Zoledronate stimulates gamma delta T cells in prostate cancer patients.

Androgen deprivation therapy is the mainstay of treatment for prostate cancer. Given its frequent failure, new therapy that reduces prostate cancer progression would be a breakthrough in treating this disease. Bisphosphonates are well-established agents for treating skeletal-related events (SREs) in prostate cancer patients with bone metastases. Exposure to bisphosphonates may not only reduce the incidence of SREs, but also have anticancer effects by modulating a patient's immunity. The purpose of this study was to examine the effect of zoledronate (ZOL) on gamma delta T cells, serum prostate-specific antigen (PSA) levels, and velocities. The effect of ZOL, with and without IL-2, on gamma delta T cell activation was examined in vitro. Furthermore, the activated state and the number of gamma delta T cells and changes in serum PSA levels were examined for patients who received ZOL infusion for the prevention of SREs. We found that ZOL activated gamma delta T cells, and the number of gamma delta T cell was increased when IL-2 was administered with ZOL in vitro. Comparisons before and after the first ZOL infusion revealed that gamma delta T cells in peripheral blood were activated by ZOL. Moreover, after the first ZOL treatment, reduction in serum PSA was observed in 3 of 11 patients, and reduction in PSA velocity was observed in 5 of 10 patients. Our findings indicate that ZOL stimulates gamma delta T cells in vivo and in vitro. This study provides further insight into the ability of gamma delta T cells to induce an antitumor immune response.

Expression of the fluoropyrimidine-metabolizing enzymes in bladder cancers as measured by the Danenberg tumor profile.

5-Fluorouracil (5-FU) and its prodrugs are used to treat various cancers. Response to 5-FU-based chemotherapy and expression of 5-FU-related enzymes differ among cancers. The objective of the present study was to investigate the relationship between the expression of 5-FU-related enzymes and clinicopathologic factors in bladder cancer. Formalin-fixed, paraffin-embedded sections of 44 bladder cancers and 27 normal bladders were included in this study. After laser capture microdissection, "Danenberg tumor profile," was performed for the measurement of 5-FU-related enzymes. There was no significant difference between dihydropyrimidine hydrogenase (DPD) mRNA expression in bladder cancer and in normal bladder. On the contrary, mRNA expressions of orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS), and thymidine phosphorylase (TP) in bladder cancer were higher than those in normal bladder. Compared with previously reported DPD mRNA expressions in other types of cancer, DPD mRNA expression in bladder cancer was relatively low. The 5-FU-related enzymatic condition of bladder cancer is favorable for 5-FU.

Use of the CellSearch Circulating Tumor Cell Test for monitoring urothelial cancer: two case reports of metastatic urothelial cancer.

Until now, there have been no reliable serum markers for monitoring urothelial cancers. However, it has been reported that it is possible to detect circulating urothelial cancer cells using the CellSearch Circulating Tumor Cell (CCTC) Test (Immunicon, Huntingdon Valley, PA). This case report is a representative example of the good correlation between the change of the number of circulating tumor cells in the blood and the state of an urothelial cancer after therapies. Detection of circulating tumor cells might be a valuable serum marker for urothelial cancer patients with distant metastasis.

TNF-alpha drives human CD14+ monocytes to differentiate into CD70+ dendritic cells evoking Th1 and Th17 responses.

Many mechanisms involving TNF-alpha, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-alpha to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14(+) monocytes cultured in the presence of TNF-alpha and GM-CSF converted to CD14(+) CD1a(low) adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-alpha, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (M phi). The mature DC (CD83(+) CD70(+) HLA-DR (high) CD14(low)) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-gamma and TNF-alpha, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-alpha added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the M phi (CD14(high)CD70(+)CD83(-)HLA-DR(-)) produced large amounts of MMP-9 and TNF-alpha without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated M phi. Therefore, additional stimulation of monocytes with TNF-alpha may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated M phi-mediated innate immunity.

Bacillus Calmette-Guérin-pulsed dendritic cells stimulate natural killer T cells and gammadeltaT cells.

BACKGROUND: Immunotherapy with bacillus Calmette-Guérin (BCG) for bladder cancer is successful, although the precise mechanism is unclear. Natural killer (NK) cells are a candidate for BCG-activated killer cells, but the roles of other T lymphocytes, such as NKT cells and gammadeltaT cells, are not fully understood. Mycobacterium tuberculosis is a potent activator of both NKT cells and gammadeltaT cells. However, it is known that the patient's prognosis is good if there are increased numbers of dendritic cells (DCs) in the urine after BCG therapy. Therefore, we investigated whether DCs are matured by BCG and whether BCG-pulsed DCs stimulate NKT cells and gammadeltaT cells. METHODS: Naïve Pan T cells were isolated form peripheral blood mononuclear cells (PBMCs) and DCs were obtained by culturing CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs were pulsed with BCG and their maturation was measured by fluorescence-activated cell sorter analysis using the CD86 antibody. Naïve T lymphocytes were stimulated by coculture with BCG-pulsed DCs in vitro, followed by FACS analysis to estimate the ratio and activation of NKT cells and the ratio of gammadeltaT cells. The (51)Cr (chromium) release assay was used to estimate the cytotoxic activity of the stimulated T cells. Cytolytic proteins in the patient's PBMCs were measured during BCG therapy using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: The DCs were matured by BCG stimulation and the number of NKT cells and gammadeltaT cells increased after culturing with BCG-pulsed DCs. The BCG-pulsed DCs also activated the NKT cells and gammadeltaT cells. Also, the lymphocytes that were cocultured with the BCG-pulsed DCs showed unspecific cytotoxic activity against a bladder cancer cell line. CONCLUSION: Sensitization of NKT cells and gammadeltaT cells by BCG-pulsed DCs might be one of the mechanisms of BCG immunotherapy.

Detection of circulating urothelial cancer cells in the blood using the CellSearch System.

BACKGROUND: Circulating tumor cells (CTCs) have been shown to aid in the therapeutic management of patients. But, only a few attempts have been made at the detection of urothelial cancer cells in the blood. The purpose of this study was to test the hypothesis that CTCs are detected in patients with urothelial cancers using newly developed CellSearch Assay. METHODS: Firstly, the bladder cancer cell lines were used to evaluate the reagents for immunocytochemical detection. After, mixed with peripheral blood mononuclear cells (PBMCs) of healthy volunteers, bladder cancer cells were stained with antibodies then multiparameter flow cytometric analysis was performed for the identification of bladder cancer cells in the PBMCs. Secondary, recovery of known numbers of spiked bladder cancer cells from whole blood was examined using CellSearch Assay. Finally, blood samples from nonmetastatic and metastatic urothelial cancer patients were investigated for CTC detection using CellSearch Assay. RESULTS: 1: Flow cytometric analysis revealed that it is possible to identify bladder cancer cells in PBMCs. 2: Sensitivity examination for detection of urothelial cancer cells with CellSearch Assay: Single regression analysis of the spiked number of cells vs. the recovered number of cells yielded a good correlation in this experiment. 3: Urothelial cancer cells were detected in 8 of fourteen patients (57.1%) with distant metastasis. Despite, no patient with nonmetastatic urothelial cancers showed positive result for this assay. CONCLUSION: This is the first report of attempt to detect circulating urothelial cancer cells in the peripheral blood of the patients with metastatic and nonmetastatic urothelial cancers by CellSearch Assay.

TCR ligand avidity determines the mode of B-Raf/Raf-1/ERK activation leading to the activation of human CD4+ T cell clone.

The interactions between peptide/MHC complexes and their cognate TCR are essential for various T cell responses. However, the relationship between the avidity of TCR ligand and the subsequent intracellular signaling through the TCR is still unclear. To investigate the effects of TCR ligand avidity on TCR-mediated signaling, we established L cells expressing HLA-DR4 molecules covalently linked with agonistic peptide (high-affinity ligand) or altered peptide ligand (APL; low-affinity ligand) at various densities as APC for a cognate human CD4(+) T cell clone. Using this system, we demonstrated that the T cell clone stimulated with APL/HLA-DR4 complexes presented at an excessive density provoked the up-regulation of CD69, IL-2 production and proliferation, but no detectable phosphorylation of ZAP-70/LAT/SLP-76. Furthermore, in contrast to the high-affinity stimulation, the low-affinity stimulation evoked delayed and sustained activation of the B-Raf/extracellular signal-regulated kinase (ERK) pathway without Raf-1 activation. The strength and duration of B-Raf/ERK activations closely correlated with the density of the TCR ligand. A knockdown approach confirmed that B-Raf activation was indispensable for the APL-induced T cell responses. These observations suggest that the differences in TCR-peptide/MHC interactions reflect the strength and duration of B-Raf/Raf-1/ERK activation in the human CD4(+) T cells.

Evolutionarily conserved and divergent regulatory sequences in the fish rod opsin promoter.

Fish have multiple types and subtypes of opsin genes that are expressed in a highly regulated manner in retinal photoreceptor cells. In the rod opsin proximal promoter region (RPPR) of zebrafish (Danio rerio), the BAT 1 regulatory region contains highly conserved OTX (GATTA) and OTX-like (TATTA) sequences that can be recognized by the mammalian cone-rod homeobox (CRX) protein. However, binding of zebrafish crx to the OTX sequence has remained elusive. In contrast to the BAT 1 region, the Ret 1 region, located approximately 20 bp upstream of the BAT 1 region in mammals, is not conserved in zebrafish. In the Ret 1 region, even the core OTX-like sequence (AATTA sequence in mammals) is destructed. We show in this study that a region between Ret 1 and BAT 1 (denoted IRB, Inter-Ret 1-BAT 1) is highly conserved among fish species. Using electrophoretic mobility shift assay (EMSA), we show that zebrafish crx binds to the conserved OTX sequence and that the fish-specific IRB region specifically binds elements present in both retinal and brain nuclear extracts of zebrafish. These results imply that the regulatory mechanisms of opsin gene expression consist not only of evolutionarily conserved but also of divergent machinery among different animal taxa.