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33 protocols using intralipid

1

In Vivo Efficacy of HNK in TNBC

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MDA-MB-231 (5×106) cells in 0.1ml of HBSS were injected subcutaneously into the right gluteal region of 4–6-week-old female athymic nude mice, procured from Harlan Laboratories Inc (Indianapolis, IN). Two weeks after initial implantation, animals were placed into two experimental groups. Mice were treated with intraperitoneal injections of 1) control (saline and Intralipid); 2) HNK, at 3 mg/mouse/day in 20% Intralipid (Baxter Healthcare), three times per week for the duration of the experiment. The dose and route of HNK administration was selected from our previous studies documenting in vivo efficacy (Nagalingam et al., 2012 (link)). Tumors volume were measured using vernier calipers, and the formula (V= a/2 × b2), where V is the tumor volume in mm3, a and b are the largest and smallest diameters in mm, respectively. All animals were sacrificed after 5 weeks of treatment, tumors were collected and weighed. One part of tumor was fixed in 10% neutral-buffered formalin for further analysis by Immunohistochemistry (IHC), other parts were utilized for RNA and protein isolation for analysis using RT-PCR or western blotting. All animal studies were in accordance with the guidelines of Johns Hopkins University IACUC.
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2

Tumor Growth Inhibition via Honokiol and Leptin

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MDA-MB-231 cells xenografts were generated; as previously described [28 (link)], grouped in four experimental groups. Mice were treated with intraperitoneal (IP) injections of 1) control (saline and Intralipid); 2) HNK, at 3 mg/mouse/day in 20% Intralipid (Baxter Healthcare, Deerfield, IL), three times per week; 3) recombinant leptin (dosage of 5 mg/kg), 5 days a week; 4) leptin and HNK for 4 weeks. The dose and route of HNK and leptin administration was selected from our previous studies documenting in vivo efficacy of honokiol and leptin [17 (link), 28 (link)]. Tumors were regularly measured; collected after 4 weeks of treatment, weighed, and subjected to further analysis. These tumors were utilized for examining the expression of Vimentin, Fibronectin, Zeb1, Zeb2, Slug and actin. All animal studies were in accordance with the guidelines of Johns Hopkins University IACUC.
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3

Xenografts Mouse Model for HNK Treatment

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MDA-MB-231-pLKO.1 and MDA-MB-231-LKB1sh cells xenografts were generated; as previously described,25 (link) grouped in two experimental groups (8 mice/group). Mice were oral gavaged three times per week for 6 weeks with (1) Vehicle (saline and Intralipid); (2) HNK, at 3 mg/mouse/day in 20% Intralipid (Baxter Healthcare, Deerfield, IL, USA). The dose and route of HNK administration was in accordance with our previous studies showing in vivo efficacy of HNK.25 (link) Tumors were regularly measured, collected after six weeks of treatment, weighed and subjected to further analysis. These tumors were utilized for examining the expression of Ki67 and phospho-Stat3 in immunoblot and immunohistochemistry analyses. For immunohistochemistry, multiple nonoverlapping representative images from each tumor section from all the tumors of each group were captured using ImagePro software for quantitation of Oct4, Nanog, Sox2, Ki-67 and phospho-Stat3 expression. All animal studies were in accordance with the guidelines of Johns Hopkins University IACUC.
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4

ANGPTL8 Inhibition Enhances Triglyceride Clearance

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Example 9

The effect of ANGPTL8 inhibition with the mAb H4H15341P on triglyceride clearance was evaluated by acute fat loading. Humanized ANGPTL8 mice were pre-bled 8 days before the experiment and put into groups of 6 mice each for each mAb tested. H4H15341P and isotype-matched control Ab were administered at 10 mg/kg by single-dose subcutaneous injection on Day 0 of the study. On day 4 of the study mice were fasted for 4 hours following intravenous administration of 20% intralipid (Baxter Healthcare, IL) at 2.5 μl/g body weight. TG level was evaluated in blood collected from the tail vein at subsequent time points. Results, expressed as (mean±SEM) of TG concentration are shown in FIG. 4. Control Ab refers to mice that received an isotype-matched negative control Ab.

Results

Administration of H4H15341P (anti-hANGPTL8) to humanized ANGPTL8 mice leads to a significantly lower TG level after acute fat load compared to control antibody. These data suggest that H4H15341P, by blocking ANGPTL8, promotes accelerated TG clearance from the circulation.

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5

Phantom-based Validation of FPGA Analyzer

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The FPGA analyzer was also tested against a software analysis scheme in phantoms. Phantoms with a range of viscosities (and therefore effective diffusion constants DB) were produced utilizing glycerol, 20% Intralipid (Baxter Healthcare, Deerfield, IL), and water, following recipes from Cortese [31 (link)] to maintain a constant μs . We produced four phantoms with four different flow levels. We measured the flow levels using the DCS system with the FPGA analyzer and an independent DCS system with offline DCS analysis in a semi-infinite geometry for ~5 minutes for each phantom. BFI of the lowest viscosity phantom (no glycerol) was set as baseline. BFI is normalized to baseline using equation 5 as the relative BFI or rBFI. The phantom test was conducted in the room temperature of 25 °C. The rBFI of each phantom was measured by both DCS systems. Bland Altman plot was used to compare the consistency of the BFI measurement with 50 pairs of measurements for each phantom.
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6

Optical Phantom for COMSOL Validation

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An optical phantom was used to validate the COMSOL simulations (Fig. 6). The same CAD file used for the COMSOL simulations was also used to make a replica of the synovial space using 3D printing (Cube 2, Cubify). A silicone mold of a hand and the 3D printed synovial space (Fig. S7) was used to generate optical phantoms, which consisted of 3% agarose gel (for structure), 1% Intralipid (Baxter, Deerfield, IL) and 50 ppm India ink to mimic the absorption and scattering properties of human tissue25 (link). IRDye 800CW carboxylic acid (LI-COR Biosciences; Lincoln, NE) was added at a concentration of 10 nM to the hand to represent background signal and 50 nM or 100 nM to the synovial cavity to represent specific uptake. The synovial cavity was placed ~2.5 mm from the surface of the hand. Once the gel solidified, it was imaged on an IVIS Spectrum (Perkin Elmer; Waltham, MA) and the TBR was calculated using the LivingImage software (Perkin Elmer; Waltham, MA).
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7

Tissue Optical Phantoms Characterization

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The tissue optical phantoms used for characterization (Supplementary Information: Tissue phantoms), contained fluorescein (20 μM) (Sigma) which brought up in 1% Intralipid (10% Solution, Baxter Healthcare) in PBS with varying concentrations of India ink following a well established protocol37 (link). The corresponding scattering coefficient μ's was equal to 11 cm-1, a value typically considered for mouse tissue phantoms37 (link). Optical densities of ink concentrations in PBS were determined by measuring the absorbance spectrum at 910 nm. Fluorescent images of the solution were taken at 10-micron intervals through the depth of the phantoms.
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8

Lipid/Heparin Infusion and Insulin Resistance

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Participants underwent saline-control or lipid/heparin infusions (Liposyn II or Intralipid; 20% lipid emulsion at 45 cc/hour, heparin at 0.4 U/kg/min), in the presence or absence of a HEC, conducted at 4 separate visits, for 6 hours commencing at approximately 6AM. The study was initiated with Liposyn II (Abbott Laboratories, North Chicago, IL; 10% safflower oil, 10% soybean oil, 1.2% egg phosphatides and 2.5% glycerin; major component fatty acids: approximately 65.8% linoleic, 17.7% oleic, 8.8% palmitic, 3.4% stearic, and 4.2% linolenic acid), but due to a product recall, some participants received Intralipid (Baxter Healthcare Corporation, Deerfield, IL; 20% Soybean oil, 1.2%egg yolk phospholipids, 2.25% glycerin; major component fatty acids: linoleic (44-62%), oleic (19-30%), palmitic (7-14%), linolenic (4-11%) and stearic (1.4-5.5%) instead. Liposyn and Intralipid are reported to induce similar degrees of insulin resistance (20 (link), 21 (link)) and this was confirmed within this study (see results).
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9

Lipid/Heparin Infusion and Insulin Resistance

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Participants underwent saline-control or lipid/heparin infusions (Liposyn II or Intralipid; 20% lipid emulsion at 45 cc/hour, heparin at 0.4 U/kg/min), in the presence or absence of a HEC, conducted at 4 separate visits, for 6 hours commencing at approximately 6AM. The study was initiated with Liposyn II (Abbott Laboratories, North Chicago, IL; 10% safflower oil, 10% soybean oil, 1.2% egg phosphatides and 2.5% glycerin; major component fatty acids: approximately 65.8% linoleic, 17.7% oleic, 8.8% palmitic, 3.4% stearic, and 4.2% linolenic acid), but due to a product recall, some participants received Intralipid (Baxter Healthcare Corporation, Deerfield, IL; 20% Soybean oil, 1.2%egg yolk phospholipids, 2.25% glycerin; major component fatty acids: linoleic (44-62%), oleic (19-30%), palmitic (7-14%), linolenic (4-11%) and stearic (1.4-5.5%) instead. Liposyn and Intralipid are reported to induce similar degrees of insulin resistance (20 (link), 21 (link)) and this was confirmed within this study (see results).
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10

Insulin Resistance Protocol: Lipid and Glycerol

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Participants were studied on 2 occasions in random order, 2 weeks apart. On each occasion, participants were admitted to the clinical research unit at 1700 h on the day before the study. At 1800 h, they consumed a standard 10 kcal/kg meal (55% carbohydrate, 30% fat, 15% protein) followed by an overnight fast. At 0630 (−210 min) the following morning, a forearm vein was cannulated for infusions. In addition, a cannula was inserted retrogradely into a vein of the contralateral dorsum of the hand, which was placed in a heated Plexiglas box maintained at 55°C to allow sampling of arterialized venous blood. On one occasion, at 0700 (−180 min), an infusion of Intralipid (20%, 0.011 mL/kg/min; Baxter Healthcare, Deerfield, IL) and heparin (200 units prime, 0.2 units/kg/min continuous) was commenced to raise FFA concentrations and create an insulin resistant state.17 (link) This was continued till the end of the study. At time 0 (1000 h), participants ingested a glucose drink (1 g/kg body weight).
The Glycerol study day differed from the Intralipid study day in that at 0700, glycerol was infused at 5 μmol/kg/min to match the amount of glycerol present during Intralipid infusion. Blood samples were obtained at periodic intervals for hormone and substrate measurement over the course of the experiment.
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