Thymidylate synthase (TS) plays a major role in the response to 5‐fluorouracil (5‐FU) by binding directly to the 5‐FU metabolite, 5‐fluoro‐dUMP (FdUMP). The change in the TS expression levels after ...5‐FU administration was examined in parallel to 5‐FU responsiveness in six human gastric adenocarcinoma cell lines to elucidate the source of variability of 5‐FU sensitivity. MKN‐1, SH‐10‐TC and MKN‐74 cells were more resistant to 5‐FU than MKN‐28, KATO III and MKN‐45 cells. Western blotting analysis revealed that the 5‐FU sensitivity of these cells did not correlate with the basal TS expression levels but did correlate with rapid detection of the TS‐FdUMP complex after exposure to 5‐FU. In 5‐FU‐resistant cells, very low levels of the TS‐FdUMP complex early after 5‐FU exposure were elevated by pretreatment with calpain inhibitors such as benzyloxycarbonyl‐leucyl‐leucinal (ZLLH), benzyloxycarbonyl‐leucyl‐leucyl‐leucinal (ZLLLH) and ALLN, but not by other protease inhibitors. In contrast, ONO‐3403, which causes calpain activation, stimulated downregulation of the TS‐FdUMP complex in 5‐FU‐sensitive cells. The expression levels of calpastatin, an endogenous calpain inhibitor, were higher in 5‐FU‐sensitive cells than in 5‐FU‐resistant cells. ZLLH increased the 5‐FU sensitivity of 5‐FU‐resistant cells, whereas ONO‐3403 decreased the sensitivity of 5‐FU‐sensitive cells. In addition, knockdown of m‐calpain by siRNA increased the 5‐FU sensitivity in 5‐FU‐resistant cells, while knockdown of calpastatin reduced the sensitivity in 5‐FU‐sensitive cells. These results suggest that calpain might reduce the chemosensitivity of human gastric cancer cells to 5‐FU possibly by rapid degradation of the TS‐FdUMP complex, a finding that is considered to have novel therapeutic implications. (Cancer Sci 2011; 102: 1509–1515)
Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs ...from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies.
Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.
The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.
We recently demonstrated efficient antitumor immunity against murine tumors using dendritic cells (DCs) activated by recombinant Sendai viruses (rSeVs), and proposed a new concept, "immunostimulatory ...virotherapy," for cancer immunotherapy. However, there has been little information on the efficacy of this method in preventing metastatic diseases. In this study, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV (rSeV/dF) using a murine model of lung metastasis. Bolus and i.v. administration of DCs harboring rSeV/dF-expressing GFP without pulsation of tumor Ag (DC-rSeV/dF-GFP) 2 days before tumor inoculation showed efficient prevention against lung metastasis of c1300 neuroblastoma, but not of RM-9 prostatic cancer. We found that the timing of DC therapy was critical for the inhibition of pulmonary metastasis of RM-9, and that the optimal effect of DCs was seen 28 days before tumor inoculation. Interestingly, the antimetastatic effect was sustained for over 3 mo, even when administered DCs were already cleared from the lung and organs related to the immune system. Although NK cell activity had already declined to baseline at the time of tumor inoculation, Ab-mediated depletion studies revealed that CD4+ cells as well as the presence of, but not the activation of, NK cells were crucial to the prevention of lung metastasis. These results are the first demonstration of efficient inhibition of lung metastasis via bolus administration of virally activated DCs that was sustained and NK/CD4+ cell-dependent, and may suggest a potentially new mechanism of DC-based immunotherapy for advanced malignancies.
Glioblastoma multiforme (GM), the most frequent primary malignant brain tumor, is highly invasive due to the expression of proteases, including urokinase-type plasminogen activator (uPA). Here, we ...show the potential of our new and powerful recombinant Sendai virus (rSeV) showing uPA-specific cell-to-cell fusion activity rSeV/dMFct14 (uPA2), named “BioKnife” for GM treatment, an effect that was synergistically enhanced by arming BioKnife with the interferon-β (IFN-β) gene. BioKnife killed human GM cell lines efficiently in a uPA-dependent fashion, and this killing was prevented by PA inhibitor-1. Rat gliosarcoma 9L cells expressing both uPA and its functional receptor uPAR (9L-L/R) exhibited high uPA activity on the cellular surface and were highly susceptible to BioKnife. Although parent 9L cells (9L-P) were resistant to BioKnife and to BioKnife expressing IFN-β (BioKnife-IFNβ), cell–cell fusion of 9L-L/R strongly facilitated the expression of IFN-β, and in turn, IFN-β significantly accelerated the fusion activity of BioKnife. A similar synergy was seen in a rat orthotopic brain GM model with 9L-L/R in vivo; therefore, these results suggest that BioKnife-IFNβ may have significant potential to improve the survival of GM patients in a clinical setting.
Glioblastoma multiforme (GM), the most frequent primary malignant brain tumor, is highly invasive due to the expression of proteases, including urokinase-type plasminogen activator (uPA). Here, we ...show the potential of our new and powerful recombinant Sendai virus (rSeV) showing uPA-specific cell-to-cell fusion activity rSeV/dMFct14 (uPA2), named "BioKnife" for GM treatment, an effect that was synergistically enhanced by arming BioKnife with the interferon-β (IFN-β) gene. BioKnife killed human GM cell lines efficiently in a uPA-dependent fashion, and this killing was prevented by PA inhibitor-1. Rat gliosarcoma 9L cells expressing both uPA and its functional receptor uPAR (9L-L/R) exhibited high uPA activity on the cellular surface and were highly susceptible to BioKnife. Although parent 9L cells (9L-P) were resistant to BioKnife and to BioKnife expressing IFN-β (BioKnife-IFNβ), cell-cell fusion of 9L-L/R strongly facilitated the expression of IFN-β, and in turn, IFN-β significantly accelerated the fusion activity of BioKnife. A similar synergy was seen in a rat orthotopic brain GM model with 9L-L/R in vivo; therefore, these results suggest that BioKnife-IFNβ may have significant potential to improve the survival of GM patients in a clinical setting.
Placozoans are marine invertebrates found in tropical and subtropical waters. Their body plan is among the simplest of free-living animals. The present study determined the mitochondrial genome ...sequence of a placozoan collected on the coast of Shirahama, Wakayama, Honshu, Japan, and compared it with those of Trichoplax adhaerens from the Red Sea and of three strains from the Caribbean Sea. The sequences of mitochondrial respiratory chain of the Japanese placozoan genes are very similar to those of the BZ49 strain from the Caribbean Sea. However, there are distinct differences in gene arrangement, such as the location of two open reading frames. This Japanese placozoan is therefore distinguishable from the other strains. Based on current knowledge of placozoan 16S diversity our 'Shirahama' strain most likely represents the H15 lineage, known from the Philippines. In the mitochondrial genome of placozoans, substitution rates are slower than in bilaterians, whereas the rate of rearrangements is faster.
We have constructed an automatic in situ observation system for monitoring the behavior of small fatigue cracks at the microstructural level that, when used in conjunction with a digital-image ...correlation (DIC) technique, permits the continuous and automatic tracking and recording of microscopic deformation behavior. To verify the effectiveness of this system, we applied it to the evaluation of small fatigue cracks in heat-treated low-carbon steel. The results confirmed that our system can be used in the automatic tracking and recording of the initiation and early growth behavior of microstructurally small fatigue cracks. By the use of DIC analysis, we also succeeded in visualizing the opening-and-closing behavior of small fatigue cracks as well as the behavior of microscopic microstructural deformations, such as inhomogeneous strain concentrations, that caused the fatigue cracks. Although the early-stage growth of fatigue cracks propagates faster than that of long cracks, it is consistent with long-crack data if the effective stress intensity factor range ΔKeff which calculated by crack opening stress measured by DIC is used.
Porcine circovirus associated diseases (PCVAD) have multiple manifestations that have been attributed to porcine circovirus type 2 (PCV2). Recently, a novel porcine circovirus, PCV type 3 (PCV3), was ...identified in pigs with systemic inflammation of unknown etiology. In this study, we tried to detect the PCV3 genome in tissue samples collected from Japanese pig herds in 2016. The PCV3 genome was detected by PCR in 7 of 73 samples. The homology between each Japanese strain was 99.5% for the full-length sequence and 98.9 to 99.2% for the open reading frame 2. These results suggest that PCV3 has already invaded Japanese pig farms.
In this study, gigacycle fatigue properties were investigated for several microstructures prepared by heat treatment designed to simulate the heat-affected zone (HAZ) that results from welding. The ...results showed that internal matrix crack origin gigacycle fatigue becomes dominant in coarse-grained microstructures in spite of low tensile strength of only about 600 MPa. It was found that, high material strength is not always necessary and that microstructure plays an important role in the development of internal-origin gigacycle fatigue fractures.
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