IntroductionMetastatic renal cell cancer (RCC) has a very poor prognosis with a median survival of only 6 to 12 months from the time of diagnosis. 1,2 Historically, there were no established effective treatment approaches for metastatic RCC because of its resistance to radiation and chemotherapy. 3 Until recently, cytokine-based immunotherapy using interferon-␣ (IFN-␣) and/or interleukin-2 (IL-2) was the only effective treatment resulting in response rates of 10% to 20%. 4 The understanding of RCC pathogenesis and identification of molecular mechanisms responsible for the malignant transformation and metastatic spread led to the development of drugs that target cancer-specific pathways, such as the PI3K/AKT and Ras/Raf/MAPK pathways. 5 RCC is often associated with up-regulated Raf1, EGFR, and VEGFR activity. 5,6 Furthermore, in a high proportion of RCC, mutational aberrations of the von Hippel-Lindau (VHL) gene were identified. The loss-of-function of this tumor suppressor gene results in an accumulation of hypoxia inducible factor (HIF)-␣ subunits and stimulation of angiogenesis via VEGF-and PDGF-receptors.Consequently, 2 novel tyrosine kinase inhibitors, sorafenib (Bayer HealthCare, Leverkusen, Germany) 5 and sunitinib (Pfizer, New York, NY), were introduced in the treatment of RCC patients.Sorafenib is a multikinase inhibitor initially developed to inhibit the Raf1-kinase pathway. 2 However, besides the RAF/MEK/ERK pathway, sorafenib targets receptor tyrosine kinases (RTKs), such as VEGFR-2 and -3, PDGFR-␤, Flt-3, and c-KIT. 2,7 In several clinical and preclinical trials, sorafenib was revealed to be a promising anticancer therapeutic, which negatively regulates tumor growth, cell proliferation, and angiogenesis 8,9 and additionally induces apoptosis in tumor cells. 10 In December 2005, sorafenib was approved by the FDA for treatment of patients with advanced/ metastatic RCC. In a randomized trial, sorafenib doubled the median duration of progression-free survival up to 24 weeks in patients refractory to or relapsed during cytokine treatment. 2,11,12 Sunitinib inhibits multiple split kinase domain RTKs, including VEGFR-1 and -2, PDGFR-␣ and -␤, c- 7,13 In 2 phase 2 studies, application of sunitinib resulted in response rates up to 40% and in a randomized phase 3 trial it showed an improved response rate and progression-free survival in comparison to 14,15 However, until now the effects of sorafenib and sunitinib on development and function of normal nonmalignant hematopoetic cells have not been evaluated in detail. It is known that PBLs isolated from patients receiving clinically relevant doses of sorafenib show inhibition of ERK phosphorylation on ex vivo PMA stimulation. 16 We therefore analyzed the immunomodulatory functions of these compounds using T cells and monocyte-derived dendritic cells (MDDCs), which were activated with ligands for TLR3 or 4. We found that sorafenib, but not sunitinib, has a detrimental effect on DC phenotype and inhibits cytokine secretion, migration ability, and T-cell stimulator...

Evidence from the animal model suggests that proteasome inhibitors may have immunosuppressive properties; however, their effects on the human immune system remain poorly investigated. Here, we show that bortezomib, a proteasome inhibitor with anticancer activity, impairs several immune properties of human monocyte-derived dendritic cells (DCs). Namely, exposure of DCs to bortezomib reduces their phagocytic capacity, as shown by FITC-labeled dextran internalization and mannose-receptor CD206 down-regulation. DCs treated with bortezomib show skewed phenotypic maturation in response to stimuli of bacterial (lipopolysaccharide [LPS]) and endogenous sources (including TNF-␣ and CD40L), as well as reduced cytokine production and immunostimulatory capacity. LPS-induced CCL-2/MCP-1 and CCL5/ RANTES secretions by DCs were prevented by DC treatment with bortezomib. Finally, CCR7 up-regulation in DCs exposed to LPS as well as migration toward CCL19/MIP-3␤ were strongly impaired. As a suitable mechanism for these effects, bortezomib was found to down-regulate MyD88, an essential adaptor for TLR signaling, and to relieve LPS-induced activation of NF-B, IRF-3, and IRF-8 and of the MAP kinase pathway. In summary, inhibition of DC function may represent a novel mechanism by which proteasome inhibitors exert immunomodulatory effects. IntroductionPharmacologic inhibitors of the proteasome have recently received much attention in the light of their potent antitumor activity. 1,2 In this setting, bortezomib (Velcade) was demonstrated to be effective in the treatment of multiple myeloma, when taken alone or in combination with traditional anticancer drugs. 3,4 Besides, preliminary evidence indicates that also patients with non-Hodgkin lymphoma, acute leukemia, or some type of solid tumors may take advantage of treatment with this kind of drugs. [5][6][7][8][9] The mechanism through which proteasome inhibitors prevent tumor growth is not completely understood even though inhibition of NF-B was suggested to play a relevant role, especially in those malignancies in which this transcription factor is constitutively activated, such as multiple myeloma. 1,2 Cumulating evidence indicating the importance of the ubiquitinproteasome pathway in different aspects of the immune response, including antigen processing, apoptosis, cell cycle, costimulation, adhesion, and chemotaxis, has fostered the evaluation of proteasome inhibitors as immunosuppressive agents. 10 Evidence from the animal model indicates a potential role for proteasome inhibitors in the treatment of graft-versus-host disease. [11][12][13][14] Similarly, these drugs proved useful in the animal model for the prevention of allograft rejection, arthritis, experimental autoimmune encephalomyelitis, and psoriasis. 10,[15][16][17] Whether proteasome inhibitors affect the immune function in humans remains controversial, since this kind of evaluation is hampered by the pre-existing immunodepression or by heavy pretreatment of the patients in the clinical studies carried out to date. Howe...

Gluconobacter oxydans, a biotechnologically relevant species which incompletely oxidizes a large variety of carbohydrates, alcohols, and related compounds, contains a gene for pyruvate decarboxylase (PDC). This enzyme is found only in very few species of bacteria where it is normally involved in anaerobic ethanol formation via acetaldehyde. In order to clarify the role of PDC in the strictly oxidative metabolism of acetic acid bacteria, we developed a markerless in-frame deletion system for strain G. oxydans 621H which uses 5-fluorouracil together with a plasmid-encoded uracil phosphoribosyltransferase as counter selection method and used this technique to delete the PDC gene (GOX1081) of G. oxydans 621H. The PDC deletion mutant accumulated large amounts of pyruvate but almost no acetate during growth on D-mannitol, D-fructose or in the presence of L-lactate. This suggested that in G. oxydans acetate formation occurs by decarboxylation of pyruvate and subsequent oxidation of acetaldehyde to acetate. This observation and the efficiency of the markerless deletion system were confirmed by constructing deletion mutants of two acetaldehyde dehydrogenases (GOX1122 and GOX2018) and of the acetyl-CoA-synthetase (GOX0412). Acetate formation during growth of these mutants on mannitol did not differ significantly from the wild-type strain.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by excessive granulopoiesis due to the formation of the constitutively active tyrosine kinase BCR-ABL. An effective drug against CML is imatinib mesylate, a tyrosine kinase inhibitor acting on Abl kinases, c-KIT, and plateletderived growth factor receptor. Recently, a study revealed that patients treated with imatinib showed impaired CTL responses compared with patients treated with IFN-A, which might be due to a treatment-induced reduction in immunogenicity of CML cells or immunosuppressive effects. In our study, we found that inhibition of BCR-ABL leads to a down-regulation of immunogenic antigens on the CML cells in response to imatinib treatment, which results in the inhibition of CMLdirected immune responses. By treating CML cells with imatinib, we could show that the resulting inhibition of BCR-ABL leads to a decreased expression of tumor antigens, including survivin, adipophilin, hTERT, WT-1, Bcl-x L , and Bcl-2 in correlation to a decreased development of CML-specific CTLs. In contrast, this reduction in immunogenicity was not observed when a CML cell line resistant to the inhibitory effects of imatinib was used, but could be confirmed by transfection with specific small interfering RNA against BCR-ABL or imatinib treatment of primary CML cells. [Cancer Res 2007;67(11):5489-97]