Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor γII)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon γ, but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.     

Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice

Immunocytological studies in this laboratory have suggested that NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in developing organs of rice (Oryza sativa L. cv. Sasanishiki) is involved in the utilization of glutamine remobilized from senescing organs through the phloem. Because most of the indica cultivars contained less NADH-GOGAT in their sink organs than japonica cultivars, over-expression of NADH-GOGAT gene from japonica rice was investigated using Kasalath, an indica cultivar. Several T0 transgenic Kasalath lines over-producing NADH-GOGAT under the control of a NADH-GOGAT promoter of Sasanishiki, a japonica rice, showed an increase in grain weight (80% as a maximum), indicating that NADH-GOGAT is indeed a key step for nitrogen utilization and grain filling in rice. A genetic approach using 98 backcross-inbred lines (BC(1)F(6)) developed between Nipponbare (a japonica rice) and Kasalath were employed to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), which is probably involved in the export of nitrogen from senescing organs and those of NADH-GOGAT. Immunoblotting analyses showed transgressive segregations toward lower or greater contents of these enzyme proteins in these BC(1)F(6). Seven chromosomal QTL regions were detected for GS1 protein content and six for NADH-GOGAT. Some of these QTLs were located in QTL regions for various biochemical and agronomic traits affected by nitrogen recycling. The relationships between the genetic variability of complex agronomic traits and traits for these two enzymes are discussed.     

The Mechanism of Salt Activation of Thermolysin: Relation with Pressure Activation and Implications of Hydration Change Coupled with Rate Process

Thermolysin (E.C. 3.4.24.4) shows a remarkable increase in catalytic activity at elevated salt concentrations or hydrostatic pressures. Salt effected K_cat, only, whilst the effect of pressure was related to both K_cat, and K_m. The turnover, derived from k_cat/K_m(V), of the hydrolysis of an N-acyldipeptide amide substrate was scarcely affected by addition of salt. These results were interpreted in terms of the stabilization of increased (or exposed) charges at the transition state of the reaction.     

Bovine adrenal cytochrome P-450(11 beta)-mediated conversion of 11-deoxycortisol to 18- and 19-hydroxy derivatives; structural analysis by 1H-NMR.

Incubation of 11-deoxycortisol with a cytochrome P-450(11β)-reconstituted system yielded, in addition to cortisol, several new steroid products. In this study, the structures of the three steroid products were elucidated. Retention time of the first product (Peak 2 substance) coincided with that of authentic 18-hydrocortisol on reverse phase HPLC. To further confirm the chemical identity of this product, the purified sample was subjected to ¹H-NMR analysis. The spectrum was essentially identical to that of 18-hydrocortisol. The retention time of the second product (Peak 3 substance) did not coincide with those of commonly occuring steroids. The one- and two-dimension ¹H-NMR spectra provided strong evidence for its structure of 19-hydroxy-11-deoxycortisol. The retention time of the third product (Peak 4 substance) did not coincide with those of commonly occuring steroids. The ¹H-NMR spectrum showed the presence of signals of 19-CH₃ and 18-CH₂ protons. There was also evidence that this product is not hydroxylated at the 11-position. Further analysis of the COSY spectra identified its structure as 18-hydroxy-11-depxycortisol. From these results, we conclude that bovine P-450(11β)deoxycortisol can catalyze the hydroxylation of 11-deoxycortisol at 11β-18- and 19-positions and produce cortisol, 18-hydroxy-11-deoxycortisol, 18-hydroxycortisol and 19-hydroxy-11-deoxycortisol.     

Myoblast adhesion and proliferation on biodegradable polymer films with femtosecond laser‐fabricated micro through‐holes

Controlling cell adhesion and cell differentiation is necessary to fabricate a tissue with arbitrary properties for tissue engineering applications. A substrate with a porous structure as a cell scaffold allows the diffusion of the cell culture medium through the scaffold. In this work, we show that the femtosecond laser fabricated micro through‐holes in biodegradable polymer films, enhance myoblast adhesion, and accelerates proliferation and differentiation. ChR2‐C2C12 and UT‐C2C12 cells were seeded on the films with micro through‐holes each fabricated by a single femtosecond laser pulse. Cell adhesion was enhanced on films with holes fabricated by laser irradiation. In addition, cell proliferation was accelerated on films with micro through‐holes that penetrate the film, compared to on films with micro craters that do not penetrate the film. On films with arrays consisting of micro through‐holes, cells aligned along the arrays and cell fusion was enhanced, indicating the acceleration of cell differentiation.     

A quantitative comparison of rates of phagocytosis and digestion of apoptotic cells by macrophages from normal (BALB/c) and diabetes-prone (NOD) mice.

Macrophages play an important role in clearing apoptotic debris from tissue. Defective or reduced clearance, seen, for instance, in non-obese diabetic (NOD) mice, has been correlated with initiation of autoimmune (Type 1) diabetes (T1D) (O'Brien BA, Huang Y, Geng X, Dutz JP, Finegood DT. Diabetes 51: 2481-2488, 2002). To validate such a link, it is essential to quantify the reduced clearance (for example, by comparison to BALB/c control mice) and to determine which elements of that clearance are impaired. Recently, we fit data for the time course of in vitro macrophage feeding experiments to basic models of macrophage clearance dynamics, thus quantifying kinetics of uptake and digestion of apoptotic cells in both mouse strains (Marée AFM, Komba M, Dyck C, ?abe?ki M, Finegood DT, Edelstein-Keshet L. J Theor Biol 233: 533-551, 2005). In the cycle of modeling and experimental investigation, we identified the importance of 1) measuring short-, intermediate-, and long-time data (to increase the accuracy of parameter fits), and 2) designing experiments with distinct observable regimes, including engulfment-only and digestion-only phases. Here, we report on new results from experiments so designed. In comparing macrophages from the two strains, we find that NOD macrophage engulfment of apoptotic cells is 5.5 times slower than BALB/c controls. Significantly, our new data demonstrate that digestion is at least two times slower in NOD, in contrast with previous conclusions. Moreover, new data enable us to detect an acceleration in engulfment (after the first engulfment) in both strains, but much smaller in NOD macrophages.     

Diurnal variation of dopamine content in the rat pineal gland

Levels of norepinephrine and dopamine in the rat pineal gland were determined by a radioenzymatic assay with modifications to separate the reaction products. Catecholamines were converted to 3-O-methylated derivatives in the presence of catechol-O-methyltransferase (EC 2.1.1.1) and S-adenosyl-L-[methyl-³H]-methionine. Following solvent extraction of the labelled normetanephrine and 3-methoxytyramine, the amines were separated by high-performance liquid chromatography. Contents of both catecholamines in the pineal gland varied with a 24-hr rhythm. The content of norepinephrine was maximal at about 6 A.M. (lights on from 8 A.M. to 8 P.M.) and declined gradually thereafter. In contrast to the level of norepinephrine, the dopamine level was highest at about 0 A.M. and fell rapidly to reach a trough after the lights were turned on. These observations suggest that the diurnal variation of norepinephrine is generated by changes in the contents of dopamine in sympathetic nerve terminals innervating the pineal.     

EPR quantification of vascular nitric oxide production in genetically modified mouse models.

With increasing use of genetically modified mice to study endothelial nitric oxide (NO) biology, methods for reliable quantification of vascular NO production by mouse tissues are crucial. We describe a technique based on electron paramagnetic resonance (EPR) spectroscopy, using colloid iron (II) diethyldithiocarbamate [Fe(DETC)2], to trap NO. A signal was seen from C57BL/6 mice aortas incubated with Fe(DETC)2, that increased 4.7-fold on stimulation with calcium ionophore A23187 [3.45+/-0.13 vs 0.73+/-0.13au (arbitrary units)]. The signal increased linearly with incubation time (r(2) = 0.93), but was abolished by addition of N(G)-nitro-l-arginine methyl ester (L-NAME) or endothelial removal. Stimulated aortas from eNOS knockout mice had virtually undetectable signals (0.14+/-0.06 vs 3.17+/-0.21 au in littermate controls). However, the signal was doubled from mice with transgenic eNOS overexpression (7.17+/-0.76 vs 3.37+/-0.43 au in littermate controls). We conclude that EPR is a useful tool for direct NO quantification in mouse vessels.