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Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral nonstructural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host species barrier. We characterized the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionarily distinct NS1 proteins counteract the IFN response using different and mutually exclusive mechanisms: while the NS1 proteins of early EIVs block general gene expression by binding to cellular polyadenylation-specific factor 30 (CPSF30), NS1 proteins from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and were rapidly selected for during EIV evolution, highlighting the importance of evolutionary processes in immune evasion mechanisms during IAV adaptation. IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remain unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that successfully established a new lineage in horses in the early 1960s and is currently circulating worldwide in the equine population. Here, we analyzed the molecular evolution of the virulence factor nonstructural protein 1 (NS1) and show that NS1 proteins from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation., (Copyright © 2018 Chauché et al.)

Metastatic pancreatic ductal adenocarcinomas (PDAC) are incurable due to the rapid development of resistance to all current therapeutics. Oncolytic adenoviral mutants have emerged as a promising new strategy that negates such resistance. In contrast to normal tissue, the majority of PDACs express the αvβ6 integrin receptor. To exploit this feature, we modified our previously reported oncolytic adenovirus, AdΔΔ, to selectively target αvβ6 integrins to facilitate systemic delivery. Structural modifications to AdΔΔ include the expression of the small but potent αvβ6-binding peptide, A20FMDV2, and ablation of binding to the native coxsackie and adenovirus receptor (CAR) within the fiber knob region. The resultant mutant, Ad5-3Δ-A20T, infected and killed αvβ6 integrin-expressing cells more effectively than the parental wild-type (Ad5wt) virus and AdΔΔ. Viral uptake through αvβ6 integrins rather than native viral receptors (CAR, αvβ3 and αvβ5 integrins) promoted viral propagation and spread. Superior efficacy of Ad5-3Δ-A20T compared with Ad5wt was demonstrated in 3D organotypic cocultures, and similar potency between the two viruses was observed in Suit-2 in vivo models. Importantly, Ad5-3Δ-A20T infected pancreatic stellate cells at low levels, which may further facilitate viral spread and cancer cell elimination either as a single agent or in combination with the chemotherapy drug, gemcitabine. We demonstrate that Ad5-3Δ-A20T is highly selective for αvβ6 integrin-expressing pancreatic cancer cells, and with further development, this new and exciting strategy can potentially be extended to improve the systemic delivery of adenoviruses to pancreatic cancer patients. Mol Cancer Ther; 17(2); 575-87. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Cadherin-17 (CDH17) has been implicated as protumorigenic for many years, but mechanisms have been unclear. A Spanish team has generated antibodies to an RGD motif in CDH17 that inhibits integrin α2β1 binding to CDH17 and thereby inhibits integrin activation, tumorigenesis, and metastasis. These reagents may have therapeutic potential. Clin Cancer Res; 24(2); 253-5. ©2017 AACR See related article by Bartolomé et al., p. 433 ., (©2017 American Association for Cancer Research.)

Super-resolution microscopy methods enable resolution of biological molecules in their cellular or tissue context at the nanoscale. Different methods have their strengths and weaknesses. Here we present a method that enables correlative confocal, structured illumination microscopy (SIM) and single-molecule localization microscopy (SMLM) imaging of structures involved in formation of invadopodia on the same sample. This enables up to four colors to be visualized in three dimensions at a resolution of between 120 and 10 nm for SIM and SMLM, respectively.

The integrin αvβ6 is up-regulated in numerous carcinomas, where expression commonly correlates with poor prognosis. αvβ6 promotes tumour invasion, partly through regulation of proteases and cell migration, and is also the principal mechanism by which epithelial cells activate TGF-β1; this latter function complicates therapeutic targeting of αvβ6, since TGF-β1 has both tumour-promoting and -suppressive effects. It is unclear how these different αvβ6 functions are linked; both require actin cytoskeletal reorganization, and it is suggested that tractive forces generated during cell migration activate TGF-β1 by exerting mechanical tension on the ECM-bound latent complex. We examined the functional relationship between cell invasion and TGF-β1 activation in pancreatic ductal adenocarcinoma (PDAC) cells, and confirmed that both processes are αvβ6-dependent. Surprisingly, we found that cellular functions could be biased towards either motility or TGF-β1 activation depending on the presence or absence of epidermal growth factor receptor pathway substrate 8 (Eps8), a regulator of actin remodelling, endocytosis, and GTPase activation. Similar to αvβ6, we found that Eps8 was up-regulated in >70% of PDACs. In complex with Abi1/Sos1, Eps8 regulated αvβ6-dependent cell migration through activation of Rac1. Down-regulation of Eps8, Sos1 or Rac1 suppressed cell movement, while simultaneously increasing αvβ6-dependent TGF-β1 activation. This latter effect was modulated through increased cell tension, regulated by Rho activation. Thus, the Eps8/Abi1/Sos1 tricomplex acts as a key molecular switch altering the balance between Rac1 and Rho activation; its presence or absence in PDAC cells modulates αvβ6-dependent functions, resulting in a pro-migratory (Rac1-dependent) or a pro-TGF-β1 activation (Rho-dependent) functional phenotype, respectively. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Integrins are transmembrane receptors that are central to the biology of many human pathologies. Classically mediating cell-extracellular matrix and cell-cell interaction, and with an emerging role as local activators of TGFβ, they influence cancer, fibrosis, thrombosis and inflammation. Their ligand binding and some regulatory sites are extracellular and sensitive to pharmacological intervention, as proven by the clinical success of seven drugs targeting them. The six drugs on the market in 2016 generated revenues of some US$3.5 billion, mainly from inhibitors of α4-series integrins. In this review we examine the current developments in integrin therapeutics, especially in cancer, and comment on the health economic implications of these developments., Competing Interests: Simon Goodman and Sabine Raab-Westphal are employed by Merck KGaA who have been active in the field of integrin pharmaceutical development. They are co-inventors on patents granted on integrin inhibitors, including abituzumab.

The 20-residue linear peptide A20FMDV2 has been shown to exhibit high selectivity and affinity for the tumour-related αvβ6 integrin and has potential as a vector for therapeutic drugs. However, it exhibits poor half-life in plasma in part due to its high susceptibility to serum proteases. In this study fourteen A20FMDV2 analogues incorporating non-proteinogenic substitutes of the native Lys16 and Leu13 residues and six A20FMDV2 analogues containing modified N- and C-termini were synthesised to increase the half-life and activity of A20FMDV2. The analogues incorporating modified terminal motifs of A20FMDV2 were found to strongly bind to the αvβ6 integrin and were subsequently functionalized with the diethylenetriaminepentaacetic acid chelating agent to facilitate coupling with radioactive indium-111 for human plasma stability and in vivo biodistribution studies. A20FMDV2 peptide variants incorporating an N-terminal d-Asn and C-terminal d-Thr exhibited improved relative activity in vitro and were less susceptible to plasma degradation., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: As antimicrobial resistant bacterial strains continue to emerge and spread in human and animal populations, understanding prescription practices is key in benchmarking current performance and setting goals. Antimicrobial prescription (AP) in companion veterinary species is widespread, but is neither monitored nor restricted in the USA and Canada. The veterinary use of certain antimicrobial classes is discouraged in some countries, in the hope of preserving efficacy for serious human infections., Objectives: The aim of this study was to ascertain the rate of prescription of a number of 'reserved' antimicrobials in a first-opinion US and Canadian horse cohort, and identify trends in their empirical use., Study Design: Retrospective cohort study., Methods: A large convenience sample of electronic medical records (2006-2012) was interrogated using text mining to identify enrofloxacin, clarithromycin and ceftiofur prescriptions. Time series analysis and logistic regression were used to identify trends and risk factors for prescription., Results: Prescription of these antimicrobials as a first-line intervention, without culture and sensitivity testing (CST), was common in this population. Enrofloxacin prescriptions were found to increase over the study period, and there was evidence of either a reducing, or static trend in the proportion of reserved APs informed by CST., Main Limitations: Dose adequacy could not be included due to the nature of the data used., Conclusions: Empirical use of reserved antimicrobials was common in this population, and further advice and guidance should be issued to first-opinion veterinarians to safeguard antimicrobial efficacy., (© 2016 EVJ Ltd.)

Background: The use of electronic medical records (EMRs) offers opportunity for clinical epidemiological research. With large EMR databases, automated analysis processes are necessary but require thorough validation before they can be routinely used., Objective: The aim of this study was to validate a computer-assisted technique using commercially available content analysis software (SimStat-WordStat v.6 (SS/WS), Provalis Research) for mining free-text EMRs., Methods: The dataset used for the validation process included life-long EMRs from 335 patients (17,563 rows of data), selected at random from a larger dataset (141,543 patients, ~2.6 million rows of data) and obtained from 10 equine veterinary practices in the United Kingdom. The ability of the computer-assisted technique to detect rows of data (cases) of colic, renal failure, right dorsal colitis, and non-steroidal anti-inflammatory drug (NSAID) use in the population was compared with manual classification. The first step of the computer-assisted analysis process was the definition of inclusion dictionaries to identify cases, including terms identifying a condition of interest. Words in inclusion dictionaries were selected from the list of all words in the dataset obtained in SS/WS. The second step consisted of defining an exclusion dictionary, including combinations of words to remove cases erroneously classified by the inclusion dictionary alone. The third step was the definition of a reinclusion dictionary to reinclude cases that had been erroneously classified by the exclusion dictionary. Finally, cases obtained by the exclusion dictionary were removed from cases obtained by the inclusion dictionary, and cases from the reinclusion dictionary were subsequently reincluded using Rv3.0.2 (R Foundation for Statistical Computing, Vienna, Austria). Manual analysis was performed as a separate process by a single experienced clinician reading through the dataset once and classifying each row of data based on the interpretation of the free-text notes. Validation was performed by comparison of the computer-assisted method with manual analysis, which was used as the gold standard. Sensitivity, specificity, negative predictive values (NPVs), positive predictive values (PPVs), and F values of the computer-assisted process were calculated by comparing them with the manual classification., Results: Lowest sensitivity, specificity, PPVs, NPVs, and F values were 99.82% (1128/1130), 99.88% (16410/16429), 94.6% (223/239), 100.00% (16410/16412), and 99.0% (100×2×0.983×0.998/[0.983+0.998]), respectively. The computer-assisted process required few seconds to run, although an estimated 30 h were required for dictionary creation. Manual classification required approximately 80 man-hours., Conclusions: The critical step in this work is the creation of accurate and inclusive dictionaries to ensure that no potential cases are missed. It is significantly easier to remove false positive terms from a SS/WS selected subset of a large database than search that original database for potential false negatives. The benefits of using this method are proportional to the size of the dataset to be analyzed., (©Marco Duz, John F Marshall, Tim Parkin. Originally published in JMIR Medical Informatics (http://medinform.jmir.org), 29.06.2017.)

The αvβ6 integrin receptor has been shown to be overexpressed on many types of cancer cells, resulting in a more pro-invasive and aggressive phenotype, this makes it an attractive target for selective drug delivery. In tumours that over-express the αvβ6 receptor, cellular uptake of liposomes can be enhanced using ligand-targeted liposomes. It has previously been shown in both in vitro and in vivo studies that liposomal alendronate (L-ALD) can sensitise cancer cells to destruction by Vγ9Vδ2 T cells. It is hypothesised that by using the αvβ6-specific peptide A20FMDV2 as a targeting moiety for L-ALD, the therapeutic efficacy of this therapy can be increased in αvβ6 positive tumours. Targeted liposomes (t-L) were formulated and the targeting efficacy of targeted liposomes (t-L) was assessed by cell uptake and cytotoxicity studies in the αvβ6 positive cells line A375Pβ6. Bio-distribution of both L and t-L were carried out in αvβ6 positive (A375Pβ6 and PANC0403) and αvβ6 negative (A375Ppuro and PANC-1) subcutaneous tumour mouse models. Immuno-compromised mice bearing A375Pβ6 experimental metastatic lung tumours were treated with L-ALD or t-L-ALD as monotherapies or in combination with ex vivo-expanded Vγ9Vδ2 T cells. In vitro, αvβ6-dependant uptake of t-L was observed, with t-L-ALD being more effective than L-ALD at sensitising A375Pβ6 to γδ T cells. Interestingly, t-L-ALD led to slightly higher but not significant reduction in tumour growth compared to L-ALD, when used as monotherapy in vivo. Moreover, both L-ALD and t-L-ALD led to significant reductions in tumour growth when used in combination with γδ T cells in vivo but t-L-ALD offered no added advantage compared to L-ALD., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Electronic medical records from first opinion equine veterinary practice may represent a unique resource for epidemiologic research. The appropriateness of this resource for risk factor analyses was explored as part of an investigation into clinical and pharmacologic risk factors for laminitis. Amalgamated medical records from seven UK practices were subjected to text mining to identify laminitis episodes, systemic or intra-synovial corticosteroid prescription, diseases known to affect laminitis risk and clinical signs or syndromes likely to lead to corticosteroid use. Cox proportional hazard models and Prentice, Williams, Peterson models for repeated events were used to estimate associations with time to first, or subsequent laminitis episodes, respectively. Over seventy percent of horses that were diagnosed with laminitis suffered at least one recurrence. Risk factors for first and subsequent laminitis episodes were found to vary. Corticosteroid use (prednisolone only) was only significantly associated with subsequent, and not initial laminitis episodes. Electronic medical record use for such analyses is plausible and offers important advantages over more traditional data sources. It does, however, pose challenges and limitations that must be taken into account, and requires a conceptual change to disease diagnosis which should be considered carefully., (Copyright © 2016 Elsevier B.V. All rights reserved.)

OBJECTIVE To characterize and describe the compensatory load redistribution that results from unilateral hind limb lameness in horses. DESIGN Retrospective case series. ANIMALS 37 client-owned horses. PROCEDURES Medical records were reviewed to identify horses with unilateral hind limb lameness that responded positively (by objective assessment) to diagnostic local anesthesia during lameness evaluation and that were evaluated before and after diagnostic local anesthesia with an inertial sensor-based lameness diagnosis system. Horses were grouped as having hind limb lameness only, hind limb and ipsilateral forelimb lameness, or hind limb and contralateral forelimb lameness. Measures of head and pelvic movement asymmetry before (baseline) and after diagnostic local anesthesia were compared. The effect of group on baseline pelvic movement asymmetry variables was analyzed statistically. RESULTS Maximum pelvic height significantly decreased from the baseline value after diagnostic local anesthesia in each of the 3 lameness groups and in all horses combined. Minimum pelvic height significantly decreased after the procedure in all groups except the hind limb and contralateral forelimb lameness group. Head movement asymmetry was significantly decreased after diagnostic local anesthesia for horses with hind limb and ipsilateral forelimb lameness and for all horses combined, but not for those with hind limb lameness only or those with hind limb and contralateral forelimb lameness. CONCLUSIONS AND CLINICAL RELEVANCE Results supported that hind limb lameness can cause compensatory load redistribution evidenced as ipsilateral forelimb lameness. In this population of horses, contralateral forelimb lameness was not compensatory and likely reflected true lameness. Further studies are needed to investigate the source of the contralateral forelimb lameness in such horses.

TGFβ1 is the most pleiotropic of all known cytokines and thus, to avoid uncontrolled TGFβ-activated processes, its activity is tightly regulated. Studies in fibrosis have led to the discovery that αv integrins are the major regulators of the local activation of latent TGFβ in our tissues. Since all cells can express one or more types of αv integrins, this raises the possibility that, in the complex milieu of a developing cancer, multiple cell types including both cancer cells and stromal cells activate TGFβ. In normal tissues, TGFβ1 is a tumour suppressor through its ability to suppress epithelial cell division, whereas in cancer, in which tumour cells develop genetic escape mechanisms to become resistant to TGFβ growth suppression, TGFβ signalling creates a tumour-permissive environment by activating fibroblast-to-myofibroblast transition, by promoting angiogenesis, by suppressing immune cell populations and by promoting the secretion of both matrix proteins and proteases. In addition, TGFβ drives epithelial-to-mesenchymal transition (EMT) increasing the potential for metastasis. Since αv integrins activate TGFβ, they almost certainly drive TGFβ-dependent cancer progression. In this review, we discuss the data that are helping to develop this hypothesis and describe the evidence that αv integrins regulate the TGFβ promotion of cancer. Graphical Abstract Mechanisms of integrin-mediated transforming growth factor beta (TGFβ) activation and its effect on stromal processes. 1 Matrix-bound latent LAP-TGFβ1 binds αv integrins expressed by epithelial cells or fibroblasts (LAP latency-associated peptide). TGFβ1 becomes exposed. 2 Active TGFβ1 binds the TGFβ receptor in an autocrine or paracrine fashion. 3 TGFβ1 signalling increases integrin expression, LAP-TGFβ1 secretion and trans-differentiation of fibroblasts into contractile cells that secrete collagens and collagen cross-linking proteins. By contracting the matrix, latent TGFβ1 is stretched making the activation of latent TGFβ1 easier and creating a continuous cycle of TGFβ1 signalling. TGFβ1 promotes cancer progression by promoting angiogenesis, immune suppression and epithelial-to-mesenchymal transition (EMT).

The average age of the global human population is increasing, leading to increased interest in the effects of chronic disease and multimorbidity on health resources and patient welfare. It has been posited that the average age of the general veterinarian-attended horse population of the UK is also increasing, and therefore it could be assumed that chronic diseases and multimorbidity would pose an increasing risk here also. However, evidence for this trend in ageing is very limited, and the current prevalence of many chronic diseases, and of multimorbidity, is unknown. Using text mining of first-opinion electronic medical records from seven veterinary practices around the UK, Kaplan-Meier and Cox proportional hazard modelling, we were able to estimate the apparent prevalence among veterinarian-attended horses of nine chronic diseases, and to assess their relative effects on median life expectancy following diagnosis. With these methods we found evidence of increasing population age. Multimorbidity affected 1.2% of the study population, and had a significant effect upon survival times, with co-occurrence of two diseases, and three or more diseases, leading to 6.6 and 21.3 times the hazard ratio compared to no chronic disease, respectively. Laminitis was involved in 74% of cases of multimorbidity. The population of horses attended by UK veterinarians appears to be aging, and chronic diseases and their co-occurrence are common features, and as such warrant further investigation., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alpha-v beta-6 (αvβ6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the αvβ6 integrin in a number of disease models. In a radioligand binding assay using a radiolabelled form of the peptide ([3H]A20FMDV2), its high affinity (K(D): 0.22 nmol/l) and selectivity (at least 85-fold) for αvβ6 over the other members of the RGD integrin family was confirmed. [3H]A20FMDV2 αvβ6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of αvβ6 by A20FMDV2 and latency-associated peptide-1 was shown to be fast (t(1/2): 1.5 and 3.1 min, respectively), concentration-dependent (EC50: values 1.1 and 3.6 nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on αvβ6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of αvβ6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane αvβ6 following RGD ligand engagement., (© 2016 S. Karger AG, Basel.)

Background: Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGFβ signaling and angiogenesis in vitro and in vivo., Methods: Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGFβ pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided., Results: Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between α2β1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFβ signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFβ markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFβ markers, n = 6; VEGF, n = 21). Inhibition of TGFβ receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siTβRII = 29.7, SD = 6.1, P = .02)., Conclusions: Type VII collagen suppresses TGFβ signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Reasons for Performing Study: Triamcinolone is commonly used in equine practice for the treatment of orthopaedic conditions. A serious potential adverse effect of triamcinolone is laminitis. However, evidence for the risk of laminitis associated with triamcinolone use is limited., Objectives: To determine the risk of laminitis within 90 days of triamcinolone administration and compare with the risk of laminitis in a veterinary-attended horse population., Study Design: Retrospective study of clinical records., Methods: Text mining and data extraction was performed using content analysis software (SimStat-WordStat v.6) on a database of anonymous digital clinical records from a convenience sample of North American equine practices (n = 9). Medical records were retrieved using a dictionary of keywords for 3 groups of horses: 1) treated with triamcinolone, 2) age and practice matched control population (no triamcinolone) and 3) all laminitic horses. Records of horses within Groups 1 and 2 were mined for evidence of laminitis within a 90-day period of treatment or a random date respectively. Data manipulation and analysis was performed using R v3.0.0 (R Development Core Team). The prevalence of laminitis within all groups was determined and relative risk of developing laminitis determined by single logistic regression., Results: The clinical records of 225,777 horses were examined. Overall prevalence of laminitis within the database was 1.1% (n = 2533). Triamcinolone was administered to 12.4% (n = 27,898) horses and 0.07% of treated horses (n = 20) developed laminitis. In the control population (n = 56,695), 0.2% of horses (n = 134) developed laminitis. The risk of developing laminitis was significantly lower in the triamcinolone treatment group than the control population (OR 0.3 95%CI, 0.18-0.48 P<0.001)., Conclusions: Triamcinolone treatment does not increase the overall risk of a horse developing laminitis. However, further investigation of risk factors for laminitis in the 20 horses identified by this preliminary study is warranted to aid development of evidence-based treatment guidelines. Ethical animal research: This study was approved by the Ethics and Welfare Committee of the School of Veterinary Medicine at the University of Glasgow. Owners gave informed consent for their horses' inclusion in the study. Sources of funding: John Crawford Endowment Fund, University of Glasgow. Competing interests: None declared., (© 2015 The Author(s). Equine Veterinary Journal © 2015 EVJ Ltd.)

This study aimed to quantify the compensatory response to naturally-occurring forelimb lameness on load redistribution. Data from lameness investigations using an inertial sensor based system to monitor the response to forelimb diagnostic anaesthesia were reviewed. Horses with primary forelimb lameness were grouped for analysis as (1) all horses combined (n= 28), (2) forelimb-only lameness (n= 8/28), (3) forelimb-contralateral hindlimb lameness (n= 14/28), (4) forelimb-ipsilateral hindlimb lameness (n= 6/28). The effect of diagnostic anaesthesia on measures of head and pelvic movement asymmetry was determined using the Wilcoxon signed rank test. Spearman's correlation analysis was performed between forelimb and hindlimb variables. Statistical significance was set at P< 0.05. Forelimb diagnostic anaesthesia resulted in a decrease in pelvic movement asymmetry among all horses and the forelimb-only and forelimb-contralateral hindlimb lameness groups. Pelvic movement asymmetry associated with the contralateral hindlimb decreased by a median of 38% (interquartile range [IQR] 10-65%), 43% (IQR 28-60%) and 28% (IQR 12-67%) in all horses, forelimb-only and forelimb-contralateral hindlimb groups respectively (P< 0.05). Maximum pelvic height difference (PDMax) significantly decreased in all horses combined and the forelimb-contralateral hindlimb lameness group by a median of 66% (IQR 24-100%) and 78% (IQR 27-100%, P< 0.01), respectively. Change in head movement asymmetry and vector sum was significantly positively correlated with PDMax in all horses combined and the forelimb-contralateral hindlimb group (P< 0.05). Forelimb lameness had a significant effect on hindlimb and pelvic movement in horses with clinical lameness resulting in compensatory load redistribution and decreased push-off from the contralateral hindlimb., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: To evaluate ex vivo cyclooxygenase (COX) inhibition and compare in vitro and ex vivo COX-1 inhibition by flunixin meglumine and firocoxib in horses., Animals: 4 healthy horses for in vitro experiments and 12 healthy horses (6 males and 6 females; 5 Thoroughbreds, 5 Warmbloods, and 2 ponies) undergoing elective surgery for ex vivo experiments., Procedures: 12 horses received flunixin meglumine (1.1 mg/kg, IV, q 12 h) or firocoxib (0.09 mg/kg, IV, q 24 h). Blood samples were collected before (baseline) and 2 and 24 hours after NSAID administration. Prostanoids (thromboxane B2, prostaglandin E2, and prostaglandin E metabolites) served as indicators of COX activity, and serum drug concentrations were measured by use of high-performance liquid chromatography. An in vitro coagulation-induced thromboxane B2 assay was used to calculate drug concentration-COX-1 inhibition curves. Effect of time and treatment on COX activity was determined. Agreement between in vitro and ex vivo measurement of COX activity was assessed with Bland-Altman analysis., Results: At 2 and 24 hours after NSAID administration, COX-1 activity was reduced, compared with baseline activity, for the flunixin meglumine group only and relative COX-1 activity was significantly greater for the firocoxib group, compared with that for the flunixin meglumine group. There was no significant change in COX-2 activity after surgery for either group. Bland-Altman analysis revealed poor agreement between in vitro and ex vivo measurement of COX-1 activity., Conclusions and Clinical Relevance: Compared with flunixin meglumine, firocoxib had COX-1-sparing effects ex vivo in equine patients that underwent elective surgery.

Background: Improved usage of the repertoires of pancreatic ductal adenocarcinoma (PDAC) profiles is crucially needed to guide the development of predictive and prognostic tools that could inform the selection of treatment options., Methods: Using publicly available mRNA abundance datasets, we performed a large retrospective meta-analysis on 466 PDAC patients to discover prognostic gene signatures. These signatures were trained on two clinical cohorts (n = 70), and validated on four independent clinical cohorts (n = 246). Further validation of the identified gene signature was performed using quantitative real-time RT-PCR., Results: We identified 225 candidate prognostic genes. Using these, a 36-gene signature was discovered and validated on fully independent clinical cohorts (hazard ratio (HR) = 2.06, 95% confidence interval (CI) = 1.51 to 2.81, P = 3.62 × 10(-6), n = 246). This signature serves as a good alternative prognostic stratification marker compared to tumour grade (HR = 2.05, 95% CI = 1.45 to 2.88, P = 3.18 × 10(-5)) and tumour node metastasis (TNM) stage (HR = 1.13, 95% CI = 0.66 to 1.94, P = 0.67). Upon multivariate analysis with adjustment for TNM stage and tumour grade, the 36-gene signature remained an independent prognostic predictor of clinical outcome (HR = 2.21, 95% CI = 1.17 to 4.16, P = 0.01). Univariate assessment revealed higher expression of ITGA5, SEMA3A, KIF4A, IL20RB, SLC20A1, CDC45, PXN, SSX3 and TMEM26 was correlated with shorter survival while B3GNT1, NOSTRIN and CADPS down-regulation was associated with poor outcome., Conclusions: Our 36-gene classifier is able to prognosticate PDAC independent of patient cohort and microarray platforms. Further work on the functional roles, downstream events and interactions of the signature genes is likely to reveal true molecular candidates for PDAC therapeutics.

The association of environmental cues with drugs of abuse results in persistent drug-cue memories. These memories contribute significantly to relapse among addicts. While conditioned place preference (CPP) is a well-established paradigm frequently used to examine the modulation of drug-cue memories, very few studies have used the non-preference-based model conditioned activity (CA) for this purpose. Here, we used both experimental approaches to investigate the neural substrates of cocaine-cue memories. First, we directly compared, in a consistent setting, the involvement of cortical and subcortical brain regions in cocaine-cue memory retrieval by quantifying activity-regulated cytoskeletal-associated (Arc) protein expression in both the CPP and CA models. Second, because NMDA receptor activation is required for Arc expression, we investigated the NMDA receptor dependency of memory persistence using the CA model. In both the CPP and CA models, drug-paired animals showed significant increases in Arc immunoreactivity in regions of the frontal cortex and amygdala compared to unpaired controls. Additionally, administration of a NMDA receptor antagonist (MK-801 or memantine) immediately after cocaine-CA memory reactivation impaired the subsequent conditioned locomotion associated with the cocaine-paired environment. The enhanced Arc expression evident in a subset of corticolimbic regions after retrieval of a cocaine-context memory, observed in both the CPP and CA paradigms, likely signifies that these regions: (i) are activated during retrieval of these memories irrespective of preference-based decisions, and (ii) undergo neuroplasticity in order to update information about cues previously associated with cocaine. This study also establishes the involvement of NMDA receptors in maintaining memories established using the CA model, a characteristic previously demonstrated using CPP. Overall, these results demonstrate the utility of the CA model for studies of cocaine-context memory and suggest the involvement of an NMDA receptor-dependent Arc induction pathway in drug-cue memory interference., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The tumor microenvironment dynamically regulates the progression of cancer. In the breast, a unique component of the microenvironment is the myoepithelial cell. Normal myoepithelial cells act as "natural tumor suppressors"; however, more recent evidence suggests that these cells develop phenotypic changes, which may contribute to loss of tumor suppressor activity. We have shown that myoepithelial cells in a subset of preinvasive ductal carcinoma in situ (DCIS) upregulate expression of the integrin αvβ6, switching on tumor promoter activity through activation of TGFβ and MMP9. This makes the tumor microenvironment more permissive to invasion, seen both in vitro and in vivo. In human tissue samples, increased myoepithelial αvβ6 expression correlated with increased risk of disease progression and recurrence. Current estimates suggest that as many as 50% of DCIS cases will never progress in the patient's lifetime, but there are no markers to predict the outcome of individual cases. The identification of αvβ6 in a subset of DCIS presents a unique way to stratify patients with DCIS into those who may or may not progress to more serious disease. As αvβ6 is not expressed on most normal adult tissues, this finding may also provide novel targets for therapy in this high-risk group., (©2014 American Association for Cancer Research.)

Learning from mistakes and prospectively adjusting behavior in response to reward feedback is an important facet of performance monitoring. Dopamine (DA) pathways play an important role in feedback learning and a growing literature has also emerged on the importance of serotonin (5HT) in reward learning, particularly during punishment or reward omission (negative feedback). Cognitive impairments resulting from psychostimulant exposure may arise from altered patterns in feedback learning, which in turn may be modulated by DA and 5HT transmission. We analyzed long-term, off-drug changes in learning from positive and negative feedback and associated striatal DA transporter (DAT) and frontocortical 5HT transporter (SERT) binding in rats pretreated with methamphetamine (mAMPH). Specifically, we assessed the reversal phase of pairwise visual discrimination learning in rats receiving single dose- (mAMPHsingle) vs. escalating-dose exposure (mAMPHescal). Using fine-grained trial-by-trial analyses, we found increased sensitivity to and reliance on positive feedback in mAMPH-pretreated animals, with the mAMPHsingle group showing more pronounced use of this type of feedback. In contrast, overall negative feedback sensitivity was not altered following any mAMPH treatment. In addition to validating the enduring effects of mAMPH on early reversal learning, we found more consecutive error commissions before the first correct response in mAMPH-pretreated rats. This behavioral rigidity was negatively correlated with subregional frontocortical SERT whereas positive feedback sensitivity negatively correlated with striatal DAT binding. These results provide new evidence for the overlapping, yet dissociable roles of DA and 5HT systems in overcoming perseveration and in learning new reward rules., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Repeated administration of methamphetamine (mAMPH) to rodents in a single-day "binge" dosing regimen produces long-lasting damage to forebrain dopaminergic nerve terminals as measured by decreases in tissue dopamine (DA) content and levels of the plasmalemmal DA transporter (DAT). However, the midbrain cell bodies from which the DA terminals arise survive, and previous reports show that striatal DA markers return to control levels by 12 months post-mAMPH, suggesting long-term repair or regrowth of damaged DA terminals. We previously showed that when rats engaged in voluntary aerobic exercise for 3 weeks before and 3 weeks after a binge regimen of mAMPH, exercise significantly ameliorated mAMPH-induced decreases in striatal DAT. However, these data left unresolved the question of whether exercise protected against the initial neurotoxicity from the mAMPH binge or accelerated the repair of the damaged DA terminals. The present experiments were designed to test whether exercise protects against the mAMPH-induced injury. Adult male Sprague-Dawley rats were allowed to run in wheels for 3 weeks before an acute binge regimen of mAMPH or saline, then placed into nonwheel cages for an additional week before autoradiographic determination of striatal DAT binding. The autoradiographic findings showed that prior exercise provided no protection against mAMPH-induced damage to striatal DA terminals. These results, together with analyses from our previous experiments, suggest that voluntary exercise may accelerate the repair of mAMPH-damaged DA terminals and that voluntary exercise may be useful as therapeutic adjunct in the treatment mAMPH addicts., (© 2014 Wiley Periodicals, Inc.)

Unlabelled: Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity., Importance: Influenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as "mixing vessels" in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Integrin αvβ6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer., Methods: Protein expression of integrin subunit beta6 (β6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student's t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni's Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided., Results: High expression of either the mRNA or protein for the integrin subunit β6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of β6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts., Conclusions: Targeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients., (© The Author 2014. Published by Oxford University Press.)

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. αvβ6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported αvβ6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and αvβ6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased αvβ6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-β1 activation. Gli1 inhibited αvβ6 expression by suppressing TGF-β1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high αvβ6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between αvβ6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and αvβ6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-β1/Smad2/3-dependent αvβ6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types. Cell binding to fibronectin through either α5β1 integrins (constitutively expressed) or αvβ6 integrins (selectively expressed in cancer and development) adapts force generation, actin flow and integrin recruitment to rigidities associated with healthy or malignant tissue, respectively. In vitro experiments and theoretical modelling further demonstrate that this behaviour is explained by the different binding and unbinding rates of both integrin types to fibronectin. Moreover, rigidity sensing through differences in integrin bond dynamics applies both when integrins bind separately and when they compete for binding to fibronectin.

Induced abortion is a controversial topic among obstetricians. "100 Professors" extolled the benefits of elective abortion in a Clinical Opinion published in AJOG. However, scientific balance requires the consideration of a second opinion from practitioners who care for both patients, and who recognize the humanity of both. Alternative approaches to the management of a problem pregnancy, as well as short and long term risks to women as published in the peer reviewed medical literature are discussed. Maintaining a position of "pro-choice" requires that practitioners also be given a right to exercise Hippocratic principles in accordance with their conscience.

The treatment of cancer has changed significantly over the past decade, from a treatment paradigm based primarily on surgery, chemotherapy and radiotherapy, to the development of targeted therapies involving tumor-specific signaling pathways, as well as the immune system. Recent developments have demonstrated the significant clinical benefits of immunotherapy that are quickly being established as standard treatments for certain malignancies. In this study, we demonstrated that clinical practice is not keeping pace with advancing immuno-oncology principles and research. We further demonstrated that the incorporation of immunotherapeutic approaches into the treatment of cancer patients varies widely, not only between developed and developing countries, but also between countries in specific geographical areas that have experienced similar cultural and economic development. We developed a survey to assess the deficiencies in scientific understanding, access to information and treatment options and investigated the differences in clinical practice behaviors among oncologists from six European countries: France, Germany, Greece, Italy, Spain and the United Kingdom. Our data demonstrated significant differences in all the categories assessed. Therefore, we concluded that there is a need to develop similar assessment tools to identify care imbalances, so that initiatives may be developed to correct care inequities that ultimately affect patient outcomes worldwide.

Purpose: This study investigated the functional and clinical significance of integrin αvβ6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS)., Experimental Design: Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvβ6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvβ6 expression, were used to measure the effect of αvβ6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model. Involvement of TGFβ signaling was established using mink lung epithelial cell (MLEC) assay and antibody inhibition, and expression and activation of matrix metalloproteinase (MMP)-9 established by Real Time-PCR and zymography., Results: Expression of αvβ6 is significantly associated with progression to invasive cancer (P < 0.006) and with recurrence over a median follow-up of 114 months in a series of matched DCIS cases treated with local excision. We show that expression of αvβ6 drives myoepithelial cells to promote tumor cell invasion in vitro and enhances mammary tumor growth in vivo. The tumor-promoting effect of αvβ6-positive myoepithelial cells is dependent on TGFβ-driven upregulation of MMP9 and can be abrogated by inhibiting this pathway., Conclusion: These findings indicate that altered myoepithelial cells in DCIS predict disease progression and recurrence and show that upregulation of αvβ6 on myoepithelial cells generates a tumor promoter function through TGFβ upregulation of MMP-9. These data suggest that expression of αvβ6 may be used to stratify patients with DCIS., (©2013 AACR.)

Reasons for Performing Study: In order for changes in lameness to be accurately and repeatably detected and recorded during diagnostic investigations, an objective measure of lameness is required., Objectives: To ascertain whether an inertial sensor-based system can distinguish between a positive and negative response to diagnostic anaesthesia of the foot and objectively assess the effect of a positive response on the trot., Study Design: Restrospective clinical study., Methods: Data obtained during lameness investigations undertaken between August 2011 and December 2012 in which either a palmar digital or abaxial sesamoid nerve block was performed were retrospectively reviewed. Response to diagnostic anaesthesia was categorised as positive (n = 14) or negative (n = 9) by one of 2 evaluators before analysis of kinematic data (i.e. blinded). Changes in maximum and minimum head difference (ΔHDMax and ΔHDMin) and change in head movement asymmetry/change in pelvic movement asymmetry (ΔHMA/PMA, measure of asymmetry) allocated to each limb were calculated. A Kruskal-Wallis one way analysis of variance on ranks was performed. Receiver operating characteristic (ROC) curves were generated for ΔHDMax, ΔHDMin and ΔHMA for the blocked limb to identify cut-off values to distinguish positive and negative responses to the block., Results: Median ΔHDMax and ΔHDMin were significantly greater after a positive response to diagnostic anaesthesia (P<0.01). Change in head movement asymmetry allocated to the blocked limb and contralateral forelimb and ΔPMA allocated to the contralateral hindlimb were significantly greater in the positive response group (P<0.05). Change in head movement asymmetry allocated to the blocked limb and ΔHDMax and ΔHDMin are useful diagnostic tests for identifying positive response to anaesthesia (area under the curve = 0.98, 0.83 and 0.96 respectively)., Conclusions: An inertial sensor-based system can identify a positive response to diagnostic anaesthesia of the foot. Symmetry of movement allocated to the blocked limb, contralateral forelimb and contralateral hindlimb significantly improve, and head movement significantly decreases in horses with a positive response to the block. Cut-off values for a positive response have been identified with good sensitivity and specificity. Forelimb lameness significantly affects contralateral hindlimb movement, which has implications for the investigation of multi-limb lameness., (© 2013 EVJ Ltd.)

Unlabelled: Transforming growth factor β activation by the αvβ6 integrin is central to the pathogenesis of idiopathic pulmonary fibrosis. Expression of the αvβ6 integrin is increased in fibrotic lung tissue and is a promising therapeutic target for treatment of the disease. Currently, measurement of αvβ6 integrin levels in the lung requires immunohistochemical analysis of biopsy samples. This procedure is clinically impractical for many patients with pulmonary fibrosis, and a noninvasive strategy for measuring αvβ6 integrin levels in the lungs is urgently required to facilitate monitoring of disease progression and therapeutic responses., Methods: Using a murine model of bleomycin-induced lung injury, we assessed the binding of intravenously administered (111)In-labeled αvβ6-specific (diethylenetriamine pentaacetate-tetra [DTPA]-A20FMDV2) or control (DTPA-A20FMDVran) peptide by nanoSPECT/CT imaging. Development of fibrosis was assessed by lung hydroxyproline content, and αvβ6 protein and itgb6 messenger RNA were measured in the lungs., Results: Maximal binding of (111)In-labeled A20FMDV2 peptide to αvβ6 integrins was detected in the lungs 1 h after intravenous administration. No significant binding was detected in mice injected with control peptide. Integrin binding was increased in the lungs of bleomycin-, compared with saline-, exposed mice and was attenuated by pretreatment with αvβ6-blocking antibodies. Levels of (111)In-labeled A20FMDV2 peptide correlated positively with hydroxyproline, αvβ6 protein, and itgb6 messenger RNA levels., Conclusion: We have developed a highly sensitive, quantifiable, and noninvasive technique for measuring αvβ6 integrin levels within the lung. Measurement of αvβ6 integrins by SPECT/CT scanning has the potential for use in stratifying therapy for patients with pulmonary fibrosis.

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells., Methods: We used tissue microarray analysis to compare immune cell infiltration of different pancreaticobiliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis) and juxtatumoral stromal (<100 μm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We also analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice and the effects of all-trans retinoic acid (ATRA) on these processes., Results: Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase(+) and CD68(+) cells compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8(+), FoxP3(+), CD56(+), or CD20(+) cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8(+) T cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8(+) T cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. In vitro migration assays showed that CD8(+) T cells, from patients with PDAC, had increased chemotaxis toward activated PSCs, which secrete CXCL12, compared with quiescent PSCs or tumor cells. These effects could be reversed by knockdown of CXCL12 or treatment of PSCs with ATRA., Conclusions: Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8(+) T cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective antitumor immune response., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Introduction: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice., Methods: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account., Results: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working., Conclusions: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

αvβ6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvβ6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvβ6, and inhibits binding to all ligands including the latency-associated peptide of TGF-β. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvβ8, but not the integrins α5β1, αvβ3, αvβ5 and α4β1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvβ6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-β-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-β by NCI-H358 tumour cells in a tumour cell-fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvβ6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvβ6 integrin, with some activity against αvβ8 integrin, that reduces both tumour growth and metastasis.

The αvβ6 integrin is up-regulated in cancer and wound healing but it is not generally expressed in healthy adult tissue. There is increasing evidence that it has a role in cancer progression and will be a useful target for antibody-directed cancer therapies. We report a novel recombinant diabody antibody fragment that targets specifically αvβ6 and blocks its function. The diabody was engineered with a C-terminal hexahistidine tag (His tag), expressed in Pichia pastoris and purified by IMAC. Surface plasmon resonance (SPR) analysis of the purified diabody showed affinity in the nanomolar range. Pre-treatment of αvβ6-expressing cells with the diabody resulted in a reduction of cell migration and adhesion to LAP, demonstrating biological function-blocking activity. After radio-labeling, using the His-tag for site-specific attachment of (99m)Tc, the diabody retained affinity and targeted specifically to αvβ6-expressing tumors in mice bearing isogenic αvβ6 +/- xenografts. Furthermore, the diabody was specifically internalized into αvβ6-expressing cells, indicating warhead targeting potential. Our results indicate that the new αvβ6 diabody has a range of potential applications in imaging, function blocking or targeted delivery/internalization of therapeutic agents.

We have developed a structurally-guided scaffold phage display strategy for identification of ligand mimetic bio-therapeutics. As a proof of concept we used the ligand of integrin αvβ6, a tumour cell surface receptor and a major new target for imaging and therapy of many types of solid cancer. NMR structure analysis showed that RGD-helix structures are optimal for αvβ6 ligand-interaction, so we designed novel algorithms to generate human single chain fragment variable (scFv) libraries with synthetic VH-CDR3 encoding RGD-helix hairpins with helices of differing pitch, length and amino acid composition. Study of the lead scFv clones D25scFv and D34scFv and their corresponding VH-CDR3 derived peptides, D25p and D34p, demonstrated: specific binding to recombinant and cellular αvβ6; inhibition of αvβ6-dependent cell and ligand adhesion, αvβ6-dependent cell internalisation; and selective retention by αvβ6-expressing, but not αvβ6-negative, human xenografts. NMR analysis established that both the D25p and D34p retained RGD-helix structures confirming the success of the algorithm. In conclusion, scFv libraries can be engineered based on ligand structural motifs to increase the likelihood of developing powerful bio-therapeutics.

Rationale: Environmental stimuli or contexts previously associated with rewarding drugs contribute importantly to relapse among addicts, and research has focused on neurobiological processes maintaining those memories. Much research shows contributions of cell surface receptors and intracellular signaling pathways in maintaining associations between rewarding drugs (e.g., cocaine) and concurrent cues/contexts; these memories can be degraded at the time of their retrieval through reconsolidation interference. Much less studied is the consolidation of drug-cue memories during their acquisition., Objective: The present experiments use the cocaine-conditioned place preference (CPP) paradigm in rats to directly compare, in a consistent setting, the effects of N-methyl-D-aspartate (NMDA) glutamate receptor antagonists MK-801 and memantine on the consolidation and reconsolidation of cocaine-cue memories., Methods: For the consolidation studies, animals were systemically administered MK-801 or memantine immediately following training sessions. To investigate the effects of these NMDA receptor antagonists on the retention of previously established cocaine-cue memories, animals were systemically administered MK-801 or memantine immediately after memory retrieval., Results: Animals given either NMDA receptor antagonist immediately following training sessions did not establish a preference for the cocaine-paired compartment. Post-retrieval administration of either NMDA receptor antagonist attenuated the animals' preference for the cocaine-paired compartment. Furthermore, animals given NMDA receptor antagonists post-retrieval showed a blunted response to cocaine-primed reinstatement., Conclusions: Using two distinct NMDA receptor antagonists in a common setting, these findings demonstrate that NMDA receptor-dependent processes contribute both to the consolidation and reconsolidation of cocaine-cue memories, and they point to the potential utility of treatments that interfere with drug-cue memory reconsolidation.

Objectives: A wireless, inertial sensor-based system has previously been validated for evaluation of equine lameness. However, threshold values have not been determined for the assessment of responses to flexion tests. The aim of this investigation was to evaluate a sensor-based system for objective assessment of the response to flexion., Methods: Healthy adult horses (n = 17) in work were recruited prospectively. Horses were instrumented with sensors on the head (accelerometer), pelvis (accelerometer) and right forelimb (gyroscope), before trotting in a straight line (minimum 25 strides) for 2 consecutive trials. Sensors measured 1) vertical pelvic movement asymmetry (PMA) for both right and left hindlimb strides and 2) average difference in maximum and minimum pelvic height (PDMax and PDMin) between right and left hindlimb strides in millimetres. A hindlimb was randomly selected for proximal flexion (60 s), after which the horse trotted a minimum of 10 strides. Response to flexion was blindly assessed as negative or positive by an experienced observer. Changes in PMA, PDMax and PDMin between baseline and flexion examinations were calculated for each test. Statistical analysis consisted of a Pearson's product moment test and linear regression on baseline trials, Mann-Whitney rank sum test for effect of flexion and receiver operator curve (ROC) analysis of test parameters., Results: There was a strong correlation between trials for PMA, PDMin and PDMax measurements (P < 0.001). A positive flexion test resulted in a significant increase in PMA (P = 0.021) and PDMax (P = 0.05) only. Receiver-operator curve analysis established cut-off values for change in PMA and PDMax of 0.068 and 4.47 mm, respectively (sensitivity = 0.71, specificity = 0.65) to indicate a positive response to flexion., Conclusions: A positive response to flexion resulted in significant changes to objective measurements of pelvic symmetry., Potential Relevance: Findings support the use of inertial sensor systems to objectively assess response to flexion tests. Further investigation is warranted to establish cut-off values for objective assessment of other diagnostic procedures.

Rationale: Protracted use of methamphetamine (mAMPH) can result in long-term impairments in cognitive function in humans. A previous study reported reversal-specific learning impairments in rats after a binge administration of mAMPH. Several studies show that extended exposure to mAMPH may confer protection against cognitive impairments and the insult to monoamine systems typically observed after larger binge doses., Objectives: To explore this issue, we compared the effects of escalating and single doses of mAMPH (and saline, SAL) on retention, reversal learning, and post-mortem analysis of dopamine and serotonin transporters, DAT and SERT., Methods: Rats learned to discriminate equiluminant stimuli and then were treated with either: (1) 4 weeks of mAMPH increasing by 0.3 mg/kg, culminating in 6 mg/kg (mAMPH(escal)); (2) 4 weeks of SAL with a single dose of 6 mg/kg on the last day of treatment (mAMPH(single)); or (3) 4 weeks of SAL. Following treatment, rats were tested on retention and reversal learning, with subsequent analysis of DAT and SERT binding across subregions of the striatum and frontoparietal cortex, respectively., Results: Retention of the pretreatment discrimination was not significantly impaired in either mAMPH treatment group. A significant decrease in ventrolateral striatal DAT binding was observed only in the mAMPH(single) group and frontoparietal SERT was unaffected by either mAMPH treatment. Both treatment groups demonstrated attenuated reversal learning, particularly on measures of accuracy and effort., Conclusions: These results show that extended and single-dose pretreatment with mAMPH similarly and selectively affect reversal learning, even in the absence of significant DAT or SERT changes.

Integrin αvβ6 is an important emerging target for both imaging and therapy of cancer that requires specific ligands based on Arg-Gly-Asp (RGD) peptides. There remains little correlation between integrin-RGD ligand specificity despite studies suggesting an RGD-turn-helix ligand motif is required. Here, we describe the application of 15 N NMR relaxation analyses and structure determination of αvβ6 peptide ligands in the presence and absence of trifluoroethanol (TFE) to identify their critical molecular nature that influences specificity, interaction and function. Two linear peptides; one known to demonstrate αvβ6 specificity (FMDV2) and the other based on a natural RGD ligand (LAP2), were compared to two additional peptides based on FMDV2 but cyclised in different positions using a disulphide bond (DBD1 and DBD2). The cyclic adaptation in DBD1 produces a significant alteration in backbone dynamic properties when compared to FMDV2; a potential driver for the loss in αvβ6 specificity by DBD1. The importance of ligand dynamics are highlighted through a comprehensive reduced spectral density and ModelFree analysis of peptide 15 N NMR relaxation data and suggest αvβ6 specificity requires the formation of a structurally rigid helix preceded by a RGD motif exhibiting slow internal motion. Additional observations include the effect of TFE/water viscosity on global NMR dynamics and the advantages of using spectral density NMR relaxation data to estimate correlation times and motional time regimes for peptides in solution.

Methamphetamine damages monoamine-containing nerve terminals in the brains of both animals and human drug abusers, and the cellular mechanisms underlying this injury have been extensively studied. More recently, the growing evidence for methamphetamine influences on memory and executive function of human users has prompted studies of cognitive impairments in methamphetamine-exposed animals. After summarizing current knowledge about the cellular mechanisms of methamphetamine-induced brain injury, this review emphasizes research into the brain changes that underlie the cognitive deficits that accompany repeated methamphetamine exposure. Novel approaches to mitigating or reversing methamphetamine-induced brain and behavioral changes are described, and it is argued that the slow spontaneous reversibility of the injury produced by this drug may offer opportunities for novel treatment development., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Several S100 proteins are up-regulated in pancreatic ductal adenocarcinoma (PDAC), the most significant being S100P. We previously reported on S100PBP, a binding partner of S100P, that shows no homology to any described protein and whose functions are completely unknown. To determine S100PBP expression across human tissues and organs, immunohistochemistry was performed using both multiorgan- and in-house-constructed pancreatic tissue microarrays. To establish S100PBP functions, cell lines with either stably overexpressed or silenced S100PBP were generated and investigated using Affymetrix gene expression arrays and complementary functional assays. We show that S100PBP is differentially expressed in various healthy and tumor specimens, which is both cancer- and tissue-type dependent. In healthy pancreas, S100PBP is expressed in the nuclear/perinuclear region of both exocrine and endocrine compartments. In early precancerous lesions, S100PBP is translocated to the cytoplasm, whereas in PDAC and metastatic lesions, its expression is significantly diminished. The most pronounced phenotypic change after manipulation of S100PBP expression was seen in adhesion; this was significantly reduced after S100PBP up-regulation and increased after S100PBP silencing. Up-regulation or silencing of S100PBP also led to a concomitant change in the levels of the protease cathepsin Z, the silencing of which significantly reduced PDAC cell adhesion. We further demonstrate that the interaction of cathepsin Z with arginine-glycine-aspartic acid-binding integrins, specifically αvβ5, mediates the changes seen in adhesion of PDAC cells., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Repeated administration of methamphetamine (mAMPH) to rodents in a single-day "binge" produces long-lasting damage to dopaminergic and serotonergic terminals. Because previous research has demonstrated that physical activity can ameliorate nigrostriatal injury, this study investigated whether voluntary exercise in rats can alter the monoaminergic damage resulting from a neurotoxic mAMPH binge. Adult male rats were allowed constant access to running wheels or kept in nonwheel cages for three weeks, then given a binge dosing regimen of mAMPH or saline. The rats were returned to their original environments for three additional weeks post-mAMPH. [(125) I]RTI-55 binding and autoradiography was used to quantify dopamine transporters (DAT), and radioimmunocytochemistry was used to quantify striatal tyrosine hydroxylase (TH). Binge mAMPH treatment significantly reduced striatal DAT and TH in a regionally specific pattern; with greatest effects in ventral caudate-putamen (CP) and relative sparing of the nucleus accumbens septi (NAc). The effects of mAMPH on striatal DAT and TH were ameliorated in the running, compared to the sedentary, animals. Also, mAMPH was found to reduce [(125) I]RTI-55 binding to serotonin transporters (SERT) in frontoparietal cortex, and this too was significantly attenuated by exercise. Additional correlational analyses showed that the post-mAMPH running of individual animals predicted the amelioration of striatal DAT and TH as well as frontoparietal SERT. Overall, voluntary exercise significantly diminished mAMPH-induced forebrain monoaminergic damage. The significant correlations between post-mAMPH exercise and markers of monoaminergic terminal integrity provide novel evidence that voluntary exercise may exert beneficial effects on behavior in recovering mAMPH addicts., (Copyright © 2011 Wiley Periodicals, Inc.)

Increasingly, research suggests a role for dopamine D1 receptors in the consolidation of extinction of both appetitive and aversive memories. However, a role for D1 receptors in extinction of memories involving drug reward has yet to be established. Here we show that post-retrieval, but not delayed, systemic administration of the D1 receptor antagonist SCH23390 results in prolonged extinction of cocaine conditioned place preference (CPP), suggesting a critical role for D1 receptors in the consolidation of extinction of cocaine-cue memories., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Rationale: Methamphetamine (mAMPH) administration in animals can lead to a variety of cognitive and behavioral deficits. We previously reported non-acute reversal learning impairments after a single-day administration of mAMPH, providing evidence of this drug's selective effects on inhibitory control. Effortful decision-making (i.e., how much effort to invest in rewards) is an aspect of cognition that has not yet been explored after mAMPH., Objectives: Given that frontostriatal circuitry mediating this type of choice is vulnerable to the effects of mAMPH, we tested the hypothesis that mAMPH may also affect decision-making involving effort, another form of cognitive flexibility., Methods: We examined the non-acute effects of an experimenter-administered single day of mAMPH on effort discounting. In this task, rats previously treated with mAMPH or saline (SAL) could select a high reward at the cost of climbing over a tall barrier or a low reward with no barrier impeding its procurement., Results: Following treatment, mAMPH rats were more work-averse than SAL rats. A control task showed there were no treatment group differences when the high and low rewards involved equal work: all rats chose the high reward preferentially. There were no significant treatment group differences in [(125)I]RTI-55 binding to dopamine and serotonin transporters (DAT, SERT) in any of the regions assayed; however, there were significant correlations of accumbens DAT and cingulate SERT with post-treatment performance., Conclusions: These findings suggest that even modest dose mAMPH exposure has long-lasting effects on effortful decision-making and may do so through influences on forebrain monoaminergic systems.

Binge administration of the psychostimulant drug, methamphetamine (mAMPH), produces long-lasting structural and functional abnormalities in the striatum. mAMPH binges produce nonexocytotic release of dopamine (DA), and mAMPH-induced activation of excitatory afferent inputs to cortex and striatum is evidenced by elevated extracellular glutamate (GLU) in both regions. The mAMPH-induced increases in DA and GLU neurotransmission are thought to combine to injure striatal DA nerve terminals of mAMPH-exposed brains. Systemic pretreatment with either competitive or noncompetitive N-methyl-D-aspartic acid (NMDA) antagonists protects against mAMPH-induced striatal DA terminal damage, but the locus of these antagonists' effects has not been determined. Here, we applied either the NMDA receptor antagonist, (dl)-amino-5-phosphonovaleric acid (AP5), or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, dinitroquinoxaline-2,3-dione (DNQX), directly to the dura mater over frontoparietal cortex to assess their effects on mAMPH-induced cortical and striatal immediate-early gene (c-fos) expression. In a separate experiment we applied AP5 or DNQX epidurally in the same cortical location of rats during a binge regimen of mAMPH and assessed mAMPH-induced striatal dopamine transporter (DAT) depletions 1 week later. Our results indicate that both ionotropic glutamate receptor antagonists reduced the mAMPH-induced Fos expression in cerebral cortex regions near the site of epidural application and reduced Fos immunoreactivity in striatal regions innervated by the affected cortical regions. Also, epidural application of the same concentration of either antagonist during a binge mAMPH regimen blunted the mAMPH-induced striatal DAT depletions with a topography similar to its effects on Fos expression. These findings demonstrate that mAMPH-induced dopaminergic injury depends upon cortical NMDA and AMPA receptor activation and suggest the involvement of the corticostriatal projections in mAMPH neurotoxicity., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over-expression of the αvβ6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix-degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9β1 integrin., Methods: H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild-type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry., Results: Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin-C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase-2 and metalloproteinase-9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9-expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well-differentiated lesions., Conclusions: The α9β1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix-degrading proteases., (© 2011 John Wiley & Sons A/S.)