Loss of the laminin subunit alpha-3 induces cell invasion and macrophage infiltration in cutaneous squamous cell carcinoma.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. OBJECTIVES: To study the effect that loss of the laminin subunit alpha-3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. METHODS: We examined the role of the basement membrane proteins laminin subunits α3, ß3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples. RESULTS: Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, ß3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples. CONCLUSIONS: These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment.

Pancreatic Cancer UK Grand Challenge: Developments and challenges for effective CAR T cell therapy for pancreatic ductal adenocarcinoma.

BACKGROUND: Death from pancreatic ductal adenocarcinoma (PDAC) is rising across the world and PDAC is predicted to be the second most common cause of cancer death in the USA by 2030. Development of effective biotherapies for PDAC are hampered by late presentation, a low number of differentially expressed molecular targets and a tumor-promoting microenvironment that forms both a physical, collagen-rich barrier and is also immunosuppressive. In 2017 Pancreatic Cancer UK awarded its first Grand Challenge Programme award to tackle this problem. The team plan to combine the use of novel CAR T cells with strategies to overcome the barriers presented by the tumor microenvironment. In advance of publication of those data this review seeks to highlight the key problems in effective CAR T cell therapy of PDAC and to describe pre-clinical and clinical progress in CAR T bio-therapeutics.

Proportion of nonsteroidal anti-inflammatory drug prescription in equine practice.

BACKGROUND: There is little knowledge of the prescription of nonsteroidal anti-inflammatory drugs (NSAIDs) and whether their prescription varies between countries. OBJECTIVE: To describe prescription practices of NSAIDs in equids in the United Kingdom (UK), United States of America (USA) and Canada. STUDY DESIGN: Descriptive observational study. METHODS: Free-text electronic medical records from 141,543 equids from 10 equine practices in the UK, 255,777 equids from 7 equine practices with 20 branches from the USA and 2 practices with 7 branches from Canada were evaluated. A validated text-mining technique was used to describe the proportion of equids prescribed NSAIDs at least once in these countries. The choice of NSAIDs in orthopaedic and colic cases was evaluated. RESULTS: The prescription of NSAIDs is more common in the USA (42.4%) and Canada (34.2%) than in the UK (28.6%). Phenylbutazone and flunixin meglumine were the drugs mostly prescribed in all countries. While flunixin meglumine was most prescribed with colic cases in all countries, a proportion received phenylbutazone despite this drug being licensed for use only with musculoskeletal disease. Phenylbutazone was the most commonly prescribed drug in cases with orthopaedic disease followed by flunixin meglumine in all countries. Only a small proportion of cases received meloxicam, ketoprofen or firocoxib. MAIN LIMITATIONS: The retrospective design might have resulted in an unknown number of incomplete records, particularly in the reporting of colic and orthopaedic disease. Although the data set is large, the relatively small number of practices recruited from each country may introduce bias. CONCLUSIONS: Clinical practice can differ between countries although the influence of individual practitioners and practice-specific policy on apparent intercountry differences requires further research. Despite several other NSAIDs being available and a substantial effort being made to evaluate their efficacy, the prescription of NSAIDs other than phenylbutazone and flunixin meglumine remains rather limited.

Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1.

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.

Use of large-scale veterinary data for the investigation of antimicrobial prescribing practices in equine medicine.

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.

Suppression of TGFß and Angiogenesis by Type VII Collagen in Cutaneous SCC.

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 TGFß 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 TGFß 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 α2ß1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFß signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFß markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFß markers, n = 6; VEGF, n = 21). Inhibition of TGFß 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 siTßRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS: Type VII collagen suppresses TGFß signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

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.

An inertial sensor-based system can objectively assess diagnostic anaesthesia of the equine foot.

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.

A human monoclonal antibody 264RAD targeting αvß6 integrin reduces tumour growth and metastasis, and modulates key biomarkers in vivo.

αvß6 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 αvß6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvß6, and inhibits binding to all ligands including the latency-associated peptide of TGF-ß. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvß8, but not the integrins α5ß1, αvß3, αvß5 and α4ß1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvß6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-ß-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-ß 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 αvß6 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 αvß6 integrin, with some activity against αvß8 integrin, that reduces both tumour growth and metastasis.

Use of a wireless, inertial sensor-based system to objectively evaluate flexion tests in the horse.

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.

Cortical ionotropic glutamate receptor antagonism protects against methamphetamine-induced striatal neurotoxicity.

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.

The effects of a novel anti-inflammatory compound (AHI-805) on cyclooxygenase enzymes and the recovery of ischaemia injured equine jejunum ex vivo.

REASONS FOR PERFORMING STUDY: Flunixin meglumine is used for treatment of equine colic despite evidence of inhibited recovery of mucosal barrier function following small intestinal ischaemic injury. This study aimed to characterise an alternative treatment (AHI-805) for abdominal pain in the horse. OBJECTIVE: To determine the effect of AHI-805, an aza-thia-benzoazulene derivative, on the cyclooxygenase enzymes and the recovery of mucosal barrier function following ischaemic injury. METHODS: Effect of AHI-805 on in vitro COX-1 and COX-2 activity was determined by measuring coagulation-induced thromboxane B(2) (TXB(2)) and lipopolysaccharide-stimulated prostaglandin E(2) concentrations in equine whole blood. Horses (n = 6) were anaesthetised and jejunum subjected to ischaemia for 2 h. Control and ischaemia injured mucosa was placed in Ussing chambers and treated with Ringer's solution containing control treatment (DMSO), flunixin meglumine (27 µmol/l), or AHI-805 (27 µmol/l). Transepithelial electrical resistance (TER), mucosal-to-serosal flux of (3) H-mannitol, and bathing solution TXB(2) and prostaglandin E metabolites (PGEM) were measured over a 4 h recovery period. RESULTS: Treatment with AHI-805 had no significant effect on TXB(2) production but significantly inhibited production of PGE(2) at a concentration of 1 µmol/l or greater. TER of flunixin or AHI-805 treated ischaemia-injured jejunum was significantly lower than control treated injured tissue over the recovery period. Mannitol flux and grade of histological damage were significantly increased by ischaemic injury only. There was a significant increase in PGEM and TXB(2) in control tissues over the 240 min recovery period, but not in flunixin or AHI-805 treated tissues. CONCLUSIONS: Flunixin meglumine and AHI-805 inhibit recovery of barrier function in ischaemic-injured equine jejunum in vitro through inhibition of the COX enzymes. POTENTIAL RELEVANCE: The novel compound AHI-805 may not be suitable for the treatment of equine colic associated with ischaemic injury.

The effect of nonsteroidal anti-inflammatory drugs on the equine intestine.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in the management of pain and endotoxaemia associated with colic in the horse. While NSAIDs effectively treat the symptoms of colic, there is evidence to suggest that their administration is associated with adverse gastrointestinal effects including right dorsal colitis and inhibition of mucosal barrier healing. Several studies have examined the pathophysiology of NSAID associated effects on the large and small intestine in an effort to avoid these complications and identify effective alternative medications. Differences in the response of the large and small intestines to injury and NSAID treatment have been identified. Flunixin meglumine has been shown in the small intestine to inhibit barrier function recovery and increase permeability to lipopolysaccharide (LPS). A range of NSAIDs has been examined in the small intestine and experimental evidence suggests that those NSAIDs with cyclooxygenase independent anti-inflammatory effects or a COX-2 selective mode of action may offer significant advantages over traditional NSAIDs.

The pharmacokinetics and in vitro cyclooxygenase selectivity of deracoxib in horses.

The purpose of this study was to determine the pharmacokinetics of deracoxib following oral administration to horses. In addition, in vitro equine whole blood cyclooxygenase (COX) selectivity assays were performed. Six healthy adult horses were administered deracoxib (2 mg/kg) orally. Plasma samples were collected prior to drug administration (time 0), and 10, 20, 40 min and 1, 1.5, 2, 4, 6, 8, 12, 24, and 48 h after administration for analysis with high pressure liquid chromatography using ultraviolet detection. Following PO administration, deracoxib had a long elimination half-life (t(1/2) k(10) ) of 12.49 ± 1.84 h. The average maximum plasma concentration (C(max) ) was 0.54 µg/mL, and was reached at 6.33 ± 3.44 h. Bioavailability was not determined because of the lack of an IV formulation. Results of in vitro COX selectivity assays showed that deracoxib was selective for COX-2 with a COX-1/COX-2 ratio of 25.67 and 22.06 for the IC(50) and IC(80) , respectively. Dosing simulations showed that concentrations above the IC(80) for COX-2 would be maintained following 2 mg/kg PO q12h, and above the IC(50) following 2 mg/kg PO q24h. This study showed that deracoxib is absorbed in the horse after oral administration, and may offer a useful alternative for anti-inflammatory treatment of various conditions in the horse.

Psoriasin (S100A7) associates with integrin ß6 subunit and is required for αvß6-dependent carcinoma cell invasion.

Expression of the integrin αvß6 is upregulated in a variety of carcinomas where it appears to be involved in malignant progression, although the biology of this integrin is not fully explored. We have generated oral carcinoma cells that express αvß6 composed of wild-type αv and a mutant ß6 that lacks the unique C-terminal 11 amino acids (aa). We found that these residues, although not required for αvß6-dependent adhesion or migration, are essential for αvß6-dependent invasive activity. We have used a proteomic approach to identify novel binding partners for the ß6 subunit cytoplasmic tail and report that psoriasin (Psor) (S100A7) bound preferentially to the recombinant ß6 cytoplasmic domain, though not in the absence of the unique C-terminal 11aa. Endogenous cellular Psor co-precipitated with endogenous ß6 and colocalised with αvß6 at the cell membrane and intracellular vesicles. Knockdown of Psor, with small interfering RNA, had no effect on αvß6-dependent adhesion or migration but abrogated αvß6-mediated oral carcinoma cell invasion both in Transwell and, the more physiologically relevant, organotypic invasion assays, recapitulating the behaviour of the ß6-mutant cell line. Membrane-permeant Tat-peptides encoding the unique C-terminal residues of ß6, bound directly to recombinant Psor and inhibited cellular Psor binding to ß6; this blocked αvß6-dependent, but not αvß6-independent, invasion. These data identify a novel interaction between Psor and ß6 and demonstrate that it is required for αvß6-dependent invasion by carcinoma cells. Inhibition of this interaction may represent a novel therapeutic strategy to target carcinoma invasion.

Striatal dopamine and glutamate receptors modulate methamphetamine-induced cortical Fos expression.

Methamphetamine (mAMPH) is a psychostimulant drug that increases extracellular levels of monoamines throughout the brain. It has previously been observed that a single injection of mAMPH increases immediate early gene (IEG) expression in both the striatum and cerebral cortex. Moreover, this effect is modulated by dopamine and glutamate receptors since systemic administration of dopamine or glutamate antagonists has been found to alter mAMPH-induced striatal and cortical IEG expression. However, because dopamine and glutamate receptors are found in extra-striatal as well as striatal brain regions, studies employing systemic injection of dopamine or glutamate antagonists fail to localize the effects of mAMPH-induced activation. In the present experiments, the roles of striatal dopamine and glutamate receptors in mAMPH-induced gene expression in the striatum and cerebral cortex were examined. The nuclear expression of Fos, the protein product of the IEG c-fos, was quantified in both the striatum and the cortex of animals receiving intrastriatal dopamine or glutamate antagonist administration. Intrastriatal infusion of dopamine (D1 or D2) or glutamate [N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)] antagonists affected not only mAMPH-induced striatal, but also cortical, Fos expression. Overall, the effects of the antagonists occurred dose-dependently, in both the infused and non-infused hemispheres, with greater influences occurring in the infused hemisphere. Finally, unilateral intrastriatal infusion of dopamine or glutamate antagonists changed the behavior of the rats from characteristic mAMPH-induced stereotypy to rotation ipsilateral to the infusion. These results demonstrate that mAMPH's actions on striatal dopamine and glutamate receptors modulate the widespread cortical activation induced by mAMPH. It is hypothesized that dopamine release from nigrostriatal terminals modulates activity within striatal efferent pathways, thereby disinhibiting thalamo-cortical circuits. By extension, these results suggest processes through which repeated exposure to mAMPH might influence cortical function in mAMPH abusers.

Running wheel exercise enhances recovery from nigrostriatal dopamine injury without inducing neuroprotection.

Forced use of the forelimb contralateral to a unilateral injection of the dopaminergic neurotoxin 6-hydroxydopamine can promote recovery of motor function in that limb and can significantly decrease damage to dopamine terminals. The present study was conducted to determine (1) whether a form of voluntary exercise, wheel running, would improve motor performance in rats with such lesions, and (2) whether any beneficial effects of wheel running are attributable to ameliorating the dopaminergic damage. In experiment 1, rats were allowed to run in exercise wheels or kept in home cages for 2 1/2 weeks, then given stereotaxic infusions of 6-hydroxydopamine into the left striatum. The rats were replaced into their original environments (wheels or home cages) for four additional weeks, and asymmetries in forelimb use were quantified at 3, 10, 17, and 24 days postoperatively. After killing, dopaminergic damage was assessed by both quantifying 3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester ([(125)I]RTI-55) binding to striatal dopamine transporters and counting tyrosine hydroxylase-positive cells in the substantia nigra. Exercised 6-hydroxydopamine-infused rats showed improved motor outcomes relative to sedentary lesioned controls, effects that were most apparent at postoperative days 17 and 24. Despite this behavioral improvement, 6-hydroxydopamine-induced loss of striatal dopamine transporters and tyrosine hydroxylase-positive nigral cells in exercised and sedentary groups did not differ. Since prior studies suggested that forced limb use improves motor performance by sparing nigrostriatal dopaminergic neurons from 6-hydroxydopamine damage, experiment 2 used a combined regimen of forced plus voluntary wheel running. Again, we found that the motor performance of exercised rats improved more rapidly than that of sedentary controls, but that there were no differences between these groups in the damage produced by 6-hydroxydopamine. It appears that voluntary exercise can facilitate recovery from partial nigrostriatal injury, but it does so without evident sparing of dopamine nerve terminals.

Alphavbeta6 integrin in wound healing and cancer of the oral cavity.

Integrins are a family of heterodimeric cell surface receptors, which are expressed on most cells where they mediate cell-cell and cell-extracellular matrix (ECM) interactions. The alphavbeta6 integrin is epithelial-specific and binds to the ECM proteins fibronectin, vitronectin and tenascin, and also to the latency associated peptide of TGF-beta. Unlike most epithelial integrins, alphavbeta6 is not expressed constitutively by healthy oral epithelia, but is up-regulated during tissue remodelling, including that accompanying wound healing and carcinogenesis. Although, the data at present have been generated principally from in vitro studies, there is increasing evidence to suggest that alphavbeta6 may promote carcinoma progression: alphavbeta6 has been shown to modulate invasion, inhibit apoptosis, regulate protease expression and activate TGF-beta1. This review examines the current literature, and discusses the possible role of alphavbeta6 in wound healing, and in the development and progression of oral squamous cell carcinoma.

Alpha v beta 6 integrin down-regulates the MMP-13 expression in oral squamous cell carcinoma cells.

The integrin alphavbeta6, a receptor for fibronectin, vitronectin, tenascin and TGF-beta latency-associated peptide (LAP), is not detectable on normal oral epithelium but is neo-expressed in oral squamous cell carcinomas (OSCC) and epithelial dysplasia. Previously it has been shown that alphavbeta6 integrin can up-regulate MMP-3 and -9 expression in OSCC cells. Using beta6-transfected and control OSCC cells we demonstrate that alphavbeta6 integrin down-regulates MMP-13 expression at both mRNA and protein level. Although expressing less MMP-13, beta6-transfected cells were found to have similar collagenolytic activity as control cells and invade at similar levels through type I collagen. Growth of the tumour cells in organotypic culture and confocal microscopy confirmed low levels of MMP-13 in cells with high alphavbeta6 expression. Furthermore, human squamous cell carcinomas of the tongue with high expression of alphavbeta6 showed lower MMP-13 levels than carcinomas with low levels of alphavbeta6. Our results suggest that alphavbeta6 down-regulates MMP-13 expression in OSCC cells and that MMP-13 is not essential for the degradation of type I collagen by OSCC cells.

Development of a quantitative method to analyse tumour cell invasion in organotypic culture.

Tumour invasion is a dynamic process occurring in three dimensions and involving interactions between both tumour and stromal cells. Experimental analysis of squamous carcinoma cell invasion has often used the organotypic gel culture system, which is generated by plating tumour cells on to a synthetic stroma composed of a collagen gel embedded with fibroblasts. Unfortunately, quantitation of invasion in these organotypic gels has relied largely on subjective pathological opinion, which may be influenced by different patterns of tumour cell infiltration. Therefore a computer-assisted digital image analysis system that assesses invasion objectively and provides a numerical 'Invasion Index' was developed. The Invasion Index, by combining depth and pattern of invasion in a single value, establishes a quantitative value that allows assessment of the influences of positive and negative regulation of tumour invasion. These data demonstrate that the organotypic gel system is a robust, accurate, and reproducible method for measuring tumour cell invasion. They also show that the Invasion Index can be used after organotypic gels have been implanted in mice for up to 6 weeks. Illustrative examples of how various factors influence the invasion of squamous carcinoma cells in three dimensions both in vitro and in vivo are provided.