Indirect traumatic optic neuropathy (ITON) refers to optic nerve injury resulting from impact remote to the optic nerve. The mechanism of injury is not understood, and there are no confirmed protocols for prevention, mitigation or treatment. Most data concerning this condition comes from case series of civilian patients suffering blunt injury, such as from sports- or motor vehicle-related concussion, rather than military-related ballistic or blast damage. Research in this field will likely require the development of robust databases to identify patients with ITON and follow related outcomes, in addition to both in-vivo animal and virtual human models to study the mechanisms of damage and potential therapies. Indirect traumatic optic neuropathy (ITON) is a condition in which a patient suffers head trauma and is found to have reduced vision and an afferent pupillary defect despite a normal acute slit lamp examination and normal acute magnetic resonance imaging (MRI) and computer tomography (CT) of the optic nerve and canal. ITON differs substantially from direct traumatic optic neuropathy (DTON); in DTON, the optic nerve axons or vascular supply are directly damaged due to strain (as may be observed in an avulsion of the optic nerve), compression  or transection . Patients suffering either ITON or DTON can experience profound vision loss after trauma. Prognosis is guarded in either situation; however, some patients with ITON may experience full recovery with no intervention. Few randomized controlled studies have explored therapies for ITON, and as discussed below, therapies employed to treat DTON appear to be ineffective for ITON. A computerized search of PubMed  revealed 195 publications on this topic from 1905-present; notably, a similar search without the word “indirect” produced 4,495 entries. Most publications concerning ITON can be divided into studies of diagnosis, therapeutic approaches and pathogenesis. Additionally, the vast majority of publications report on civilian blunt injuries rather than blast or ballistic injuries that may be experienced more commonly by military personnel.The incidence of ITON has not been directly reported in any large population. However, very good surveillance studies of traumatic optic neuropathy of any type have been reported for pediatric  and adulBy definition, ITON is a condition in which injury to the optic nerve can be clinically confirmed, i.e., patients demonstrate reduced visual acuity, color vision, and/or visual field, as well as a relatively afferent pupillary defect. Notably, this latter finding may not be present in bilateral cases. Direct ophthalmoscopy of the nerve is also expected to appear normal, though optic atrophy or pallor are expected to develop. Automated visual field testing should be offered; however, the vision of subjects may be too poor to glean useful results. In most cases, testing with visual evoked potentials (VEP) is not needed to establish the diagnosis. However, in questionable cases, VEP may provide confirmatory data. VEP may also have predictive value; patients with better responses on VEP may be more likely to regain some or all of their vision , 12. VEPCT and plain radiographic findings in ITON would, by definition, exclude fractures in the optic canal because they would be suggestive findings of DTON. However, patients with ITON may suffer other skull fractures. A study of 39 patients with both ITON and peri-orbital fractures indicated that posterior orbital fracture is associated with poorer visual outcomes than those with anterior fractures, suggesting that CT may help in guiding decisions to offer therapy . MRI, paNo therapy has been confirmed to effectively treat ITON, but observation is considered an acceptable option.Observational studies of ITON have demonstrated wide variability in visual function, the rate of recovery and long-term outcomes. Most patients in the pediatric TON surveillance study improved or at least did not worsen from baseline visual acuity . RecoverTwo pilot studies exploring the efficacy of intravenous (IV) erythropoietin have been published , 20. In Corticosteroids have also been offered to patients for ITON. A Cochrane review from 2013 found one double-masked, placebo-controlled and randomized study in which high-dose IV corticosteroids were offered within 1 week of the injury causing ITON; there was no significant benefit over observation . This fiA 2010 study investigated the potential additive effect of providing levodopa-carbidopa to improve the visual outcomes of patients with indirect traumatic optic neuropathy (ITON) . The ratOptic canal decompression represents the primary surgical therapy attempted in treating ITON. A number of retrospective studies provided anecdotal data suggesting that this surgery can be effective even if offered after a few weeks of observation or medical therapy , 31–37. Transcorneal electrical stimulation (TES) has been reported to maintain more normal morphology and increase the survival of rat retinal ganglion cells after nerve crush , to promNotably, one reported case series found that acupuncture, a traditional Chinese medicine, was successfully applied to treat ITON . AcupuncResearch on novel neuro-protective medical therapies for optic nerve injury, particularly against glaucoma –54, optiStudies suggest that the biomechanical response of the cranial contents to traumatic loads is an important aspect in understanding the etiology of ITON. A seminal and frequently referenced study using holographic interferometry on human skulls reported that frontal loading results in the deformation of the ipsilateral orbital roof near the optic foramen . One mayA more recent anatomic study of cadaveric orbits and optic nerves with the intent of identifying possible etiologies for ITON was reported in 2002 . Forty-oDiffuse axonal damage should also be considered as a mechanism underlying ITON. Diffuse axonal damage due to injurious inertial force to the head has been associated with poor neurological outcomes. Rapid deformations of axons in the white matter tracts of the brain can lead to damage to the axonal cytoskeleton and the impairment of axoplasmic transport. Subsequent swelling and calcium entry into damaged axons can lead to further dysfunction and physical breakage and additional neuropathological changes in the brain tissue . There iLittle is known with certainty about the etiology of indirect traumatic optic neuropathy. This sight-threatening condition may be a relatively common comorbidity of blunt craniofacial head trauma. Unfortunately, there are neither proven strategies for prevention, mitigation or cure, nor are there satisfactory laboratory models to explore this condition. The authors strongly support the development and analysis of clinical databases to better understand the natural history of this condition. Further, the authors support efforts to create models, both virtual and real, to clarify the biomechanics of ITON.


Despite the availability of recently developed chemotherapy regimens, survival times for pancreatic cancer patients remain poor. These patients also respond poorly to immune checkpoint blockade therapies , which suggests the presence of additional immunosuppressive mechanisms in the pancreatic tumour microenvironment (TME). CD40 agonist antibodies (αCD40) promote antigen presenting cell (APC) maturation and enhance macrophage tumouricidal activity, and may therefore alter the pancreatic TME to increase sensitivity to immune checkpoint blockade. Here, we test whether αCD40 transforms the TME in a mouse syngeneic orthotopic model of pancreatic cancer, to increase sensitivity to PD-L1 blockade. We found that whilst mice bearing orthotopic Pan02 tumours responded poorly to PD-L1 blockade, αCD40 improved overall survival. αCD40 transformed the TME, upregulating Th1 chemokines, increasing cytotoxic T cell infiltration and promoting formation of an immune cell-rich capsule separating the tumour from the normal pancreas. Furthermore, αCD40 drove systemic APC maturation, memory T cell expansion, and upregulated tumour and systemic PD-L1 expression. Combining αCD40 with PD-L1 blockade enhanced anti-tumour immunity and improved overall survival versus either monotherapy. These data provide further support for the potential of combining αCD40 with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer. Tumours employ a number of mechanisms to escape detection and elimination by the adaptive immune system . Tumour reg) [The PD-1 (programmed cell death protein 1) and CTLA-4 (cytotoxic lymphocyte-associated antigen 4) immune checkpoint pathways can both contribute to tumour immune evasion. PD-L1 (programmed death ligand 1) expressed on tumour cells and infiltrating myeloid cells engages PD-1 on activated T cells, downregulating T cell effector functions . CTLA-4 reg) . Antibodreg) . Howeverreg) , 9.+ effector T cell infiltration in their tumours [+ effector T cell immune response is actively being restrained by PD-L1 expression [reg [Melanoma patients that respond to PD-1 blockade are reported to show baseline PD-L1 expression and CD8 tumours –12. Thispression . The panion [reg . In contion [reg , 15. Theion [reg .LSL-G12D/+ / Trp53LSL-R172H/+ / Pdx-1-Cre) macrophage depletion with CSF1R inhibitors, or blockade of fibroblast-derived CXCL12 activity with a CXCR4 inhibitor, enhanced T cell infiltration and anti-tumour activity of αPD-L1/αPD-1 and αCTLA4 [Various strategies have therefore been developed to transform the immunosuppressive pancreatic TME, and so enhance the response of pancreatic cancer patients to immune checkpoint blockade therapies. A cell-based cancer vaccine, GVAX, was able to induce the formation of tertiary lymphoid aggregates in pancreatic cancer patient tumours, and resulted in objective clinical responses in combination with anti-CTLA-4 , 18. In d αCTLA4 , 20.CD40 agonistic antibodies (αCD40) in combination with gemcitabine have been reported to show early signs of clinical activity in pancreatic cancer patients . CD40 agHere we build on the above results by investigating the mechanism by which αCD40 improves sensitivity to PD-L1 blockade in pancreatic tumours. Since s.c. Pan02 tumour-bearing mice respond well to immune checkpoint blockade , 29, we A syngeneic orthotopic pancreatic tumour model was developed whereby Pan02 cells were implanted surgically into the pancreas tail of immunocompetent mice. Orthotopic Pan02 tumours diffusely invaded into the normal pancreas and metastasised to the peritoneal cavity. Primary tumours had a dense desmoplastic stroma highly infiltrated with macrophages (data not shown). We found that orthotopic Pan02 tumour-bearing mice responded poorly to gemcitabine, PD-L1 blockade and CTLA-4 blockade Figure . This alIn contrast to the minimal effects of immune checkpoint blockade, we found that a CD40 agonist antibody, FGK4.5, led to a significant reduction in early tumour growth and peritoneal metastatic spread (day 29) as measured by endpoint imaging of a luciferase-overexpressing Pan02 tumour cell line . In separate studies . Isotype treated tumours were invasive, diffusely infiltrating the surrounding normal pancreas Figure . In conts Figure . Whilst g Figure . There w+ T cells. The Th1 mediators IFNγ . Mice were sacrificed 24h after the final dose of αCD40 .Since induction of PD-L1 expression was reported to mediate acquired resistance to αCD40 in breast tumour models , we nexts Figure . Further− Figure  and CD8++ Figure  effector+ T cell infiltration and also upregulation of PD-1 and PD-L1, we tested the hypothesis that a combination of αCD40 and αPD-L1 would induce systemic anti-tumour immunity in this model. Pan02 tumour-bearing mice were treated with αCD40  and αPD-L1 , and monitored for tumour growth and overall survival  and breast tumour models , 34. We + cytotoxic T cells were not excluded from isotype treated Pan02 tumours, suggesting that Pan02 tumour-infiltrating CD8+ T cells are either naïve or have become inactivated. Both exclusion of effector T cells from tumours and inactivation of tumour infiltrating T cells have been proposed to be responsible for the failure of some patients to respond to immune checkpoint blockade [+ T cells are excluded from many pancreatic cancer patient tumours, and from KPC mice with spontaneous pancreatic tumours [+ T cell infiltration in tumours is observed, CD8+ T cells become inactivated through loss of CD3ζ [We found that orthotopic Pan02 tumour-bearing mice were resistant to PD-L1 and CTLA-4 blockade. However, CD8 tumours , 15. How of CD3ζ .+ T cell-dependent anti-tumour immunity [+ T cell infiltration and tumour PD-L1 expression. Our finding that αCD40 induced an increase in CD8+ cells in orthotopic Pan02 tumours is in accord with data reported by Zippelius et al. [Multiple effector mechanisms have previously been reported to contribute to αCD40-mediated inhibition of tumour growth and metastasis. αCD40 can induce both T cell-independent anti-tumour effects, enhancing macrophage tumouricidal activity and stromal remodelling, and CD8immunity . Our dats et al.  in s.c. The majority of αCD40-treated animals developed progressive disease, indicating that αCD40-induced adaptive immune activation was insufficient. Our data suggest that upregulation of the PD-L1 / PD-1 immune checkpoint, both in the TME and systemically on myeloid cells, contributes to the failure of this adaptive immune response. Thus, whilst PD-L1 blockade alone had minimal effects on overall survival, combining CD40 agonism with PD-L1 blockade improved overall survival in comparison to either monotherapy alone.PD-L1 upregulation on tumour cells (including Pan02 cells) and myeloid cells can be induced by IFNγ , 28. HowSurvival times for pancreatic cancer patients treated with even the more recently developed chemotherapy regimens  remain poor , 40. CheIn summary, our data provide further preclinical support for the potential benefits of combining immunotherapies such as αCD40 that transform the TME with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer.Pan02 cells, a SMAD4-null chemically induced mouse pancreatic tumour line, was from the National Cancer Institute , 46. PanThe following antibodies from eBioscience, BD Bioscience and Biolegend were used for flow cytometry: anti-CD45 (clone 30-F11), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14), anti-CD3 (clone 145-2C11), anti-CD4 (Clone RM4.5), anti-CD8 (clone 53-6.7), anti-F4/80 (clone BM8), anti-MHCI (clone 28-8-6), anti-CD80 (clone 16-10A), anti-CD86 (clone GL-1), anti-MHCII (clone M5/114.15.2), anti-Gr-1 (clone RB6-8C5), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone6D5), anti-PD-L1 (clone 10F.9G2), anti-PD-1 (clone 29F.1A12), anti-FoxP3 (FJK-16S).5 Pan02 or Pan02-CAG-luc2 pancreatic tumour cells in chilled matrigel (Corning) were surgically implanted in the pancreas tail [Experiments using 6 - 8 week old female C57BL/6J mice (Charles River) were conducted under a U.K. Home Office Project Licence in accordance with the U.K. Animal (Scientific Procedures) Act 1986 and in accordance with EU Directive EU 86/609. 30 minutes after prophylactic analgesia , mice were shaved and anaesthetised (isoflurane). 5 × 10eas tail  and the Group sizes for tumour growth studies were determined based on existing data for orthotopic Pan02 tumour growth, to test for greater than 45 % tumour growth inhibition. The power calculation was a one-sided 2 sample t-test carried out at the 5 % significance level. Animals were randomised to treatment groups based on body weight prior to the first treatment. Due to the technical challenges of in vivo imaging of orthotopic tumours in C57/BL6 mice, we developed and validated a blinded scoring system based on abdominal palpation and animal welfare . This inFor imaging studies, luciferase expression in Pan02-CAG-luc2 tumours was assessed 29 days after cells were implanted. Animals were injected with 3.3 mg of luciferin (Perkin Elmer) i.p., euthanised and the peritoneal cavity exposed. The peritoneal cavity was imaged 20 minutes after luciferin injection on the IVIS100 (Perkin Elmer) with an acquisition time of 10 seconds.Animals were euthanized and spleens were removed and dissociated to a single cell suspension through a 70 μm nylon mesh. Splenocytes were stained with a fixable live dead dye (Life Technologies) followed by block with anti CD16/32 antibody (eBiosciences) and staining with cocktails of fluorescence conjugated antibodies prior to fixation in 1 % formaldehyde / PBS. FoxP3 nuclear staining was carried out on fixed and permeabilised cells . Stained cells were analysed by flow cytometry  with data analysis in FlowJo (Tree Star). Positive staining gates were identified by comparison to cells stained with the full antibody panel minus the antibody of interest (FL-1 control).2O2 in methanol prior to immunostaining. Following a protein/serum block, sections were immunostained with primary rat monoclonal antibodies to FoxP3 (eBioscience), CD8 (eBioscience) and F4/80 (AbD Serotec) for 1 hour at room temperature. Primary antibodies were detected with goat anti-rat IgG (Jackson ImmunoResearch) followed by anti-goat Ig ImmPRESS-HRP polymer (Vector). CD8 was detected directly with mouse adsorbed anti-rat ImmPRESS-HRP polymer (Vector). Staining was then visualised using DAB+ substrate (Dako). Sections were then counterstained with Haematoxylin, dehydrated, cleared and permanently coverslipped. Tissue morphology and collagen were also demonstrated using Haematoxylin & Eosin and Masson's Trichrome staining respectively. Slides were digitally scanned using an Aperio Scanscope and whole slide image analysis was performed in the tumour region only with algorithms created in Definiens Tissue Studio version 4.1.Tumour samples were harvested, fixed in 10 % neutral buffered formalin, processed and embedded into paraffin wax blocks. Sections were taken at 4 μm and then de-waxed, rehydrated and antigen retrieved using either a pressure cooker with low or high pH buffer (Vector / Dako) or enzymatic retrieval with proteinase K. Sections were then blocked for endogenous peroxidase with HAnimals were euthanized and pancreatic tumours dissected out from normal pancreas, before being either snap frozen or transferred to RNALater. Snap frozen tumours were homogenised in tissue lysis buffer  and phosphatase inihibitor cocktails II and III (Sigma Aldrich)) using a GentleMACS tissue dissociator (Miltenyi) followed by sonication and clarification of homogenates by centrifugation at 10,000 g for 10 minutes. Homogenate protein concentrations were quantified by BCA assay (ThermoFisher Scientific). Cytokine and chemokine levels in homogenates and plasma were quantified by multiplex ELISAs according to manufacturer's instructions .RNA was extracted from tumours stored in RNALater using RNeasy mini kits (Qiagen), followed by mRNA reverse transcription with multiScribeTM MuLV enzyme (Life Technologies). Chemokine, cytokine and PD-L1 mRNA expression was quantified using a mouse immune gene signature array (Applied Biosystems #4367786) and a specific PD-L1 Taqman qPCR primer set (Applied Biosystems Mm00452054_m1). qPCR was run on a QuantStudio7 Flex real time PCR system (Applied Biosystems).3 Pan02 cells were co-cultured with 8.3 × 104 disaggregated splenocytes from Pan02 tumour-bearing mice (day 29 post-implantation) in RPMI (Life technologies) with 10 % fetal bovine serum (SAFC Biosciences) and 10 U/mL IL-2 for 96 hours in MultiscreenHTS IP plates (Millipore) coated with anti-IFNγ capture antibody (eBiosciences). The number of IFNγ-producing T cells was then quantified by ELISPOT assay according to manufacturer's instructions (eBiosciences).Pan02 cells are MHCI negative (data not shown), and were therefore IFNγ-treated to upregulate MHCI , before mitomycin C treatment  and extensive washing. 8.3 × 10https://cran.r-project.org/web/packages/statmod/index.html) in R version 1.4.18. All other statistical analyses were performed in Graphpad Prism v6.03.The effect of αCD40 on tumour growth was analysed using a statistical permutation test comparing tumour growth over the whole course of model progression, using the method of  with ana


Optical molecular imaging is an emerging novel technology with applications in the diagnosis of cancer and assistance in image-guided surgery. A high tumour-to-background (T/B) ratio is crucial for successful imaging, which strongly depends on tumour-specific probes that rapidly accumulate in the tumour, while non-bound probes are rapidly cleared. Here, using pre-invasive breast cancer as a model, we investigate whether the use of combinations of probes with different target specificities results in higher T/B ratios and whether dual-spectral imaging leads to improvements in tumour characterization.We performed optical molecular imaging of an orthotopic breast cancer model mimicking ductal carcinoma in situ (DCIS). A combination of carbonic anhydrase IX (CAIX)- and human epidermal growth factor receptor 2 (HER2)-specific variable domains of the heavy chain from heavy-chain antibodies (VHHs) was conjugated either to the same fluorophore (IRDye800CW) to evaluate T/B ratios or to different fluorophores  to analyse the expression of CAIX and HER2 simultaneously through dual-fluorescence detection. These experiments were performed non-invasively in vivo, in a mimicked intra-operative setting, and ex vivo on tumour sections.Application of the CAIX- and HER2-specific VHH combination resulted in an increase of the T/B ratio, as compared to T/B ratios obtained from each of these single VHHs together with an irrelevant VHH. This dual tumour marker-specific VHH combination also enabled the detection of small metastases in the lung. Furthermore, dual-spectral imaging enabled the assessment of the expression status of both CAIX and HER2 in a mimicked intra-operative setting, as well as on tumour sections, which was confirmed by immunohistochemistry.These results establish the feasibility of the use of VHH ‘cocktails’ to increase T/B ratios and improve early detection of heterogeneous tumours and the use of multispectral molecular imaging to facilitate the assessment of the target expression status of tumours and metastases, both invasive or non-invasively.The online version of this article (doi:10.1186/s13550-016-0166-y) contains supplementary material, which is available to authorized users. Molecular imaging of cell surface markers has become an increasingly important strategy for the imaging of cancer, which may be used for diagnosis, for assessment of therapy response and for guidance during surgical resection . OpticalThe basis of optical imaging lies in the detection of light emitted from fluorophore, making it a cost-effective, non-radioactive imaging modality for the detection of cancer, both in the screening and intra-operative setting. Recent advances in optical imaging probes are focused on the development of near-infrared (NIR) fluorophores. Fluorophores emitting light in the NIR range of the spectrum (e.g. 700 and 800 nm) allow deeper tissue penetration than the fluorophores which emit in the standard 400–600-nm range. These advantages are a result of lower light absorption by blood and other tissue components, as well as of minimal tissue autofluorescence in this range of the spectrum –4.Besides an appropriate NIR fluorophore and an imaging system able to detect the light emitted by this fluorophore, optical molecular imaging requires high tumour specificity, which can be obtained by employing fluorescent probes targeting tumour-specific markers that are ideally (over)expressed strictly in cancerous, and not in normal, tissues. Several biomarkers have been identified and their (over)expression has been associated with cancer development. Among these are the human epidermal growth factor receptor 2 (HER2) and the hypoxia marker carbonic anhydrase IX (CAIX) , 6. VariSufficient signal  and a clear delineation of the tumour are essential for successful and accurate tumour detection through imaging. Taking into account the heterogeneity of cancers (e.g. breast cancers ), it mayIn this study, we investigate whether a combination of two optical probes that specifically recognize two independent breast cancer markers could improve tumour detection by optical imaging. For this, we have employed the VHH B9 targeting CAIX, which localizes to peri-necrotic regions of tumours , and thev/v) fetal calf serum (FCS), 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM l-glutamine at 37 °C in a humidified atmosphere containing 5 % CO2. The generation of luciferase overexpressing MCF10DCIS cells has been described before [MCF10DCIS.com (further referred to as MCF10DCIS) cells , mimicking DCIS of breast, were cultured according to the supplier’s guidelines in Dulbecco’s modified Eagle medium nutrient mixture F-12 (DMEM/F12) supplemented with 10 %  when bacteria reached log phase. HER2- and CAIX-specific VHHs were purified from the periplasmic fraction by protein A affinity chromatography using a HiTrap protein A HP column  on the ÄKTAxpress system . Irrelevant VHH (R2) was purified using immobilized metal affinity chromatography .The HER2-specific VHH, 11A4, and the CAIX-specific VHH, B9, were produced as described before , 11. BriN-hydroxysuccinimide (NHS) esters of either IRDye800CW or IRDye700DX  in twofold molar excess of NIR fluorophore to protein, for 2 h at RT. All fluorophores were purchased at LI-COR Biosciences . After conjugation, the remaining unconjugated fluorophores were removed using sequentially two (in case of IRDye800CW) or three (in case of IRDye680RD or IRDye700DX) Pierce Zeba™ Desalting Spin Columns . The degree of conjugation was determined according to the instructions given by the provider for each of the fluorophores.Conjugation of the fluorophores was performed as described before . BrieflyEthics statements: All procedures performed in studies involving animals were in accordance with the ethical standards and approved by the Animal Experimental Committee of Utrecht University (DEC-Utrecht no. 2012.III.02.015).The orthotopic mouse model of breast cancer (MCF10DCIS cells) used in this study was based on a previously described model . In brien = 7), 11A4-800 and R2-800 (n = 7) or B9-800 and R2-800 (n = 7). After the injections, mice were imaged with the Pearl Impulse Small Animal Imaging System (LI-COR Biosciences) at the following time points: 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 24 h and 48 h postinjection (p.i.). Regions of interest (ROIs) were drawn in the tumour and in the upper leg region as normal tissue areas. The upper leg region was chosen as the background due to the proximity to the abdominal area and the reduced risk for the mice, as compared to the risk of shaving the thoracic breasts. T/B ratios were calculated by dividing the mean intensity of tumour ROI by the mean intensity of background ROI  determined with Pearl Impulse Software .In the first part of the study, where the advantage of using two probes instead of one was tested with respect to obtaining higher tumour-to-background ratios (T/B ratios), mice were divided into three groups. Each group was injected intravenously with one of the following combinations of VHHs : 11A4-800 and B9-800 (n = 8), 11A4-800 and R2-700 (n = 8) or B9-800 and R2-700 (n = 7). After the injections, mice were imaged as described above and the fluorescence intensities of IRDye680RD and IRDye700DX were detected in the 700-nm channel, whereas the fluorescence intensity of IRDye800CW was detected in the 800-nm channel. T/B ratios were calculated as described above.In the second part of the study, mice were divided into three groups and then injected intravenously with combinations of VHHs conjugated to different NIR fluorophores to study whether the expression status of HER2 and CAIX could be detected simultaneously in vivo : 11A4-680 and B9-800 , mice were injected intravenously with the combination  of 11A4-680 and B9-800 (n = 3). As controls, one mouse was injected with R2-800 and R2-700 and a healthy mouse with 11A4-680 and B9-800. Mice were sacrificed 5 h p.i. by cervical dislocation, and lung metastases were imaged with the Pearl Impulse Small Animal Imaging System (LI-COR Biosciences) upon surgical removal of lungs.In the third part of the study, the potential of VHH-IR to detect lung metastasis was evaluated. For the development of the lung metastasis model, 1 × 10Mice injected with the combination of VHHs with either IRDye800CW, IRDye680RD or IRDye700DX were sacrificed 5 h p.i., and their tumours were collected. These were first imaged (i.e. ex vivo) and, immediately thereafter, fixed in neutral buffered formalin, routinely processed to paraffin blocks and stored in the dark until further processing. Four-micrometre-thick sections were scanned using the Odyssey imaging system at the highest (21 μm) resolution and quality. Immunohistochemistry (IHC) for detection of CAIX and HER2 combined with a haematoxylin and eosin (H&E) staining were performed as described before . Slides p values of <0.05 were considered to be statistically significant.Statistical analysis was performed using GraphPad Prism (version 5.02). Comparisons of T/B ratios were analysed using a Mann-Whitney test. In order to investigate whether a combination of two fluorescent VHHs specifically recognizing two separate and validated breast cancer biomarkers could (a) improve tumour detection through optical imaging with an increase in T/B ratio and/or (b) facilitate tumour characterization and observation of different areas of the tumour, we used the HER2-specific VHH, 11A4 , and theTo investigate whether the targeting of the tumour with two tumour-specific VHHs conjugated to the same fluorophore results in an increased contrast, we injected intravenously seven mice bearing two MCF10DCIS breast cancer xenografts in the fourth mammary glands with either 11A4-800 and B9-800 or the controls 11A4-800 and R2-800 or B9-800 and R2-800. The imaging was performed up to 48 h postinjection (p.i.). Already 3 h p.i., a clear accumulation of the fluorescent probes was seen in the tumours for each of the three probe combinations . Significant differences were also observed between 11A4-800 and the irrelevant VHH, R2-700 . The difference between B9-800 and the irrelevant VHH, R2-700, at 6 h p.i. was of borderline significance .The T/B ratios were calculated and plotted in time, revealing that the maximal T/B ratios were obtained already 2 h p.i. Fig. . The T/BTo investigate the possible application of dual-spectral imaging in the surgical setting, a number of mice bearing MCF10DCIS xenografts were injected with the indicated fluorescent VHH combinations, and 5 h p.i. mice were sacrificed, the skin was removed thereby uncovering the xenografts and optical imaging was performed Fig. . The folCompared to the results obtained with in vivo imaging Fig. , the T/BMCF10DCIS cells are known to have the capacity to metastasize into the lungs. In this part of the study, the VHH combination B9-800 and 11A4-680 was evaluated for its potential to detect metastasis in the surgical setting. This could provide valuable information on their tumour marker expression status and its molecular resemblance to the primary tumour. When metastases of 1–2 mm were detected through BLI, mice were injected with the B9-800 and 11A4-680 combination. As controls, one mouse with developed lung metastases was injected with R2-800 and R2-700 irrelevant VHH combination and a healthy mouse without metastases was injected with B9-800 and 11A4-680 combination. Five hours p.i., all mice were sacrificed and their chest wall was removed in order to facilitate the imaging of the lungs. No NIR fluorescence was found in the healthy lung controls, thus confirming the tumour marker specificity of both B9-800 and 11A4-680. A clear fluorescent signal was observed at the lung bearing metastasis when the mouse was injected with the B9-800 and 11A4-680 VHH combination, which was twice the intensity of the R2-800 and R2-700 irrelevant VHH combination Fig. .Fig. 5OpTo investigate the possibility of the ex vivo assessment of the molecular status of the tumour by employing the 11A4-680 and B9-800 combination, dual-spectral imaging of tumour sections was conducted and compared to conventional haematoxylin and eosin (H&E) staining. The HER2-targeted probe, 11A4-680, was observed to be homogenously distributed throughout the tumour section, while the CAIX-targeted one, B9-800, was only confined to the peri-necrotic areas of the tumour Fig. . These rDespite many efforts made over the years to facilitate cancer detection, improvements are still eagerly awaited. At the moment, six imaging modalities can be used to detect and stage cancer, namely the following: x-ray , magnetic resonance imaging (MRI), single-photon emission tomography (SPECT), positron emission tomography (PET), ultrasound (US) and, more recently, clinical evaluation initiated with optical imaging. Each of these techniques has advantages but also limitations. Recent advances in targeted probe development stimulated further improvements of PET, SPECT and more recently also of optical imaging , 15. AltTaking into account the heterogeneity of (pre-invasive) breast cancers  as a modThe current diagnostic ‘gold standard’ in biomarkers’ expression assessment relies exclusively on ex vivo methods, such as immunohistochemistry (IHC) and gene amplification-based fluorescent in situ hybridization (FISH). To obtain tissue for these assessments, a biopsy is required. This invasive procedure may have side effects, such as bleeding, and it is difficult to take many biopsies or repeat the procedure for monitoring the treatment response . FurtherTo our knowledge, this is the first study in which VHHs were used in a dual-spectral imaging setting. The idea of imaging with a combination of probes has been earlier investigated by Barrett et al. with the use of mAbs targeting epidermal growth factor receptor (EGFR) and HER2 conjugated to NIR fluorescent dyes such as Rhodamine green, Cy5.5 or Cy7 . The tumCrucial for image-guided surgery is the clear delineation of the tumour and determination of the margins. As could be expected, after removal of the skin from the fourth mammary glands, higher T/B ratios were obtained as compared to T/B ratios obtained in a non-invasive imaging setting. These observations highlight the potential of dual- or even multispectral imaging in the surgical setting, allowing for complete tumour visualization in order to enable radical tumour resections. However, at wavelengths shorter than 800 nm, more autofluorescence can be expected. It is therefore essential that the development of probes suitable for dual- or even multispectral imaging is followed by improvement in imaging systems that would provide imaging with accurate separation of each signal.Similarly to many other cancers that can evolve into metastatic disease, breast cancer is a clinically heterogeneous disease, which develops distant metastasis in 10–15 % of breast cancer patients during 3 years postdetection of the primary tumour . Based oFurthermore, multispectral optical imaging is a step forward towards implementation in the clinic of the recently introduced concept of molecular fluorescence pathology, as a cost-effective, time-efficient and sensitive technique, complementary to conventional IHC. Direct fluorescence imaging of the VHHs conjugated to NIR fluorophores already present in the tumour tissue would require no further costly lab processing, but only sectioning of the tissue blocks and subsequent imaging. This molecular fluorescent pathology would, however, strongly depend on the imaging target and probably be restricted to cell surface markers that are accessible to VHHs.With this feasibility study, we show the successful optical molecular imaging of CAIX- and HER2-positive DCIS xenografts employing a combination of two tumour-specific VHHs, in vivo, in the mimicked surgical setting, as well as ex vivo on tumour sections. The simultaneous determination of the expression status of multiple, clinically relevant tumour markers could lead to a better detection of the primary tumour and its metastases, providing optimal tumour delineation during surgery under image guidance. Furthermore, this approach could provide a more rapid assessment of tumour marker expression in the pathological setting. Altogether, although this is a proof of principle study and further studies are certainly needed to assess the potential of this approach in the clinic, multispectral optical imaging could possibly improve the current management of patients from early diagnosis of cancer to surgical procedures, treatment decision-making and monitoring of treatment response.


We discuss some aspects of the fluid dynamics of vitreous substitutes in the vitreous chamber, focussing on the flow induced by rotations of the eye bulb. We use simple, yet not trivial, theoretical models to highlight mechanical concepts that are relevant to understand the dynamics of vitreous substitutes and also to identify ideal properties for vitreous replacement fluids. We first recall results by previous authors, showing that the maximum shear stress on the retina grows with increasing viscosity of the fluid up to a saturation value. We then investigate how the wall shear stress changes if a thin layer of aqueous humour is present in the vitreous chamber, separating the retina from the vitreous replacement fluid. The theoretical predictions show that the existence of a thin layer of aqueous is sufficient to substantially decrease the shear stress on the retina. We finally discuss a theoretical model that predicts the stability conditions of the interface between the aqueous and a vitreous substitute. We discuss the implications of this model to understand the mechanisms leading to the formation of emulsion in the vitreous chamber, showing that instability of the interface is possible in a range of parameters relevant for the human eye. Retinal detachment is a serious, sight threatening condition that occurs when fluid enters the potential space between the neurosensory retina and the retinal pigment epithelium. Posterior vitreous detachment is primarily responsible for the generation of tractions on the retina that might produce retinal tears. These can possibly evolve into retinal detachment, since the detached vitreous often displays tight attachment points with the retina, where concentrated mechanical stimuli occur . In the Surgery is the only viable way to treat retinal detachment . One of Various vitreous substitutes are employed in the surgical practice, with largely different mechanical properties , 5. In pAt present, the only long-term vitreous substitutes widely employed in the clinical practice are silicone oils. They have suitable properties of chemical stability and transparency and have a high surface tension with the aqueous humour, which is a desirable property. The rational of using silicone oil as intraocular tamponade is to interrupt the open communication between the subretinal space/retinal pigment epithelial cells and the preretinal space with the aim of securing, in the first few days after surgery, chorioretinal adhesion induced by cryo- or laser treatment. Depending on the location of the retinal break oils with different densities (either higher or lower than the aqueous) can be adopted , 7. PropThe mechanical properties of tamponade fluids  influence the efficiency of the treatment and, therefore, a full understanding of the mechanical implications associated with the surgery is desirable. With the present work we aim at clarifying, from a purely mechanical point of view, the implications of adopting tamponade fluids with different mechanical properties. The problem is extremely complex even if only mechanics is accounted for, and, therefore, we proceed in this paper by introducing simple theoretical models that shed some light on specific, yet crucial, aspects of the problem.We start by considering the effect of viscosity of the tamponade fluid on the mechanical actions exerted on the retina during eye rotations.Due to the limited tamponade effect of silicone oils we then investigate further factors leading to the successful surgery. In particular, we investigate the changes of the maximum wall shear stress when silicone oils are used, accounting for the possible presence of a thin layer of aqueous separating the retina from the tamponade fluid.The success rate of surgery when silicone oils are used is about 70%. One of the common problems after vitrectomy, especially in the long run, is the formation of an oil emulsion. The reasons why this happens when silicone oils are used as tamponades are still unclear. A further aim of this paper is to present a simple theoretical model that predicts the role of oil properties  in the process of emulsion formation. To this end we study the stability of the interface between two superposed immiscible fluids set in motion by movements of the eye.The results presented in this paper are based on solutions of the mathematical equations that govern the motion of fluids. Fluid dynamics is a very well developed branch of physics, the modern foundations of which date back to the 19th century. The so-called Navier-Stokes equations, named after Claude-Louis Navier and George Gabriel Stokes, are known to accurately model the motion of a viscous fluid described as a continuum body. These equations are mathematically very complex and admit closed-form solutions, that is, solutions that can be expressed analytically in terms of known functions, only in very special cases. If an analytical solution of a problem can be found, its dependency on the controlling parameters  can be easily determined, without the need of computational simulations, and physical insight on the problem is therefore effectively obtained. In this paper we discuss some analytical solutions of the Navier-Stokes equations, which are relevant to understanding the dynamics of vitreous substitutes.ρ (mass per unit volume) and the (dynamic) viscosity μ (which is a measure of resistance to flow) and in which the stress is linearly proportional to the rate of deformation. Water, aqueous humour, and oils fall into this category.We consider purely viscous fluids, that is, fluids whose mechanical properties are completely characterized by the density Fluid motion in the vitreous chamber can be driven by different mechanisms, in particular, rotations of the eyeball or thermal differences between the anterior and posterior segments of the eye. However, it can be shown by simple order-of-magnitude arguments that the motion induced by eye rotations is much stronger than the thermally driven flow  and, theWe consider three different, relatively simple, models that shed light on important aspects of the dynamics of vitreous substitutes in the vitreous chamber. Proper interpretation of results from experimental or more complex theoretical models requires a full understanding of the results presented here. The details of the mathematical models are briefly reported in the appendices.Model 1. We first review results obtained by previous authors concerning the case of a rigid hollow sphere of radius R, modelling the vitreous chamber, filled with a fluid and study fluid motion generated by small-amplitude, periodic, torsional oscillations of the sphere  and has the same velocity of the wall at r = R. In the two cases the frequency is kept constant and is equal to 20 rad/s, which is a realistic value for real eye rotations. In μ = 0.96 Pa·s [μ = 0.001 Pa·s). In the two cases the velocity profiles are significantly different. In the high viscosity case they are almost straight lines; in other words the fluid moves almost as if it was a rigid body. On the other hand, when the viscosity is small, a thin layer forms at the wall in which the fluid moves and the velocity in the core of the domain is vanishingly small. This layer is referred to as an oscillatory boundary layer. The thickness of the oscillatory boundary layer at the wall is of order δ ~ √(μ/ρω). This means that similar results could have been obtained by keeping fixed the viscosity of the fluid and changing the frequency of oscillations. In fact, the problem is governed by a single dimensionless parameter α, the Womersley number, defined as α = √((ρR2ω)/μ), which can be physically interpreted as the ratio R/δ, between the radius of the sphere and the thickness of the oscillatory boundary layer. Flows characterized by the same value of the Womersley number have identical velocity profiles.We first consider the motion of a fluid contained in a sphere of radius rotation , 11. In .96 Pa·s ), wherear = R). We note that the real healthy vitreous is a viscoelastic fluid [In purely viscous fluids, whatever the value of the viscosity, the maximum of the velocity is invariably attained at the wall  [In see also ). This ilso [·s) . It appeFinally, we report in In the previous section we have discussed how the stress on the retina depends on the viscosity of a vitreous substitute, under the assumption that the fluid completely fills the vitreous chamber. In particular, we have shown that the mechanical actions on the retina grow with increasing fluid viscosity. In reality the situation is more complicated than this because, owing to the hydrophobic nature of vitreous substitutes, a thin layer of aqueous may form between the retina and the vitreous substitute.We therefore now consider how the scenario described in the previous section is modified when we account for the presence of a thin layer of aqueous close to the retina.μa = 10−3 Pa·s for the aqueous and μvs = 1 Pa·s for the vitreous substitute, e.g., a silicon oil). Figures d of the aqueous layer has been assumed. In the first case . In this case the motion of the wall is also felt in the vitreous substitute, which moves with a significant velocity. On the other hand, when d > δ, most of the motion keeps confined within the aqueous layer and the velocity in vitreous substitute is very small  and using the viscosity of the aqueous. When d/δ tends to zero, the scaled stress obviously tends to 1  and the stress on the wall is maximum. However, the figure shows that it is sufficient for a thin layer of aqueous to be present to make the maximum shear stress at the wall drop significantly. When d/δ ≈ 1 or greater, the presence of the vitreous substitute is not felt by the wall and the stress drops to the value it would attain in the presence of aqueous alone. This simple model highlights the importance of accounting for the possible presence of the thin layer of aqueous at the wall in the calculation of the stress on the retina.This has important implications for the wall shear stress at the wall, as it is shown in The presence of an aqueous layer separating the vitreous substitute from the retina was shown in the previous section to have an important effect on the shear stress on the retina. It is also known that one of the main complications after injection of long-term vitreous replacement fluids (particularly silicon oils) is the possible occurrence of emulsification. This implies that the oil-aqueous interface might break, eventually leading to the formation of oil droplets dispersed in the aqueous. There are several possible causes of generation of an emulsion, with one of them being introduction of mechanical energy into the system that breaks down the oil aqueous interface . Many auIn order to investigate the feasibility of this assumption and determine which parameters play a role in the breakdown of the interface, we present in this section results from an idealized, yet informative, theoretical model. As discussed in m = μvs/μa, which is the ratio between the viscosities of the two fluids, and S = σ/(ρdU2), which represents a dimensionless surface tension at the interface, where σ denotes the dimensional surface tension between the two fluids, ρ denotes fluid density, d is the thickness of the aqueous layer, and U is the maximum wall velocity. We note that, for the sake of simplicity, we neglect possible differences between the densities of the two fluids, thus effectively neglecting the role of gravity. The other dimensionless parameters that govern the stability problem are set to values that are reasonable for real eye rotations.The problem of the stability of the interface is governed by the four dimensionless parameters introduced and described in the appendices. Here we discuss the role of two of them: S and m.Our stability analysis shows that very long waves on the interface are invariably unstable during certain phases of the oscillation cycle. In other words the amplitude of very long disturbances always grows in time. We note that in the absence of an interface this stability problem consists in the stability of the so-called “Stokes boundary layer,” that is, the flow of a single fluid over an oscillating wall. This problem has been largely studied in the literature  and it im between the viscosities of the two fluids. Note that the viscosity of silicon oils is much larger than that of water. The figure shows that as m increases the system becomes more stable, again meaning with this statement that only very long waves are expected to possibly grow in time. Conversely, for relatively small values of m progressively shorter waves are found to be unstable.In In the present paper we have discussed theoretical results from three different idealized mathematical models that, in our view, help in understanding some of the basic features of the fluid mechanics of vitreous substitutes in the eye. We have focused our attention on the flow generated in the vitreous chamber by rotations of the eye globe, which is by far the most important mechanism generating fluid motion.We first have considered the case in which the whole vitreous chamber is filled with a single fluid and have modelled the chamber as a rigid sphere, performing sinusoidal small amplitude torsional oscillations, similar to what was done by previous authors , 11. We We have also briefly recalled how flow characteristics change when a viscoelastic fluid fills the vitreous chamber. The real healthy vitreous has viscoelastic properties, and there is a large body of research devoted to the identification of vitreous replacement fluids with viscoelastic properties. We have recalled that the motion of a viscoelastic fluid can be resonantly excited by eye rotations and, if this happens, large values of the shear stress are expected to develop on the retina. This has important implications for the choice of the ideal properties of vitreous substitutes. Soman and Banerjee  and SwinIn the second part of the paper we considered the effect of a thin layer of aqueous separating the vitreous substitute from the retina. Since vitreous substitutes are normally hydrophobic fluids and complete filling of the vitreous chamber can be hardly obtained, a layer of aqueous in correspondence with the retina is likely to form. We have shown that, when this is the case, the maximum stress on the retina can be significantly reduced, even if the viscosity of the vitreous replacement fluid is very large. Therefore, the possible existence of an aqueous layer should be accounted for when estimating the mechanical stresses on the retina after injection of a vitreous substitute.The presence of an aqueous layer and, consequently, of an interface between the aqueous and the vitreous substitute also has a crucial effect in the possible development of an emulsion, which is one of the main drawbacks associated with the use of silicon oils. Making use of a simple mathematical model we have studied the stability of the aqueous-vitreous substitute interface. The results show that the interface becomes more unstable if the surface tension decreases and it becomes more stable if the viscosity of the vitreous substitute is higher. Both results are in agreement with clinical observations. In fact there is evidence that the tendency to emulsification is significantly enhanced by the presence of surfactants that decrease the surface tension between the two fluids . Moreove


The presence of a tumour is very often associated with wasting in the host, affecting both skeletal muscle and adipose tissue. In the present study we used sorafenib, a multi-kinase inhibitor with anti-tumour activity, in order to investigate the effects of chemotherapy on wasting. Three different experimental mouse tumour models were included: C26 colon carcinoma, B16 melanoma and Lewis lung carcinoma (LLC). The results obtained clearly show that sorafenib was effective in reducing tumour growth in LLC and B16 models, while it had no effect on C26. Interestingly, sorafenib treatment reduced the signs of muscle wasting and improved the physical activity in the LLC model and also in the C26, despite the absence of antineoplastic action in the latter. Our results discard a role for IL-6 in the action of sorafenib since the drug did not affect the levels of this cytokine. Conversely, sorafenib seems to act by influencing both STAT3 and ERK activity at muscle level, leading to reduced accumulation of Pax7 and atrogin-1. Sorafenib may interfere with muscle wasting by decreasing the activation of these signal transduction pathways. Development of cachexia is often found in cancer patients. It occurs in 50 to 80% of these patients The loss of body weight results from both decreased adipose tissue and muscle; however, muscle wasting should be regarded as the most important since it plays a key role in recovery from cancer cachexia. Thus, it is an important determinant of survival and muscle strength and function, central to the recovery process Many efforts have been involved in designing a treatment for the cachectic syndrome, but unfortunately there is not a single one fully satisfactory in reversing weight loss. The development of different therapeutic strategies has focused on two targets: counteracting anorexia and neutralizing metabolic disturbances During the last decades several experimental models were established to study cancer cachexia New focused cancer treatments are less toxic than traditional chemotherapy. Such therapies ought to make tumour cells more susceptible, without increasing host toxicity. From this point of view, sorafenib is a multikinase inhibitor that has shown efficacy against a wide variety of tumours in preclinical models The aim of the present study was to characterize the cachectic phenotype of three commonly used experimental tumours in mice treated with sorafenib, in order to provide a reliable tool for translational research.Policy on Humane Care and Use of Laboratory Animals (ILAR 2011). The Bioethical Committee of the University of Barcelona approved the experimental protocol. All animal manipulations were made in accordance with the European Community guidelines for the use of laboratory animals.C57BL/6 (for LLC and B16 tumour models) or Balb/C (for C26 tumour model) mice weighing about 20 g  were maintained on a regular dark-light cycle (light from 08:00 to 20:00), with free access to food and water during the whole experimental period. They were cared for in compliance with the 5 B16 or C26 cells or i.m. (hind leg) with 5×105 LLC cells (see below for cell expansion). TB animals were divided into two sub-groups: untreated (n = 8) and treated (intragastric administration) every day with sorafenib . Mice were sacrificed under anaesthesia 14 days after tumour implantation. The day of sacrifice, the animals were weighed and anaesthetized with an intraperitoneal injection of ketamine/xylazine mixture . Several tissues and the tumour were rapidly excised, weighed, and frozen in liquid nitrogen.The mice were randomized and divided into two groups, namely controls  and tumour bearers (TB). TB mice were inoculated alternatively subcutaneously in the back with 5×10in vitro in Dulbecco's Modified Eagle's Medium  supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 100 µg/mL sodium pyruvate, 2 mM L-glutamine, at 37°C in a humidified atmosphere of 5% CO2 in air. The day of tumour implantation the cells were trypsinized, re-suspended in sterile saline and rapidly injected in the mice. Lewis lung carcinoma (LLC) cells were obtained from previous tumour hosts harvesting the cells in the exponential growth phase. The LLC is a cachexia-inducing, rapidly growing murine metastatic tumour composed by poorly differentiated cells, with a relatively short doubling time. The number of lung metastases was evaluated by light microscopy after necropsy. The metastases weight was evaluated according the methodology used by Donati et al Colon26 carcinoma (C26) and B16 melanoma (B16) cells were maintained Total blood was withdrawn from anaesthetized mice by cardiac puncture and collected in heparinized tubes. A drop was used to fill haematocrit capillary tubes that were centrifuged in a haematocrit centrifuge for 5 min at 800 x g. Haematocrit was calculated as percentage of packed cell volume of the total blood.Total physical activity  was determined during the last 24 hours before the sacrifice of the animals in control mice and tumour-bearing mice (both non-treated and treated with sorafenib), using activity sensors which translate individual changes in the infrared pattern caused by movements of the animals into arbitrary activity counts. For the measurements, animals remained in their home cage. A frame containing an infrared beam system was placed on the outside of the cage; this minimised the stress of the animals.IL-6 serum levels were detected by a commercially available mouse ELISA kit, used according to the manufacturer instructions . Serum from each animal (50 µL) was assayed in duplicate. Quantitative calibration was obtained performing a standard curve with recombinant mouse IL-6.2, 5 mM KCl, 0.1 mM EDTA, pH 8.0, 0.1% Triton X-100, with freshly added protease and phosphatase inhibitors. Samples were then centrifuged , the supernatant collected as the cytosolic fraction and the pellet resuspended in 20 mM HEPES, pH 7.9, containing 25% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH 8.0, protease and phosphatase inhibitor and incubated on ice for 30 min. Cell debris were removed by centrifugation  and the supernatant (nuclear fraction) collected. Protein concentration was assayed using BSA as working standard. Equal amounts of protein (30 µg) were heat-denaturised in sample-loading buffer , resolved by SDS-PAGE and transferred to nitrocellulose membranes. The filters were blocked with Tris-buffered saline (TBS) containing 0.05% Tween and 5% non-fat dry milk and then incubated overnight with antibodies directed against: p-ERK (Tyr204), total-ERK, total-STAT3 and GAPDH , FoxO3a and p-STAT3 , Atrogin-1  and the monoclonal antibody against Pax7 developed by Atsushi Kawakami, obtained from the Developmental Studies Hybridoma Bank (University of Iowa). Peroxidase-conjugated IgG  was used as secondary antibody. Membrane-bound immune complexes were detected by an enhanced chemiluminescence system  on a photon-sensitive film . Protein loading was normalized according to GAPDH expression. Quantification of the bands was performed by densitometric analysis using the ImageJ software.50 mg of gastrocnemius muscle were homogenized in 10 mM HEPES, pH 7.5, containing 10 mM MgClStatistical analysis of the data was performed by means of one-way analysis of variance (ANOVA). Statistically significant differences was analysed by post hoc Duncan test. All statistical tests were performed using SPSS version 21.In order to ascertain the effects of sorafenib on tumour-bearing animals we used three distinct experimental models: C26 colon carcinoma, B16 melanoma and Lewis lung carcinoma (LLC). All of them are associated with a clear cachectic response. From this point of view, as can be seen in Treatment with the anti-tumour sorafenib-inhibitor of the MAPK cascade- resulted in a huge reduction in tumour mass in the B16-bearing mice (94%) while it exerted only a small effect in the LLC  . RemarkaIn the B16 model, sorafenib treatment increased food intake in comparison with the non-treated B16-bearing mice . ConversTumour growth led to a decrease in haematocrit in all the models tested, being especially marked in the B16 (56%) and the LLC (56%) mice, where the tumour mass was bigger . It is iThe decreased body weight was associated with a concomitant loss of muscle mass. As can be seen in As can be seen in Taking into consideration the fact that the rescue of muscle mass does not necessarily mean a concomitant recovery of muscle function In order to explain the mechanisms accounting for the tumour-independent sorafenib effects, we determined the circulating concentrations of IL-6, a cytokine that has been reported to be affected by sorafenib, which reduces its release In order to further investigate the mechanisms by which sorafenib exerts its effects, we determined the intramuscular concentration and activation state of several proteins that might be involved in the sorafenib action. As far as we are concerned, there are no reports suggesting a direct kinase-inhibiting action of sorafenib in the skeletal muscle. For this part of the study we only used the C26 model, since in this setting, despite the absence of drug effects on tumour burden, sorafenib improved both muscle weight and activity parameters. The results depicted in per se induces wasting In conclusion, the results presented here reinforce the idea that sorafenib is an efficient drug for the treatment of tumours in addition to being a candidate for anti-cachectic therapy. This is based on the fact that any reduction in tumour mass results in a reduction in cachexia and also in the fact that, even without affecting the tumour, it does behave as an anti-cachectic molecule, as observed in the C26 tumour model. Yet, previous studies actually conclude that sorafenib Figure S1Correlation between tumour mass (expressed as % of the mean) and haematocrit in the three tumour models. Pearson analysis shows a significant correlation of tumour versus haematocrit in B16  and a tendency in LLC . No correlation is present in C26 .(TIF)Click here for additional data file.


The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The following information is missing from the Funding section: This study was supported by an unrestricted grant from UCB Pharma (The authors have also provided an updated Competing Interests statement: This study was supported by an unrestricted grant from UCB Pharma. João Eurico Cabral da Fonseca received this grant as the head of Rheumatology Research Unit at Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This does not alter our adherence to PLOS ONE policies on sharing data and materials.


Epithelial organs are almost universally secretory. The lung secretes mucus of extremely variable consistency. In the early prenatal period, the secretions are of largely unknown composition, consistency, and flow rates. In addition to net outflow from secretion, the embryonic lung exhibits transient reversing flows from peristalsis. Airway peristalsis (AP) begins as soon as the smooth muscle forms, and persists until birth. Since the prenatal lung is liquid-filled, smooth muscle action can transport fluid far from the immediately adjacent tissues. The sensation of internal fluid flows has been shown to have potent morphogenetic effects, as has the transport of morphogens. We hypothesize that these effects play an important role in lung morphogenesis. To test these hypotheses in a quantitative framework, we analyzed the fluid-structure interactions between embryonic tissues and lumen fluid resulting from peristaltic waves that partially occlude the airway. We found that if the airway is closed, fluid transport is minimal; by contrast, if the trachea is open, shear rates can be very high, particularly at the stenosis. We performed a parametric analysis of flow characteristics' dependence on tissue stiffnesses, smooth muscle force, geometry, and fluid viscosity, and found that most of these relationships are governed by simple ratios. We measured the viscosity of prenatal lung fluid with passive bead microrheology. This paper reports the first measurements of the viscosity of embryonic lung lumen fluid. In the range tested, lumen fluid can be considered Newtonian, with a viscosity of 0.016 ± 0.008 Pa-s. We analyzed the interaction between the internal flows and diffusion and conclude that AP has a strong effect on flow sensing away from the tip and on transport of morphogens. These effects may be the intermediate mechanisms for the enhancement of branching seen in occluded embryonic lungs. The prenatal lung is completely fluid-filled, and epithelial cells lining the lung continuously produce lumen fluid throughout gestation. The fluid is mainly secreted in the distal end and then flows to the upper airway where it is either absorbed or released into the amniotic space. Fluid production increases till mid gestation period and then decreases as the lung reaches full term . The proThus, mechanical inputs strongly affect developing lung—but the details matter. What is the load on a tissue? What parts of the tissue are deformed, how much is each location deformed, in what directions is it deformed, and is there a dynamic signature? How might mechanical inputs be sensed by the tissue, in different locations, in different directions, and what might that mean for the adaptive functioning of the organ? How do varying tissue and fluid characteristics affect the mechanical aspects?Lung lumen fluid is moved by secretion, airway peristalsis (AP) and, later, fetal breathing movements. Airway peristalsis and tonic contractions begin as soon as airway smooth muscle (SM) forms. AP is initially weak and uncoordinated, but settles into a rhythmic wave moving proximodistally  Fig 1)..Fig 1). Peristalsis in the broader sense has been thoroughly studied in open-ended tubes such as the gut and ureter, where it is an essential method of transport –10. HoweIn vitro, a trachea excised distal to the larynx will close up in a wound healing response, creating a sealed lumen. The trachea can be maintained in an open configuration by insertion of a stiff tube. These situations of open or closed trachea are mechanically distinct, and will have different characteristics of tissue stretch [There is a third distinct case of peristalsis, when both ends of the tubule are closed. In the intact embryonic airway, the larynx is pinched closed, but opens under sufficient lumen pressure.  stretch , 5 and fThis goal of this study was to analyze the flows of AP in the prenatal lung, using estimates based on standard results from fluid dynamics, measurements of viscosity of prenatal lung lumen fluid, and a computational model of the fluid-structure interactions in a simplified unbranched tubule, with a closed distal end (tip) and either open or closed proximal end (trachea). Our analysis leads us to suggest morphogenetic roles for AP in the two separate arenas of flow sensing and enhancement of transport of solutes such as growth factors. In both cases, we are able to quantify observable measures and suggest expected experimental outcomes.net flow into or out of the lumen must be due to other processes such as secretion (causing outward flow) and growth (causing inward flow). Although AP thus does not serve to remove fluid, it strongly affects diffusive transport, as detailed below.In the embryonic lung, the Reynolds number is relatively low (Re = 1), and thus, inertial effects can be neglected. Furthermore, in vitro studies have shown that flows due to AP can be completely reversible, as suspended debris in the lumen has been shown to move in response to AP and return to its original location . In a tucomplete occlusion (CO) and partial occlusion (PO). Most significantly, the direction of flow is different in CO and PO: For CO, fluid moves with the peristaltic wave, reversing only when the wave is released. For PO, fluid moves counter to the peristaltic wave  because the distal tip is closed; if both ends of the tube were open, or in a closed circulation (loop), fluid could flow forwards or backwards.In complete occlusion, fluid moves ahead of the peristaltic wave, in the direction of the wave. In incomplete  occlusion, that flow still occurs at a smaller level, but there is substantial flow in the direction vper and the average reflux velocity at the pinch O: We estimate the relationship between the velocity of the peristaltic SM wave )2(1−O)2 . If thervmid the midline velocity, a the initial tube radius, O the occlusion, and μ the viscosity [Flow is coupled to a pressure gradient. If the trachea is closed, the pressure is spatially uniform . If the iscosity .A closed trachea blocks flow out of the lung, so flow inside the lung is substantially reduced. Because a closed trachea does not permit pressure release, lumen pressure is spatially uniform, and occlusion is substantially reduced for the same SM force , Fig 4. In viscous flow, as in the prenatal lung, the fluid touching the lumen surface does not move relative to the surface (no slip). However, the lumen surface is where we find the maximal fluid shear. Conversely, in a symmetric tubule, the maximal fluid velocity is at the midline of the tubule, where fluid shear is zero. Flow velocities at the midline almost perfectly track shear rates at the epithelial surface .O is linear in the ratio FT/E, where F is SM force density, T is epithelial thickness, and E is tissue stiffness. For a closed trachea, our simulations gave O = 0.46FT/E (R2>0.99); for open trachea, O = 0.67FT/E (R2>0.98), independent of viscosity. Pressure buildup from AP depends on fluid viscosity only if the trachea is open. For closed trachea, maximal pressure depends only on smooth muscle force density F and epithelial thickness T: pmax = 0.087FT (R2>0.99); for open trachea, maximum pressure is roughly linear in viscosity: pmax∝(μ/μw)0.8 (R2>0.96), consistent with our estimates illustrated in We performed a parametric study of our computational model to determine the relationship between model inputs and outputs. Some flow features are independent of the viscosity of the lumen fluid. Flow is driven by pressure buildup due to occlusion. Maximal occlusion -1) did not measurably differ from 1 (R2 > 0.95). Thus, in the prenatal mouse lungs measured, the lumen fluid was Newtonian up to 30 s-1. We found that the viscosity of the lung fluid is an order of magnitude higher than that of water (log10(μ/μw) = 1.1±0.3). Specifically, water has a viscosity of 0.001 Pa-s and we measured embryonic mouse lung lumen viscosity at 0.016 ± 0.008 Pa-s. Variability between individuals was significantly greater than variability within an individual or between frequencies 2) . For exaThus AP can substantially enhance diffusive transport even for a small occlusion. The enhancement is greater, the larger the molecule and the more viscous the fluid. The effect is small for diffusion in water, but for diffusion in mucus, the effect can be very large.Branching in the pseudoglandular period proceeds on a consistent length scale. It is only in later developmental stages that differential growth rates and oriented cell divisions  lead to A cell cannot directly detect bulk fluid velocity, but it can infer bulk velocity from the surrogate measure of shear rate at the lumen surface. In a straight tube, the maximal fluid velocity is on the midline, and there is zero flow at the lumen surface. Conversely, fluid shear is zero on the midline, and maximal on the lumen surface. At a given location along the airway, for our assumption of a Newtonian fluid, surface shear rate is nearly proportional to midline velocity , except In all cases, shear rates from AP are negligible in the tip region, only becoming significant in the part of the airway experiencing SM contraction . PrimaryWe also predict that any flow-sensing PC would be shorter proximally, where the SM is most strongly contractile and flows are fastest, and longer distally, where SM, SM contraction, and flows are less developed. If there were flow in the distal tip, it could not be due to AP, but must be due to secretion. If flow-sensing primary cilia are found distal to the SM , we canThus, if PC are found along the entire intraluminal surface of a growing bud, they may perform two separate functions in the developing airway, depending on their position: either probing AP-driven flow in the SM-wrapped epithelium; or detecting secretion-driven flow at the distal tip. In particular, because shear from AP is greater where SM is more developed , intraluminal PC should also be progressively shorter if SM-associated epithelial cells are to perform equal physiological functions, since signaling at PC depends on the scale of bending and stretching of its plasma membrane that are proportional to fluid shear. By the same principle, distal tip epithelial cells should have PC with a mechanotransduction machinery fine-tuned to secretion instead of AP-driven flow. This suggests that signaling networks in distal tip epithelia would be functionally distinct from those in SM-associated epithelia and, consequently, that epithelial cells from either of those two regions should be phenotypically distinct.We speculate that PC serve to sense flow in developing ducts, and in turn to regulate morphogenesis based on sensed flows (some examples are reviewed in ). BecausIf an epithelial cell were able to detect stretch at its basal or apical end, and flow at its apical surface, it could, in principle, infer information from far away in the lung, using only local information. It could, in effect, remotely detect, from hundreds of microns away, whether the trachea is closed or open. For example, an airway epithelial cell that sensed stretch could infer its position from geometric cues (discussed in ), and ifto proximal tissues, but also of enhancing transport from distal tissues.Smooth muscle forms in the lung at the same time as the lung becomes too large for diffusion to remain an efficient means of transport. At the same time, the cardiovascular system is developing, and is also moving fluid by peristalsis. Peristalsis represents an ancient and widespread mechanism for bulk transport. We have shown that even a small, weak peristaltic wave in the embryonic airway can dramatically increase the efficiency of transport, especially for large molecules, and especially in viscous fluids, such as may develop in late gestation. Thus the known effects of AP in stimulating and regulating morphogenesis may be based not just in mechanisms involving tissue stretch, but also in mechanisms involving transport of morphogens. Enhanced transport especially of large molecules, such as growth factors, in the lumen would have the effect not just of enhancing transport -1), lumen fluid could be considered Newtonian. Our model and analysis were built on an assumption of the unknown properties of the lumen fluid being Newtonian. Shear rates in AP can be high (100-1000/s), as we find both from our estimates without elastic energy storage and also from our simulations with elastic energy stored in the tissues. The highest shear rates are at the stenosis; the closer the occlusion is to complete, the higher will be the shear rates at the stenosis. If the lumen contains mucus, its non-Newtonian properties will be significant in this flow regime. If further testing reveals non-Newtonian behavior at higher shear rates or in other model organisms, we might then conclude that one of the many functions of prenatal AP is the same function as coughing and sneezing in the postnatal lung: to exploit the shear-thinning properties of mucus, to clear it from the lung.This paper reports the first measurements of the viscosity of embryonic lung lumen fluid. We found that in the range tested . AP We know that mechanical inputs strongly affect developing organs—but the mechanical details matter, and are studied far more rarely than the molecular details: what is the load on a developing tissue, what parts of the tissue are deformed, how much is each location deformed, in what directions is it deformed, and is there a dynamic signature? Might mechanical inputs be sensed by the tissue, in different locations, in different directions, and what might that mean for the adaptive functioning of the organ? Do varying tissue and fluid characteristics affect the mechanical aspects? In the developing lung, for example, it has long been appreciated that fetal lung liquid plays a key role in morphogenesis and development: draining the liquid leads to tissue hypoplasia, whereas blocking the exit leads to dysplastic overgrowth of the tissue. There have even been clinical trials in humans to evaluate whether the latter approach might be useful to rescue hypoplastic lungs in utero, some with positive and some with mixed results. This paper thus considers the development of the lung from the point of view of fluid mechanics and provides some important insights from first principles in this field. While fluid mechanics is not on the usual syllabus for pediatricians or pediatric surgeons, clearly it has been co-opted by evolution to help form the branched structure and gas diffusion surface of the developing lung.The embryonic lung was modeled as a single unbranched radially symmetric tubule with three tissue layers and a lumen .The tissues were modeled as uniform, isotropic, Hookean, undergoing finite strain. The lumen fluid was modeled as uniform, Newtonian, and creeping. The trachea was modeled as either open or closed. To our knowledge, there are no published studies of the mechanical properties of embryonic lung tissues or lumen fluid; we used the estimated parameter values in The dynamic model was implemented in a finite element package, COMSOL, with its Fluid-Structure module, bidirectional coupling, large-strain formulation, ALE moving mesh, and particle tracing. Poisson's ratio in the tissue was approximated by 0.45. Convergence generally required occlusion to be < 95% and maximal lumen pressure < 15 Pa. The FEM model was verified against analytical results for a hollow sphere and an open tube.Mice were maintained as a colony in a facility that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All animal procedures were approved by the North Carolina State University institutional animal care and use committee, #12-044-B. Mouse lungs were extracted from discarded C57BL/6J embryos decapitated for a separate study, and kept in PBS. Viscosity of lumen fluid was measured by passive bead microrheometry , 33. LunS1 FileRaw data from particle-tracking velocimetry.(RAR)Click here for additional data file.S1 VideoWhen trachea is closed, pressure in lumen is uniform as peristaltic wave progresses.(AVI)Click here for additional data file.S2 VideoWhen trachea is open, pressure builds distal to stenosis, but remains low proximal to it, which permits greater occlusion than with closed trachea.(AVI)Click here for additional data file.S3 VideoClosed trachea restricts flow of lumen fluid, resulting in minimal mixing of contents. Tracer particles return almost to original positions.(AVI)Click here for additional data file.S4 VideoPeristalsis with open trachea creates high shear, resulting in substantial fluid mixing as packets of fluid from near the midline make large excursions, in some cases exiting the lung before lung refills.(AVI)Click here for additional data file.


North Pacific subtropical high (NPSH) is permanent high-pressure system over the Northern Pacific Ocean and it extends to the western North Pacific during the boreal summer (June-July-August), which is so called the western North Pacific subtropical high (WNPSH). Here, we examine the covariability of the NPSH-WNPSH during summer using both observation and Coupled Model Intercomparison Project phase 5 (CMIP5) model data. The statistical analyses indicate that the NPSH-WNPSH covariability shows significant decadal variability in the observations, in addition, the in-phase relationship of NPSH-WNPSH is enhanced after the mid-to-late 1990s. A dipole-like sea surface temperature (SST) pattern, i.e., a warming in the western Pacific and a cooling in the eastern Pacific, is dominant after the mid-to-late 1990s, which acts to enhance the covariability of NPSH-WNPSH by modulating the atmospheric teleconnections. However, the covariability of NPSH-WNPSH in the future climate is not much influenced by the anthropogenic forcing but it is largely characterized by the natural decadal-to-interdecadal variability, implying that the enhancement of NPSH-WNPSH covariability after the mid-to-late 1990s could be considered as part of decadal-to-interdecadal variability. The sliding correlation coefficients between the intensities of the WNPSH and NPSH reveal a gradual increase with decadal fluctuation . Furtherr ∼ 0.84 for SVD1 and r ∼ 0.79 for SVD2), verifying a critical role of the tropical SST variability on the NPSH-WNPSH covariability. Furthermore, the SST variability due to the SVD1 and SVD2 explains substantial variance of 58% (42%) for NPSH (WNPSH).311The spatial structure of the SVD1 and its PC time series indicate that an in-phase relationship of the NPSH and WNPSH is associated with a dipole-like SST pattern, i.e., a warming (cooling) in the IO-WP and a cooling (warming) in the EP . Note thr ∼ 0.54 in SST and r ∼ 0.74 in Z850), which indicates that the SVD2 represents an out-of-phase in the two subtropical high-pressures. Recent studies have effectively described how the IO warming and WP cooling generate the meridional dipole height pattern over western North Pacific through positive atmosphere-ocean feedback processes3On the other hand, the spatial structure of the SVD2 reflects an out-of-phase height variance over North Pacific, which is associated with the triple-like structure in the tropical basins . The triHow a dipole-like SST pattern in the tropical basins, i.e., a warming (cooling) in the WP and a cooling (warming) in the EP, acts to enhance the covariability of NPSH-WNPSH after the mid-to-late 1990s? To examine this statistically, we display the 13-yr sliding standard deviation of the SST time series projected onto SST pattern of SVD1 and SVD2 during 1920–2010, respectively . While tThe IO-WP SST  has warmed by 0.39 °C during the last 32 years . Such ra20Coherent statistical evidences suggest the recent intensification of the covariability between NPSH and WNPSH along with considerable decadal variabilities. The strengthening of covariability in the two subtropical highs after the mid-to-late 1990s is associated with the dipole-like SST pattern in the tropical basins, i.e., a warming (cooling) in the WP and a cooling (warming) in the EP. The CMIP5 future climate simulation shows no significant change in the relationship of NPSH-WNPSH covariability. These reflect that a considerable portion in strengthening of covariability in the two subtropical highs in the observations is derived from the natural variability on multi-decadal timescales.We speculate that the interdecadal change in covariability of WNPSH-NPSH is significantly associated with the change in the air-sea interactions in the WP such as the SST-convective forcing. For example, the convective response to the anomalous SST forcing in the WP is quite different before and after the mid-to-late 1990s see . After t1723The present study used monthly mean SST from the Met Office Hadley Centre Sea Ice and Sea Surface Temperature version 1.1 (HadISST1.1)The Z850 and SLP were used to identify the NPSH and WNPSH. The NPSH and WNPSH indices were calculated as the area-averaged anomalies of Z850 and SLP over  and . To investigate the major coupled patterns between tropical SSTs and mid-latitude North Pacific, we applied a SVD analysis to the JJA seasonal mean tropical SST and extratropical North Pacific atmospheric system, i.e., IO-WP-EP SST anomaly over  and North Pacific geopotential height anomaly at 850 hPa over . The statistical significance for the composite and regression analysis was estimated with a two-tailed Student’s t test with the degree of freedom of n−2.2 due to both anthropogenic and natural forcing, were imposed on the historical run. The RCP4.5 run, in which radiative forcing stabilized at ~4.5 Wm−2 after 2100, was chosen for future climate projectionst century .To investigate future changes of tropics-midlatitudes atmospheric covariability under anthropogenic global warming, we used the 20 coupled general circulation models (CGCMs) that participated in CMIP5. The used model, institution and horizontal resolution of the atmospheric components are represented in How to cite this article: Yun, K.-S. et al. Covariability of western tropical Pacific-North Pacific atmospheric circulation during summer. Sci. Rep.5, 16980; doi: 10.1038/srep16980 (2015).


After publication of the original article , it cameThis work was supported in part by a grant to BH: European Union’s Seventh Framework Program (FP/2007/2013)/European Research Council (ERC) Grant Agreement 322820.