Visual loss in glaucoma is associated with pathological changes in retinal ganglion cell (RGC) axons and a slow decline in the RGC population. Age and elevated intraocular pressure (IOP) are the main risk factors for glaucomatous loss of vision. Several studies have implicated the proinflammatory cytokine tumor necrosis factor- α (TNF-α) as a link between elevated IOP and RGC death, but the cellular source of TNF-α and its causative role in RGC death remain uncertain. Here, using a rat model of glaucoma, we investigated the source of elevated TNF- α and examined whether Etanercept, a TNF-α blocker that is in common clinical use for other indications, is protective against RGC death.Episcleral vein cauterization (EVC) caused intraocular pressure (IOP) to be elevated for at least 28 days. IOP elevation resulted in a dramatic increase in TNF-α levels within a few days, axonal degeneration, and a 38% loss of RGCs by 4 weeks. Immunostaining coupled with confocal microscopy showed that OHT induced robust induction of TNF-α in Iba-1-positive microglia around the optic nerve head (ONH). Despite persistent elevation of IOP, Etanercept reduced microglial activation, TNF-α levels, axon degeneration in the optic nerve, and the loss of RGCs.Ocular hypertension (OHT) triggers an inflammatory response characterized by the appearance of activated microglia around the ONH that express TNF-α. Blocking TNF-α activity with a clinically approved agent inhibits this microglial response and prevents axonal degeneration and loss of RGCs. These findings suggest a new treatment strategy for glaucoma using TNF- α antagonists or suppressors of inflammation. Retinal ganglion cell (RGC) death and subsequent visual field defects that progress to blindness are the underlying pathophysiology of glaucoma Considerable evidence suggests that the damage begins within the optic nerve due to structural changes within the lamina cribrosa TNF-α is a proinflammatory cytokine that is secreted in response to infection and trauma, and can lead to apoptosis in susceptible cells through the activation of caspases We induced OHT in the right eyes of rats (n = 40) by cauterizing the episcleral vein, leaving the left eye as a control. Whereas the average IOP in the control eye was 14.4±0.3 mm Hg, IOP rose to 47.6±12.7 mm Hg immediately after cauterization and remained elevated for the duration of the study in 80% (n = 32) of the eyes at 4 weeks after EVC; 12.5% (n = 5) fell into phthisis and 7.5% (n = 3) did not meet the criteria for successful OHT induction. In the OHT groups, Etanercept (0.3 or 1.0 mg/kg) or saline was injected intraperitoneally three times a week from the time of initial IOP elevation until the time of euthanasia. Over the next 4 weeks, IOP remained stable at approximately 2.2-fold above baseline in the group treated with 0.3 mg/kg Etanercept, 1.9 -fold in the group treated with 1.0 mg/kg Etanercept, and 2-fold in the sham-operated control group; these values did not differ from one another  and astrocytes (GFAP). In sham-operated (control) retinal whole-mounts, we observed resting microglia with ramified processes distributed in a mosaic pattern around the ONH, with very few amoeboid-shaped cells and minimal GFAP expression , C′. Fiv2, and this number decreased by 40.2% in OHT eyes . Etanercept (0.3 mg/kg) maintained axon density at considerably higher levels than were seen in saline-treated controls  and loosely organized myelin with multilayered, whorled masses . In the retina, both NF-L and NF-M antibodies stained axon fascicles around the ganglion cell layer . Protein2, whereas the number in OHT retinas treated only with saline was 38% lower  loss in glaucoma to TNF-α. Our results demonstrate this link using a different method to induce ocular hypertension (OHT) and a different species than our earlier study, while for the first time identifying microglia at the optic nerve head (ONH) as the source of TNF-α. In addition, we show that Etanercept (Enbrel®), a widely used TNF-α antagonist, attenuates inflammation and RGC loss.In the present study, we elevated intraocular pressure (IOP) by episcleral vein cauterization (EVC), which led to axon degeneration in the optic nerve and eventual loss of RGCs. Elevated IOP caused levels of TNF-α to increase within 3 days, and by 7 days, levels were 17-fold higher than normal and remained elevated for at least 4 weeks. However, compared to proinflammatory eye disease such as experimental uveitis induced by lipopolysaccharide Retinal microglia are resident macrophages derived from myeloid progenitor cells that are capable of phagocytic activity in multiple conditions Another finding in our study was the early dramatic decrease in retinal levels of NF-L and NF-M 14 days after OHT induction , which pIn conclusion, our results suggest that elevation of the pro-inflammatory cytokine TNF-α and ONH inflammation play an important role in linking elevated intraocular pressure to the loss of optic nerve axons and RGCs that are the hallmark features of glaucoma . LowerinAll animal experiments followed the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee of The Massachusetts Eye and Ear Infirmary. Male Brown Norway rats (8–10 weeks) were purchased from Charles River Laboratories . A total of 140 rats between 200 and 300 g weight were used. Animals were housed under a 12-h light/dark cycle with free access to water and food. For general anesthesia, a mixture of ketamine  and xylazine  was administered intramuscularly.n = 140) by episcleral vein cauterization (EVC) according to published procedures with minor modifications Chronic ocular hypertension (OHT) was induced in the right eye  with rats under topical anesthesia with 0.5% proparacaine hydrochloride eye drops. IOP was measured before EVC, immediately after cauterization, and then on days 1, 7, 14, 21, and 28. The TonoLab acquires six valid rebounds of the device from the eye and takes the mean of the middle four readings to make one summary measurement. The instrument indicates the reliability of the reading by the position of a bar next to the reported value. A single set of readings was collected with the best reproducibility indicator (no bar) at each time point during the IOP measurements. Rats were excluded from further analysis if the peak IOP was <6 mm Hg above baseline or if the treated eye developed corneal ulceration, hyphema, or other ocular pathology. In total, 13 rats were excluded based on these criteria.Etanercept, a soluble 75 kDa TNF-α receptor/Fc fusion protein , was reconstituted with sterile water according to the manufacturer’s instructions. Rats were separated into three groups. Starting one day after induction of chronic glaucoma, two groups (n = 10 each) were given subcutaneous injection of Etanercept at either 0.3 mg/kg or 1 mg/kg three times a week until the day of sacrifice. A third group (n = 10) received intraperitoneal injection of physiological saline three times a week. These doses were selected based upon previous studies that demonstrated the effectiveness of the drug in other disease models For retinal cryosection immunostaining, sections through the retina (10 µm) with the optic nerve attached were pre-blocked  and then incubated with rabbit anti-Iba-1 antibody  as a marker for microglia, anti-neurofilament light chain monoclonal antibody , and/or anti-neurofilament middle chain monoclonal antibody  overnight at 4°C. Alexa Fluor 488-conjugated goat anti-mouse IgG  or Alexa Fluor 546-conjugated donkey anti rabbit IgG (Invitrogen) were used as secondary antibodies and incubated at room temperature for 1 hour prior to mounting in Vectashield mounting medium .For whole-mount immunostaining, rat eyes were enucleated immediately after transcardiac perfusion. Post-fixed retinas were washed several times in fresh PBS, transferred to multi-well plates, and blocked in 10% normal goat serum  with 0.5% Triton X-100 in 0.1 ml PBS overnight. The retinas were then incubated in the same medium containing antibody TUJ1 , anti-NeuN , mouse monoclonal antibody to TNF-α , and/or rabbit anti- Iba-1 antibody  for 1 day at 4°C on a shaking platform. After three further washes, retinas were incubated with a goat antibody to mouse IgG conjugated to Alexa Fluor 546 and a donkey antibody to rabbit IgG conjugated to Alexa Fluor 488 diluted in blocking medium  overnight at 4°C and flat-mounted onto slides after making four radial slits and coverslipped in Vectashield mounting medium.2.Surviving RGCs were quantified in retinal whole mount preparations that were double-immunolabeled with the TUJ1 antibody directed against βIII tubulin and anti-NeuN. Within the ganglion cell layer, βIII tubulin is expressed only in RGCs, and thus immunostaining for this antigen can be used to distinguish RGCs from the many amacrine cells that are also in this layer 2 overnight at 4°C. The tissue was washed in 0.1 M cacodylate buffer and postfixed in 2% aqueous OsO4. Segments were dehydrated in graded alcohols and embedded in Epon. One-micron sections were cut and stained with 1% toluidine blue in 1% borate buffer. Five regions from each section were chosen for quantitation of axon density. In brief, 100× photomicrographs of each section were collected using an Olympus Povis AX70 microscope . Axons were counted semi-automatically with a method described previously RGC axons were quantified in the retrobulbar optic nerve 10 mm from the globe under light microscopy. At least three sections were used from each of six rats for each condition. Optic nerves were placed immediately into fixative consisting of 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate buffer with 0.08 M CaClUsing whole-mounted retinas, we classified the activation of Iba-1-positive microglia as described Retinal microglia and astrocytes were visualized in retinal whole-mounts by immunostaining with antibodies directed against Iba-1 and GFAP as previously described. The antibody to TNF-α was used for colocalization studies as specified in the manufacturer’s recommendations. Whole-mounts were visualized using a Leica SP5 confocal microscope with 10× and 63× objectives. The fluorescence of individual fluorochromes was captured separately in sequential mode  after optimization to reduce bleed-through between channels using Leica software. A stack of optical sections was taken at intervals of 0.16 µm.Retinas from experimental eyes with chronic OHT and control eyes were dissected from the RPE-choroid at designated time points after EVC. Samples were separated electrophoretically on NuPAGE 4–12% Bis-Tris Gels (Invitrogen) and transferred onto PVDF membrane . After blocking with 3% nonfat dried milk, membranes were incubated overnight with a primary antibody against TNF-α , neurofilament light chain , and neurofilament middle chain . The blotted membranes were then incubated for 30 min at room temperature with an HRP-conjugated secondary antibody directed against rabbit IgG . Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) and exposed onto Fuji RX film .To investigate whether we could detect Etanercept in the rodent retina, immunoprecipitation (IP) was performed as previously described. 2 value >0.98, demonstrating the sensitivity and reliability of the method.The tissue complex containing the vitreous and neural retina was collected at 3, 7, 14, 21, or 28 days after EVC. Proteins were extracted from each retina individually in 200 µl PBS containing a protease inhibitor mixture  and sonicated . The supernatant was collected after centrifugation at 14,000×g for 20 min at 4°C , and the total protein concentration was measured with the DC protein assay kit . The level of TNF-α was determined with rat TNF-α enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer’s protocols with slight modification. TNF-α standards were reconstituted down to 1.5625 pg/ml. The absorbance at 450 nm was measured using a 96-well plate spectrophotometer . Absorbance values were converted to TNF-α concentration by comparison with a simultaneously generated standard curve and then normalized by the level of protein per retina. Standard curves showed an RResults are expressed as mean values ± standard error of mean (SEM). Statistical analysis was performed nonparametrically using Mann-Whitney U-test and Kruskal-Wallis tests . A p-value of less than 0.05 was considered statistically significant.Figure S1Immunoprecipitation of Etanercept from retinal lysates. Five hundred mg of retinal lysate was incubated with anti-human IgG1 antibody and protein A/G agarose beads. Etanercept and mouse IgG were used as a positive control. Mice treated with Etanercept showed immunoprecipitation of Etanercept, whereas control mice did not. OHT, ocular hypertension; Etan., Etanercept.(TIF)Click here for additional data file.


Differences have been seen among the anti-TNFs in mediating reverse signalling of membrane TNF-α (mTNF-α). Natural killer (NK) cells express high levels of mTNF-α and may be involved in rheumatoid arthritis pathogenesis. We examined the effect of certolizumab pegol (CZP) and the other anti-TNFs, adalimumab (ADA), etanercept (ETA) and infliximab (IFX), on cellular activities of NK cells.1k and Fab′ PEG) for a further 4 h. Antibody-dependent cellular cytotoxicity (ADCC) was measured by loss of cell membrane integrity, by binding of 7-amino-actinomycin D to DNA. For analysis of soluble cytokine production and β-hexosaminidase release as an index of cell degranulation, NK cells were incubated with rhIL-2 and anti-TNF agents. The concentration of the soluble cytokine interferon-γ (IFN-γ) was determined by enzyme-linked immunosorbent assay. β-hexosaminidase release was quantified upon enzymatic cleavage of 4-methylumbelliferyl N-acetyl-β-D-glucosaminide in citrate buffer  by spectrophotometric analysis.Peripheral blood mononuclear cells from healthy volunteers were isolated and incubated in the presence of 100 U/mL recombinant human (rh) interleukin-2 (IL-2) for 20 h and 10 μg/mL anti-TNF agents or isotypic controls  and not with CZP (1%).Anti-TNF agents may result in increased NK cell–mediated cytotoxicity by promoting the release of multiple cytotoxic effector molecules and inflammatory cytokines via reverse signalling through constitutively expressed mTNF-α. CZP can activate NK cells but, in contrast to conventional anti-TNFs, does not mediate ADCC due to its unique structure (lacking an Fc region).


Colymbetes, sive de arte natandi dialogus et festivus et iucundus lectu” ). At its inception, swimming was understood simply as moving in water – first, in order to survive, and then, to compete. Today, the term clearly connotes swimming sports, and as such can be defined as an activity in which a person practices a regulated Olympic sport in order to move as fast as possible through the water thanks to the propulsive forces generated by arm, leg, and body movements overcoming the resistance the water presents to progress . Each article has been rigorously peer reviewed by at least two referees from 11 countries in addition to the nine above , involving over 40 researchers and 25 institutions. The articles are divided into two main blocks – competitive swimming (12 papers), and other aquatic activities (9 papers) corresponding to the following themes: aquatic exercise (3 papers), swim education (2 papers), synchronized swimming (2 papers), water polo (1 paper), and diving (1 paper). By scientific area, the papers may be categorized as: Biomechanics (7), Anthropometric (2), Education (2), Evaluation (2), Physiology (2), Training (2), Aquatic exercise (1), Psychology (1), Sociology (1) and others (1).Finally, we wish to thank the Editor-in-Chief, Professor Dr Adam Zając, and the Technical and Statistical Editor, Professor Dr Adam Maszczyk for the trust and responsibility deposited in us for the preparation of this Special Issue. We hope and trust that it will meet with the approval of the readers of the Journal of Human Kinetics.


The main problem both in type 1 (T1DM) and type 2 (T2DM) diabetes is the development of chronic vascular complications encompassing micro- as well as macrocirculation. Chronic complications lower the quality of life, lead to disability, and are the cause of premature death in DM patients. One of the chronic vascular complications is a diabetic retinopathy (DR) which leads to a complete loss of sight in DM patients. Recent trials show that the primary cause of diabetic retinopathy is retinal neovascularization caused by disequilibrium between pro- and antiangiogenic factors. Gaining knowledge of the mechanisms of action of factors influencing retinal neovascularization as well as the search for new, effective treatment methods, especially in advanced stages of DR, puts special importance on research concentrating on the implementation of biological drugs in DR therapy. At present, it is antivascular endothelial growth factor and antitumor necrosis factor that gain particular significance. Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), 3.6% of patients with T1DM diagnosed at a younger age and 1.6% of those in whom T2DM developed in a later stage of life were considered blind [Dynamic increase in morbidity both for type 1 (T1DM) and type 2 (T2DM) diabetes has reached the state of an epidemic in developed countries and in the developing ones; the incidence of diabetes is increasing at still a quicker pace . At presed blind . Moreoveed blind .Pathogenesis of DR is complex, and despite a lot of research, it has not been completely elucidated. The disease process encompasses characteristic changes, namely, thickening of the basement membrane coupled with its increased permeability, loss of pericytes leading to diminished vessel wall tone and development of protruding micro aneurysms, and proliferation of mesangium causing obstruction and obliteration of capillaries. Now, we know that diabetic retinopathy is the effect of genetic, environmental, and immunological factors acting together –18. The α (TNF-α) and vascular endothelial growth factor (VEGF), adhesive molecules, and the activation of a nuclear transcription factor NFκB [Up till now, both our research and the work of other authors show significantly higher AGEs level both in the serum of children with T1DM and late vascular complications , 30 and tor NFκB –36. Thistor NFκB , 21, 23,Research on the drug which would protect amino groups of proteins exposed to glycation in the first step or prevent the cross-links formation in the third step of the reaction is still ongoing. Drugs preventing the formation of AGE complexes probably block the carbonyl groups of Amadori rearrangement products. This prevents the formation of cross-links with other proteins by binding with their amino groups. Aspirin also exhibits some protecting properties , 38. AnoVEGF, also known as the vascular permeability factor—VPF or vasculotropin, is nowadays considered the main angiogenesis-controlling factor. VEGF is produced by endothelial cells, macrophages, CD4 lymphocytes, plasma cells, myocytes, megakaryocytes, and neoplastic cells . It is aCurrently, there are four anti-VEGF agents which have been used in the management of diabetic retinopathy, including pegaptanib , ranibizumab , bevacizumab , and VEGF Trap-Eye . is an aptamer, which was the first of anti-VEGF factors to be approved for the treatment of neovascular AMD, as an inhibitor of the 165 VEGF isomer . A phaseγ [  is 93% of a human immunoglobulin Ig1 and 7% of a murine fragment. A specific antibody domain recognises all VEGF-A isoforms , 55. Bevγ . Researcγ , 61. is a humanized antibody fragment directed at all isoforms of VEGF-A and is fabricated specifically for intravitreal use. Ranibizumab is now FDA approved for the treatment of age-related macular degeneration as well as macular edema associated with retinal vein occlusion. For diabetic macular edema, an initial small pilot study showed efficacy of intravitreal injections of ranibizumab in reducing macular thickness and improving visual acuity , 63. NCT00473382) and RISE (NCT00473330), trials of ranibizumab in diabetic macular edema [In a recent study, authors presented a two-year observation of patients after dosing ranibizumab in diabetic macular edema. After the initial 6 months, all patients were followed up every 2 months. Patients in group 1 could be reinjected if they had persistent or recurrent DME, patients in group 2 could receive either ranibizumab alone or laser only, and patients in group 3 could receive ranibizumab alone or in combination with laser. After 24 months, patients gained 7.7, 5.1, and 6.8 letters in each of the groups, respectively, and the percentage of patients who gained three or more lines of visual acuity was 24, 18, and 26%, respectively . A recenar edema . Patientar edema .μm versus −67.9 μm) compared with laser alone [VEGF Trap is a 115 kDa recombinant fusion protein consisting of the VEGF binding domains of human VEGF receptors 1 and 2 fused to the Fc domain of human IgG1 . The resIt is worth noting that all the above mentioned drugs have their side effects depending on the drug and the method of administration itself , 69.IGF is a polypeptide showing likeness to insulin. There are two such factors: insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II). IGF-1 polypeptide circulates in the blood as an IGF-binding protein (IGF-BP), probably inhibiting the activity of the free IGF. IGF-I is the main growth factor secreted under the influence of a human growth hormone hGH. In vivo and in vitro studies indicate that IGF-1 acts as an antiapoptotic and anti-inflammatory factor , 71. In κb and a Fas ligand, a cytokine belonging to the TNF and its receptor (FasFas/CD95) superfamily, by PEDF action. Volpert et al. have proven that anti-FasL antibodies and the use of caspase inhibitors inhibit PEDF. So, PEDF can prevent cell apoptosis by activating transcription factor NF-κb, as well as activate programmed cell death by increasing the expression of a Fas ligand [PEDF is a 50 kDa protein with neuroprotective, neurotrophic, and antiangiogenic activities , 82. Pigs ligand . In expes ligand . In the s ligand . γ activation [PEDF may exhibit antiangiogenic effect through its antioxidant action. The authors have shown that by its antioxidant effects PEDF can block the effects of proangiogenic factors , 92. In β belongs to the family of transforming growth factors with immunoregulatory properties [β (TGF-β) is present in three inactive isoforms bound with latent associated protein (LAP) and latent TGF-beta binding protein (LTBP). TGF-β1 is present in endothelial cells, hemopoietic cells, and connective tissue cells, TGF-β2 in epithelial tissue and in neurons, and TGF-β3 in connective tissue cells. The activation takes place with the use of plasmin or cathepsin D, after cleavage from nonactive complex. It exerts its action through type I, II, and III TGF-β receptors and in conjunction with a specific SMAD protein present in the cytoplasm [β inhibits proliferation of T and B lymphocytes, NK cells and the expression of class II MHC particles as well as the formation of cytotoxic T-lymphocytes [β can inhibit enzymes that take part in the degradation of these proteins—heparinase, collagenase, and stromelysin. Moreover it increases the production of tissue inhibitors of metalloproteinases (TIMP). The increased level of TGF-β1 has been found in the idiopathic pulmonary fibrosis, diabetic retinopathy, and glaucoma [β. Moreover, in the presence of trans-resveratrol showed inhibition of TGF-β1 and VEGF, COX-2, IL-6 and IL-8 [β function can have adverse effects, such as the exposure of intraocular tissue to the damaging effects of local and systemic immune responses. Therefore, a new anti-TGF-β therapy is to selectively block its activation at sites where excess TGF-activation occurs, without affecting its basic function [TGF-operties . In humaytoplasm –96. Its phocytes . Moreoveglaucoma –99. Amonglaucoma , 101. Thfunction .natural killer) cells, as well as the production of INFγ and TNF-α by these cells. On one hand, IL-12 contributes to the development of autoimmunological diseases, such as rheumatoid arthritis, multiple sclerosis, and type 1 diabetes [α was significantly higher and the level of IL-12 was significantly lower than in the control group without the symptoms of diabetic retinopathy [α production may be the result of insufficient IL-12 level. Maybe the balance between the pro- and antiangiogenic cytokines is one of the factors preventing the development of diabetic nephropathy and retinopathy in those children. It has been shown that, in T1DM patients, IL-12 serum concentration was the highest in subgroup with > or = 3 mg/L hsCRP (P < 0.05) [IL-12 is a multipurpose cytokine and in physiological conditions, it is produced mainly by macrophages, dendritic cells, keratinocytes, granulocytes, and mast cells . It stimdiabetes , 104; ondiabetes . Few studiabetes . In vitrdiabetes , 108. In < 0.05) . Moreove < 0.05) . The att < 0.05) , 112.α is one of the main inflammatory response cytokines. It is produced mainly by monocytes and macrophages and interacts with them by endo-, para-, and autocrine mode of action. It acts chemotactically on monocytes and neutrophils and activates them as macrophages. It enhances the cytotoxicity of monocytes and macrophages, being at the same time one of the mediators of this cytotoxicity. Biological effects largely depend on the quantity and intensity of TNF secretion [α is one of the cytokines inducing the interruption of the blood-retina barrier by loosening tight junctions between individual endothelial cells of the retina and also between the cells of the retinal pigment epithelium. Apart from taking part in the inflammatory processes, TNF-α plays an important role in neovascularisation and vasomotor reactions [α expression. This cytokine fulfills its numerous duties thanks to the ability to stimulate the synthesis of other cytokines, functionally connected with TNF-α, extracellular matrix proteins, modulation of monocyte, and macrophage chemotaxis, as well as the effect on the expression of adhesion molecules in the retinal vessels [α has been found in the serum of children and adolescents with T1DM and NPDR [α level can be an independent predictive factor for NPDR development in children with T1DM [α have been found in adult patients with T1DM and PDR [α has also been found in the vitreous body of patients with T2DM and PDR as compared with the control group [TNF-ecretion . TNF-α ieactions , 115. Fa vessels . TNF-α hand NPDR , 116. Th and PDR , 118. Inol group .Currently, five anti-TNF agents are commercially available: etanercept, infliximab, adalimumab, ceertolizumab pegol, and golimumab –136.α and TNF-β to the surface TNF receptors, inactivating TNF and suppressing neutrophil migration and proinflammatory cytokine synthesis. Clinical studies have been indeterminate regarding the efficacy of etanercept for the treatment of ocular inflammation [It is a fusion (hybrid?) protein composed of a TNF receptor and the Fc fragment of human IgG antibody. It inhibits the binding of TNF-ammation –122.α but not TNF-β [It is an IgG1 chimeric monoclonal antibody with a constant human region and a variable murine one. This agent binds both the soluble and the cell-bound TNF-ot TNF-β . It has ot TNF-β . Howeverot TNF-β .α, which binds both its soluble and cell-bound forms [It is a fully humanized IgG1 monoclonal antibody, specifically directed against TNF-nd forms . Adalimund forms , 128. It is a chimeric monoclonal antibody that binds to CD20 antigen on the surface of B cells and suppresses B-cell differentiation resulting in reduced IgG and IgM production . It has α), preventing TNF-α binding with its receptors. Human TNF binding by golimumab neutralizes TNF-α-induced expression of adhesive selectin E particles, vascular cell adhesion molecules (VCAM-1), and intercellular adhesion molecules (ICAM-1) on the surface of endothelial cells. In in vitro studies, golimumab inhibits the TNF-induced secretion of interleukin IL-6, IL-8 and the granulocyte-macrophage colony stimulating factor (GM-CSF). In patients receiving golimumab, there was an improvement in C-reactive protein concentration, which in turn significantly decreased concentrations of interleukin 6 (IL-6), ICAM-1 particles, metalloproteinase (MMP-3), and vascular endothelial growth factor (VEGF). Moreover, in patients with RA or ankylosing spondylitis, it was the TNF-α concentration that decreased, while in those with psoriatic arthritis, it was the IL-8 concentration that decreased [It is a human monoclonal antibody forming stable complexes with high affinity to the soluble and transmembrane form of human tumor necrosis factor  and antiangiogenic, PEDF is a good material for study; although at present, the success of monoclonal antibody therapy can be judged to be merely moderate. The administration of all the above mentioned drugs is associated with many complications, drugdependent as well as linked with the method of drug administration itself. The latter can give transient complications, such as subconjunctival hemorrhage, the feeling of a foreign body under the eyelid, and increased intraocular pressure. Serious complications, with about 0.05% frequency, encompass the intraocular inflammation, retinal detachment, damage to the lens, or hemorrhage into the vitreous body. However, ongoing energetic studies in many centers around the world suggest that most probably in the near future we will achieve therapeutic success in preventing loss of sight in DM patients. The search for new, efficient treatment methods, especially in advanced stages of diabetic retinopathy, makes the research on biological drugs especially important. Conducted studies suggest that among known factors, both pro-angiogenic (such as TNF-


ADAMTS5 knockout mice compared with wild-type mice. The purpose of this study was to evaluate the suppressive effects of injections of ADAMTS5 small interference RNA (siRNA) oligonucleotide on intervertebral disc degeneration in the rabbit anular needle-puncture model.The etiology of degenerative disc disease is unknown. Several investigators have reported the presence of proteolytic enzymes, such as the matrix metalloproteinase (MMP) and ADAMTS  families, in degenerated human discs. Glasson and colleagues recently reported that a significant reduction occurs in the severity of cartilage destruction in ADAMTS5 or the control. The suppression of the ADAMTS5 gene by siRNA transfection was assessed by using real-time polymerase chain reaction (PCR), both in monolayer and alginate bead cultures with or without interleukin-1β (IL-1β) stimulation. The effect of siRNA was determined in vivo by using the rabbit anular needle-puncture model . One week after the initial anular puncture, the animals received an injection of the control or anti-ADAMTS5 oligonucleotide . Disc height, magnetic resonance imaging (MRI) , and safranin-O staining (histologic grade) were assessed.Rabbit nucleus pulposus (NP) cells were transfected with siRNA oligonucleotides specific for ADAMTS5 mRNA level in NP cells (P < 0.01). ADAMTS5 gene suppression was 70% compared with the control oligonucleotide in both monolayer and alginate bead culture with or without stimulation with IL-1β. The injection of anti-ADAMTS5 oligonucleotide in vivo resulted in improved MRI scores with increased signal intensity and improved histologic grade scores with statistical significance (P < 0.05). No significant change in disc height was observed.IL-1β treatment significantly increased the ADAMTS5 siRNA induced the suppression of degradation in NP tissues, as shown by significantly improved MRI and histologic grades. The mechanism of response to siRNA may be worthy of exploration for possible therapeutic purposes.A single injection of  However, the dual deletion of ADAMTS4 and ADAMTS5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine osteoarthritis in vivo [The intervertebral disc (IVD) encompasses two structures, the anulus fibrosus (AF) and the nucleus pulposus (NP). The predominant matrix component of the AF is collagen type I, whereas the NP contains randomly organized collagen fibers (mainly type II) and highly hydrated proteoglycans, primarily aggrecan, which impart compressive resistance to the tissue. Aggrecan is cleaved at a specific "aggrecanase" site ,2; this ) family -6. In a ype mice . Glassonype mice  also rep in vivo .ADAMTS4 or ADAMTS5 is the major aggrecanase responsible for degradation of aggrecan in the human IVD. Modulating the enzymatic activity or gene expression of the responsible enzymes might be a valid approach for protecting human IVD tissues from degradation.Compared with cartilage, the NP has a higher content of aggrecan that is more degraded and a higher proportion of molecules not bound to hyaluronan . A recenADAMTS5 mRNA was expressed in human normal and OA cartilage [ADAMTS4 mRNA was very low in vivo and was induced in vitro only after stimulation with IL-1β. These results suggest that investigating both ADAMTS4 and ADAMTS5 may shed light on the mechanism of IVD degeneration.IVDs of patients with lumbar disc herniation have been shown to express proinflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) ,13, whicartilage , ADAMTS4in vivo rabbit anular puncture model of disc degeneration showed the anabolic effects of bone morphogenetic protein-7 [in vitro.Biologic treatment strategies for human IVD degeneration include increasing the levels of anabolic growth factors or blocking the catabolic cascade or both. On the anabolic side, an rotein-7  and growrotein-7 . The antrotein-7 , tissue rotein-7 , IL-1 rerotein-7 ,21,22, aADAMTS4 and ADAMTS5 in human cartilage was recently found by using a small interfering RNA (siRNA) approach in normal and osteoarthritic explants [in vivo. The purpose of this study was to prove our hypothesis that a single injection of ADAMTS5 siRNA inhibits the production of ADAMTS5 and suppressed IVD degeneration in the rabbit anular needle-puncture model.The anticatabolic role of the specific inhibition of explants . To dateM NaCl; Cambrex CC-3234, Charles, Iowa, USA) at a density of 5 × 105 cells/ml. The beads were maintained for up to 14 days with Chondrocyte Differentiation Media (Cambrex CC-3225) and seeded in a 12-well plate at a density of 1 × 105 cells/well.Lumbar IVDs from four consecutive levels  were dissected from the spines of adolescent Japanese white rabbits  after killing by injection of an excess amount of sodium pentobarbital . Tissues were separately harvested from the NP and the outer layer of the AF, and the cells were isolated by using a sequential proteinase and collagenase digestion, as previously described . PrimaryADAMTS5 gene was constructed from a completely homologous region of sequences in the ADAMTS5 gene of the human, rat, and mouse from the NCBI website [ADAMTS5 gene was cloned by using the TA Cloning Kit  and confirmed by sequencing by using the ABI PRISM 310. Sequences for primers used in these analyses were as follows: 5'-CTCCCAGGACAAACCTACGA-3' and 5'-CCTCTTCCCTGTG CAGTAGC-3' for ADAMTS5 cDNA amplification. SiRNA for the ADAMTS5 oligonucleotide was constructed by using the Takara Website [ADAMTS5 oligonucleotides of the siRNA used in these analyses were as follows: sense oligonucleotide 5'-CGAUCCUCAAAGCACUACUTT-3', anti-sense oligonucleotide 5'-AGUA GUGCUUUGAGGAUCGTT-3' for the control, sense oligonucleotide 5'-CCACCAUCACG GAAUUCCUTT-3', and anti-sense oligonucleotide 5'-AGGAAUUCCGUGAUGGUGGTT-3' for ADAMTS5. Sense and antisense oligonucleotide siRNAs were separately dimerized for the control and ADAMTS5.The siRNA oligonucleotide for the rabbit  website . The rev Website . SequencADAMTS5 gene was constructed from the sequence originally analyzed. For transient transfection, NP cells were seeded in a 12-well plate at a density of 1 × 105 cells/well and cultured in complete media.A standard for the rabbit ADAMTS5 oligonucleotide or control oligonucleotide, added directly to media without transfection reagents. After 48 hours' incubation, the cells were harvested, and the mRNA level of ADAMTS5 was assessed.After 48 hours, the NP cells were transiently transfected with the anti-ADAMTS5 oligonucleotide or control oligonucleotide was added to the culture for transfection. After 24 hours' incubation with siRNA, NP cells were collected and subjected to mRNA analysis.After 48-hour preculture, cells were cultured with or without IL-1β (10 ng/ml)  in serum-free DMEM. After 24 hours of IL-1 treatment, anti-ADAMTS5 oligonucleotide  was prepared for in vivo experiments, but first tested in an in vitro alginate culture system. NP cells were cultured in alginate beads, as described earlier. After 14 days, NP cells in alginate beads were transfected with anti-ADAMTS5 oligonucleotide or control oligonucleotide (Dharmacon) without transfection reagents. Results are reported normalized to GAPDH.Freshly prepared stable anti-Total RNA was extracted from transfected cells by using Isogen  and purified with the SV Total RNA Isolation System .ADAMTS5 was calculated by using the comparative threshold (CT) method, as previously described [GAPDH).Random-primed cDNAs were synthesized by using Multiscribe reverse transcriptase . Quantitative real-time PCR was carried out by using a PRISM 7700 sequence detector with the QuantiTect SYBR Green PCR kit  according to the manufacturer's instructions. The relative expression of escribed . ResultsADAMTS5 siRNA oligonucleotide  per disc). The timing of injection was earlier than that used in other studies [ADAMTS5 siRNA during the acute phase of disc degeneration. Nine weeks after the initial anular puncture (8 weeks after the injection), all rabbits were killed.An anular puncture model was established by using an 18-gauge needle at a defined depth of puncture (5 mm), as previously reported . New Zea studies ,18 to reRadiographs were taken at time 0 and at weeks 1, 2, 3, 5, 7, and 9 after the puncture. Extreme care was taken to maintain a consistent level of anesthesia during radiography of each animal at each time point to obtain a similar degree of muscle relaxation, which may affect the disc height. Therefore, the preoperative radiograph was always used as a baseline measurement. Radiographs were digitally scanned and digitally stored by using an image-capture software program.All radiographic images were independently analyzed by using a custom program for MATLAB software  by an orthopedic researcher who was blinded to the treatment groups. Data are reported as the IVD height expressed as the disc-height index (DHI)  . ChangesMRI examinations were performed on all rabbits in the study by using a 0.3-T imager  with a quadrature extremity coil receiver. After killing, the spinal columns with surrounding soft tissue were isolated and subjected to MRI analysis . An obseAfter MRI assessment, the experimental IVDs were excised from the vertebral body-disc-vertebral body unit, and each IVD was fixed in 10% formalin, decalcified, embedded in paraffin, sectioned, and assessed with conventional histology and immunostaining. Midsagittal sections (5 μm) of each IVD were stained either with hematoxylin and eosin or with safranin-O. An observer, blinded to the experiment, analyzed the histologic sections and graded them by using our recently established protocol .post hoc test . Other statistical analyses were assessed with the Mann-Whitney U test. In vitro experiments were performed 3 times.The %DHI was statistically analyzed by using a two-way repeated analysis of variance and Fisher protected least significant difference as a ADAMTS5 siRNA might be influenced by the rich positively charged matrix in NP tissues.Rabbit NP cells were chosen because their response to IL-1β was more consistent than that of AF cells (as determined from previous experiments) and because the injection site in the NP has a higher concentration of proteoglycans. The effectiveness of administration of ADAMTS5 gene in rabbit NP cells. At 48 hours after transfection, the NP cells that received the ADAMTS5 siRNA oligonucleotide showed approximately a 75% knockdown of constitutive expression of ADAMTS5 mRNA  in a dose-dependent manner in rabbit NP cells  was administered into the NP of the rabbit IVDs. After the siRNA injections, no significant differences were found in the %DHI between the control siRNA and the ADAMTS5 siRNA groups during the observation period   Figure .ADAMTS5 siRNA significantly affected the histochemical changes found with IVD degeneration. Eight weeks after the control or ADAMTS5 siRNA injections, the control siRNA group displayed a complete loss of NP tissues, which had been replaced by a fibrocartilaginous tissue  than those in the control siRNA group  .The injection of e Figure  and 7c. e Figure  and 7g. s Figure  and 7d; s Figure  and 7h. p Figure . The tot) Figure . The scoADAMTS5 siRNA into the NP on the delay or attenuation of disc degeneration. Our results demonstrate that the designed ADAMTS5 siRNA was (a) active in vitro and (b) effective in suppressing the degeneration of the NP tissue in the in vivo rabbit model. However, the injection of ADAMTS5 siRNA did not induce the anticipated recovery of disc height.With the rabbit anular puncture model, this study explored the efficacy of a direct injection of ADAMTS5 gene. ADAMTS5 siRNA-transfected rabbit NP cells showed approximately a 75% knockdown of ADAMTS5 mRNA compared with the control siRNA. Although we demonstrated that IL-1β treatment significantly increased the ADAMTS5 mRNA level in NP cells, the suppression of the expression of the ADAMTS5 gene by ADAMTS5 siRNA was 70% compared with the control oligonucleotide in both monolayer and alginate bead culture under stimulation with IL-1β.We successfully designed and constructed an siRNA oligonucleotide with biologic activity for the rabbit ADAMTS5 siRNA during the acute phase of disc degeneration after anular puncture in the rabbit delayed the progression of disc degeneration, as assessed by MRI scores, signal intensity of NP on MRI, and histologic scores. MRI findings of a high T2 signal intensity in the NP indicate that the NP in the punctured discs treated with ADAMTS5 siRNA was hydrated. Therefore, ADAMTS5, which cleaves the core protein of aggrecan, may significantly contribute to the loss of water content of the NP after anular puncture. However, the reason that the disc height loss was not reversed by the injection of ADAMTS siRNA remains to be determined. One possible explanation is that the treatment with siRNA for ADAMTS5 is an anticatabolic one, not anabolic. In addition, a possibility remains that the injected siRNA was retained in the NP area, where the siRNA was injected, by some mechanism, or mainly internalized by NP cells and did not distribute to the AF area. In a previous study using the rabbit anular puncture model, the injection of osteogenic protein-1 into the NP induced an increased proteoglycan content of both the AF and the NP and the recovery of disc-height loss by 6 weeks [ADAMTS5 siRNA in the anular puncture model, nor did we assess the half-life of injected siRNA. The limited effects of ADAMTS5 siRNA may point to a complex involvement of multiple enzymes in disc degeneration. Nevertheless, the strong suppression of the ADAMTS5 gene by siRNA in vitro and in vivo, especially in NP tissues, indicates that ADAMTS5 might play an important role in IVD degeneration.The intradiscal injection of  6 weeks . One couADAMTS5 siRNA-treated discs, as observed with MRI. The improving histologic scores associated with the NP might indicate that ADAMTS5 is more involved in matrix degeneration of the NP than that in the AF. In addition, the direct injection of siRNA into the NP may induce a localized improvement. It is worth noting that the histologic scores for the AF and the border between the AF and NP in the ADAMTS5 siRNA-treated discs showed a trend to be lower (improved) when compared with those for the control discs. These findings might indicate that the inhibition of degeneration or improved reparative activity of the NP may have contributed to the improved histologic grading for the AF and the border between the AF and NP.Histologic findings from safranin-O staining were supportive of the maintenance of NP tissues in the ADAMTS4, 5), matrix metalloproteinase-3 (MMP-3), and MMP-13, gene expression was increased in cells derived from the human NP cells [ADAMTS4 and -5 mRNA occurred downstream of NF-κβ activation in NP cells. These results, and the recent reports on the contribution of IL-1 in disc degeneration [The treatment of human disc cells with IL-1 induced an imbalance between catabolic and anabolic events, responses that represent the changes seen during disc degeneration ,13,29. ANP cells  reportedneration , may indADAMTS5 in turn suppressed IVD degeneration; this suggests the possible contribution of ADAMTS5 to disc degeneration, especially in the NP of the rabbit anular puncture model of disc degeneration.In summary, we have shown evidence that the suppression of ADAMTS5 siRNA suppressed disc degeneration in the NP, as shown by the significantly improved MRI and histologic grades. The results may suggest that ADAMTS5 contributes to the degeneration of NP tissues in the rabbit anular puncture disc-degeneration model. The mechanism for the differences in response to siRNA in disc height and MRI findings may be worthy of exploration.A single injection of T: comparative threshold; DHI: disc height index; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IL-1β: interleukin-1β; IVD: intervertebral disc; MMP: matrix metalloproteinase; MRI: magnetic resonance imaging; NP: nucleus pulposus; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; RT-PCR: reverse transcriptase-polymerase chain reaction; siRNA: small interference RNA; TNF-α: tumor necrosis factor-α.ADAMTS: a disintegrin and metalloprotease with thrombospondin-like repeats; AF: anulus fibrosus; CThe authors declare that they have no competing interests.in vitro assay of ADAMTS5 siRNA.SS conceived this study and made substantial contributions to the study design and to writing the manuscript. SS also acquired and interpreted the data. YA and KA performed data acquisition, statistical analysis, and interpretation of data. KM participated in the design of the study and finalized the manuscript. YK and TK participated in the design of the study and performed the statistical analysis. CM performed the histologic analyses. AI carried out the


Certolizumab pegol (CZP) has a different mode of action from the other anti-TNFs, infliximab (IFX), adalimumab (ADA) and etanercept (ETA), that may be due both to its structure and how it signals through membrane TNF-α (mTNF-α). Different effects of this signalling could relate to the exact epitopes to which the anti-TNFs bind. We investigated the binding epitope for CZP and how it differs from the other anti-TNFs.Epitope mapping for CZP binding to TNF was achieved using minimal shift analysis of nuclear magnetic resonance) data on the non-PEGylated Fab′/TNF complex. Hydrogen/deuterium (H/D) exchange mass spectrometry was used to identify TNF peptides containing amide protons that are protected in the Fab′/TNF complex. A Biacore assay was used to determine the difference in affinity between cynomolgus monkey and human TNF for each of the 4 anti-TNFs. This technique involved the use of cross-species mutagenesis of the 4 residues that differ between cynomolgus monkey and human TNF . The epitope identified by these methods was confirmed by further site-directed mutagenesis and Biacore-based studies.13 amino acids  were identified as comprising the CZP epitope on TNF. These residues formed a cluster on the outer face of each monomer. Biacore studies on TNF peptides expressed with these residues mutated showed that, although there was some overlap, the pattern of binding for each of the anti-TNFs was different.All 4 anti-TNFs tested bind to similar but not identical epitopes on the TNF molecule. These differences between the exact epitope could explain the variations in the downstream signalling properties through mTNF-α of the anti-TNFs and may help to elucidate some of the mode of action differences emerging for these agents.


The present study further characterizes synoviocyte TRP expression and activation responses to thermal and osmotic stimuli after pre-treatment with proinflammatory mediator tumor necrosis factor alpha . Initial temperature induced rapid transient spikes and subsequent slower rise reflect TRPV1 and TRPV4 channel activation, respectively. Only after prolonged TNF-alpha exposure (12 and 16 hr) is recruitment of synoviocytes observed with sensitized TRPV4 responses to hypoosmolarity (3–4 fold increase). TNF-alpha increases TRPV1 (8 hr peak) and TRPV4 (12 hr peak) immunostaining, mRNA and protein expression, with a TRPV1 shift to membrane fractions.Fluorescent imaging of Fura-2 loaded human SW982 synoviocytes reveals immediate and delayed cytosolic calcium oscillations elicited by (1) TRPV1 agonists capsaicin and resiniferatoxin (20 – 40% of cells), (2) moderate and noxious temperature change, and (3) osmotic stress TRPV4 activation (11.5% of cells). TNF-alpha pre-treatment  significantly increases (doubles) capsaicin responsive cell numbers and [Ca2+]i spike frequency, as well as enhances average amplitude of temperature induced [Ca2+]i response dependent on the TRP stimulus and time after exposure. Augmented relevance of TRPV1 and TRPV4 as inflammatory conditions persist would provide calcium mediated cell signaling required for pathophysiological responses of synoviocytes in inflammatory pain states.TNF-α provides differentially enhanced synoviocyte TRPV1 and TRPV4 expression and [Ca Temperature sensitive transient receptor potential (TRP) channels belonging to the V- (or vanilloid related) subfamily are widely expressed in mammalian cells. Four members of this subfamily, TRPV1–4 conduct mono- and di-valent cations when activated by temperatures ranging from > 23°C (TRPV3 and TRPV4) to > 43°C (TRPV1) or > 53°C (TRPV2). In addition, TRPV1–4 function as important membrane sensors for extracellular chemical, osmotic, or mechanical stimuli. TRPV1 channels are activated by low pH (< 5.9) and endovanilloids. TRPV4 and TRPV2 respond to cellular swelling and mechanical stimulation. TRPV1 and TRPV4 are activated by anandamide and arachidonic acid metabolites i) mobilization in control (vehicle treated) and TNF-α pretreated cells. Numerical values for the percent of cells responding, average peak amplitude and duration are provided in Additional file -13 – 10-6 g/L). Figures 2+]i changes were completely abolished by the TRPV1 antagonist capsazepine in both control and TNF-α- treated samples, thus confirming the specificity of TNF-α-mediated vanilloid receptor sensitization. The TNF-α sensitizing effect on TRPV1 activation was evident in the concentration range of 1–50 ng/ml TNF-α after 8 to 16 hr incubations  was tested by monitoring intracellular calcium . TRPV4 responses were characterized with hypoosmolar physiological saline (see below).TNF-α induced sensitization of TRPV1 was further demonstrated by observing increased synoviocyte response to high-degree bath temperature. As shown in our earlier experiments , synovio2+]i [2+]i during prolonged exposure to 30 or 36°C in the presence or absence of 2-aminoethoxydiphenyl borate (2-AB) which activates and sensitizes TRPV3, but not TRPV4 [2+]i changes caused by 30 or 35°C temperature , and is not significantly different from the temperature threshold of vehicle treated cells (29 ± 2°C) (n = 4). However, as shown in Figure 2+]i rise and several large peaks were observed. These calcium spikes superimposed on an elevated [Ca2+]i of significantly smaller amplitude are occasionally observed in temperature stimulated synoviocytes that were not treated with TNF-α. Both average and peak [Ca2+]i elevations induced by 36°C are larger in TNF-α treated cells, with maximal effect observed at 8 hr  n = . However8 hr Fig . There wIn contrast to TRPV1 thermal responses, pretreatment with TNF-α did not induce changes in synoviocyte responsiveness to "cold" activator menthol  or irritant icilin . Neither the numbers of cells responding to menthol (16–21%) or icilin (96–100%), nor the average amplitude and duration of the calcium response for the TNF-α-treated  group were significantly altered from responses of the control group (vehicle) , the average fluorescent ratio is not significantly changed by switching from isotonic (300 mosmol/l) to hypotonic (150 mosmol/l) conditions  after 2, 4, or 8 hr exposure to TNF-α induces somewhat increased i and Western blot cell fraction data. After 12 hr TNF-α treatments, SW982 synoviocytes assumed a more flattened appearance with an increase in cell area (1772 ± 203.1 vs. 1246 ± 106.1 um2)  Fig  and obseum2) Fig  and cellThe SW982 synoviocyte staining for TRPV1 is coincident with the peak physiological responses (8 hr). The delay in increased responsivity to TRPV4 Fig  occurs aIn the present study we demonstrated up-regulation of two members of the TRP cation channel family in SW982 human synovial cells after preincubation with TNF-α. The TNF-α induced increases in TRPV1 and TRPV4 gene and protein expression at 8 and 12 hr respectively, are coincident with significant enhancement of responses to increasing temperature and decreasing osmolarity mediated by TRPV1 and TRPV4. These two TRP channels appear to be the major thermal and osmosensors in synoviocytes. Marginal facilitation of the calcium response at 30°C, in synoviocytes incubated with 2-AB demonstrates the potential for TRPV3 mediated response to thermal stimuli in some circumstances, although no response to camphor was generated. TNF-α did not induce a change in synoviocyte responsiveness to TRPA1 (icilin) or TRPM8 (menthol) activation.A number of endogenous factors can activate TRPV1 and TRPV4 during inflammation . Thi2+]i in response to noxious temperature and icilin. It is also unclear why the synoviocytes readily responded to hypoosmolarity but not to TRPV4 agonist 4α PDD. This heterogeneous response suggests that these TRP channels exist in multi-potential functional states, that non-neural cellular responses are not identical to neuronal responses, and/or that tertiary protein structure plays a role in activation properties. Other mechanisms regulating TRP channel sensitization may be involved, such as PKC or PKA dependent phosphorylation i responses and cell recruitment.The present data indicate that synoviocytes express functional thermal and osmotic receptors inducible under inflammatory conditions. The TNF-α mediates up-regulation of TRPV1 and TRPV4, doubling mRNA and protein expression at 8 and 12 hr, respectively. Microarray data has been published detailing downstream targets for TNF-α in synoviocytes  and demo2+]i signaling responses of synoviocytes during inflammation would participate in the TNF-α mediated functions that promote the painful inflammatory and joint destruction processes locally and impact subsequent responses of the immune system. The data suggest TRPV1 and TRPV4 play important roles mediating the intracellular Ca2+ increase that drives critical functions such as associated proliferative and secretory responses of synoviocytes during joint inflammation thus promoting TRPV1 and TRPV4 as attractive potential therapeutic targets.The literature suggests that TRP expression in epithelial lining cells may be extremely important in cells that interface with dramatic changes in the external and internal environment induced by thermal and mechanical stress, acidity, hypoosmolarity, foreign chemicals and endogenous mediators. Synoviocytes are exposed to all of the above stimuli in the course of routine joint function and in acute and chronic inflammatory states. The synoviocytes are like other fibroblasts that secrete the inflammatory mediator, TNF-α , as wellThis paper confirms a physiological role for TRP cation channels in transduction of relevant information to synovial lining cells. The inflammatory milieu includes thermal, osmolar, acidic and mediator stimuli, all potent activators of TRP channels. Our study supports an inducible responsivity for TRP cation channels TRPV1 and TRPV4 in synoviocytes to inflammatory mediator, TNF-α. Pretreatment of human synoviocytes with TNF-α produces (1) increased TRPV1 and TRPV4 mRNA expression at 8 hr, (2) increased TRPV1 protein expression at 8 hr in the membrane fraction, (3) increased TRPV4 cellular expression and increased cell size at 12 hr, and (4) differentially enhanced calcium responsiveness and cell recruitment in response to thermal and osmolarity challenges. Responses of synoviocytes depend on the TRP stimuli and time point after TNF-α exposure. The delayed transduction and responsivity are highly suggestive of downstream transcriptional regulation. This study emphasizes the need for studies designed with prolonged time courses to better understand clinically relevant physiological responses of local cells that impact inflammatory pain states. These findings further support augmented relevance of synoviocyte TRP ion channels in arthropathies, and potentially other non-neuronal TRP responses in inflammatory states.2). Low passage cells (3–15×) were used in this study.The fibroblast-like synovial SW982  cells we2+]i in cells exposed to chemical activators of different TRP channels a fluorescent calcium imaging approach was used. Cells were harvested with 0.25% trypsin-0.02% EDTA disruption, plated on 15-mm circular quartz glass coverslips at a density of 200,000 cells/mm3 and incubated at 37°C for 24–48 h before an experiment. On the day of the experiment, the cells were loaded with the calcium-sensitive fluorescent dye fura-2 as previously described [2+]i with the formula [Ca2+]i = Kd [(R-Rmin)/(Rmax - R)](Sf2/Sb2)i was clearly seen at the appropriate time and when the [Ca2+]i returned to baseline in the absence of treatment. Detection threshold of [Ca2+]i changes was dictated by signal to noise ratio and was at least 10 nM over the baseline. Peak and average amplitude of [Ca2+]i changes over a baseline were used for quantitative estimation of calcium response.For measurement of i in large numbers of cells exposed to chemical activators of different TRP channels, human SW982 synoviocytes were plated onto 12 mm coverslips, cultured for 24 hr, then pre-treated with TNF-α (1 ng/ml). The cells were loaded with the calcium-sensitive fluorescent dye fura-2 for 30–60 min before calcium recording. The activation was done on 3 plates with over 60 cells selected. The imaging setup included a Nikon 200E microscope with a 20× SuperFluo lens, and a computer-controlled illumination system  equipped with a digital monochrome-cooled charge-coupled device RoperCoolsnap HQ camera . Fluorescent emission at 510 nm from regions of interest (corresponding to a single cell) was acquired online with the MetaFluor software . The signal was obtained in dual, 340 and 380 nm, excitation mode and average intensity of fluorescence in each region after were used to estimate 340-to-380 ratios. The elevation in calcium influx for the individual and population data was considered an increased response if statistically different from the baseline.For simultaneous measurement of -amino}-ethyl)-amino]-methylchromen-4-one) was obtained from Cayman Biologicals . Thapsigargin used to test cytosolic calcium release at the end of each study was obtained from Calbiochem .The low passage SW982 cell cultures were grown in 75 mm flasks until they reached 70–80% confluency. Leibovitz's L-15 Medium  with 10% FBS, 1% P/S was used for culture medium and the cells were incubated at 37°C, room air, per ATCC recommendations. For the experiments, TNF-α (1 ng/ml) was added to the designated flasks. The cells were harvested by disruption (see below) over a time course of baseline (immediately after TNF-α addition), and at 4 and 8 hours after addition. Flasks containing untreated cells were also harvested at the same time as baseline controls. These studies were performed in three separate experiments.® III First-Strand kit  with random hexamer primers as described by the manufacturer. Two microgram of total RNA from each individual sample was used to synthesize cDNA for TRPV1, TRPV4 and β-actin, respectively.Total RNA was isolated using Trizol reagent  in accordance with manufacturer's protocol. First-strand cDNAs were synthesized from total RNA by RT using the SuperScriptQuantitative real-time PCR analysis was performed by the use of an Applied Biosystems 7000 Sequence Detection System . Each sample was analyzed in triplicate and the means were used for statistical purposes. The thermal profiles were obtained by using 2 min incubation at 50°C, followed by an initial 10 min denaturation step at 95°C, and by 40 cycles of 1 min each at 60°C plus 15 sec at 95°C. Significant contamination with genomic DNA was excluded by amplifying non-reverse-transcribed RNA.Hs00218912_m1), TRPV4 (Hs00540967_m1) and β-Actin (Hs99999903-m1) were ordered from Applied Biosystem Inc. The threshold cycle (Ct), i.e. the cycle number at which the amount of amplified gene of interest reached a fixed threshold, was determined subsequently. Relative TRPV1 and TRPV4 mRNA expression was calculated by the comparative Ct method described elsewhere [-ΔΔCt (fold), where ΔCt = Ct of target gene (TRPV1 or TRPV4)-Ct of endogenous control gene (β-actin), and ΔΔCt = ΔCt of samples for target gene-ΔCt of the control for the target gene. 2 μl of synthesized cDNA from each individual sample was used to amplify for TRPV1, TRPV4 and β-actin, respectively.The probes and primers for TRPV1  for 8 hours. Western Blot analysis was performed on whole cell protein and subcellular protein fractions extracted from synovial SW982 cells with ProteoExtract Subcellular Proteome Extraction kit . Protein transferred to membranes were probed with anti-TRPV1 antibody  and TRPV4  antibody, at 4°C overnight. Amounts of total protein loaded per lane: 15 μg for cytosolic and membrane fractions, 10–15 μg for whole cell and 5 μg for rat spinal cord lysate as a positive control. The protein analysis was performed with protein from three experiments.Cells on glass coverslips were fixed with 4% freshly mixed paraformaldehyde:methanol  for 30 min at room temperature. The coverslips were washed 3× in PBS and incubated at room temperature for 30 minutes with 3% neutral serum. The primary antibody  or TRPV4  was diluted in TBS with 1.0% neutral serum (0.02% Triton X-100). The primary antibody was allowed to incubate on the glass coverslips for 48–72 h at 4°C. The glass coverslips were then washed 3× with 1% serum, 0.02% Triton X-100 and then incubated for 30–60 min at room temperature with the appropriate secondary IgG antibody , using donkey anti-goat (for TRPV1) and goat anti-rabbit (for TRPV4). Each had a FITC fluorescent tag . After final washes 3× with PBS, the stained coverslips were inverted onto a dot of VectaShield mounting media containing blue nuclear counterstain DAPI. Immunocytochemical controls included the absence of the primary or secondary antibodies, or were done in the presence of matched serum at the same dilution. The specificity of the antibodies was confirmed for TRPV1 by adsorption controls with P-19 N-terminus peptide provided by Santa Cruz, and for TRPV4 with peptide 853–871 supplied by Alomone Labs . There was no staining in either the method controls or the peptide blocked controls. TNF-α stimulated increase in TRPV1 was greatly reduced by co-incubation with a TNF-α inhibitor, CAY10500. Stained cells were visualized with a Nikon FXA microscope equipped with MetaVue software .Sigma Plot and Sigma Stat scientific software  were used for conversion and analysis of acquired data. Data are reported as means ± SE. MetaMorph software  were used for off-line analysis of calcium imaging data. Student's t-tests or ANOVA for multiple comparisons with Tukey's multiple comparison post hoc test or Dunnett's method were used to compare data obtained from TNF-α treated vs. control series, unless otherwise indicated.The authors declare that they have no competing interests.All authors read and approved the manuscript. MYK was responsible for calcium imaging responses to capsaicin, thermal and osmotic challenges, as well as the initial manuscript draft; TAM was responsible for experimental and primer design for synoviocyte culture studies; HY was responsible for RT-PCR; LZ and FM were responsible for all calcium imaging studies with TNF-α primed synoviocytes; SA and LP were responsible for western blots of whole cell and cell fractions of control and TNF-α primed synoviocytes; SA was responsible for immunolocalization studies, microscopy, cell area and MetaMorph analyses; and KNW was responsible for experimental design of the studies, data synthesis, data figures and manuscript editing for publication.Responses of human SW982 synoviocytes to capsaicin, icilin and menthol after TNFα pre-treatment. Dose response curve for TNF-α pre-treatment (8 hr) and stimulation with capsaicin (1 μM).Click here for fileSW982 synoviocytes responses to hypotonic saline, with and without pre-treatment with TNF-α, with and without inhibitor ruthenium red (3 μM).Click here for file


In vitro, 200 nM D1(A12) inhibited shedding of ADAM17 substrates TNF-α, TNFR1-α, TGF-α, amphiregulin (AREG), HB-EGF and IL-6Rα, from IGROV1-Luc cells, (4.7 nM IC50 for TNF-α shedding). In IGROV1-Luc xenografts in vivo, D1(A12) IgG showed pharmacokinetic properties suitable for efficacy studies, with a single i.p. dose of 10 mg/kg D1(A12) sufficient to maintain IgG plasma and ascites fluid concentrations above 100 nM for more than 7 days. The plasma half life was 8.6 days. Next, an efficacy study was performed, dosing D1(A12) or anti-human TNF-α antibody infliximab at 10 mg/kg q7d, quantifying IGROV1-Luc tumour burden by bioluminescence. D1(A12) IgG showed a significant reduction in tumour growth (p = 0.005), 56% of vehicle control. Surprisingly, D1(A12) did not reduce the concentration of circulating human TNF-α, suggesting that another enzyme may compensate for inhibition of ADAM17 in vivo (but not in vitro). However, D1(A12) did show clear pharmacodynamic effects in the mice, with significant inhibition of shedding from tumour of ADAM17 substrates TNFR1-α, AREG, and TGF-α . Thus, D1(A12) has anti-ADAM17 activity in vivo, inhibits shedding of EGFR ligands and has potential for use in EGF ligand-dependent tumours.ADAM 17  is a potential target for cancer therapy, but the small molecule inhibitors reported to date are not specific to this ADAM family member. This membrane-bound metalloproteinase is responsible for ectodomain shedding of pathologically significant substrates including TNF-α and EGFR ligands. The aim of this study was to evaluate the pharmacokinetics, pharmacodynamics and anti-tumour efficacy of the first specific inhibitor, an anti-human ADAM17 IgG antibody, clone D1(A12). We used intraperitoneal xenografts of the human ovarian cancer cell line IGROV1-Luc in Balb/c nude mice, chosen because it was previously reported that growth of these xenografts is inhibited by knock-down of TNF-α.  TNF-α converting enzyme  is a membrane-bound metalloproteinase responsible for cleavage and ectodomain shedding of TNF-α, EGFR ligands and other pathologically significant substrates. Dysregulation of ectodomain shedding is associated with autoimmune and cardiovascular diseases, neurodegeneration, infection, inflammation and cancer TM) in breast cancer ADAM17 has been implicated in many cancers. It is overexpressed in ovarian cancer Small molecular inhibitors of ADAM17 have been developed but none have yet shown complete specificity for ADAM17. For example, INCB3619 and INCB7839 inhibit ADAM10, ADAM17 and matrix metalloproteinases MMP2, MMP12 and MMP15 in vitroA specific human ADAM17 inhibitory antibody, D1(A12), has recently been developed, which inhibits the proteolysis of ADAM17 substrates  in cancer cells TM), has been investigated for its efficacy against ovarian cancer in vivo. For comparison, a group of mice were treated with infliximab, which was expected to directly reduce functional human TNF-α, also leading to inhibition of growth of the IGROV1-Luc tumours.High levels of expression of TNF-α have been observed in ovarian carcinoma TM, Janssen Biotech Inc.), a kind gift from Prof. Peter Taylor (Kennedy Institute of Rheumatology), was dissolved in sterile saline. Control human plasma IgG (R&D Systems 1-001-A) was used as a control in some cell-based assays. N-TIMP-3 was prepared as described by Lee etalIsolation and purification of human anti-ADAM17 antibody D1(A12) has been described previously The IGROV1 human ovarian cancer cell line 6 IGROV1-Luc cells and were observed daily for tumour growth and clinical signs. Tumour burden was quantified weekly by bioluminescent imaging, as described below.All mouse studies were performed in accordance with the UK Animals (Scientific Procedures) Act 1986, under project licence number 80/2346, with approval from the CRUK Cambridge Research Institute Animal Ethics Committee and following current UK Guidelines Both antibodies were diluted in sterile PBS, pH 7.4 for dosing into mice at 10 mg/kg. For the PK study at least 2 mice per time point were dosed i.p. with 10 mg/kg in a volume of 9.5 ml/kg. The tumour-bearing mice were dosed for PK studies either 34 days after tumour implantation (for shorter time points), or 27 days after implantation for 7 – 9 day time points. The concentrations of D1(A12) and infliximab in plasma, ascites fluid and tumour homogenates were measured by quantitative ELISA as described below. For the efficacy study the mice were randomised into 3 groups and dosed i.v. with the antibodies at 10 mg/kg in a dose volume of 4.76 ml/kg, or with sterile PBS as a vehicle control. The first dose was given on day 4 after tumour cell injection, immediately after the first bioluminescent imaging session, and every 7 days thereafter until day 32. On day 32 the mice were imaged for the last time and given a final dose, then they were killed the following day, day 33, 20 hours after the last dose.Blood was collected in lithium heparin tubes, centrifuged at 18,800 g for 5 minutes and plasma collected. Ascites fluid was collected from the abdominal cavity and centrifuged at 18,800 g for 5 minutes to remove any cells. Plasma and ascites fluid were then snap-frozen and stored at −80°C. Organs and tumour were collected post-mortem with part snap-frozen and part fixed in Neutral Buffered Formalin (Sigma). Formalin-fixed tissues were paraffin embedded, sectioned and stained with haematoxylin and eosin. For analysis of tumour tissue by ELISA, weighed pieces of frozen tissue (approximately 20 mg) were homogenised in MK-28R tubes containing metal beads using a Precellys 24 homogeniser  at 6000 rpm for 50 seconds, twice. The samples were homogenised at a concentration of 50 mg of tissue per ml of buffer  and phosphatase inhibitor cocktail 2 (Sigma) each diluted 1 in 100). The homogenates were centrifuged at 10,000 g for 10 minutes at 4°C and the supernatant was used for ELISAs (see below).Mice were anaesthetised using isoflurane then injected i.p. with 150 μg/g body weight D-Luciferin in PBS  and bioluminescent imaging with a charge-couple device camera  was initiated 10 min after injection. Images were obtained for groups of 3 mice with a 13 cm field of view, binning (resolution factor) of 8 (medium), F stop  = 1, exposure  = 1 to 10 seconds.2/steradian) for each mouse, from a constant sized Region of Interest drawn over the mouse abdomen. The mean and standard deviation were expressed for each treatment group. Significance between treated and vehicle groups was calculated using a t Test.Data were analysed using Living Image 3.2 Software (Xenogen) and presented as Average Radiance (units: photons/sec/cmFor both D1(A12) and infliximab human IgGs, ELISA plates were coated with 50 μl/well 100 nM anti-human IgG (Abcam ab700) in PBS. After washing with PBS-Tween (0.1% v/v) and blocking with PBS-Milk (5% w/v), 50 μl of sample was added to each well  or infliximab IgG for the standard curves ). After incubation and washing the human IgG was detected by incubating with anti-human-kappa light chain-HRP antibody (Bethyl A80-115P) (1∶2000 in PBS-milk), followed, after washing, by 50 μl of 3,3′,5,5′-tetremethylbenzidine (TMB) incubated at room temperature for 2 mins then quenched with 50 μl 1M HCl. Absorbance was measured at 450 nm. Pharmacokinetic parameters were calculated using a non-compartmental model in WinNonlin v5.1 (Pharsight).ELISAs for ADAM17 substrates were performed using R&D Systems Duoset kits: human TNF-α , human soluble TNFR1-α , human TGF-α (cat. No. DY239), human AREG (cat. No. DY262), human IL-6Rα (cat. No. DY227) and human HB-EGF (DY259). The DY210 kit was confirmed to be specific for human TNF-α by testing recombinant mouse TNF-α with this kit, there was no cross-reactivity.Where tumour homogenates were used, raw nM concentrations in homogenate solution were converted to nmoles per mg tissue, and then using the assumption that tissue density is 1 g/ml, the molar concentration in tissue was calculated.Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of tumour and liver after antigen retrieval by enzyme digestion (proteinase k) at 37°C for 10 minutes. The human IgG with which the mice had been dosed (D1(A12) or infliximab) was detected by binding of a Biotin-SP-conjugated AffiniPure F(ab')2 fragment donkey anti-human IgG (H+L) , followed by staining with DAB+ chromogen. Slides were scanned using a ScanScopeXT  at 20X magnification and scanned images were viewed using Spectrum v10 software (Aperio Technologies Inc). No image manipulation was performed.in vitro secreted TNF-α, TNFR1-α, TGF-α, AREG, HB-EGF and IL-6Rα, all known substrates for the shedding activity of ADAM17, into the culture medium even when unstimulated (50 in IGROV cells of 4.7 nM (50 of 72 nM), a natural metalloproteinase inhibitor previously shown to inhibit ADAM17 in vitro in the presence of normal growth medium (data not shown), which is consistent with the effect of the TNF-α shRNA on IGROV1-Luc cells as previously reported IGROV1-Luc tumour cells imulated . When stimulated . The inhf 4.7 nM . D1(A12)The IGROV1-Luc cells grew and disseminated intraperitoneally in nude mice , as prevmax = 523+/−58 nM, Tmax 2 days, half life 8.6 days. More limited sampling was then performed in mice bearing IGROV1-Luc tumours  concentrations of above 100 nM can be maintained by dosing 10 mg/kg once every 7 days. 100 nM D1(A12) is sufficient concentration to cause maximal inhibition of ADAM17 function in IGROV1 cell culture (The pharmacokinetics (PK) of D1(A12) antibody were investigated using a single 10 mg/kg dose i.p., in non-tumour-bearing mice . PK para tumours , in whic culture .The detection of D1(A12) antibody by ELISA does not necessarily mean that the antibody had retained its activity, as it could be partially denatured, so the binding activity of the plasma D1(A12) antibody was assessed . The D1(6 p/s/cm2/Sr) 2.71+/−1.35, 2.92+/−1.23 and 2.65+/−0.91 for vehicle, infliximab and D1(A12) groups, respectively. Tumour size at the endpoint on day 32 is presented in 6 p/s/cm2/Sr) on day 32 of 23.3+/−9.1 compared to the vehicle group . The mean tumour burden in the D1(A12) group at the endpoint was 56% of the vehicle control. In contrast there was no inhibition of tumour growth in mice treated with infliximab compared to vehicle, with the tumour burden of 39.1+/−11.6 in the infliximab group (p = 0.68).Having established that the D1(A12) antibody has suitable PK characteristics, we tested the effect of weekly dosing in IGROV1-Luc xenografts with 10 mg/kg D1(A12) (n = 11), in comparison with 10 mg/kg infliximab (n = 8) and PBS vehicle (n = 12). Prior to the first dose on day 4 after cell injection there was no significant difference in the tumour burden between the groups: Avg Radiance  and infliximab was determined in the plasma and ascites fluid  and in tTo investigate the distribution of D1(A12) and infliximab in the tumour tissue, anti-human IgG immunohistochemistry was performed on paraffin sections from the tumours . The twoTo determine whether either antibody had pharmacodynamic effects we analysed the concentrations in plasma and ascites fluid of the products of ADAM17 sheddase activity, i.e. soluble human TNF-α, soluble TNFR1-α, TGF-α and amphiregulin (AREG) . All fouin vitro.We have investigated the biological activity of D1(A12), a monoclonal antibody specific for human ADAM17, which inhibits ADAM17 function by cross-domain inhibition of the ectodomain. We have demonstrated that the antibody (at nanomolar concentrations) inhibits shedding of ADAM17 substrates including TNF-α and AREG in IGROV1-Luc cells max were lower in the tumour bearing mice than the plasma Cmax in the mice without tumours. However, this is not surprising given the larger total body fluid volume in the tumour-bearing mice due to the ascites fluid compartment. The plasma and ascites fluid concentrations of D1(A12) IgG were very similar within each individual mouse, from both the PK and efficacy studies, suggesting that the antibody is able to equilibrate between these two compartments after either IP or IV dosing. We have demonstrated that D1(A12) antibody is structurally stable in the mouse circulation, retaining the ability to bind to human ADAM17 up to 9 days after dosing. However, the ability of D1(A12) to bind to human FcγR1 decreased with time in the mouse plasma, such that only 32% of the binding activity remained after 9 days in the mouse. The reduced FcγR1 binding capacity may be due to soluble Fc-receptors present in mouse serum. For example, mouse FcRn binds human IgG1 with high affinity in vivo half-life of antibodies The PK properties of the D1(A12) IgG in mice were found to be suitable for efficacy studies. The plasma half life is consistent with published values for the half life of human IgG antibodies in mouse plasma in vivo, so efficacy studies were performed in the IGROV1-Luc model. D1(A12) antibody showed anti-tumour effects, with the tumour burden at the end of the study approximately 56% of the vehicle control group. This was a modest anti-tumour effect, and some possible explanations for this are discussed below.The PK properties and stability of D1(A12) IgG in the mouse circulation indicated that weekly dosing should maintain concentrations high enough to be biologically active 50, but analysis by IHC showed limited penetration beyond the tumour blood vessels. Similar limited penetration has been observed in xenograft tumours for the high affinity anti-HER2 antibody trastuzumab in vivo. Distribution of monoclonal antibodies into tumour tissue has been shown to be limited by penetration through capillary walls and also by additional factors once the antibody has crossed the blood vessel wall Tumour homogenates had concentrations of D1(A12) IgG above the cellular ICTo determine whether the antibodies were hitting their targets and having pharmacodynamic effects, ELISA assays of ADAM17 substrates were performed on plasma and ascites fluid samples at the end of the efficacy study. Vehicle-treated mice showed much higher concentrations of soluble TNF-α, TNFR1-α, AREG and TGF-α in ascites fluid than in plasma, suggesting that these proteins do not freely equilibrate between the plasma and ascites compartments, in contrast to the dosed IgGs which did equilibrate between the compartments.in vivo, which was surprising because TNF-α shedding was inhibited by D1(A12) in the IGROV1-Luc cells in vitro. The lack of inhibition of TNF-α shedding in vivo might explain the relatively modest antitumour effect of the antibody, because TNF-α was thought to be a key driver of tumour growth in this model, based on the shRNA work of Kulbe, etalComparison of the D1(A12) treated group with the vehicle controls showed that D1(A12) did significantly inhibit shedding of ADAM17 substrates, the EGFR ligands TGF-α and AREG, and TNFR1-α. However, D1(A12) did not significantly inhibit TNF-α shedding in vivo (but not in vitro). This may be ADAM10: ADAM10 has shown TNF-α sheddase activity in ADAM17-deficient murine fibroblasts in vivo, inhibition of both ADAM17, ADAM10 and ADAM19 may be required. In the in vivo studies the tumour cells would have been exposed to D1(A12) for 28 days, and if the compensatory mechanisms were slow to develop, this could explain why this compensation was not observed in the in vitro assays which were performed over 48 hours or less. Another possible explanation for the lack of inhibition of TNF-α shedding in vivo is that shedding in trans could be occurring, where murine ADAM17 on host cells, which would not be regulated by D1(A12), could cleave TNF-α on adjacent human tumour cells. To our knowledge trans-shedding has not been reported for ADAM17, but it has for ADAM10 cleavage of ephrin A5 in vivo, it is striking that shedding of the other ADAM17 cleavage products were inhibited by D1(A12). It is possible that the difference relates to the type of transmembrane proteins involved: pro-TNF-α is a Type II transmembrane protein, whose C-terminus is extracellular, whereas pro-AREG and the other EGFR ligands are Type I transmembrane proteins, whose N-termini are extracellular. This might confer a difference in specificity for other metalloproteinases in certain contexts, or the ability to be a substrate for shedding in trans. Another possible explanation for the difference between the ADAM17 substrates is that TNF-α shedding might occur deeper within the tumour mass (where antibody penetration may be limiting) than the shedding of the EGFR ligands. If the D1(A12) was unable to inhibit TNF-α shedding by ADAM17 deep in the tumour this pharmacokinetic feature could also explain the discrepancy between the in vitro and in vivo studies, as the cells in vitro would all be exposed to the antibody.ADAM17 has been considered the key enzyme responsible for shedding of TNF-α, but our results with the D1(A12) antibody suggest that another enzyme may compensate when ADAM17 activity is inhibited in IGROV1-Luc cells grown in vivoetal., have recently confirmed that infliximab did not reduce tumour growth in this IGROV1 model, even when co-administered with an anti-mouse TNF-α antibody in vitro and in vivo results.We used the specific human TNF-α antibody infliximab in our efficacy studies as a positive control, expecting it to inhibit growth of IGROV1-Luc tumours. TNF-α shedding was significantly inhibited in the infliximab group compared to the vehicle, but surprisingly infliximab did not inhibit tumour growth, in contrast to the published study showing that TNF-α shRNA knockdown did inhibit IGROV1-Luc tumour growth in vivo. Thus, the D1(A12) specific ADAM17 inhibitor which showed modest antitumour effects in the IGROV1-Luc model may show more effective antitumour activity in tumours that are dependent on EGFR ligand signalling.While inhibition of ADAM17 by D1(A12) did not significantly inhibit TNF-α shedding it did efficiently inhibit shedding of other ADAM17 substrates, the EGFR ligands TGF-α and AREG, suggesting that other ADAMs do not compensate for loss of ADAM17 activity on these proteins Figure S1Analysis of the binding capacity of the D1(A12) IgG in mouse plasma. Each line represents the plasma from one mouse, taken from the tumour-bearing mice used in the PK study . Using the D1(A12) plasma IgG concentrations as determined in (TIF)Click here for additional data file.


Thioredoxin (Trx) and thioredoxin reductase 1 (TR1) are among the major redox regulators in mammalian cells and have a wide variety of roles, including removal of intracellular reactive oxygen species (ROS) and prevention of cell death. Tumor necrosis factor-α (TNF-α) induces cancer cell death. Although ROS have been proposed to participate in this process, the role of the thioredoxin system in TNF-α stimulated cell death remains unclear. We investigated the possibility that the thioredoxin system protects against TNF-α-induced cancer cell death by examining whether TR1/Trx1 status controls TNF-α-induced apoptosis in EMT6 murine breast cancer cells. TR1-deficient cells were more sensitive to TNF-α than control cells. Increased sensitivity to TNF-α was most pronounced in Trx1-deficient cells. TNF-α-induced nuclear localization of phosphorylated ERK 1/2 (p-ERK 1/2) correlated with increased apoptosis in TR1- and Trx1-deficient cells, suggesting a pro-apoptotic role for nuclear p-ERK 1/2 in TNF-α-induced apoptosis. In addition, phosphoinositide 3-kinase (PI3K) inhibition dramatically reduced TNF-α-stimulated apoptosis and nuclear localization of p-ERK 1/2. In contrast, inhibition of ROS, MEK, JNK, or p38 did not significantly alter p-ERK 1/2 localization or apoptosis in TR1- and Trx1-deficient cells compared to control cells. Further, NF-κB p65 localization was not changed in TR1- and Trx1-deficient cells in response to TNF-α relative to control cells. Our data suggest that the thioredoxin system plays a critical role in protecting against TNF-α-induced apoptosis by regulating the levels of nuclear p-ERK 1/2 in a PI3K-dependent manner. The thioredoxin system is one of the most important cellular redox regulatory systems. Thioredoxin (Trx) and thioredoxin reductase 1 (TR1) are the principal components of this system. The thioredoxin system regulates the redox state of protein thiols and controls many cellular processes, including proliferation, defense against oxidative stress, and apoptosis . Trx wasMammalian TR1 is a selenium-containing protein that has selenocysteine (Sec), the 21st amino acid, at its catalytic site. Sec is essential for the activity of TR1 ,12. TR1 Extracellular signal-regulated kinases 1/2 (ERK 1/2) are part of one of the mitogen-activated protein kinase (MAPK) cascades, which regulate numerous cellular processes . ExogenoTo elucidate the role of TR1 and Trx1 in tumor necrosis factor (TNF)-α-induced apoptosis in cancer cells, we examined the sensitivity of TR1- or Trx1-deficient breast cancer cells to TNF-α. TR1 deficiency sensitized cells to TNF-α, and Trx1 deficiency resulted in an extremely high sensitivity to TNF-α. Levels of nuclear phospho-ERK 1/2 (p-ERK 1/2) were induced by TNF-α. The most significant increases in nuclear localization of p-ERK 1/2 were observed in cells that lacked TR1 and Trx1. In contrast, PI3K inhibition diminished levels of nuclear p-ERK 1/2 and rescued cells from TNF-α-induced apoptosis. Thus, abrogation of the thioredoxin system increased nuclear localization of p-ERK 1/2 and significantly increased apoptosis in response to TNF-α stimulation.Recombinant mouse TNF-α, propidium iodide (PI), NuPage 4-12% Bis-Tris gels, polyvinylidene difluoride (PVDF) membrane, hygromycin B, Lipofectamine 2000, Waymouth’s MB 752/1 medium, antibiotic-antimycotic solution, and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies. PD98059, SB203580, JNK inhibitor II, and LY294002 were purchased from EMD Millipore Bioscience. The siRNA vector pSilencer 2.1-U6 Hygro was from Ambion, Inc. Antibodies against cleaved caspase-3 and phosphorylated and total ERK 1/2, p38, and JNK 1/2 were obtained from Cell Signaling Technology, Inc. The antibody against TR1 was from Epitomics. An anti-Trx1 antibody was from Abcam, Inc. The anti-p65 antibody was from Santa Cruz Biotechnology, Inc. Qproteome Mitochondria Isolation kit was purchased from QIAGEN. The BCA protein assay reagent and SuperSignal West Dura Extended Duration Substrate were from Thermo Fisher Scientific Inc. Protease inhibitor and phosphatase inhibitor cocktails were from Roche Diagnostics.°C with 5% CO2. Cells were stably transfected with pU6m3 (control), siTR1 (TR1 knockdown), and siTrx1 (Trx1 knockdown) constructs and were designated EMT/pU6m3, EMT/siTR1, and EMT/siTrx1, respectively, as previously described [6 cells/dish were seeded onto 60-mm dishes and incubated overnight. Cells were left untreated or treated with TNF-α (10 ng/ml) for 48 h and harvested after washing with phosphate-buffered saline (PBS). Cell viability was measured by the trypan blue exclusion method. A Student’s t test was used to calculate the statistical significance of the experimental data.The mouse mammary carcinoma cell line, EMT6, was obtained from the American Type Culture Collection. Cells were grown in Waymouth’s MB 752/1 media supplemented with 15% FBS and an antibiotic-antimycotic solution in a humidified incubator at 37escribed . To meas6/60-mm dish) and incubated overnight before treating with TNF-α (10 ng/ml). Cells were incubated for 10, 30, or 60 min and harvested after washing twice with PBS. To examine the effects of MAPK and PI3K inhibitors on the cellular localization of p-ERK 1/2, cells were treated with ERK inhibitor , p38 inhibitor , JNK inhibitor II (10 μM), PI3K inhibitor , or buffer for 1 h and treated with TNF-α for 10 min. Cells were harvested after washing twice with PBS. All western blots were carried out in duplicate and representative data are shown in the figures.Cells were seeded onto a cell culture dish , incubated on ice for 30 min with intermittent vortexing, and centrifuged at 13,000 × g for 15 min at 4°C. Mitochondrial extracts were prepared with Qproteome mitochondria isolation kit following the manufacturer’s instructions. Protein concentrations were quantified with BCA protein assay reagent. Protein samples were electrophoresed on NuPAGE 4-12% Bis-Tris gels, and the separated protein samples were transferred to a PVDF membrane. The membrane was incubated with a primary antibody and washed with TBS-T. The membrane was incubated with a horseradish peroxidase-conjugated secondary antibody, exposed to SuperSignal West Dura Extended Duration Substrate, and visualized on X-ray film. The corresponding uncropped figures of western blots presented in the text , protease inhibitor cocktail, and phosphatase inhibitor cocktail) as described elsewhere . Nuclearthe text  and 4B a6/60-mm dish) were seeded onto culture dishes and incubated overnight. Cells were pre-treated with ERK inhibitor , p38 inhibitor , JNK inhibitor II (10 μM), PI3K inhibitor , or buffer for 1 h and treated with TNF-α for 24 h. Cells were harvested and fixed with cold 70% ethanol overnight at 4°C. Fixed cells were centrifuged, re-suspended in PBS containing RNase A, and incubated for 30 min at 37°C. Cells were stained overnight in the dark with PI (50 μg/ml) and analyzed by flow cytometry on a FACS Calibur 2 Sorter (Becton Dickinson). The apoptotic cell populations were quantified by analyzing data with FlowJo .Cells  activation. Pre-treatment with NAC decreases ROS induction in response to TNF-α stimulation ,46. HoweOur data further indicated that cells with Trx1 knockdown showed a more significant reduction in cell survival (15%) than TR1-deficient cells (50%). A similar trend was observed with caspase-3 activation and apoptosis. Previous studies have shown that TR1 knockdown led to a significantly higher level of selenium cytotoxicity in mouse DT cells in comparison to Trx1 loss in these cells . The enhPIK3CA hyper-activating mutations or loss of the PTEN phosphatase, resulting in constitutively activated PI3K [A potential limitation of the study was the use of a single murine breast cancer cell line. However, EMT6 cells are a well-established model system for studying molecular abnormalities in breast cancer. Further studies will need to be performed in human breast cancer cell lines. In particular, and due to the critical role of PI3K signaling in the apoptotic response of TR1- and Trx1-deficient cells, breast cancer cell lines that harbor constitutive activation of the PI3K signaling pathway will also need to be examined. A subset of human breast cancers are known to express ted PI3K ,48. Our In summary, the following novel findings were made with the cancer cell lines with stable knockdown of the thioredoxin system(1). Loss of thioredoxin function increased the apoptotic response of murine breast cancer cells to TNF-α(2). Levels of nuclear p-ERK 1/2 were increased in association with apoptosis in TNF-α-treated thioredoxin-defective cells(3). TNF-α-induced apoptosis and localization of nuclear p-ERK 1/2 were dependent upon PI3K signaling. Thus, abrogation of the thioredoxin system enhanced sensitivity to TNF-α through a PI3K-dependent mechanism involving localization of phosphorylated ERK 1/2 to the nucleus. Our results provide novel insights into the mechanisms by which inhibition of TR1 and Trx1 promotes apoptosis of cancer cells and strongly support thioredoxin system blockade as a novel therapeutic strategy for cancer.Figure S1Nuclear translocation of p65 by TNF-α treatment and the effect of MAPK inhibitors on TNF-α-induced caspase-3 activation.(A) EMT/pU6m3, EMT/siTR1, and EMT/siTrx1 cells were untreated or treated with TNF-α for the indicated time intervals. Cytosolic and nuclear extracts were prepared as stated in the Materials and Methods section. Expression of p65 was measured by western blotting. (B) Cells were pre-treated with PD98059 (PD) or SB203580 (SB) for 1 h and treated with TNF-α. Cleaved caspase-3 was examined after 24 h by western blotting.(PDF)Click here for additional data file.Figure S2Effect of PD98059 and SB209580 on TNF-α-induced apoptosis.EMT/pU6m3, EMT/siTR1, and EMT/siTrx1 cells were pre-treated with MEK inhibitor, PD98059 , and p38 inhibitor, SB203580 , for 1 h and incubated with TNF-α. The percentages of apoptotic cells were measured by examining DNA contents after staining with PI and are shown in the graphs. Experimental details are given in the Materials and Methods section.(PDF)Click here for additional data file.Figure S3Effect of MAPKs and PI3K inhibitors on the nuclear translocation of p65 by TNF-α.Cells were pre-treated with PD98059, SB203580, JNK inhibitor II, or LY294002 and treated with TNF-α. Nuclear extracts were western blotted for p65. Experimental details are given in the Materials and Methods section.(PDF)Click here for additional data file.Figure S4Uncropped figures of western blots presented in the text .(PDF)Click here for additional data file.