Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile virus and Japanese encephalitis virus, which host range includes mainly mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Circulation of USUV in Africa was documented more than 50 years ago, but it was not until the last decade that it emerged in Europe causing episodes of avian mortality and some human severe cases. Since autophagy is a cellular pathway that can play important roles on different aspects of viral infections and pathogenesis, the possible implication of this pathway in USUV infection has been examined using Vero cells and two viral strains of different origin. USUV infection induced the unfolded protein response, revealed by the splicing of Xbp-1 mRNA. Infection with USUV also stimulated the autophagic process, which was demonstrated by an increase in the cytoplasmic aggregation of microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagosome formation. In addition to this, an increase in the lipidated form of LC3, that is associated with autophagosome formation, was noticed following infection. Pharmacological modulation of the autophagic pathway with the inductor of autophagy rapamycin resulted in an increase in virus yield. On the other hand, treatment with 3-methyladenine or wortmannin, two distinct inhibitors of phosphatidylinositol 3-kinases involved in autophagy, resulted in a decrease in virus yield. These results indicate that USUV virus infection upregulates the cellular autophagic pathway and that drugs that target this pathway can modulate the infection of this virus, thus identifying a potential druggable pathway in USUV-infection. The identification of cellular components and metabolic pathways involved in virus replication provides valuable information for the development of new antiviral strategies. Autophagy is one of these metabolic pathways with multiple implications during viral replication. Autophagy literally means self-digestion and constitutes a cellular process by which intracellular components are enclosed by membrane structures and degraded. Interestingly autophagy can contribute either positively or negatively to viral infections. For instance, several viruses hijack these autophagic membranes to build their replication complexes or take advantage on metabolic rearrangements induced following autophagy, while in other cases autophagy contributes to viral clearance and innate immunity. In this study, we explored the possible implication of the autophagic pathway during Usutu virus infection (USUV). USUV is an African mosquito-borne flavivirus that mainly infects mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Our results indicate that infection by USUV of different origins triggers an autophagic response within infected cells. Even more, drugs that target components from the autophagic pathway modulate USUV-infection. These results provide the basis for the design of new antiviral research lines against this pathogen. Flavivirus genus in the Flaviviridae family. USUV was first described in South Africa in 1959, and since then, it has been reported in several African countries including Senegal, Central African Republic, Nigeria, Uganda, Burkina Faso, Cote d'Ivore, and Morocco Culex mosquitoes and birds, although two isolations of USUV from human serum, including one severe case, have been documented The variety of factors that have contributed to the emergence of the flavivirus West Nile virus (WNV) in the Americas and its re-emergence in other parts of the world could also provide a suitable scenario for the emergence of other arboviruses A detailed knowledge of the cellular processes involved in pathogen and host cell interactions is desirable to design effective strategies to combat arboviral diseases. In the case of USUV, the role of many aspects of the interaction between the virus and the host cell, for instance its relationship with the autophagic pathway, remains to be explored. Macroautophagy (thereafter referred as ‘autophagy’) is a cellular process by which cytoplasmic components are sequestered into double-membrane vesicles and degraded to maintain cellular homeostasis. In addition to this, autophagy constitutes an evolutionarily ancient process for survival during different forms of cellular stress, including infection with viruses Flaviviridae, autophagy has been associated to different aspects of the replication and pathogenicity of some members of this virus family, including Dengue virus (DENV) Regarding the In this study we have analyzed the induction of autophagy following infection with a prototypic African strain of USUV and a recent European isolate All animals were handled in strict accordance with the guidelines of the European Community 86/609/CEE at the biosafety animal facilities of the Centro de Investigación en Sanidad Animal of the Instituto Nacional de Investigación Agraria y Alimentaria (CISA-INIA). The protocols were approved by the Committee on Ethics of Animal Experimentation of INIA (permit number 2013–015).Mouse monoclonal antibody J2 against double-stranded RNA (dsRNA) was purchased from English & Scientific Consulting (Hungary). Rabbit monoclonal anti-LC3B, rabbit anti-p62/SQSTM1 and mouse monoclonal anti-β-actin antibodies were from Sigma-Aldrich . Rabbit anti-calnexin antibody was from ECM Biosciences . Polyclonal serum from a mice experimentally infected with USUV SAAR 1776 (INIA permit number 2013–015) was also used to detect USUV proteins. Secondary antibodies against Mouse or Rabbit IgGs coupled to Alexa Fluor-488, -594 or -647 were purchased from Life Technologies . Anti-rabbit and anti-mouse secondary antibodies coupled to horseradish peroxidase were from Dako and Sigma, respectively.All manipulations of infectious virus were carried out in Biosafety level 3 (BSL-3) containment facilities. USUV strain SAAR 1776 (GenBank acc: AY453412.1), the reference South African strain of USUV, and the Austrian strain of USUV Vienna 2001-blackbird , a recent European isolate of USUV 4Cl, Merck) was used at 25 mM. Cells were infected, or mock-infected, and drugs were added to the medium after the first hour of infection. Stock solutions of wortmannin and rapamycin were prepared in dimethyl sulfoxide (DMSO), and DMSO was also used as control in non-treated cells (drug vehicle). Tunicamycin (Sigma), an inducer of unfolded protein response, was also dissolved in DMSO and used at 10 µg/ml. The viability of cells with or without treatment was tested with CellTiter-Glo Luminiscent Cell Viability Assay (Promega).Autophagy inhibitors 3-methyladenine (3-MA) and wortmannin, and autophagy inducer rapamycin, were purchased from Sigma and used at the concentrations of 2.5 mM, 0.5 µm and 100 ng/ml, respectively. Amonium chloride (NHA plasmid encoding GFP-LC3 http://rsbweb.nih.gov/ij/) and Adobe Photoshop CS2.Assays were carried out as described 2. Cells were scrapped from the flasks and post-fixed in 1% osmium tetroxide-1% potassium ferricyanide (1 h at 4°C), washed three times with bidistilled water and treated with 0.15% tanic acid (1 min). Cells were washed with the buffer and with bidistilled water and stained with 2% uranyl acetate (1 h). Samples were washed and then dehydrated in ethanol and embedded in the resin. Samples were examined using a Jeol JEM-1010 electron microscope  operated at 80 kV and images were acquired using a digital camera 4K×4K TemCam-F416 .Vero cells infected with USUV (MOI of 5 PFU/cell) were washed and fixed 24 h p.i. (30 min at 37°C) in 4% paraformaldehyde-2% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 plus 5 mM CaClWestern blot were performed as reported RNA was extracted from control and infected cells with TriPure Isolation Reagent  as indicated by the manufacturer. Reverse transcriptions PCR reactions (RT-PCR) were carried out using the SuperScript One Step RT-PCR (Life Technologies). Unspliced or spliced Xbp-1 mRNA was amplified as described Data are presented as mean ± standard error of the mean (SEM). To test the significance of the differences, analysis of the variance (ANOVA) was performed with statistical package SPSS 15  applying Bonferroni's correction for multiple comparisons. Statistically significant differences were considered at P<0.05.As a first approach, cells infected with USUV strain SAAR 1776 were analyzed by transmission electron microscopy. Infected cells exhibited morphological characteristics associated to flavivirus infection. These included electron dense virions located inside endoplasmic reticulum cisternae  and membFollowing induction of autophagy, microtubule-associated protein 1 light chain 3 (LC3), a mammalian homolog of yeast Atg8 (autophagy-related protein 8) is conjugated to phosphatidylethanolamine and targeted to autophagic membranes labelling autophagic vacuoles As commented above, following induction of autophagy the lipidated form of LC3 is targeted to autophagic membranes labelling autophagic vacuoles Flaviviridae family may be based on autophagosome membranes labelled with LC3 It has been proposed that replication of several members of the 4Cl    [41], [4ic flux) , indicat mCherry  that ind mCherry . Cells i mCherry . This in mCherry . This isThe interaction of flavivirus with endoplasmic reticulum during viral replication can result in induction of endoplasmic reticulum stress Overall, our results pointed to an upregulation of the autophagic pathway in USUV-infected cells and, thus, the possibility of manipulating the autophagic pathway to modulate infection with USUV was addressed. Hence, to evaluate the potential of pharmacological modulation of autophagy as a candidate for antiviral approach design against USUV, the effect of two inhibitors of autophagy, 3-MA and wortmannin The findings reported above were obtained using the prototypic USUV strain SAAR 1776 whose pathogenic capability has not been conclusively proven even in the birds. To analyze if the USUV circulating in Europe displayed similar interactions with the autophagic pathway, the USUV strain Vienna 2001 (a recent isolate of USUV which pathogenicity has been extensively proven at least in birds) was included in the study Flavivirus genus comprises more than 50 viral species that include well long known arthropod-borne pathogens as DENV, WNV, JEV, St. Louis encephalitis virus, Murray Valley encephalitis virus, Yellow fever virus or tick-borne encephalitis virus (http://www.ictvonline.org/virusTaxonomy.asp?version=2012). But this viral genus also contains other neglected viral pathogens of currently increasing interest. Among these recently considered potential threats is USUV, a flavivirus endemic from Africa that emerged in Europe during the last decade  or a recent European strain (Vienna 2001) triggered an autophagic response in the host cell. This is consistent with findings obtained for other flaviviruses Flaviviridae family as DENV or HCV. While several studies have been pointed that viral replication may be based on membranes of autophagosomal origin that contain LC3 There is a controversy related to the autophagic origin or not of the structures that provide the platform for replication of distinct members of the Flaviviridae family Autophagy constitutes a major metabolic pathway that is currently being explored for treatment of multiple human disorders that include certain types of cancer and metabolic diseases, neurological disorders or viral infections Overall this study provides the first evidence for a role of autophagy during the infection of the mosquito-borne USUV. Our results indicate that pharmacological inhibition of the autophagic pathway can reduce infection by this virus in cultured cells. These observations identify autophagy as a metabolic pathway involved on USUV-infection, thus opening a potential new research line for the design of antiviral therapies against this pathogen.Figure S1Specific staining of USUV-infected cells with a polyclonal serum. Vero cells were infected or not (mock) with USUV SAAR 1776 (MOI of 5 PFU/cell) and fixed and processed for immunofluorescence (24 h p.i.) using the serum obtained from a mouse experimentally infected with USUV. AF-488 anti-mouse IgG was used as secondary antibody. Both images were acquired using the same microscope settings. Scale bars: 10 µm.(TIF)Click here for additional data file.


Vascular calcification and reduced bone density are prevalent in chronic kidney disease and linked to increased cardiovascular risk. The mechanism is unknown. We assessed the relationship between vascular calcification, femoral bone density and left ventricular mass in patients with stage 3 non-diabetic chronic kidney disease in a cross-sectional observational study.2). Abdominal aortic calcification was assessed using lateral lumbar spine radiography and was present in 48%. Mean femoral Z-score measured using dual energy x-ray absorptiometry was 0.60±1.06. Cardiovascular magnetic resonance imaging was used to determine left ventricular mass. One patient had left ventricular hypertrophy. Subjects with aortic calcification had higher left ventricular mass compared to those without , as did patients with femoral Z-scores below zero . In univariate analysis presence of aortic calcification correlated with left ventricular mass ; mean femoral Z-score inversely correlated with left ventricular mass . In a multivariate regression model that included presence of aortic calcification, mean femoral Z-score, gender and 24-hour systolic blood pressure, 46% of the variability in left ventricular mass was explained (P<0.001).A total of 120 patients were recruited (54% male, mean age 55±14 years, mean glomerular filtration rate 50±13 ml/min/1.73 mIn patients with stage 3 non-diabetic chronic kidney disease, lower mean femoral Z-score and presence of aortic calcification are independently associated with increased left ventricular mass. Further research exploring the pathophysiology that underlies these relationships is warranted. Cardiovascular morbidity and mortality are increased in patients with chronic kidney disease (CKD) and have an independent inverse relationship with glomerular filtration rate (GFR) Patients with CKD have deranged bone-mineral metabolism, with increased levels of the phosphaturic hormone fibroblast growth factor 23 (FGF-23) and parathyroid hormone (PTH) arising from impaired renal excretion of phosphate and impaired vitamin D activation To determine the relationship between femoral bone density, presence of vascular calcification and LVM in patients with stage 3 non-diabetic CKD.2) Subjects were recruited from the renal clinics at the Queen Elizabeth Hospital Birmingham, UK, from 2009 to 2011. Patients were included if aged 18–80 years with stage 3 CKD  using the four-variable Modification of Diet in Renal Disease formula with serum creatinine recalibrated to be traceable to an isotope derived mass spectroscopy method Dual energy x-ray absorptiometry scanning  was used to assess BMD of both proximal femurs . All scans were reported by an experienced bone densitometry clinical scientist blinded to clinical data. T-scores that refer to the young adult reference mean calculated from the manufacturer's database  population) The presence and extent of abdominal aortic calcification were assessed using a lateral lumbar spine radiograph and validated semi-quantitative scoring method as previously described 2) =  √((weight (kg) × height (cm))/3600). Left ventricular hypertrophy was defined as an LVMI greater than age and gender corrected limits Cardiovascular magnetic resonance imaging was performed on a 1.5 Tesla scanner . Serial contiguous short axis cines were piloted from the vertical long axis and horizontal long axis of the left ventricle  in accordance with previously validated methodologies Declaration of Helsinki and the principles of Good Clinical Practice.West Midlands Research Ethics Committee approved the study and written informed consent was obtained from each participant. The study was conducted in accordance with the 2. Variables not normally distributed were log transformed prior to analysis . Analysis of variance with post hoc Tukey was conducted to assess the impact of mean hip Z-score and presence of vascular calcification on LVMI. Pearson correlation and linear regression were used to assess the relationship between two continuous variables. Colinearity between variables was assessed by examining the variance inflation factor; a value >5 indicated colinearity. A Type I error rate below 5% (P<0.05) was considered statistically significant.All data were analysed using SPSS version 19 . Data are shown as mean±standard deviation, frequency (percentage) or median (interquartile range). Data distribution was tested using the Kolmogorov-Smirnov test and normality plots. Normally distributed variables were analysed using unpaired t-tests and χIntraobserver variability for LVMI was determined by random selection of twelve scans (10% of the cohort) for repeat analysis by the same observer. Interobserver variability for aortic calcification score was determined by comparing calcification scores between two observers for all 120 subjects. Variability of LVMI and aortic calcification score was determined using an intraclass correlation coefficient and two-way random effects model.Of the 1297 patients with non-diabetic CKD screened in the outpatient renal clinics, 602 met the study entry criteria. In total 494 patients were approached to take part in the CRIB-PHOS study; 374 declined to participate and therefore 120 were recruited. Demographic data for these 120 subjects are shown in 2, P = 0.001). There was no significant difference in parameters of femoral bone density between the groups.A comparison of demographics between patients with calcification and those without is shown in 2, P = 0.02). There were no other demographic or biochemical differences between the groups; in particular there were no significant differences in markers of bone metabolism or inflammation. A two-way analysis of variance was used to explore the effect of high or low Z-scores and the presence or absence of aortic calcification on LVMI. Both Z-score grouping and presence of aortic calcification had significant effects on LVMI . Patients in the high mean femoral Z-score group were older , heavier  and had significantly lower LVMI than those in the low Z-score group . Interobserver variability for aortic calcification was also low with an intraclass correlation coefficient of 0.917 .In this cross-sectional observational study of patients with stage 3 non-diabetic CKD a significant independent inverse relationship between femoral bone density Z-scores and LVMI was demonstrated. Furthermore, the presence of aortic calcification was associated with increased aortic stiffness and significantly greater LVMI independent of bone density. Our findings highlight important links between bone disease, vascular calcification and LVM, a prognostically important cardiovascular variable.Several population studies have linked reduced bone density and osteoporotic fractures with cardiovascular disease, especially heart failure, although the mechanisms are unknown Coronary artery calcification independently correlated with LVMI in a study of 118 haemodialysis patients Arterial stiffness is a powerful predictor of poor cardiovascular outcome 2), Toussaint et al demonstrated an inverse correlation between femoral arterial calcification  assessed using computed tomography (CT) and femoral BMD measured using DEXA scanning et al divided 72 patients with stage 2–4 CKD into tertiles of vertebral bone density measured using CT and found those in the lowest bone density tertile to have significantly higher coronary artery calcification than those in the other tertiles 2) together with our use of a less sensitive semi-quantitative technique for detection of vascular calcification.Several reports from the general population have demonstrated an inverse association between severity of vascular calcification and bone density Abnormalities of mineral metabolism, such as elevated serum phosphate and calcium-phosphate product, have been identified as risk factors for the presence or progression of vascular calcification in CKD patients in a number of studies Our study was observational and cross-sectional in design and thus subject to potential confounding from missing variables. We can only report associations and not infer causality. The use of lateral lumbar spine radiography and a semi-quantitative scoring method to assess severity of aortic calcification is not as sensitive as other modalities such as CT scanning and is therefore likely to have underestimated the prevalence of vascular calcification in our cohort. Nevertheless we were still able to demonstrate an independent relationship between severity of vascular calcification and LVM using this method.In patients with stage 3 non-diabetic CKD lower femoral bone density Z-scores and presence of vascular calcification are independently associated with increased LVM. Further studies into the mechanisms and potential effect of treatments that maintain bone density and attenuate progression of vascular calcification on LVM are warranted.


The ability of subjects to respond to nutritional challenges can reflect the flexibility of their biological system. Nutritional challenge tests could be used as an indicator of health status but more knowledge on metabolic and immune responses of different subjects to nutritional challenges is needed. The aim of this study was to compare the responses to high-fat challenges varying in fat type in subjects with different metabolic risk phenotypes.MCP1 and IL1β in PBMCs. Obese and obese diabetic subjects had different PBMC gene expression and metabolic responses to high-fat challenges compared to lean subjects. The MUFA challenge induced the most pronounced TAG response, mainly in obese and obese diabetic subjects.In a cross-over design 42 men (age 50–70 y) consumed three high-fat shakes containing saturated fat (SFA), monounsaturated fat (MUFA) or n-3 polyunsaturated (PUFA). Men were selected on BMI and health status  and phenotyped with MRI for adipose tissue distribution. Before and 2 and 4 h after shake consumption blood was drawn for measurement of expression of metabolic and inflammation-related genes in peripheral blood mononuclear cells (PBMCs), plasma triglycerides (TAG), glucose, insulin, cytokines and ex vivo PBMC immune response capacity. The MUFA and n-3 PUFA challenge, compared to the SFA challenge, induced higher changes in expression of inflammation genes The PBMC gene expression response and metabolic response to high-fat challenges were affected by fat type and metabolic risk phenotype. Based on our results we suggest using a MUFA challenge to reveal differences in response capacity of subjects.NCT00977262ClinicalTrials.gov  In the western world food is generally continuously available and most of the day is spent in the postprandial state. Food intake can elicit a transient metabolic and low inflammatory response, especially when high fat is consumed Elevated postprandial triglyceride concentrations are considered as risk factors for cardiovascular disease and were higher associated with cardiovascular events compared to fasting triglyceride concentrations Postprandial meal studies showed that the type of fat consumed could affect the metabolic and inflammatory response The objective of this study is to characterize the PBMC, metabolic and immune response to high-fat challenges in subjects with different metabolic risk phenotypes. Responses to different types of fat  were compared in order to reveal which type of fat may be best used in future high-fat challenges to test response capacity of subjects.The protocol for this trial and CONSORT checklist are available as supporting information (Protocol S1 and Checklist S1).Male Caucasian volunteers aged 50–70 years participated in the study. The study population consisted of three groups; 1) lean subjects, 2) obese subjects and 3) obese type 2 diabetic subjects. Subjects were excluded if they were vegetarian, regular tobacco smoker, allergic to dairy products or fish oil, current or recent user of fish oil supplements, consumed more than four times fish/wk, had an unstable body weight, used antibiotics or anti-inflammatory medication or had a long-term medical condition that could interfere with the study outcome. Lean and obese subjects were excluded when using cholesterol-lowering medication; and lean subjects were excluded when using blood pressure lowering medication. Obese diabetic subjects were all diagnosed with diabetes mellitus in the past, and did not use insulin and/or thiazolidinediones. During a screening visit an oral glucose tolerance test (OGTT) was performed and fasting urinary glucose concentration was measured in healthy subjects to exclude the presence of (unknown) diabetes mellitus . Subjects were informed about the design and purpose of the study and all subjects provided written informed consent. The Medical Ethical Committee of Wageningen University (the Netherlands) approved the study and the study was registered at clinicaltrials.gov as NCT00977262. The study was conducted according to the principles of the declaration of Helsinki and in accordance with the Medical Research Involving Human subject Act.Sample size was determined based on the sample size in a comparable study with lean male subjects in which differences in PBMC gene expression after consumption of three different high fat shakes were detected All subjects consumed three high-fat shakes enriched with SFA, MUFA or n-3 PUFA in a crossover design, on three different days with at least one week between each study day. Participants in each subject group were randomly allocated to one of the six possible sequences of shake consumption. The possible sequences were equally distributed over the participants within a subject group. A research assistant generated the random allocation sequence and assigned the subjects to the interventions. Shakes were given a letter code and both subjects and researchers were blinded to the intervention.The evening before the study day, subjects consumed an identical low-fat meal and were not allowed to eat or drink anything after 8 pm except water. The next morning, subjects came to Wageningen University, the Netherlands, and a fasting blood sample was collected. Blood was drawn into EDTA-containing tubes for plasma isolation and into BD Vacutainer Cell Preparation Tubes containing sodium citrate  for PBMC isolation. After the first blood sample subjects received a high-fat shake, which they had to consume within 15 min. Blood samples were collected 2 h and 4 hours after consumption of the high-fat shake. During a study day, subjects were physically inactive and did not eat or drink anything except water.All high-fat shakes were isocaloric and differed only in fat composition. The shakes contained low-fat yoghurt, low-fat milk, strawberry flavour, 7.5 g of sugar and 95 g of the test fat. The SFA shake contained 95 g palm oil  and the MUFA shake contained 95 g high-oleic acid sunflower oil . The n-3 PUFA shake contained 40 g palm oil and 55 g Marinol D-40 , of which 40% was docosahexanoic acid (DHA). Vitamin E  was added to Marinol D-40 by the manufacturer to prevent oxidation. The same amount of vitamin E was added to the SFA and MUFA shakes. The macronutrient composition of the shakes was calculated based on the database of the Dutch Nutrient Databank and shown in IL1b, IL8, Monocyte chemotactic protein 1 (MCP1), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFκB1), TNFα) and lipid metabolism related genes (ATP-binding cassette sub-family A member 1 (ABCA1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), sterol regulatory element binding transcription factor 1 (SREBP1), LDL receptor (LDLr), liver X receptor alpha (LXRα), cytochrome P450 family 27 subfamily A polypeptide 1 (CYP27A1) in PBMCs. These genes were selected because we previously showed that their expression was affected by high-fat consumption The primary outcomes of the study were postprandial gene expression changes of immune-related genes (http://pga.mgh.harvard.edu/primerbank/index.html). Q-PCR data were normalized by measuring cycle threshold ratios between candidate genes and a housekeeping gene, human ribosomal protein LP0, which was shown to be consistent within PBMCs PBMCs were isolated from whole blood by using BD Vacutainer Cell Preparation Tubes according to the manufacturer’s instructions. PBMC RNA was isolated by using the Qiagen RNeasy Micro kit  and reverse transcribed using a cDNA synthesis kit . Standard Q-PCR was performed using SensiMix real-time PCR reagents  and a Bio-Rad CFX384 machine . Primer sequences used were chosen based on the sequences available in PRIMERBANK  on a SECTOR Imager 2400 reader  for the measurement of IL1β and TNFα.Immediately after blood was drawn in EDTA-containing tubes it was centrifuged , and plasma was stored at −80°C until analysis. Plasma FFA concentrations were analysed by the ACS-ACOD Method . Plasma triglyceride and glucose concentrations were measured using the Dimension Clinical Chemistry System . Glucose was measured by the Synchron LX20 System using hexokinase and glucose-6-phosphate dehydrogenase . Insulin concentrations were measured by enzyme-linked immunosorbent assay .Serum fatty acid composition in the triglyceride fraction was determined in pooled samples per group and per high-fat challenge at 0 h and 4 h and measured by gas-liquid chromatography as previously described 4 per ml were stimulated for 2 h at 37°C with 1 ng/ml lipopolysaccharide (LPS). Subsequently, cells were centrifuged and supernatants collected and stored at −80°C until analysis. TNFα produced by the PBMCs was measured in the supernatants by enzyme-linked immunosorbent assay according to manufacturer’s instructions . LPS-stimulated TNFα production was corrected for TNFα production in non-stimulated cells.PBMC immune response capacity in the fasted (0 h) condition and 4 h after the high-fat challenge was tested ex vivo and used as a measure of PBMC functionality. Ex vivo immune stimulation experiments were performed in a random subgroup of 13 lean and 15 obese subjects. Immediately after isolation PBMCs were re-suspended in RPMI 1640 culture medium with 10% heat-inactivated fetal calf serum and 1% penicillin and streptomycin. Cells in a concentration of 2.5×10Body weight was measured at screening and monitored during the study period. Body composition was determined on one study day by air-displacement plethysmography  The statistical packages PASW  and SAS  were used for analysis. Differences in baseline characteristics between subject groups were analysed by analysis of variance (ANOVA) for single measures  or by linear mixed model for repeated measures  and followed by post-hoc LSD tests. Differences in responses for the different high-fat shakes and subject groups were analysed by linear mixed model. Delta values (changes from baseline) were used as dependent variables in the analysis and baseline values were included as covariables and time and shake were included as repeated factors in the model. If statistical significance was found, post-hoc LSD tests were performed to identify differences between shakes or groups. The number of obese diabetic subjects included in our trial was lower than intended, which may influence the chance of finding significant differences between subject groups. Therefore all statistical analyses were also performed without including the obese diabetic subject group. If outcomes differed between both analyses this is marked in the tables and indicated in the table footnotes.The effect of VAT, SAT and VAT/SAT ratio on baseline and postprandial measures was only investigated within subject groups because of expected large differences in VAT and SAT between subject groups due to the predefined BMI categories. Obese diabetic subjects were excluded for this analysis due to the low number of subjects that was measured in the MRI scanner. For the analysis of the effect of body fat distribution VAT, SAT and VAT/SAT ratio were included as continuous covariables in the linear mixed model.Between September 2009 and December 2009 42 men  were recruited and enrolled in the trial . BaselinBaseline values and changes in fatty acid composition of the TAG fraction in pooled serum samples 4 h after the high-fat challenge are shown in table S1. The percentage palmitic acid was 1.1-fold higher after the SFA challenge, the percentage oleic acid was 1.6-fold higher after the MUFA challenge and the percentage DHA was 9.5-fold higher after the n-3 PUFA challenge, reflecting the composition of the shakes.PDK4, LDLr, IL8 and MCP1 expression were depending on the type of fat in the challenge. The MUFA challenge induced a lower decrease in PDK4 expression at 2 h (p<0.001) if compared with the other challenges. The MUFA and n-3 PUFA challenge induced a higher increase in MCP1 and IL8 expression at 4 h (p<0.05) compared with the SFA challenge.Gene expression levels at baseline (fasting state) were not different between the subject groups. Changes in expression of ABCA1 and LDLr and the inflammation-related genes IL1b and MCP1 depended on whether the subjects were lean, obese or obese diabetic. Changes in expression of ABCA1 and LDLr were less pronounced for obese and obese diabetic subjects (p<0.05) compared with lean subjects. Differences between groups in expression changes of IL1b and MCP1 were most likely due to higher increases of expression of these genes in obese diabetic subjects compared with lean and obese subjects, since the effects were not significant when obese diabetic subjects were excluded from analysis.The high-fat challenge altered expression of several metabolic genes and inflammation-related genes (table S2). Changes in expression of the metabolic genes There were no interaction effects between subject groups and shakes for the measured genes.Fasting plasma IL1β concentration was higher in obese diabetic subjects compared to lean and obese subjects . ChangesFasting levels and changes in PBMC immune response capacity after the high-fat challenges, measured as ex vivo LPS-stimulated TNFα production of PBMCs, were neither depending on the type of fat in the challenge, nor depending on the metabolic risk profile of the subjects . HoweverChanges in FFA, TAG, insulin and glucose concentration after the different high-fat challenges in the subject groups are depicted in The changes in TAG, FFA, insulin and glucose concentrations were also different between lean, obese and obese diabetic subjects. For TAG response an interaction effect between group and shake (p = 0.048) was observed, meaning that the postprandial TAG response was different for lean, obese and obese diabetic subjects and depending on the type of high-fat shake consumed.Within the group of lean subjects and within the group of obese subjects there was no significant influence of abdominal fat distribution  on changes in plasma FFA, TAG, insulin or glucose concentrations, changes in PBMC gene expression, or changes in cytokine concentrations in response to the high-fat challenges.ABCA1, LDLr and inflammatory genes IL1β and MCP1 differed between lean, obese and obese subjects with diabetes. Moreover, plasma TAG, FFA, insulin and glucose concentrations after the challenge differed between subjects with distinct metabolic phenotypes. Comparison of responses to high-fat challenges high in SFA, MUFA or n-3 PUFA showed that high MUFA induced the most pronounced response, especially for TAG.This study shows that in response to a high-fat challenge, the expression of the metabolic genes ABCA1 and the increase in LDLr expression in lean subjects in response to the challenge were comparable with observations in lean subjects in earlier studies performed by us and others ABCA1 and LDLr in response to a high-fat challenge might thus be considered as potential markers of health status.The decrease in IL1β and MCP1 in response to the challenge was also different between groups, mainly due to larger increases in obese diabetic subjects, and not in obese subjects. We might consider these gene expression changes as indicators of a more severe metabolic phenotype; however we should interpret the study outcomes for the diabetic group with caution, since only a low number of diabetic subjects were included in the study. The greater change in IL1β expression in obese diabetic subjects was not reflected in plasma IL1β concentrations. This could be explained by the fact that IL1β is also produced by other cells than PBMCs. The low power to detect significant effects in the small obese diabetic subject group with high variation in cytokine response may also play a role.The change in expression of the inflammatory genes MCP1 and IL8 expression compared to the SFA challenge. This is in line with findings from a previous study showing that acute high n-3 PUFA intake induced a pro-inflammatory PBMC gene expression profile HIF1α) were seen among the fat challenges (unpublished data). Another explanation may be that palmitic acid, which is generally more regularly consumed, poses less stress to PBMCs resulting in lower expression changes of inflammation genes than high doses of oleic acid or DHA. A high-fat palm oil shake may thus be less suitable to acutely challenge the system compared to shakes containing unsaturated fats.PBMC gene expression responses to the challenges were also depending on the type of fat consumed. The n-3 PUFA and MUFA challenge induced higher increases in After all high-fat challenges PBMC immune response capacity was reduced, but whether this reduction is an indication of lower inflammatory status or diminished cell immune functioning cannot be distinguished. Moreover, an effect of circadian rhythm on PBMCs cannot be excluded. Since PBMC immune response capacity was not different between subject groups, this ex vivo test after a challenge may not be a sensitive indicator for differences in health status.The secondary study outcomes, plasma TAG, FFA, insulin and glucose concentrations after the challenge, clearly differed between subjects with distinct metabolic phenotypes. The differential changes in TAG and insulin concentrations in lean, obese and obese diabetic subjects are in line with results from postprandial studies showing higher TAG and insulin responses in obese and diabetic subjects after high-fat consumption The plasma metabolic responses to the challenges were also depending on the type of fat consumed. Comparison of responses to high in SFA, MUFA or n-3 PUFA challenges showed that high MUFA induced the most pronounced response, especially for TAG. The TAG-raising effect after a MUFA challenge was more pronounced in obese and obese diabetic subjects, suggesting that acute high MUFA intake might be a stronger metabolic challenge for subjects with a metabolic risk phenotype. It should be further investigated whether the plasma TAG response to a MUFA challenge could be used as a marker to detect small differences in health status in subjects with more comparable phenotypes.The MUFA challenge may have been the most challenging for the biological system because it contained almost exclusively oleic acid  while the other shakes contained a mixture of fatty acids. Our test shakes were not reflecting habitual meal intake, but used as a metabolic challenge test. However, because habitual intake of fish n-3 PUFA is very low we prepared an n-3 PUFA shake consisting of a mixture of palm oil and 55 gr fish oil, a well-tolerated dose in a former study at our group Remarkably, our n-3 PUFA shake induced a less pronounced increase in insulin concentration compared with the other shakes. Only few other studies have reported acute insulin-lowering effects of n-3 PUFA intake The amount of VAT, SAT or the VAT/SAT ratio did not influence the response to a high-fat challenge within subject groups. Although the number of subjects per group might have been too small to detect a significant effect of fat distribution, it is also arguable whether high VAT is strongly affecting metabolic health; some studies did find an effect of VAT, mainly on TAG response In our study we monitored different aspects of the response to high-fat challenges, i.e. plasma metabolites, inflammatory proteins, PBMC gene expression and immune cell functioning. We were able to detect differential changes in expression of metabolic parameters and certain genes between the selected subject groups differing in BMI and health status. However, for identification of small differences between responses of subjects that are phenotypically more similar, more sensitive monitoring of the response is needed. Whole genome transcriptome, proteome or metabolome profiling tools could be used for more extensive characterization of the challenge response. As shown in previous studies, extensive profiling could identify subtle changes of genes, proteins or metabolites in pathways and clusters In conclusion, of the three fat types studied most pronounced changes were seen for the high oleic sunflower oil (MUFA). Therefore this fat type seems the most promising challenge to test metabolic response capacity. We identified several genes expressed in PBMCs and plasma metabolic measures that were differently responding to a high-fat challenge in subjects with distinct metabolic risk phenotypes. These potential markers are likely candidates to be further tested and used in high-fat challenge tests to define metabolic response capacity of subjects.Table S1Serum fatty acid composition of the TAG fraction  of pooled samples for group and time point.(DOC)Click here for additional data file.Table S2Changes in PBMC gene expression of lean subjects (n = 18), obese subjects (n = 18) and obese diabetic subjects (n = 6) at 2 h and 4 h after high-fat shake.(DOC)Click here for additional data file.Checklist S1CONSORT checklist.(DOC)Click here for additional data file.Protocol S1Trial protocol.(DOC)Click here for additional data file.


To present genome-wide association analyses of genotypic and environmental risks on age-related macular degeneration (AMD) using 593 subjects from the age-related eye disease study (AREDS), after adjusting for population stratification and including questionable controls.Single nucleotide polymorphism (SNP) associations with AMD for the non-Hispanic white population were investigated using a log-additive model after adjusting for population stratification. Replication of possible SNP-disease association was performed by genotyping an independent group of 444 AMD case and 300 control subjects. Logistic regression models were used to assess interaction effects between smoking and SNPs associated with AMD. Independent genetic risk effects among the disease-associated SNPs were also investigated using multiple logistic regression models.CFH], complement factor B [CFB], the age-related maculopathy susceptibility 2 locus containing the hypothetical gene [LOC387715]/the high-temperature requirement A-1 [HTRA1], and complement component 3 [C3]) that were already known to be associated with AMD were identified. An additional 26 novel SNPs potentially associated with AMD were identified, but none were definitely replicated in a second independent group of subjects. Smoking did not interact with known AMD loci, but was associated with late AMD. Statistically independent genetic signals were observed within the Pleckstrin homology domain-containing family A member 1 (PLEKHA1) region near LOC387715/HTRA1 and within a haplotype spanning exon 19 of the C3 gene.Population stratification was observed among the individuals having a self-reported race of non-Hispanic white. Risk allele frequencies at established AMD loci demonstrated that questionable control subjects were similar to control subjects in the AREDS, suggesting that they could be used as true controls in the analyses. Genetic loci (complement factor H [ARMS2]) locus spanning PLEKHA1-LOC387715-HTRA1 and the C3 gene may contain multiple independent genetic risks contributing to AMD.Population stratification among Caucasian subjects from the multicentered AREDS was observed, suggesting that it should be adjusted for in future studies. The AREDS questionable control subjects can be used as control subjects in the AREDS genome-wide association study (GWAS). Smoking was an independent risk factor for advanced AMD in the AREDS subjects. There continues to be evidence that the 10q26 (age-related maculopathy susceptibility 2 gene [ Age-related macular degeneration (AMD) is a progressive neurodegenerative disease that is the leading cause of vision loss in older individuals . The morHLA) locus, mitochondrial genome, interleukin 8 (IL8), apolipoprotein E (ApoE), and factor I (FI), may alter the risk of AMD, but additional studies are needed due to inconsistent or absent replication in independent study groups , complement factor I [CFI], complement component 2/complement factor B [C2/CFB], age-related maculopathy susceptibility 2 [ARMS2], and complement component 3 [C3] loci) after adjusting for the first two principal components, and we found the most significant SNP for each locus. Second, we calculated the extent of linkage disequilibrium (using r2) between the most significant SNP in each locus and the rest of the SNPs in that locus to determine if there were any significant SNPs not in high linkage disequilibrium (r2 less than 0.2). Third, we performed logistic regression to see if such SNPs had any additional effect after conditioning on the most significant SNP for each locus. Seven SNPs were thus obtained, statistically accounting for the genetic risk contributed to AMD by the known loci as follows: the CFH locus, rs1853883; CFI locus, rs6848178; C2/CFB locus, rs541862; ARMS2 locus, rs2014307 and rs4565845; and C3 locus, rs2230199 and rs433594. The two SNPs from the ARMS2 locus (and the C3 locus) were selected to represent that locus, as they were in low linkage disequilibrium (r2<0.2), they statistically significantly contributed to AMD, and they each made an independent contribution to AMD conditioning on the other SNP. To understand the independent genetic effects within the ARMS2 locus (and C3 locus), haplotype analyses were performed for using the score test implemented in R package haplo.stats [We wished to determine if any SNPs would be associated with AMD after adjusting for known loci. The process of choosing SNPs to exclude known loci was as follows: First, we calculated single SNP p values for all SNPs in each known locus  in the replication subject group, but the p value (0.02) would not be considered significant after Bonferroni correction for multiple testing. The value of incorporating population stratification and using the log-additive model is illustrated by the SNP (rs2230199) in C3 in SNPs associated with AMD at a p value of less than 0.0001 before or after adjustment for SNPs already known to be associated with AMD are shown in rs11208590, rs572515, rs7529589, rs12038333, and rs203674) reached nominal significance levels , but none of the interactions would be considered significant after Bonferroni correction for multiple testing.Smoking was not associated with early AMD , exudative AMD , and advanced AMD  compared to control groups among the AREDS GWAS subjects. The replication sample showed similar results: early AMD , geographic atrophy , exudative AMD , and advanced AMD . All 59 SNPs associated with AMD at p values less than 0.0001 or already known to be associated were investigated for possible interactions with smoking status. Five SNPs (rs4565845 and rs2014307 across the ARMS2 locus at chromosome 10q26 (see Correction for known SNPs in Methods section). SNP rs4565845 is located within the pleckstrin homology domain-containing family A member 1 (PLEKHA1) gene, while rs2014307 is located between LOC387715 and the high-temperature requirement A-1 (HTRA1) promoter. We inspected the haplotypes formed by the six SNPs genotyped in the AREDS GWAS  across 62 kb of the 10q26 locus (PLEKHA1-LOC387715-HTRA1 promoter). The SNP most highly associated with AMD was rs2014307, which captures information from both risk and protective haplotypes across the ARMS2 locus. Formation of haplotypes with and without rs4752692 did not appear to lead to any refinement of the association of haplotypes with AMD. Specifically, the addition of rs4752692 split the haplotype formed by the other five SNPs (GC-TAC) that were highly associated with AMD (p=7.4×10−12) into two haplotypes  that were both associated with AMD.We observed statistically independent effects of both rs433594 and rs2230199) across the C3 locus. The non-synonymous SNP rs2230199 forms a high-risk haplotype with another non-synonymous SNP (rs1047286), and the effects of these two SNPs cannot be clearly distinguished [rs433594 is only modestly associated with AMD in the AREDS GWAS (p=0.01). However, four SNPs in this region near exon 19  form a haplotype  that is associated with AMD (p=3×10−4) at a level similar to the non-synonymous SNP rs2230199 (3.4×10−5). The GGTC haplotype spanning exon 9 does not appear to be in linkage disequilibrium with the haplotype tagged by rs2230199 that spans exons 3–9.We also observed independently, statistically significant effects of two SNPs (nguished . SNP rs4A genome-wide scan performed on 593 subjects from the AREDS was released in the database of Genotypes and Phenotypes (dbGaP) without an associated publication describing the genetic features of the data set. The AREDS genome-wide scan data set has been used extensively for genetic studies on AMD, as detailed in the introduction. Because of the ongoing analyses using the AREDS subjects and the AREDS genome-wide scan data set, we felt that a detailed analysis of the genome-wide scan data set might provide valuable guidance on its use in AMD research.The AREDS data set has been studied previously in two reports ,26. ThesTLR3 locus [Prior to initiating the analysis of the genome-wide scan data set, we sought to determine the heterogeneity within the various categories of control subjects within the AREDS. The phenotypic range of drusen that has been allowed for control subjects in AMD studies has been controversial. Many groups use an extreme phenotype approach in which only a few hard drusen (without more advanced features such as pigment changes) are allowed in control subjects, based on fundus photography ,30,31. OR3 locus ,34. HoweR3 locus .A gold standard for defining a control could be based on clinical criteria  or some combination of biomarkers contributing to disease risk. Although genetic variation accounting for approximately 80% of the risk for AMD has now been identified, this information was not available at the time that the AREDS AMD severity scales were developed ,37. For We evaluated the AREDS GWAS for problems that could lead to faulty conclusions in genetic association studies. Genetic heterogeneity can inflate the significance of the association between SNPs and disease (type I errors) ,39-41. IAnother problem in GWAS studies is the observation that differences in heterozygote frequencies between cases and controls can lead to type I errors, or false positive associations . Thus, wrs4565845 and rs2014307) across the ARMS2 locus at chromosome 10q26 and of two SNPs (rs433594 and rs2230199) across the C3 locus. The independent effect of rs4565845 that we observed in the PLEKHA1 gene adjacent to the ARMS2 locus does not seem consistent with a well defined genomic region . Others also have reported independent association with AMD in the PLEKHA1 region, but it has not yet been possible to convincingly demonstrate an independent biologic effect, given the modest association with AMD, compared to the strong association in the haplotype block spanning the hypothetical gene LOC387715 and the promoter of HTRA1 [ARMS2 locus becomes better understood.We observed statistically independent effects of two SNPs  that increases the risk of AMD. Re-sequencing studies are underway that may help address this question.On the other hand, the independent effect of We identified 29 SNPs with p values greater than 0.0001 either before or after correction for known loci associated with AMD . We atteARMS2 locus may mediate [Smoking is well established as a risk factor for both incident early AMD and advanced AMD -47. In t mediate -50 or in mediate ,52 with C3 gene. Additional studies will be needed to determine if this observation can be replicated. There continues to be evidence for independent risks in the ARMS2 locus.In summary, we reported the value of correcting for population stratification when working with the AREDS GWAS and have provided evidence supporting the use of questionable control subjects as controls in the AREDS GWAS study. Further, we presented further evidence that the contribution of smoking to the development of AMD is independent of genetic risks. Finally, we observed that the AREDS data set has evidence suggestive of a previously unknown genetic risk near exon 19 of the


GRN), is significantly higher expressed in aggressive CD38+ZAP-70+ as compared to indolent CD38−ZAP-70− chronic lymphocytic leukemia (CLL) cases. Here, we measured Pgrn plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in the Essen CLL cohort of 131 patients and examined Pgrn for association with established prognostic markers and clinical outcome. We found that high Pgrn plasma levels were strongly associated with adverse risk factors including unmutated IGHV status, expression of CD38 and ZAP-70, poor risk cytogenetics  as detected by flourescence in situ hybridization (FISH) and high Binet stage. Pgrn as well as the aforementioned risk factors were prognostic for time to first treatment and overall survival in this series. Importantly, these results could be confirmed in the independent multicentric CLL1 cohort of untreated Binet stage A patients (n = 163). Here, multivariate analysis of time to first treatment revealed that high risk Pgrn , unmutated IGHV status , high risk as defined by the study protocol  but not poor risk cytogenetics were independent prognostic markers. In summary our results suggest that Pgrn is a novel, robust and independent prognostic marker in CLL that can be easily measured by ELISA.Progranulin (Pgrn) is a 88 kDa secreted protein with pleiotropic functions including regulation of cell cycle progression, cell motility, wound repair and tumorigenesis. Using microarray based gene expression profiling we have recently demonstrated that the gene for Pgrn, granulin ( ZAP-70−CD38− patients representing the extremes of the disease spectrum, we identified a panel of CLL subtype distinction genes, the majority of which was found to be over-expressed in the prognostically unfavorable ZAP-70+CD38+ subgroup Chronic lymphocytic leukemia (CLL) is a heterogenous disease with a highly variable clinical course. In a continuing effort to investigate a molecular basis that may underlie this diverse clinical behavior and discover novel prognostic molecular markers we have performed a series of gene expression profiling studies using DNA microarray technology +CD38+ cases GRN) located on chromosome 17q21.31 and consists of tandem repeats of a 12-cysteine module termed granulin GRN is expressed in a wide range of different tissues including bone marrow stromal cells and elements of the innate and adaptive immune system GRN mutations cause frontotemporal lobar degeneration, which is a devastating neurological disease characterized by the presence of ubiquitinated inclusions of the transactivation response element DNA-binding protein-43 Our more recent work focused on progranulin (Pgrn) which is a secreted glycoprotein identified as one of the top 20 CLL subtype distinction genes over-expressed in ZAP-70in vitro to promote cell survival and confer resistance to dexamethasone treatment GRN expression in high vs. low risk CLL subgroups and explore its potential as a novel prognostic factor. To this end, we measured Pgrn concentrations in plasma and serum samples from CLL patients and investigated the relationship of Pgrn levels with established prognostic markers and clinical outcome.However, as yet data on the role of Pgrn in hematological malignancies are limited to multiple myeloma where it has been demonstrated 9/l and a typical CD19+, CD20+ CD5+, CD23+, Ig light chain (κ or λ light chain) restricted immunophenotype as revealed by flow cytometry of peripheral blood cells Unicentric cohort from Essen: Peripheral blood samples from 131 patients with CLL and 31 healthy blood donors were analyzed after obtaining written informed consent according to our institutional guidelines. This study was approved by the Ethics Commission of the University of Duisburg-Essen (reference 04–2459). Plasma samples were freshly prepared by centrifugation at 600 G for 5 min within four hours after blood collection and stored at −80°C until analysis. The diagnosis of CLL required a persistent lymphocytosis of more than 5.0×10Validation cohort provided by the German CLL study group (GCLLSG): The German CLL study group kindly provided serum samples of 163 previously untreated Binet stage A patients, representing a subgroup of the risk-stratified CLL1 trial of the GCLLSG . Median follow-up of this subgroup was 6.6 years. Serum samples had been collected centrally at study entry and stored at −80°C. All patients provided written informed consent for use of their serum samples for clinical and research purposes. This study was approved by Ethics Commission of the University of Duisburg-Essen (reference number 99-184-1315-Y). The clinical characteristics and laboratory data of the two study populations are shown in Progranulin plasma (Essen cohort), serum  and cell culture supernatant levels were measured using Quantikine Kits, according to the manufacturer`s instructions . The absorbance was recorded by a MRX microplate reader and analyzed by Revelation software version 4.22 . Intra- and inter-plate assay precision determined by coefficients of variance (CV) were 4.9% and 7.9%, as reported by the manufacturer.6/ml in RPMI1640  with 10% (v/v) fetal calf serum  supplemented with 1000 U/ml penicillin and 100 U/ml streptomycin  in the absence (controls) or presence of different concentrations of human recombinant progranulin . After two and five days incubation in a 5% CO2 and air incubator at 37°C cell culture supernatants from the control samples were frozen at −80°C until Pgrn measurement as described above. Cell viability was assessed using trypan blue exclusion.Leukemic B-cells were isolated from the PB of CLL patients employing Lymphoprep  density-gradient centrifugation resulting in a purity of >90% as determined by flow cytometry. CLL cells were cultured at a concentration of 1.5×10Comparisons of clinical and laboratory parameters between patient subgroups were performed using the Mann-Whitney U test (for continuous quantitative variables) and the Fisher’s exact test . Spearman correlation coefficients were calculated to analyze the correlation between two continuous variables. Survival time and censored waiting times measured from time of diagnosis were plotted by the Kaplan-Meier method and compared using the log-rank test. For multivariate analysis we used a Cox proportional hazards model. The statistical analyses were performed with SPSS statistics version 20  and GraphPad Prism version 6 .GRN mRNA levels in CLL cells and protein secretion we quantified the concentration of Pgrn in plasma samples from 16 CLL patients with available gene expression data from a previous microarray study 2 = 0.4329) could be observed. Furthermore, cell culture experiments revealed that the leukemic cells are capable of secreting Pgrn into the culture supernatant in a time-dependent fashion with large interindividual differences  as compared to normal controls  and 73 patients required treatment according to National Cancer Institute Working Group criteria. The median time from diagnosis to first treatment (TTFT) of the entire cohort was 64 months . As shown in Eighteen patients from the Essen cohort, nine individuals with stable and nine patients with progressive disease (defined by increasing lymphocyte counts and requiring therapy) were studied at three or more time-points (range 3–5). As illustrated in In a second set of experiments we aimed to confirm our findings in an independent multicentric prospectively followed patient series. To this end we employed serum samples collected from a representative subset of Binet stage A patients included in the CLL1 study of the German CLL study group (GCLLSG). In a pilot series of N = 5 patients from the Essen cohort we could not detect relevant differences in Pgrn levels measured in heparin plasma as compared to serum collected simultaneously from the same patient p>0.05, .Comparing Pgrn blood levels in the CLL1 cohort with our unicentric results from Essen revealed a largely similar distribution pattern, however the median Pgrn concentration was significantly higher in the latter patient group , TTFT and OS times in CLL patients with high Pgrn serum levels as compared to their low Pgrn counterparts  and plasma vs. serum samples collected from individual patients yielded remarkably similar results, suggesting that the assay used in our study was very robust. These technical aspects compare favorably with some of the established prognostic markers in CLL which are more labor- and cost-intensive  GRN mRNA concentrations in immunomagnetically purified CLL cells with Pgrn protein plasma levels in the same patients and observed a highly significant correlation. Furthermore, cell culture studies using purified CLL cells revealed a time dependent secretion of Pgrn into the culture supernatant. These results provide circumstantial evidence that progranulin concentrations measured in the plasma indeed reflect the amount of progranulin production in the leukemic cells derived from individual patients.Following up on gene expression profiling studies from our own group vs. high risk disease. We found strong associations between high Pgrn plasma levels and poor prognostic markers, such as Binet disease stage at diagnosis, mutational status, ZAP70, CD38, low smudge cell counts and high risk cytogenetic aberrations . Different from our own unicentric retrospective cohort, the CLL1 trial only included previously untreated Binet stage A patients. The median time from diagnosis to study entry was one year and the median follow-up of this series was 80 months. Comparing the distribution of progranulin levels in the two patient cohorts revealed a largely similar pattern, however the median Pgrn value was significantly lower in the CLL1 series as compared to the Essen cohort. This finding is probably due to the fact that about 30 percent of the Essen patients had advanced Binet stage B and C disease whereas only stage A patients were included into the CLL1 study. The more benign clinical characteristics of the CLL1 series is also exemplified by the longer time interval from diagnosis to first treatment as compared to the Essen patients (TTFT 101 months vs. 64 months) and the lower prevalence of adverse prognostic factors including FISH cytogenetics, IGHV and β2-microglobulin. Notwithstanding these differences, we observed the same associations between high Pgrn serum levels, poor prognostic markers, TTFT and OS as in the retrospective series from Essen. Most importantly, using TTFT as a read-out in a multivariate Cox regression model including Binet disease stage, CD38 and cytogenetic risk for the Essen cohort and IGHV status, lactate dehydrogenase (LDH), cytogenetic risk and high risk as defined by the study protocol in CLL1 series, progranulin had independent power predicting an unfavorable prognosis in both patient cohorts.Next, we evaluated the prognostic value of Pgrn in our local CLL cohort using the median Pgrn plasma level of 165.5 ng/ml to define two patient subgroups with low- GRN in CLL patients as compared to normal controls are not clear. We investigated the possibility that disrupted Pgrn expression may be caused by aberration of the GRN gene located at chromosome 17q21.31. Exploiting a panel of CLL cases which had been previously investigated for the presence of structural chromosomal abnormalities by high resolution SNP array profiling GRN locus in any of the 55 individual CLL samples analyzed (data not shown). Interestingly, Jiao et al. GRN mRNA, leading to its down-regulation in miR-29b transfected HEK293 cells. MicroRNA-29b is heterogeneously expressed in CLL and has been demonstrated to be associated with an aggressive course of the disease The molecular mechanisms underlying deregulated mRNA expression of in vitro increased cell proliferation via an Akt-dependent mechanism. Along the same line Abrhale and coworkers in vivo through stimulation of the NF-κB pro-survival pathway we are currently investigating the possibility that Pgrn enhances the effects of TLR9 agonists on CLL cells.Progranulin contributes to many important biological processes and it is tempting to speculate about its potential role as a disease propagating factor in CLL. While to date no unique Pgrn-binding cell surface receptor has been identified In summary, these results along with our previously published gene expression data Figure S1Correlation of Pgrn plasma levels and GRN mRNA concentrations in individual CLL cases. Samples from N = 16 patients were subjected to both GRN mRNA quantification using Affymetrix U133A microarrays and ELISA analysis of Pgrn plasma concentrations. mRNA expression values available from a previously published study (GSE4392) and Pgrn plasma levels were found to be correlated . The regression line in the plot was produced by linear regression analysis.(TIF)Click here for additional data file.Figure S2Secretion of Pgrn into cell culture media and survival of CLL cells in the presence of recombinant Pgrn. Freshly isolated CLL cells from five individual patients were cultured as described in patients, materials and methods section. (A) ELISA revealed that CLL cells are capable of secreting Pgrn in a time-dependent fashion with large inter-individual differences. (B) Survival of CLL cells in the presence or absence of different concentrations of human recombinant Pgrn as determined by trypan blue exclusion. Data represent mean +/− SEM.(TIF)Click here for additional data file.Figure S3Distribution of Pgrn levels in the study populations. Pgrn concentrations were measured by ELISA in plasma and serum samples collected from CLL patients in Essen (A) and the CLL1 study cohort (B), respectively. The vertical lines represent the median Pgrn concentrations which were used as a cut-off to define patient subgroups with high vs. low Pgrn levels. For comparison of the clinical characteristics of the two cohorts refer to (TIF)Click here for additional data file.Figure S4Association of established prognostic markers and clinical outcome in the CLL cohort from Essen. Kaplan-Meier curves depict the cumulative proportion of untreated patients with CLL (TTFT) grouped according to IGHV mutation status (A), cytogenetic risk (B), CD38 expression (C), ZAP-70 expression (D), Binet stage (E) and smudge cell percentage (F). Statistical analysis was conducted using the log-rank test.(TIF)Click here for additional data file.Figure S5Comparison of Pgrn levels in heparin plasma as compared to serum collected simultaneously from the same patients. The lines connect the symbols of five individual patients (A). Correlation of Pgrn plasma levels in plasma and serum samples collected simultaneously from the same patients (B). The regression line in the plot was produced by linear regression analysis.(TIF)Click here for additional data file.Figure S6Association of established prognostic markers and clinical outcome in the CLL1 cohort. Kaplan-Meier curves depict the cumulative proportion of untreated patients with CLL (TTFT) grouped according to IGHV mutation status (A), cytogenetic risk (B), thymidine kinase (C), study arm (D), LDH (E) and lymphocyte doubling time (F). Statistical analysis was conducted using the log-rank test.(TIF)Click here for additional data file.


Long-term locoregional control in locally advanced squamous cell carcinoma of the head and neck (SCCHN) remains challenging. While recent years have seen various approaches to improve outcome by intensification of treatment schedules through introduction of novel induction and combination chemotherapy regimen and altered fractionation regimen, patient tolerance to higher treatment intensities is limited by accompanying side-effects. Combined radioimmunotherapy with cetuximab as well as modern radiotherapy techniques such as intensity-modulated radiotherapy (IMRT) and carbon ion therapy (C12) are able to limit toxicity while maintaining treatment effects. In order to achieve maximum efficacy with yet acceptable toxicity, this sequential phase II trial combines induction chemotherapy with docetaxel, cisplatin, and 5-FU (TPF) followed by radioimmunotherapy with cetuximab as IMRT plus carbon ion boost. We expect this approach to result in increased cure rates with yet manageable accompanying toxicity.The TPF-C-HIT trial is a prospective, mono-centric, open-label, non-randomized phase II trial evaluating efficacy and toxicity of the combined treatment with IMRT/carbon ion boost and weekly cetuximab in 50 patients with histologically proven locally advanced SCCHN following TPF induction chemotherapy. Patients receive 24 GyE carbon ions (8 fractions) and 50 Gy IMRT (2.0 Gy/fraction) in combination with weekly cetuximab throughout radiotherapy. Primary endpoint is locoregional control at 12 months, secondary endpoints are disease-free survival, progression-free survival, overall survival, acute and late radiation effects as well as any adverse events of the treatment as well as quality of life (QoL) analyses.The primary objective of TPF-C-HIT is to evaluate efficacy and toxicity of cetuximab in combination with combined IMRT/carbon ion therapy following TPF induction in locally advanced SCCHN.NCT01245985 Clinical Trial Identifier: EudraCT number: 2009 - 016489- 10 Platinum-containing radiochemotherapy is the current standard of care in the conservative treatment approach for locally advancend squamous cell carcinoma of the head and neck (SCCHN) . The magIf choosing radiochemotherapy, the results of the MACH-NC meta-analysis indicated chemotherapy should be platinum-based -4. ThereImprovement of local control and overall survival by intensified treatment regimen comes at a price: increased toxicity of the combined approach, high number of patients unable to complete the full treatment course  versus standard cisplatin and 5-FU (PF) -28. BothThe TPF-C-HIT trial is a phase II trial evaluating radioimmunotherapy with cetuximab and radiation as intensity-modulated radiotherapy with carbon ion boost following TPF induction.The TPF-C-HIT trial is a prospective, non-randomized phase II feasibility trial evaluating locoregional control (LRC) of radioimmunotherapy with cetuximab as carbon ion boost and IMRT following standard induction chemotherapy with TPF as primary endpoint. Secondary endpoints are disease-free survival (DFS), progression-free survival (PFS), overall survival (OS), acute/late effects as well as any adverse events of radioimmunotherapy as carbon ion boost and IMRT.As explained above, long-term local control in advanced squamous cell head and neck cancer (SCCHN) remains a therapeutic challenge. In order to further improve local control for these patients after standard induction chemotherapy with TPF, raster-scanned carbon ion therapy, and radioimmunotherapy as IMRT as the so far most effective treatment components are combined in order to achieve the best possible results. This combination regimen will be tested as to efficacy and toxicity profile. TPF-C-HIT is a monocentric, open-label, non-randomised phase II trial.Patients on the trial receive 2 cycles of induction chemotherapy with docetaxel, cisplatin, and 5-FU followed by re-staging imaging. In case of treatment response, these patients receive their radiation therapy planning scan and one further cycle of TPF. In case of tumour progression after 2 cycles of TPF, according to international recommendation and guidelines, surgery should be re-evaluated as salvage treatment. After completion of the third course of TPF, patients go on to combined radioimmunotherapy: 8 fractions of carbon ion therapy at 3 GyE per fraction  to the primary tumour and involved lymph nodes are followed by IMRT to the whole neck at daily fractions of 2 Gy up to a total dose of 50 Gy. Cetuximab loading dose is given 7 days prior to commencement of radiotherapy followed by weekly doses throughout the treatment. Treatment duration will therefore be 108 days (corresponding to approximately 16 weeks). An overview of the trial schedule is provided in figure To evaluate efficacy and toxicity of radioimmunotherapy with cetuximab as carbon ion boost and IMRT following standard induction chemotherapy with TPF. Primary endpoint is local control at 12 months post completion of treatment. Secondary endpoints are disease-free survival, progression-free survival, overall survival, acute and late radiation effects as well as any adverse events according to CTCAE v. 4.0, and proteomic and genomic analyses. For development of prognostic markers, proteomic and genomic analyses are also included in the secondary endpoints.The main analysis is based on the hypothesis that an absolute increase of local-regional control rate at 1 year of 17% is considered to qualify the study treatment as very promising for additional testing. The decision rules declared by this design with the above characteristics are as follows:0: p ≤ p0 = 63% vs. H1: p ≥ p1 = 80%.H0 = 63% is the clinically "uninteresting" local-regional control rate at 1 year, corresponding to the null hypothesis H0(according to [prding to ).1 = 80% is the clinically "interesting" local-regional control rate at 1 year, corresponding to the alternative hypothesis H1.p0 = 0.63 and π1 = 0.8, a sample size of 45 patients was calculated. Assuming a drop out rate of 10%, 50 need to be recruited for the trial.Sample size calculation for the TPF-C-HIT trial is based on a Fleming's single stage design for phase II studies. With a significance level of 0.05, a power of 80%, πLocoregional control (LRC) will be assessed according to the RECIST criteria  with theDisease-free survival, progression-free survival, and overall survival are calculated from treatment start (corresponding to day 1 of induction chemotherapy) to first event  up to the follow-up visit 12 months post completion of combined radioimmunotherapy. All adverse events under standard induction chemotherapy and prior to the first administration of cetuximab will be recorded as medical history. Adverse Events will be reported using NCI CTCAE v4.0.• Histologically confirmed locally advanced (stage III or IV), non-metastatic squamous cell carcinoma of oro-, hypopharynx and larynx ,• Oral cavity or oro-, hypopharynx or laynx as the primary tumor site,• At least one uni-measurable lesion according to the RECIST,• Karnofsky performance Status > 70%,9/l, platelets > 100 × 109/l, hemoglobin > 10.0 g/dl,• Adequate bone marrow function: neutrophils > 1.5 × 10• Adequate liver function: Bilirubin < 1.5 mg/dL, SGOT, SGPT < 3 × ULN, GGT < 5 × ULN,• Adequate renal function: GFR> 70 ml/min.,• Age between 18 to 70 years,• Life expectancy of at least 6 months,• Ability of subject to understand character and individual consequences of clinical trial,• Signed written informed consent,• Negative serum/urine Beta-HCG test in women of childbearing potential,• effective contraception for patients in procreative age• Previous systemic chemotherapy, radiotherapy or surgery for carcinoma of the head and neck or and larynx,• Nasopharyngeal carcinoma,• M1 (distant metastases)• Prior exposure to EGFR pathway targeting therapy,• Participation in other interventional trials within the last 30 days,• Any surgery within the last 30 days,• Known allergic or hypersensitivity reaction to any drugs scheduled for the study treatment,• signs of active infection• Symptomatic peripheral neuropathy CTC grade 2 or higher,• ototoxicity CTC grade 2 or higher, except if due to trauma caused by tumour mass,• other serious illnesses or medical conditions: therapy-refractory unstable heart disease, congestive heart failure NYHA °III and °IV; coagulopathies• Other previous malignancy within the past 5 years except prior, adequately treated basal cell carcinoma of the skin or pre-invasive carcinoma of the cervix• Significant neurological or psychiatric condition including dementia or seizures or other serious medical condition prohibiting the patient's participation in the trial by judgement of the investigators• Legal incapacity or limited legal capacity• Positive serum/urine β-HCG/pregnancy• Drug abusePatients receive three cycles of docetaxel/cisplatin/5-FU (TPF) according to the schedule described by Vermorken et al  at the fDocetaxel 75 mg/m² body surface Cisplatin 75 mg/m² body surface 5-FU 750 mg/m² body surface As a standard, patients of course receive adequate premedication with corticosteroids (dexamethsone) prior to application of docetaxel, antiemesis, and hydration according to standard recommendations of the vendours. In addition, patients receive prophylactic antibiotic treatment with ciprofloxacine 500 mg po bid for 10 days starting one week after commencement of each cycle. Dose reductions will be performed as recommended by the vendour.9/l) persisting for ≥ 7 days was observed during cycle one or two of TPF.Pegylated G-CSF is administered prophylactically if a prior episode of febrile neutropenia or infection with either delayed recovery of absolute neutrophil count at day 21 or grade 4 neutropenia (ANC < 0.5 × 10Cetuximab is administered as 400 mg/m² body surface loading dose 7 days prior to RT-treatment start (d57) after administration of anti-histamines (dimetindene) and corticosteroids (dexamethasone).Weekly administrations of Cetuximab 250 mg/m2 body surface follow for the duration of radiotherapy from d64.Patients are immobilized using individual thermoplastic head masks with thermoplastic shoulder fixation. Planning examinations consist of a planning CT scan (3 mm slice thickness) with the patient positioned in the individual fixation device and contrast-enhanced MRI for 3D image correlation.Gross Tumor Volume (GTV) includes the gross primary tumour or involved node(s) based on clinical and endoscopic examinations, CT scan, or other imaging techniques if applicable. Involved lymph nodes are defined as any lymph nodes > 1 cm or nodes with a necrotic center.The Clinical Target Volume (CTV) is defined as GTV plus surrounding areas at risk for containing microscopic disease as delineated by the attending radiation oncologist. CTV1 includes the GTV with a margin of approximately 0.2 cm. CTV2 represents the nodal levels to receive elective irradiation. The CTV margins may be smaller if the GTV is adjacent to the spinal cord or other critical normal tissues. Lymph node levels are delineated in accordance with current guidelines and recommendations -36 as fo• Submental nodes : In cases where the floor of mouth or level IB are involved.• Submandibular nodes : All cases except primary palate tumours which do not extend to the tonsil or base of tongue. Only the ipsilateral level IB is included unless the primary tumour reaches or crosses the midline. The ipsilateral level IB must be included in all cases if upper jugular metastases are found.• Upper deep jugular  nodes: all cases .• Subdigastric (jugulodigastric) nodes, midjugular, lower neck, and supraclavicular nodes (levels II through IV): all cases, bilaterally, except for tonsillar fossa tumours > 1 cm from from the midline and surgical node negative contralateral neck.• Posterior cervical nodes (level V): all cases, at the neck side where there is evidence of jugular nodal metastases.• Retropharyngeal nodes: all cases.Planning Target Volume (PTV1 and PTV2)includes CTV1 and CTV2 with an additional margin in order to compensate for set-up variability and internal organ motion. A minimum margin of 0.2 cm around the CTV in all directions is required to define each respective PTV, except for situations in which the CTV is adjacent to spinal cord or other critical normal tissues. In such situations, the margin can be reduced judiciously if image guidance is used regularly.The Carbon ion therapy treatment planning is carried out using a dedicated treatment planning system (TPS) developed for and in co-operation with HIT (Heidelberg ion beam therapy centre). TPS offers the following functionalities also expected in conventional radiation therapy as well as methods for biological RT treatment optimization. As ion beams exhibit an increased biological effective dose depending on various factors, these need to be included within the planning algorithm. In addition, steering parameters for scanned ion beams need also be calculated by the TPS.Carbon ion treatment is given at the HIT after inverse treatment planning in active beam application (raster-scanning method). A monoenergetic ion beam with a full-width/half-maximum (FWHM) of 5 mm is extracted from the accelerator system (synchrotron) and magnetically deflected to subsequently scan all planned iso-energetic slices roughly corresponding to the tumour's radiological depth. Using this method almost any desired dose distribution can be created.In view of the tolerance dose to organs at risk a dose of 24 GyE in 8 fractions for carbon ions will be prescribed to the median of the calculated dose distribution for the target volume (PTV1). Dose specification is based on biologic equivalent dose because of the high relative biologic effectiveness (RBE) of carbon ions Treatment planning aims at covering the PTV1 with the 95%- and 90%-treatment isodose.Our policy is to avoid under-dosage within a target volume if the dose constrains for the normal tissue are not exceeded.®. IMRT hence is delivered either at a 6 MV-linear accelerator (step-and-shoot technique) or at a 6 MV tomotherapy unit to a total dose of 50 Gy in 2 Gy/fraction. IMRT image guidance consists of MV cone-beam CTs which are taken into account for the calculation of the total applied dose.Radiotherapy will be given to the patients only as intensity modulated radiotherapy (IMRT) and must be used for the entire course of treatment. Intensity-modulated RT is planned using two commercially available planning systems: KonRad (Siemens OS) for step-and-shoot applications or TomotherapyIMRT will be given in 25 fractions over approximately 5 weeks. The primary tumour, involved nodes (PTV1) and subclinical disease sites (PTV2) will receive 2.0 Gy per day. The total doses for the PTV1 and the PTV2 will thus be 50.0 Gy± 5%, respectively. Treatment breaks should be minimized, any break of more than 5 days will be considered a major protocol violation.All plans must be normalized such that 95% of the volume of the PTV1 is covered with the prescribed dose. In addition the following requirements must be met:• No more than 20% of the PTV1 should receive ≥ 110% of the prescribed dose.• No more than 5% of PTV1 or PTV2 should receive ≤ 90% of the prescribed dose.• No more than 2% or 2 cc of tissue outside the PTVs should receive ≥ 110% of the prescribed dose to the PTV1.The constraints are applicable for the summation (carbon ion and photon IMRT) plan.• Spinal cord: a margin of 0.5-1 cm around the spinal cord may be added to create a Planning Organ at Risk Volume (PRV). The dose to any point within the spinal cord should not exceed 45 Gy to any volume larger than 0.03 cc.• Parotid glands: the objective is to limit the mean dose to at least one gland to ≤ 26 Gy; alternatively at least 20 cc of the combined volume of both parotid glands to < 20 Gy or at least 50% of one gland to <30 Gy.• Glottic larynx: the dose to the larynx should be kept <45 Gy whenever feasible.• Brachial plexus: the dose to the brachial plexus must be limited to ≤ 60 Gy in patients with suspicious level IV node(s).• Mandible: 70 Gy should not be exceeded at any point.• Brain stem: the tolerated dose is 54 Gy, in volumes of appox. 1 cc the dose can be 60 Gy.• Unspecified tissue outside the target volumes: ≤ 100% of the dose prescribed to PTV2. No more than 5% of the non-target tissue can receive greater than the dose to PTV2.Portal image of each field or orthogonal images that localize the isocenter placement of IMRT must be obtained on the first day of therapy. At least weekly set-up controls using MV cone-beam CTs (cb-CTs) are required for the IMRT treatment, daily IGRT is required for carbon ion therapy. Doses applied to the patient by the acquisition of position controls may be taken into account for calculation of overall applied dose.Parotid glands:1. Mean dose to either parotid < 26 Gy or2. At least 50% of the either parotid gland will receive < 30 Gy or3. At least 20 ml of the combined volume of both parotid glands will receive < 20 Gy.Submandibular/sublingual glands and oral cavity: Reduce the dose as much as possible.Critical normal structure constraints followed by the prescription goals are the most important planning priorities. The priorities in addressing the protocol aims and constraints will be in the following order:1) Critical Normal Structure Constraints,2) Prescription Goals,3) Planning Goals: salivary glands.After inclusion into the trial and the patient's written informed conset, the patient receives two cycles of TPF followed by imaging for re-staging. Without evidence of progressive disease (PD) according to RECIST, the patient then receives an individual fixation device consisting of thermoplastic head mask and shoulder fixation as well as RT treatment planning scans (CT/MRI). Subsequently, patients are administered the third cycle of TPF Figure . CetuximCetuximab is given weekly throughout the course of radiotherapy Figure .In case of progressive disease (PD) on the re-staging examination after two cycles of TPF, surgical salvage should be re-evaluated.First follow-up examination including diagnostic, contrast-enhanced MRI will be carried out 6 weeks post completion of radiation treatment. Further controls including MRI are 3, 6, and 12 months thereafter, in 6 monthly intervals until 2 years post RT, then in yearly intervals  at each follow-up. Tumour response will be evaluated according to the RECIST-criteria .If the patient has given (separate) informed consent, 30 mL venous blood will be collected from each subject for the proteomic examinations prior to the first administration of TPF, (e.g. on day -7/day 1), at day 50 (before the first treatment with cetuximab), day 64, day 73 and 106 of the treatment phase and once at the end of therapy visit (day 108). Thus, the overall volume of blood samples used for Proteomic/Genomic investigations will be approximately 120 ml. Following parameters/pathways will be investigated:• In order to predict the efficacy of the trimodal therapy blood will be collceted during therapy and follow-up to detect and correlate the levels of well known tumor- and angiogenesis markers  using Enzyme-Linked Immunosorbent Assay (ELISA). Further, platelet protein content (i.e. tumor angiogenesis growth factors and cytokines) will be analyzed using citrate blood samples and correlated with serum- and plasma- protein results.• In order to perform the genomic analysis, patients' blood samples are collected as indicated and RNA, miRNA and DNA isolation will be performed. Based on an established platform, linear RNA-amplification, labelling and hybridization on human genome wide oligo-arrays  are planned. DNA samples are used to identify potential chromosomal aberrations or epigenetic alterations that might predict treatment response. RNA and miRNA samples are further analyzed by real time quantitative RT-PCR to confirm microarray data and to test a subset of clinical predictors.The determinations of proteomic and genomic parameters will be carried out at the Department of Radiation Oncology in Heidelberg.No further genetic investigations on the blood collected during the study will be carried out!The TPF-C-HIT trial has been designed by the Department of Radiation Oncology, and is carried out at the National Centre for Tumour disease (NCT) Heidelberg, the Heidelberg Ion Therapy Centre (HIT), and the department of Radiation Oncology, University of Heidelberg. It is an investigator-initiated trial; the Department of Radiation Oncology is responsible for co-ordination, overall trial management, registration ; EudraCT registration (EudraCT number: 2009 - 016489- 10), database management, quality assurance, monitoring, and reporting is carried out by Alcedis GmbH, Gießen, Germany.Patients are recruited by the Department of Radiation Oncology, Heidelberg, Germany. Induction chemotherapy with TPF is given and monitored by the NCT, Heidelberg, Germany, combined radioimmunotherapy is carried out by the Department of Radiation Oncology/HIT, Heidelberg, Germany.Adverse and serious adverse events are recorded using NCI common toxicity criteria for adverse events (CTCAE v. 4). Acute radiation effects are defined as effects occurring within 90 days from beginning of radiotherapy. Late effects are defined as effects observed thereafter. Safety analysis is performed with respect to frequency of serious adverse events and adverse events stratified by organ system, severity, causality.Patient accrual is completed with inclusion of the last patient and should extend for approximately 2 years from trial initiation. Regular trial participation for each patient terminates 18 months from first administration of TPF or the patient's death respectively.• Patient wish• Cetuximab treatment delay for more than 2 consecutive weeks,• Occurrence of any grade 4 toxicities related to cetuximab,• Occurrence of >/= grade 3 allergic/hypersensitivity reaction related to cetuximab.• Medical condition necessitating treatment termination and withdrawal of the patient from the trial• Pregnancy• Lack of complianceThe trial can be prematurely closed or suspended by the principal investigator (PI) in following cases:• Medical or ethical reasons relevantly affecting the risk-benefit relationship,• Difficulties in recruitment of subjects suggest unjustifiable prolongation of the study timeline,• Previously unexpected adverse events  occur with unjustifiable frequency,• Expected adverse events occur with an unexpectedly high incidence,• Relevant superiority of patients in one treatment arm of a comparable clinical trial,• Legal authorities' decisionThe Ethics Committee (EC) and the competent regulatory authorities will be informed about premature closure of the trial. Furthermore, the Ethics Committee(s) and competent regulatory authorities themselves may decide to stop or suspend the trial.If the trial is closed prematurely, the trial material such as completed, partially completed, and blank CRFs will be returned to the coordinating investigator.All involved investigators have to be informed immediately about a cessation or suspension of the trial. The decision is binding on all investigators.The final protocol was approved by the University of Heidelberg Medical School ethics committee (AFmo-167/2010), the German radiation protection authority (Bundesamt für Strahlenschutz: = BfS) and Paul-Ehrlich Institute (: = PEI). The trial complies with the Helsinki Declaration in its recent German version, the Medical Association's professional code of conduct, principles of Good Clinical Practice (GCP) guidelines and the Federal Data Protection Act. It will be carried out in keeping with local legal and regulatory requirements. Medical confidentiality and Federal Data Protection Act will be followed. Written informed consent is obtained from each patient in oral and written form.With the introduction of novel radiotherapy techniques such as IMRT and particle therapy (neutron and carbon ion therapy), higher local control rates in radioresistant tumours such as adenoid cystic carcinoma could be achieved over the last decade ,16. TaxaWith the available data, there are various options to intensify treatment for patients with locally advanced head and neck tumours: modern radiotherapy techniques provide tools for relative dose escalation within the tumour, particle therapy may further improve local control by increased biological effectiveness, and induction chemotherapy improves long-term survival in these patients. All of these treatment approaches have shown clinical feasibility and efficacy as single modalities.Current standard practice in Europe mostly consists of combined radiochemotherapy in the conservative management of locally advanced head and neck tumours (SCCHN). Management of acute treatment side-effects in clinical routine shows further intensification is rarely possible albeit feasible in terms of improved outcome. Assuming toxicity of combined radioimmunotherapy being less severe than in radiochemotherapy as demonstrated by Bonner and co-workers , this maThe TPF-C-HIT trial is a phase II trial to evaluate three modern treatment algorithms for head and neck cancer: Patients receive TPF induction chemotherapy followed by radioimmunotherapy with cetuximab as intensity-modulated radiotherapy (C12) plus carbon ion boost. IMRT is applied in conventional doses per fraction and total dose. The primary tumour and involved lymph nodes will be boosted by application of a carbon ion boost in active beam application (raster-scanning method). Due to these highly sophisticated RT techniques no increased risk of side effects associated with radiotherapy is expected as compared to conventional techniques and may even reduce potential side effects in this setting. Due to the advantages of IMRT and carbon ion therapy in receiving a better dose distribution in the target volume improved local control rates may be achieved. To our knowledge, this is the first clinical trial evaluation this trimodal treatment regimen.Prof. Debus is a member of the Merck KGaA advisory board.All authors have read and approved the final manuscript.MWM, JK, ADJ, and JD developed the study protocol and planned the trial. AH is responsible for statistical considerations/basis of the trial. All authors are responsible for conducting and co-ordination of the trial as well as patient recruitement. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/182/prepub