limitations, we applied RNA electroporation to human peripheral blood lymphocytes (PBLs) activated with anti-CD3 antibody and interleukin-2 (IL-2) for 3 days and assessed that PBL transiently expressing anti-Her-2/neu CIR (CIR-PBL) containing signaling portion of CD28 and CD3z could elicit strong cytotoxicity in vitro and antitumor responses in vivo. The CIR-PBL expressed high level of CIR in CD4 þ , CD8 þ and CD56 þ cells. Her-2/neu-specific stimulation induced secretion of type-I cytokines including interferon-g (IFN-g), IL-8 and granulocyte-macrophage colony-stimulating factor, and IFN-g secretion was mainly mediated by CD8 þ T cells. CIR-PBL specifically killed SKOV3 cell line expressing Her-2/neu. Adoptive transfer of CIR-PBL in SKOV3 xenograft model led to significant inhibition of tumor growth compared with transfer of mock-transduced PBL and showed higher inhibition than those with Herceptin, humanized monoclonal antibody specific for Her-2/neu. These results provided evidence that electroporation of CIR RNA to human PBLs could be used for rapid generation and high number of therapeutic antigen-specific T cells for adoptive immunotherapy.

Ras proteins control signalling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. BRAF, which encodes an RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers. The activating mutation of BRAF is known to play a role in tumour development. As there have been no data on the BRAF mutation in non-Hodgkin's lymphoma (NHL), we analysed the genomic DNAs from 164 NHLs by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) for the detection of somatic mutations of BRAF (exons 11 and 15). Overall, we detected BRAF mutations in four NHLs (2.4%). Whereas most BRAF mutations in human cancers involved V599 of BRAF, all of the four BRAF mutations in the NHLs involved other amino acids (one G468A, two G468R and one D593G). To our knowledge, this is the first report on BRAF mutation in NHL, and the data indicate that BRAF is occasionally mutated in NHL, and suggest that BRAF mutation may contribute to the tumour development in some NHLs.

SUMMARYThe effects of 3-deazaadenosine (DZA), 3-deaza(Ϯ)-aristeromycin (DZAri) and 3-deazaneplanocin (DZNep) on tumour necrosis factor-␣ (TNF-␣) production were examined in the mouse macrophage cell line, RAW264.7, stimulated with lipopolysaccharide (LPS). The 3-deazaadenosine analogues inhibited the TNF-␣ production and the inhibition was dependent upon the concentration of the analogue. DZA reduced the level of TNF-␣ mRNA suggesting that DZA acts at a transcriptional step. In contrast, DZAri and DZNep had little effect on mRNA levels for TNF-␣, implying that these compounds inhibit a post-transcriptional or translational biosynthetic step of TNF-␣ synthesis. The observation that homocysteine (Hcy) potentiated the DZA inhibition of TNF-␣ production and of TNF-␣ mRNA levels suggests that the inhibition of TNF-␣ production may be caused by elevated levels of 3-deazaadenosylhomocysteine (DZAHcy). The results show that the 3-deazaadenosine analogues are potent inhibitors of TNF-␣ production in the RAW264.7 cell line stimulated with LPS and suggest that these analogues may be effective agents for the treatment of diseases in which TNF-␣ plays an important pathogenic role.

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