SUPPLEMENTAL MATERIAL
SUPPLEMENTAL FIGURES AND FIGURE LEGENDS
A. Methods for supplemental figures
Native 1D gel electrophoresis and lipid stain. For each sample, 12 µl serum were mixed with 12 µl 2X Native Tris-Glycine Sample Buffer (Invitrogen, Carlsbad, CA) and 20 µl were loaded per well onto Novex Native Tris-Glycine minigels (Invitrogen) using Native Tris-Glycine Running Buffer (Invitrogen)1. Gels were run 19 hours at 35 V and then stained with Sudan Black (Sigma Chemical, St. Louis, MO) according to manufacturer’s instructions.
Native-native 2D gel electrophoresis and Western analysis for apoA-I. Serum was stored at -80oC until analyzed. HDL particle subpopulations2, 3 were analyzed by native-native 2D gel electrophoresis, according to Asztalos2 with minor modifications4. Briefly, 1.5 µl serum was run on a 0.7% agarose vertical-slab minigel in Tris-Tricine buffer2, 4 (1 hr, 100V), separating native particles in plasma by charge in the first dimension. The lane was excised and applied to a 4-25% nondenaturing linear gradient polyacrylamide minigel (Jule Inc., Milford, CT)4 and subjected to electrophoresis in 1X TBE buffer (3.5 hr, 200V), separating native particles by size in the second dimension. Particles were electrotransferred to a PVDF membrane (Immobilon-P, 0.45 um pore size, Millipore, Billerica, MA), and blots were probed with goat anti-human apolipoprotein A-I, HRP conjugated (Meridian Life Sciences, Memphis, TN). Particles were visualized by chemiluminescence using Western Lightning (Perkin-Elmer, Waltham, MA) as the substrate and exposure to Kodak MR-2 film. To better visualize the apoA-I –containing HDL particles in patient serum prior to chemotherapy, native-native 2D gel electrophoresis was repeated on this sample using 15 times as much serum (22.5 ul).
Luminex. Cytokine levels (IL-2, IL-4, IL-6, IL-8, GM-CSF, IFN-g, TNFa and IL-10) in IVLBCL patient serum were measured using the Bio-Plex ProTM Human Cytokine Assay 8-Plex Group 1 Magnetic Assay (Bio-Rad, Hercules, CA) according to manufacturer’s instructions. Samples were diluted 1:4 for the assay.
a-lipoprotein. a-lipoprotein was determined in the NIH Clinical Center using standard methods (native agarose gel electrophoresis). R2 is Pearson correlation coefficient after log10 transformation of IL-10 and a-lipoprotein values.
B. Supplemental Figures
Supplemental Figure 2S
Supplemental Figure 4S
B. Supplemental Figure Legends
Supplemental Fig. 1S. “Disappearing HDL” Case 1 patient has small, poorly lipidated HDL particles. Plasma from a healthy volunteer or Patient 1, along with purified HDL or LDL as size controls, was separated by size using native 1D gel electrophoresis. Neutral lipids were visualized by staining the gel with Sudan Black. Lane 1: LDL. Lane 2: HDL. Lane 3: Healthy Control Plasma. Lane 4: Patient 1 Plasma.
Supplemental Fig. 2S. Recovery of normal HDL subpopulation distribution after chemotherapy. Upper panel: HDL subpopulations from “Disappearing HDL” Patient 1 before (left) or after (right) chemotherapy were analyzed by native-native 2D gel electrophoresis and Western blotting for apoA-I. This patient appeared to have only very low levels of small (a4) HDL particles prior to treatment but normal HDL subpopulation size distribution after chemotherapy. Lower panel: HDL subpopulations from Patient 1 before chemotherapy as above, with 15X more serum loaded to better visualize apoA-I – containing HDL particles in pre-chemotherapy serum. Both preb and a4 HDL particles are clearly apparent in this panel.
Supplemental Fig. 3S. Inverse correlation between IL-10 and HDL-C. A.Multiplex analysis of cytokines (IL-2, IL-4, IL-6, IL-8, GM-CSF, IFNg, TNFa, IL-10) in IVLBCL Patients 1 (left) and 2 (right), before and after chemotherapy. As in Fig. 2B, the first time point (“Day 0”) represents the last lipid time point before initiating chemotherapy. B. Multiplex analysis of cytokines for Patient 3. In a separate experiment, cytokines were determined for Patient 3 as for Patients 1 and 2.
Supplemental Fig 4S. a-lipoprotein inverse correlation with IL-10 in 8 ALPS patients. a-lipoprotein and IL-10 were quantified in serum from 8 ALPS patients. R2 is Pearson correlation coefficient after log10 transformation of IL-10 and a-lipoprotein values.
3. SUPPLEMENTAL TABLE I
(adjusted for d1) -- using MIXED MODELS
Parameter / Interval / Mean change Placebo
/ Mean change IL10
/ DIFFERENCE of changes between IL-10 and placebo
(mixed model) / SE of Difference
(mixed model) / 2-sided P-value)
Cholesterol (mg/dL) / d1-d29 / -4.8333 / -77.5833 / -72.75 / 13.9349 / <0.0001
d1-d57 / -1.5 / -12.0833 / -10.5833 / 13.9349 / 0.44756
d1-d85 / -2.3333 / -7.5446 / -5.2113 / 14.0989 / 0.71166
HDL (mg/dL) / d1-d29 / 0.8333 / -35.5833 / -36.4167 / 4.4023 / <0.0001
d1-d57 / 4.6667 / -5.8333 / -10.5 / 4.4023 / 0.01707
d1-d85 / 3.3333 / -3.0076 / -6.3409 / 4.475 / 0.15649
LDL (mg/dL) / d1-d29 / -0.8333 / -71.5833 / -70.75 / 16.5618 / 0.00002
d1-d57 / -1.6667 / -13.8333 / -12.1667 / 16.5618 / 0.46257
d1-d85 / 3.6667 / -14.0507 / -17.7173 / 16.7175 / 0.28923
Triglycerides (mg/dL) / d1-d29 / 2.8333 / 153.9167 / 151.0833 / 51.9906 / 0.00366
d1-d57 / 28 / 41.5833 / 13.5833 / 51.9906 / 0.79389
d1-d85 / -6.6667 / 9.4825 / 16.1492 / 53.1421 / 0.76121
LPa / d1-d29 / 2.8 / -21.9685 / -24.7685 / 10.4119 / 0.01737
d1-d57 / 3.4 / 3.9465 / 0.5465 / 10.6108 / 0.95892
d1-d85 / -0.4681 / -3.72 / -3.2526 / 11.5007 / 0.77732
ApoA-I / d1-d29 / -1.5 / -66.5455 / -65.0455 / 8.3116 / <0.0001
d1-d57 / 2.8333 / -0.9091 / -3.7424 / 8.3116 / 0.65252
d1-d85 / -2.5 / -6.3502 / -3.8502 / 8.4042 / 0.64686
ApoB / d1-d29 / -7.3333 / -26.5455 / -19.2121 / 9.4269 / 0.04155
d1-d57 / -4.6667 / -11.2727 / -6.6061 / 9.4269 / 0.48345
d1-d85 / -6.7882 / -10.2423 / -3.4541 / 9.6704 / 0.72095
ApoE / d1-d29 / -1.36 / 1.0889 / 2.4489 / 6.3939 / 0.70172
d1-d57 / 5.0652 / 5.7092 / 0.644 / 6.8312 / 0.92489
d1-d85 / 8.2879 / -2.1717 / -10.4596 / 7.4986 / 0.16305
4. SUPPLEMENTAL REFERENCES
1. Freeman LA. Western blots. Methods Mol Biol. 2013;1027:369-385
2. Asztalos BF, Sloop CH, Wong L, Roheim PS. Two-dimensional electrophoresis of plasma lipoproteins: Recognition of new apo a-i-containing subpopulations. Biochim Biophys Acta. 1993;1169:291-300
3. Asztalos BF, Tani M, Schaefer EJ. Metabolic and functional relevance of hdl subspecies. Curr Opin Lipidol. 2011;22:176-185
4. Freeman LA. Native-native 2d gel electrophoresis for hdl subpopulation analysis. Methods Mol Biol. 2013;1027:353-367