Inhibition of antigen-specific immune responses by co-application of an indoleamine 2,3-dioxygenase (IDO)-encoding vector requires antigen transgene expression focused on dendritic cells.

We have previously shown that particle-mediated epidermal delivery (PMED) of plasmids encoding ß-galactosidase (ßGal) under control of the fascin-1 promoter (pFascin-ßGal) yielded selective production of the protein in skin dendritic cells (DCs), and suppressed Th2 responses in a mouse model of type I allergy by inducing Th1/Tc1 cells. However, intranasal challenge of mice immunized with pFascin-ßGal induced airway hyperreactivity (AHR) and neutrophilic inflammation in the lung. The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been implicated in immune suppression and tolerance induction. Here we investigated the consequences of co-application of an IDO-encoding vector on the modulatory effect of DNA vaccination by PMED using pFascin-ßGal in models of eosinophilic allergic and non-eosinophilic intrinsic airway inflammation. IDO-encoding plasmids and pFascin-ßGal or pCMV-ßGal were co-applied to abdominal skin of BALB/c mice without, before or after sensitization with ßGal protein. Immune responses in the lung were analysed after intranasal provocation and airway reactivity was determined by whole body plethysmography. Co-application of pCMV-IDO with pFascin-ßGal, but not pCMV-ßGal inhibited the Th1/Tc1 immune response after PMED. Moreover, AHR in those mice was attenuated following intranasal challenge. Therapeutic vaccination of ßGal-sensitized mice with pFascin-ßGal plus pCMV-IDO slightly suppressed airway inflammation and AHR after provocation with ßGal protein, while prophylactic vaccination was not effective. Altogether, our data suggest that only the combination of DC-restricted antigen and ubiquitous IDO expression attenuated asthma responses in mice, most probably by forming a tryptophan-depleted and kynurenine-enriched micromilieu known to affect neutrophils and T cells.

New parallel wavelength-dispersive spectrometer based on scanning electron microscope.

A new wavelength - dispersive X-ray spectrometer for scanning electron microscopy (SEM) has been developed. This spectrometer can cover an energy range from 50 eV to 1120 eV by using an array made of seventeen reflection zone plates. Soft X-ray emission spectra of simple elements of Li, Be, B, C, N, Ti, V, O, Cr, Mn, Fe, Co, Ni, Cu, Zn and Ga were measured. The overall energy resolving power on the order of E/ΔE ~80 to 160 has been demonstrated. Spectrometer with 200 reflection zone plates has been used as a multi-channel analyser in the energy range of 100 - 1000 eV for quasi - continuous spectra measurements. The predicted energy-resolving power on the order of E/ΔE = 50 has been achieved in the entire energy range.

Dendritic cell-specific biolistic transfection using the fascin gene promoter.

The transcriptional targeting of gene expression to selected cells by cell type-specific promoters displays a fundamental tool in gene therapy. In immunotherapy, dendritic cells (DCs) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection. Here we report on transcriptional targeting of murine skin DCs using plasmids which include the promoter of the gene of the cytoskeletal protein fascin to control antigen production. Fascin, which is mandatory for the formation of dendrites, is synthesized among the hematopoietic cells exclusively by activated DCs. The activity of the promoter of the fascin gene reflects the endogenous production of the protein, being high in mature DCs but almost absent in immature DCs or other cutaneous cells. Here we describe the analysis of transgene-specific immune responses after DC-focused biolistic transfection. In conclusion, the murine fascin promoter can be readily used to target DCs in DNA immunization approaches and thus offers new opportunities for gene therapy.

Individual dose and scheduling determine the efficacy of combining cytotoxic anticancer agents with a kinase inhibitor in non-small-cell lung cancer.

PURPOSE: To investigate the combination of conventional cytotoxic anticancer agents and a small molecule kinase inhibitor in preclinical models of non-small-cell lung cancer (NSCLC). METHODS: We compared the induction of apoptosis by DNA-damaging anticancer drugs and PKC412, a predominantly protein kinase C (PKC)-specific small molecule inhibitor, in six NSCLC cell lines of different histologic and genetic backgrounds. The outcome of various combinations and schedules of DNA-damaging agents and PKC412 was studied, and isobolograms were calculated. Conditional expression of pro-apoptotic BAK was applied to specifically target apoptotic signal transduction in combination with drug therapy. RESULTS: Resistance of NSCLC cells to DNA damage-induced apoptosis was mainly determined at the mitochondrial step of the intrinsic pathway of caspase activation. PKC412 effectively inhibited the growth factor signal transduction, but failed to induce apoptosis in NSCLC cells resistant to DNA-damaging agents. Combining conventional anticancer drugs with PKC412 at different doses and schedules resulted in unpredictable outcomes, including synergistic, additive, and antagonistic interactions. In contrast, conditional expression of BAK reliably sensitized drug-resistant NSCLC cells to apoptosis induced by cytotoxic agents or PKC412. CONCLUSIONS: Combining DNA-damaging anticancer drugs with a pharmacologic inhibitor of growth and survival factor signaling in NSCLC may result in unpredictable treatment outcomes. In contrast, targeting specific death effector mechanisms, such as apoptotic signal transduction, is a promising strategy to sensitize NSCLC to cytotoxic agents or kinase inhibitors.

Induction of tolerogenic lung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitor ameliorated experimental allergic asthma.

Signal transducer and activator of transcription (STAT)-3 inhibitors play an important role in regulating immune responses. Galiellalactone (GL) is a fungal secondary metabolite known to interfere with the binding of phosphorylated signal transducer and activator of transcription (pSTAT)-3 as well of pSTAT-6 dimers to their target DNA in vitro. Intra nasal delivery of 50 µg GL into the lung of naive Balb/c mice induced FoxP3 expression locally and IL-10 production and IL-12p40 in RNA expression in the airways in vivo. In a murine model of allergic asthma, GL significantly suppressed the cardinal features of asthma, such as airway hyperresponsiveness, eosinophilia and mucus production, after sensitization and subsequent challenge with ovalbumin (OVA). These changes resulted in induction of IL-12p70 and IL-10 production by lung CD11c(+) dendritic cells (DCs) accompanied by an increase of IL-3 receptor α chain and indoleamine-2,3-dioxygenase expression in these cells. Furthermore, GL inhibited IL-4 production in T-bet-deficient CD4(+) T cells and down-regulated the suppressor of cytokine signaling-3 (SOCS-3), also in the absence of STAT-3 in T cells, in the lung in a murine model of asthma. In addition, we found reduced amounts of pSTAT-5 in the lung of GL-treated mice that correlated with decreased release of IL-2 by lung OVA-specific CD4(+) T cells after treatment with GL in vitro also in the absence of T-bet. Thus, GL treatment in vivo and in vitro emerges as a novel therapeutic approach for allergic asthma by modulating lung DC phenotype and function resulting in a protective response via CD4(+)FoxP3(+) regulatory T cells locally.

Dendritic cells lentivirally engineered to overexpress interleukin-10 inhibit contact hypersensitivity responses, despite their partial activation induced by transduction-associated physical stress.

BACKGROUND: Dendritic cells (DCs) constitute an attractive target for immunotherapeutic approaches. Because DCs are largely refractory to transfection with plasmid DNA, several viral transduction protocols were established. The potential side-effects of lentiviral transduction on the phenotype and activation state of DCs left unstimulated after transduction have not been assessed. There is a need to analyse these parameters as a result of the requirement of using DCs with a low activation state for therapeutic strategies intended to induce tolerance. METHODS: Lentivirally-transduced bone marrow (BM)-derived DCs (LV-DCs) in comparison with mock-transduced (Mock-DCs) and untreated DCs were analysed with regard to the induction of maturation processes on the RNA, protein and functional level. BM-DCs engineered to overexpress interleukin (IL)-10 were analysed for therapeutic potential in a mouse model of allergic contact dermatitis. RESULTS: Compared with untreated DCs, Mock-DCs and LV-DCs displayed an altered gene expression signature. Mock-DCs induced a stronger T cell proliferative response than untreated DCs. LV-DCs did not further augment the T cell proliferative response, but induced a slightly different T cell cytokine pattern compared to Mock-DCs. Accordingly, the gene promoter of the DC maturation marker fascin mediated efficient expression of the model transgene IL-10 in unstimulated-transduced BM-DCs. Nevertheless, IL-10 overexpressing BM-DCs exerted tolerogenic activity and efficiently inhibited the contact hypersensitivity response in previously hapten-sensitized mice. CONCLUSIONS: Lentiviral transduction of BM-DCs results in their partial activation. Nevertheless, the transduction of these DCs with a vector encoding the immunomodulatory cytokine IL-10 rendered them tolerogenic. Thus, lentivirally-transduced DCs expressing immunomodulatory molecules represent a promising tool for induction of tolerance.

Development of a nondestructive method for underglaze painted tiles--demonstrated by the analysis of Persian objects from the nineteenth century.

The paper presents an analytical method developed for the nondestructive study of nineteenth-century Persian polychrome underglaze painted tiles. As an example, 9 tiles from French and German museum collections were investigated. Before this work was undertaken little was known about the materials used in pottery at that time, although the broad range of colors and shades, together with their brilliant glazes, made these objects stand out when compared with Iranian ceramics of the preceding periods and suggested the use of new pigments, colorants, and glaze compositions. These materials are thought to be related to provenance and as such appropriate criteria for art-historical attribution. The analytical method is based on the combination of different nondestructive spectroscopic techniques using microfocused beams such as proton-induced X-ray emission/proton-induced gamma-ray emission, X-ray fluorescence, 3D X-ray absorption near edge structure, and confocal Raman spectroscopy and also visible spectroscopy. It was established to address the specific difficulties these objects and the technique of underglaze painting raise. The exact definition of the colors observed on the tiles using the Natural Color System helped to attribute them to different colorants. It was possible to establish the presence of Cr- and U-based colorants as new materials in nineteenth-century Persian tilemaking. The difference in glaze composition (Pb, Sn, Na, and K contents) as well as the use of B and Sn were identified as a potential marker for different workshops.