Functional profiling and gene expression analysis of chromosomal copy number alterations Contrarily to the traditional view in which only one or a few key genes were supposed to be the causative factors of diseases , we discuss the importance of considering groups of functionally related genes in the study of pathologies characterised by chromosomal copy number alterations . Recent observations have reported the existence of regions in higher eukaryotic chromosomes -LRB- including humans -RRB- containing genes of related function that show a high degree of coregulation . Copy number alterations will consequently affect to clusters of functionally related genes , which will be the final causative agents of the diseased phenotype , in many cases . Therefore , we propose that the functional profiling of the regions affected by copy number alterations must be an important aspect to take into account in the understanding of this type of pathologies . To illustrate this , we present an integrated study of DNA copy number variations , gene expression along with the functional profiling of chromosomal regions in a case of multiple myeloma . Background Genomic copy number alterations such as gains or losses of chromosomal regions have been shown to be on the basis of many human pathologies . Classical approaches to characterize these genetic aberrations used comparative genomic hybridisation -LRB- CGH -RRB- , in which genomic DNA was hybridised to metaphase chromosomes . -LSB- 1 -RSB- Recently , the use of different types of microarrays to directly study genomic variations in DNA copy number is becoming more and more popular . Such massive genomic approaches are known as array comparative genomic hybridisation , or Array CGH . -LSB- 2 -RSB- These new technologies along with the use of expression arrays allow for a highly accurate characterisation of the dependence of gene expression on alterations in genomic copy number . -LSB- 3 -RSB- As in many genome-scale methodologies data analysis and , in particular , the biological interpretation of the results constitutes a well-known bottleneck . Specific problems related to the analysis of Array CGH can be circumscribed mainly to two types : appropriate mapping and visualisation of the data onto the chromosomes , and efficient copy number estimation . This last aspect has been the motivation for a number of analytical approaches recently proposed -LSB- 4 -RSB- , that can be considered the first generation of algorithms for Array CGH analysis . Obviously , copy number variations are expected to have a strong effect on gene expression . -LSB- 5,6 -RSB- Nevertheless , the ultimate aim of studies of copy number chromosomal alterations is to understand what is the effect produced in functional terms . In the classical vision one or a few key genes are the causative factors for the this type of pathologies , and the problem consisted in identifying such genes within the region amplified or deleted . The existence of regions in the chromosomes containing coexpressing genes -LSB- 7 -RSB- which , in addition , are functionally related has recently been reported even in higher eukaryotes . -LSB- 8 -RSB- Actually , regional arrangements of genes have found to be regulated not only by copy number alterations but also by different mechanisms such as epigenetic modifications . -LSB- 9 -RSB- This reinforces the functional role of chromosomal regions including groups of functionally related genes and its possible impact on diseases such as cancer . -LSB- 10 -RSB- These observations give credence to a new vision in which chromosomal alterations can be causing effects not by altering single key genes but by acting on complete molecular sub-systems such as pathways of functionally related genes . Recently , different approaches have focused on the functional aspects of the results of microarray experiments . -LSB- 11,12 -RSB- Nevertheless , the possible functional significance at regional level of copy number alterations has been largely ignored . Here we present a combined approach to the study of copynumber alterations , gene expression and functional profiling , exemplified in a case of multiple myeloma . -LSB- 13 -RSB- Methodology Functional profiling of Array-CGH experiments under this new perspective would require of three steps : 1 -RRB- detection of regions with copy number variations -LRB- the origin of the disease -RRB- , 2 -RRB- detection of regional alterations in gene expression -LRB- the causes of the disease -RRB- and 3 -RRB- analysis of enrichment in functional terms in the detected regions -LRB- the consequences of the alteration or the functional basis of the disease -RRB- . While copy number alterations can be detected by means of different methods , alterations in the levels of gene expression are not always easy to be detected using the typical methods -LRB- t-test or similar -RRB- due several factors such as small sample sizes . For this reason here we will only use plots to visualize the effect of one variable -LRB- copy number -RRB- into the other one -LRB- expression level -RRB- . The third step , the functional profiling , becomes then the most important aspect of the analysis given that it will provide a functional explanation of the molecular basis of the disease caused by copy number alterations . Detection of copy number alterations We have used a segmentation method which is a variant of the circular binary segmentation method -LSB- 14 -RSB- , for copy number change detection -LRB- isowindow -RRB- . The isowindow method tries to identify boundaries between regions with a significant change in the values of intensity of hybridisation of the probes by some consecutive steps . Firstly a t-test is used to determine differences between regions around all possible boundary points . Once all the candidate boundaries have been selected -LRB- a liberal p-value is used at this stage -RRB- there are sorted from small to high minimum p-values . In a second step the boundary candidates in the list with overlapping neighbourhoods are filtered to obtain a refined list of optimal non-overlapping boundary candidates . All the p-values are recalculated for the redefined neighbourhoods and a more stringent threshold is applied here . Finally , regions at both sides of each boundary candidate are again compared with a t-test . If they are not significantly different in their average hybridisation values , then they are merged as a unique region . Otherwise they define two regions with different copy number value . This is a simple and quick procedure that allows for easily changing from fine to coarse resolution by modifying the thresholds for the p-values . We have compared isowindow to other two methods for breakpoint detection , GLAD -LSB- 15 -RSB- and circular binary segmentation -LRB- CBS -RRB- -LSB- 14 -RSB- , which are among the best performers . -LSB- 4 -RSB- In the GLAD method a likelihood function with weights determined adaptively is used to solve the copy number estimation problem locally based on data smoothed . Then , the algorithm finds , for each probe , the maximal neighbourhood in which the local constant assumption holds . Each of the constant pieces of the line define a block of probes with similar copy number among them and different copy number from that of the nearby regions . On the other hand , the CBS method selects firstly a segment of the data -LRB- a group of probes that are all consecutively arranged in the genome or in a chromosome -RRB- . The copy number measures of the probes in that segment are compared to those in the reminder dataset using a t-statistic . Hence , the method can distinguish whether the segment chosen has a copy number that is higher or lower than the overall copy number in the data , assumed to be the normal reference . This scheme is iterated exhaustively for all possible segments in the dataset , spotting those that correspond to regions of altered copy number . An approximation to the relative performances of the methods used was obtained by means of simulated data sets . Such datasets were generated by means of a piecewise constant function plus random alterations normally distributed with mean value and three different levels for the standard deviation -LRB- corresponding to noise levels 0.2 , 0.5 and 1 -RRB- . A mean value of 0 would correspond to a normal region , without copy number alterations , while mean values lower and higher would correspond to deletions or amplifications at different degrees , respectively . Amplified and deleted regions of different sizes are randomly situated within the simulated normal chromosome and the methods have to locate them at different noise levels . The method proposed here performs at least as well as the GLAD and CBS -LRB- Table 1 -RRB- while being more efficient in terms of runtimes . Isowindow shows a better performance in finding small amplicons . Functional profiling of regions with copy number alterations The final aim of a Array-CGH experiment is to find a molecular explanation for the effects of the detected copy umber alterations . The interpretation of genomescale data is usually performed in two steps : in a first step genes of interest are selected in this case because they are located in the amplified -LRB- or lost -RRB- region detected . In a second step , the selected genes of interest are compared to the background -LRB- here the rest of genes in the chromosome -RRB- in order to find enrichment in any functional category -LRB- gene ontology , KEGG pathways , etc. -RRB- This comparison to the background is required because otherwise the significance of a proportion -LRB- even if high -RRB- can not be determined . Different approaches have been developed to this end . -LSB- 11 -RSB- Here we will use the FatiGO + -LRB- 16 -RRB- program , which uses a Fisher 's exact test to determine the enrichment in different functional categories including gene ontology , KEEG pathways , Interpro functional motifs , Swissprot keywords and some regulatory elements such as transcription factor binding sites or other regulatory motifs . -LSB- 17 -RSB- Discussion We have implemented all the described functionalities in a program , ISACGH -LRB- an acronym for In Silico Array CGH -RRB- , which is used to illustrate the concept of functional profiling of CGH arrays with an example of multiple myeloma -LRB- MM -RRB- , an incurable form of haematological neoplasia . Nine MM cell lines were obtained from the DSMZ -LRB- Deuche Sammlung von Mikroorganismen und Zelkuturen GmbH , Braunschweig , Germany -RRB- and were cultured under recommended conditions . DNA and RNA were extracted using supplier 's protocols . Microarray assays were performed using the CNIO OncoChip , which contains 7657 different cDNA clones of cancer related genes . -LSB- 18 -RSB- CGH experiments onto cDNA arrays and hybridisation were performed as described in -LSB- 13 -RSB- and quantified using the GenePix Pro 5.0 software -LRB- Axon Instruments Inc. , Union City , CA -RRB- . Cy3/Cy5 ratio values were normalized using the DNMAD tool from the GEPAS -LSB- 19,20 ,21 -RSB- and the resulting data were transformed to log2 ratios . Our purpose was to identify any possible region that contained copy number gains -LRB- amplifications -RRB- , to study the expression of the genes included in that particular region and to understand the possible functional consequences of such alterations . Using the segmentation method as implemented in the ISACGH we could detect a putative amplicon in the chromosome 18 -LRB- which remained undetected with both GLAD and CBS , because of the low density of the array , although the effect would have been the same in a high density arrays with a small amplicon -RRB- The figure shows the region -LRB- left -RRB- and the slight , although appreciable , differences in gene expression levels within the amplicon -LRB- right -RRB- . A unique feature offered by ISACGH is the possibility of obtaining a functional profile of the detected chromosomal regions . When the amplicon is analysed through the FatiGO + program -LSB- 16,17 -RSB- a number of GO terms arise as over-represented in the genes contained in such region . Thus , the GO terms regulation of cellular process -LRB- GO : 0050794 -RRB- and regulation of physiological process -LRB- GO : 0050791 -RRB- were significantly overrepresented in the amplicon -LRB- FDR adjusted p-value = 0.0336 -RRB- . Genes annotated with these terms were : BCL2 , MALT1 , NEDD4L , MBD2 , TNFRSF11A and TCF4 . Some of them have annotations at more detailed levels in GO , although the number of genes was too small as to produce statistically significant results . For example BCL2 and MALT1 are annotated as negative regulation of programmed cell death -LRB- GO : 0043069 -RRB- . These observations suggest that some processes altered , that ultimately lead to diseases , are not produced by the deregulation of one unique gene , but are the combined result of simultaneous deregulations of genes involved in a pathway or a particular biological function . In addition , these findings stress the importance of the use of functional profiling methods for the proper understanding and interpretation of the results of the genome-scale experiments . This unique feature included in ISACGH is of extreme importance since growing evidence suggests the existence of clusters of functionally related genes in the chromosomes -LSB- 8 -RSB- and the possible impact on diseases such as cancer . -LSB- 10 -RSB- Although ISACGH -LSB- 22 -RSB- can be used alone , it is tightly integrated in the GEPAS package . -LSB- 19 , 21,23 -RSB- GEPAS , that stands for Gene Expression Profile Analysis Suite -LRB- GEPAS -RRB- , constitutes one of the most complete resources for microarray data analysis available over the web . GEPAS includes facilities for normalisations , clustering , gene selection , predictors and functional profiling . Thus , different operations -LRB- including pre-processing or normalization -RRB- can directly be performed within the same environment , without the necessity of any file reformatting step . Conclusion Despite a number of applications dealing with the estimation of genomic copy number have been recently published -LSB- 4 -RSB- , there are different aspects of the analysis of Array CGH data that have been poorly addressed or even ignored . Recent evidences strongly support the existence of regional arrangements of functionally related genes -LSB- 8 -RSB- , with obvious consequences for the understanding of diseases characterised by copy number alterations , such as an important number of cancers . -LSB- 10 -RSB- This fact reduces the validity to the classical vision , in which one or a few key genes would be the causative factors of the disease , and urges to take into consideration the functional dimension in the interpretation of the effects of copy number alterations . In this new scenario , the deregulation of blocks of functionally related genes located in the chromosomal regions with copy number alterations would be behind the disease phenotype . The methods for functional profiling have proven in many scenarios its usefulness . An obvious challenge is to increase our knowledge in different aspects of function and cooperation between genes in order to be able of applying this methods in a way that allows us to unravel new unknown functional aspects of the biology of the cell and their connections to pathologies .

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Oxidation of chylomicron remnant-like particles inhibits their uptake by THP-1 macrophages by apolipoprotein E-dependent processes The influence of the oxidative state of chylomicron remnants -LRB- CMR -RRB- on the mechanisms of their uptake and induction of lipid accumulation by macrophages derived from the human monocyte cell line , THP-1 , during foam cell formation was investigated using chylomicron-remnant-like particles -LRB- CRLPs -RRB- at 3 different levels of oxidation . The oxidative state of CRLPs was varied by exposure to CuSO4 -LRB- oxCRLPs -RRB- or incorporation of the antioxidant , probucol -LRB- pCRLPs -RRB- into the particles . oxCRLPs caused significantly less accumulation of triacylglycerol in the macrophages than CRLPs , and their rate of uptake was lower , while pCRLPs caused more lipid accumulation and were taken up faster . Uptake of all 3 types of particles was inhibited to a similar extent when entry via the low density lipoprotein -LRB- LDL -RRB- receptor related protein -LRB- 80 -- 90 % -RRB- , LDL receptor -LRB- − 30 -- 40 % -RRB- , CD36 -LRB- − 40 % -RRB- and phagocytosis -LRB- − 35 -- 40 % -RRB- was blocked using lactoferrin , excess LDL , anti-CD36 and cytochalasin D , respectively , but blocking scavenger receptors-A or - B1 using poly inosinic acid or excess HDL had no effect . These findings show that oxidation of CRLPs lowers their rate of uptake and induction of lipid accumulation in macrophages . However , oxidation does not change the main pathways of internalisation of CRLPs into THP-1 macrophages , which occur mainly via the LRP with some contribution from the LDLr , while CD36 and phagocytosis have only a minor role , regardless of the oxidative state of the particles . Thus , the effects of CMR oxidation on foam cell formation contrast sharply with those of LDL oxidation and this may be important in the role of dietary oxidized lipids and antioxidants in modulating atherosclerosis . 1 Introduction Chylomicron remnants -LRB- CMR -RRB- carry lipids of dietary origin from the gut to the liver for processing -LSB- 1 -RSB- and there is now a large and growing body of evidence indicating that these lipoproteins are strongly atherogenic . They have been shown to be taken up into the artery wall as efficiently as LDL -LSB- 2 -- 5 -RSB- ; remnant-like particles containing apolipoprotein E -LRB- apoE -RRB- have been isolated from human aortic intima and atherosclerotic plaque -LSB- 6,7 -RSB- ; and delayed clearance of CMR from the circulation correlates with lesion development -LSB- 8,9 -RSB- . Moreover , we and others have shown that CMR induce extensive lipid accumulation causing foam cell formation in human monocyte-derived macrophages -LRB- HMDM -RRB- -LSB- 10 -RSB- and in human and murine monocyte/macrophages cell lines -LSB- 11,12 -RSB- . Low density lipoprotein -LRB- LDL -RRB- plays a major role in atherogenesis and in foam cell generation , but oxidation of the lipoprotein particles , a process which can occur within the artery wall , is necessary before extensive lipid accumulation is induced -LSB- 13 -RSB- . In striking contrast , CMR do not require prior oxidation to cause macrophages to form foam cells -LSB- 10 -- 12 -RSB- . However , our studies have demonstrated that incorporation of lipophilic antioxidants into the particles enhances , rather than inhibits , lipid uptake and accumulation in the cells -LSB- 14,15 -RSB- , suggesting that the oxidative state of CMRs may play a role in their induction of foam cell formation , but in the opposite way to that of LDL . Oxidized CMR could occur either in the artery wall by the action of the cell-associated lipoxygenase and myeloperoxidase which are believed to oxidize LDL , or in the circulation , because dietary oxidised lipids , which are produced when fat is cooked at high temperatures , have been shown to be transported in these lipoproteins -LSB- 16,17 -RSB- . Clearly , therefore , it is important for the understanding of the atherogenicity of CMR to establish how their oxidation influences their uptake and induction of foam cell formation and the pathways by which CMR are internalised by the cells . It has been demonstrated that CMR are taken up by the liver by apolipoprotein E -LRB- apoE -RRB- - dependent pathways mediated by the LDL receptor -LRB- LDLr -RRB- and the LDL receptor-like protein -LRB- LRP -RRB- -LSB- 1 -RSB- . The exact mechanisms by which CMR are taken up by macrophages , however , are not yet definitively established , and nothing is known about the effects of oxidation of the particles on the routes by which they are internalised . The LDL receptor -LRB- LDLr -RRB- appears to play a part -LSB- 1 -RSB- , but as it is down-regulated by the influx of cholesterol into cells , native LDL does not induce foam cell formation -LSB- 16 -RSB- , and our studies -LSB- 18 -RSB- have suggested that the delivery of cholesterol to macrophages by CMR has a similar effect . Thus , other mechanisms are also likely to be involved , and evidence from experiments using antibodies to the LDLr and animals lacking the LDLr supports this view -LSB- 10 -RSB- . Candidates include the LRP -LSB- 19 -RSB- , the apoB48 triacylglycerol-rich lipoprotein receptor -LRB- apoB48r -RRB- -LSB- 20 -RSB- , an as yet unidentified 43 kDa protein described by Elsegood et al. -LSB- 21 -RSB- and scavenger receptors such as scavenger receptor A -LRB- SR-A -RRB- and CD36 -LSB- 1 -RSB- . Phagocytosis has also been suggested as a possible mechanism -LSB- 22 -RSB- . Since the route of uptake of LDL by macrophages is profoundly changed after oxidation from the regulated LDLr to the unregulated scavenger receptors -LSB- 16 -RSB- , it is important to establish whether the oxidative state also alters the mechanisms of uptake of CMR by the cells . The aim of this study is to investigate the effects of the oxidative state of CMR on their uptake by macrophages and on the accumulation of lipid within the cells , and to determine how oxidation affects the pathways involved in the internalisation of the particles . Chylomicron remnant-like particles -LRB- CRLPs -RRB- at three different levels of oxidation -LRB- CRLPs , oxidized CRLPs -LRB- oxCRLPs -RRB- and CRLPs containing the antioxidant probucol -LRB- pCRLPs -RRB- -RRB- and macrophages derived from the human monocyte cell line THP-1 were used as the experimental model , and the mechanisms of uptake were evaluated using specific inhibitors of the processes believed to be involved . The findings clearly demonstrate that oxidation of CRLPs reduces the rate of their uptake by THP-1 macrophages and decreases lipid accumulation in the cells , and further show that this is due to differential interaction with apoE dependent receptors . 2 Materials and methods RPMI 1640 medium , fetal bovine serum -LRB- FBS -RRB- , l-alanyl-l-glutamine -LRB- glutamax -RRB- penicillin/streptomycin and β-mercaptoethanol were obtained from Gibco -LRB- Paisley , UK -RRB- . FBS was heat inactivated -LRB- 56 °C , 30 min -RRB- before use . Trypan blue , fatty acid-free bovine serum albumin -LRB- BSA -RRB- , phospholipids , cholesterol , cholesteryl oleate , phorbol 12-myristate 13-acetate -LRB- PMA -RRB- , Oil red O , probucol , poly inosinic acid -LRB- poly I -RRB- , lactoferrin and cytochalasin D were supplied by Sigma -LRB- Poole , UK -RRB- . 1 ′ 1 ′ - dioctadecyl-3,3-3 ′ ,3 ′ - tetramethylindo-carbocyanide perchlorate -LRB- DiI -RRB- was from Cambridge Bioscience -LRB- Cambridge , UK -RRB- . The blocking antibody for CD36 was obtained from Immunodiagnostic Systems Ltd -LRB- Tyne and Wear , UK -RRB- and cholesterol oxidase from Merck Biosciences Ltd -LRB- Nottingham , UK -RRB- . DiI-labelled acetylated LDL -LRB- acLDL -RRB- was purchased from Molecular Probes -LRB- Paisley , UK -RRB- . 2.1 Preparation of lipoproteins LDL and high density lipoprotein -LRB- HDL -RRB- were isolated from human plasma -LRB- National Blood Service , London UK -RRB- by ultracentrifugation . Plasma was layered under 0.9 % NaCl -LRB- d = 1.006 g/ml -RRB- , centrifuged for 5 h at 100,000 × g -LRB- 4 °C -RRB- , and the top fraction discarded . The density of the bottom layer was raised to 1.063 g/ml with KBr , layered under KBr -LRB- d = 1.063 g/ml -RRB- and centrifuged for 16 h at 175,000 × g -LRB- 4 °C -RRB- . LDL was collected from the top fraction by tube slicing . For the preparation of HDL , the bottom fraction was then adjusted to d 1.21 g/ml with KBr , layered under KBr -LRB- d 1.21 g/ml -RRB- and centrifuged at 175,000 × g/ml at 4 °C for 18 h and the top fraction containing HDL was collected by tube slicing . LDL and HDL were dialysed against saline for 48 -- 72 h prior to used . CRLPs were prepared by sonication -LRB- power setting 22 -- 24 μm , 20 min , at 56 °C -RRB- of a lipid mixture containing 70 % trilinolein , 2 % cholesterol , 3 % cholesteryl ester and 25 % phospholipids in 0.9 % NaCl -LRB- w : v -RRB- in Tricine Buffer -LRB- 20 mM , pH 7.4 -RRB- followed by stepwise density gradient -LRB- 2.5 ml d 1.065 g/ml , 2.5 ml d 1.020 g/ml , 3 ml d 1.006 g/ml -RRB- ultracentrifugation as described by Diard et al. -LSB- 23 -RSB- at 17,000 × g for 20 min at 20 °C . The upper layer of grossly emulsified lipids was then removed and replaced with an equal volume of NaCl solution -LRB- d = 1.020 g/ml -RRB- and tubes were centrifuged at 70,000 × g for 1 h -LRB- 20 °C -RRB- . For apoE binding , lipid particles collected from the top layer were incubated with the dialysed d 1.063 -- 1.21 g/ml fraction of human plasma -LRB- National Blood Transfusion Service , North London Centre , UK -RRB- prepared as described above at 37 °C with shaking for 4 h -LRB- 1 volume of particles : 2 volumes plasma -RRB- . The CRLPs containing apoE were then isolated by ultracentrifugation at d = 1.006 × g/ml -LRB- 120,000 × g for 12 h at 4 °C -RRB- , harvested from the top layer , purified by a second centrifugation at the same density -LRB- 202,000 × g for 4 h at 4 °C -RRB- and stored at 4 °C under argon until required . All preparations were used within 1 week . For DiI-labelled CRLPs and pCRLPs , probucol -LRB- 1 mg -RRB- and/or DiI were added to the lipid mixture prior to sonication . CRLPs were oxidized by incubation with CuSO4 -LRB- 20 μM -RRB- with shaking for 5 h at 37 °C and the CuSO4 was then removed by dialysis -LRB- 0.9 % NaCl , 24 h , 4 °C -RRB- . The oxidation process had no effects on the fluorescent properties of the DiI label . 2.2 Culture of THP-1 cells THP-1 monocytes were maintained in RPMI 1650 culture medium containing 10 % -LRB- v/v -RRB- FBS , glutamax -LRB- 2 mM -RRB- , penicillin/streptomycin -LRB- 100 U/ml/100 μg/ml -RRB- and β-mercaptoethanol -LRB- 20 μM -RRB- -LRB- culture medium -RRB- . The cells were differentiated into macrophages by incubation with PMA -LRB- 200 ng/ml -RRB- for 72 h at 37 °C in 5 % CO2/95 % air . The medium containing PMA and any non-adherent cells were then removed and the macrophages were washed with PBS -LRB- 3 × 1 ml -RRB- and incubated with CRLPs or DiI-labelled CRLPs -LRB- 30 μg cholesterol/ml -RRB- in the presence or absence of specific inhibitors as indicated in the text . The inhibitors were added 1 h prior to the addition of the CRLPs . Cell viability as assessed by Trypan blue exclusion was > 95 % and was not affected by any of the CRLP types or conditions used . After the incubation , the macrophages were washed -LRB- culture medium 3 × 1 ml -RRB- and lipid accumulation was assessed by staining with Oil red O , or harvested for lipid analysis -LSB- 24 -RSB- . For studies with DiI-labelled CRLPs , the fluorescence associated with the cells was assessed by viewing with a Zeiss LMS 510 laser - scanning confocal microscope and quantified by absorbance volume analysis , or by fluorescence-activated cell analysis -LRB- FACS -RRB- using a BD FACS Calibur flow cytometer -LRB- BD Biosciences , Oxford , UK -RRB- . For FACS analysis , cells were harvested in PBS containing EDTA -LRB- 5 mM -RRB- and lidocaine - HCl -LRB- 8 mg/ml -RRB- , incubated for 20 min at 37 °C and centrifuged at 10,000 × g -LRB- 2 min -RRB- . The cell pellet was then resuspended and fixed in PBS containing 4 % formalin . 2.3 Analytical methods For mRNA analysis , total RNA was extracted from THP-1 macrophages before or after incubation with CRLPs -LRB- 30 μg cholesterol/ml -RRB- using a kit from Sigma -LRB- Poole , UK -RRB- and first strand synthesis was carried out using a kit supplied by Promega -LRB- Southampton , UK -RRB- according to the manufacturer 's instructions . mRNA levels for the LDLr , LRP and the housekeeping gene glyceraldehyde phosphate dehydrogenase -LRB- GAPDH -RRB- were determined by real time polymerase chain reaction using a SYBR green quantitative PCR kit -LRB- Sigma , Poole , UK -RRB- with an Opticon light cycler under the following conditions ; denaturation at 94 °C for 2 min followed by 40 cycles of 94 °C for 15 s , 58 °C for 1 min , with a final extension at 72 °C for 1 min . The primers used and the product sizes are shown in Table 1 . The Ct values were determined by automated threshold analysis using Opticon Monitor 2 software . Data were normalized with the values obtained for GAPDH and the fold change in mRNA expression in CRLP-treated as compared to untreated macrophages was determined by the method described by Pfaffl -LSB- 25 -RSB- . Lipids were extracted from cell samples by the addition of chloroform : methanol -LRB- 2:1 v : v , 20 volumes -RRB- followed by 0.88 % KCl -LRB- v : v -RRB- -LRB- 40 % total volume -RRB- . The mixture was shaken and the chloroform layer containing the lipids was collected and dried under nitrogen . The triacylglycerol -LRB- TG -RRB- content of CRLPs and lipid extracts was determined using a fully enzymatic assay kit -LRB- Alpha Laboratories , Eastleigh , Hants , UK -RRB- in which glycerol released from triacylglycerol by a lipase is measured using glycerol phosphate oxidase and peroxidase . The total cholesterol -LRB- cholesterol + cholesteryl ester -RRB- content of CRLPs and lipid extracts was determined using cholesterol oxidase and cholesteryl esterase -LRB- kit supplied by Sigma , Poole , UK -RRB- , and the cholesterol content of lipid extracts was determined enzymatically using cholesterol oxidase -LSB- 26 -RSB- . Cholesteryl ester concentrations were calculated by subtracting the values for cholesterol from those for total cholesterol . The extent of oxidation of CRLPs was determined by measuring the level of thiobarbituric acid-reacting substances -LRB- TBARS -RRB- in the preparations -LSB- 27 -RSB- . The apolipoprotein content of CRLPs was assessed by SDS-PAGE using 10 % gels -LSB- 28 -RSB- . Proteins were visualised by staining with Coomassie blue and the bands corresponding to apoE were quantified by optical density volume analysis . Significance limits were calculated using ANOVA repeated measures or one way ANOVA followed by Student 's paired t test for experiments with multiple time points or a single time point , respectively , except where indicated otherwise . 3 Results 3.1 Characteristics of CRLPs The lipid composition of CRLPs , oxCRLPs and pCRLPs is shown in Table 2 . The concentration of TG and TC in CRLPs and pCRLPs was similar , but that of oxCRLPs was a little lower , reflecting the dilution of the preparations during the oxidation procedure . The mean TG : TC ratio , however , was between 5 and 6 for all 3 CRLP types . Previous work in our laboratory has shown that the phospholipid content of CRLPs , oxCRLPs and pCRLPs prepared by the methods used here are not significantly different -LSB- 15,29 -RSB- . Analysis of the TBARS content of the particles showed that there were significant differences in their oxidative states , with values being higher in oxCRLPs as compared to CRLPs , while those for pCRLPs were lower -LRB- one way ANOVA , Bonferroni 's test post hoc -RRB- -LRB- Table 2 -RRB- . The apoE content of CRLPs , oxCRLPs and pCRLPs was evaluated by SDS PAGE electrophoresis . The results showed that the particles contained apoE , but no apoE was detected in the top fraction of the d 1.063 -- 1.21 g/ml fraction of human plasma incubated and centrifuged in the absence of lipid particles -LRB- Fig. 1A -RRB- , indicating that the apoprotein was bound to the CRLPs during the incubation . No apoCs or other apolipoproteins were detectable . There were no significant differences in the apoE content of the CRLPs , oxCRLPs or pCRLPs as assessed by optical density volume analysis -LRB- Fig. 1B -RRB- . 3.2 Effect of the oxidative state of CRLPs on the induction of lipid accumulation in macrophages Incubation of THP-1 macrophages with CRLPs or oxCRLPs for 5,24 or 48 h caused a marked increase in the total lipid found in the cells -LRB- Fig. 2A -RRB- at all time points , but lipid accumulation was greater with CRLPs as compared to oxCRLPs -LRB- P < 0.001 -RRB- . This effect was mainly due to greater accumulation of TG in the presence of CRLPs -LRB- P < 0.001 -RRB- -LRB- Fig. 2B -RRB- , as the trend for an increase in TC in CRLP - as compared to oxCRLP-treated macrophages did not reach significance -LRB- Fig. 2C -RRB- . 3.3 Effect of the oxidative state of CRLPs on their uptake by macrophages The uptake of DiI-labelled CRLPs , oxCRLPs and pCRLPs by THP-1 macrophages was assessed by confocal microscopy and FACS analysis -LRB- Fig. 3 -RRB- . Examination of cells incubated with DiI-labelled particles for periods up to 24 h with the confocal microscope showed that the fluorescence associated with the cells increased with time in all cases , but that there was clearly more in pCRLP - and less in oxCRLP-treated as compared to CRLP-treated cells -LRB- Fig. 3A -RRB- . Quantification of the cell-associated fluorescence and analysis by ANOVA repeated measures showed that the rate of uptake of CRLPs over 24 h was significantly higher than that of oxCRLPs -LRB- P < 0.01 -RRB- and significantly lower than that of pCRLPs -LRB- P < 0.01 -RRB- -LRB- Fig. 3B -RRB- , and a similar result was obtained using FACS -LRB- P < 0.05 , CRLPs vs. oxCRLPs or pCRLPs -LRB- Fig. 3C -RRB- . 3.4 Role of apoE in the uptake of CRLPs of different oxidative states To investigate whether apoE is necessary for uptake of CRLPs , THP-1 macrophages were incubated with DiI-labelled CRLPs or CRLPs without apoE for 2 h and the fluorescence associated with the cells was determined by FACS analysis . In the absence of apoE , the uptake of CRLPs was reduced by about 90 % -LRB- fluorescence values : CRLPs , 98.6 ± 7.1 ; CRLPs without apoE , 8.4 ± 0.9 , n = 3 -RRB- . The potential role of apoE dependent receptors in the uptake of CRLPs by the cells was studied using excess LDL and the LRP ligand , lactoferrin , to block entry via the LDLr and LRP , respectively . The effects of lactoferrin -LRB- 2 mg/ml -RRB- on lipid accumulation in macrophages exposed to CRLPs or oxCRLPs as assessed by Oil red O staining are shown in Fig. 4 . Lactoferrin had no effect on the lipid content of cells incubated in the absence of CRLPs , but reduced that in macrophages treated with either CRLPs or oxCRLPs -LRB- P < 0.05 , both cases -RRB- . When the effects of excess LDL -LRB- 200 μg cholesterol/ml -RRB- and lactoferrin -LRB- 2 mg/ml -RRB- , on the fluorescence associated with the macrophages after incubation for 1 , 4 , 16 and 24 h with CRLPs , oxCRLPs or pCRLPs were assessed by confocal microscopy -LRB- Fig. 5A -- C -RRB- , no significant inhibition was observed in the presence of excess LDL , but lactoferrin caused a decrease of > 90 % in experiments with CRLPs -LRB- Fig. 5A -RRB- and oxCRLPs -LRB- Fig. 5B -RRB- and > 80 % with pCRLPs -LRB- Fig. 5C -RRB- -LRB- P < 0.0001 , all cases -RRB- . Addition of LDL and lactoferrin together completely abolished detectable uptake of all three types of particles -LRB- Fig. 5A -- C -RRB- . FACS analysis of macrophages treated with CRLPs , oxCRLPs or pCRLPs in the presence or absence of lactoferrin -LRB- 2 mg/ml -RRB- , LDL -LRB- 300 μg cholesterol/ml -RRB- or LDL + lactoferrin for 2 h also showed that lactoferrin caused a marked decrease of about 75 % in the uptake of all three types of CRLPs -LRB- P < 0.01 -RRB- , and in this case significant inhibition -LRB- − 30 -- 40 % -RRB- was observed in the presence of LDL -LRB- P < 0.05 -RRB- -LRB- Fig. 5D -RRB- . Moreover , in a further separate experiment in which the excess of LDL added was increased by lowering the concentration of CRLPs to 10 μg cholesterol/ml and raising that of LDL to 500 μg cholesterol/ml , the uptake of both CRLPs and oxCRLPs was also significantly inhibited -LRB- fluorescence values -LRB- n = 3 -RRB- : CRLPs , 94.8 ± 6.2 ; CRLPs + LDL , 58.7 ± 5.8 -LRB- P < 0.05 -RRB- ; oxCRLPs 56.7 ± 1.5 ; oxCRLPs + LDL , 29.2 ± 7.3 -LRB- P < 0.05 -RRB- -RRB- . As observed in the experiments using confocal microscopy , adding both LDL and lactoferrin to the incubations caused a further decrease to similar minimal levels in the uptake of all three types of CRLPs . Assessment of the fluorescence associated with THP-1 macrophages after incubation with DiI-labelled acetylated LDL -LRB- acLDL -RRB- -LRB- 10 μg cholesterol/ml -RRB- for 2 h showed that acLDL uptake , in contrast to that of CRLPs , was not significantly inhibited by excess LDL -LRB- 500 μg cholesterol/ml -RRB- or lactoferrin -LRB- 2 mg/ml -RRB- -LRB- Fig. 6 -RRB- . 3.5 Role of scavenger receptors and phagocytosis in the uptake of CRLPs of different oxidative states The effects of blocking the class A scavenger receptor SR-A , the class B receptors CD36 and SR-B1 or phagocytosis on the uptake of CRLPs by THP-1 macrophages was investigated using poly-I , a known ligand for SR-A , a blocking antibody to CD36 , excess HDL , which binds to SR-B1 , and cytochalasin D , which blocks the polymerization of actin microfilaments -LSB- 30 -RSB- . Evaluation of the fluorescence associated with the cells after incubation with DiI-labelled CRLPs , oxCRLPs or pCRLPs for 1 , 4,16 or 24 h by confocal microscopy -LRB- Fig. 7A -- C -RRB- show no significant change in the presence of poly-I -LRB- 5 μg/ml -RRB- or excess HDL -LRB- 300 μg/ml -RRB- . Significant decreases , however , were observed in the presence of anti-CD36 -LRB- 1 μg/ml -RRB- -LRB- − 38 % after 24 h -RRB- -LRB- P < 0.05 -RRB- and cytochalasin D -LRB- 10 μg/ml -RRB- -LRB- − 27 % after 24 h -RRB- -LRB- P < 0.01 -RRB- in experiments with CRLPs -LRB- Fig. 7A -RRB- , although no change in the uptake of oxCRLPs or pCRLPs was detected with these treatments -LRB- Fig. 7B , C -RRB- . FACS determinations after incubation of macrophages with CRLPs , oxCRLPs or pCRLPs for 2 h confirmed that poly-I and HDL did not inhibit the uptake of the particles -LRB- Fig. 7D -RRB- . In contrast , however , the uptake of DiI-labelled acLDL was decreased by about 90 % in the presence of poly-I -LRB- 50 μg/ml -RRB- -LRB- Fig. 6 -RRB- . As found in the experiments using confocal microscopy , anti-CD36 and cytochalasin D significantly inhibited the uptake of CRLPs , but in this case effects of a similar magnitude were also seen with oxCRLPs and pCRLPs -LRB- After 24 h : anti-CD36 ; CRLPs , − 38 % , oxCRLPs − 40 % , pCRLPs , − 39 % : cytochalasin D ; CRLPs − 39 % , oxCRLPs − 35 % , pCRLPs − 38 % -RRB- , although the changes with oxCRLPs did not reach significance -LRB- Fig. 7D -RRB- . 3.6 Expression of mRNA for the LDLr and LRP in THP-1 macrophages In THP-1 macrophages exposed to CRLPs -LRB- 30 μg cholesterol/ml -RRB- for 24 h , LRP mRNA levels were increased in CRLP - treated THP-1 cells -LRB- fold change , 2.7 ± 1.01 , n = 3 -RRB- , while LDLr mRNA abundance was decreased -LRB- 0.27 ± 0.11 -LRB- range -RRB- , n = 2 -RRB- . 4 Discussion Since homogeneous CMR can not be obtained easily from human blood without contamination with other lipoproteins of a similar density such as chylomicrons and very low density lipoprotein -LRB- VLDL -RRB- , model CRLPs were used in this study . These particles are similar in size , density and lipid composition to physiological remnants -LSB- 23 -RSB- , and also contained human apoE , thus they differ from physiological CMR only in lacking apoB48 . Importantly , extensive previous studies in both humans and experimental animals have demonstrated that chylomicron - and chylomicron remnant-like particles without apoB48 are cleared from the blood and metabolised in a similar way to the corresponding physiological lipoproteins -LSB- 31 -- 34 -RSB- . In addition , CRLPs lacking apoB48 , but containing apoE from the appropriate species , have been found to have effects which mimic those of physiological remnants in rat hepatocytes and pig endothelial cells -LSB- 23,35 -RSB- , and our earlier work has shown that CRLPs cause extensive lipid accumulation in THP-1 macrophages and human monocyte derived macrophages -LRB- HMDM -RRB- which is comparable to that found in experiments with physiological CMR from rats and the murine macrophage cell line J774 -LSB- 24,36 -RSB- . For the current investigation , the use of CRLPs facilitated the manipulation of the oxidative state of the particles by inclusion of the lipophilic antioxidant probucol or by exposure to oxidising conditions similar to those used extensively in studies with oxLDL -LSB- 16 -RSB- . In previous work , we have shown that incorporation of probucol into CRLPs increases their uptake by THP-1 macrophages -LSB- 15 -RSB- , and that this is due to protection of the particles from oxidation , since lycopene , a chemically unrelated antioxidant , has a similar effect -LSB- 14 -RSB- . For the present study , therefore , we used pCRLPs in addition to CRLPs and oxCRLPs , so that 3 different oxidative states were tested . Assessment of TBARS in the particles used showed that the oxidative states of CRLPs , oxCRLPs or pCRLPs were significantly different , and there were no significant differences in their apoE content , thus they provided a convenient and suitable model for the study . Earlier work in our laboratory and others has demonstrated that CMR cause the extensive lipid accumulation associated with foam cell formation in HMDM , THP-1 macrophages and murine macrophage cell lines -LSB- 24,36,37 -RSB- . In experiments with THP-1 macrophages and HMDM , we found that CRLPs induced a greater increase in the intracellular total lipid content than oxLDL -LRB- at the equivalent cholesterol level -RRB- , and that , as might be expected , this was mainly due to greater accumulation of TG , while TC levels were raised to a comparable extent with both types of lipoprotein -LSB- 24 -RSB- . In the current study we compared the effects of oxidized and non-oxidized particles and , although both caused rises in cellular lipid content compared to untreated cells , the increase was clearly smaller with oxCRLPs as compared to CRLPs , mainly because of decreased accumulation of TG -LRB- Fig. 2 -RRB- . These results suggest that the uptake of CRLPs by the cells may be inhibited by oxidation , and this was confirmed in confocal microscopy and FACS studies -LRB- Fig. 3 -RRB- , which showed that the rate of uptake of CRLPs was decreased by oxidation and increased by incorporation of probucol into the particles to protect them from oxidation -LRB- Table 2 -RRB- . These results clearly demonstrate that oxidation of CMR inhibits their uptake by macrophages and attenuates foam cell formation . This is an important new finding , since this effect is strikingly different from that found with LDL , where oxidation is required to induce macrophages to form foam cells . CMR are known to be cleared from the circulation by the liver mainly by apoE-dependent pathways involving the LDLr and the LRP , and the present study shows that apoE also plays an important role in the entry of CRLPs into THP-1 macrophages , since a markedly reduced rate of uptake was observed when apoE was not present in the particles . These finding are in agreement with earlier work which has suggested that both the LDLr and the LRP are involved in the uptake of CMR by macrophages -LSB- 10,19,38 -RSB- . Our results indicate that both receptors are able to mediate the uptake of CRLPs regardless of their oxidative state , although lactoferrin , a ligand for the LRP , caused a marked reduction in lipid accumulation and uptake after exposure of THP-1 macrophages to all three types of CRLPs -LRB- Figs. 4 , 5 -RRB- , while the maximum effect of excess LDL on uptake observed was more modest in all cases -LRB- Fig. 5 -RRB- . This lesser role of the LDLr may be due to its down-regulation on the influx of lipoprotein into cells . We have shown previously that the expression of mRNA for the LDLr in THP-1 macrophages is decreased by CRLPs while the expression of LRP mRNA is increased -LSB- 18 -RSB- , and the present study , which shows a decrease of about 73 % in LDLr mRNA and a 2.7 fold rise in LRP mRNA after exposure of the macrophages to CRLPs , is in agreement with these findings . The differences in rates of uptake of oxCRLPs , CRLPs and pCRLPs were retained in the presence of inhibitors of both the LDLr and the LRP , although when both the LDLr and the LRP were blocked , uptake of oxCRLPs , CRLPs and pCRLPs was reduced to a similar low level -LRB- Fig. 5 -RRB- , suggesting that the difference in their rate of uptake is apoE-dependent . Thus , an important novel finding of this study is that oxidation of CRLPs , unlike LDL , does not change their major routes of uptake by macrophages , with apoE-dependent receptors being of major importance and the LRP playing the predominant role , regardless of the oxidative state of the particles . ApoE does not bind to the LDLr family in its lipid-free state , as interaction with lipid is necessary to induce a conformational change which promotes high affinity for the receptors -LSB- 39 -RSB- . In addition , apoE has been shown to adopt different conformations when complexed to different lipids . Thus , changes in the lipid composition of lipoproteins such as an increased content of oxidized lipids or the presence of lipophilic compounds such as probucol may alter the conformation of the protein on their surface , and not all apoE molecules on a particular remnant particle may be able to act as ligands -LSB- 39,40 -RSB- . The different rates of uptake of CRLPs oxCRLPs and pCRLPs by macrophages demonstrated here , therefore , could be explained by effects on interaction with the LDLr and the LRP caused by differences both in the conformation of apoE and in the number of apoE molecules able to bind to the receptors , even though the total amount of apoE associated with the particles is not changed . Although we can not rule out the possibility that the faster internalization of pCRLPs is due a specific effect of probucol on the surface structure of the particles , the finding that CRLPs are taken up more slowly after oxidation and more rapidly when the antioxidant is present suggests the changes are more likely to be related to the amounts of oxidized lipid in the particles . The expression of scavenger receptors such as SR-A and the class B receptors CD36 and SR-B1 is a characteristic feature of macrophages , and SR-A and CD36 are know to play a part in the induction of foam cell formation by oxidized or chemically modified LDL -LSB- 41 -RSB- . It is possible , therefore , that this type of receptor may also be involved the uptake of oxCMR by macrophages . In the present study , poly I , a ligand for SR-A , reduced the uptake of acLDL by THP-1 cells by about 90 % as expected , but had no significant effect on the uptake of CRLPs , irrespective of their oxidative state . In addition , excess HDL , which binds to SR-B1 , did not prevent the entry of CRLPs , oxCRLPs or pCRLPs into the cells . In contrast , in the presence of anti-CD36 , the uptake of all three CRLP types was inhibited by about 35 -- 40 % so that the differences in their rates of uptake were retained -LRB- Fig. 7 -RRB- . Thus , SR-A and SR-B1 do not appear to play a significant part in the uptake of oxCRLPs by macrophages , although CD36 may have a role which is unaffected by the oxidative state of the particles . Mamo et al. -LSB- 22 -RSB- have reported previously that the phagocytosis may be important in the uptake of CMR by rabbit alveolar macrophages , although in a later electron microscopy study with HMDM they found no evidence for entry via this route -LSB- 42 -RSB- . In our experiments with THP-1 macrophages , however , cytochalasin D inhibited the uptake of all CRLPs types tested by 35 -- 40 % , suggesting that some phagocytosis of CRLPs of all oxidative states does occur in these cells -LRB- Fig. 7 -RRB- . It has been suggested that the apoB48r may be involved in the uptake of chylomicron remnants by macrophages -LSB- 1,20 -RSB- , and Kawakami et al. -LSB- 43 -RSB- have reported that it is responsible for the induction of macrophage foam cell formation by remnant lipoproteins from hyperlipidemic patients . Since apoB48 is an integral protein incorporated during the assembly of chylomicrons in intestinal cells , it is not possible to bind it to model CRLPs in a physiological way . The particles used in the present work , therefore , do not contain apoB48 and we were unable to study involvement of this receptor in the uptake of CMR by macrophages . However , antibodies to apoB48 have been found not to inhibit the uptake of chylomicron remnants by rat macrophages -LSB- 44 -RSB- , and Elsegood et al. -LSB- 21 -RSB- who were unable to detect binding of chylomicron remnants to a protein with a molecular weight corresponding to the apoB48 receptor in THP-1 macrophages have suggested that it may be specific for VLDL remnants rather than chylomicron remnants . The results of this study demonstrate that oxidative modification of CMR as compared to LDL has profoundly different effects on the uptake of the particles and the subsequent induction of lipid accumulation in macrophages . Instead of markedly enhancing foam cell formation , oxidation of CMR slows their uptake by macrophages and reduces the amount of lipid subsequently accumulated in the cells . This difference may be due to the different receptor mechanisms involved , since oxidation of LDL shifts the main route of uptake from the regulated LDLr to the unregulated scavenger receptors , while our experiments suggest that CMR are taken up mainly by the LRP with some contribution from the LDLr , with CD36 and phagocytosis playing only minor roles , irrespective of their oxidative state . These findings provide important new information about the way in which oxidation of CMR influences their induction of foam cell formation and the mechanisms involved , and has important implications for the role of dietary factors such as oxidized lipids and antioxidants which are transported in CMR in the promotion of atherosclerosis .

O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O

chylomicron remnants
foam cells
macrophages
atherosclerosis