Effects of CUX1 and sh-CUX1 overexpression on TNF-α and IL-6 protein expression in THP-1 cells and monocytes stimulated with LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−), assessed by ELISA.

<p><b>A, C.</b> Effects of CUX1 and sh-CUX1 overexpression on TNF-α protein expression in THP-1 cells and monocytes stimulated with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−). THP-1 cells (5×10⁵) or monocytes (5×10⁵) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), with or without CUX1 or sh-CUX1 overexpression, and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were cultured for the indicated periods, and serum was then obtained from the well and subjected to ELISA analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 5, *<i>P</i><0.05 vs. control. **<i>P</i><0.05 vs. stimulation with LPS alone. B, D. Effects of CUX1 and sh-CUX1 overexpression on IL-6 protein expression in THP-1 cells and monocytes stimulated with a combination of LPS and either sCD26/DPPIV(+) or sCD26/DPPIV(−). THP-1 cells (5×10⁵) or monocytes (5×10⁵) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), with or without CUX1 or sh-CUX1 overexpression, and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were cultured for the indicated periods, and serum was then obtained from the well and subjected to ELISA. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 5, *<i>P</i><0.05 vs. control. **<i>P</i><0.05 vs. stimulation with LPS alone. Statistical analysis was performed using Student’s <i>t</i>-test.</p

Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on the transcriptional activities of the human TNF-α and IL-6 gene promoters.

<p>A, C. Effects of stimulating THP-1 cells and monocytes with a combination of LPS and either sCD26/DPPIV(+) or sCD26/DPPIV(−) on the transcriptional activity of the human TNF-α (−1.8 kb) gene promoter. THP-1 cells (5×10⁵) and monocytes (5×10⁵) were incubated in culture medium with or without LPS or various concentrations of either sCD26/DPPIV(+) or sCD26/DPPIV(−), with or without overexpression of CUX1 or sh-CUX1, and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were cultured for the indicated periods and then subjected to reporter assays. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *<i>P</i><0.05 vs. control. **<i>P</i><0.05 vs. stimulation with LPS alone. B, D. Effects of stimulating THP-1 cells and monocytes with a combination of LPS and either sCD26/DPPIV(+) or sCD26/DPPIV(−) on the transcriptional activity of the the human IL-6 gene promoter. THP-1 cells and monocytes (5×10⁵) were incubated in culture medium with or without LPS, with or without various concentrations of either sCD26/DPPIV(+) or sCD26/DPPIV(−), with or without CUX1 or sh-CUX1 overexpression, and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were cultured for the indicated periods and then subjected to reporter assays. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *<i>P</i><0.05 vs. control. **<i>P</i><0.05 vs. stimulation with LPS alone. Statistical analysis was performed using Student’s <i>t-</i>test.</p

Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on TNF-α and IL-6 mRNA expression, assessed by quantitative RT-PCR.

<p><b>A, B.</b> Effects of stimulating THP-1 cells with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on TNF-α and IL-6 mRNA expression. THP-1 cells (1×10⁵) were incubated in the culture medium with or without LPS or various concentrations of sCD26/DPPIV(+) and sCD26/DPPIV(−). The cells were then cultured for the indicated periods. Total RNA was prepared and subjected to quantitative RT-PCR analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *<i>P</i><0.05 vs. control (stimulation with LPS alone). **<i>P</i><0.05 vs. Mock. C, D. Effects of stimulating monocytes with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on TNF-α and IL-6 mRNA expression. Monocytes (1×10⁵) were incubated in the culture medium with or without LPS or various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−). Then, the cells were cultured for the indicated periods. Total RNA was prepared and subjected to quantitative RT-PCR analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *<i>P</i><0.05 vs. control (stimulation with LPS alone). **<i>P</i><0.05 vs. Mock. Statistical analysis was performed using Student’s <i>t-</i>test.</p

Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on the binding affinity of transcription factors with the human TNF-α and IL-6 gene promoters, assessed by ChIP assay.

<p>A, C. Effects of stimulating THP-1 cells with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on the binding affinity of transcription factors to the human TNF-α and IL-6 gene promoters. THP-1 cells (5×10⁵) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), and with or without the DPPIV inhibitor P32/98 (10 µM). The cells were then cultured for the indicated periods. They were fixed and subjected to ChIP analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *<i>P</i><0.05 vs. control. **<i>P</i><0.05 vs. stimulation by LPS alone. ^#<i>P</i><0.05 vs. sCD26/DPPIV(+). B, D. Effects of stimulating monocytes with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on the binding affinity of transcription factors to the human TNF-α and IL-6 gene promoters. Monocytes (5×10⁵) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), and with or without the DPPIV inhibitor P32/98 (10 µM). The cells were then cultured for the indicated periods. They were fixed and subjected to ChIP analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *<i>P</i><0.05 vs. control. **<i>P</i><0.05 vs. stimulation by LPS alone. ^#<i>P</i><0.05 vs. sCD26/DPPIV(+). Statistical analysis was performed using Student’s <i>t-</i>test.</p

Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on ERK1/2, AKT, c-Fos, NF-κB p65, NF-κB p50, and CUX1 protein expression, assessed by Western blotting.

<p>A, B. Effects of stimulating THP-1 cells with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on ERK1/2, AKT, c-Fos, NF-κB p65, NF-κB p50, and CUX1 protein expression with or without the DPPIV inhibitor sitagliptin. THP-1 cells (1×10⁵) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were then cultured for the indicated periods. Cytoplasmic and nuclear fractions were prepared as described in the Methods section and subjected to Western blotting. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *<i>P</i><0.05 vs. control. C, D. Effects of stimulating monocytes with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on ERK1/2, AKT, c-Fos, NF-κB p65, NF-κB p50, and CUX1 protein expression with or without the DPPIV inhibitor sitagliptin (1 µM). Monocytes (1×10⁵) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were then cultured for the indicated periods. Cytoplasmic and nucleic fractions were prepared as described in the Methods section and subjected to Western blotting. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *<i>P</i><0.05 vs. control. Statistical analysis was performed using Student’s <i>t</i>-test.</p

Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on TNF-α and IL-6 protein secretion, assessed using ELISAs.

<p>A, B. Effects of stimulating THP-1 cells with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on TNF-α and IL-6 protein secretion. THP-1 cells (1×10⁵) were incubated in the culture medium with or without LPS or various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−). The cells were then cultured for the indicated periods. Serum was obtained from the well and subjected to ELISA. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 5, *<i>P</i><0.05 vs. control (stimulation with LPS alone). **<i>P</i><0.05 vs. Mock. C, D. Effects of stimulating monocytes with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on TNF-α and IL-6 protein secretion. Monocytes (1×10⁵) were incubated in the culture medium with or without LPS or various concentrations of either sCD26/DPPIV(+) or sCD26/DPPIV(−). The cells were then cultured for the indicated periods. Serum was obtained from the well and subjected to ELISA. Each panel shows a typical example of the results. Values represent the mean ± S.E.M., n = 5, *<i>P</i><0.05 vs. control (stimulation with LPS alone). **<i>P</i><0.05 vs. Mock. Statistical analysis was performed using Student’s <i>t</i>-test.</p

Effect of the addition of complement in 21 fresh and 227 stocked serum samples.

<p>Distribution of serum samples is shown for neutralizing antibody titers assayed without inactivation and for those assayed after inactivation with the addition of complement, using 21fresh serum samples (left panel). 227 stocked serum samples were assayed in a similar manner (right panel).</p

Genome construction of the recombinant mumps Hoshino strain expressing GFP and expression of GFP.

<p>Vero cells were infected with GFP Hoshino mumps strain at m.o.i. = 0.02 and subjected to experiments for GFP expression with fluoro EIA and microscopic examination on day 1, 3 and 5 of infection in comparison with mock-infection. Infectivity was assayed in culture supernatants on day 1, 3, and 5 of infection.</p

Relationship between the appearance of CPE and GFP expression.

<p>Serial two-fold dilutions from 1∶4 to 1∶256 were mixed with an equal volume of challenge virus. In the left panel, the schematic results of two neutralization methods are shown. CPE was observed in one of the two wells at 1∶32, and the conventional neutralizing antibody titer was 1∶16 by 100% inhibition of CPE. The mean FU value of the two cell control wells was 202 and that of the 1∶32 dilution was 450, showing 1∶16 of neutralizing antibody titer. Using 1,452 serum samples, the consistency of neutralizing antibody titers was compared based on different cut-off values for GFP expression: 1.5-fold, 2.0-fold, and 2.5- fold of FU values of the cell control wells.</p

Effects of freeze-thawing and inactivation at 56°C for 30 min on neutralizing antibody titers.

<p>Upper panel shows the neutralizing antibody titers of eight fresh sera (A–H), without inactivation and after three or five rounds of freeze-thawing. Lower panel shows the results of neutralizing antibody titers after inactivation.</p