Osteoarthritis (OA) is the most common form of arthritis and has multiple risk factors including joint injury. The purpose of this study was to characterize the histologic development of OA in a mouse model where OA is induced by destabilization of the medial meniscus (DMM model) and to identify genes regulated during different stages of the disease, using RNA isolated from the joint “organ” and analyzed using microarrays. Histologic changes seen in OA, including articular cartilage lesions and osteophytes, were present in the medial tibial plateaus of the DMM knees beginning at the earliest (2 week) time point and became progressively more severe by 16 weeks. 427 probe sets (371 genes) from the microarrays passed consistency and significance filters. There was an initial up-regulation at 2 and 4 weeks of genes involved in morphogenesis, differentiation, and development, including growth factor and matrix genes, as well as transcription factors including Atf2, Creb3l1, and Erg. Most genes were off or down-regulated at 8 weeks with the most highly down-regulated genes involved in cell division and the cytoskeleton. Gene expression increased at 16 weeks, in particular extracellular matrix genes including Prelp, Col3a1 and fibromodulin. Immunostaining revealed the presence of these three proteins in cartilage and soft tissues including ligaments as well as in the fibrocartilage covering osteophytes. The results support a phasic development of OA with early matrix remodeling and transcriptional activity followed by a more quiescent period that is not maintained. This implies that the response to an OA intervention will depend on the timing of the intervention. The quiescent period at 8 weeks may be due to the maturation of the osteophytes which are thought to temporarily stabilize the joint. Osteoarthritis (OA) affects over 27 million people in the United States and is similarly prevalent across the globe, making it the most common cause of chronic disability in adults Studies of human OA are limited by its multifactorial nature, the lack of tissue that can be obtained at various stages of the disease and difficulties in defining disease onset. Basic mechanistic studies often are performed with tissue obtained at the time of joint replacement, which represents end-stage disease. These studies have focused largely on changes present in the articular cartilage; however, OA is a condition that affects the joint as an organ including not only the cartilage but also subchondral bone, synovium, ligaments and, in the knee, the meniscus  model where OA is surgically induced by transection of the medial meniscotibial ligament. In this model, OA results from altered joint biomechanics and the pathologic changes of cartilage destruction, subchondral bone thickening, and osteophyte formation are similar to the changes seen in human OA The present study was designed as a time-course experiment to evaluate changes in gene expression during the development of OA and to compare these changes to the histologic progression of the disease. The recently reported comparison of gene expression at the 8 week time point in young adult and older adult mice Male C57BL/6 mice (n = 129) were purchased from Charles River Inc. and were 12 weeks of age at the time of DMM surgery or sham surgery performed as a control. Animal use was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Wake Forest School of Medicine Animal Care and Use Committee (protocol #A09-622). All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering. Butorphanol (2.5mg/kg) was administered in the event of pain.Details of animal housing and induction of OA have been recently published Hind limbs from both the operated side and the contralateral side of each animal were dissected and processed for histological analysis. The details of fixation, processing, and sectioning, as well as the histological analysis, were as recently published Immunohistochemistry was performed on sections from 3 DMM and 4 sham control mice at the 16-week time point in order to examine tissue distribution of type III collagen, fibromodulin and proline-arginine-rich end leucine-rich repeat protein (Prelp). Antigen retrieval and blocking steps were as recently described Knee joint tissue from the medial joint compartment was obtained from the time 0 group and the operated hind limbs from the other time points and processed for RNA isolation as described RNA was pooled prior to microarray analysis such that 3 randomly selected samples from each surgical group and time point were pooled to create a each biological replicate. Because 9 mice were used for each experimental group, a total of three biological replicates per group were analyzed using the Affymetrix Mouse Genome 430 2.0 oligonucleotide arrays as described 2 of the signal intensity and the Affymetrix detection p-value for each probe set. Because some genes had more than one probe set, we provide results for both numbers of genes and probe sets when the numbers differed. The complete dataset has been provided to the Gene Expression Omnibus (GEO) repository (accession number GSE41342). Relative gene expression in the samples collected from the sham-operated control animals was calculated to identify those genes that changed as the animals aged over the 16 week time course independent of DMM-induced OA. For this analysis, sham gene expression was calculated as the signal log ratio (SLR) of the average time 0 intensity (baseline un-operated controls) to each sham replicate pool for each time point. The average of the time 0 intensity was used to ensure a consistent baseline, and each biological replicate of the sham arrays was analyzed individually to allow biological consistency to be measured in the analysis. The SLR is the log2 of the fold change and so an SLR of 0.5 =  1.4-fold. After SLR calculations, each probe set had 12 associated SLR values , which together are referred to as the “sham time course”.Microarrays were imaged and the resulting data normalized using systematic variation normalization (SVN) as previously described Relative gene expression changes for the DMM time course were calculated to identify those genes that changed over time due to the DMM-induced changes in the joints. For this analysis, the sham results were used as a time-matched control (sham 2 weeks used as control for DMM 2 weeks etc.). Sham replicate signal intensities for each time point were averaged to ensure a consistent baseline for each replicate. DMM gene expression was then calculated as the time-matched signal log ratio of the average sham intensity to each DMM replicate for each time point, where each DMM replicate was analyzed individually. After SLR calculations, each probe set had 11 associated SLR values , which are referred to as the “DMM time course”.Relative gene expression for the sham time course and DMM time course was filtered for differentially expressed genes with consistent expression changes over time as previously described 2ATmd 2ATmd’s functionality since its original release. This was implemented by converting the consensus matrix to an adjacency matrix and then using MATLAB’s depth first search algorithm to identify consensus clusters. A figure of merit (FOM) analysis was run to determine the optimal number of clusters present in each replicate data set and to identify which clustering algorithm formed the most homogeneous clusters with respect to Euclidean distance Filtered DMM time course gene expression data were clustered using the updated consensus clustering option provided by version 3 of SCAnnotation enrichment analysis was performed using the Database for Annotation, Visualization, Integration and Discovery (DAVID) Real-time PCR was performed using samples of RNA from the same pools used for the microarrays as previously described Analysis of the microarray data is presented above. The histological data and the real time PCR data were found to be normally distributed. Histological data from both the lateral and medial tibial plateaus was evaluated using ANOVAs  and repeated measures analyses  using SPSS version 17.0 . The real time PCR results at each individual time point for sham controls and DMM animals were analyzed using t tests with StatView 5.0 software .The sham operated joints and the contralateral control  demonstrated minimal to no pathological changes over the 16 week time course. Among the earliest changes observed in the DMM joints at 2 weeks after surgery were abaxial osteophytes in the medial tibial plateau. Osteophytes were primarily cartilaginous at 2 weeks, were ossified at 4 weeks, and were composed of trabecular bone that contained marrow spaces occupied by hematopoietic tissue at 8 and 16 weeks  significantly and consistently regulated over time in the sham joints. A complete list of these genes is provided in A small subset of 25 genes remained up-regulated over the time course . FunctioThe time course results for changes in expression in the DMM joints relative to time-matched sham joints revealed 371 genes (427 probe sets) that were significantly and consistently regulated over the three biological replicates . ExaminaThese results were much different from the time course results noted in the sham joints, consistent with changes in gene expression due to induction of OA by the DMM surgery. An overlap analysis revealed 94 genes (97 probe sets) that were in common in the DMM/sham and sham/baseline control datasets . Many ofThe 94 genes in common between DMM/sham and sham/baseline were analyzed by DAVID. This returned 10 significant annotation groups  that incConsensus clustering of genes with altered expression after DMM surgery identified 27 clusters with 2 or more probe sets and 38 genes (39 probe sets) that were classified as outliers (singletons). The complete list of the genes found in each cluster and the DAVID analysis for the clusters are found in Only clusters 5, 7, 10 and 12 exhibited significant down-regulation at specific time points. Cluster 5 contained genes down-regulated at week 4; cluster 10, genes down-regulated at weeks 4 and 8; and clusters 7 and 12, genes down-regulated at week 8. Cluster 5 genes were not associated with any significantly over-represented annotations, but this cluster of 27 genes (32 probe sets) included Cytl1 (cytokine-like 1), which has been shown to be expressed during chondrogenesis and to promote chondrocyte differentiation through stimulation of Sox9 transcriptional activity Significant up-regulation of gene expression at 16 weeks was seen in clusters 15 and 16, both of which also exhibited high up-regulation at week 4. Cluster 15 consisted of 7 probe sets covering three genes; Prelp, Col3a1 , and Fmod (fibromodulin). Prelp and fibromodulin were also present in the DMM/sham-Sham/baseline overlap gene list reported above, where they were found to be highly down-regulated at 16 weeks in the sham joints; however, these genes were highly up-regulated in the DMM joints. Cluster 16 was comprised of four probe sets, representing 3 genes: Col14a1 , Bgn (biglycan), and Fbln7 (fibulin 7), all associated with the extracellular matrix. Col14a1 was identified in a microarray analysis of human tissue Genes in clusters 3 and 11 displayed temporal profiles of up-regulation only at 4 weeks, with little significant change in expression from control at any other time point. Cluster 3 contained 45 genes (48 probe sets), with significant annotations that included regulation of transcription, DNA binding, regulation of RNA metabolic process, zinc ion binding, and transcription. Potential genes of interest were NFκB activating protein, Atf2 and Erg. Cluster 11 was composed of 11 genes, which exhibited no significantly over-represented functions. One of the genes in this cluster was Asb13, an ankyrin repeat and SOCS box-containing protein.Clusters 1, 2, 4, 9, and 14 contained genes that exhibit strong up-regulation at 4 weeks, with less up-regulation at 2 weeks. Cluster 1 contained 48 genes (54 probe sets) representing genes whose significant annotations include embryonic organ morphogenesis, positive regulation of cell differentiation and developmental process. This cluster included several genes thought to be involved in joint biology and/or OA including syndecan 4, Bmp-1, and Timp-2. Cluster 2 contained 39 genes (52 probe sets) for genes also up-regulated at 2 and 4 weeks. The cluster included over-represented annotations for collagen, signal peptide, extracellular region, extracellular matrix, cell adhesion and glycosaminoglycan binding. Genes of potential interest in this cluster included collagen genes such as Col5a1 and Col16a1, growth factor-related genes such as Igf1 and Egfr, MMP-14, and biglycan. . Clusters 4, 9, and 14 contained 33, 16 and 8 genes , respectively. DAVID did not identify any function annotations over-represented any of these clusters. Cluster 4 genes included two glutathione peroxidases, Gpx7 and Gpx8, perhaps suggesting some redox-related event.Clusters 6, 8, and 13 also displayed the most up-regulation at week 4, but to a lesser extent than clusters 1, 2, 4, 9, and 14. Cluster 6 included 23 genes (24 probe sets) whose DAVID-identified over-represented annotations included signal peptide, glycoprotein, cell adhesion, serine/threonine kinase signaling pathway, skeletal system development and growth factor activity. Genes of particular interest included TGFβ2 and TGFβ3, latent TGFβ binding protein and Ddr2. Cluster 8 contained 19 genes for which the significant gene annotations included cell adhesion and contained COMP and Mmp13 which are thought to be important markers of the OA process. Cluster 13 included 9 genes for which there were no significantly over-represented functions. One of these, Enpp3, a pyrophosphatase, has been previously associated with osteoarthritis Overall, the analysis of gene clusters based on the metrics of both shape and magnitude of the time profile provided insight into the temporal process of osteoarthritis development. Only a few genes were down-regulated as part of this process, and those were down-regulated mainly at 4 and/or 8 weeks. Many more genes were up-regulated and most of the up-regulated genes displayed up-regulation at 4 weeks, with lesser up-regulation at 2 and 16 weeks. The phasic process was clearly visualized in these clusters, with the gene expression at 8 weeks being significantly different from gene expression at the other time points. This difference was consistent across all replicates.Based on the results of the consensus clustering, we choose to immunostain for type III collagen, fibromodulin, and Prelp, in order to determine which tissues might be providing the signals noted by gene expression at week 16. A total of 3 DMM and 4 sham control mice were studied and representative images are shown in We selected a set of 16 genes to evaluate using RT-PCR based on genes tested in our previous study Both the histologic and gene expression analysis support a phasic process for the development of OA in this model. The early phases at 2 and 4 weeks after DMM surgery were the most active in terms of gene expression at a time when the earliest cartilage lesions in the medial tibial plateau were very mild but abaxial chondrophytes had already formed. The chondrophytes matured to osteophytes as the articular cartilage lesions became more severe but, interestingly, this was accompanied by a significant decline in overall gene expression at the 8 week time point. In addition to the differences in overall gene expression, the findings from cluster analysis showed that each time point studied had a unique gene signature that also supports a phasic process.Perhaps most striking was the decline in gene expression at 8 weeks after surgery. There was a significant down-regulation of genes regulating cell proliferation while matrix remodeling genes that were up-regulated at 2 and 4 weeks were mostly off, suggesting that a quiescent phase had been reached. However, at 16 weeks this pattern had changed and was accompanied by significant cartilage loss in the medial tibial plateau while cartilage damage and chondrocyte death was beginning to be evident in the lateral tibial plateau along with the appearance of lateral axial osteophytes. Because RNA for the gene arrays was only isolated from the medial side of the joint, the changes in gene expression observed at 16 weeks were not due to the disease progressing laterally but rather reflect more advanced disease medially. The lack of time points between 4 and 8 weeks and 8 and 16 weeks prevents any conclusions about the length of the quiescent phase.The significant expression of Prelp, Col3a1, and fibromodulin at 16 weeks suggests that a matrix repair or a “wound healing response” was active. At 16 weeks, the articular cartilage from the DMM group had severe lesions in the medial tibial plateau with some loss of cartilage. This suggested that the signal at 16 weeks for these genes may have come from either early changes on the femoral side, since some femoral tissue was included for RNA extraction, or more likely, from other joint structures such as the meniscus, synovium, ligaments and subchondral bone. Immunostaining revealed that ligaments and the fibrocartilage found over osteophytes were common locations for all three proteins. These results are consistent with previous studies that examined the location and function of these proteins. Prelp is thought to play a role in matrix organization through interactions with collagen, as does the small leucine-rich proteoglycan fibromodulin Increased expression of type III collagen has been previously noted in human OA cartilage The phasic changes in gene expression noted in the DMM knees were not the result of aging of the animals over the 16 week time course. Changes in gene expression were noted when we compared the 12 week old animals at baseline (un-operated) and the sham controls over the 16 week time course but these differed significantly from the changes in gene expression noted in the DMM relative to sham joints. This was seen in the overlap analysis where the changes over time in expression in the DMM joints were most often in the opposite direction to changes over time in the sham joints. This was most evident for Prelp and fibromodulin which were significantly down-regulated at week 16 in the sham controls but increased in the DMM joints. Interestingly, immunopositivity for both of these proteins was notably stronger and more extensive in fibrocartilage over osteophytes than in articular or meniscal cartilage. In addition, immunopositivity for Prelp was increased in degenerative cartilage matrix compared with normal cartilage. In the sham time course analysis, most genes exhibited an up-regulation of expression at 2 weeks followed by a progressive decline out to 16 weeks, compared to time zero. Many of these genes were involved in morphogenesis and development, suggesting the animals were more actively growing at the start of the study and then growth likely slowed. Importantly, the progressive increase in subchondral bone area and thickness were the only histologic measures that changed with time in all groups .We have recently reported a comparison of the 8 week time point results included in the present study with results from the same time point in animals that were 12 months-old at the time of DMM surgery A phasic progression of OA was suggested by a previous study in humans where radiographic progression was related to changes in serial measures of serum COMP, as an OA biomarker There are important limitations to the present study. Because RNA was not isolated from individual tissues it is not possible to determine which specific tissue  contributed to the changes in gene expression. Also, if a gene is expressed in a single tissue, pooling of tissues will reduce the signal to a lower level than what would have been observed in the individual tissue. That is the likely explanation for the overall lower levels of gene expression observed in the present study when compared to work using a single tissue. Based on the immunostaining for type III collagen, fibromodulin, and Prelp, as well as immunostaining for IL-33, periostin, and CCL21 reported in our previous study Further studies will be needed to determine the importance of these findings for human OA. An important implication of the results is that the degree of response to an intervention given during a phasic process will depend on the timing of the intervention. Pre-clinical studies using animal models most often start the intervention at the same time or just after the start of the OA process while in human trials participants are likely to be at various stages of the disease process when the intervention is initiated. Finding markers of various disease stages in OA could be used to direct targeted therapy to the proper phase of the disease when the target is most active.Table S1Filtering results for the sham time course.(DOCX)Click here for additional data file.Table S2Filtering results for the DMM time course.(DOCX)Click here for additional data file.File S1Excel file of 406 Sham filtered probe sets and DAVID results. Results are presented as signal log ratio (SLR) which is the log2 of the fold change and so an SLR of 0.5 =  1.4-fold. A positive number would be up-regulation and negative number down-regulation.(XLSX)Click here for additional data file.File S2Excel file of 427 DMM filtered probe sets and DAVID results. Results are presented as signal log ratio as described for supplemental file 1.(XLSX)Click here for additional data file.File S3Excel file of 97 common probe sets and DAVID results.(XLSX)Click here for additional data file.


This method, which doesn’t require special hardware modifications, can be used to overcome partial volume effect, the major limitation of MR-based approaches that are currently being investigated for the detection of BAT in humans. With this method we can exploit the characteristic cellular structure of BAT to selectively image it, even when (as in humans) it is intimately mixed with other tissues. We demonstrate and validate this method in mice using PET scans and histology. We compare this methodology with conventional 1H MR fat fraction methods. Finally, we investigate its feasibility for the detection of BAT in humans.The recent discovery of active Brown Adipose Tissue (BAT) in adult humans has opened new avenues for obesity research and treatment, as reduced BAT activity seem to be implicated in human energy imbalance, diabetes, and hypertension. However, clinical applications are currently limited by the lack of non-invasive tools for measuring mass and function of this tissue in humans. Here we present a new magnetic resonance imaging method based on the normally invisible intermolecular multiple-quantum coherence  Obesity is rapidly spreading across most developed countries and is thought to be more harmful to health than alcohol or smoking because of its association with many other medical conditions 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) and computed tomography (CT) scans have identified active BAT in adults and shown a strong correlation between BAT activity and the basal metabolic rate While in small animals malfunction of this tissue is known to cause obesity, in humans the role and the incidence of tissue is less clear. In fact until recently, BAT was thought to exist in humans only in infancy and early childhood.18F-FDG-PET is highly nonspecific. Moreover, confounding factors such as blood glucose levels and room temperature conditions Although this tissue is a clear target for obesity treatments, the current modality of choice for imaging metabolically active BAT in humans, PET/CT, presents significant limitations. BAT metabolism relies on fatty acid consumption, not glucose consumption, so 1H MR can be used to differentiate these two tissues, at least in rodents MRI is very attractive for BAT studies since it is non-invasive, does not deliver mutagenic radiation, and has no limitations in imaging penetration depth. More interestingly, the difference in chemical shift between water protons and fat protons makes it possible to differentiate lean from fatty tissues as well as normal white fat (WAT) from brown fat. While brown fat is characterized by multilocular brown adipocytes with average water content of about 50%, white fat is characterized by unilocular adipocytes with water content of less than 10%. As a consequence, fat fraction measurements performed with In this paper we show that we can overcome these weaknesses of conventional MRI by using the non-linear MR signal generated by intermolecular zero-quantum coherences (iZQCs) that originates from closely spaced water and fat spins 18FDG-PET scanning, that this signal does indeed originate from brown adipose tissue and can therefore be used to specifically detect and map this tissue in rodents. Finally, we investigate the feasibility to use this signal, which we call BATSCI (BAT-Specific Coherence Imaging), to detect human BAT, using both in vitro and in vivo experiments.Building upon our previous spectroscopy studies in rodents This sequence is used to collect 2D iZQC spectra as well as to map the BATSCI signal in both animals and humans. BATSCI maps are collected by a CSI type acquisition scheme. Briefly, a series of 2D iZQC images are acquired with a different iZQC evolution times, t1. A Fourier transform along the t1 dimension allows us to display the iZQC spectrum from which a BATSCI map can be obtained.3) used in this experiment, which is still higher than the resolution typically used for human brown fat MRI studies (∼1 mm3), the BAT and the WAT/muscle mixture look exactly the same since they have a similar water/fat faction (2.B). On the other hand, the water-fat iZQC signal for these samples is very different (2.C). The water-fat iZQC signal was present only in the BAT sample, while it was close to the noise level in the WAT-muscle mixture, despite the similar water-fat content of these two samples and the much lower resolution used for this imaging experiment (8 mm3). This is because the intensity of the water-fat iZQC signal depends not only on the relative concentration of water and fat spins, but also on their relative distribution over a distance smaller or equal to the selected correlation distance. The correlation distance for these experiments was selected to be ∼80 µm, much smaller than the nominal image resolution, but big enough to allow the detection of BAT of different sizes. At this scale, water and fat spins are still mixed together in the BAT sample , while in the WAT/muscle mixture they are not since they are part of different tissues.In µm. As expected, lower values lead to a strong diffusion weighting and to a reduction of the BATSCI signal intensity, while higher values, though seemingly resulting in a slightly higher BATSCI signal, lead to a contamination from other coherences that may not be completely dephased by the correlation gradients.The dependence of the BATSCI signal intensity on the correlation distance is shown in In vivo imaging studies on several mouse strains  also show the possibility to map BAT in vivo in the mouse body. In In 18F-FDG-PET scans are presented. As expected from previous studies 18F-FDG uptake in BAT, in the Harderian glands and in the myocardium. 18F-FDG uptake in BAT is lower than in the myocardium and higher than in brain (below the image threshold). The BATSCI map reveals a strong signal only from areas corresponding to BAT: the interscapular, the axillary, and the cervical area, further confirming the suitability of this method to specifically detect BAT.In In The ability of BATSCI to detect and distinguish active and inactive BAT mass is an important advantage as compared to PET. We demonstrate this with a series of BATSCI experiments before, during, and post BAT stimulation by norepinephrine (NE), a catecholamine with predominant alpha-1 and some beta-1 adrenergic stimulation.2* weighted signal decreases by 22%, the BATSCI signal decreases by almost 25%, as the result of broadening of the BATSCI resonance line due to a microscopic increase in magnetic susceptibility. This reduction lasts during the entire BAT activity followed by a slow signal recovery as the efficacy of norepinephrine on BAT starts to diminish. On the other hand no appreciable signal variation was noticed in the nearby muscle and white fat, consistently with what we have previously observed Injection of NE has largely been used in rodents to stimulate BAT thermogenesis We have also used this method to examine the presence of BAT in fatty tissues excised from 11 human autopsy cases of both sexes, aged from 28 to 95 years old, using the small animal imaging scanner. For each subject, fatty tissues were excised from both the abdominal and the supraclavicular areas. All samples were then analyzed with conventional NMR spectroscopy methods, to evaluate their overall fat fraction, and with BATSCI, to detect their overall BAT content. To determine the % of adipose tissue containing brown fat cells in the tissues analyzed we then used immunohistochemical staining for UCP1. UCP1 is a marker gene for brown adipose tissue. It plays a crucial role for BAT thermogenesis since it uncouples ATP synthesis from electron transport so that energy is dissipated to produce heat. The presence of this protein is therefore essential for the identification of a fat depot as BAT depot 1H-MRS spectra , between the two supraclavicular samples (40±5% and 50±5% respectively), the relative ratio between the BATSCI signal and the nearby fat-fat signal . For these studies we enrolled 12 healthy volunteers with a BMI ranging from 19 to 28. For each subject we measured the intensity of the water-fat iZQC signal normalized to the intensity of the nearby fat-fat iZQC signal in a 20 cm slice covering the entire cervical–supraclavicular area and, in two cases, the abdominal area. The feasibility to use BATSCI to detect BAT in humans All human in vivo studies were approved by the Institutional Review Board of the University of North Carolina at Chapel Hill (IRB # 12–1229) and the University of Minnesota. All subjects gave written informed consent to the study.All animals studies were performed with approval of the Duke University Institutional Animal Care and Use Committee .http://plastic.surgery.duke.edu/education-and-training/advanced-training/human-fresh-tissue-laboratory) and were exempt from IRB approval.Human tissue samples from cadavers were kindly donated by the Duke Human Fresh Tissue Laboratory .For the in vivo imaging studies we used 14 C57 3-month old male mice, 4 nude mice, and 4 balb/c and 3 Cav1-mutated female mice at 12 weeks of age (Jackson Labs). Before the MR study, the animals were first induced with Nembutal (70 mg/kg) in a single intra-peritoneal injection. A catheter was then inserted in the intraperitoneal cavity to administer a maintenance dose of anesthesia (20 mg/Kg every 45 minutes or as needed). The animals were then placed in a supine position inside a birdcage 1H coil with a 35 mm inner diameter. Bore temperature was then monitored using MR-compatible temperature probes. At the end of the study, animals were euthanized through an overdose of pentobarbital. At the time of dissection the BAT areas were readily recognizable by gross inspection in the interscapular, axillary, and cervical portions of the anterior subcutaneous depots. They were dissected and fixed in formaline for histophatology evaluation.For the in vitro MR studies were performed on a 7T small animal magnetic resonance tomograph with a 210 mm inner bore diameter, interfaced to a Bruker Biospec console . The system is equipped with a gradient coil system (maximum gradient strength 42 Gauss/cm) and a 1H transmitter-receive coil with a 35 mm inner diameter.All animals and in vivo imaging studies, coronal respiratory-gated spin echo images  were acquired.For the The BATSCI maps were acquired with a modified version of the 2D iZQC spectroscopic sequence outlined in Fat fraction measurements were performed using frequency selective water and fat spin echo sequences  preceded by a saturation module . More specifically, water images were acquired using a fat saturation suppression module followed by a water selective spin echo sequence. Similarly, fat images were acquired by using a water suppression saturation module followed by a fat selective spin echo sequence. Fat fraction images were then obtained using homemade Matlab code that performed a pixel by pixel calculation of fat fraction (SIfat/(SIfat+ SIwater)), where SIfat represents the fat image signal intensity and SIwater represents the water image signal intensity.in vivo detection of BAT activity we used 6 C57 (Charles River) male mice. For these experiments we intubated and mechanically ventilated the animals at 60 breaths/min with a tidal volume of 0.3–0.4 ml and 21% O2, using an MR compatible ventilator. Two catheters were inserted in the intraperitoneal cavity, one to administer a maintenance dose of anesthesia  and the other to administer norepinephrine  (2.5 mg/kg) dissolved in 2 mM ascorbic acid solution in a single injection.For the We detected BAT activity by collecting a series of 11 BATSCI images with very low spatial resolution (3.745 mm×3.745 mm×20 mm), every 15–30 minutes before, during and after stimulation of BAT. The acquisition of the BATSCI images was interleaved with the acquisition of high resolution T2* weighted images  with a very short echo time (TE = 6 ms) to preserve signal intensity in BAT. We then evaluated the intensity of the BATSCI signal and the signal intensity of the T2* weighted images coming from interscapular brown fat region as function of time.For the BAT temperature studies we used 6 C57 male mice and 6 balb/c (Charles River) female mice. After inducing anesthesia, we continuously monitored but did not maintain room, rectal, and interscapular BAT temperatures by the minute using fiber-optic temperature probes. For the placement of the interscapular BAT temperature probe, we made a surgical incision above the iBAT pad. The temperature probe was then slid underneath the iBAT pad and secured with 3 skin sutures and surgical tape. The rectal probe was inserted 1 cm beyond the anus and secured to the tail with surgical tape. Room temperature was measured with a temperature probe taped on the surgical pad next to the animal.18F-FDG-PET experiments were performed on 2 C57 male mice within 32 hours from the MR study, using a dedicated Siemens micro-PET scanner for small animals with a reconstructed image resolution of 2 mm. For the 18F-FDG-PET scans, stimulation of BAT was induced by acute cold exposure , and by leaving the animal in a cold bag of ice during radiotracer uptake. The animals were then transported to the imaging room for the injection of the radiotracer, which was given to the non-anesthetized animal in a single intra peritoneal injection of 18F-FDG (200 µCi in 0.2 ml). To prevent inhibition of BAT metabolic activity during 18F-FDG uptake the animals were kept conscious in their cages on a cold bag of ice and at a room temperature of 18°C. Before the scans the animals were anesthetized with a single I.P. injection of ketamine, which is known to stimulate the symphatetic nervous system 1H MRS methods. At the end of the MR study, the tissues were fixed in 4% paraformaldehyde for 24 hours at 4°C, embedded in paraffin, and sectioned in 4 µm slices. The sections were de-waxed and rehydrated in a series of graded alcohols. Heat-induced epitope retrieval was performed in citrate buffer (pH 6.0) in a steamer for 20 minutes. The blocking procedure was carried out using Cyto-Q Background Buster  for 30 minutes followed by UCP1 primary antibody incubation for 1 hour at room temperature . Secondary antibody incubation and detection was performed for 30 minutes with biotinylated goat anti-rabbit  and Vectrstain Elite ABC complex respectively . The slides were then incubated in diaminobenzidine and counterstained in hematoxylin. Brown adipose tissue from a mouse was used as a positive control. Normal rabbit serum was used as a negative control.Human tissue samples were collected from 11 cadavers  of age ranging between 28 and 95 years old. The brown adipose tissue was excised from the supraclavicular region, lateral to the sternocleidomastoid muscles and the edge of the posterior scalenus. The white adipose tissue was harvested from the subcutaneous region of the abdomen. Specimens were placed in a vial and analyzed with the BATSCI method as well as with conventional in vitro human tissue data to identify correlation between UCP1 expression and BATSCI signal intensity.Sections were evaluated for abundance of UCP1-positive adipocytes and for UCP1 stain intensity by two pathologists, blinded to tissue group identity and MR results. Tissue scoring scale for both the adipose cells expressing the antibody and the intensity of the stain was: none/minimal = 1, mild = 2, moderate = 3, marked = 4. Simple linear regression analysis was performed on the In vivo human studies were performed on two 3-T horizontal bore magnet (Tim-Trio Siemens). Both systems were equipped with a body coil for signal transmission and a spinal array coil for signal reception. The spinal array coil was located such that the entire neck-supraclavicular area was covered by the receiver. No respiratory gating was applied to the sequence. Before the iZQC experiment a localization experiment was performed to position the area of interest at the iso-center. After the localization experiment a B1 mapping was used to accurately calibrate the RF pulses in the area of interest. For these experiments both 2D-iZQC spectra and 3D-iZQC data sets for iZQC water-fat signal mapping were acquired. For these experiments the spectroscopy sequence was slightly modified to reduce contaminations coming from imperfect spin excitation. Specifically, all excitation pulses were substituted with adiabatic bir4-pulses (5.12 ms duration). For the acquisition of the 2D spectrum the t1 delay (8.62 ms) was then stepped 32 times in increments of 900 microseconds. Each scan was acquired with a repetition time of 3.2 s a number of averages of 8, resulting in a total scan time of about 15 minutes. For the acquisition of the BATSCI maps a total of 16 images  were acquired with different values of t1 for a total scan time of 50 minutes.This study describes and evaluates the use of a novel, noninvasive MRI method named BATSCI to image BAT mass and function in both mice and humans. The main hypothesis is that BATSCI signal originates exclusively from BAT and can therefore be used to map this tissue in the body. This is because BATSCI signal comes primarily from correlations between water and fat spins separated by a user-controlled distance, the “correlation distance”. By choosing a correlation distance less than 100 microns, we can probe the relative concentration of water and fat spins at a this “cellular level”, a scale which goes far beyond that commonly accessible by standard magnetic imaging methods. This eliminates confounding partial volume effects that make conventional fat fraction MR measurements, currently under investigation for the detection of BAT in humans, less specific.in vivo imaging experiments performed here, along with the histological and 18F-FDG-PET evaluations, allow us to conclude that the BATSCI signal originates, at least in rodents, from BAT. Therefore, in these animals, BATSCI can be used to identify and map BAT. Our animal experiments clearly show that BATSCI identifies the major BAT locations . In addition, BATSCI maps are very similar to the BAT maps obtained by 18F-FDG-PET scans, and correlate well with BAT maps obtained with conventional fat-fraction MR measurement methods, despite the very different image resolution used for these scans.This study builds upon our previous spectroscopy studies on BAT in vivo mice studies we show the possibility to detect BAT activity. BATSCI signal is very sensitive to the local changes in blood oxygenation that occur at the cellular level during BAT activity, suggesting a new noninvasive way to investigate BAT function. At the same time, the strong local change in susceptibility and intrinsic low BAT T2* values In addition, with our 18F-FDG-PET scans, although the enhanced intensity of the BATSCI signal in those areas that are known to contain BAT is encouraging. Moreover, the strong positive correlation found between BAT mass and BATSCI signal intensity in the in vitro experiments on human brown adipose tissue also suggests the possibility to use the BATSCI signal to uniquely identify BAT in humans. In these in vitro experiments we observed a strong positive association between BATSCI signal intensity and UCP1 staining intensity, but at the same time an absence of correlation between BATSCI signal intensity and number of BAT cells. This apparent discrepancy is not surprising and simply reflects the expected variation in tissue fat content This study also reports on the feasibility to conduct these studies in humans. Unlike in mice, in humans the association between BATSCI and BAT cannot be uniquely established without guided biopsy or combined in vitro human studies have shown, samples with similar fat fraction can give rise to completely different BATSCI signal intensities, whose strength depends exclusively on the water-fat distribution at the cellular level, and therefore, on their cellular composition. This extreme sensitivity of BATSCI to BAT cellular composition was observed in in vitro studies of human BAT, and more likely was the major cause of the large variation in BATSCI signal intensity found in our in vivo human studies. In this small study we didn’t find any increased incidence of BAT in females versus males. On the other hand, consistent with the previously observed inverse correlation between BMI and BAT mass, we noticed a negative correlation between the intensity of the BATSCI peak and the subject BMI. Clearly this correlation cannot entirely be attributed to an enhanced BAT content, or to the presence of BAT with the classical morphology , since a lower white fat content easily leads to a lower fat-fat iZQC peak and a higher normalized BATSCI signal.Undoubtedly, the most significant advantage of this methodology with respect to conventional fat fraction measurements is its immunity to partial volume effects. As the Aside from its immunity to partial volume effects, it is important to point out that this methodology presents the same limitations of other fat fraction measurement methods that rely on the peculiar water-fat content of BAT for detection. Knowing that in BAT water-fat composition can vary substantially from a nearly fat depleted stage to a more lipid-full stage BATSCI method is a research tool that still needs extensive technical improvements before it can be widely utilized in clinical work. Currently the major limitation is the very low SNR of the BATSCI images. Because of the intrinsically low T2 and T2* of this tissue, which limit the use of long sequence delays needed for BATSCI signal growth in vivo human imaging study is the lack of a validation or comparison with conventional 18F-FDG-PET scans or fat-fraction imaging methods that are gaining popularity for the detection of BAT. This comparison was not done because of the already long imaging examination, which overall was already close to 2 hours. In addition, before these comparisons can be made, the BATSCI image resolution should be considerably improved. Because of the very different image resolution between BATSCI and fat fraction measurements  and because of the very diffuse distribution of BAT in humans, such comparison is not expected to be as straightforward as in mice.The present study did not conclusively show that in humans, as in mice, BATSCI maps correspond to BAT maps. A major limitation of our Our studies only suggest the possibility to use BATSCI to detect human BAT but clearly more experiments are needed to validate this methodology in humans. Further investigations should also be considered to assess its sensitivity to different “shades” of BAT, as well as the extent of possible contaminations from intramyocellular lipid or other tissues that closely resemble BAT structures, like bone marrow.18F-FDG-PET scans. This will facilitate future studies in which we aim to determine the true prevalence of this tissue in the adult human population and to detect its metabolic dysfunction.Despite these limitations, the possibility to detect BAT with MR, independently from its activity, is an important feature that will inherently lead to fewer false negative results than


New ideas and approaches will be needed in the future to address the pressing questions of how ROS as signaling molecules can participate in the coordination of stress adaptation and development and how they are involved in the chatter of the organelles.The production of reactive oxygen species (ROS) in different plant subcellular compartments is the hallmark of the response to many stress stimuli and developmental cues. The past two decades have seen a transition from regarding ROS as exclusively cytotoxic agents to being considered as reactive compounds which participate in elaborate signaling networks connecting various aspects of plant life. We have now arrived at a stage where it has become increasingly difficult to disregard the communication between different types and pools of ROS. Production of ROS in the extracellular space, the apoplast, can influence their generation in the chloroplast and both can regulate nuclear gene expression. In spite of existing information on these signaling events, we can still barely grasp the mechanisms of ROS signaling and communication between the organelles. In this review, we summarize evidence that supports the mutual influence of extracellular and chloroplastic ROS production on nuclear gene regulation and how this interaction might occur. We also reflect on how, and  During their life plants face a vast set of environmental challenges: extreme changes in ambient illumination, temperature, and humidity, differences in soil salinity, attack by pathogens and herbivores, mechanical wounding, etc. To withstand all these challenges, plants have developed a repertoire of signaling pathways that is unparalleled in its complexity among living organisms. Signaling through plant hormones , cell su1O2), superoxide (2O2), and hydroxyl radical (•OH) are unavoidable by-products of aerobic metabolism . For example, ROS that are produced in the chloroplast have been implicated as intermediates in retrograde signaling from chloroplast to nucleus during acclimation of photosynthesis  can also be regarded as a part of a larger network where apoplastic signals induce the generation of ROS in the chloroplast, which in turn leads to regulation of nuclear gene expression by several still uncharacterized, but at least partially chloroplast-derived, ROS-dependent retrograde signals.A critical role in this signal integration and decision-making is played by a class of reactive forms of the molecular oxygen, collectively referred to as reactive oxygen species ROS; . ROS, intabolism  which hatabolism  as well tabolism . During tabolism , the roltabolism  and is ntabolism . Yet uncynthesis . Intriguynthesis . Thus, tFigure 2; 2O2, either spontaneously or in a reaction catalyzed by the superoxide dismutase  under physiological conditions and in many respects resembles a molecule of water. Its dipole moment, similar to that of H2O, limits passive diffusion of H2O2 through biological membranes. Possible candidates for the import of apoplastic H2O2 are aquaporins , a ubiquitous family of channel proteins that has undergone an extensive expansion in vascular plants . Those putative receptors or other sensory systems for extracellular ROS in plants have so far remained elusive, but for example, several classes of receptor-like protein kinases (RLKs) with cysteine-rich extracellular domains  have been suggested to be involved in ROS perception .What happens in the plant cell after an extracellular oxidative burst has been triggered? A connection of apoplastic and chloroplastic ROS into common signaling networks during the plant stress response is evident in various model systems and processes , althougFigure 2). The second increase in ROS production happens several hours after the pathogen attack  varies according to changing photosynthetic electron transfer and CO2 fixation rate. Extracellular stimuli, such as recognition of bacterial components by the plasma membrane receptors, can rapidly regulate chloroplastic functions. During plant–pathogen interactions the cues perceived in the apoplast trigger MAPK cascades  and result in fast down-regulation of photosynthetic genes and accumulation of H2O2 in chloroplasts that is necessary for initiation of HR-mediated cell death  at an early stage of pathogen recognition. This decrease in NPQ makes chloroplasts more predisposed to the production of ROS, which might be a priming mechanism for chloroplast ROS signaling at later stages of immune response  and on the other hand to a retrograde signal (soldat8 mutation in the chloroplastic RNA polymerase SIGMA SUBUNIT 6 (SIG6) gene increases the tolerance of seedlings to 1O2  biosynthesis, and hormonal control . For exall death . Anotherresponse . Severalresponse . For exae signal . Anothere signal . The sols to 1O2 , which ls to 1O2 . GUN1, as to 1O2 . The hems to 1O2 . The dis1O2) as a chloroplastic signal involved in the regulation of plant cell death. PS II and its light-harvesting antennae produce 1O2 when light-excited chlorophylls adopt the rare triplet state and then reduce triplet oxygen (1O2 is enhanced when the light-excited electrons cannot escape PS II chlorophylls because the downstream components of electron transfer chain (mainly the plastoquinone pool) are already over-reduced, a situation typical of excessive illumination. 1O2 readily reacts with lipids, proteins, and pigments and is rapidly quenched by water, which makes its diffusion distance from the site of production shortest among all ROS  conserved in higher plants are involved in this process, although their exact role is unknown . Regulation of CAS activity is linked to the state of photosynthetic electron transfer chain. CAS has earlier been shown to be involved in high light acclimation of the green alga Chlamydomonas reinhardtii , peroxiredoxin, and glutathione peroxidase. H2O2 produced in chloroplasts gives rise to retrograde signals. The signaling is not well understood and might be a combination of passive diffusion of H2O2with indirect pathways including hormonal (abscisic acid) signaling  seem to be involved in this H2O2 leakage  have similarities to the profiles induced by ozone, while 12 h after flg22 treatment the expression profile resembled that of chloroplastic ROS production induced by MV . It is unlikely that, for example, merely a change in the cytoplasmic redox state could carry the information about the subcellular source of H2O2. Therefore, as proposed  the apoplast, the chloroplast, and the nucleus. To achieve the elaborate and fine-tuned responses to biotic and abiotic stimuli that we observe on transcriptional, biochemical, and physiological level, intense and strictly controlled communication between the subcellular “crime scenes” must take place. While some components of this information exchange have been proposed, we still lack a thorough understanding on how the apoplast, the chloroplast, and the nucleus keep in touch. It will be perhaps one of the major challenges of ROS research in plants to understand ROS-induced signaling pathways between different organelles. Once we find out which components transmit information under specific conditions, we will be able to generate an integrated view of ROS signaling and its role in environmental adaptation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Research in the Plant Stress group of Professor Jaakko Kangasjärvi is supported by Biocentrum Helsinki, the Academy of Finland, the University of Helsinki, and the ERA-PG.


VEGF gene have been associated with a susceptibility to several disorders, no genome-wide search on VEGF serum levels has been reported so far. We carried out a genome-wide linkage analysis in three isolated populations and we detected a strong linkage between VEGF serum levels and the 6p21.1 VEGF region in all samples. A new locus on chromosome 3p26.3 significantly linked to VEGF serum levels was also detected in a combined population sample. A sequencing of the gene followed by an association study identified three common single nucleotide polymorphisms (SNPs) influencing VEGF serum levels in one population (Campora), two already reported in the literature  and one new signal (rs3025020). A fourth SNP (rs41282644) was found to affect VEGF serum levels in another population (Cardile). All the identified SNPs contribute to the related population linkages (35% of the linkage explained in Campora and 15% in Cardile). Interestingly, none of the SNPs influencing VEGF serum levels in one population was found to be associated in the two other populations. These results allow us to exclude the hypothesis that the common variants located in the exons, intron-exon junctions, promoter and regulative regions of the VEGF gene may have a causal effect on the VEGF variation. The data support the alternative hypothesis of a multiple rare variant model, possibly consisting in distinct variants in different populations, influencing VEGF serum levels.Vascular Endothelial Growth Factor (VEGF) is the main player in angiogenesis. Because of its crucial role in this process, the study of the genetic factors controlling VEGF variability may be of particular interest for many angiogenesis-associated diseases. Although some polymorphisms in the  VEGF gene have been associated with a variation of protein levels Angiogenesis, or the growth of new blood vessels, is required for any process that results in the accumulation of new tissue as well as many processes involving tissue remodelling. When the regulation of angiogenesis fails, blood vessels are formed excessively or insufficiently. It is thus a characteristic of multiple pathologies including cancer, cardiovascular disease, arthritis, psoriasis, macular degeneration, and diabetic retinopathy. In particular, insufficient angiogenesis can be a cause of ischemia, and excessive angiogenesis can result in tumor neovascularization and growth. The angiogenesis process is highly controlled through the balance of pro- and anti-angiogenic factors. VEGF is a crucial player in angiogenesis as it represents the principal pro-angiogenic factor. Throughout development, VEGF orchestrates the process of angiogenesis by regulating the growth, development, and maintenance of a healthy circulatory systemIn this work we searched for new quantitative trait loci (QTLs) and polymorphisms influencing VEGF serum levels, in three isolated populations, each living in a different village in the remote hilly region of the Cilento and Vallo di Diano National Park, South Italy. As we recently reported VEGF gene region as the main QTL for VEGF serum level variation, with a strong and consistent effect in all three populations. An additional and new QTL was detected on chromosome 3p26.3. With a weaker effect, this QTL was detected only in the combined sample of the three populations.We identified the 6p21.1 VEGF gene was conducted in sub-samples from each of the three villages. Three SNPs were found to be significantly associated with VEGF serum levels in the village of Campora and a fourth SNP (rs41282644) was significantly associated with VEGF serum levels in the village of Cardile.Focusing on the 6p21.1 signal, an extensive sequencing analysis of the VEGF polymorphisms for tumors in the general population.Altogether, the combination of information on linkage and association in these three population isolates with a common origin allows us to reject the hypothesis of a direct effect on VEGF serum levels of the four SNPs identified. The data suggest an effect of rarer variants, possibly different among the three populations. These results raise a crucial issue in the search for predictive and prognostic The characteristics of the study samples are reported in VEGF gene that is exactly located at 0.5 Mb from the D6S282 marker and at 2 Mb from the D6S459 marker.Genome-wide linkage analysis was performed in the three population samples on the sub-pedigree sets generated by the breaking procedure applied to each population genealogy. A very strong signal was found on chromosome 6p21.1, with the highest LOD score at the marker D6S459 in Campora , in Gioi , and in the combined sample , and at the nearest marker D6S282 in Cardile  see . The 6p2An additional linkage was detected on chromosome 3p26.3  at marker D3S4559, able to reach statistical significance in the combined sample . AdditioVEGF gene was carried out in a total group of 136 individuals. In detail, the exons, intron-exon junctions, promoter and regulative regions were analyzed in 42 individuals from Campora, 49 individuals from Gioi and 45 individuals from Cardile. The individuals included in these three hereafter denoted “detection samples” were chosen to best represent the population's genetic diversity. Data from NCBI (Assembly GRCh37) report 77 SNPs (64 SNPs and 13 Ins/Del) in the regions of the VEGF gene included in our analysis. In our detection samples, 36 out of the 77 (32 SNPs and 4 Ins/Del) were detected in at least one population and 18 new polymorphisms (17 SNPs and 1 Ins/Del) were identified. Two SNPs (rs3025020 and rs833070) outside the sequencing regions but available from previous studies were included in the analysis. The SNP characteristics for the three population “detection samples” are presented in detection sample” sizes, all but two of the 18 new SNPs were detected in only one individual , the two remaining SNPs being detected in two individuals from different populations  above 5% were tested for association with the VEGF serum levels. detection sample” and for the SNPs repeatedly reported as associated with VEGF serum levels or correlated phenotypes in the literature. Significant associations were found between the VEGF serum levels and three common SNPs in Campora: the rs25648 variant located in the 5′UTR, the rs3025020 placed in the intron 6 and the rs3025039 located in the 3′UTR.In each “vs TT = 295.2 pg/ml) whereas the TT genotype of the rs25648 and rs3025020 variants was associated with higher levels of VEGF . No linkage disequilibrium was observed among these three SNPs .These associations were confirmed in the large population sample of Campora . The TT Surprisingly, no significant associations were found between these three SNPs and the VEGF levels in the two population samples from Gioi and Cardile . Howeverdetection sample” of Cardile and the association was confirmed in the population sample of this village but not in the population sample of Campora nor in that of Gioi (vs GG = 391.2 pg/ml). In contrast to the rs25648, rs3025039 and rs3025020 SNPs, the rs41282644 SNP has a very low frequency in the Caucasian reference population (MAF = 1% in the pilot 1 CEU sample from the 1000 Genome Project) but has become more frequent in the Cilento villages . Note that SNP rs41282644 is not strongly correlated with the rs25648, rs3025020 and rs3025039 SNPs .Nonetheless, a variant located in the 3′UTR, rs41282644, was significantly associated with the VEGF serum levels in the “ of Gioi . In Carddetection sample  among the four SNPs associated in at least one population was not significantly different across the populations, as suggested by the results of the global LD comparison test proposed by Zaykin et al Haplotypes were also analyzed in the three population samples using successively the three SNPs associated in Campora and all the four associated SNPs (three associated in Campora and one associated in Cardile) for haplotype reconstruction. More frequent haplotypes were tested for association with the VEGF serum levels. The results show that when the three SNPs associated in Campora were considered, two haplotypes were found to be associated with the VEGF serum levels in Campora, but were not associated in Gioi and in Cardile . InteresWhen all the four associated SNPs (three associated in Campora and one associated in Cardile) were used for haplotype reconstruction, only the T-T-C-G haplotype was still associated with the VEGF levels in Campora although only at the nominal level. No association was found between any of the haplotypes tested and the VEGF levels in Gioi and Cardile.To evaluate the contribution of the associated SNPs to the linkage signals detected on 6p21.1, the linkage statistics were recomputed conditional on the associated SNPs. In Campora, the original linkage peak dropped from a LOD score of 7.52 to a LOD score of 6.82 when the rs3025039 genotypes were taken into account, to a LOD score of 6.35 in the case of the rs3025020 variant and to a LOD score of 6.47 in the case of rs25648. When the linkage statistics was computed conditional on the three SNP genotypes, the LOD score dropped to LOD = 5.00, highlighting the independence of these three association signals . A compaSimilarly, the LOD score of 6.56 detected in Cardile, dropped to a LOD = 5.58 when the linkage statistics was computed conditional on the rs41282644 SNP genotypes.The same conditional analyses were carried out in the other population samples, respectively Gioi and Cardile for SNPs rs3025039, rs3025020 and rs25648 and Gioi and Campora for SNP rs41282644. As expected, no variation in the LOD score was observed in these samples (data not shown).VEGF gene region. Our genome-wide search detected three additional linkage signals outside the VEGF gene region. A signal on chr3p26 was observed but only reached significance when the three population samples were combined to increase the power, which suggests a weaker effect of this QTL. Further, no clear candidate genes could be identified in this region. Two additional signals were found on 2p16.3 and 20q13.13. Although not consistent across the populations and not detected in the combined sample, these might be of interest since interesting candidate genes are located in these regions. In fact, the EPAS1 gene, located on 2p16.3, is known to be involved in the transcriptional regulation of VEGF VEGF gene In this study, we reported a high heritability of VEGF serum levels in our three samples  and a very consistent and strong linkage of this trait with the VEGF gene region on chr6p21.Altogether, the genome-wide linkage results suggest that most of the genetic variability accounting for the VEGF heritability comes from the VEGF gene have been associated with VEGF protein levels and/or with a susceptibility to (or the severity of) several cancers such as breast, lung, colorectal, bladder prostate and gastric VEGF SNPs, those frequently reported to be associated are: rs699947, rs833061, rs1570360, rs2010963, rs3025039 and rs25648 Several SNPs in the In our study, associations between rs3025039 and rs25648 and VEGF levels were detected in Campora but not in the two other villages, although these two SNPs have a similar frequency and LD pattern in all three villages. Associations with the other reported SNPs  could not be identified and new association signals were discovered: rs3025020 in Campora and rs41282644 in Cardile.From the analysis of haplotypes involving the rs25648- rs3025020- rs3025039 SNPs, we note that in Campora the T-T-C haplotype is more strongly associated with the VEGF levels than the C-C-T haplotype and that it is still associated when the rs41282644 G allele was added (T-T-C-G) to the haplotype. However, the overall haplotype association results, although interesting, are less significant then the single SNP association results, as expected given that all of these are common SNPs and there is a very low LD between them.All the associated SNPs in our study contribute to the linkage signal, but none of them explains the majority of the signal, even when considered together but independently  or as a haplotype. The detection of different association signals in populations with a very similar genetic background and in which a strong linkage was detected, strongly suggests that these cannot point to functional variants, but only to proxies correlated to the functional variants.Whether these variants are more likely to be rare or common, different or similar among populations remains an open question. Still, given that the LD patterns among common SNPs in the region are relatively similar, if common causal variants were involved, their association with rs3025039, rs25648, rs3025020 or rs41282644 should not be specific to Campora or Cardile. These SNPs should be proxies in all three populations. On the contrary, rare variants, more sensitive to genetic drift, could well display a discordant LD pattern with common variants among the three populations, explaining the discordant association results. A further study of the region, including a sequencing of the whole linkage region in larger sets of individuals, will be required to elucidate this hypothesis.From a more methodological perspective, this work suggests that our study design, able to provide complementary information on linkage and association in three isolated populations with similar common genetic variations but possibly divergent rarer variations, is particularly powerful in a discrimination between causal and non causal variants.The study includes 1,957 individuals, recruited through a population-based sampling strategy in three small isolated villages of the Cilento region, South Italy: 656 individuals from the village of Campora, 852 from the village of Gioi and 449 from the village of Cardile. The recruited sample represents about 85% of the living population of each village. Blood samples were collected in the morning after the participants had been fasting for at least 12 h. Aliquots of serum were immediately prepared and stored at −80°C, and were subsequently used for the assessment of VEGF levels. VEGF (pg/ml) was measured using an enzyme-linked immunosorbent assay, according to the manufacturer's instructions . The study design was approved by the ethics committee of Azienda Sanitaria Locale Napoli 1. The study was conducted according to the criteria set by the declaration of Helsinki and each subject signed an informed consent before participating in the study.Mann-Whitney U test to compare median values in independent samples was performed to compare the VEGF serum levels among population samples. Kruskal-Wallis test was applied to assess the influence of age on VEGF serum variation. These analyses were performed with the SPSS software.A genome-wide scan of 1,122 microsatellites (average marker spacing of 3.6 cM and mean marker heterozygosity of 0.70) was performed by the deCODE genotyping service. All subjects having a VEGF measurement were genotyped. Mendelian inheritance inconsistencies were checked with the Pedcheck programIn each village, the vast majority of the phenotyped individuals were connected through a unique deep pedigree. In Campora, 627 out of the 656 phenotyped individuals were included in a 3,049-member pedigree. In Gioi, 798 out of the 852 phenotyped individuals were related through a 4,190-member pedigree. In Cardile, a pedigree of 2,384 members connected 425 individuals out of the 449 phenotyped individuals.The heritability of VEGF serum levels was estimated using the SOLAR software The linkage analysis was performed following a procedure based on a multiple splitting of the genealogy, that we developed and already applied to various complex traits A linear regression model of the log-transformed VEGF on age was applied and the residuals were used as a quantitative trait in the multipoint quantitative linkage analysis on each sub-pedigree set using the regression-based approach implemented in MERLIN-REGRESS The contribution of the associated polymorphisms to the linkage signal on chromosome 6 was assessed by performing the linkage analysis on a new phenotype: the VEGF levels adjusted for age and SNP genotypes with a genotypic modeling of the SNP effect.To take into account the multiple testing problem created by both the number of markers tested and the number of pedigree sets analyzed, we considered a parametric false discovery rate (FDR) approach.For each marker in each population, the mean LOD score statistics over all the sub-pedigree sets was transformed into a test statistic with a theoretical null distribution following a standard normal 0 first components (K0≤K). K0 was chosen such that the L1 distance between the estimated null density and the density of the theoretical null distribution  was the minimum. Finally, we report here the markers with a q-value below 5%A K-components Gaussian mixture model with equal variances was chosen to modelize the marginal distribution of the transformed test statistics, as such a mixture model efficiently separates the empirical null distribution . All the individuals included in the “detection samples” were among the oldest individuals for which DNA was available with children, grand-children and great-grandchildren included in the population sample. The mean number of direct descendants  was 5.8 for the 42 individuals included in the Campora detection sample, 8.3 for the 45 individuals included in the Cardile detection sample and 9.12 for the 49 individuals included in the Gioi detection sample.To identify polymorphisms in the 2, and 40 ng of genomic DNA as template, with 2 units of recombinant Taq DNA polymerase. The cycling conditions were as follows: 95°C for 3 min, followed by 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec for 35 cycles, and by a final extension at 72°C for 7 min. The PCR products were purified by using MultiScreen PCRµ96 Filter Plates (Millipore) and were sequenced on both strands using the Applied Biosystems BigDye v3.1 sequencing kit according to the manufacturer's recommendations on an Applied Biosystems 3730 DNA Analyzer Sequencer. The sequences were then analyzed using the SeqAnalysis and BioEdit softwares. The SNP discovery accuracy was assured by sequencing in two replicates the fragments including the new SNPs.Altogether, 9.8 kb, corresponding to 50% of the entire gene, were analyzed. The oligonucleotide primers for the amplification and sequencing of these regions were designed using the primer prediction program Primer3 . The PCRdetection samples” . The rate of successful genotypes was above 95% for each SNP.As mentioned in the VEGF gene were tested for association with the log-transformed VEGF adjusted for age phenotype in the detection samples. The genotype frequencies of the tested SNPs are reported in a supplementary table (All frequent SNPs (MAF>5%) identified in the ry table . SignifiTo correct for multiple testing, we applied the procedure proposed by Nyholt population sample, 104 individuals for the Cardile population sample and 111 individuals for the Gioi population sample.The global comparison of LD among the rs25648, rs3025039, rs3025020 and rs41282644 SNPs in the population samples was conducted using the approach proposed by Zaykin et al The haplotypes were reconstructed taking advantage of family information and tested for association with the VEGF levels using the software FBAT. A biallelic test was performed in which each haplotype was tested against all the others pooled together and an additive model was applied. Two analyses were carried out successively. One used the haplotypes made of the three SNPs associated in Campora and the other used the haplotypes made of the four associated SNPs (three associated in Campora and one associated in Cardile). Only haplotypes having a frequency >1% were tested for association with the VEGF levels.Figure S1VEGF gene. The exons are reported in black, introns in white, regulative regions in dark grey, and promoter region in light grey. The position of the 56 SNPs identified in the gene is also indicated. The four SNPs associated with the VEGF levels are framed. B) LD patterns between the four associated SNPs in Campora, Gioi and Cardile. R-squared values are indicated.A) Schematic representation of the (TIF)Click here for additional data file.Table S1Characteristics of the sub-pedigree sets used in the linkage study for the VEGF serum levels in Campora, Gioi and Cardile(DOC)Click here for additional data file.Table S2VEGF geneList of the primers designed to sequence (DOC)Click here for additional data file.Table S3detection samples of the 26 SNPs analyzed for association with the VEGF levels.Genotype frequencies in the (DOC)Click here for additional data file.


Bordetella pertussis strain, was developed as a live attenuated nasal pertussis vaccine by genetically eliminating or detoxifying 3 toxins.Acellular pertussis vaccines do not control pertussis. A new approach to offer protection to infants is necessary. BPZE1, a genetically modified 3, 105 or 107 colony-forming units as droplets with half of the dose in each nostril. 12 controls received the diluent. Local and systemic safety and immune responses were assessed during 6 months, and nasopharyngeal colonization with BPZE1 was determined with repeated cultures during the first 4 weeks after vaccination.We performed a double-blind, placebo-controlled, dose-escalating study of BPZE1 given intranasally for the first time to human volunteers, the first trial of a live attenuated bacterial vaccine specifically designed for the respiratory tract. 12 subjects per dose group received 10Colonization was seen in one subject in the low dose, one in the medium dose and five in the high dose group. Significant increases in immune responses against pertussis antigens were seen in all colonized subjects. There was one serious adverse event not related to the vaccine. Other adverse events were trivial and occurred with similar frequency in the placebo and vaccine groups.BPZE1 is safe in healthy adults and able to transiently colonize the nasopharynx. It induces immune responses in all colonized individuals. BPZE1 can thus undergo further clinical development, including dose optimization and trials in younger age groups.NCT01188512ClinicalTrials.gov  Pertussis is today the most prevalent vaccine-preventable disease in the developed world Bordetella pertussis circulation, and a cocooning strategy of vaccinating people in close contact with infants to prevent transmission to this group. However, they all suffer from their inherent limitations The disease is most severe and fatal in infants too young to be protected by the current vaccination programs B. pertussis induces strong immune responses, even in very young infants B. pertussis strain has therefore been constructed as a vaccine candidate to be administered by the nasal route, in order to mimic natural infection without inducing disease. In this strain, named BPZE1, dermonecrotic toxin and tracheal cytotoxin have been genetically removed, whereas pertussis toxin (PT) has been genetically detoxified by two independent mutations, each deactivating the toxic activity of PT, without affecting the immunogenic properties However, in contrast to immunization, natural infection by Here, we tested BPZE1 for the first time in man in a placebo-controlled, double-blind, dose-escalation trial to provide proof of concept of its safety, its ability to colonize and its immunogenicity when given nasally to healthy young adult male volunteers in a single dose. This is the first time that a live attenuated bacterial vaccine specifically designed for the nasal route has ever been tested in humans.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see The study was conducted in accordance with the protocol, ICH Good Clinical Practices standards, Declaration of Helsinki and applicable regulatory requirements as well as any European and Swedish applicable laws and regulations relating. The trial is registered with ClinicalTrials.gov, NCT01188512. The Swedish Medical Product Agency (MPA) and the regional ethical review board in Stockholm approved the study and the amendments. Since this was a first-in-man study of a genetically modified microorganism it was a requirement from the MPA, that only males should be enrolled in the study. The volunteers signed the informed consent form after receiving written and oral information. Information was given before and during visit 1, so the subject had sufficient time to consider participation, which started on visit 2.http://www.karolinskatrialalliance.se/en).The study was performed at the phase I unit, Karolinska Trial Alliance, Karolinska University Hospital, Stockholm, Sweden ; Group 2 (medium dose), 105 cfu; Group 3 (high dose), 107 cfu] and 4 volunteers/group receiving the diluent as placebo. Thus the 12 placebo recipients were evenly distributed throughout the whole study period. The administration of vaccine or diluent alone was performed in a double-blind fashion with administration of coded vials with exception for the two first study subjects in each dosage group, who received active substance (unblinded) as required by the Swedish MPA.Sixteen individuals were included in each of the three dose groups , with 12th, 2010 and January 20th, 2011.A liquid formulation of three different BPZE1 batches  in phosphate-buffered saline containing 5% saccharose  was used. Vaccine or placebo was given as single administration by nasal application of 0.1 ml in each nostril. The dose of the active ingredient or placebo was administered within 2 hours after thawing. The volunteers were vaccinated between September 9The volunteers provided written informed consent and were screened 2–6 weeks before vaccination. The Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Trials Group 1 was followed for at least 7 weeks before group 2 was vaccinated. Group 2 was followed for at least 9 weeks before group 3 was vaccinated. Interim safety meetings were held with IDMC and the sponsor before administering the next dose.Venous blood samples were collected at screening 2–6 weeks prior to vaccination, on the day of vaccination and at 1, 2 and 4 weeks and 5–6 months after vaccination.B. pertussis was confirmed by Gram stain and Catalase and Oxidase test. For further details see Supporting information Nasopharyngeal aspirates were collected to determine colonization by BPZE1 of the nasopharyngeal mucosa 4, 7, 11, 14 and 28 days and, if necessary, 45 days after vaccination. The aspirates were grown on charcoal agar plates . The plates were read daily for up to 7 days. The presence of Serum IgG antibodies against PT, filamentous hemagglutinin (FHA), pertactin and fimbriae types 2/3 were analyzed by standardized ELISA as described in Supporting information Haemoglobin and cell blood count  were analyzed on a Sysmex XE-5000 instrument.Case report forms, patient journals and raw data collected at the clinic are archived at the Karolinska Trial Alliance, Karolinska University Hospital. Raw data from the laboratory testing and all other documentation from the trial are archived at the Swedish Institute for Communicable Disease Control for at least 15 years. Samples collected for isolation and immunogenicity testing are stored in the biobank at the Swedish Institute for Communicable Disease Control.The difference between the vaccine dose groups  in the number of local and systemic solicited AE, and the proportion of subjects colonized with BPZE1 was evaluated using Fisheŕs exact test for contingency tables.Due to the limited number in each placebo group (n = 4) the power of any test between the three placebo groups would be very low. Therefore only comparisons were performed between the dosage groups and the whole placebo group (n = 12).Due to non-normally distributed data for the specific antibody levels, Friedman’s one-way analysis of variance for repeated measures was used to compare the overall differences in serum IgG distribution over time. In case of a significant overall test, pairwise comparisons with day 0 were performed between time-points within respective dose group.For the same reason, Kruskal-Wallis one-way analysis of variance was used to compare the overall differences in IgG levels between dose groups. The dose groups were compared at all time points in separate analyses. Pairwise comparisons between dose groups were performed if the overall test showed a significant test result.All hypothesis tests performed were two-sided and p<0.05 was considered significant.All statistical analyses were done using the software R (R Core Team (2012). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.) There were no differences in baseline data between the volunteers assigned to vaccine or placebo except for the volunteers in the low dose group, who had lower systolic blood pressure than other groups, but still within normal limits . In totaThere was one SAE. The first subject vaccinated openly with the low dose died 7 weeks after vaccination. The SAE was reported to the sponsor and the sponsor notified the Swedish MPA, the European Medical Agency Eudravigilance database and the Ethics Committee. After careful assessment, the death was judged by the IDMC as not vaccine-related. Out of consideration for the deceased and his family it was decided not to give more information about the death.Immediate AE within 6 hours of vaccination were all trivial and short-lasting with no differences between the placebo and different dosage groups.Local and systemic solicited AE, which were noted in the diary and confirmed with interviews at subsequent visits, were all mild to moderate. The most common were rhinorrhoea, sneezing and cough. There were no differences between placebo and any of the treatment groups  and S3 oThere were no abnormalities in vital signs or blood haematology in any volunteer during the study (data not shown).All volunteers were asked unsolicited questions about AE at all visits, including the last 5–6 months after the vaccination. There was no SAE and only mild to moderate episodes of upper respiratory tract infections and short-lasting fever with no differences between groups.BPZE1 was isolated 1–3 times from one subject in the low-dose group, one in the medium-dose group and five in the high-dose group between days 4 and 14 . On day Serum antibodies against PT, FHA, pertactin and fimbriae appeared between days 14 and 28 and remained high during the 6 months follow up  and 4. PB. pertussis antigens was detected in the placebo group and among non-colonized subjects in any dose group, while pronounced increases against all four tested antigens occurred among the colonized subjects. Overall significant increases were seen in the colonized group for IgG to PT (p = 0.007), FHA (p<0.001), pertactin (p<0.001), and fimbriae (p<0.001). Pairwise comparisons between culture positive and negative subjects showed significant differences for anti-PT, anti-FHA and anti-pertactin but not for anti-fimbriae . 3 cfu on, were used sequentially. The doses chosen were based on the results of pre-clinical studies in mice Live attenuated mucosal vaccines have a number of advantages over injectable subunit or inactivated vaccines, including the relatively low production cost and the ease of needle-free administration, which increases compliance. With a few exceptions B. pertussis, colonization was detected between days 4 and 28 after administration Except for one, which was not related to the vaccine, no SAE was noted. All other symptoms, mostly from the upper respiratory tract, headache and short-lasting fever, were trivial and evenly distributed in the placebo and three dosage groups. Seven of the 36 vaccinated individuals were colonized, and colonization was dose-dependent, with five of the seven in the high-dose group. Similar to what is seen with natural infection by th dose (manuscript in preparation). The PT antibodies induced in the culture positive individuals in this study were of the same magnitude, 27 IU/ml, one month after vaccination. Interestingly, five out of seven culture positive subjects responded against fimbriae 2/3, and one had an increased level but was defined as a non-responder, because he had a high pre-vaccination antibody level to fimbriae. This is in contrast to what has been shown after infection with B. pertussis when only 14% developed low levels of antibodies against fimbriae, unless vaccinated with vaccine containing fimbriae A strong correlation was found between colonization and the induction of serum antibody responses to PT, FHA, pertactin and fimbriae. All colonized individuals mounted an antibody response to these antigens, most often to all four of them in contrast to the non-colonized volunteers. Interestingly, the antibody titers in the colonized low-dose vaccinee were not inferior to those of the high-dose subjects, suggesting that as long as colonization is established, regardless of the dose, antibodies to all B. pertussis antigens are induced. The genetic alteration of PT did not appear to affect its immunogenicity, since the median PT antibody levels were of the same magnitude as those that we have observed after a fifth dose of a combined tetanus, low dose diphtheria and acellular pertussis vaccine . That study included 115 adolescents aged 14–15 years primed with a five-component acellular pertussis vaccine at 3 and 5 months of age and boostered at 12 months and 5.5 years of age. In that study the level of PT antibodies was 20 IU/ml one month after the 57 cfu is still not high enough, and that a higher dose is needed. A dose of 109 cfu of virulent B. pertussis was necessary to establish an infection in non-human primates 8 cfu/dose of live attenuated Bordetella bronchiseptica are used for the vaccination of dogs against kennel cough B. pertussis or Bordetella parapertussis or to cross-reactivity with antigens from other bacteria. No relation between anti-PT IgG and colonization was found, but it should be noted that volunteers with pre-existing anti-PT IgG ≥20 IU/ml were excluded from the study. Whether pre-existing immunity prevents BPZE1 uptake and whether a higher dose or vaccine volume may overcome this will be addressed in future studies. These observations may also be relevant for the understanding of protective immunity against B. pertussis infection and support results from previous clinical studies Low anti-pertactin IgG levels have been related to susceptibility to B. pertussis infection while high anti-pertactin and high anti-PT antibody levels indicated protection following household exposure Although increasing doses led to increased colonization and immune response frequencies, even at the highest vaccine dose not all volunteers were colonized. Several non-mutually exclusive explanations can account for this and will be the subject of future studies. It is possible that a dose of 10In conclusion, this first-in-man study shows that live BPZE1 given intranasally is safe and able to colonize the nasopharynx of subjects with low pre-vaccination antibodies to FHA, pertactin and fimbriae. It induces serum IgG antibody responses in all colonized but not in non-colonized subjects. However, before BPZE1 can be used in the target population, the newborns, it will be necessary to first conduct further trials in older populations, such as adolescents and children of both sexes. The steps required to reach the target population strongly depend on the decisions made by the regulatory agencies of countries in which these trials can be performed. Discussions with several of these are currently under way. As a first live attenuated bacterial vaccine specifically designed for the respiratory tract, this proof-of-concept study may have a broad impact. Furthermore, recombinant BPZE1 derivatives producing heterologous antigens can be used for the development of multivalent vaccines The sponsor Inserm and the Strategic Planning and Clinical Trial Monitoring Committee (COSSEC) of Inserm represented by Sebastien Boy and Anne Puech and the European Clinical Research Infrastructure Network (ECRIN) for their support; Dominique Raze, Linda Kostic, Eva Hansson Pihlainen, Carina Bengtsson, Siri Wahlquist for skilful technical assistance at the laboratory; Ria Bral and Geert Deschamps from Innogenetics, Martine Vandermarliere and Marijke Verhaeghe from Innogenetics and Q-Biologicals for their input and work during the development and cGMP production; The study team at the Karolinska Trial Alliance (KTA) for vaccination, sample collection, and excellent surveillance of the volunteers at the clinic. The members of the Scientific Advisory Board and the Independent Data Monitoring Committee, Patrick Olin, Ragnar Norrby, Roland Dobbelaer, Odile Launay, Marta Granström and Micha Roumianzeff for invaluable advice during the study. Jérome Weinbach, Delphine Sondaz and Marie Clotteau from Inserm Transfert for management of the Child Innovac project.Table S1Frequency and Timing of Safety and Immunogenicity Measurements.(DOCX)Click here for additional data file.Table S2No Differences were found between Vaccine Groups Regarding Local and Systemic Solicited Adverse Events.(DOCX)Click here for additional data file.Table S3Differences versus High-Dose Group in Antigen Specific Serum IgG Levels.(DOCX)Click here for additional data file.Table S4Number of Local Solicited Adverse Events during Week 1 and 2 in the Placebo Group, the Culture Negative Subjects and the Culture Positive Subjects.(DOCX)Click here for additional data file.Study Protocol S1(PDF)Click here for additional data file.Consort Checklist S1(DOC)Click here for additional data file.Method S1Inclusion and Exclusion Criteria.(DOCX)Click here for additional data file.Method S2Nasopharyngeal Aspirate Cultivation.(DOCX)Click here for additional data file.Method S3Serum IgG Antibody Analyses.(DOCX)Click here for additional data file.