Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region. If proven, and not controlled in the early stages, the emergence of ML drug resistance threatens to become a widespread problem in the US that may limit the effectiveness of current preventive drug treatment methods.Strains of 3 in Aedes aegypti for experimental infection of two groups of dogs, each of which contained two laboratory dogs, one treated with prophylactic ivermectin (12 μg/kg) monthly for 6 months at twice the label dose (6 μg/kg), and one untreated control.To validate practice reports, a statewide survey of Louisiana veterinarians was done to define the extent of the problem and identify focal ‘hotspots’ of reported ML LOEs using Geographic Information Systems (GIS) methods. The present study then utilized microfilariae (Mf) from two canine field cases from different state locations that fit criteria for a high index of suspicion of LOE against heartworms by ML drugs. Blood containing Mf from the canine field cases was used to infect and produce LD. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and microfilaremia by the Knott’s test. Mf counts gradually increased post-patency in test and control dogs. Infective larvae raised from microfilariae from the treated Group I dog were used to successfully establish a second generation isolate, confirming heritability of resistance in the face of a monthly ivermectin challenge dose of 24 μg/kg, given monthly for 3 months.Both treated and untreated dogs from Group I and Group II developed patent in vivo evidence of the existence of ML drug resistance in dogs infected by D. immitis L3 from suspect field LOE cases in the Lower Mississippi Delta. Results encourage further work on mechanisms underlying the emergence of ML resistance in D. immitis and development of evidence-based resistance management strategies for heartworm preventives in order to extend the useful life of current drugs.These experimental infection studies provide  In 2005, an evaluation of the efficacy of heartworm preventive products was published by the Center for Veterinary Medicine, US Food and Drug Administration (FDA) that reported concerns of an increased number of reports by practicing veterinarians on lack of efficacy (LOE) of macrocyclic lactone (ML) preventive drugs . Eighteein vitro indicators of resistance [in vivo experiments, the gold standard, to establish confirmed resistant strains from canine field cases in the laboratory [In response to this, and anecdotal reports of increasing LOE cases from the veterinary community in heartworm endemic areas, pharmaceutical firms initiated reimbursement programs in 2004 to cover costs of treatment of LOE cases if owners and their veterinarians could document full compliance with recommended preventive treatment practices. Concurrently, several research efforts were initiated, investigating the spatio-temporal scale and dynamics of LOE reports [sistance ,4, possisistance -10 and iboratory ,12.D. immitis, and 2) establishment of ML resistant isolates of D. immitis in experimentally infected laboratory dogs using L3 raised in Aedes aegypti fed on microfilaremic blood from 2 dogs with a ‘high index of suspicion of resistance’.The objectives of studies reported here are to record results of: 1) a 2009 survey to document the experience of veterinarians in Louisiana on potential emerging ML resistance by In August 2009, a one-page ‘check-off’ questionnaire survey was sent to all Louisiana veterinarians listed in the Louisiana Veterinary Medical Association (LVMA) database and in the 2008 billing records of the Louisiana Animal Disease Diagnostic Laboratory (LADDL). Fifteen survey questions queried whether ML LOE cases had been seen in client dogs, including year of the first ML LOE case, specific drug used on suspected LOE cases, and the number of ML LOE cases reported. A total of 855 surveys were sent.Survey data results were entered into a Microsoft® excel  database and linked to a map of Louisiana within a geographic information system (GIS) according to the longitude/latitude geographic point location and Zip Code of each responding veterinarian’s clinic using ArcGIS 9.3 software  [D. immitis:History of failure of efficacy and full monetary compensation by a commercial pharmaceutical firm;Residence in an area identified as a ‘hotspot’ of suspected ML drug resistance in the 2009 statewide veterinary practitioner survey of LOE cases [Persistence of circulating Mf following an accepted microfilaricidal dose of ML  and;High frequency of a genotype marker previously reported to be correlated with potential ML resistance, single nucleotide polymorphism at sites 11 and 618 (GG-GG) of a gene encoding for P-glycoprotein .Microfilariae (Mf) positive blood from canine field cases from different state locations was collected from client-owned dogs for use in the current study. After review and approval of experimental protocols by the Louisiana State University Institutional Animal Care and Use Committee, owner consent forms informing clients of the study purpose and design were agreed upon and signed prior to enrollment in the study. Based on practitioner and client cooperation, two dogs were selected based on the following criteria for a ‘high index of suspicion of ML resistance’ by D. immitis L3 at 12–15 months-of-age.Four male hound-cross dogs of 4–5 months-of-age were purchased from a USDA approved commercial supplier for experimental infections. All dogs were housed strictly in indoor runs in a mosquito-free kennel facility confirmed by periodic overnight sampling using a CDC Miniature Light Trap, Model 512 . All dogs tested negative for adult heartworm antigen  in serum samples collected prior to experimental infection and at 1, 2, 3, and 4 months post-infection. None of the dogs were treated with any ML in the holding period before experimental infection of dogs with Dogs were randomly assigned to two groups of two dogs for experimental infection by the two field isolates . At 27–33 days after infection, one dog in each group was treated with 12 μg/kg ivermectin diluted 1:9 in propylene glycol by the subcutaneous (SC) route. The untreated control dog received a similar volume of only propylene glycol by the SC route. At monthly intervals thereafter, dogs received respective ivermectin (12 μg/kg) or propylene glycol sham treatment for a total of 6 monthly treatments. Although not required to confirm resistance, the ivermectin monthly treatment was repeated six times, at double the recommended dose, in order to ensure the presence of resistance. Ivermectin drug  was from batch number BA132/10 .Aedes aegypti mosquitos using a membrane feeding apparatus . Mosquitoes were infected and held in an insectary for 14–16 days  prior to harvesting L3 according to the procedure of the NIAID/NIH Filariasis Research Reagent Repository Center (FR3), Athens, GA, USA [D immitis. Recovered L3 were held in RPMI tissue culture media at room temperature for up to 3 hours until SC inoculation. Group I (LSU 10) dogs each received 69 L3 (31 on 23rd March 2012 and 38 on 30th March 2012) and Group II (LSU 13) dogs each received 75 L3 (test) or 114 L3 (untreated control) on 15th June 2012. Group I dogs were infected twice, one week apart, because an adequate number of L3 larvae were not obtained following the initial harvest.Blood containing Mf from the two canine field cases was collected and used to infect laboratory-raised  GA, USA . Each do, and at two week intervals thereafter, to determine D. immitis infection status and the onset of patency. Blood samples were analyzed for adult heartworm antigen  and the presence of microfilariae was monitored using a 20 μL direct blood smear and the modified Knott’s technique [Whole blood samples were collected from each dog at monthly intervals for four months following experimental infectionechnique . Results3) and monitor the dog used to establish this second generation isolate of the LSU 10 strain (LSU 10-II). An ivermectin treatment dose at 4 times the recommended preventive level was used to further confirm strain resistance, which had previously been assessed at twice the recommended level.In separate follow-up studies, infective larvae raised from microfilariae from the treated Group I dog (LSU 10 strain) were used to confirm heritability of resistance in the face of a monthly ivermectin challenge dose of 24 μg/kg for 3 monthly treatments. Similar procedures to those previously described were used to infect .Of the 855 one-page mail surveys sent to all Louisiana practitioners in August 2009 regarding their opinions of ML LOEs reported within their clinics, 221 were returned (25.8% response rate). Of the 221 surveys submitted, 70% were from clinics classified as ‘small animal’ and 25% were from ‘mixed animal’ facilities. GIS analyses of survey results indicated there were focal locations with veterinarian perceived high rates of reported LOEs of ML drugs against heartworms and that numbers increased from 2005 to 2008. Pertinent survey questions with results are shown in Figure D. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and microfilaremia by the modified Knott’s test. Mf counts gradually increased post-patency in test and control dogs (Table Both treated and untreated dogs from Group I LSU 10) and Group II (LSU 13) developed patent 0 and GroMicrofilaremia in one of the dogs (LSU 10a), initially found in moderate numbers, became sporadic with biweekly counts of 2–8 Mf per ml of blood. Microfilariae from this dog (LSU 10a) were used to 1) maintain an adequate Mf level of LSU 10 strain to infect mosquitos for second generation passage of the to a new laboratory dog (LSU 10-II); 2) to confirm resistance in the face of challenge treatment by a 24 μg/kg dose of ivermectin given monthly for 3 months, and 3) to demonstrate heritability of the LSU 10 resistant strain. Monthly monitoring of the LSU 10-II dog showed it became positive for circulating antigen by 204 DPI and became patent with circulating Mf by 259 DPI.D. immitis isolates from two separate client-owned dogs  are thus currently available for further study:Five laboratory dogs harboring resistant strain LSU 10a ivermectin LSU 10b untreated controlLSU 10-II ivermectin LSU 13a ivermectin LSU 13b untreated controlD. immitis.We propose that we have successfully isolated two strains from proven cases of resistance in client-owned dogs in Louisiana that can be used to further study and characterize the emergence of ML resistance by Genomics studiesD. immitis infected dogs, were collected, preserved in isopropyl alcohol, and analyzed blindly in order to genotype the circulating Mf population for two single nucleotide polymorphism loci on a P-glycoprotein gene previously shown to be correlated with a loss of efficacy of macrocyclic lactone heartworm anthelminthics [www.qiagen.com). Also, individual Mf DNA samples had been amplified using Repli-g screening kit from Qiagen®, to increase DNA concentration of individual samples. Individual genotypes had been identified after PCR, Sanger sequencing, and analysis of chromatograms using Sequencher® (http://genecodes.com/). Table Samples of 20–30 individual Mf, from Group I and Group II minthics -7. The iD. immitis exist in the general population of Louisiana dogs, and these resistant strains can be identified by case history, in vivo tests and genetic markers to enable development of effective alternative drug therapies to extend the useful life of current preventive drugs’.The aim of the current study was to provide evidence to test the following hypothesis – ‘Resistant strain(s) of et al. [D immitis. Finally, a statewide survey was done of Louisiana veterinary practices in 2009 to identify the extent and geospatial distribution of company reimbursed cases  clinical history of failure of efficacy and full monetary compensation by a commercial pharmaceutical firm; b) residence in an area identified as a ‘hotspot’ of suspected drug resistance in the 2009 statewide veterinary practitioner survey of LOE cases [We adopted a four point ‘high index of suspicion’ criteria to enable identification and isolation of resistant strains of OE cases  and d) hOE cases .3 raised in A. aegypti from circulating microfilariae in blood from candidate dogs [Using these criteria for high index of suspicion, LADDL began searching for cases in high prevalence foci of infection in Louisiana by retrospective study of positive confirmatory antigen-based tests and Knott’s examination done on LOE cases referred to LADDL by veterinary practitioners in order to qualify for LOE case reimbursement by pharmaceutical firms. Two candidate dogs (LSU 10 and LSU 13) were identified in separate geographic locations Figure  that fitD. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and detection of microfilariae by the Knott’s test. Mf counts gradually increased in number of Mf after patency in experimentally infected dogs  [1) Wider use of the Mf reduction test in veterinary practices to identify suspect resistant cases ; 2) Deveant dog) ; 4) Use ant dog) ; and 5) ant dog) -30 or seant dog)  pending Additionally, a time-space surveillance system may be warranted to alert the veterinary community on the need for intervention by mapping the location of reported LOE cases and ‘high index of suspicion’ LOE cases that can be confidently attributed to true drug resistance, such as by future genetic marker testing. Regular updates by the FDA on the status of required LOE reports, with other records collected by pharmaceutical industry partners, may serve this purpose. The value of an effective surveillance system may be further enhanced by development of biology-based predictive models on the potential for spread of ML resistance based on climate/environmental suitability determinants, and use of ecological niche models for surveillance using geographic information systems (GIS) risk analysis methods -34.


AtMYB12 transcription factor on bone health including longitudinal growth. Constitutive expression of AtMYB12 in tomato led to a significantly enhanced biosynthesis of flavonoids in general and the flavonol biosynthesis in particular. Pre-pubertal ovary intact BALB/c mice received daily oral administration of vehicle and ethanolic extract of wild type (WT-TOM) and transgenic AtMYB12-tomato (MYB12-TOM) fruits for six weeks. Animal fed with MYB12-TOM showed no inflammation in hepatic tissues and normal sinusoidal Kupffer cell morphology. MYB12-TOM extract significantly increased tibial and femoral growth and subsequently improved the bone length as compared to vehicle and WT-TOM. Histomorphometry exhibited significantly wider distal femoral and proximal tibial growth plate, increased number and size of hypertrophic chondrocytes in MYB12-TOM which corroborated with micro-CT and expression of BMP-2 and COL-10, marker genes for hypertrophic cells. We conclude that metabolic reprogramming of tomato by AtMYB12 has the potential to improve longitudinal bone growth thus helping in achievement of greater peak bone mass during adolescence.Externally visible body and longitudinal bone growth is a result of proliferation of chondrocytes. In growth disorder, there is delay in the age associated increase in height. The present study evaluates the effect of extract from transgenic tomato fruit expressing  Both childhood and adolescence are the important periods for the growth of skeleton. During these periods, bone growth leads to increased size and strength of individual bones16et al., (2008) demonstrated that GAG synthesis and sodium [35S] sulfate incorporation in female bovine articular chondrocytes is affected by flavonoids, daidzein and genisteinPhytoestrogens have shown to have positive effect on chondrocytes8Arabidopsis, AtMYB1215AtMYB12 led to an increase in the longitudinal height of bones by supplementation to growing female mice. This growth is the resultant of endochondral proliferation of cells in growth plate region and that led to conversion of proliferating chondrocytes into hypertrophic cells and then to new bone formation.Flavonoids are plant secondary metabolites that act as phytoestrogens and exhibit a range of health beneficial properties including positive effect on bone health12AtMYB12 under control of constitutive promoter CaMV35S and WT plants were analyzed to confirm the reported modulation in flavonoid accumulation−1 FW; rutin, 1 ± 0.06 mg g−1 FW) as compared to the fruits of WT-TOM  (−1 FW) and kaempferol (0.7 ± 0.06 mg g−1 FW) in MYB12-TOM fruits as compared to WT-TOM fruits. As a reflection of enhanced polyphenol and flavonoid content, the total antioxidant capacity of MYB-TOM in terms of trolox equivalents was also enhanced more than 5 fold as compared to WT-TOM Homozygous transgenic lines expressing  g−1 FW) . To quano WT-TOM . Taken t−1.d−1) to growing female BALB/c mice daily for 6 weeks and different parameters were analysed  compared to control group and 28.57% (P < 0.01) compared to WT-TOM . MarkingGene expression analysis at the site of growth plate in MYB12-TOM group of animals shows significantly increased expression of BMP2 compared with WT-TOM and control group . Further-TOM group as compared to control group. Significant increase (P < 0.05) in the overall OPG/RANKL ratio, indicator of bone formation rather than bone resorption, was also observed in MYB12-TOM treatment group , Runt related transcription factor (RUNX2) and osteocalcin (OCN) in MYB12-TOM group of animals as compared to other groups –e. RUNX2nt group .At the end of the treatment period, trabecular architecture of isolated femur and tibia in all the groups was studied using 3D-μCT. μCT changes were observed in the tibial metaphyseal region, trabecular structural changes were clearly visible in control, WT-TOM and MYB12-TOM groups as represented in Height and growth rates are important indicators of health in infants and adolescents. Much more bone is deposited by the process of modeling versus remodeling as the skeleton grows in both size and density in children and adolescents. Most people of short stature do not have a pathological condition per se but merely exhibit some deviations from the normal parameters of growth. Our previous studies using phytopharmacological approach have shown beneficial effects for bone healthFlavonoid biosynthesis has been manipulated in different plant species through homologous or heterologous expression of regulatory and structural genes of flavonoid pathway1720Therefore in this study, we explored the effect of nutritionally rich transgenic tomatoes for longitudinal height attainment in growing female rodents. Contrary to males, in females the attainment of peak bone mass is restricted only to the pre-pubertal stage. When there is commencement of estrogen hormone at the time of puberty, it marks the reduce in longitudinal bone growth222324The epiphyseal plate region is mainly responsible for the elongation of the long bones. The bone elongation function is carried out by interstitial expansion of chondrocyte cells while one end is continuously replaced by a calcified matrix to convert to bone. This interstitial expansion happens through a combination of chondrocyte proliferation and enlargement of the lacunae that surround these cells. Our results show that MYB12-TOM treatment increased the expression of COL10 indicating increase in number of hypertrophied cells which in turn contributed to the expansion of the lacunae in the growth plate. From this data, it is apparent that MYB12-TOM may be responsible for the interstitial growth of the epiphyseal plate which, in turn, determined the rate of bone elongation .Peak Bone Mass (PBM) is influenced by a variety of factors including nutrition and physical activity which can improve bone health2689Through this study, we demonstrate that development of nutritionally-rich transgenic food can positively affect growing animals. We observed that the transgenic fruit extract rich in flavonoids affected cellular processes at the growth plate, metaphyseal bone, and bone marrow which directly impacted bone lengthening, bone mass accumulation, and modulation of cells towards osteoblastic lineage ,b. It is29In conclusion we demonstrate the effect of naturally occurring phytoestrogens on growing rodents through pathway engineering of flavonoid biosynthesis. This will address the problem of bone growth defects such as short height and delay in peak bone mass achievement in females. In the present study performed in pre-pubertal female mice, we show that MYB12-TOM significantly improves bone growth by increasing the height of growth plate via increased number and size of hypertrophic chondrocytes that promotes longitudinal growth with markedly improved bone microarchitecture.et al., (2014)Pusa early dwarf (tomato) WT as well as AtMYB12 expressing transgenic lines have been used in this studyad libitum. After acclimation for 48 hr, 10 mice were randomly assigned to three groups. The proposed study was performed to compare the in vivo effect of the continuous administration of CONTROL and WT-TOM (tomato fruit crude extracts) versus MYB12-TOM (transgenic tomato standardized fruit crude extracts) (100 mg kg−1 d−1) for a period of 6 weeks.All experiments were performed in accordance with IAEC  guidelines. Experimental protocols were approved by CSIR-Central Drug Research Institute, Lucknow. We obtained 21 days old female BALB/c mice weighing 12–15 gm each from animal house. All animals were individually housed at 22-24 °C in 12hr/12hr light-dark cycles. Normal chow diet and water were provided Following treatment with WT-TOM and MYB12-TOM, liver tissue from different groups were collected and fixed in 4% paraformaldehyde. Sample were dehydrated in ascending grades of isopropanol, cleared in xylene and embedded in paraffin wax using standard procedures. Transverse sections of 5 μm were stained with haematoxylin and eosin and representative images were captured using Nikon Eclipse 80i−1), trabecular connection density (mm−3), and trabecular separation (Tb.Sp) (mm), trabecular porosity were directly measured on three-dimensional images. The trabecular bone pattern factors (Tb.pf) (mm−1) and the structure model index were computed using software provided with the μCT machine.Micro–computed topographic  determination of excised bones was carried out using the Sky Scan 1076 μCT scanner  as described in previously published reports313233The bones were excised during autopsy, cleaned and collected in RNA later. For RNA isolation, bones were cut in two parts according to one upper head part with growth plate region for chondrogenic genes and below growth plate portion for osteo- and adipogenic genes expression study. We marked the bones and cut them by fine bone cutter blade . After separation the bones were separately crushed in liquid nitrogen and RNA was isolated by trizol method for qPCR experiments. cDNA was synthesized with a RevertAid cDNA synthesis kit , using 2 μg of total RNA. SYBR green  chemistry was used to perform the quantitative determination of relative expression of all genes. Real Time PCR (qPCR) was performed to assess the expression of osteoblast, adipocytes, and chondrocyte specific genes from femur and tibia of mice from our previously published protocol3536353637During the time of autopsy, femur and tibia were dissected out, cleaned and decalcified in 2% EDTA. For morphometric analysis of the growth plate region, three sections of 5 µm size of bone were obtained from tibia after decalcification and were mounted on a glass slide. Paraffin sections were de-waxed re-hydrated and were stained with haematoxylin and eosin (H&E). The total width of the growth plate at the proximal end of each tibia was measured along longitudinal axis oriented 90° to the transverse plane of the growth plate. At least ten measurements were obtained from each epiphyseal growth plate and final width determination in individual animals indicated the average of these values3840Mice treated with CONTROL, WT-TOM and MYB12-TOM groups were used for immunohistochemistry of COL10a. Immunohistochemistry was performed as per previously published protocolAll of the COL10a immuno-stained sections were reviewed and scored independently in a blinded manner, based on the H-score method, which reflects the staining intensity collected with the percentage of cells staining positively. For H-score method, 10 fields were chosen randomly at 40× magnification. The staining intensity in the cells was scored as 0, 1, 2 and 3 corresponding to the zero, weak, intermediate and strong staining, respectively. In each field, the total number of cells as well as cells stained at each intensity was calculated. The H-score was calculated following the formula: (% of cells stained at intensity category 1 × 1) + (% of cells stained at intensity category 2 × 2) + (% of cells stained at intensity category 3 × 3). H-scores varied from 0 to 300 where 300 represented 100% of cells strongly stained (3+)43Data are expressed as mean ± SEM unless otherwise indicated. The data obtained in experiments with multiple treatments were subjected to one-way ANOVA followed by post hoc Newman-Keuls multiple comparison test of significance using GraphPad Prism 5.0 software. Qualitative observations have been represented following assessments made by three individuals blinded to the experimental designs.How to cite this article: Choudhary, D. et al. Genetically engineered flavonol enriched tomato fruit modulates chondrogenesis to increase bone length in growing animals. Sci. Rep. 6, 21668; doi: 10.1038/srep21668 (2016).


A major question in neuroscience is how diverse subsets of synaptic connections in neural circuits are affected by experience dependent plasticity to form the basis for behavioral learning and memory. Differences in protein expression patterns at individual synapses could constitute a key to understanding both synaptic diversity and the effects of plasticity at different synapse populations. Our approach to this question leverages the immunohistochemical multiplexing capability of array tomography (ATomo) and the columnar organization of mouse barrel cortex to create a dataset comprising high resolution volumetric images of spared and deprived cortical whisker barrels stained for over a dozen synaptic molecules each. These dataset has been made available through the Open Connectome Project for interactive online viewing, and may also be downloaded for offline analysis using web, Matlab, and other interfaces. This complexity at first seems daunting, but may in fact enable researchers to better characterize synaptic populations and their experience-dependent modification. In particular, the definition of different synapse types based on characteristic expression of distinct molecular constituents by may enable identification of subpopulations involved in learning, while identifiable patterns of molecular modifications at existing connections may be used to reveal functional changes in synaptic strength8.Many hundreds of distinct proteins are involved in the development of synapses and the mechanics of synaptic signaling10. The ability to measure the molecular composition of many individual synapses in the context of the larger circuits they comprise should greatly enhance our understanding of the roles of diverse synapse subsets in neuronal information storage and plasticity. (See Array tomography (ATomo) is uniquely well suited to proteomic mapping of synaptic circuits. Ultrathin sectioning of resin-embedded tissue samples enables immunohistochemical multiplexing and high-resolution imaging of millions of synapses in situ, followed by computational reconstruction into precisely aligned image volumes12 (see 13. Theneurons immediately above and below a given barrel (the ‘barrel column’)normally respond primarily to stimulation of a single principal whisker (PW), but alsoexhibit weaker surround-whisker (SW) responses. Loss of input from the column’s PWdepresses the column’s response to this whisker, while potentiating responses tospared SWs, which can come to dominate circuit activity in the deprived column15.This produces a remapping of the barrel field such that the representations of sparedwhiskers expand into neighboring deprived columns. Plasticity of many different components of the barrel column circuit have been implicated in this remapping, including excitatory and inhibitory synapses in thalamorecipient L4, superficial L2/3 and deep L5 . The whiskerregion of S1 is organized as a grid-like spatial map, with rows of thalamically-innervatedlayer 4 (L4) ‘barrels’ corresponding to the layout of whiskers on theface12 see . ThecirL5 refs.  and 18.19–21. We developed a method to precisely dissect pairs of neighboring barrel columns for ATomo imaging, and produced volume reconstructions of stains against many functionally important synaptic molecules in these adjacent spared and deprived columns. Additionally, this data contains cell-type specific transgenic labeling of layer 5 pyramidal neurons with YFP23, as well as immunohistochemical labeling of PV+ interneurons24–26. Thus, synaptic subpopulations may be classified based on characteristic molecular signatures or association with YFP+ pyramidal neurons or PV+ interneurons, and the prevalence of these populations as well as changes in expression of molecules thought to play a role in experience-dependent plasticity may be compared between spared and deprived columns.In our experiments, we trimmed alternating facial whiskers of adult mice in a ‘chessboard’ pattern, every 2-3 days for 7 days, which has been shown to produce significant functional and structural plasticity in barrel cortex well into adulthood8. We make this dataset publicly available alongside two supplementary datasets (see Data Records) in the hopes that other researchers will apply their own creative approaches to discovering patterns of synapse molecular diversity and novel signatures of experience dependent cortical learning.The Chessboard Dataset described here comprises over 6 million cubic microns of neocortical tissue within a well-defined microanatomical structure, stained for over a dozen different synaptic proteins and imaged at synaptic resolution. As such, it is likely to be a valuable resource for researchers interested in statistical characterization of the diversity of protein expression patterns in mouse neocortical synapses and the effects of experience-dependent plasticity on these patterns22 purchased from Jackson Labs (Strain Name: B6.Cg-Tg(Thy1-YFP)HJrs/J, Stock number: 003782). Mouse Ex1 was excluded from the Chessboard Dataset (see Data Records) because it was found not to express YFP. Thereafter, we used the Transnetyx genotyping service to restrict experiments to YFP-positive individuals based on the results of automated rtPCR comparison with JAX gene sequences.All procedures related to the care and treatment of animals were approved by the Administrative Panel on Laboratory Animal Care at Stanford University. Animals used to generate these datasets were derived from hemizygous transgenic founder mice of the thy1-yfp H line27. Mice were excluded from these dataset if any whisker loss was observed in addition to the chessboard trimming.We induced experience dependent map plasticity in 8 adult male mice (P63-70) by unilaterally trimming facial whiskers on the right side of the face in a chessboard pattern  every 2-3 days for 7 days . We trim28 to localize barrel columns C1 and C2 for dissection. Mice were sedated with intraperitoneal injections of 0.05 ml chlorprothixene and anesthetized with isoflurane (0.8% in O2). Mice were kept on a heating pad, and we ensured that mice remained at a stable level of anesthesia by monitoring their core temperature and tracking their respiration rate. We also locally anesthetized the scalp with subcutaneous injections of ~0.01 ml lidocaine. We covered the eyes with petroleum jelly and provided hydration with a pre-operative subcutaneous injection of 0.5 ml physiological saline.Following 7 days of chessboard deprivation, we imaged whisker-evoked intrinsic optical signals (IOS)We stabilized the heads of the mice with ear-bars and surgically exposed a region of the skull overlying the left somatosensory cortex. We then affixed a custom metal head-plate with a 2 mm round imaging window to the exposed skull approximately centered on the location of left barrel cortex, and clamped this head plate to a metal post attached to the imaging apparatus. Once the mice were thus head-fixed, we removed the ear-bars, filled the imaging window with warm low-melting-point agarose, and covered the window with a round glass coverslip.29. We measured IOS with the camera focused 500 microns beneath the cortical surface to maximize signals originating L4 barrels.To identify the location of barrel-columns C1 and C2 without producing additional plasticity in these columns by direct stimulation, we used IOS imaging to map the representations of adjacent spared whiskers . In C2-s30. Clear IOS peaks were generally detected within 15 bouts (~5 min) of stimulation. For each whisker, once an IOS peak was established, we photographed the vascular pattern at the surface of the cortex with contrast enhanced by 530 nm green LED illumination (LumiLEDs). Because the position of the camera relative to the imaging window could shift slightly as we switched whiskers, we registered the vascular images and their paired IOS peaks to map the location of each whisker-associated peak relative to the surface vasculature  of changes in reflectance at this frequency. This FFT-based method allows fast and robust detection of stimulus-associated IOS peaks with little interference from other cardiovascular rhythms, which occur at much higher frequenciesculature .31. We estimated the locations of the C-row barrel columns by registering in vivo images of the surface vasculature collected under 530 nm illumination to the visible vasculature of the fixed somatosensory cortex tissue. We used tissue paint (Polysciences) to help reveal additional vascular features in the fixed tissue and to highlight the estimated position of the C-row of the barrel field el field . We embeel field , as estiWe confirmed that our punch contained the C1 and C2 barrel columns by making 80-micron sections of the somatosensory cortex tissue remaining after the tissue punch procedure (hereafter ‘remainder tissue’), and stained these for CO to reveal the pattern of the barrel field in layer 4. We then registered our standard barrel field map to the CO stain of remainder tissue sections to reveal which barrels overlapped with the punched-out hole in these sections .The CO-stained sections also allowed us to estimate the precise position and orientation of the columns contained within the embedded tissue-punch in order to plan the sectioning of ribbons for ATomo . For thi32.We dehydrated the tissue punches containing C1 and C2 columns in ascending alcohols up to 80% to retain YFP fluorescence, and embedded them in LR White resin according to previously published methods33. We collected 40–100 ribbons from each embedded block, enough to contain all or most of the targeted pairs of barrel columns. Although we could in principle attempt to reconstruct the columns in their entirety, the scale of such a project was outside the capabilities of our current image acquisition and reconstruction pipeline. Instead, for each block we identified the ribbon that best approximated a plane passing through the centers of both neighboring barrels for imaging and reconstruction  while others were cut from the B-side , producing different left-right orientations of the columns in the final data volumes See .ATomo Image Processing and Reconstruction, below), which aided in selecting ribbons taken from closest to the barrel centers, based on their larger barrel hollows visible as lower DAPI and YFP density. Once a ribbon was selected, the 25x DAPI/YFP images were also used to identify boundaries between columns and layers to guide higher magnification imaging of layers 3–5a in columns C1 and C2. Column boundaries could be detected by the higher density of YFP dendrites running between columns. The centers of L4 barrels were identified by their low DAPI density compared to higher density ‘septal’ regions between barrels, which is a characteristic feature of mouse barrel cortex15.For all imaging experiments, we first create a low-resolution overview of the ribbon to be imaged by making a mosaic image at low magnification (10x) of a DAPI stain for cell nuclei included in the mounting medium (molecular probes ref S36939) . To ensu35. For all sections in each ribbon, we imaged a region of the section corresponding to layers 3-5a in all of the columns present . Automated mosaic imaging of the selected region was targeted using custom software . For several ribbons, to minimize focus artifacts resulting from deviations of the 70 nm sections from the objective’s limited focal plane, z-stacks of each mosaic position were imaged and then computationally merged using an extended depth of field algorithm (Zeiss Axiovision).For selected ribbons from each chessboard-deprived animal, we performed 6–8 rounds of triple or quadruple immunostaining, paired with high-resolution imaging with a 63x plan-apochromat objective (Zeiss)All custom code described in this section is available for download athttps://smithlabsoftware.googlecode.com.Following imaging, we used plugins available in the FIJI image processing software as well as custom routines to create high-dimensional volume reconstructions of the imaged tissue with all of the antibody stains in registry. First, all images were background subtracted using a 20 pixel rolling ball filter (FIJI/ImageJ) and deconvolved (Matlab) based on empirical point-spread functions measured with fluorescent tetraspeck beads. Following deconvolution, the effective resolution of the images was 100×100×70 nm ref. .We used a DAPI nuclear stain present in our mounting medium in all imaging sessions as a fiducial marker both for stitching together adjacent microscope fields of view and registering images of the same tissue sections taken in different imaging sessions (see below).37. These features were filtered using RANSAC38 to detect inliers specifying the model for rigid transformation between sessions, which could then be applied to images from the other channels of that session to bring them into registry with the first session.Minor shifts in the position of the coverslip-affixed ribbon between imaging sessions can be simply modeled as rigid linear transformations to achieve appropriate session registration using custom FIJI/ImageJ plugins. These transformations were calculated based on SIFT features detected in the corresponding DAPI images from each imaging session39. This channel was used for fine alignment because it densely and reliably stains synapses across multiple 70 nm sections. The alignment calculated from Synapsin1 was then applied to all of the other channels already in registry to produce full multi-channel reconstructed data volumes.Unlike the rigid transformations used for 2d registration between imaging sessions, mechanical deformations of the tissue during the process of sectioning make the transformations needed for 3d alignment of ultrathin serial-section images significantly nonlinear. Synapse-scale alignment was achieved with another custom FIJI/ImageJ plugin by applying the elastic algorithm developed by Saalfeld and colleagues to the Synapsin1 channelThere are 3 data records associated with this data descriptor, all derived from serial-section ribbons from 9 chessboard-deprived mice , imaged The primary Chessboard Dataset consists of 12 reconstructed data volumes  Individual data files for each channel in each volume reconstruction are also available through FigShare (Data Citation 1), as files 8–295 described in The Chessboard Dataset is hosted for interactive browsing and download through the Open Connectome Project (OCP) at Please note that the number of animals, imaged ribbons, and derived data volumes differ because (1) two ribbons from mouse Ex12 were imaged and (2) multiple barrel columns from ribbons Ex2R18, Ex3R43, and Ex6R15 were reconstructed separately into 2-3 columnar data volumes per ribbon. Also, note that animal ribbon Ex14-R58 was subject to several significant problems during antibody staining (see http://openconnecto.me/#!services/chru.There are two ways to access the Chessboard Dataset through OCP: 1) interactively browsing through entire data volumes over the web through OCP’s CATMAID interface, which is ideal for quick visual inspection of the data, or 2) by downloading specific data regions  through OCP’s HDF5 interface, which is ideal for handling the data for analysis. OCP also has its own API for those wishing to interact more deeply with the project’s data structures. For the most up-to-date descriptions of OCP’s services, visit http://catmaid.org/). OCP uses this interface to enable fast, browsable online data viewing as well as collaborative data annotation. To view the data, visit http://openconnecto.me/catmaid and browse projects labeled ‘Array Tomography ’. You may view these projects only by selecting Home> Array Tomography in the upper left-hand corner of the browser window. We have curated several useful multi-channel volumes to explore, but users may also construct their own combinations of channels to visualize different molecular relationships using hdf5 data cutouts (see below).CATMAID stands for Collaborative Annotation Toolkit for Massive Amounts of Image Data .http://tinyurl.com/OCP-cutout-wiki. We have prepared sample scripts for data access using MATLAB at https://gist.github.com/ncweiler. The cutout service also enables collaborative annotation of the data . These metadata annotations are registered to the original data and can be either used privately or shared publicly. See more details on data annotations at http://tinyurl.com/OCP-annotation-wiki.For additional documentation of the cutout service, see the OCP wiki page at the following address: Prior to the high-resolution, high-magnification imaging that generated the Chessboard Dataset image volumes, we first imaged intrinsic YFP and DAPI-stained cell bodies in each ribbon at low magnification. These images allowed us to create a roughly aligned reconstruction of the complete ribbons, which we then used to help identify anatomical layers and columns for the subsequent high-magnification imaging that generated the Chessboard Dataset image volumes. 2d projections of these whole-ribbon reconstructions, created using either a standard deviation projection or maximum intensity projection (FIJI), are also made available through FigShare as RGB tiff images to support the identification and analysis of specific anatomical regions (Data Citation 2).The supplementary Elution-Test Dataset consists of data volumes derived from seven short ribbon segments derived from mouse Ex1  was imaged at four non-consecutive tissue sections, preventing volumetric reconstruction.The Elution-Test Dataset may be downloaded from Figshare (Data Citation 1), as files 1–7 described in See Note that the OCP web addresses as written will display the full extent of the first slice (z-section) of a multi-slice volume image. To view a smaller region in x or y, adjust the values in the second-to-last and third-to last terms in the HDF5 web address. To view a different slice, adjust the final term in the HDF5 address. All antibodies used in this study have been validated by their originators as reliable and specific to the antigens of interest, and tested within our laboratory for reliable and repeatable staining of ATomo ribbons of LR-white embedded tissue sections. We make sure all antibodies stain in patterns that correspond to their expected synaptic localization. That is, for example, each GABAergic antibody was screened to confirm that it co-stained a significant number of putative GABAergic synapses defined by multiple other GABAergic antibodies, and that it did not excessively stain within cell bodies or blood vessels, where synapses would not be expected.We have quantified these expected staining patterns by performing a pairwise 2-dimensional cross-correlation analysis to quantify spatial correlations  between the principal antibodies used in these experiments. Pairwise cross-correlation plots were generated by shifting pairs of registered antibody channels against each other in x and y and calculating 2d image correlations for each spatial shift.These pairwise cross-correlations demonstrated sharp central peaks for colocalized antibodies, and broader noisier values for poorly colocalized antibodies. Examining the cross-correlations at small 2d shifts between images reveals that pairs of antibodies which are expected to colocalize within either pre- or postsynaptic compartments  have sharp peaks of correlation, while pairs of antibodies which represent associated pre- and postsynaptic compartments  have broader, more diffuse cross-correlation peaks .To quantify these correlations, we measured the absolute peak of the cross-correlation, andcalculated a half-maximum value based on the average background correlation. Using Matlab, wealso calculated a 2d contour of the cross-correlation peak at the half-maximum value, andmeasured the area of this contour and calculated the diameter of a circular contour with thesame area. These values are displayed in 9. In the present datasets, we also observed comparable staining quality irrespective of the multiple rounds of stripping and staining.In previously published work, we demonstrated that multiple rounds of elution have minimal effect on tissue antigenicity as quantified by the consistency of staining with an antibody against Synapsin1 across multiple rounds of imagingTo further test the consistency of our staining procedure, we have performed three rounds of repeated staining with fifteen key antibodies on ribbons equivalent to those used to produce the Chessboard Dataset. We used 2d image correlation to assess the similarity of staining between the first and third rounds of imaging for each antibody . To compSince the large proportion of dark background pixels could be contributing significantly to these image correlations, we also computed a ‘percent consistent’ metric that measured the proportion of bright pixels that were bright in both images. We set an approximate threshold between foreground and background at 1,000 a.u., and quantified the proportion of pixels above this threshold in either imaging session that were also above threshold in the other imaging session. As with the correlation metric, we compared this ‘percent consistent’ metric between the observed colocalization and the ‘random’ colocalization achieved by rotating one image 180° . This piWe find that the antibodies against Synapsin1 and PSD95 produce highly consistent stainingbetween sessions, while the rest (with the exception of NR2A) ranged from relatively good torelatively poor consistency, when compared with the extremely low values expected by randomcolocalization . This anAn important result of this analysis was that the NR2A antibody appeared to be completely unreliable, performing much more poorly than expected compared to random colocalization. Previous observations by our lab (data unpublished) and the correlation analysis presented in The stochasticity observed in some antibodies will be an important consideration in anyanalysis of the Chessboard Dataset. We do not yet have an adequate biophysical model to explain the source of this variability, which is why we have made this Elution-Test dataset available through Figshare for download and further analysis http://openconnecto.me/ocp/ca/[data token]/[orthogonal-view]/[channel token]/[resolution]/[x-range]/[y-range]/[z-range]/Template: http://openconnecto.me/ocp/ca/Ex6R15C2/xy/DAPI-1/0/700,1700/500,1500/15/Example: Produces cutout of DAPI-1 channel from data volume Ex6R15C2 at full resolution (‘0’) for x-range 0–1400, y-range 0–700, and z-slice 15.Result: Multi-Channel 2d Web-View http://openconnecto.me/ocp/ca/[data token]/mcfc/[orthogonal-view]/[channel tokens]/[resolution]/[x-range]/[y-range]/[z-range]/Template: http://openconnecto.me/ocp/ca/Ex6R15C2/mcfc/xy/0,0,0,Synapsin1-2,PSD95-1,DAPI-1/0/700,1700/500,1500/20/Example: Produces image of Synapsin1-2 (red), PSD95-1 (green), and DAPI-1 (blue) channels from data volume Ex6R15C2 at resolution 0 for x-range 0–1400, y-range 0–1400, and z-slice 20.Result: Channels are displayed as Cyan, Yellow, Magenta, Red, Green, Blue in order listed. Use placeholder channel token ‘0’ as above to skip colors.Note: Multi-Channel 2d Matlab Download https://gist.github.com/ncweiler/8075665Script: This script downloads a specified 2d data cutout as a false-color png file. It also creates a data object in Matlab containing an RGB image of the cutout and displays the data as a false-color image. Channel colors are determined as in CYMRGB multi-channel false color web view above.Result: Multi-channel Volume Matlab Download (hdf5 data file)https://gist.github.com/ncweiler/7953445Script: This script downloads a specified multichannel 3d data cutout as an hdf5 file. It creates a data object in Matlab containing one image stack for each channel. A user can then display specified slices of each channel stack.Result: As in the above examples, the following arguments will be needed to access data cutouts through the HDF5 interface:Data tokenTo create a data cutout from a particular data volume, you must use the associated data volume token. For example, the token ‘Ex10R55’ would be used to access the volume reconstruction of that ribbon.Channel Token(s)Desired protein channels may be specified either by name or by numeric channel token . Alternatively, see A list of available public data tokens is available here: ATomo’s ability to sequentially stain multiple different synaptic proteins and image their localization at high resolution presents the opportunity to define synapses by the spatial colocalization of multiple synaptic molecular constituents, whereas measurements of single antibodies could be noisy or misleading. See Synapsin1 is a vesicle-associated actin-binding protein. Staining for Synapsin1 is found at the majority of glutamatergic and GABAergic synapses in cortex, although we have observed it to be brighter at glutamatergic synapses10. This highly robust stain (see 40. Expression of distinct isoforms of the vesicular glutamate transporter (vGluTs) has commonly been used to distinguish between axonal boutons of vGluT1-expressing intracortical synapses and vGluT2-expressing thalamocortical synapses. Some presynaptic terminals stain for both vGluT1 and vGluT2, which are likely to originate from a subpopulation of thalamocortical neurons expressing vGluT1 in addition to the typical thalamically expressed vGluT2 , an enzyme responsible for synthesizing the neurotransmitter GABA, and vGAT, a vesicular GABA transporter, both stain presynaptic terminals of inhibitory synapses and can be used to differentiate these from excitatory synapses56–58. vGAT protein is thought to be translated locally at GABAergic and glycinergic boutons, making this stain highly specific for inhibitory synaptic (as opposed to extrasynaptic) loci58.Antibodies against glutamate decarboxylase Ra1, has been stained for in these experiments. This subunit is present in one of the largest subclasses of GABA(A) receptors and so can be used in combination with gephyrin staining to identify and subclassify the postsynaptic elements of GABAergic synapses62.GABAergic synapses express a distinct postsynaptic scaffolding protein, Parvalbumin (PV) is a calcium binding protein expressed by a large subclass of GABAergic neurons, including fast-spiking basket cells and chandelier cells. This stain can therefore be used to identify presynaptic GABAergic terminals belonging to these cell classes63. PV+ basket cell and chandelier cell synapses can be readily distinguished from each other, as basket cells make en passant and terminaux synapses primarily onto postsynaptic cell bodies, whereas chandelier cells make distinctive long ‘cartridge’ synapses onto the axon initial segments of postsynaptic neurons65.Synapsin1 and PSD95 staining could be used as measure of synaptic potentiation or deprivation as a result of experience66–68. Whisker deprivation in adult mice has recently been shown to increase the total density of Synapsin1 staining and postsynaptic spines in spared cortex, but only after complete unilateral whisker trimming69. Chessboard deprivation, which we chose to promote Hebbian circuit-level plasticity rather than the wholesale homeostatic plasticity expected from a complete removal of whisker input71, may not have such drastic effects. Indeed, several long-term in vivo imaging studies have shown significant dendritic spine turnover, but not wholesale changes in total spine number in particular populations of L5 pyramidal neurons72, implying that chessboard map plasticity may have more to do with altering specific circuit connectivity than massively altering connectivity per se.The size of both pre- and post-synaptic compartments has been correlated with synaptic strength, and so differences in the size and intensity of 73–75. The relative brightness of immunostaining for the different receptor subunits at individual synapses could be used to define postsynaptic receptor ratios, which are thought to be highly important for regulating Hebbian, homeostatic and meta-plasticity. In particular, the ratio of NMDARs and AMPARs can been used as a proxy for synaptic potentiation77. In addition, changes in AMPA receptor subunits GluR1 and GluR2 are associated with Hebbian and homeostatic synaptic plasticity78–81, while changes in expression of NMDA receptor subunits NR2A and NR2B are associated with developmental and adult metaplasticity82–88. GluR1, in particular, has been implicated as a key player in map plasticity driven by chessboard deprivation89.Differential expression of glutamate receptor subunits is also indicative of synaptic state, and can be used to characterize synaptic strength and explore the mechanisms of experience dependent synaptic changeNR2A antibody we have used exhibits a very low signal to noise ratio. As a result, we recommend being highly cautious in interpreting this particular antibody channel.We should also note that although we have frequently observed good spatial colocalization between all of these glutamate receptor subunit stains and PSD95-stained postsynaptic compartments R concentration at GABAergic synapses have been linked to forms of inhibitory plasticity, which could be detected by changes in staining intensity of these molecules at synaptic subsets91–97. Shifts in inhibition of spared versus deprived whisker responses are thought to be involved in certain types of whisker map plasticity99, and chessboard deprivation, in particular, is thought to keep open cortical critical periods through an effect on PV+ intracortical inhibition71.Alterations in Arc is an immediate early gene product whose synaptic expression is modified by neural activity. It is thought to have a role in synaptic depression and potentially other forms of synaptic plasticity such as synaptic homeostasis100–103. However, the antibody we used here proved extremely noisy in the initial ribbons we stained for it, and we discontinued its use in later ribbons. Any analysis of this channel should be approached with caution.YFP expression, in some cases amplified by an antibody against GFP, labels a subpopulation of layer 5 pyramidal neurons in the thy1-yfp line-H mice used in these experiments104. In vivo two-photon microscopy studies have observed increased dendritic spine turnover in the distal dendritic tufts of these neurons following chessboard deprivation similar to that performed in this experiment16–18. Analysis of this ATomo dataset may permit quantification of additional spine plasticity in deeper layers of the cortex. Synaptopodin is a protein associated with the spine apparatus, a structure observed at a subset of large dendritic spines and thought to be involved in synaptic plasticity106.Intrinsic transgenic 108, and ourown analysis of the antibodies used in this study .


Protein misfolding cyclic amplification (PMCA) and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease . However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods -  Prnp gene. While TSE present sporadic and familial origins, they also can be acquired. Transmission can be achieved by several ways. In humans, a novel form of CJD, named variant (v) CJD and first reported in 1996, likely results from the infection by BSE agent through alimentation Prnp and possibly other polymorphisms. Importantly, prions replicate in tissues other than the nervous system, mainly lymphoid tissues. It is notably the case for vCJD and eventually the clinical phase could even not appear resulting in an asymptomatic carriage of vCJD infection Transmissible spongiform encephalopathies (TSE) or prion diseases are deadly infectious neurodegenerative diseases naturally affecting a broad range of mammalian species including humans (Creutzfeldt-Jakob disease-CJD), goats, sheep (scrapie), cattle  and cervids (chronic wasting disease-CWD). Mammalian prions are thought to consist in aggregated and misfolded forms of a host protein, the prion protein (PrP), encoded by the Distribution of prion infectivity among blood components may substantially vary depending on the host species and/or the prion agent. While previous studies in rodents in vitro prion amplification to optimize the detection ex vivo assays have been developed to detect brain-derived prion infectivity without resorting to animal inoculation Sensitive and accurate detection of blood prions are required to design pertinent tests for non invasive early diagnostic, to improve the safety of blood transfusion and blood-derived products and to study the species/agent-dependant blood prionemia in infected animals or individuals. In the recent years, blood detection tests targeting abnormal PrP have been developed, some of which - Protein Misfolding Cyclic Amplification (PMCA) In this work, we aimed at comparing in the same experimental model the relative sensitivity and specificity of these different assays to detect prion in blood. We chose sheep experimentally infected with classical scrapie to isolate WBC and platelets at different stages of the incubation period. Infectivity of the different samples was tested by PMCA and bioassay in ovine PrP transgenic mice (tg338) mice. WBC were also subjected to the ovRK13 cell-based assay and to the Cerebellar Organotypic Slice Culture Assay (COSCA). Our data show that cell-based assay and COSCA are sensitive enough to detect prion infectivity in WBC from asymptomatic sheep. This study confirms the earlier finding that WBC are a suitable target for preclinical detection of infection and extends the number of complementary tools for prion detection in blood cells.All animal experiments have been performed in compliance with our institutional and French national guidelines, in accordance with the European Community Council Directive 86/609/EEC. The experimental protocols were approved by the INRA Toulouse/ENVT ethics committees, and were performed in the approved animal facilities (C 31 555 27) of author's institution by an agreed experimentator (311055503). All efforts were made to minimize suffering.Oral inoculation of the PG127 sheep isolate has been described in detail previously Whole blood was collected from the jugular vein using citrate dextrose (35 mL) in a 250-mL blood collection pouch . Preparation of white blood cells and platelet samples were as described previously res was confirmed in clinically-affected mice.Mouse bioassays were carried out in ovine VRQ PrP transgenic mice (tg338), which are highly sensitive for detection of PG127 sheep scrapie infectivity nd round reactions were seeded with 1/10 of the first round. Samples were than processed for PrPres analysis by immunoblotting.PMCA in a Misonix 4000 sonicator was carried out as previously described Cerebella were dissected from 10- to 12-day-old tg338 transgenic mice (tg338) over-expressing the VRQ allele of the ovine prion protein 5 cells/μL. For quantification purposes, serial dilutions of brain stock prepared from terminally ill tg338 mice experimentally infected with PG127 scrapie strain and a 10−2 dilution of a brain from a non-infected tg338 mouse (negative control) were prepared in SCM. After 7 to 10 days in vitro (DIV), WBC suspensions (106 cells/slice) and brain dilutions (2 μL/slice) were applied on slices (8 to 10 slices per condition). The slices were then culture for an additional 42 DIV.Frozen pellets of WBC were resuspended in SCM at 5.10Res was extracted by the Bio-Rad test protocol, by using 20 µg/mL proteinase K (PK) for 10 min at 37°C. After denaturation, the samples were run on 12% Criterion XT Bis-Tris gels (Bio-Rad), electrotransferred onto nitrocellulose membranes (Bio-Rad), and immunoblotted with biotinylated anti-PrP antibody Sha31b Slices were harvested by scrapping and homogenized at 20 µg/μL in 5% sterile glucose solution with a Rybolyser . For analysis, samples corresponding to 50μL of homogenate  were processed then loaded on the gel. Briefly, PrP7 cells) were homogenized in 200 μL of a 5% sterile glucose solution with a high-speed homogenizer (TeSeE Precess 48 system) and diluted to 800 μL with cell culture medium . The cell-based assay procedure has been described previously g, 1 min) and cellular proteins in the post-nuclear supernatants were quantified by bicinchoninic acid . Digestion of 750 µg of proteins with PK  was performed for 2 h at 37°C with a mass ratio of 4 µg of PK per mg of cellular proteins and the reaction was stopped by addition of Pefabloc (Sigma-Aldrich) to 4 mM. PK-digested samples were centrifuged for 30 min at 20,000×g and pellets were analyzed by western blot. Samples separated by 12% SDS-PAGE electrophoresis were transferred to PVDF membranes (Bio-Rad). The western blots were stained for PrP with Sha31 mAb WBC , previously end-point titrated in tg338 mice, has a titer of 10res amplification was observed for all platelets samples collected 80 dpi onwards. Notably, consistent and robust detection was achieved even for platelets samples that showed incomplete transmission to tg338 mice (see below). This is a further illustration that PMCA is more sensitive than bioassay for detection of prions res amplification was not achieved and no PrPres was observed with platelets collected before inoculation. For the bioassay, groups of six tg338 transgenic mice over-expressing the VRQ allele of the ovine prion protein res) deposition in brain. Platelet homogenates corresponding to 15 mL of plasma prepared at the different time points from the 5 donor sheep were inoculated and the results are presented in Platelets samples were used to seed PMCA reactions using tg338 mice brain homogenate as substrate. Each sample, run in 5 replicates, was subjected to 2 successive rounds. The results are shown in res amplification   after 100-fold dilution. These results confirm the early and persistent prionemia associated with WBC already observed in this experimental model. In PMCA as in bioassay experiments, apparent infectivity associated with platelets is weaker than apparent infectivity associated with WBC, thus WBC only were used to compare the four different methods.WBC homogenates were tested by PMCA in the same conditions than platelets. All WBC samples collected at 50 dpi and later lead to PrP and D5) . At late and D5) . Howeverres as a marker of prion infection. The large majority (9 out of 10) of the WBC samples assayed and collected at days 80, 130 and at the terminal stage of the disease (180 dpi) lead to infection of the recipient ovRK13 cells, as assessed by western blot detection of PrPres  and the presence of PrPres was assessed by western blot analysis. We first compared, by endpoint titration the sensitivity of the tg338-derived COSCA to that of the tg338 mouse bioassay, by using a pool of 127S brain homogenate . The equivalent of 4 nanograms of 127S-infected tg338 brain was sufficient to elicit PrPres detection in slice cultures after 42 DIV  r 42 DIV , n = 2. cultures . Only oncultures . Slices uivalent .ex vivo methods (cell-based assay and COSCA) are sensitive enough to detect prion infectivity in WBC of preclinical scrapie sheep, and as such represent an ethical alternative to mouse bioassay to study blood prionemy. Studies with cell culture models have reported that live cells transmit infection much more efficiently than dead cells All of the methods tested in this work confirm that WBC are the most suitable candidates to screen for blood prion infectivity during the asymptomatic stage of the disease. Among the different methods, PMCA (2 rounds) and mouse bioassay are the most sensitive as they consistently detect abnormal PrP and infectivity, respectively, at the first time point (50 dpi) tested in this study. Our results demonstrate that Figure S1Sensitivity of the Cerebellar Organotypic Slice Culture Assay. Immunoblots of PK-treated slice culture homogenates probed with anti-PrP antibody Sha31, showing PrPres accumulation in slice cultures. Cerebellar organotypic slices were prepared from tg338 pups and maintained in culture during 42 days in vitro after exposure to serial dilutions of PG127 scrapie-infected brain stock prepared from terminally ill tg338 mice, previously used (TIF)Click here for additional data file.


Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly, carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately within few months of initial treatment, tumor relapse occurs because of platinum-resistance. This is attributed to chemo-resistance of cancer stem cells (CSCs). Herein we show for the first time that withaferin A (WFA), a bioactive compound isolated from the plant Withania somnifera, when used alone or in combination with cisplatin (CIS) targets putative CSCs. Treatment of nude mice bearing orthotopic ovarian tumors generated by injecting human ovarian epithelial cancer cell line (A2780) with WFA and cisplatin (WFA) alone or in combination resulted in a 70 to 80% reduction in tumor growth and complete inhibition of metastasis to other organs compared to untreated controls. Histochemical and Western blot analysis of the tumors revealed that inclusion of WFA (2 mg/kg) resulted in a highly significant elimination of cells expressing CSC markers - CD44, CD24, CD34, CD117 and Oct4 and downregulation of Notch1, Hes1 and Hey1 genes. In contrast treatment of mice with CIS alone (6 mg/kg) had opposite effect on those cells. Increase in cells expressing CSC markers and Notch1 signaling pathway in tumors exposed to CIS may explain recurrence of cancer in patients treated with carboplatin and paclitaxel. Since, WFA alone or in combination with CIS eliminates putative CSCs, we conclude that WFA in combination with CIS may present more efficacious therapy for ovarian cancer. Epithelial ovarian cancer (EOC) remains the leading cause of death in women among gynecologic cancers and is the 5th highest cause of cancer-related deaths in women in the United States In our previous studies WFA has been a part of Indian traditional medicine for centuries. It is available in US over-the-counter as a dietary supplement and is known to treat various disorders due to its anti-inflammatory Development of drug resistance and recurrence of ovarian cancer has been a major clinical problem. A number of mechanisms that induce drug resistance have been proposed. Over the last several years, there has been increasing evidence that “cancer stem cells (CSCs)”, are the most important trigger of tumor progression, chemo-resistance and relapse after initial treatment In our current study, we show for the first time that WFA alone or in combination with CIS if employed to treat mice bearing human orthotopic ovarian tumors not only suppresses tumor growth but targets cells expressing CSC markers as well as inhibits Notch1 and its downstream signaling genes (Hes1 and Hey1) that have been reported to play a crucial role in self-renewal and maintenance of CSCs (33).Ovarian epithelial cancer cell line A2780 was initially obtained from Denise Connolly (Fox Chase Cancer Center) and was maintained in RPMI 1640 medium containing insulin and supplemented with penicillin/streptomycin (100 IU/ml and 100 µg/ml) and 10% fetal bovine serum (FCS)  as described previously 5 cells were transferred to the top chamber. The medium containing 5% FBS was added to the lower chamber. The cells were incubated at 37°C for 24 h and allowed to migrate through the membrane. Non-migrated cells were removed with a clean cotton swab. Migrated cells on other side of the membrane were stained with crystal violet and counted in three different fields under Olympus microscope. The experiments were repeated for three times. The values represented are the mean ± SEM of three independent experiments.Cell migration in vitro was assayed by determining the ability of cells to migrate through a synthetic basement membrane. The procedure used was as described previously 6) cells were directly injected into left ovary of 5 to 6 weeks old nu/nu female mice (Jackson Laboratory) under aseptic conditions and under light anesthesia. After 10 days of post-cell injection, mice were treated with 1) vehicle control (10% DMSO and 90% glyceryl trioctanoate), 2) WFA 2 mg/kg, 3), CIS 6 mg/kg, and 4) WFA 2 mg/kg plus CIS 6 mg/kg. Five animals randomly were included in each group. CIS in saline was injected i.p. once a week, whereas WFA was injected i.p. every other day. After 4 weeks of treatment, animals were sacrificed; tumor and other tissues such as un-injected ovary, lung, kidney, liver, adrenal and heart were collected from each mouse. Tumors were weighted at the time of collection. The tumors and other tissues were divided into two parts, one part was snap frozen, and second part was fixed in 10% buffered formalin. The animals' experiments were approved by the University of Louisville, Institutional Animal Care and Use Committee (IACUC) (protocol # 12063).Orthotopic ovarian tumors were generated by injecting ovarian cancer cell line A2780 directly into ovary as described by Nunez Cruz et al. Formalin fixed tumor and tissues were processed and embedded in paraffin using standard protocols as described previously Formalin fixed paraffin embedded tissues were deparaffinized in xylene and rehydrated in a decreasing graded series of ethanol as described previously 3VO4, and 1 mM NaF) supplemented with Complete Mini Protease Inhibitor tablet . To prepare extract from normal and tumor tissues, tissues were suspended in lysis buffer and homogenized on ice using Polytron homogenizer followed by centrifugation at 10,000 rpm for 5 min. The supernatants were collected and protein concentration in each sample was determined using Bradford method (BioRad Laboratories) according to supplier's instructions. Forty µg of protein from each sample was fractionated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes as described previously 2O2 and probed with β-actin antibody in order to serve as an internal control.A2780 cells were plated into 6 well plates. After 24 h of plating, cells were treated with WFA and CIS both alone and in combination as described previously (10). After 48 h of treatment, cells were lysed in chilled lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% NP-40, 1 mM NaStatistical comparison of data was carried out by the student's t test (for single comparison). Probability of p<0.05 determined from the two-sided test was considered significant. The statistical analysis was carried out by using SPSS 10.0 software.Various steps are involved in tumor progression and metastasis including detachment of tumor cells from the primary tumor site, transmigration into lymph- or blood vessels, attachment to endothelium at distant sites of metastasis followed by seeding into new location and subsequent expansion. To examine the effect of WFA and CIS on A2780 cell migration, we treated the A2780 cells with WFA and CIS both alone and in combination for 48 h. As shown in in vitro studies, we showed that treatment of CIS-sensitive cell lines (A2780 and CaOV3) as well as CIS-resistance cell line (A2780/CP70) with WFA and CIS both alone and in combination inhibited cell proliferation in a time- and dose-dependent manner and induced cell apoptosis and DNA damage. Moreover, the combined effect of WFA and CIS was synergistic In our H&E histo-pathological analysis of un-injected opposite ovaries, livers, and lungs showed metastasis to livers and ovaries in mock- treated animals only. Metastatic cells comprised ∼10% of cells in those organs . In contChemo-resistance and recurrence of ovarian cancer is a major problem and cause of death. In recent years, a concept of CSCs in solid cancers including ovarian cancers has been proposed To confirm our immuno-histochemical analysis of CD44, CD24, CD34, CD117 and Oct4 positive cells in orthotopic tumors, we performed Western blot analysis of the tumor extracts using specific antibody for markers detected by immuno-histochemical staining. As shown in Increase in number of cells expressing markers of putative CSCs in tumors collected from animals treated with CIS as analyzed by immuno-staining as well as Western blot analysis suggests that treatment by CIS may increase number of cells expressing these markers and may explain development of chemo-resistance and reoccurrence of ovarian cancer in patients treated with CIS or its derivative such as carboplatin in combination with paclitaxel that are commonly used in chemotherapy. In contrast elimination of cells expressing CSC markers in tumors on treatment with WFA alone or in combination with CIS (6 mg/kg) demonstrates that WFA is highly effective in eliminating cells expressing CSC markers.Self-renewal, drug resistance and differentiation are key characteristics of CSCs. Sonic Hedgehog (Shh), Notch1, Twist1, Snail and Wnt1 signaling transduction pathways play major roles in the self-renewal of these cells The most common first line chemotherapy used for ovarian cancer after cytoreductive surgery is carboplatin in combination with paclitaxel. Initial response rate to this combination is very high (70 to 80%), however within 6 to 20 months after initial treatment tumor relapse and patients become resistance to CIS In recent years, several investigators have reported a presence of small population of CSCs in tumor tissues to be responsible for induction of chemo-resistance and recurrence of cancer Increase in number of CSCs in tumors inoculated in nude mice followed by CIS treatment is result of amplification of CSCs present in human cancer cell line A2780. On other hand growing tumor will attract host-derived normal stem cells that will provide stroma and vasculature for expanding tumor tissue. These cells could provide trophic signals for CSCs, and this is currently investigated in our laboratories.In the past years a great deal of efforts has been devoted to develop drugs that can kill cancer cells as well as CSCs in order to reduce chemo-resistance and recurrence of cancer after treatment. WFA as reported exhibits an inhibitory effect against several different types of cancer cells. However, its effect on CSCs has not been explored so far. In our previous study Self-renewal, drug resistance and differentiation are key characteristics of CSCs and several developmental pathways such as Sonic Hedgehog (Shh), Notch, Wnt and TGFβ, Twist, and Snail which have been shown to be crucial in these processes In our present study, we show for a first time highly significant inhibition of Notch1 and its downstream signaling proteins Hes1 and Hey1 in tumors collected from animals treated with WFA (2 mg/kg) as compared to tumors from mock-treated animals. In contrast, animals treated with CIS (6 mg/kg) alone showed a significant increase in levels of Notch1, Hes1 and Hey1 genes which is consistent with the increase in number of CSCs, suggesting an important role of Notch1 transduction pathway in amplification of those cells. More importantly, treatment of animals with WFA (2 mg/kg) + CIS (6 mg/kg) prevented increase of Notch1, Hes1 and Hey1 expression, suggests that such combined therapy ameliorates unwanted effect of CIS treatment alone and unwanted expansion of CSCs. Thus, treatment of patients that have become resistance to CIS and have developed recurrence cancer could be benefited by WFA treatment alone or in combination with CIS.The silent observation from this study is that treatment of mice bearing human ovarian tumors with CIS results in an unwanted expansion of cells that express CSC markers, what may lead to CIS resistance and recurrence of ovarian tumor. In contrast, WFA if employed alone or in combination with CIS ameliorates this unwanted effect. The data obtained from our study suggest that WFA alone or in combination with CIS may serve as a safer and more efficacious therapy for both first line and second line options for ovarian cancer.


HU+), decreased (PRMHU−), or unchanged (PRMHU0) attenuation. A total of 7 PVCFs were compared to undiseased vertebrae in each patient serving as controls. A receiver operator curve (ROC) analysis identified optimal imaging times for group stratification. BMD changes were apparent by an elevated PRMHU+ as early as 1 year before fracture. ROC analysis showed poor performance of PRMHU− in stratifying PVCFs versus controls. As early as 6 months before PVCF, PRMHU+ was significantly larger (12.9 ± 11.6%) than control vertebrae (2.3 ± 2.5%), with an area under the curve of 0.918 from an ROC analysis. Mean HU changes were also significant between PVCF (26.8 ± 26.9%) and control (−2.2 ± 22.0%) over the same period. A PRM analysis of BMD changes using standard CT imaging was sensitive for spatially resolving changes that preceded structural failure in these patients.Pathologic vertebral compression fractures (PVCFs) cause significant morbidity in patients with metastatic bone disease. Limitations in existing clinical biomarkers leave clinicians without reliable metrics for predicting PVCF, thus impeding efforts to prevent this severe complication. To establish the feasibility of a new method for defining the risk of a PVCF, we retrospectively analyzed serial computed tomography (CT) scans from 5 breast cancer patients using parametric response mapping (PRM) to quantify dynamic bone miniral density (BMD) changes that preceded an event. Vertebrae segmented from each scan were registered to the same spatial frame and voxel classification was accomplished using a predetermined threshold of change in Hounsfield units (HU), resulting in relative volumes of increased (PRM Bone metastases occur in approximately 70% of patients with metastatic breast cancer, and bone is the most common site of metastasis for patients with estrogen receptor-positive (ER+) disease . The spiBisphosphonates such as zoledronic acid and the monoclonal antibody-targeting receptor activator of nuclear factor kappa-β ligand denosumab are the current bone-directed therapies that function to inhibit osteoclast activity. Bisphosphonates decrease the risk of SREs, including PVCFs, by approximately one-third, and denosumab may further improve the control of SREs by another 20% , 11. AltPredicting individuals who are at the greatest risk for developing a PVCF would provide opportunities for earlier treatment and the design of clinical trials to prevent fractures. However, existing biomarkers for predicting pathologic fractures have limited efficacy. Biochemical markers such as serum alkaline phosphatase and urinary deoxypyridoline have shown some promise for predicting pathological fractures . Bone miWe previously developed the image analysis technique parametric response mapping (PRM) to quantify temporal changes in imaging data on a voxel-by-voxel basis –19. In aRetrospective imaging and clinical data were obtained from 5 patients at the University of Michigan and approved by an institutional review board. Patients were included in the study based on confirmed PVCF using CT and more than 3 scans pre-PVCF. An additional 2 cases of osteolytic lesions were found for anecdotal analysis despite the fact that fractures were not observed. All images were reviewed by a musculoskeletal radiologist to confirm the location and date of the fracture, to assess for other fractures, and to identify the vertebral body with the least amount of metastatic disease for use as a control. Clinical information was extracted from medical records, including patient characteristics , oncologic history , history of oncologic treatment , bone-modifying agents or procedures , clinical characteristics proximal to compression fracture , and treatment of vertebral fracture.HU) were generated between time points by first calculating the difference between HU values for each voxel. Individual voxels were classified based on the extent of change observed in attenuation (ΔHU). Voxels that yielded a ΔHU greater than a receiver operating curve (ROC)-optimized threshold (ξ) were designated red (ΔHU > ξ), those that decreased by more than the threshold were designated blue (ΔHU≤-ξ), and those that did not significantly change from baseline were designated green. Because of a lack of true control imaging data, 100 HU was arbitrarily chosen for longitudinal PRM analysis. Volume fractions of the total bone volume were calculated for the 3 classifications: PRMHU+ (red voxels denoting increased HU), PRMHU− (blue voxels denoting decreased HU), and PRMHU0 . For longitudinal tracking of PRM values, each time point was analyzed against the baseline image.PRM analysis was performed as previously described using software developed in MATLAB  . Brieflyt1 = 3, 6, 9, or 12 months; t0 and t1, for PRM analysis  was calculated as a summary statistic for selecting the optimal combination of PRM threshold and imaging time points, with a greater value indicating more accurate results. The cutoff value for discriminating between groups was then determined using the accuracy measure (ACC), defined as ACC = (TP + TN)/n, where TP is number of true positive results, TN is true negative, and n is the total number of samples. A higher value indicates a more accurate cutoff. Group comparisons (fracture versus control) were performed using a paired 2-tailed Student's t test, and significant difference between groups was defined as P < .05. All group statistics and plots are depicted as group mean with errors representing the SD.To assess the ability of PRM measurements to stratify the PVCF cases versus their respective controls, 3 variables were explored: (1) the threshold that designated a significant change in HU within a voxel ; (2) the second time point relative to the date of fracture . Attributed to the limited number of datasets analyzed, 2 cutoffs were identified for PRMHU+ stratification between fracture and control vertebrae: 1.5% (sensitivity/specificity = 1.00/0.71) or 6.3% (sensitivity/specificity = 0.71/1.00), with ACC = 0.857. PRMHU− was not found to be useful for determining fracture risk in this patient population.The timing of the analysis and PRM threshold was explored for optimal predictability of fracture and sensitivity to bone density changes by varying δve value , with a HU+ (P =.047) between PVCF (12.9 ± 11.6%) and controls (2.3 ± 2.5%), but PRMHU− was not significant (P =.288). The percentage change in mean bone attenuation (δHU) was also found to be significant (P = .046) between PVCF (26.8 ± 26.9%) and controls (−2.2 ± 22.0%).In addition to the ROC analysis, group means were also compared , D. A siHU in the presence of osteolytic lesion bone resorption. Because these lesions did not result in PVCFs, they were not included in the previous analysis. In 1 case, shown in HU−, toward predominantly sclerotic remodeling, as evidenced by a large PRMHU+ in the final time point. This sclerotic progression falls in line with the clinical evaluation, with evidence of disease progression in the lumbar spine as demonstrated by (1) worsening lower back pain, (2) a rise in tumor blood markers, (3) magnetic resonance imaging results that showed further lumbar vertebral collapse and increased associated soft tissue, and (4) increased uptake of the L3 and skull lesion via bone scan.Two cases of osteolytic lesions were also identified for anecdotal analysis in the thoracic spine, neither of which resulted in fracture within the time limits of the study. These vertebrae served to demonstrate the utility of PRMHU revealed no focal progression; however, a diffuse loss of bone density throughout the bone was apparent starting at about 19 months (fifth time point), as evidenced by the increased number of disseminated blue voxels. This loss in bone mass may be caused by any combination of (1) osteoporosis, (2) tumor infiltration, (3) fatty infiltration, or (4) hemangioma.In another clinical case study, the patient had preexisting bone deterioration before the imaging study; the red arrow in the first image of The goal of this study was to apply the PRM methodology to standard CT imaging as a proof of concept for evaluating fracture risk in patients with bone metastases. The availability of regularly acquired longitudinal CT images provided a unique opportunity for the first clinical application of PRM for predicting risk of PVCFs in patients with metastatic breast cancer. Although limited to cases of PVCFs in this study, there are a multitude of bone-involved diseases resulting in SREs, such as osteoporosis. We expect that PRM also will be a robust metric for changes in BMD associated with other diseases.The current standard for determining fracture risk in patients with bone metastases is BMD measured by dual x-ray absorptiometry. This method has seen widespread clinical use and has shown an impressive predictive value with a relative risk of 2.3 per SD change . HoweverHU+ (red). Although vertebrae with known lesions and resulting fractures generally showed an increasing trend in PRMHU+ toward fracture  sensitivity to localized changes in density that may be masked when using whole-volume statistics, especially at early time points, and (2) resulting classification PRM maps that provide spatial context to these changes. PRM analysis of a rat model of osteoporosis revealed an early drop in bone density that preceded whole-bone measurements, with observable trabecular deterioration as well as cortical expansion for compensation . Althougfracture , controlfracture . Becausehis case , we belihis case  and 5. FThe balance between image resolution and acquisition time  is a well-known tradeoff. Finite element analysis requires very high resolution and low noise to accurately model the mineralized structure and determine bone strength, which in turn requires greater acquisition time and a significantly higher dose. In vivo or clinical application of this method is therefore not often feasible with current CT technology. The PRM method does not require such high resolution and thus, as shown in this study, can provide sensitive results on relatively low-resolution images. Moreover, through simultaneous quantification and spatial display of both increasing and decreasing bone density, an indication of the degree of osteolytic/osteoblastic activity of the lesion may be ascertained. In addition to risk assessment, the spatial maps of density change may prove useful for planning orthopedic strategies if necessary.Further studies are necessary to fully explore the quantification of fracture risk progression using this technique, which would ideally monitor all lumbar and thoracic vertebrae simultaneously. This would necessitate an automated segmentation routine for efficient processing and clinical accessibility and scalability. In addition, the acquired image resolution as well as reconstructed image noise may substantially affect detected density changes as a result of the partial-volume effect and limited ability of the coregistration process to accurately interpolate bone tissue interfaces. Although a comprehensive assessment is beyond the scope of this preliminary study, the results presented are very promising and present PRM as a potentially novel image biomarker for early detection of skeletal-related events in cancer patients.The overall purpose for accurately predicting vertebral fracture risk is to provide clinicians with the required evidence for undertaking intervention. Bone density analysis through PRM provided a significant indication of an impending fracture with an imaging frequency of twice per year—ample time for adjusting patient care and possibly improving patient quality of life when applied prospectively. Spatial maps of density changes revealed focal bone remodeling and could provide much-needed context for corrective intervention and potentially a greater sensitivity to fracture-related bone changes than whole-bone analysis, especially in the case of mixed osteolytic/osteoblastic lesions. Ultimately, PRM is a sensitive and flexible image biomarker that can be developed for clinical use as an indicator of disease and fracture risk progression.


Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a median survival of only about two years even in the 20% of patients that present early enough to be eligible for surgical resection and adjuvant chemotherapy. With this urgent medical need in mind, we are exploring the use of adoptive T cell transfer in patients with PDAC, as this therapy has shown remarkable clinical success in patients with advanced melanoma.+cells/mm2) T cell infiltration in 60% of patient biopsies analyzed by immunohistochemistry (n=66). Flow cytometric analysis shows that tumor-infiltrating T lymphocytes (TILs) predominantly display an activated effector memory phenotype with signs of exhaustion or prolonged antigen exposure, such as high PD1 (n≥30).Despite the notion that, different from melanoma, PDAC is a non-immunogenic tumor, we observe at least moderate (>150 CD3in vitro culture TILs can be expanded from 85% of PDAC patients (n=96) and show growth capacity and phenotypes similar to melanoma patient TIL (n=62). The majority of tested PDAC TIL cultures produce IFN-γ in response to autologous tumor cells in an MHC-I dependent manner, but cross-reactivity and a low response magnitude are common observations.Upon in situ expansion after tumor-antigen encounter; and iii) in most patients not maintained during the TIL expansion period, likely leading to a loss of important T cell clones and a shift in tumor-reactivity in the TILs available for patient treatment after in vitro expansion.In order to gain insight into the original tumor-reactivity of TILs before expansion, we studied the T cell receptor (TCR) repertoire by deep sequencing and made the following observations: the TIL TCR repertoire is i) distinct from the broad repertoire observed in the blood; ii) usually dominated by large T cell clones, possibly due to ex vivo and to ‘rescue’ TIL-TCRs with valuable reactivities that could be reintroduced in the form of genetically engineered T cells. Whether PDAC TILs are reactive towards shared antigens and/or mutation-derived neo-antigens is currently being investigated based on exome and RNA sequencing data from a subset of patients.Deep sequencing of the TCR repertoire in combination with TCR cloning provides us with a tool to study TIL reactivity directly