Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-alpha/IL-1/IL-6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)-gamma and no IL-4 or IL-10. Production of IFN-gamma was significantly induced by addition of PGE2, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN-gamma(low), IL-4(neg) cells, TNF-alpha/IL-1/IL-6 promoted growth of IFN-gamma(intermediate), IL-4(neg) CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN-gamma production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-alpha/IL-1/IL-6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF-alpha/IL-1, IL-6 + PGE2-cultured DC seem to be optimal for generation of IFN-gamma-producing CD4/CD8+ T cells.

Indoleamine 2,3-dioxygenase (IDO) is an interferon-g (IFN-g)-induced enzyme, which is suggested to play an important role in the prevention of allogeneic fetal rejection. IDO effects the suppression of T-cell activity by catabolizing the essential amino acid L-tryptophan. We studied IDO expression by reverse transcription polymerase chain reaction (RT-PCR) in dendritic cells and by real-time RT-PCR in monocytes of patients undergoing allogeneic transplantation for leukaemia, who developed acute graft-versus-host disease (aGvHD), and compared the IDO expression with that of pregnant women and healthy volunteers.A spontaneous IDO expression was detected in the monocytes of 20 pregnant women with an IDO/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio at a median of 1.0%, whereas none of 15 healthy volunteers or patients after allogeneic transplant had any detectable spontaneous IDO expression. The IDO expression increased by in vitro IFN-g stimulation in pregnant women (median 116%), healthy volunteers (median 11.7%) and patients with a lowgrade aGvHD (grades 0±II) 28 days after transplant (median 433%) but not in patients with a severe aGvHD (grades III±IV) (median 0%), which was highly significant (P < 0.01).IDO expression was also measured in dendritic cells by qualitative RT-PCR, where a spontaneous IDO expression was detected in 16 of 31 (52%) pregnant women versus none of 17 healthy volunteers and none of 62 studied patients after transplant. IFN-g-induced IDO expression was detected in all pregnant women, all volunteers and 47 of 49 (96%) patients with a low-grade aGvHD (grades 0±II) after transplant, whereas only in two of 13 (16%) patients with aGvHD grade III±IV was IFN-g-induced IDO expression observed.These data suggest that IDO expression might be involved in the development of allogeneic immune tolerance.

The block ciphers MISTY1 and MISTY2 proposed by Matsui are based on the principle of provable security against differential and linear cryptanalysis. This paper presents attacks on reduced-round variants of both ciphers, without as well as with the key-dependent linear functions FL. The attacks employ collision-searching techniques and impossible differentials. KASUMI, a MISTY variant to be used in next generation cellular phones, can be attacked with the latter method faster than brute force when reduced to six sounds.

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture. Achieving persistent and high-level gene expression in a significant percentage of target cells is important for establishing successful clinical applications of gene therapy. For gene therapy of the skin, a renewable tissue undergoing constant turnover, and for achieving long-term expression in a significant percentage of keratinocytes, gene targeting to keratinocyte stem cells (KSC) or long-lasting keratinocyte progenitor cells will be required (36). In ex vivo skin gene therapy, keratinocytes are removed from the donor and transduced with retroviral vectors in vitro without selectively targeting KSC. The genetically modified keratinocytes can then be grafted back onto the donor. Even though progress in skin gene therapy has enabled gene expression from retroviral vectors to be detected for sustained periods in grafted keratinocytes (9,24,32,56), transgene expression in vivo is frequently decreased over time, either in the duration of expression or in the percentage of keratinocytes expressing the gene (12,14,18,35,50). The factors contributing to this decreased expression may be similar to those for other tissues (14,27,41,51) and may include in...