Effect of NCS 613 on LPS-induced and basal TNFαsecretion by PBLs from SLE patients.

<p>PBLs from three patients with SLE (SLE1, SLE2 and SLE3) were purified and cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028899#s4" target="_blank">Materials and Methods</a>. PBLs from SLE1 patient were incubated for 45 min with increasing doses of NCS 613 and stimulated with 5 µg/mL of LPS (<b>A</b>). PBLs were incubated for 45 min with (□) or without (▪) 10 µM of NCS 613 and stimulated (<b>B</b>; SLE1, SLE2 and SLE3) or not (<b>C</b>; SLE2) with 5 µg/mL of LPS. TNFα secretion was determined by ELISA. The results are expressed as the mean concentration (pg/mL) ±s.e.m. of duplicate cultures. There was no basal TNFα secretion in the culture of PBMCs from SLE1 and SLE3 patients. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Pentoxifylline, denbufylline and NCS 613 treatment effects on proteinuria and survival rate of MRL/lpr mice.

<p>Four groups of MRL/lpr mice were injected via the i.v. route at 5, 7, 9 and 13 weeks with either 100 µL of PBS-10% ethanol (control, n = 10), or 100 µg pentoxifylline in 100 µL PBS-10% ethanol (n = 10), or 100 µg denbufylline in 100 µL PBS-10% ethanol (n = 10), or 30 µg NCS 613 in 100 µL PBS-10% ethanol (n = 8). Proteinuria was measured as described in the Methods section at weeks 10, 14, 18 and 20 are represented for each mouse. Each symbol represents an individual mouse and horizontal lines indicate the median. Closed symbols represent PBS-treated mice and open symbols represent inhibitors-treated mice (pentoxifylline, denbufylline and NCS 613). Because PBS-treated-MRL/lpr mice present a high mortality, dead mice were considered to have a proteinuria >2000 mg/dL (the level measured the week before their death). Survival rate is expressed as the percentage of surviving mice with time expressed in weeks. *, <i>P</i><0.05; **, <i>P</i><0.01, ns; not significant.</p

Protein expression pattern of PDE4 in the kidneys of 8 week-old CBA/J control mice.

<p>PDE4A, PDE4B, PDE4C, and PDE4D protein expression was assessed on 8 week-old CBA/J control mice as described in the Methods section. The apparent size of protein bands is expressed in kDa.</p

Protein expression pattern of PDE4 in kidneys of MRL/lpr mice of 8 and 18 weeks.

<p>PDE4A, PDE4B, PDE4C, PDE4D and GAPDH protein expressions were assessed on 8 week-old (C8) and 18 week-old (C18) CBA/J control mice and 8 week-old (L8) and 18 week-old (L18) MRL/lpr mice. Data are the mean±s.e.m. of three mice.*, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

PDE inhibitor treatment effects on <i>ex vivo</i> LPS-induced TNFα secretion by PBLs from MRL/lpr mice.

<p>Four groups of 7 MRL/lpr mice each were injected i.v. at 5, 7, 9 and 13 weeks with either 100 µL of PBS-10% ethanol (control), or 100 µg pentoxifylline in 100 µL PBS-10% ethanol, or 100 µg denbufylline in 100 µL PBS-10% ethanol, or 30 µg NCS 613 in 100 µL PBS-10% ethanol. Blood samples were collected from 14 week-old treated mice. PBLs pooled from seven mice of each group were purified and cultured in the presence of LPS, and TNFα secretion was determined by ELISA 24 h later. The results are expressed as the mean concentration (pg/mL) ±s.e.m. of duplicate cultures. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

IC₅₀ (µM) values for pentoxifylline, denbufylline and NCS 613 on PDE1-PDE5 isozymes.

<p>IC₅₀ values were determined at 1 µM substrate concentration in the presence of the modulator for PDE1 and PDE2 and represent the mean±s.e.m. of three independent experiments. CaM = calmodulin.</p

Evolution with the disease of cAMP-PDE activities in the kidneys of MRL/lpr mice.

<p>cAMP-PDE specific activities in total homogenate (A) and contribution of PDE2, PDE3 and PDE4 (B–D) were assessed on 8 week-old (C8) and 18 week-old (C18) CBA/J control mice and 8 week-old (L8) and 18 week-old (L18) MRL/lpr mice as described in the Methods section. Data are expressed as pmol/min/mg of protein and are the mean±s.e.m. of the data obtained from three individual mice. **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Pattern of cAMP-PDE activities in the kidneys of 8 week-old CBA/J control mice.

<p>cAMP-PDE specific activities in total homogenate and contribution of PDE2, PDE3 and PDE4 were assessed as described in the Methods section. Data are expressed as pmol/min/mg of protein and are the mean±s.e.m. of the data obtained from three individual mice.</p

IC₅₀ (µM) values for pentoxifylline, denbufylline and NCS 613 on human recombinant PDE4 subtypes.

<p>IC₅₀ values were determined at 1 µM cAMP substrate concentration and are given with their confident intervals.</p