The aim of the study was to explore Fc mutations of a humanised anti-Lewis-Y antibody (IgG1) hu3S193 as a strategy to improve therapeutic ratios for therapeutic payload delivery.Four hu3S193 variants  were generated via site-directed mutagenesis and radiolabelled with diagnostic isotopes iodine-125 or indium-111. Biodistribution studies in Lewis-Y-positive tumour-bearing mice were used to calculate the dose in tumours and organs for therapeutic isotopes .111In-labelled I253A and H435A showed similar slow kinetics  and a maximum tumour uptake of 33.11 ± 4.05 and 33.69 ± 3.77 percentage injected dose per gramme (%ID/g), respectively. 111In-labelled I253A/H310A cleared fastest  with the lowest maximum tumour uptake (23.72 ± 0.85 %ID/g). The highest increase in tumour-to-blood area under the curve (AUC) ratio was observed with the metal-labelled mutants (90Y and 177Lu). 177Lu-CHX-A" DTPA-hu3S193 I253A/H310A (6:1) showed the highest tumour-to-blood AUC ratio compared to wild type (3:1) and other variants and doubling of calculated dose to tumour based on red marrow dose constraints.90Y- and 177Lu-based therapy, with the best candidate being hu3S193 I253A/H310A for 177Lu-based therapy.These results suggest that hu3S193 Fc can be engineered with improved therapeutic ratios for The online version of this article (doi:10.1186/s13550-016-0180-0) contains supplementary material, which is available to authorized users. The use of monoclonal antibodies (mAbs) to deliver radioisotopes to tumour sites (radioimmunotherapy (RIT)) has been very successful in the treatment of haematologic tumours as the targeted radiation is able to kill the highly radiosensitive leukemias and lymphomas. Treatment of solid tumours that are generally more radioresistant has been less successful due to several factors including long serum persistence, poor tumour penetration and slow diffusion rates of mAbs . The lar131I , suitable for tumours <1 cm, and 90Y , suitable for tumours >1 cm, are both β-particle-emitting isotopes and have been used in >95 % of clinical RIT trials [131I, 177Lu  has been identified as having favourable characteristics for treatment of tumours particularly with heterogeneous antigen expression [90Y and 177Lu labels are retained intracellularly after endocytosis, the 131I label is rapidly released from the cells [The choice of the optimal radionuclide for RIT depends on its intended use. Physical properties such as path length, emission energy and physical half-life must correlate with a particular tumour size. he cells , 9.2 and tetrameric scFv constructs without an Fc fragment. These constructs showed a rapid elimination phase compared to wild-type IgG1, more rapid tumour targeting with the maximum tumour uptake observed at an earlier time point but strongly reduced maximal tumour uptake [Early engineering approaches to reduce the serum half-life of antibodies and improve therapeutic ratios were based on reduction in antibody size by deletion of constant domains , 10, 11.r uptake –14. Anotr uptake .The FcRn receptor recycles IgG antibodies into the blood circulation. During the last decade, the Fc-FcRn binding site has been well characterised, with I253, H310 in the CH2 domain and H435 in the CH3 domain identified as the key residues involved in the Fc-FcRn binding –19. The y) humanised IgG1 variants carrying specific mutations in the Fc-FcRn binding region and investigated their binding properties to murine FcRn (muFcRn) and human FcRn (huFcRn) in vitro and in vivo [2) without an Fc fragment. In contrast, two alanine mutations in hu3S193 Fc (e.g. I253A/H310A) were necessary to completely abrogate binding to muFcRn. Single non-alanine variants of hu3S193 that were able to completely abrogate muFcRn binding were I253D, I253P, H310D and H310E [We recently produced anti-Lewis-Y . pEE6.4/hu3S193 HC was used as a template for site-directed mutagenesis (GeneTailor™ Site-Directed Mutagenesis System (Invitrogen) or QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene) to introduce the following substitutions in the CH2 and CH3 domains: I253A, H310A, H435A and I253A/H310A [HindIII and EcoRI double digest. For the simultaneous expression of each mutated hu3S193 antibody, light and mutated heavy chain genes were cloned into a double-gene vector (pDGV) using a NotI and PvuI double digest. Freestyle 293-F cells (1 × 106 cells/mL) were transfected with the pDGV constructs according to the manufacturer’s instructions (Invitrogen). Supernatants were harvested at 96 h post transfection. Hu3S193 antibodies were purified using HiTrap KappaSelect columns . Purified proteins were analysed by SDS-PAGE under non-reducing conditions and evaluated by size exclusion chromatography on a Superdex 200 HR 10/30 column  using 0.01 mol/L sodium phosphate and 0.15 mol/L NaCl (pH 7.2) as elution buffer.The construction and production of hu3S193 and huA33 has been described before , 32. TheThe Lewis-Y binding activity of hu3S193 antibodies was determined by BIAcore analysis using a synthetic Lewis-Y tetrasaccharide coupled to BSA  on a CM5 chip using a BIAcore 2000 as described  . Hu3S1935 cells were incubated with hu3S193 antibodies (200 nmol/L) in DMEM/F12 medium containing 10 % foetal bovine serum on ice. After washing the cells with PBS, cells were incubated with a goat phycoerythrin-conjugated anti-human IgG (Sigma) and incubated on ice for 30 min. Cells were washed with phosphate-buffered saline and resuspended in a final volume of 300 μL. In control samples, primary antibody was omitted. Flow cytometric analysis was done using a Guava EasyCyte Plus flow cytometer. Cancer cell populations were gated based on forward and side scatter variables. Data analysis was done using WinMDI (Joseph Trotter).Fluorescence-activated cell sorter (FACS) analysis was done on Lewis-Y-positive A431 skin cancer cells. Aliquots of 2 × 10125I, 131I, 111In and 177Lu. Iodine-125 and lutetium-177 were obtained from PerkinElmer , iodine-131 was obtained from ANSTO  and indium-111 was obtained from MDS Nordion (Canada).Hu3S193 antibodies were radiolabelled with four isotopes: Radioiodination was performed using pH-neutralised isotopes, catalysed by iodogen-coated glass beads as previously published , 33. Afttrans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A″ DTPA) [Radiolabelling of the hu3S193 antibody constructs with indium-111 and lutetium-177 was done by using the bifunctional metal ion chelate C-functionalized A″ DTPA) , 35. A c6 Ley-positive A431 cells were incubated with 20 ng of radiolabelled antibody constructs for 45 min at room temperature with continuous mixing throughout to keep the cells in suspension. Cells were washed three times, and pellets were measured in a gamma counter . Three samples of radiolabelled antibody at the same concentration as that initially added to the cells were measured at the same time of the cell pellets, and immunoreactivity was calculated: (cpm cell pellet/mean cpm radioactive antibody standards) × 100. Serum stability was analysed by determination of immunoreactivity on the day of injection, at 48 h, and 7 days after 20 ng of radiolabelled antibody was incubated in human serum at 37 °C.Radiolabelling was performed on the day of injection into mice. Prior to injection, the percentage of unbound radionuclide content was determined by ITLC as previously described . Determinu/nu; 4–6 weeks; Animal Resources Centre) were injected in the tail vein with 0.185 MBq 125I-mutant  and 0.185 MBq 131I-hu3S193  wild type antibody (n = 5). A separate group of athymic mice were injected with 0.185 MBq 125I- hu3S193 mutant  and 0.185 MBq 111In-hu3S193  mutant (n = 5). All animal studies were approved by the Austin Hospital Animal Ethics Committee and were conducted in compliance with NHMRC Australian code of practice for the care and use of animals for scientific purposes. Blood samples (10–20 μL) were collected from groups of five at 5 min, 1, 2, 4, 8, 24, 48, 72, 120, 168, 240 and 336 h after injection of radioactive antibodies. Samples were counted in a gamma counter (Cobra II). Standards prepared from injected material were counted each time with blood samples enabling calculations to be corrected for physical decay of isotope.Female athymic mice  or mice bearing A431 tumours . To study the tumour uptake and biodistribution in normal tissues of radioiodinated hu3S193 variants, A431 tumour-bearing BALB/c nu/nu mice were co-injected intravenously with 0.185–0.74 MBq (5–20 μCi) 131I-hu3S193 variants (2–6 μg protein) and 0.185–0.74 MBq 125I-hu3S193 wild type (2–6 μg protein). In a second study, 0.185–0.74 MBq 111In-CHX-A″ DTPA-labelled mutant (2–6 μg protein) and 0.185-0.74 MBq 125I-labelled hu3S193 wild type (2–6 μg protein) were co-injected. 111In-CHX-A″ DTPA-labelled wild type and 125I-hu3S193 wild type were also injected as a control group. 125I-hu3S193 wild type was used as an internal control in all injected animals to enable direct comparison between the different mutants. Typically, groups of four to five mice were sacrificed at 4, 24, 48, 72, 120, 168, and 240 or 288 h after injection of radiolabelled antibodies. For evaluation of 177Lu-labelled antibodies, mice were sacrificed at 48 h after injection. At the designated time points, groups of mice (n = 4–5) were humanely sacrificed by over-inhalation of isoflurane. Mice were bled by cardiac puncture, and tumours and organs , lungs and heart) were immediately removed and blotted dry. All samples were weighed and counted in a dual gamma scintillation counter . Triplicate standards prepared from the injected material were counted at each time point with tissue and tumour samples enabling calculations to be corrected for the physical decay of the isotopes. Results of the radiolabelled antibody distribution over time were calculated as the mean percentage injected dose per gramme (%ID/g ± SD) for each mutant and parental hu3S193 per time point.Biodistribution studies were done with athymic non-tumour-bearing mice (BALB/c t1/2α), beta half-life (t1/2β), area under the curve extrapolated to infinity (AUC0-∞) and mean residence time (MRT). Alpha half-lives of mutant hu3S193 were constrained to be smaller or equal to the alpha half-life of hu3S193 wild type. Significant differences in these values were examined by comparing the coefficient of variation (CV %) for the estimated parameters.Antibody serum concentrations were expressed as percentage injected dose per millilitre (%ID/mL), and the blood concentrations (μg/mL) were calculated. A two-compartment IV bolus model with macro-parameters, no lag time and first order elimination (WinNonlin Model 8) was fitted to serum data obtained from blood clearance studies for each animal using unweighted non-linear, least squares with WinNonLin version 5.2 . Estimates were determined for the pharmacokinetic parameters: alpha half-life . Time-activity curves generated from biodistribution data were corrected for radiodecay. Therefore, the pharmacokinetic values calculated from such data refer to pharmacological values of the antibodies in the absence of radioisotope. Appropriate physical half-life corrections were applied to convert %IDpharmacological/g to %IDradioisotope/g, and the time-activity curves were fit to either a two or three exponential function from which the AUC is determined for 90Y, 177Lu and 131I. AUC integration from zero to infinity was done by the sum of a trapezoidal integration of the measurement range (AUC0–288 h) plus an extrapolated model fit for the extrapolated range (AUC288 h–∞). To calculate accumulated activity, photon dose and edge effects were ignored. Dose to red marrow was determined from blood concentrations using a baseline value of 0.1 [To estimate radioimmunotherapeutic applications for e of 0.1 .PM, Mediso Medical Imaging Systems, Budapest, Hungary) individually. Groups of two mice were injected with 3.7 MBq (270 μg) 177Lu-CHX-A″ DTPA-labelled antibody  and serially imaged at 2 days post injection. Imaging procedures involved anaesthesia of mice by isoflurane. Each mouse was scanned in supine position with its head secured via ear and tooth bars. Respiration was monitored by a pressure-sensitive pad adhered to the abdomen. The imaging study started with a whole body T1-weighted MR scan, followed by a 60-min SPECT scan which was reconstructed into a volumetric image with the voxel size of 0.3 mm × 0.3 mm × 0.3 mm using the Mediso Tera-TOMO® Monte Carlo-based algorithm. Subsequently, reconstructed SPECT and MR images were transferred to a research PACS system where the images could be retrieved for further processing and analysis.All the SPECT and MR scans were performed on a small animal nano-SPECT/CT imaging system and a small animal nano-PET/MR imaging system  and sacrificed at 48 h post injection. Organs and blood were collected as described before, and the radiolabelled antibody distribution over time was calculated as the mean %ID/g ± SD for each antibody per time point.To confirm data obtained from imaging, groups of five mice bearing A431 tumours were injected with 6.29 MBq (100 μg) www.graphpad.com. Tumour-to-tissue ratios or tissue uptake at specific time points were analysed using one-way ANOVA with Tukey’s multiple comparison post test. When less than three groups were compared, a non-parametric t test (one-tailed) was used.To compare tumour uptake at different time points for the mutants versus hu3S193 wild type, two-way ANOVA with the Bonferroni post test was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA To investigate whether hu3S193 antibodies with reduced serum persistence would benefit payload delivery, three hu3S193 single variants  and one double variant (I253A/H310A) were generated. All antibodies were expressed in transiently transfected freestyle 293-F cells. Expression yields of antibodies ranged from 15 to 40 mg/L in shake flasks containing 60 mL medium. HiTrap KappaSelect columns were used to purify antibodies. Quality control using SDS-PAGE and HPLC on a Superdex 200 column showed highly pure antibody preparations with >98 % purity. All mutants maintained effective antigen binding in FACS analysis and BIAcore  using 125I-labelled antibody co-injected with their 111In-CHX-A″ DTPA-labelled counterpart, and blood clearance parameters were calculated (Table t1/2β) of radioiodinated or radiometal-chelated hu3S193 wild type. Radioiodinated hu3S193 variants showed shorter half-lives than their radiometal counterpart; the shorter the half-life the smaller the differences in half-lives observed between radioiodinated and radiometal-chelated hu3S193 mutants. Although different terminal serum half-lives were seen dependent on the choice of isotope, a similar ranking order of area under the curve (AUC) was observed: wild type>H435A = I253A>H310A>I253A/H310A  and 32.81 ± 7.10 for I253A/H310A (P < 0.001). Tumour uptake was significantly lower for all 131I-labelled mutants compared to wild type (P < 0.0001) at all time points except 4 h post injection , all hu3S193 variants exhibited a hepatic clearance. This is evidenced by the 131I-, 90Y-, 177Lu-) antibodies and tumour-to-blood AUC ratios were calculated  and 177Lu-labelled (1.9) hu3S193 I253A/H310A. Tumour-to-kidney ratios were lowest for 90Y-hu3S193 I253A (2.4) and 90Y-hu3S193 I253A/H310A (2.5) followed by 90Y-hu3S193 H310A (3.1) and 177Lu-hu3S193 H310A (3.1). Tumour-to-liver AUC ratios were superior for the 131I-labelled variants with the highest tumour-to-liver AUC ratio observed with 131I-hu3S193 I253A/H310A (5.6).AUC ratios were also calculated for tumour-to-liver and tumour-to-kidney as these clearing organs could be dose limiting, especially for radiometal-labelled mutants. The lowest tumour-to-liver AUC ratios were observed with the 90Y-CHX-A″ DTPA-hu3S193 H310A reached a tumour-absorbed dose 1.2-fold higher than hu3S193 wild type   gradually decreasing to 25.0 ± 2.5 % (wild type) versus 18.4 ± 1.4 % (I253A/H310A) after 6 days of incubation in human serum at 37 °C.Qualitative imaging was performed with hu3S193 wild type, hu3S193 I253A/H310A and huA33 control in A431 tumour-bearing mice. Mice were injected with 3.7 MBq  of radiolabelled antibody. HuA33 wild type was used as a negative control as the A431 cell line does not express A33 antigen. Quality controls showed that at day 0, only 3.4 % of 177Lu-CHX-A″ DTPA-labelled hu3S193 wild type . The tumour-to-liver ratio was higher for wild type  compared to I253A/H310A .Whole body scans were obtained at 48 h post injection Fig. . By 48 hype Fig. , d and Iype Fig. , e. No type Fig. , 3f. Hig177Lu-CHX-A″ DTPA-labelled hu3S193 wild type or I253A/H310A (n = 5). After 48 h post injection, mice were sacrificed and organs were counted for radioactivity (Table P < 0.01). Tumour uptake of 177Lu-CHX-A″ DTPA-labelled I253A/H310A was specific and significantly higher than 177Lu-CHX-A″ DTPA-labelled huA33 . Importantly, tumour-to-blood ratios were 2.93 ± 0.65 (hu3S193 wild type), 0.49 ± 0.30 (huA33 control) and 16.56 ± 3.71 (hu3S193 I253A/H310A). Tumour-to-liver ratios were 7.71 ± 1.26 (hu3S193 wild type), 6.37 ± 0.62 (huA33 control) and 1.20 ± 0.23 (hu3S193 I253A/H310A).In a separate biodistribution study, mice were injected with 177Lu-CHX-A″ DTPA-labelled hu3S193 antibodies (270 μg) and biodistribution (100 μg). Tumour uptake with 5 μg (~0.25 mg/kg) 111In-CHX-A″ DTPA-labelled hu3S193 antibodies was not significantly different from tumour uptake with 100 μg (~5 mg/kg) 177Lu-CHX-A″ DTPA-labelled hu3S193 antibodies as shown in Additional file Higher protein doses were used for imaging with 90Y- and 177Lu-based therapy, with the best candidate being hu3S193 I253A/H310A for 177Lu-based therapy, achieving almost double tumour-to-blood AUC ratios compared to wild-type hu3S193.Fc engineering is a promising technique to increase the therapeutic ratio for payload delivery. This approach aims to increase the therapeutic efficacy by reducing the terminal half-life of antibodies or Fc-containing antibody constructs, thus reducing the dose-limiting toxicity to the blood and bone marrow of intact radiolabelled therapeutic antibodies. In this study, four variants of an intact IgG1 antibody, hu3S193, with a broad range in half-life were created by site-directed mutagenesis and analysed in blood clearance studies and biodistribution studies. Our results show that hu3S193 can be engineered with improved therapeutic ratios for Four Fc variants of an intact IgG1 antibody, hu3S193, with a broad range in serum half-life were created by site-directed mutagenesis and analysed in preclinical blood clearance studies and biodistribution studies. Non-specific uptake of hu3S193 wild type in a Lewis-Y-negative cell line SW1222 is known to be low ; in addi131I-labelled scFv-Fc H310A/H435Q could deliver >7000 cGy, limited by red marrow toxicity. 90Y-labelled scFv-Fc I253A was best suited for 90Y delivery, although dose predictions did not match the intact cT84.66 antibody. The current study extends their findings to an intact IgG1 and includes predictions for 177Lu-based therapy. In contrast to the anti-CEA study by Kenanova et al., the radioiodinated anti-Ley hu3S193 variants cleared much faster than the radioiodinated scFv-Fc fragments, and therefore, no therapeutic benefit could be obtained from the radioiodinated hu3S193 variants. The hu3S193 variants might be more sensitive to deiodination in vivo compared to the scFv-Fc variants. To study the benefit for RIT of using radioiodinated hu3S193 variants with faster blood clearance, other radioiodination methods such as site-specific radioiodination of cysteine-containing hu3S193 variants could be explored [Early engineering approaches using deletion of constant domains to reduce the serum half-life of antibodies have shown that tumour-to-normal dose ratios can be improved , 11, 37.explored .y-expressing tumours such as colon, lung, ovarian and breast cancer require radiation doses of at least 60 Gy to eradicate solid tumours in patients [Whilst a doubling in dose to the maximum predicted dose calculated for hu3S193 wild type was predicted and a doubling in the tumour-to-blood AUC ratios achieved, the dose needed for tumour eradication in patients is likely to be higher than that achievable with a single infusion. Lepatients , use in patients  or combipatients , 29. To 177Lu-labelled I253A/H310A. The imaging results reflect the biodistribution data; the highest tumour uptake was seen with hu3S193 wild type, but specific tumour uptake was still achievable with the fastest clearing hu3S193 variant I253A/H310A, and tumour-to-blood ratios were far higher at earlier time points with the mutant hu3S193 constructs. However, because 177Lu can only be imaged using SPECT cameras, a PET equivalent such as 89Zr-labelled I253A/H310A might be a better alternative and may allow better quantification of tumour uptake and saturability in vivo. We are currently evaluating 89Zr-labelled hu3S193 variants in tumour-bearing mice in small animal PET studies.Due to the toxicity limitations of RIT and patient tumour variability, it is useful to develop theranostics to guide dosimetry in patients, exemplified in this study by 177Lu-imaging (270 μg) and biodistribution (100 μg) studies versus 111In-biodistribution (5 μg) studies. We demonstrated that tumour uptake at 5 and 100 μg protein doses were comparable. Lewis-Y expression is very high on A431 cells and similar to MCF-7 cells, estimated to express around 106–107 antibody binding sites [A higher protein dose was used for ng sites . A recenng sites . EGFR isAlthough significant increases in therapeutic doses were predicted with some variants compared to hu3S193 wild type, increased toxicities to normal tissue such as the liver and kidneys were also predicted. A shift in toxicity from the bone marrow to liver as toxicity limiting organ for radiometal-labelled antibody variants with a faster blood clearance is not surprising. As the antibody variants clear faster from the body, radioactivity exposure to the blood is reduced. In addition, due to the reduced binding affinity of the variants for FcRn, an increase in catabolism is expected as the variants are no longer recycled in the blood circulation and degraded in the lysosomes (e.g. hu3S193 I253A/H310A) , and thu90Y- and 177Lu-based therapy, with the best candidate being hu3S193 I253A/H310A for 177Lu-based therapy. These results are highly relevant to utilising Fc engineering as a tool to improve the clinical application of hu3S193 and other intact antibodies especially for payload delivery.Four Fc variants of an intact IgG1 antibody, hu3S193, with a broad range in serum half-life were created by site-directed mutagenesis and analysed in preclinical blood clearance studies and biodistribution studies. We have shown that hu3S193 can be engineered with improved therapeutic ratios for


Incorporation of hypoxia into our model with FMISO-PET increases the model–data agreement by an order of magnitude. This improvement was robust to our definition of hypoxia or the degree of radiation resistance quantified with the FMISO-PET image and our computational model, respectively. This work demonstrates a useful application of patient-specific modelling in personalized medicine and how mathematical modelling has the potential to unify multi-modality imaging and radiation treatment planning.Glioblastoma multiforme (GBM) is a highly invasive primary brain tumour that has poor prognosis despite aggressive treatment. A hallmark of these tumours is diffuse invasion into the surrounding brain, necessitating a multi-modal treatment approach, including surgery, radiation and chemotherapy. We have previously demonstrated the ability of our model to predict radiographic response immediately following radiation therapy in individual GBM patients using a simplified geometry of the brain and theoretical radiation dose. Using only two pre-treatment magnetic resonance imaging scans, we calculate net rates of proliferation and invasion as well as radiation sensitivity for a patient's disease. Here, we present the application of our clinically targeted modelling approach to a single glioblastoma patient as a demonstration of our method. We apply our model in the full three-dimensional architecture of the brain to quantify the effects of regional resistance to radiation owing to hypoxia  Followi1.1.Clinically, glioblastoma progression and response to therapy are monitored with MRI ,5. For t18F]-fluoromisonidazole (FMISO) positron emission tomography (PET) -FMISO-PET study on an IRB-approved research protocol. MRI and PET protocols can be found in the electronic supplementary material. The patient underwent an MRI study 3 days following the completion of RT, and subsequent images were taken at two-month intervals. The MRI and PET images were spatially coaligned to a common coordinate system using a rigid body transformation to the BrainWeb phantom [atlab software suite [The patient's diagnostic and pre-operative MRIs were performed 13 days apart. Three days after the biopsy procedure and 2 days prior to the first fraction of RT, the patient underwent an [ phantom  using th phantom  and statre suite .2.4.atlab. The accuracy and reproducibility of this method is comparable to manual tumour delineation [V (cm3) was calculated numerically using the formulaN two-dimensional slices, acquired in the axial plane, and where slice i has thickness idz and tumour area iA. The tumour area iA is determined by summing the number of pixels containing tumour on slice i. The tumour growth velocity, shown in Tumour volumes were measured for the T1Gd and T2 sequences for MRI studies  using a ineation . Specifi2.5.+; p < 0.0001).The co-registered FMISO-PET images were scaled to the average venous blood concentration of FMISO activity  to produce a tumour/blood (T/B) ratio image seen in 2.6.−2 given daily during course of RT and continued adjuvantly for a total of 14 months [Five days after the diagnostic biopsy, the patient was treated with RT delivered with a three-dimensional conformal treatment plan using 6 MV photons. The target volumes were defined as the T2-defined abnormality with a 2.5 cm margin, which had a planned target dose of 54 Gy delivered in 30 daily fractions of 1.8 Gy per fraction. An additional dose was delivered to a smaller volume defined by the gadolinium-enhancing region plus a 2 cm margin of 7.2 Gy delivered in an additional four daily fractions of 1.8 Gy per fraction for a total of 61.2 Gy to this region. The target volumes are labelled as isodose curves of 61.2 and 54 Gy on the planning MRI in 2.7.c = c at time t and location x =  in units cells per mm3, in terms of diffuse invasion and density-dependent logistic growth. Logistic growth relates the per capita growth rate to available space for the cells to grow, so that if there are few cells in a unit volume of tissue, the overall growth rate is higher than if there are many cells per unit volume. The maximum number of tumour cells that can fit in a cubic millimetre of tissue is known as the carrying capacity, denoted K, and is computed to be 1.91 × 106 (cells mm−3) assuming a 10 µm diameter tumour cell. Once the tumour cell density reaches the carrying capacity (c = K) at a particular spatial location, the tumour cells are space-restricted and therefore do not proliferate. Because we do not incorporate the clearance of dead cells, when the tumour cell density reaches the tissue carrying capacity, we assume the tumour cells are dead or become quiescent in this region.ρ, per year) and invasion (D(x), mm2 per year). The invasion of malignant tumour cells into the brain parenchyma is influenced by the anatomy of the brain, codified in equation (The PIRT model (equations  and 2.22.2) quanequation  represenequation  and , The invasive migration of tumour cells throughout the brain presents a challenge to understanding the true extent of the subclinical disease, as tumour dispersal speeds can vary up to 100-fold between pioneering cells in white matter compared with the more random motion in the core of the tumour and in grey matter ,27,28. T = 100Dg ,30. The 2.9.S) from the linear-quadratic dose–response model. Equation ) as determined by the patient's clinical radiation treatment plan into a biological effect of each radiation treatment [reatment ,15,32,33reatment . First-oS) from the linear-quadratic model , there is no radiation effect. This situation corresponds to an unresponsive necrotic core, where we assume that there is no radiation-induced cell killing, because the cells are already dead. A necrotic core is a histologic and radiographic hallmark of glioblastoma that develops when the tumour growth exceeds the tissue carrying capacity.The radiation loss term in the PIRT model (equation ) is nonlequation ) determiequation ). For lo2.10.α/β fixed. This allows us to regard α as the single parameter to define radiation sensitivity. For each point in space and time, an effective dose and probability of cell survival can be calculated that corresponds uniquely to the individual patient's treatment plan and the linear-quadratic dose–response model parameter α. Increasing α decreases the probability of cell survival, S, and therefore increases the probability of RT-induced cell death. Increasing values of α correspond to increasing treatment effect and deviation from untreated growth. With the ratio α/β fixed, the single parameter α can be uniquely determined using either the T1Gd or T2 post-chemoradiation tumour size, using a bootstrap optimization technique, yielding a one-to-one relationship between α and model prediction error, as described in Rockne et al. [α owing to the localization of FMISO-PET activity within the T2+ region , which determines relative resistance to RT in regions of low oxygen [α and β to be spatially defined, so that the OER is applied in regions of hypoxia, as follows:The ratio of the parameters ous work ,25,34–36w oxygen –39. The nd three , corresp2.12.(1) Clinical radiation dose delivered with spatially uniform treatment response. This corresponds to a value for the OER equal to one (OER = 1) at all locations in the brain, and assumes that all tumour cells are equally susceptible to RT damage.(2) Clinical radiation dose with localized radio-resistance owing to hypoxia via a binary relationship. This quantifies focal radio-resistance defined by the patient's FMISO-PET scan, where the tissue to blood value is greater than or equal to 1.2 , definesThe PIRT model is used to investigate the role of FMISO-PET-defined hypoxia in determining model-predicted radiation response and to test the sensitivity of our definition of hypoxia in influencing radiographic response to RT. Each simulation was run using an anatomically accurate three-dimensional brain phantom  and the 2.13.D/ρ [D/ρ) are more hypoxic than diffuse tumours (large ratio D/ρ) [To establish a histopathological correlation of cellular density to imaging, we associate enhancing features on T1Gd and T2-weighted MRIs with high and low tumour cell densities, respectively ,42. One D/ρ ) which htio D/ρ) . This caD) and proliferation (ρ) [The PIRT model (equation ) predict ,45. A co  computedtion (ρ) ,42.2.14.D and ρ were calculated based on baseline tumour growth kinetics using pre-biopsy MRI. Specifically, within the spectrum of dynamics observed in glioblastoma, the patient's net rates of invasion and proliferation that characterize the tumour growth lie within 1 standard deviation of the population mean observed in 63 patients [α, the calculated value of α = 0.055 (per Gy) reflects neither an exceptionally resistant nor sensitive response (D/ρ = 0.93 mm2) reflects a tumour growth pattern which is balanced between cooptive and angiogenic vascularity and therefore predicts a modest hypoxic burden.Patient-specific model parameters patients . Similarresponse . The rel2.15.In order to assess the accuracy of model predictions, we compare the simulated and actual tumour regions on T1Gd and T2-weighted MRI using spatially defined similarity metrics. Each metric returns a value indicating the quality of agreement between model and observed tumour growth. This analysis was performed on the two MRIs prior to RT and on the first MRI following RT on both the T1Gd and T2 tumour volumes. Model-predicted RT response is defined with the first MRI performed post-RT . BecauseIn addition to voxel-wise metrics of concordance, we quantified morphological similarity by computing the distance between the predicted and observed tumour surfaces in three dimensions. This measure returns a distribution of distances, so that a value of zero in this distribution indicates intersection of the simulated and actual tumour surfaces. We report the median and standard deviation of this distribution—the closer to zero and smaller the variance, the better the model prediction. Reporting the median and standard deviation allows us to evaluate both variance and bias in our model predictions and avoid a cancelling effect of including both positive and negative distances that would be reflected in the mean of the distribution.3.Using our patient-specific model for glioma growth and response to RT, we find the incorporation of hypoxia-mediated radiation resistance defined with FMISO-PET leads to an order of magnitude decrease in relative volumetric error, from 14.6% to 1.1% . IncorpoSpatial concordance between the model-predicted disease burden prior to and following RT was measured through voxel-wise similarity metrics. These metrics are challenged by the spatial alignment of the clinical MRI with the atlas and by a large number of true negatives for pixels far outside the gross tumour region. With this limitation in mind, we observed an increase in voxel-wise concordance by incorporation of localized hypoxia-modulated radiation resistance for most metrics. Volume similarity was increased from 0.844 to 0.91, and the Jaccard metric was increased from 0.72 to 0.83. Sensitivity increased from 99.75 to 99.87, although specificity decreased from 30.25 to 15.61.The distance between surfaces analysis was performed for all three MRI studies, two prior to biopsy and one immediately following the full course of RT on T1Gd and T2 sequences are illustrated in 3.1.To investigate the sensitivity of our predictions to definitions of hypoxic regions and parameter selection, we considered a range of OER values in the HV ranging from 1.5 to 3.0. This produced a parabolic relationship between relative volumetric error and OER using T1Gd size such that the minimum model–data error occurred at approximately OER = 2.5. OER values between 2.0 and 3.0 yielded relative volumetric errors at or below approximately 1%, and all resulted in an order of magnitude improvement to the model prediction. This analysis demonstrates the robustness of the model predictions to variations in OER, and by extension, the volume fraction of hypoxic cells the T/B FMISO-PET image represents.D, ρ) or response rates  is discussed in detail in the supplement to Rockne et al. [To further investigate the role of spatial localization of radiation resistance, simulations were also performed in isotropic three-dimensional spherical symmetry without anatomical complexity, using the T1Gd isodensity as a lower bound threshold to determine the hypoxic region. In these simulations, we observed a similar outcome, that is, that model–data agreement is substantially improved with the inclusion of hypoxia-mediated radiation resistance, where HV is within the bulk tumour region. However, the model predictions were not as accurate with this simplification. This suggests that the contrast-enhancing region is not a suitable surrogate for hypoxic tumour, and that a more specific indicator such as FMISO-PET in three dimensions not only improves the model prediction, but is also needed in a spatially heterogeneous tumour such as GBM. Sensitivity of model-predicted tumour burden following RT based on variability in the tumour growth  of the tumour. The model predictions are improved by the incorporation of an OER to approximate the degree of radio-resistance created by hypoxic conditions within the tumour. The large improvements in model–data agreement attributable to a modest volume of hypoxia-mediated resistance to radiation effect underscores the significance of spatial heterogeneity in delivery and response to RT in glioblastoma and the complexity of a three-dimensional model.in vivo clinical data. Ideally, a more patient-specific, biologically driven model of the process of angiogenesis, hypoxia and necrosis using a modelling approach which integrates multi-modality imaging is desired [Without the incorporation of OER and localized radiation resistance, the model is unable to fully capture post-RT tumour size in regions of high cellular density, motivating the addition of the OER parameter derived from  desired ,54. In c desired .in vivo quantitatively. By providing quantitative metrics of a patient's response to radiotherapy, our model has the potential to unify multi-modality imaging and treatment planning and establish a useful application of patient-specific modelling in personalized medicine.The goal of modelling biological response to therapy has been long sought after, but there have been no demonstrable successes with the potential for impacting individual treatment planning . Other e


To compare total fluid intake (TFI), defined as the sum of water and all other fluid types, assessed with a 24-h dietary (food and fluid) recall with mean TFI assessed with a 7-day fluid-specific record among adolescents and adults.This repeated cross-sectional study compared TFI as assessed by two fluid assessment instruments using a crossover approach. 290 adolescents  and 289 adults  from Indonesia completed the study.r = 0.333; p < 0.001). The Bland–Altman method, however, showed an underestimation (bias) of mean TFI by a 24-h recall when compared with the 7-day fluid record . The mean difference also increased with increasing TFI: Mean difference for the lowest and highest quartiles of TFI was 139 versus −1265 mL/day. The 7-day fluid record recorded two  extra drinking acts compared with the 24-h recall, whereas the mean volume per drinking act was significantly higher with the 24-h recall .Significant correlations were observed between fluid intake assessed with the 24-h recall and the 7-day fluid record (Compared with a 7-day fluid record, a 24-h dietary recall significantly underestimated TFI. Subjects recalled two less drinking acts, while estimating the volume consumed per drinking act to be larger. Since the adequate intakes for total water intake are based on median intakes observed in national surveys that most frequently used the 24-h recall method, they may potentially be underestimated. Widyakarya Nasional Pangan dan Gizi 2012) set adequate total daily water intake recommendations (RDI) for adolescent aged 16–18 years at 2.1 L/day for girls and 2.2 L/day for boys, while for adults aged 19–64 years, consuming 2.3 and 2.5–2.6 L/day was recommended for women and men, respectively   382 mL/day; A Bland–Altman plot for mean TFI recorded with the 24-h dietary recall and 7-day fluid record is presented in Fig. r = 0.333; p < 0.001) .The Spearman rank correlation test showed a significant correlation between the fluid intake data estimated with the 7-day consecutive fluid record and the 24-h dietary recall  and adults (19–64 years). The results indicated that, at group level, the 24-h dietary recall significantly underestimated TFI compared with the 7-day fluid record, and that there was poor agreement between both methods.any day of the 7-day fluid record was comparable to the difference in TFI between the 24-h recall and the mean of the 7 days of the fluid record. This means that the intake of a group estimated by the 24-h recall cannot be used/interpreted as the intake on any given day. A last possible explanation for the difference in TFI between both instruments could be a social desirability bias. When performing the 24-h recall, the subject was face to face with a nutritionist and might have over- or underreported their TFI or intake of a certain fluid type  to meet a socially desired health standard. The over- or underestimating of a certain fluid type was, however, considered to be unlikely as the subjects consumed 75–76 % of their TFI as water. Moreover, the contribution of the different fluid types to TFI was comparable between both methods. An over- or underreporting of the volume consumed was likely as the volume per drinking act significantly differed between both methods, with a higher volume per drink act observed with the 24-h recall. These observations suggest that (1) with a 24-h recall, subjects recall two drinking acts less than with a 7-day fluid record, but they estimated their volume consumed per drinking act to be larger and (2) a 24-h recall seems to be an acceptable method if the aim was to describe the contribution (in %) of different fluids to TFI of a group. The latter observation should, however, be confirmed in a sample with a more diverse fluid intake pattern than the one of this Indonesian sample (75–76 % of TFI was water).There are several possible explanations for the differences in TFI at group level estimated with the two methods. Firstly, the 7-day fluid record is a prospective method, whereas a 24-h dietary recall is a retrospective method that depends on respondents’ memory, recall, and conceptualization abilities. The underestimation by the 24-h recall compared with the 7-day fluid diary might thus be due to subjects not recalling some drinking acts. This hypothesis is supported by the current results showing two extra drinking acts with the 7-day fluid record compared with the 24-h recall. A second explanation could be linked to the specificity of the instruments: With the 24-h dietary recall, both foods and fluids were recorded; therefore, subjects might have focused on their food and fluid intake at meal time. Depending on the country, fluids are more frequently consumed outside of meals ; thus, dDespite the disagreement in TFI measured with both instruments, the TFI data of the 24-h recall and TFI of 7-day fluid record were significantly correlated. Yet, according to the Dancey and Reidy’s  categoriSome limitations of the study need to be acknowledged. Since both instruments were compared in the absence of an objective biomarker of TFI, this is a relative comparison. In nutrition however, when different instruments are compared, a 7-day dietary record is frequently used as the reference method , 14. SinThis study also had several strengths. Firstly, a large sample was recruited, with an equal distribution of gender and an equal coverage of both age groups. Moreover, the study was completed with a high compliance rate . The crossover approach is a second strength to highlight. This enabled a pairwise comparison between both instruments, but it also allows to conclude that the systematically higher fluid intake estimated with the 7-day fluid record is not due the nutritionists improving their interview skills or participants learning over time to report more fluid intake. A third strength worthy of mentioning is the use of the Bland–Altman method for data analysis, which is the most recommended validation procedure , 13. ThiThe results of this study have implications in several contexts. The current recommendations on total water intake in Indonesia, but also in the USA defined by the Institute of Medicine, are adequate intakes based solely on median observed intakes , 20. TheWhen compared with a 7-day fluid record, a 24-h dietary recall significantly underestimated TFI with the difference between TFI assessed with these two instruments increased with increasing mean TFI. Both instruments showed disagreement in 23 % of the study population on the classification of the adequacy of their water intake. These results suggest that the evaluation of the public health risk associated with inadequate or over consumption of different types of fluids based on 24-h recall data may not be accurate, and that the adequate intake for total water (currently based on 24-h intakes) may require revision in the future.


Thus, DCs represent the immune systems’ Achilles heel. Uniquely among the human herpesviruses, VZV infects both DCs and T cells, and exploits both as Trojan horses. During primary infection VZV-infected DCs traffic to the draining lymph nodes and tonsils, where the virus is transferred to T cells. VZV-infected T cells subsequently spread infection throughout the body to give the typical varicella skin rash. The delicate interplay between VZV and DCs and its consequences for viral immune evasion and viral dissemination will be discussed in this article.Varicella zoster virus (VZV), a human alphaherpesvirus, causes varicella and subsequently establishes latency within sensory nerve ganglia. Later in life VZV can reactivate to cause herpes zoster. A reduced frequency of VZV-specific T cells is strongly associated with herpes zoster illustrating that these immune cells are central to control latency. Dendritic cells (DCs) are required for the generation of VZV-specific T cells. However, DCs can also be infected  In 1943 Helmut Ruska discovered that the virus particles isolated from varicella lesions and those isolated from zoster lesions had identical morphology . The virAfter primary infection has been resolved VZV establishes latency in the dorsal ganglia of nerve cells that innervate the affected skin. It can reactivate, mostly in immunosuppressed individuals and in elderly adults, resulting in herpes zoster (commonly known as shingles) after anterograde transport to the corresponding dermatome. Skin blisters that occur during varicella and zoster harbor high numbers of infectious particles. The main route of transmission is through cell free virus particles that are carried by aerosols of infectious respiratory tract secretions or vesicular fluid. Pathological consequences of VZV include congenital varicella syndrome  and postMore than 30 years ago a live attenuated vaccine (vOka) was developed from the Oka parental strain pOka; . IntriguIn the absence of inflammation (steady state) two main groups of DCs are observed in the skin and mucosa of the respiratory tract: conventional DCs (cDCs) and Langerhans cells LCs; . They miin vivo into so-called inflammatory DCs  and interleukin (IL)-4 and subsequently exposing them to maturation stimuli such as CD40L or tumor necrosis factor (TNF)-α -α . These me to VZV . VZV-infe to VZV  but can e to VZV . The opee to VZV .ex vivo are susceptible to VZV infection  after injection of VZV-infected human T cells . Furtherin vivo, although the absence of LC in VZV skin lesions and the ability to infect primary LCs ex vivo strongly suggest this , epidermal and/or dermal DCs, transfers VZV to T cells in vivo. Finally, it has not been investigated whether mature DCs are more effective in transferring VZV to T cells as it is the case with HIV  such as toll-like receptors TLRs; . Intact em cells . Similarem cells .Herpesviruses have developed diverse and numerous strategies to interfere with innate immune responses . VZV preT cells not only transport VZV to the skin but also play a pivotal role in controlling VZV during primary infection . Decreascis/medial Golgi (Varicella zoster virus interferes with T cell responses similar to most other herpesviruses . For thial Golgi . By whical Golgi .in vitro (Central to viral immune evasion is the third signal which can be blocked only by VZV clinical strains but not by vOka, the VZV vaccine strain . Efficiein vitro . This isin vitro .In this article we have discussed how VZV, one of the most successful human pathogens, exploits DCs to disseminate in the human organism, evade the antiviral immune responses and cause disease. In contrast, vOka, the only live attenuated vaccine available against human herpesviruses, does not cause significant disease. Although both wild type VZV and vOka target DCs only DCs infected with vOka can be instructed to become a Th1-promoting antigen-presenting cell. It will be important in the future to precisely define on the molecular level how DCs infected with wild type VZV differ from those infected with vOka. Moreover, it is necessary to pinpoint the viral proteins responsible. A better understanding of these differences could allow to design desperately needed vaccines against other human herpesviruses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Accumulating evidence indicates that fractionated radiotherapy (RT) can result in distant non-irradiated  tumour regression. Although preclinical studies indicate the importance of T cells in this infrequent phenomenon, these studies do not preclude that other immune mechanisms exhibit an addition role in the abscopal effect. We therefore addressed the question whether in addition to T cell mediated responses also humoral anti-tumour responses are modulated after fractionated RT and whether systemic dendritic cell (DC) stimulation can enhance tumour-specific antibody production. We selected the 67NR mammary carcinoma model since this tumour showed spontaneous antibody production in all tumour-bearing mice. Fractionated RT to the primary tumour was associated with a survival benefit and a delayed growth of a non-irradiated  secondary tumour. Notably, fractionated RT did not affect anti-tumour antibody titers and the composition of the immunoglobulin (Ig) isotypes. Likewise, we demonstrated that treatment of tumour-bearing Balb/C mice with DC stimulating growth factor Flt3-L did neither modulate the magnitude nor the composition of the humoral immune response. Finally, we evaluated the immune infiltrate and Ig isotype content of the tumour tissue using flow cytometry and found no differences between treatment groups that were indicative for local antibody production. In conclusion, we demonstrate that the 67NR mammary carcinoma in Balb/C mice is associated with a pre-existing antibody response. And, we show that in tumour-bearing Balb/C mice with abscopal tumour regression such pre-existing antibody responses are not altered upon fractionated RT and/or DC stimulation with Flt3-L. Our research indicates that evaluating the humoral immune response in the setting of abscopal tumour regression is not invariably associated with therapeutic effects. However certain radiation-induced bystander effects of non-irradiated cells exhibit biological effects which can be beneficial for treatment outcome –3. Alreaadiation , 5. Althadiation , 7. Thesadiation –12, lungadiation , 14, renadiation , hepatocadiation , and chradiation . Until nadiation , 18.Over the last decades significant progress was made in our knowledge on how the immune system can be activated by injured tissue and various molecular pathways triggered by damage-associated molecular patterns (DAMPs) . Therefoet al. described an animal model (67NR mammary carcinoma) in which RT also influenced the tumour outside the field of irradiation [nude) mice, indicating that T cells were necessary for radiation-induced bystander effects [In an effort to gain fundamental insight into the immunological mechanism of the abscopal effect, Demaria adiation . In this effects . These a effects .et al. recently showed that the concept of DC stimulation extends beyond animal models and they provided proof-of-principle that stimulating the endogenous DC compartment with GM-CSF combined with RT resulted in objective abscopal responses in more than 20% of patients with metastatic cancer. The presence of abscopal effects was also associated with a survival benefit in these patients [In line with observations on the importance of T cells, also evidence was provided that stimulating the endogenous DC compartment augments the efficacy of the RT-induced activity against metastasis –25. Treapatients .et al. was not aimed for immune monitoring, they showed differences in baseline blood parameters in abscopal responders and non-responders [et al. observed that the abscopal effect was paralleled with the induction of antibody responses against the tumour in a patient with metastatic melanoma [Taken together these findings clearly indicate the importance of DC-induced T cell responses in abscopal tumour regression. This concept is further supported by the fact that abscopal effects can be enhanced by immune checkpoint inhibitors such as CTLA-4 blockade in animal models  as well sponders . Additiomelanoma . In thismelanoma .Nowadays, it remains to be established whether the induction of antibody responses against the tumour is directly involved in abscopal tumour inhibition, or whether the induction of such antibodies only serves as a biomarker indicating the development of novel immune responses in a subset of patients. Alternatively, it is possible that the presence of such antibody responses is a prerequisite for abscopal effects.In the current study, we aimed to assess whether the abscopal tumour growth delay that is observed after fractionated RT is paralleled with the modulation of a pre-existing humoral anti-tumour response in the 67NR mammary carcinoma animal model. In addition, we assessed whether stimulation of plasmacytoid (pDC) and conventional (cDC) DC with Flt3-L alone or combined with fractionated RT can modulate a pre-existing humoral immune response.2 and 5% CO2 chamber at 37°C.The murine  67NR mammary carcinoma cell line was kindly provided by S. Demaria . The 67NR cell line was cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal calf serum (Greiner Bio One) and 1% penicillin-streptomycin (Gibco). The metastatic murine 4T1 mammary carcinoma (a differentiated subclone of the non-metastatic 67NR cell line) and C26 colon carcinoma cell lines (both obtained from the ATCC) were cultured in Roswell Park Memorial Institute Medium  supplemented with 10% fetal calf serum and 1% penicillin-streptomycin. The cell lines were incubated in a humidified 20% Oet al. [5 67NR cells were subcutaneously injected in immunocompetent Balb/C mice . The mice were anesthetized with isoflurane  prior to cell injections. The cells were injected in the right (primary tumour) and left flank (secondary tumour) at day 0 and day 2 respectively. At day 14, tumour-bearing mice were randomised based on primary tumour size and assigned to four groups: untreated, Flt3-L, RT, RT+Flt3-L. The tumour-bearing mice were housed separately, had free access to food and water, and were checked every day for animal well-being according to local institutional guidelines for animal welfare. The tumour size was assessed three times a week by palpation , and corrected using the following formula: L*W*H*(π/6) [3 for both tumours) or at the end of the experiment (day 162), mice were anesthetized and sacrificed by cervical dislocation. None of the mice died without euthanasia. The blood and both tumours were collected for further analysis. The blood was incubated with heparin (LEO Pharma) and centrifuged to separate the plasma. Half of the tumour was homogenized and used as a single cell suspension for flow cytometric analysis and the other half of the tumour was embedded in TissueTec and further stored at -80°C for immunohistochemistry (IHC). The animal experiments were performed according to Dutch government guidelines, approved by the Animal Ethical Committee of Maastricht University .In order to investigate abscopal tumour regression, we took advantage of the mouse model previously generated and optimized by Demaria et al. . In shor*H*(π/6) . At the i.p. injection of pentobarbital (60mg/mL in NaCl 0.9%). The mice (four per session) were positioned in a customized Plexiglas tray. The animals’ body was protected by lead shielding except for the primary tumour that was placed under a linear accelerator  as is schematically illustrated in Only the primary tumour was irradiated with three times 8 Gy (±45.5 mGy/s) in three consecutive days . The tumi.p. injections of 10μg Flt3-L  for ten consecutive days as previously described [The tumour-bearing mice in groups Flt3-L and RT+Flt3-L received escribed . The adm6 67NR cells were cultured, harvested in PBS, and heat-killed for 30min at 60°C. The heat-killed cells were resuspended in incomplete Freund’s adjuvant  containing 200μg DNAse, and 0.5mg/mL CpG ODN 1826 . The mixture was injected in immunocompetent Balb/C mice. Primary immunized mice received a booster injection containing IFA, CpG ODNs and heat-killed 67NR cells after 3 weeks. The immunized mice were anesthetized and sacrificed by cervical dislocation 1 to 3 weeks after the boost injection. The blood was collected by cardiac puncture and centrifuged to separate the plasma.In order to obtain heat-killed 67NR cells, a total of 10 *10We optimized two independent methods for demonstrating the presence of TBAs in the plasma of 67NR tumour-bearing mice. For both methods we intentionally used different antibody clones for detection. In this manner, we used fluorescent detection with flow cytometry (FC), which has the advantage of a higher dynamic range in samples with a higher concentration. The second method used enzymatic amplification for a more sensitive detection in the lower concentration range.5 67NR cells were transferred to a 96 wells v-bottom plate. The cells were incubated with 10% FCS for 30min (Greiner Bio One) to prevent non-specific binding. Then, diluted plasma (1:20) samples were added for 30min at room temperature (RT). TBAs in the plasma were stained with APC labelled anti-mouse pan-Ig  for 20min (RT). In case of immunoglobulin (Ig) isotype determination in the plasma, APC labelled anti-mouse IgG1 , PE labelled anti-mouse IgG2a , FITC labelled anti-mouse IgG2b , and PE-Cy7 anti-mouse IgM  were used. In between all steps, cells were washed with buffer  and centrifuged for 3min, 800g at 4°C. The stained samples were measured and analysed using the FACSCanto II (BD) and FACSDiva software v6.0 (BD) respectively.1 *105 67NR cells were added to a flat-bottom 96 wells cell culture plate and cultured for 18hrs at 37°C. The adherent cells were washed with buffer  and fixated with 3.8% formaldehyde (Sigma) for 45min at RT. To prevent non-specific binding, the plate was subsequently incubated with 1% BSA (Sigma) for 1h at RT. Then, diluted plasma (1:20) samples of the intervention mice were added for 1h at RT. TBAs in the plasma were detected with HRP labelled anti-mouse pan-Ig  and TMB substrate (Thermo Scientific). The enzymatic reaction was stopped by 0.9M H2SO4. In between all steps, 67NR cells were washed with buffer. The absorbance was measured at 450nm (BioTek Synergy HTX).1 *10The immunoglobulin isotypes that were present in the tumour tissue were determined using the cytometric bead array (CBA) kit (BD). TissueTec embedded tumour tissue was homogenized using a mini-bead beater (BioSpec) for 2x20s and 1x30s in CellLytic MT Mammalian Tissue Extraction buffer (Sigma). The total protein concentration in μg/mL was measured using the BCA (bicinchoninic acid assay) method (Thermo Scientific). The tumour lysates were incubated with microbeads coated with capture antibodies for the different Ig isotypes. The bound Ig isotypes were stained with fluorescent-labelled detection antibodies. Subsequently, the samples were measured using flow cytometry according to the manufacturers protocol. FCAP Array analysis software v3.0 (BD) was used for data analysis. The concentration of all Ig isotypes was normalized for total protein quantity of the tumour lysates.6 tumour cells were added to a 96 wells v-bottom plate. The cells were incubated with mouse serum (Dako) to prevent non-specific binding (20min). Upon incubation, APC labelled anti-mouse CD45 (clone 30-F11), PE-Cy7 labelled anti-mouse CD11c (clone HL3), V500 labelled anti-mouse CD4 (clone RM4-5), FITC labelled anti-mouse CD19 (clone 1D3), V450 labelled anti-mouse CD8α  were added for 15min at RT. The life-death marker 7-AAD (BD) was added before measurement. All fluorescent labelled antibodies were purchased from BD, unless otherwise specified. In between all steps, the cells were washed with buffer  and centrifuged for 3min, 800g at 4°C. The stained cells were measured and analysed .1.5 *10Half of the tumours embedded in TissueTec were sectioned (10μm) using a cryostat (Leica) and fixated with acetone. The unconjugated rat anti-mouse B220-CD45r, CD3, CD4, and CD8 antibodies (hybridoma cell lines) were added for 1h at RT. Then, peroxidase blocker and biotinylated anti-rat Ig  were added for 30min at RT. These biotinylated antibodies were detected with HRP labelled streptavidin (30min) and DAB substrate (BD). The tumour sections were dehydrated in 50%, 70%, 96%, and 100% ethanol (5min) and analysed using the Carl Zeiss Axioskop 40 microscope.The graphs, figures, and statistical analyses were made with GraphPad Prism Pro version 6 (GraphPad Software) and combined in Adobe Illustrator CS6/CC (Adobe Systems). The Kaplan-Meier survival curves were compared using the log-rank (Mantel-Cox) test. The Mann-Whitney t-test was used to determine statistical significance between the treatment groups. For all data, a P value of < 0.05 (*) was considered to be statistically significant. P-values < 0.01 were graphically presented as (**).et al., in which RT of the primary tumour resulted in growth delay of the distant tumour outside the field of irradiation [In an effort to gain fundamental insight into the immunological mechanisms of the abscopal effect, we took advantage of the animal model that was developed and optimized by Demaria adiation . Further5 67NR tumour cells in both flanks with a temporal separation of 2 days between the primary (right flank) and the secondary (left flank)  . At day t flank) . Immunot3; secondary 3.9 ±4.5 mm3)  the tumour volume that they had after randomization (day 14). The groups RT and RT+Flt3-L (RT) demonstrated a delay in primary (30 ±10 days) and secondary (23 ±7 days) tumour growth as compared to the groups untreated and Flt3-L  ; P<0.05.4.5 mm3) .To determine whether fractionated RT and DC stimulation with Flt3-L is associated with a survival benefit, we followed the untreated and treated animals for 162 days. As shown in In order to investigate whether inoculation of the 67NR tumour itself results in the induction of antibodies we measured the immunoglobulin (Ig) levels in the plasma of tumour-bearing Balb/C mice using two detection methods. The optimization of both detection methods is shown in In order to determine whether the detected antibodies were specific for 67NR, we incubated plasma from healthy immunocompetent (-control) and vaccinated (+control) mice with murine carcinoma cell lines 67NR, 4T1 (mammary), and C26 (colon) and visualized antibody binding by means of flow cytometric detection. The detected antibodies in the plasma of vaccinated Balb/C mice showed cross-reactivity with the 4T1 cell line , as the level of antibody binding was similar to the 67NR cell line  . In contTaken together these data indicate that 67NR inoculation is sufficient to drive the induction of a humoral immune response against the tumour in all mice. These antibodies bind specifically to the 67NR tumour and its related cell line 4T1 but not to the C26 cell line.To assess whether the abscopal tumour growth inhibition that is observed after fractionated RT is paralleled with the modulation of the humoral anti-tumour response, we evaluated the quantity of TBAs in the plasma of sacrificed tumour-bearing mice that were either untreated or treated with Flt3-L, RT, or RT+Flt3-L. As shown in 1, IgG2a, IgG2b, and natural isotype IgM were measured in plasma of vaccinated (+control), untreated and treated  tumour-bearing mice. Vaccinated (+control) mice showed a different percentual composition of isotypes IgG  and IgM (23%) as compared to tumour-bearing mice in non-RT and RT groups    . In partlute MFI . Notably0 ±1940)  between 1, IgG2a, IgG2b, and natural isotype IgM) of TBAs in homogenized tumour tissues using a cytometric bead assay. We detected Ig isotypes IgG1, IgG2a, IgG2b, and IgM in the primary and secondary tumour of mice in non-RT and RT groups (2a (65 ±46 pg/mL) as compared to kappa IgG1 (14 ±26 pg/mL), IgG2b (12 ±22 pg/mL) and IgM (17 ±14 pg/mL). The Ig isotypes kappa IgG3, IgA, and IgE were absent in both tumours (data not shown).In order to determine whether antibodies and immune cells are present at the site of the tumour, we measured the presence and Ig isotype composition  in the tumours. First, we evaluated the localisation of these cells using IHC. Therefore, we performed IHC and visualized B cells and CD3+, CD4+, and CD8+ T cells in both the primary and secondary tumours ; S7A FigSecond, in order to quantitate the infiltration of CD19+ B cells and CD4+, CD8+ T cells we performed flow cytometric stainings on single tumour cell suspensions. Using this approach, we confirmed and quantitated the presence of CD19+ B cells as well as CD4+, CD8+ T cells in the tumour at the end of the experiment . NotablyTaken together these data indicate that we did not find evidence indicating tertiary lymphoid structures or local production of TBAs at site of the tumours. In cases where primary tumours could be collected in the irradiated animals at the end of the experiment there were less T cells infiltrating the tumour as compared to the primary tumours of non-irradiated animals. It should, however, be emphasized that this evaluation takes place after reaching the endpoint of the experiment that is often characterized by exponential growth of the tumour. In cases where one of the tumours was resolved these specimens could not be collected.et al. that abscopal tumour regression depended totally on the presence of T cells [et al. further showed that predominantly cytotoxic CD8+ T cells were associated with abscopal tumour regression [Undebatable T cells play a crucial role in abscopal tumour regression as illustrated by several lines of evidence in literature. The most compelling proof of concept was the demonstration of Demaria  T cells . In subsgression . Howevergression , 27. CerBased on two clinical case studies that reported an association between abscopal tumour regression and the induction of humoral responses , 11, we Based on our findings we cannot specify the antigens that are expressed on the cell surface of 67NR tumours. Presumably the antibodies in the plasma bind to epitopes that are shared between various tumours that target tumour-associated rather than tumour-specific antigens. Nevertheless these antigens are clearly more abundant on 67NR tumour cells and its derived cell line 4T1 selected for being more metastatic , 30 as cet al. They demonstrated that fractionated RT was more effective than a single dose of RT. This latter finding is in line with previous research of Demaria et al. who indicated that a single dose of RT did not result in abscopal effects [In order to address a potential role of this humoral immune response we evaluated how this was altered upon fractionated RT. We showed that abscopal tumour growth inhibition is observed after fractionated RT in the 67NR mouse model. Fractionated RT in combination with or without Flt3-L administration resulted in growth delay of both irradiated and non-irradiated tumours, indicating abscopal effects. The groups receiving RT also showed an increased number of surviving tumour free mice. However, several of these irradiated tumour-bearing mice demonstrated exponential tumour growth, suggesting the importance of the immunogenic state of the Balb/C mice as well as the 67NR tumour. We applied fractionated RT to the primary tumour to reduce harmful side effects to the surrounding healthy tissue for clinical translation. This approach resulted in abscopal effects (RT groups), which was also observed by Dewan  effects , 24. Imp effects , 32. Not2a, which is the most effective at complement fixation and antibody-dependent cellular cytotoxicity (ADCC) [et al. who showed the crucial role of T cells in abscopal tumour regression [et al. demonstrated in a subset of patients with abscopal effects that not only the pre-existing antibody response was enhanced in magnitude but also antibodies targeting different epitopes of the same antigen and seromic analysis demonstrated that in one patient a novel antibody response against ten new antigens was observed [The lack of modulating the antibody response upon RT suggests that antibodies do not play a key role in abscopal tumour regression in the 67NR mouse model. However, current immunotherapy approaches that use monoclonal antibodies targeting immune checkpoints show significant anti-tumour responses in multiple cancer types, so monitoring B cell- as well as antibody responses might be of great value . Althougy (ADCC) –36, it iy (ADCC) . This isgression , 24. Furobserved . Based oet al. showed CD8+ T cell-mediated abscopal effects in a pre-clinical animal model in which fractionated RT was combined with CTLA-4 blockade [Until now abscopal tumour regression after RT alone is only anecdotally observed in patients with cancer. However, recent studies have demonstrated that combination therapy with fractionated RT and CTLA-4 blockade (ipilimumab) or GM-CSF treatment, the percentage of patients showing abscopal tumour regression increased up to 20% , 13, 27.blockade . It mighIn conclusion, our study showed that fractionated RT with or without Flt3-L administration induces abscopal effects in the 67NR mouse model. Here, we observed a pre-existing antibody response that is also observed in abscopal responders in human studies. However, such humoral immune responses in terms of total quantity of plasma antibodies or the Ig isotype composition are not altered upon fractionated RT and/or upon DC stimulation with Flt3-L in this model. Taken together our data demonstrate that monitoring antibodies in the setting of abscopal tumour regression does not invariably associates with therapeutic effects.S1 Fig3) of both 67NR tumours in Balb/C mice assigned to groups untreated, Flt3-L, RT, and RT+Flt3-L. All Balb/C mice had palpable tumours at the start of the experiment (before the treatments). The stars indicate Balb/C mice that were tumour free at the end of the experiment (day 162).The size (mm(TIF)Click here for additional data file.S2 Figd (MHC-I) on 67NR tumour cells. An APC-conjugated antibody against total immunoglobulins (α Ig APC) detected the un-conjugated H-2Kd antibody (0.5 mg/mL) using flow cytometry (FC). In order to quantitatively analyse the antibody concentration without the influence of matrix effects that originate from undesired protein factors in the plasma, we performed a dilution experiment of an antibody of known concentration in different concentrations of plasma. We stained 105 cells with 5 concentrations  of a monoclonal unconjugated H-2Kd antibody in 5 concentrations of mouse plasma  or in buffer. b. Different numbers (0.75 *105 to 10 *105) of 67NR tumour cells were incubated with vaccinated (+control) or -control plasma and detected using an α Ig APC antibody to determine the optimal signal to background ratio. Iso: Ig isotype control, MFI: mean fluorescent intensity.a. The surface expression of H-2K(TIF)Click here for additional data file.S3 FigBalb/C (immunocompetent) mice were vaccinated with incomplete Freund’s adjuvant (IFA), CpG oligodeoxynucleotides (ODNs), and heat-killed 67NR cells. To boost the antibody response, a secondary vaccination cocktail of IFA, CpG ODNs, and heat-killed 67NR cells was injected in primary immunized Balb/C mice. Per week one injected mouse was sacrificed and the plasma was collected . The levels of immunoglobulins (α Ig APC) in the plasma were measured after one injection  or boost injection . The plasma samples from the boosted mice were used as positive control in the cellular flow cytometry and ELISA-based detection systems. Control immunocompetent Balb/C mice were injected with phosphate buffered saline (PBS).(TIF)Click here for additional data file.S4 Fig5 67NR tumour cells were coated and incubated with plasma from immunocompetent (-control), vaccinated (+control), untreated, and treated  Balb/C mice. An HRP conjugated antibody against total Ig detected the TBAs. Tumour-bearing mice received fractionated RT  or no RT . Mann-Whitney t-test with P<0.01 **.a. To determine the optimal concentration of the detection antibody, we titrated the α Ig HRP antibody  using vaccinated and -control plasma. To control for background staining we included the α Ig HRP only and unstained. b. After determining the optimal α Ig HRP dilution, the plasma of vaccinated and -control was titrated  and detected using a 1:2000 dilution of α Ig HRP. The background staining was determined as described previously. c. A total of 1 *10(TIF)Click here for additional data file.S5 Fig1), CD3 (IgG2a), CD158b (IgG2b), and CD158a (IgM). These mouse anti-human antibodies were detected with an antibody cocktail containing rat anti-mouse IgG1 (APC), IgG2a (PE), IgG2b (FITC), and IgM (PE-Cy7). b. FC dotplots of the gating strategy. The lymphocyte population was gated in the FSC vs. SSC dotplot. The positive IgG1 population was negative for isotypes IgG2a, IgG2b, and IgM, indicating no cross-reactivity. This strategy was repeated for all Ig isotypes. c. The MFI levels of isotypes IgG1, IgG2a, IgG2b, and IgM. These rat anti-mouse Ig isotype antibodies show minimal crossreactivity.a. Schematic representation of the optimization procedure. Freshly isolated human peripheral blood mononuclear cells (PBMCs) were incubated with unconjugated mouse anti-human antibodies specific for CD28 (IgG(TIF)Click here for additional data file.S6 Fig5 67NR tumour cells were incubated with plasma from vaccinated (+control), non-irradiated , and irradiated  Balb/C mice. The -control plasma was used to assess the background of each isotype staining. The four different isotypes were detected using fluorescent labelled IgG1 (APC), IgG2a (PE), IgG2b (FITC), and IgM (PE-Cy7) antibodies.To determine the Ig isotypes in the plasma of tumour-bearing mice, 1 *10(TIF)Click here for additional data file.S7 Figa. Immunohistochemical (IHC) staining of CD3+, CD4+, and CD8+ tumour infiltrating T cells in both the primary and secondary tumour . b. FC density plots of the gating strategy. Living haematopoietic cells (CD45+ vs. 7-AAD-) were gated. In this gate, the lymphocyte population was selected based on the FSC vs. SSC. To ensure that only lymphocytes were gated, CD11c+ cells were excluded. In the CD11c- (CD8α) gate, CD4+ and CD8α+ T cells were selected. c, d. The percentage CD4+ and CD8α+ T cells in the primary and secondary tumour of (non)- vs. irradiated animals measured with FC.(TIF)Click here for additional data file.S1 FileDevelopment of cellular read-out systems for the detection of TBAs in the plasma of Balb/C mice.(PDF)Click here for additional data file.


Energy Homeostasis Associated (ENHO) gene whose physiological role in humans remains incompletely defined. Here we investigated the impact of dietary interventions that affect systemic glucose and lipid metabolism on plasma adropin concentrations in humans. Consumption of glucose or fructose as 25% of daily energy requirements (E) differentially affected plasma adropin concentrations (P < 0.005) irrespective of duration, sex or age. Glucose consumption reduced plasma adropin from 3.55 ± 0.26 to 3.28 ± 0.23 ng/ml (N = 42). Fructose consumption increased plasma adropin from 3.63 ± 0.29 to 3.93 ± 0.34 ng/ml (N = 45). Consumption of high fructose corn syrup (HFCS) as 25% E had no effect . Overall, the effect of glucose, HFCS and fructose on circulating adropin concentrations were similar to those observed on postprandial plasma triglyceride concentrations. Furthermore, increases in plasma adropin levels with fructose intake were most robust in individuals exhibiting hypertriglyceridemia. Individuals with low plasma adropin concentrations also exhibited rapid increases in plasma levels following consumption of breakfasts supplemented with lipids. These are the first results linking plasma adropin levels with dietary sugar intake in humans, with the impact of fructose consumption linked to systemic triglyceride metabolism. In addition, dietary fat intake may also increase circulating adropin concentrations.Adropin is a peptide hormone encoded by the  Rapid effects of fasting and feeding on adropin expression in mice suggest functions related to metabolic adaptation. Consistent with this hypothesis, adropin-knockout mice are insulin resistant22The roles of adropin in human physiology are not as clear. In women, we recently reported a positive association between plasma adropin concentrations and dietary fat consumptionTo address this gap in knowledge, plasma adropin concentrations were measured in samples obtained at baseline and after consumption of glucose, fructose or high fructose corn syrup (HFCS) as 25% of energy (E) for 2 wk (Study A)111213P < 0.05, paired t test), 0.1 kg, and 0.8 kg  body weight, respectively , although the effect of time was not significant . Analysis of the change of plasma adropin concentrations (ΔAdropin) indicated that glucose and fructose consumption were having opposite effects  for a difference between the three groups. Post hoc analysis indicated a tendency (P = 0.057) for a difference, with fructose consumption increasing and glucose consumption reducing plasma adropin concentrations  with no effect of time . As before, there was a trend for a difference in ΔAdropin between the glucose (reduced) and fructose (increased) groups  that was not affected of by sex , with no significant effect of time  . A univariate analysis comparing ΔAdropin between glucose and fructose with study, sex, age, BMI and ethnicity as covariates also indicated a highly significant difference    . The oth N = 45) .Adropin for Study A and B participants consuming glucose or fructose indicated a responder/non-responder scenario (Inspection of Δscenario . With glscenario . With frscenario .Adropin) and previously collected plasma lipid and glucose metabolic parameters. To differentiate between responders and non-responders, we separated data pooled from Study A and B into quartiles ranked by ΔAdropin. To determine whether the differences in ΔAdropin in each quartile correlated with plasma lipid and lipoprotein risk factors, we performed an initial screen of the risk factors comparing baseline values in the 1st and 4th quartiles using a Student’s t-test. Factors that showed significant differences (P < 0.05) in ΔAdropin between the 1st and 4th quartile were then analyzed further using ANOVA.A large body of data related to lipid and glucose metabolism had been collected from all participants during the original experiments10st and 4th quartiles in participants consuming fructose. Further analysis of fasting TG (TGfst) (totAUC) (iAUC)  higher in individuals in the 4th relative to all other quartiles (P < 0.05). Distribution by age and sex was similar in the 4 groups . There were also significant correlations between ΔAdropin and baseline values for TGFst , TGiAUC  and TGAUC  , the tot(totAUC) , and the) (iAUC)  indicate< 0.005) .2) who also had the lowest plasma adropin concentrations measured in these studies (0.6 ng/ml) and high risk factors for cardiovascular disease. The individual had the highest TG levels at baseline . This participant also had very high levels of fasting TG- and cholesterol-rich remnant lipoproteins .Of note, the most pronounced fold-increase (3.8 fold) of adropin with fructose consumption was observed in a female in Study B . The potential responder/nonresponder situation was further analyzed by grouping the highest and lowest “responders” . Interestingly, participants exhibiting the largest increase in response to MCT (“responders) also appeared to have lower plasma adropin concentrations at baseline . However, the distinction between responders and nonresponders was less clear for LCT, with nonresponders showing a dip following breakfast , there was considerable variability between individuals suggesting interactions between sugar and other variables. In the fructose group, the participants exhibiting the largest increases of plasma adropin had higher baseline circulating TG profiles. This outcome suggests an association between elevated TG levels and the regulation of plasma adropin concentrations in humans.The consistency in the outcomes observed in the two different 2 wk and 10 wk treatment paradigms is a strength of this study. However, data interpretation is complicated by the lack of specific information regarding the molecular mechanisms by which circulating adropin levels are regulated. The observed changes are presumably indirect downstream consequences of changes in lipid and glucose metabolism associated with sugar consumption. Glucose passes through the liver and is readily oxidized as needed or converted to glycogen. In contrast, fructose is retained by the liver and is more readily converted to fatty acids and exported as very-low density lipoproteins. Consumption of diets with high fructose content has been suggested to promote risk factors for cardiovascular and metabolic risk disease. Indeed, in the studies from which these plasma samples were obtained, fructose consumption was associated with the development of dyslipidemia10de novo lipogenesis, reduced postprandial fat oxidation, and enhanced glucose oxidation, and, even though weight gain was similar for both groups, only fructose consumption increased intra-abdominal obesity1111It is of interest to note the parallels between the effects of adropin and fructose consumption on fuel selection. In mice, adropin limits fat oxidation while enhancing glucose oxidation in lean and diet-induced obese mice3Previous findings on the metabolic response of humans to MCT are, however, not consistent with this hypothesis. While MCT appear to have a stimulatory effect on plasma adropin concentrations, they are preferentially oxidized and cause a net increase in energy expenditure and fat oxidation16Whether fructose consumption would similarly affect plasma adropin concentrations in rodents as observed in humans has not been determined. It is worth noting that the provision of fructose to mice has also been observed to increase adiposity by enhancing lipogenesis and reducing energy expenditure18Enho gene is either not active or expressed at low levels. A mild increase in plasma adropin observed in physiological conditions may therefore be sufficient to alter fuel selection without producing changes in metabolism that prevent weight gain.One caveat to this model is that over expression of adropin or administration of adropin peptide to mice does not result in obesity3Finally, another perhaps simpler interpretation of this data is that there is a link between hypertriglyceridemia and plasma adropin levels. This might involve a homeostatic interaction between adropin and TG metabolism. In mice, adropin appears to be stimulated by dietary fat content. Adropin also has an inhibitory effect on the expression of genes involved in lipogenesis, although whether this effect is direct or secondary to other metabolic changes has not been determinedThe second part of this study examined plasma adropin concentrations following consumption of a high fat meal. The samples used for this experiment came from a study comparing the response of adult men to a breakfast meal supplemented with 20 g of MCT or LCTThe results of our analysis suggest a complex situation with regard to the response of plasma adropin to dietary fats in humans. The impact of dietary fat on plasma adropin concentrations appears to be dependent on baseline levels, with an inverse relationship. Strong linear associations between the postprandial AUC and baseline fasting plasma adropin concentrations were evident. While this was evident for both LCT and MCT, the latter appear to have elicited a more potent effect in those individuals who are “responders”. Indeed, one interpretation of our data is that individuals with plasma adropin concentrations at the low end of the spectrum will experience a rapid increase following ingestion of meals rich in fats.The main outcomes of this report should be considered preliminary as none of the studies from which samples were obtained were specifically designed to investigate the regulation of circulating adropin. However, these results are important in suggesting that further studies investigating the impact of macronutrient consumption on plasma adropin concentrations are needed.These results suggest an association between dietary and systemic lipid/triglyceride metabolism in the regulation of plasma adropin concentrations. Lipids originating either from the diet or from endogenous production appear to positively affect plasma adropin concentrations in humans. Further studies designed to specifically investigate the mechanisms linking lipid metabolism to plasma adropin concentrations are warranted.The protocols used for original studies were reviewed and approved by the Institutional Review Boards at UC Davis10Plasma for this experiment was from 82 participants  of mixed ethnicity . Biometric data are shown in Plasma used for this study was obtained from 32 participants  consuming 25%E from fructose or glucose for 10 wk2). In brief, this study was performed in two parts . Ten men participated in Study C1; the protocol was then revised and another 7 men enrolled into Study C2. Each study involved a 2-arm, randomized, single-blind, cross-over design. Each arm consisted of a test day spaced 3 to 14 d apart that differed in the type of oil incorporated into the meal: MCT oil  or corn oil . The nutrient composition of the breakfast meals was different . Both diets provided the same amount of test oil. For 2 d prior to the test, participants were requested not to consume alcohol or participate in structured exercise. They were asked to record their food intake at dinner the night before the test day and consume the same meal the night before the follow-up test day. The participants fasted for at least 12 h during the night before the test day.Physical characteristics have been reportedAdropin concentrations were measured using EDTA-plasma not previously been thawed in duplicate by EIA t-test was used for the analysis of the AUC.Data analysis used SPSS Statistics 22. Univariate analysis with repeated measures (within-subjects factors: time at pre- and post-intervention) was used to determine the impact of dietary intervention, with sugar type as between-subjects factors in the analysis. For comparisons between sugar groups involving single measurements , ANOVA were used followed by Bonferroni corrected multiple comparisons. For analyzing the response to MCT and LCT, which was a randomized cross-over design, a pair How to cite this article: Butler, A. A. et al. Differential Responses of Plasma Adropin Concentrations To Dietary Glucose or Fructose Consumption In Humans. Sci. Rep.5, 14691; doi: 10.1038/srep14691 (2015).


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The following grant number is missing from the Funding section: 81311120472.The complete, correct Funding section is: This study was partly supported by start-up funds from the Texas A & M Health Science Center, College of Medicine, by an American Heart Association- Grant-in-Aid (14GRNT 20490320), by Research Mentor Award by Scott & White Memorial Hospital and Texas A&M and National Natural Science Foundation of China to S. Gupta (SG) and Liling Wu (No. 81311120472). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.