IBI-SBP regression slopes of baroreflex and nonbaroreflex episodes obtained by the 1 min sliding window analysis (<i>b</i>_<i>al</i>), by the sequence method (<i>b</i>_<i>seq</i>), and by the "Z" technique (<i>b</i>_z) in control (C, •), in hemodialysis (HD, ♦) and in transplanted (TX,■) patients.

<p>IBI-SBP regression slopes of baroreflex and nonbaroreflex episodes obtained by the 1 min sliding window analysis (<i>b</i>_<i>al</i>), by the sequence method (<i>b</i>_<i>seq</i>), and by the "Z" technique (<i>b</i>_z) in control (C, •), in hemodialysis (HD, ♦) and in transplanted (TX,■) patients.</p

Correlations of baroreflex and nonbaroreflex <i>b</i>_<i>sl</i> slopes (absolute values) with age and with LFα and HFα indices in control (C), in hemodialysis (HD) and in transplanted (TX) patients.

<p>Correlations of baroreflex and nonbaroreflex <i>b</i>_<i>sl</i> slopes (absolute values) with age and with LFα and HFα indices in control (C), in hemodialysis (HD) and in transplanted (TX) patients.</p

Baroreflex and non-baroreflex function determined by three methods.

<p>SBP-systolic blood pressure; IBI-interbeat interval; HR- heart rate. (<b>a</b>) The sliding window method: SBP and IBI tracings and their correlations during a baroreflex episode (SBP and IBI change in the same direction, upper panel) and during a non-baroreflex episode (SBP and IBI change in different directions, lower panel). (<b>b</b>) The sequence technique: SBP and IBI tracings and their correlations during baroreflex episodes (SBP and IBI change in the same direction, increased or decreased SBP, upper panels) and during nonbaroreflex episodes (SBP and IBI change in opposite directions, increased or decreased SBP, lower panels). (<b>c</b>) The "Z" method: Three dimensional histogram for couples of SBP and HR. The modal class (the maximal, most frequent HR-SBP histogram value) was taken as a set point of spontaneous activity. The SBP-HR pair classes in quadrant 1 (low SBP associated with high HR values) and in quadrant 3 (high SBP associated with low HR values) are representative of baroreflex episodes, while quadrants 2 (low SBP with low HR) and 4 (high SBP with high HR) represent non-baroreflex episodes. Regression lines and correlation coefficients are shown for baroreflex episodes (quadrants 1 and 3) and for nonbaroreflex episodes (quadrants 2 and 4).</p

Representative tracings of <i>b</i>_<i>sl</i> (the slope of 1 min IBI-SBP regression line assessed by 1 min sliding window method), and r-the correlation coefficient as a function of time during the whole session of the recording in a control (upper panels, A) individual and in a transplanted patient (lower panels, B).

<p>The bold portions of <b><i>b</i>_<i>sl</i></b> tracings represent epochs with a correlation coefficient (r) between IBI and SBP greater than 0.5. In the control patient’s tracing, 55% of episodes in such epochs were baroreflex with a positive <i>b</i>_<i>sl</i>, while the remaining 45% were non-baroreflex with a negative <i>b</i>_<i>sl</i>. A similar tracing for a transplanted patient is shown in Figure 4, lower panel. In this patient, all sequences (100%) were nonbaroreflex episodes.</p

HCC and hepatocyte cell lines express TRAIL, TRAIL receptors, Fn14 and TWEAK.

<p>(<b>A</b>) The mRNA expression level of TRAIL, TRAIL receptors (DR4, DR5, DCR-1, DCR-2, OPG), Fn14 and TWEAK was determined by quantitive real-time PCR analysis. A representative experiment of three independent experiments is shown. Data are shown as average of triplicates (SD < 0.3), normalized against two endogenous control human genes, TBP and Actin-B, as calculated by Dataassist v2.0 software. (<b>B</b>,<b>C</b>) Protein expression of TRAIL, TRAIL receptors (DR4, DR5, DcR1, DcR2), Fn14 and TWEAK was determined by flow cytometeric analysis. (<b>D</b>) Fn14•TRAIL binds to HCC cells – HepG2 cells were incubated with Fn14•TRAIL, soluble TRAIL, Fn14 or the combination of the latter for 30 min at 4°c, immune-stained with PE-conjugated anti-Fn14, and analyzed by flow-cytometry. The results represent the mean +/- SD of triplicates (* <i>p</i> ≤ 0.05). </p

Fn14•TRAIL is more potent than soluble forms of Trail and Fn14 alone or in combination.

<p>(<b>A</b>) SK-HEP-1 [A, left panel], HepG2 [A, middle panel] and Huh7 [A, right panel] HCC cell lines, as well as NKNT3 [B, left panel] and FHB [B, right panel] hepatocyte cell lines, were incubated with 0, 3, 30 or 300 ng/ml of Fn14•TRAIL, TRAIL, Fn14-Fc or combination of the later two for 48 hours. Viable cells were stained with trypan blue and counted. The results represent the mean +/- SD of three independent experiments (* <i>p</i> ≤ 0.05). (<b>B</b>) HepG2 HCC cells were incubated with 30ng/ml Fn14•TRAIL for 24 hours, in the presence or absence of anti Fn14 or anti TRAIL blocking antibodies. Treated cells were stained by Annexin V-FITC and Propidium Iodide, and counted by flow cytometer (2x10⁴ cells per sample). The results represent the mean +/- SD of two independent experiments (* p ≤ 0.05).</p

No hepatotoxicty is observed in Fn14•TRAIL treated mice.

<p>HepG2 Nude mice were treated daily with subcutaneous injections of 200μg of Fn14•TRAIL (n=4) or vehicle (n=4) for 8 days. One hour after last injection blood counts (<b>A</b>) liver enzymes and urea (<b>B</b>) were measured. Livers were harvested and H&E stained sections were tested (<b>C</b>). Data are presented as mean +/- S.E. </p

Fn14•TRAIL induces death of HCC cell lines in a dose dependent manner.

<p>SK-HEP-1 (<b>A</b>) HCC cells were incubated with indicated concentrations of Fn14•TRAIL for 72 [left panel], 48 and 24 hours [right panel]. (<b>B</b>,<b>C</b>) HepG2 [B, left panel] and Huh7 [B, right panel] HCC cell lines, as well as NKNT3 [C, left panel] and FHB [C, right panel] hepatocyte cell lines, were incubated with indicated concentrations of Fn14•TRAIL for 48 and 24 hours. Viable cells were stained with trypan blue and counted. Results represent the mean +/- SE of 3 independent experiments (* <i>p</i> < 0.05 ** <i>p</i> < 0.01 vs no Fn14•TRAIL). </p

Molecular analysis of the Fn14•TRAIL.

<p>(A) The amino-acid sequence of the Fn14-TRAIL protein. The amino-acid sequence of the extra-cellular domain of human Fn14 (amino-acids 1-52 of the mature protein, marked in bold letters) are directly linked to the extra-cellular domain of human TRAIL (amino-acids 53-217 of the mature protein, non-bold letters). The underlined sequence represents the signal-peptide of the human Urokinase protein, utilized to secrete Fn14-TRAIL out of the cell and removed from the mature protein. (B) Fn14-TRAIL separated at denaturizing conditions on SDS-PAGE, Coomassie gel staining. (C) Western blot analysis with anti-TRAIL and anti-Fn14 primary antibodies. </p