C. elegans model of excitotoxicity, where a mutation in a central Glu transporter (glt-3) in a sensitizing background causes Glu-Receptor –dependent neuronal necrosis. We expand our studies on the role of the IIS cascade in determining susceptibility to excitotoxic necrosis by either blocking IIS at the level of PI3K/AGE-1 or stimulating it by removing the inhibitory effect of ZFP-1 on the expression of PDK-1. We further show that the components of the Cytohesin/GRP-1, Arf, and PIP5K/PPK-1 complex, known to regulate PIP2 production and the IIS cascade, modulate nematode excitotoxicity: mutations that are expected to reduce the complex's ability to produce PIP2 and inhibit the IIS cascade protect from excitotoxicity, while overstimulation of PIP2 production enhances neurodegeneration. Our observations therefore affirm the importance of the IIS cascade in determining the susceptibility to necrotic neurodegeneration in nematode excitotoxicity, and demonstrate the ability of Cytohesin/GRP-1, Arf, and PIP5K/PPK-1 complex to modulate neuroprotection.During ischemic stroke, malfunction of excitatory amino acid transporters and reduced synaptic clearance causes accumulation of Glutamate (Glu) and excessive stimulation of postsynaptic neurons, which can lead to their degeneration by excitotoxicity. The balance between cell death-promoting (neurotoxic) and survival-promoting (neuroprotective) signaling cascades determines the fate of neurons exposed to the excitotoxic insult. The evolutionary conserved Insulin/IGF Signaling (IIS) cascade can participate in this balance, as it controls cell stress resistance in nematodes and mammals. Blocking the IIS cascade allows the transcription factor FoxO3/DAF-16 to accumulate in the nucleus and activate a transcriptional program that protects cells from a range of insults. We study the effect of IIS cascade on neurodegeneration in a  This combination causes extensive neuronal necrosis that is dependent on Ca2+-permeable GluRs, defining it as nematode excitotoxicity C. elegans to identify the IIS cascade as a factor that can modulate the extent of neurodegeneration in both nematodes and mammalian neuronal cultures glt-3;nuIs5 worms: both a mutation in PI3K/AGE-1 that blocks IIS from expelling FoxO3/DAF-16 from the nucleus, and a drug that translocates FoxO3/DAF-16 into the nucleus reduced the extent of neuronal necrosis in nematode excitotoxicity.Cell stress resistance control by IIS is only one of the many signaling pathways conserved from nematodes to humans. Conservation of function extends also to the use of Glu and the molecular building blocks that mediate its function as an excitatory neurotransmitter in the nervous system C. elegans, showing that Cytohesin/GRP-1 and Arf can control asymmetric cell division glt-3;nuIs5 animals is neuroprotective in nematode excitotoxicity, and we establish that the IIS-regulating Cytohesin/Arf/PIP5K complex modulates this neuroprotective effect.We now look for upstream regulators of IIS in the modulation of excitotoxicity. We are especially intrigued by the function of a complex of proteins that include the Guanine Exchange Factor (GEF) Cytohesin/GRP-1, the small G-protein Arf, and the PIP2-synthesizing enzyme PIP5K/PPK-1. A number of studies in mammals and flies link the Cytohesin/Arf/PIP5K complex to insulin signaling-dependent liver metabolism, membrane transport, and cell growth, demonstrating its functions in providing PIP2 as a substrate for PI3K/AGE-1 and therefore as a stimulator of the IIS cascade C. elegans strains were generate and maintained using standard methods. Strains used in this study include: Nematode Excitotoxicityglr-1::gfp-1;Pglr-1::Gαs(Q227L) V; lin 15(+)]; Δglt-3 (bz34) IV; nuIs5  I; otIs220 Is  IV; grp-1 (tm1956) IIIotIs114 Is  X, gqIs25  I.EG3361 (lin-15(n765ts) X oxIs12 gqIs25[P animals.C. elegansglt-3;nuIs5 . Animals were mounted with an agar chunk on a cover slip and observed using an inverted DIC microscope (without anesthesia). The animals on the chunk were screened, individual animals were classified for their developmental stage, and the number of degenerating neurons for each animal was recorded. Necrotic neurodegeneration is seen as swollen neurons that look like vacuolated structures . Similarly to the stochastic nature of neuronal necrosis seen with other triggers of necrotic neurodegeneration in C. elegans , the number of degenerating neurons in the control group is not stereotypically repeated in exact values . Instead, cell death shows a very typical dynamics, as it peaks at L3 with the maturation of Glu signaling in the worm, and then goes down as cell corpses are engulfed and removed. The level of neurodegeneration in our excitotoxicity model can vary in response to growth conditions, and keeping the strain running by repeated re-chunking over very long periods can suppress its levels. Therefore, special care was given to the use of recently isolated or outcrossed strains, the use of freshly grown (non-stressed) animals in multiple sessions, and in each session, comparison of test strains to control animals exposed to identical growth conditions . Each bar in glt-3;nuIs5) used as reference in each experiment was re-isolated from the new cross, to enhance the similarity with the new strain being tested. Critical new strains were obtained in two independent crosses and neurodegeneration was scored to verify the effect in independent strains.Levels of excitotoxic neurodegeneration were quantified as described by Mano & Driscoll C. elegansLY294002 (LC Laboratories) drug was dissolved in 100% ethanol to produce a stock solution of 25 mM. 20 microliter of ethanol without (control) or with LY294002 were added to 12 well plates with MYOB agar+OP50 bacteria C. elegansglt-3(bz34);nuIs5 animals  causes a moderate reduction in the number of necrotic corpses in head neurons compared to non-treated animals  mutation indeed causes increased susceptibility to excitotoxicity, increasing the average number of necrotic neurons in the L3 stage from 4 to 6  with mutations that affect this complex. This approach is usually more productive in C. elegans than pharmacological intervention (which many time is ineffective in the worm) or RNAi (which many times is ineffective in nematode neurons), though it has its drawbacks. For example, there are a few Arf homologs in the worm, but only some can be studied by genetic elimination, since their KO strain is lethal (as is ppk-1 KO). However, we managed to study the KO of two key components grp-1     or arf-1 allele)  suppressglt-3;nuIs5 animals using LY294002, and compared animals that have WT grp-1 to animals carrying a grp-1 KO. Neurodegeneration levels in grp-1;glt-3;nuIs5 animals exposed to LY294002 was very similar to that of glt-3;nuIs5 animals exposed to LY294002  we used a strain that exhibits over-expression and excessive activity of PPK-1. Weinkove et al. found that over-expressing PPK-1 from the powerful pan-neuronal rab-3 promoter causes excessive production of PIP2, and that mature neurons are especially susceptible to PPK-1 overexpression Prab-3::PPK-1 construct to glt-3;nuIs5 animals we saw an increase in the level of necrotic neurodegeneration .To increase the validity of our conclusion that the Cytohesin/GRP-1, Arf, and PIP5K/PPK-1 complex regulates the IIS cascade to determine the level of susceptibility to excitotoxicity, we also tested other possible explanations for the neuroprotective effect of grp-1 on the level of excitotoxic neurodegeneration is based on the involvement of grp-1 in apoptosis, as seen in some post-embryonic lineages in the nematode grp-1 could reduce the extent of cell death. To test the possible involvement of apoptosis as a mediator of neurodegeneration in our excitotoxicity model we blocked apoptosis using the ced-4(n1162) mutation mec-4(d) –induced necrosis ced-4 did not affect the level of excitotoxic neurodegeneration , as apoptosis seems not to be involved in nematode excitotoxicity. It is possible that other IIS cascade-regulated processes might also be influenced by this complex. However, as the focus of our research is excitotoxicity, our data does not address those other functions of the IIS cascade. Together with our previous data on the nuclear translocation of DAF-16 as a means to induce neuroprotection, our studies are therefore in line with a model where the Cytohesin/GRP-1, Arf, and PIP5K/PPK-1 complex controls the transcriptional output of the IIS cascade to regulate susceptibility to excitotoxicity (Our data puts the spotlight on the IIS-regulating Cytohesin/GRP-1, Arf, and PIP5K/PPK-1 complex and its role in regulating susceptibility to excitotoxicity in toxicity .et al.Our initial interest in the Cytohesin/GRP-1, Arf, and PIP5K/PPK-1 complex was based on the studies that indicate its physical association with the PSD and with GluRs. Currently the subcellular localization of this complex is unknown (other than the observation by Weinkove


Introduction. Strontium salts are anti-irritants for chemically induced sensory irritation. Interstitial cystitis is a painful disease without definitive therapy. The aim of the study was to determine the effect of strontium in bladder with experimental interstitial cystitis model. Material and Methods. Rats' bladders in control group were instilled with NaCl. Second group was instilled with E. coli LPS. Third group was instilled with strontium. Fourth group was initially instilled with strontium and then LPS. Fifth group was instilled with LPS initially and then strontium. Urine of rats was collected at the beginning and end of the study. Results. Histamine and TNF-α changes were statistically significant in the second group but were not significant in the third group. When we compared the histamine levels of second via fourth and fifth groups the changes were statistically not significant. When we compared the TNF-α levels of second via fourth and fifth groups the changes were statistically significant. Conclusions. In our model, strontium did not make any significant changes in histopathology or histamine levels; however, it significantly reduced the levels of TNF-α. Given the role of TNF-α in the physiopathology of interstitial cystitis, these results suggested that further studies are required to evaluate the potential use of strontium in the management of interstitial cystitis. Interstitial cystitis/painful bladder syndrome (IC/PBS) was defined by the Society for Urodynamics and Female Urology as “an unpleasant sensation  perceived to be related to the urinary bladder, associated with the lower urinary tract symptoms for more than 6 weeks duration, in the absence of infection or other identifiable causes” . It is aThe etiology and pathogenesis of IC/PBS are unclear. Many possible pathophysiological mechanisms have been suggested for IC/PBS, including inflammatory, neurogenic, autoimmune, vascular, or lymphatic disorders; damage to the glycosaminoglycan layer and apoptosis of urothelium; and the occurrence of toxic substances in the urine .α also promotes apoptosis through binding to TNF receptor 1, resulting in many effects, ranging from inflammation to apoptosis [α in the development of the IC has been revealed in various studies [α [α induces apoptosis via TNF-related apoptosis inducing ligand (TRAIL) and its receptors in IC/PBS patient's bladder tissue samples [TNF-poptosis . The rol studies , 6. One udies [α . Anotherα and inflammatory cytokines [Strontium compounds are used in the treatment of osteoporosis, in palliative treatment of metastatic prostate cancer, and as anti-irritant in dermatologic diseases –9. Osteoytokines .α in urine in an established animal model of bladder inflammation [We investigated the effect of intravesical SC on the release of histamine and TNF-ammation  using ba Escherichia coli K-235 was purchased from Sigma-Aldrich (L2143-10 mg) and its 100 μg/mL concentration was prepared with sterile water in a culture medium.This work is supported by the Scientific Research Project Fund of Cumhuriyet University under the Project number “T-499.” Strontium chloride hexahydrate (SrCl2 6H2O) was purchased from Sigma-Aldrich (255521) and its 3% (w/v) concentration was prepared with sterile water freshly. LPS fromμL of one of the following substances in 5 groups for each rat. The first group's bladders were instilled with pyrogen-free saline, the second group's bladders were instilled with LPS on days 1 and 6, and the third group's bladders were instilled with strontium on days 1 and 6. The fourth group as preventing groups' bladders were instilled initially with strontium on days 1 and 3 following LPS on days 4 and 6. The fifth group as treatment groups' bladders were instilled initially with LPS on days 1 and 3 following strontium on days 4 and 6  were purchased from the Animal Center of the Faculty of Medicine, Cumhuriyet University, Sivas, Turkey. The Animal Research Committee of Faculty of Medicine, Cumhuriyet University, conducted the study in accordance with the guidelines for the care and use of laboratory animals. The procedure of Stein et al. was followed to instill LPS into the bladder . Procedu 4 and 6 . These sα and histamine levels were detected by ELISA method in rats' urine, using Cayman  commercial kits with Thermo Multiscan HC plate reader device.TNF-t-test when appropriate, and P values of less than 0.05 were considered to be statistically significant.All data were expressed as mean ± SEM. Statistical comparisons between groups were performed using general linear models of analysis of variance (ANOVA) followed by the Tukey test and a α values  were 9.85 ± 3.58 ng/mL and 6.9 ± 0.9 pg/mL, respectively, at the beginning of the study. The levels rose to 34.14 ± 3.02 ng/mL and 119.2 ± 13.5 pg/mL at the end of the study. These changes were statistically significant  (Second (LPS) group histamine and TNF-of both) .α levels were 9.76 ± 3.61 ng/mL and 4.3 ± 2.1 pg/mL, respectively, at the beginning of the study. The levels rose to 11.33 ± 2.57 ng/mL and 27.8 ± 14.1 pg/mL at the end of the study. These changes were not statistically significant (P: 0.995 and 0.102) (Third (strontium) group histamine and TNF-d 0.102) .P: 0.942 and 0.702) (Fourth (preventive) group end of study histamine level was 31.88 ± 2.58 ng/mL. Fifth (treatment) group end of study histamine level was 37.28 ± 4.16 ng/mL. When we compared the histamine levels of second (LPS) group (34.14 ± 3.02 ng) with those of fourth and fifth groups, the changes were not statistically significant (d 0.702) .α level was 28.0 ± 15.1 pg/mL. Fifth (treatment) group end of study TNF-α level was 25.0 ± 14.6 pg/mL. When we compared the TNF-α levels of second (LPS) group (119.2 ± 13.5 pg/mL) with those of fourth and fifth groups, the changes were statistically significant (P < 0.001 for both) (Fourth (preventive) group end of study TNF-or both) .There was no significant difference between the groups histologically .α levels in treatment and preventive groups. To our knowledge this is the first study reporting that SC may decrease urine TNF-α in response to LPS. However, SC could not inhibit LPS induced urine histamine levels in both groups.IC/PBS is a chronic condition characterized by frequent and urgent urination, pain in the pelvis and/or incontinence, and variable inflammation. There are various animal models of IC but none of these models are not reliable. LPS has been used to induce experimental cystitis  like cycα levels. We showed that SC significantly reduced the TNF-α levels of urine when applied as preventive and treatment purpose in experimental cystitis model induced by LPS. Interestingly, in the same model, strontium chloride did not affect urinary histamine levels.When SC was instilled to normal bladders, it did not make significant changes in bladder histology or urine histamine and TNF-α values or the bladder histopathology when we applied strontium to normal bladder intravesically. These findings suggested that strontium does not have any negative effect on bladder glycosaminoglycan layer in contrast to the effect on cartilage.Svensson et al. demonstrated that cartilage had a 60% lower rate of synthesis of glycosaminoglycans and collagen on the rats which received oral strontium therapy in their study . Due to α is in the regulation of immune cells and it can also induce apoptotic cell death through activation of the transcription factor, nuclear factor kappa-light-chain enhancer of activated B cells, and c-Jun N-terminal kinase [α [α mainly induces urothelium apoptosis via TRAIL pathway and TRAIL-R4 is the predominant receptor in the interstitial cystitis inflammation [The primary role of TNF-l kinase . Shie etinase [α . Anotherammation .α levels were found to be increased when the cystitis was induced with LPS [α levels increased in the urine samples of rats.In experimental studies, urine and bladder tissue TNF-with LPS , 17. In Previous studies have shown that elevated levels of urinary histamine are commonly observed in patients with IC/PBS  and are α, IL-6 and slowing down osteoclastogenesis [β and TNF-α, in regardless CaSR activation status, osteoblasts [α in large animal model of osteoporosis [Topal et al. recently demonstrated that oral strontium chloride hexahydrate (40 mg/kg and 160 mg/kg) suppressed serum TNF-alpha levels and had comparable therapeutic efficiency when compared with prednisolone in a rat ulcerative colitis model . Buache ogenesis . In addieoblasts . Furtheroporosis .α which is shown in these publications supports our finding in this study.Cytokine expressions, including lymphocytes and immune cells, depend on calcium influx through the cell membranes. Intracellular calcium increases providing calcium-calmodulin complex and calcineurin causes proinflammatory cytokine expression such as TNF-alpha and IL-1. Strontium competitively blocks the influx of calcium and may block proinflammatory cytokines expression indirectly , 25. Theα gene in the bladder tissue is the major limitation of this study.The lack of measurements of the amount of TNF and the expression of TNF-α. Given the role of TNF-α in the physiopathology of interstitial cystitis, these results suggested that SC may be a promising agent for the treatment of IC/PBS and further studies are required to evaluate the potential use of strontium in the management of interstitial cystitis.In our interstitial cystitis model, created with lipopolysaccharide, SC did not make any significant changes in histopathology of the bladders and the levels of histamine; however, it significantly reduced the levels of TNF-


Active immunization, or vaccination, with tumor necrosis factor (TNF)-Kinoid (TNF-K) is a novel approach to induce polyclonal anti-TNF antibodies in immune-mediated inflammatory diseases. This study was performed to transfer the proof of concept obtained in mice model of rheumatoid arthritis (RA) into human. We designed a pilot study to demonstrate the feasibility of therapeutic vaccination in RA.This was a phase IIa, placebo-controlled, multicenter study in adults with RA who previously experienced secondary failure of TNF antagonists. Patients were immunized intramuscularly with 2 or 3 doses of placebo (n = 10) or 90 (n = 6), 180 (n = 12), or 360 µg TNF-K (n = 12). The primary objective was to identify the best dose and schedule based on anti-TNF antibody titers. Clinical symptoms and safety were assessed during 12 months and solicited reactions for 7 days after each injection.The highest anti-TNF antibody response was detected in patients immunized with 360 µg TNF-K and with 3 injections, although this difference was not significant with all other groups. Similar proportions of patients receiving TNF-K and placebo reported adverse events up to month 12. Serious adverse events were reported by 4 patients treated with TNF-K (13.3%) and 3 treated with placebo (30.0%), all unrelated to treatment. At month 12, DAS28-CRP, tender and swollen joint counts, and HAQ scores decreased significantly more in patients who exhibited anti-TNF antibody response than in patients who did not.TNF-K therapeutic vaccination induced dose- and schedule-dependent anti-TNF antibodies in RA patients and was well tolerated. Patients who developed anti-TNF antibodies showed a trend toward clinical improvement. Although the most aggressive dose and schedule, i.e. 360 mg dose administered 3 times, did show a strong trend of higher antibody response, further studies are warranted to examine even higher and more frequent doses in order to establish the best conditions for clinical improvement.NCT01040715ClinicalTrials.gov  Rheumatoid arthritis (RA) is a chronic systemic immune mediated inflammatory disease that affects 0.5 to 1% of the population, resulting in major functional disability and increased mortality Among these, active immunization against TNF with TNF-Kinoid (TNF-K) is a promising development Based on the proof of concept established in experimental arthritis, TNF-K entered clinical development. A Phase I clinical trial, performed in Crohn’s disease patients, showed it was well tolerated and immunogenic nd World Medical Association General Assembly, Edinburgh, Scotland, October 2000). The study protocol was approved by the medical ethics committees at all participating institutions, as detailed below:This study was a phase II, randomized, double-blind, multicenter clinical trial examining the safety and immune responses of TNF-K in adults with RA who previously experienced a secondary failure of anti-TNF biologics . The primary objective was to identify the best dose and schedule of administration of TNF-K in terms of anti-TNF antibody response induced by either 2 injections (day 0 and 28) or three injections  of TNF-K at 3 dosages . The study was performed at 21 centers in Argentina, Belgium, Bulgaria, Chile, Croatia, France and Romania, in compliance with European Medicines Agency Guidelines on Clinical Evaluation of New Vaccines, International Committee for Harmonization Guidelines on Good Clinical Practice, and the Declaration of Helsinki (revision of the 52Comite Independiente de Etica para Ensayos en Farmacologia Clinica (Fefym), Buenos Aires; Comité Institucional de Etica en Investigacion en Salud (CIEIS), Sociedad de Beneficencia Hospital Italiano Cordoba city; Comité de Etica del Centro de Investigaciones Reumatologicas, San Miguel de Tucuman; Comité de Docencia e Investigacion - Centro Medico Privado de Reumatologia, San Miguel de Tucuman; Argentina. Université Catholique de Louvain Faculté de Médecine Commission d’Ethique Biomédicale Hospitalo-Facultaire, 1200 Bruxelles, Belgium. Ethics Committee for Multicenter clinical trials, Sofia, Bulgaria. Comité Etico Cientifico Del servicio de Salud Metropolitano Oriente (SSMO) Providencia, Santiago, Chile. Central Ethics Committe, agency for Medecines and Medical devices, Zagreb, Croatia. Comité de Protection des Personnes (CPP) Ile de France II, Paris, France. Ministry of Health, National Ethics Committee for the Clinical study of medecines, Bucharest, Romania. CEIC de Asturias, Hospital Universitario Central de Asturias, Oviedo, Spain. Commission Cantonale d’éthique de la recherche sur l’être humain, Lausanne, Switzerland.Patients gave written informed consent before inclusion in the trial.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Adults 18 to 70 years with RA were considered for inclusion if they had RA that was diagnosed at least 6 months earlier according to the revised 1987 criteria of the American College of Rheumatology (ACR) and a DAS28-CRP>3.2 at screening TNF-K was prepared by aldehyde crosslinking of recombinant human TNF and KLH as described previously Treatments were randomly assigned using a sequential, incremental, staggered-dose design . In eachAnti-TNF antibody titers were measured in sera collected on days 0, 17, 28, 38, 56, 84, 112, 140, 168, 252, and 336 using a direct enzyme-linked immunosorbent assay in which microtiter plates were coated with human recombinant TNF 3H]-thymidine incorporation was assessed as described previously Peripheral blood mononuclear cells from patients were isolated by Ficoll-hypaque density gradient centrifugation, added  to round-bottom 96-well plates, and incubated at 37°C with medium alone or with TNF-K, recombinant human TNF , or KLH  at 0.1, 1 and 10 µg/mL. After 72 h, ) or placebo (9/10 [90.0%]) and infections were reported by similar proportions of patients treated with TNF-K and placebo (43.3% vs. 50.0%). Treatment-related AEs were reported by 14 patients treated with TNF-K (46.7%) and 6 treated with placebo (60.0%). One non-serious AE considered possibly treatment-related (moderate polyarthritis 57 days after a second dose of 180 µg TNF-K) led to early discontinuation of the treatment and one patient treated with TNF-K reported a severe AE (headache) considered by the investigator to be treatment-related.SAEs were reported by 4 patients treated with TNF-K  and 3 treated with placebo . None of the SAEs was considered treatment-related. No case of serious infection, no tuberculosis, no malignancies, no anaphylactic reactions, no serum sickness-like reactions and no deaths related to treatment were reported up to month 12.Clinical response rates between TNF-K and placebo groups were not significantly different, but non-significant trends towards better clinical responses were seen in patients administered 360 µg TNF-K who had the most pronounced antibody responses. In post-hoc analysis, clinical responses were therefore compared between antibody responders (n = 23) and non-responders (n = 17).DAS28-CRP, mean tender joint count, swollen joint count, patient’s global assessment, physician’s global assessment, patient assessment of pain, and HAQ score improved progressively up to month 12 in antibody responders, whereas no clear trend over time was observed in patients who remained negative for anti-TNF antibodies . At montThis pilot study showed that TNF-K was able to induce anti-TNF antibodies in patients with RA who had experienced a secondary failure with anti-TNF biologics. This antibody response was dose-dependent, since only patients immunized with 360 µg TNF-K had all an antibody response. Thus active anti-cytokine immunization with Kinoid appeared to be feasible in humans against TNF as shown here and also against IFNα as previously reported in lupus patients This study also showed that TNF-K was well tolerated. The injection of TNF-K did not induce any severe local reactions and no unexpected safety concerns were identified. No cases of severe infection including tuberculosis were observed. Larger studies should be performed to evaluate the absolute risk of severe infection, including tuberculosis, and compare with other strategies of TNF blockade Despite the small size of the study and the fact it was not specifically powered to detect clinical improvements, we observed progressive and significant improvements in the group of antibody responders compared to the non-responders; this was noticed for DAS28-CRP, tender and swollen joint counts, physician global activity score, and HAQ disability/function score, suggesting a clinical effect of the polyclonal anti-TNF antibodies induced by TNF-K. A similar observation was made in mice models with an association of the immune responder status to TNF-K with the clinical response A potential bias in our study is the absence of stratification with regards to previous anti-TNF treatment. Indeed, Van Vollenhoven et al recently reported that etanercept as second anti-TNF following loss of efficacy of a first anti-TNF  yielded a better clinical response than adalimumab or infliximab following etanercept In conclusion, this pilot study showed that anti-TNF therapeutic vaccination with TNF-K was feasible, safe and promising in RA. Further studies are warranted to examine higher and repeated doses to establish the best conditions for clinical improvement.S1 FigureGeometric mean fold seroconversion by isotype. Anti-TNF antibody was detected by enzyme-linked immunosorbent assay as previously described :19442-7) but with isotype-specific secondary antibodies. The seroconversion ratio was calculated as the ratio of the extrapolated optical density for the highest dilution of tested serum divided by the mean optical density for a pool of serum from 3 healthy donors.(PDF)Click here for additional data file.S1 TableAnti-TNF antibody Titers. Individual anti-TNF antibody titer (Dil-1)(PDF)Click here for additional data file.S2 TableNeutralizing Capacity. Individual neutralization capacity (NC50 Dil-1) at D0, D17 and D56(PDF)Click here for additional data file.S3 TableLymphoproliferation assay. Stimulation index of patient PBMCs stimulated with TNF-Kinoid, TNF and KLH at 0.1, 1 and 10 µg/ml.(PDF)Click here for additional data file.S1 ProtocolProtocol of the study. A phase II, randomized, double-blind, controlled study to evaluate the immune responses, safety and clinical efficacy of three doses of TNF-Kinoid in adult patients with rheumatoid arthritis who have relapsed despite anti-TNF biological therapy.(PDF)Click here for additional data file.S1 ChecklistCONSORT 2010 checklist of the present study.(DOC)Click here for additional data file.


A new operant test for preclinical pain research, termed the Mechanical Conflict System (MCS), is presented. Rats were given a choice either to remain in a brightly lit compartment or to escape to a dark compartment by crossing an array of height-adjustable nociceptive probes. Latency to escape the light compartment was evaluated with varying probe heights  in rats with neuropathic pain induced by constriction nerve injury (CCI) and in naive control rats. Escape responses in CCI rats were assessed following intraperitoneal administration of pregabalin (10 and 30 mg/kg), morphine (2.5 and 5 mg/kg), and the tachykinin NK1 receptor antagonist, RP 67580 (1 and 10 mg/kg). Results indicate that escape latency increased as a function of probe height in both naive and CCI rats. Pregabalin (10 and 30 mg/kg) and morphine (5 mg/kg), but not RP 67580, decreased latency to escape in CCI rats suggesting an antinociceptive effect. In contrast, morphine (10 mg/kg) but not pregabalin (30 mg/kg) increased escape latency in naive rats suggesting a possible anxiolytic action of morphine in response to light-induced fear. No order effects following multiple test sessions were observed. We conclude that the MCS is a valid method to assess behavioral signs of affective pain in rodents. Pain in laboratory animals is typically inferred by measuring innate withdrawal reflexes to noxious mechanical, electrical, or thermal stimuli applied to the hindpaw or tail. Experimentally, reflex methods are easy to perform and efficient, and they have been used with great success in the study of pain mechanisms and analgesic drug development , 2. HoweConflict or motivational choice paradigms have been used for decades to assess complex decision-making behavior in animals. In the simplest form of conflict paradigm, animals, typically in a state of deprivation, voluntarily choose to perform a task that will deliver a noxious stimulus in order to receive a reward, such as food, water, or access to copulation –28. Thusescape an aversive, yet non-noxious environment (light compartment) by subjecting itself to noxious stimulation (crossing the nociceptive probes), or 2) avoid the nociceptive probes but remain in the aversive bright light compartment.The Mechanical Conflict System (MCS) is a conflict paradigm that uses noxious mechanical stimulation and bright light as opposing aversive stimuli. In this test, learned escape from a brightly lit compartment to a dark compartment is obstructed by an array of height-adjustable sharp pins referred to as nociceptive probes. The animal must choose between two opposing motivational drives: 1) 1 receptor antagonist, RP 67580, on MCS responses was also assessed in CCI rats. It was hypothesized that higher probes would increase escape latency in both naive and CCI rats, and that CCI-induced increases in escape latency would be attenuated by pregabalin and morphine, both of which are antinociceptive in the CCI model. It was predicted that RP 67580 would not alter MCS escape responses. The data presented here provide preliminary support for the utility of the MCS in preclinical pain research.The objective of the present study was to evaluate escape behavior in the MCS under variable intensity levels of mechanical stimulation produced by changing the height of the nociceptive probes. Latency to escape the light compartment was measured in the chronic constriction nerve injury (CCI) model of neuropathic pain and in naive control rats. The effect of systemic administered analgesics, pregabalin, morphine, and the tachykinin NKThis study was conducted in strict accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committees at the University of Michigan , the Veterans Affairs Ann Arbor Healthcare System (VA Protocol 0902–002), and the University of Kentucky .® paper nesting material  or red polycarbonate boxes were added to the home cages for enrichment. Water and food (Purina 5001 Rodent Chow or Harlan 2016 or 2018 Rodent Diet) were provided ad libitum. Animals were allowed one week to acclimate to the facility after arrival without experimenter contact. Rats were handled on 2–3 separate days for 10 min before the start of data collection to habituate them to human contact.A total of 73 male, Sprague-Dawley rats  were tested at two experimental sites: the stimulus-response experiment and the pharmacological assessment in CCI rats were conducted at the VA Ann Arbor Healthcare System (n = 48); the pharmacological assessment in naive control rats was conducted at the University of Kentucky (n = 25). Rats were shipped at 275–300 g and housed as groups of 2–3 in filter‐top polycarbonate cages in a climate‐controlled vivarium maintained on an alternating 12‐hour light/dark cycle (lights on at 0700 hour). All procedures were conducted between 1000 and 1600 hours. Vivarium cage racks were equipped with water dispensers and forced ventilation. The top of the rack was enclosed to prevent direct room lighting from entering the cages to maintain rats’ innate photophobia. Home cages were filled with Sani‐Chips  to a depth of approximately 3 cm. Enviro-DriAll behavioral testing was conducted in quiet rooms maintained at approximately 22–24°C and normal humidity (30–50%). Room illumination was provided only via artificial means in the form of ceiling mounted fluorescent fixtures that generated an ambient illuminance of approximately 115 fc (foot‐candle) at work level. No other behavioral testing was performed during MCS test sessions. Rats underwent room acclimation for 30–60 minutes before the start of behavioral testing on each experiment day.As shown in An array of 500 stainless steel nociceptive probes was embedded onto an aluminum plate located below the floor of the probe compartment. The probe array was lowered below the compartment floor for training sessions and “no probe” 0 mm control conditions, and elevated in 0.5–1 mm increments above floor to a maximum height of 4 mm for nociceptive testing. Probes were positioned within approximately 1 cm of each other making it improbable that an animal could cross the probe array without at least a portion of two paws making contact with the probes at any time. The probes were tapered to a tip dimension of approximately .4 mm, making them sharp but unable to puncture or damage the plantar surface of the paw.Rats were placed individually into the light compartment with the light turned off and the escape door closed. No attempt was made to position animals in any particular orientation. Following 15 s of dark acclimation, the compartment light was turned on for the duration of the test. The escape door was opened 20 s thereafter. Latency to escape the light compartment was recorded using a stopwatch starting from the time the escape door opened until all four paws exited the light compartment. Failure to escape the light compartment within 20 s resulted in the light compartment escape door being closed and the rat being returned to its home cage. Rats that did not exit the light compartment were assigned an escape latency of 20 s for that trial. A 20 s escape cut-off was selected following preliminary studies showing that a majority of trained rats exited the light compartment within this timeframe. Time of entry into the dark compartment was recorded when all four paws entered the dark compartment. The dark compartment door was closed immediately following rat entry and the rat was returned to its home cage after 20 s of darkness. Rats that escaped the light compartment but failed to enter the dark compartment after 60 s  were returned to their homecage until the next trial. The test procedure was repeated three times per test session with a minimum of 10 minutes between trials. Mean escape latency from the light compartment was the primary variable of interest for analysis.Rats underwent apparatus familiarization and training without nociceptive probes prior to data collection. The familiarization procedure consisted of 1–4 sessions conducted over two one or two days in which rats were allowed to individually explore the entire MCS apparatus for five minutes. All compartment doors were open and the light compartment was illuminated during familiarization. A training procedure was then conducted so that the animals learned that they could escape the light compartment, cross the probe compartment, and gain access to the dark compartment. Training consisted of 3–5 sessions, of three trials each, conducted on consecutive days following the procedure described above. Only one session was conducted per day. This training procedure resulted in stable escape and crossing behavior.A stimulus-response assessment using naive control rats and CCI rats was conducted to determine the effect of different probe heights on escape behavior. Following training, rats underwent six test sessions of three trials each: five sessions with elevated probes  and one session without elevated probes (0 mm). This series of probes was selected from preliminary data that showed consistent crossing behavior at these heights and significant suppression of crossing behavior with probe heights greater than 4 mm. Test sessions were counterbalanced using a quasi-Latin square design that maintained the 0 mm and the 4 mm sessions at either the beginning or the end of the testing sequence. This design permitted evaluation of the effects of multiple test sessions on escape latency. Only one test session was conducted per day and sessions were separated by 24–72 hours. One to two hours prior to each test session, rats underwent one “pretest” trial without elevated probes. This pretest served to re-familiarize the animals to the MCS task. The order in which rats were removed from their home cage for testing was randomized on each test day.1 receptor antagonist, RP 67580 (1 and 10 mg/kg), on MCS escape behavior. To mitigate potential confounding effects of these compounds on the course of neuropathic pain, each drug was tested in a separate group of rats and the order of treatments  was randomized within each group. Treatments were separated by 1 week to allow for metabolic clearance. To streamline assessment of pharmacological effects, each drug was tested with the 3 mm probe condition as the standard noxious test stimulus. The 3 mm probe was chosen for two reasons. First, the Stimulus-Response Assessment indicated that escape latency at the 3 mm condition was significantly higher than at the 0 mm (no probe) condition (see CCI rats were used to assess the effect of morphine sulfate (2.5 and 5 mg/kg), pregabalin (10 and 30 mg/kg), and the tachykinin NKtion see ; Fig 2. The effect of drug treatments on normal MCS escape behavior was also assessed using naive control rats. Each session consisted of one pretest trial followed by three stimulus trials separated by 10 minutes each. Rats first underwent two testing sessions to establish baseline escape latencies in the absence of drug. Animals were randomly presented with either the 0 mm (no probe) condition or the 3 mm probe condition during the first session. The second session began at least one hour after the completion of session 1. In session 2, animals were tested with the opposite probe presentation from session 1. Two more sessions following the same procedure were then conducted on the same day to assess drug effects on escape behavior. Separate groups of rats received the highest dose of drug followed by testing with 0 or 3 mm probes, randomly assigned. One hour after the completion of this session, a second testing session was initiated. In this session, animals were presented with the opposite probe height. Crossover pharmacology was performed a minimum of 48 hours later, with animals who received drug now receiving vehicle, and vice versa. Again, probe height presentation order was randomized.1 receptor antagonist, RP 67580 [-Octahydro-2-[1-imino-2-(2-methoxyphenyl)ethyl]-7,7-diphenyl-4H-isoindol)], was purchased from Tocris Bioscience  and dissolved in diluted HCI and dimethyl sulfoxide  in sterile isotonic saline. All drugs or their vehicle were administered intraperitoneal (ip) in a volume of 1 ml. MCS testing began 20 min after morphine administration, 60 min following pregabalin administration, and 20 min following RP 67580 administration. Doses and dosing schedules were determined following literature review and pilot testing.Morphine sulfate and pregabalin were purchased from Sigma-Aldrich  and dissolved in a vehicle of sterile isotonic saline. The tachykinin NKA chronic constriction injury was produced by ligation of the left common sciatic nerve using a method similar to that originally described by Bennett and Xie  and prevMCS training occurred prior to CCI surgery and rats were given 7 days to recover after surgery before behavioral testing resumed. Successful implementation of the CCI technique to induce peripheral neuropathic pain was confirmed at 7–8 days post-surgery by the presence of mechanical hypersensitivity of the left hindpaw to a normally innocuous mechanical stimulus (5.07 g nylon monofilament) as described previously –35.p-value of < .05 was considered statistically significant.Stimulus-response data were analyzed by a mixed Analysis of Variance (ANOVA) using the General Linear Model (GLM) procedure. Probe height  was the within-subjects factor and experimental group (2 levels: naive and CCI) was the between-subjects factor; escape latency from the light compartment measured in seconds was the dependent variable. Pairwise comparisons between groups at every probe height were performed within the GLM, with Bonferroni adjustment, to determine the effect of CCI on escape latency. A one-way, repeated measures ANOVA was then performed separately on each group followed by Dunnett’s post hoc test. Post hoc analyses compared escape latency at each probe height to the no probe (0 mm) baseline condition. Planned comparisons using independent and paired t-tests (two-tailed) were used to determine the effect of multiple test sessions and/or drug administration on escape latency and probe compartment crossing time. Statistical outliers were identified using Grubbs’ method , 37. SPSp < .01); separate analyses conducted with this animal included in the dataset revealed no significant change in results. Multivariate analysis revealed a significant main effect of probe height  and a marginally significant interaction between probe height and group , suggesting that both groups exhibited changes in escape latency as a function of probe height  and group  indicating that naive and CCI rats exhibited different escape latency profiles. The probe height X group interaction was not significant in univariate analysis (p > .05).Stimulus-response functions were obtained for both naive and CCI rats (days 12–19 post surgery) in the MCS. A mixed ANOVA was conducted to assess whether there were group (CCI and naive) and probe height  differences in latency to escape the light compartment. One rat from the naive group was identified as a statistical outlier and excluded from analysis . Pairwise comparisons were performed to further explore group differences in escape latency. A comparison between groups collapsed across all probe heights revealed that CCI rats exhibited a significantly higher mean escape latency compared to naive rats . Additional pairwise comparisons between groups at every probe height indicated that CCI rats exhibited significantly higher escape latency at 4 mm compared to naive rats . A marginal increase in escape latency was also observed in CCI rats at other probe heights compared to the naive control group . To determine whether increased escape latency was related to a CCI-induced motor impairment, 0 mm escape behavior was assessed in a separate group of rats before and after CCI surgery. A slight yet non-significant increase of 3.02 (± 1.71) s in baseline escape latency was observed following CCI surgery , suggesting that CCI has negligible impact on locomotor activity in the MCS . Post-hoc comparisons with Dunnett’s test revealed that latency to escape the light chamber was significantly higher at the 3 mm (6.62 ± 1.54 s) and 4 mm (10.18 ± 2.65 s) probe heights compared to the 0 mm condition (1.58 ± 0.45 s), both ps < .05. A significant main effect of probe height was also observed in the CCI group . Post-hoc results indicated that compared to the 0 mm condition (4.30 ± 1.21 s), escape latency was significantly increased at 3 mm  and 4 mm .A one-way repeated measures ANOVA with Greenhouse-Geisser correction was conducted to assess the effect of probe height on escape latency within each group separately. In the naive group, results indicated a significant main effect of probe height on escape latency (Fp > .05). Likewise, escape latency did not differ between rats tested first at 4 mm (12.57 ± 2.08 s) and those tested last at 4 mm . These data suggest that the effect of probe height on light-induced escape is not influenced by multiple test sessions, testing order, or other learning effects. Furthermore, as expected, there was no evidence of tissue damage on the plantar surface of the paws, abdomen, face, or tail following multiple test sessions.The effect of multiple test sessions on escape latency was assessed by independent samples t-tests. As shown in The effect of systemic administration of antinociceptive compounds on escape latency was assessed in three separate groups of rats following MCS training and CCI surgery. Tests conducted in each drug group prior to drug administration confirmed that MCS escape latencies were consistent to those observed in CCI rats in the stimulus-response assessment for the 0 and 3 mm probe conditions. Drugs showing antinociceptive effects in the MCS were then evaluated in separate naive control animals to determine the effect of each compound on normal escape behavior and light-induced aversion.13 = 3.48, p < .01) and 30 mg/kg  as compared to vehicle treatment (15.15 ± 1.52 s). In contrast, pregabalin (30 mg/kg) had no effect on escape latency in naive rats at either 0 or 3 mm probe heights compared to vehicle (both ps > .05), suggesting that pregabalin does not affect normal escape behavior or aversiveness to bright light . Escape latency was not reduced following administration of 2.5 mg/kg morphine . As shown in 11 = 2.07, p = .063), and a significant increase at 3 mm . However, morphine did not increase probe compartment crossing time in naive rats suggesting that mobility was not affected by this treatment  or 10 mg/kg . Given the failure of RP 67580 to exhibit antinociceptive effects in CCI rats, it was not evaluated in naive rats.The effect of the NK1 receptor antagonist, RP 67580, on escape latency is depicted in The MCS is an operant method of evaluating the affective-motivational dimension of nociceptive behavior in rats. In this test, noxious mechanical stimuli in the form of sharp nociceptive probes obstruct an animal’s escape route from a brightly lit compartment to a dark compartment. This scenario is somewhat analogous to the rat’s natural environment were potentially painful mechanical stimuli such as claws, teeth, or botanical thorns can deter access to food and safety . The aniEscape from the light compartment was the primary behavior of interest in the present study. When nociceptive probes are not elevated (0 mm condition), the MCS escape response is guided by the principle of negative reinforcement. Thus, escape is reinforced by the termination of the light stimulus when the rat enters the non-illuminated probe compartment. The dark compartment, which is both darker than the probe compartment and includes a narrowed entryway, provides a desirable destination that strengthens this escape response. Pragmatically, this design encourages animals to not linger in the probe compartment or return back to the light compartment. The MCS thus leverages rats’ innate photophobia and preference for dark enclosed spaces. As such, little to no training is required for the MCS. However, some degree of training is often desirable as it reduces inter- and intra-rat variability and generates fast, stable escape responding. Training also serves to identify poor performers for additional training or elimination. Here, most animals learned the task after 2–3 training sessions and escape behavior remained consistent following multiple test sessions.The MCS escape response is modified when the nociceptive probe array is elevated and obstructs the escape route between compartments. Now the animal receives a punishment when it escapes and it learns that it can avoid this punishment by not escaping. But the motivational drive to escape the light still exists even when the probes are elevated. Hence, the probes create a motivational conflict in that the desire to escape into darkness is opposed by an aversion to noxious stimulation. Viewed another way, the nociceptive probes act as a form of “resistance” against the motivation to escape .escape or avoid a stimulus in conflict paradigms depends, in part, on the relative intensities of the stimulus being escaped and the punishment being avoided, as well as the motivational state of the organism [The decision to organism . For exaorganism , 26, 29.organism . Accordiorganism  increaseIt is unlikely that impaired mobility induced by the CCI procedure contributed to increased escape latency in CCI rats. Although decreases in locomotor activity have been observed following CCI , others In the present study, most animals choose to escape, suggesting that the motivation to escape the light was ultimately stronger than that to avoid probe contact. Nevertheless, animals required more time to make the decision to escape  when probe height or mechanical sensitivity was increased. The meaning of this finding and how it relates to human pain behavior isn’t entirely clear, but it may reflect a cognitive-evaluative dimension of pain. Although poor decision-making is reported in humans and animals with chronic pain –48, therEscape latency is one of several quantifiable behaviors in the MCS. For example, we previously assessed the amount of time spent on the nociceptive probe array during crossing as a direct measure of mechanical sensitivity . Spinal It was hypothesized that if mechanical hypersensitivity induced by CCI increased escape latency, then antinociceptive treatments that reduce CCI hypersensitivity would decrease escape latency. Pregabalin, and the structurally related compound gabapentin, reduce calcium-dependent release of pronociceptive neurotransmitters, including glutamate and substance P . PregabaThe effects of pregabalin and morphine on anxiety and mobility are essential considerations when evaluating their antinociceptive action. In the MCS, anxiety, expressed as fear of light, was inferred by examining baseline escape behavior in naive animals following drug administration. If a drug were anxiolytic, it would be expected to increase escape latency by reducing the aversiveness of light and thus the motivation to escape to the dark compartment. Similarly, locomotor deficits would also be evident by increased escape latency at 0 mm following drug administration. Our data show that escape latency in naive rats was virtually unchanged between vehicle and pregabalin conditions , suggestIn contrast, morphine increased escape latency in naive rats . This suUnlike pregabalin and morphine, RP 67580 failed to decrease MCS escape latency. RP 67580 binds with high affinity to the rat and mouse NK1 receptor –74 and iThese data offer preliminary evidence that the MCS escape response is a valid behavioral measure of nociception in rat. Many experimental questions remain. Validation studies with other pain models, noxious stimuli, rodent species/strains, and analgesics are required. Some of this work has begun , 87. HowThe MCS is an ethologically relevant operant method to assess the affective-motivational dimension of nociceptive behavior in rats. MCS escape behavior exhibits stimulus-response properties in both naive and neuropathic rats, remains stable over multiple test sessions, and is altered in a dose-dependent manner by antinociceptive treatments.S1 FigEscape behavior in the 0 mm (no probe) condition was assessed in a subset of rats (n = 18) from the Pharmacological Assessment study before and after CCI surgery. A non-significant increase in escape latency was observed following CCI surgery.(EPS)Click here for additional data file.S2 FigPanel A, n = 13) and morphine  do not alter the time spent crossing the probe compartment from the light compartment to the dark compartment in the presence of 0 or 3 mm probes.Pregabalin ((EPS)Click here for additional data file.


Although toxicities related to the mechanism of action may be readily detected, there is always a concern about the ‘hidden toxicities' that occur when a drug is combined with some other microenvironmental factor in animals or in the clinic. Wolenski et al.1 described a model of synergistic cytotoxicity between the investigational agent pevonedistat (MLN4924) and the inflammatory cytokine TNF-α. The toxicity between pevonedistat and TNF-α was confirmed across multiple cultured cell lines, primary hepatocytes, and in rats. These non-clinical findings led to an improved understanding of the potential for clinical toxicities with pevonedistat that can occur in patients with a pre-existing inflammatory state.2The ability to detect and monitor potential toxicities of new clinical candidates is an absolute necessity for any pharmaceutical agent before the clinic. This safety profile is established during pre-clinical development through κB transcription factor  signaling with selected small-molecule pharmaceuticals can trigger toxicity.6Stimulation of the TNF-receptor (TNFR) results in a range of pro-survival outcomes  that are all regulated by the NF-n factor .3 Howeve2 The role of NAE is to transfer NEDD8, an ubiquitin-like protein, to downstream substrates such as cullin-RING ligases (CRLs).7 Proteins ubiquinated by the CRLs are targeted for proteasome-mediated degradation, and impaired turnover of the DNA replication factor CDT1 leads to cell cycle arrest  that has been evaluated in clinical trials for the treatment of cancer.e arrest . In addiet al.1 identified that the combination of pevonedistat and TNF-α was cytotoxic both in vitro and in vivo. A hepatoma cell line became 50-fold more sensitive to TNF-α when dosed in combination with pevonedistat. Similarly, although rats tolerated the single agents alone, the combination resulted in liver damage. Additional cell-based characterizations found that pevonedistat and TNF-α specifically activated apoptosis, and inclusion of Z-VAD (a pan-caspase inhibitor) switched the mechanism of death to necroptosis (α when combined with Z-VAD (to block apoptosis) and Necrostatin-1 (to block necroptosis). Trimeric MLKL was also validated as a biomarker of active necroptosis, which is consistent with the literature.9The manuscript by Wolenski roptosis . Cells oα cell death.1 This was best illustrated through a knockdown of caspase-8 expression that prevented the pevonedistat and TNF-α synergistic cytotoxicity. Qualitatively, caspase-8 activity in the liver was also highest in rats that received the combination treatment. In cells, pevonedistat and TNF-α treatment preferentially resulted in the accumulation of the p10 protease subunit of caspase-8. This novel finding was interpreted as evidence for increased caspase processing/activation and not due to impaired protein degradation.A single protein, caspase-8, was the driver of pevonedistat and TNF-et al. investigated a known link between the CRL member cullin-3 and caspase-8 ubiquitination.11 However, there was no evidence that caspase-8 was ubiquitinated in the pevonedistat model.1 Knockdown of cullin-3 expression, which was hypothesized to make cells sensitive to TNF-α by mimicking pevonedistat, instead limited cell death caused by TNF-α. Thus, the exact mechanism of the toxicity requires additional work to fully characterize.There remains a gap in the understanding of how pevonedistat sensitizes cells to cytotoxic TNF signaling (illustrated by a ‘?' in 12 Although pevonedistat only activated necroptosis in cultured cells under specific conditions, the results from Wolenski et al. suggest that compounds that affect the TNFR pathway may have similar effects.The clinical findings with pevonedistat necessitated an investigative effort to establish a pre-clinical model. Two key lessons were learned from this effort. First, a pre-clinical screening strategy should be employed to identify potential synergistic cytotoxicities between compounds and pro-inflammatory agents. Second, attention needs to given to determine whether regulated necrosis pathways, such as necroptosis, can contribute to drug-induced toxicities. There are multiple types of regulated necrosis, all of which cause the dysregulation of the redox metabolome.13 It is relevant to the understanding of liver toxicity to determine what activates these pathways, how these triggers and pathways influence liver toxicity, and to develop a cell-health assessment strategy for pre-clinical testing. This might be accomplished through a high-content imaging assessment such as proposed by Xu;14 an in vitro screen such as described by Cosgrove;5 and in vivo models such as those developed by Roth.15 The objectives of all of these approaches is to identify and characterize the mechanisms that drive toxicity, and to define a set of comprehensive assays that improve early safety screening. Validation of these assays with existing compounds, such as pevonedistat, could lead to a better understanding of potential toxicities before compounds enter the clinic.From a broader perspective, the dysregulation of cell death is at the center of essentially every form of liver disease.


The mechanism by which trauma initiates healing remains unclear. Precise understanding of these events may define interventions for accelerating healing that could be translated to the clinical arena. We previously reported that addition of low-dose recombinant human TNF (rhTNF) at the fracture site augmented fracture repair in a murine tibial fracture model. Here, we show that local rhTNF treatment is only effective when administered within 24 h of injury, when neutrophils are the major inflammatory cell infiltrate. Systemic administration of anti-TNF impaired fracture healing. Addition of rhTNF enhanced neutrophil recruitment and promoted recruitment of monocytes through CCL2 production. Conversely, depletion of neutrophils or inhibition of the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death. Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair. If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization. Therefore, it is critical to understand how the early inflammatory response initiates and orchestrates fracture repair, an area that remains poorly understood. A competent immune system is essential for effective wound healing, although excessive inflammation has been found to be deleterious. For example, the absence of TNF or IL-6 signaling has been found to impair fracture healing  following osteotomy and found that rhTNF was effective in augmenting fracture healing, as indicated by increased relative % callus mineralization, by day 28 after injury only at 1 ng , which also inhibits TNF expression and release   as shown by the representative sections in FigOur observation that TNF inhibition impaired fracture healing  site Fig–G to enaphils Fig. The neuphils Fig. From daFig+) Fig. NeutropAs recruitment of neutrophils represents a key early event during the inflammatory response Nathan, , we inveet al, Next, mice were treated with a validated and specific Ly6G-blocking antibody  and monocytes/macrophages   Fig.+ cells into the air pouch model by TNF was abrogated by the addition of CCL2 neutralizing antibody   and sIL-6R, the main neutrophil-derived chemokines responsible for monocyte recruitment  mobilized to the cell surface upon cell activation , it has also been shown to be important in the recruitment of neutrophils . Skeletally mature (12–14 week old) female C57/BL6 mice were obtained from Charles River, UK. Normal chow and drinking water were provided Anti-TNF: TN3-19.12, a neutralizing hamster IgG1 anti-TNF-α/β mAb (Leinco Technologies) or IgG control was given (1 mg) via intraperitoneal injections on days 0, 2, 5, and 8 after operation.rmIL10 (R&D Systems) was injected locally at the fractures site at 10 ng on days 0 and 1; PBS was used as vehicle control.Anti-IL10R or IgG isotype control (R&D Systems) was given (2 μg) via intraperitoneal injections on day 1 before operation and days 0, 1, 4, 6, and 8 after operation.2A isotype control, 2A3 (BioXCell) , 0.01 ng (n = 7), 1 ng (n = 6), 100 ng (n = 8)Fign = 9), D0 + 1 (n = 6), D0 (n = 10), D1 (n = 7), D2 (n = 8), D3 (n = 6)Fign = 10), anti-TNF (n = 6), rmIL-10 (n = 8)Fign = 6), 0.25 (n = 6), 0.5 (n = 12), 1 (n = 9), 3 (n = 10), 6 (n = 6), 12 (n = 6), 24 (n = 10), 72 (n = 6)Fign = 8), Anti-Ly6G (n = 9); (right) n = 3 per groupFign = 8), Anti-Ly6G (n = 9)Fign = 3) per groupFign = 11) media + TNF (n = 12), FS (n = 8), FS + TNF (n = 11), FS + TNF + anti-CCL2 (n = 8), FS + TNF + IgG (n = 8)Fign = 6), media + TNF (n = 6), FS (n = 6), FS + TNF (n = 6)Fign = 3 per groupFign = 6), CCR2 antagonist (n = 7)Fign = 6), TNF (n = 6), Day 28 PBS (n = 7), TNF (n = 7)FigrmCCL2 (R&D Systems) was injected locally at the fracture site 10 ng or 100 ng on day 0 and day 1; PBS was used as vehicle control.3, as previously described , 50 kV, 800 μA, and 8.3 μm isometric voxel resolution, and 0.7 degree rotation step. Bones exhibiting angulation, rotation, or hypertrophic non-union were excluded. Scans were analyzed using SkyScan CTAnalyser software, version 1.9.3.0 . Each bone was analyzed by selecting a volume of interest commencing at the fracture site and proceeding proximally for 363 slices (3 mm in height). This region was selected to avoid potential confounding effects of the fibula. Total callus volume was delineated from surrounding tissues and the original bone of the tibia using hand-contoured regions of interest. Mineralized callus volume was determined using global thresholding at a density range calibrated to bone mineral density of 350 gm/cmMurine lower limbs were fixed in 4% paraformaldehyde overnight and decalcified for 6 weeks in a 50% mixture of 20% EDTA and 4% paraformaldehyde. Care was taken during processing and section cutting to avoid RNase contamination.Tnf mRNA was accomplished by in situ hybridization using an antisense riboprobe synthesized with T7 RNA polymerase using 35S-UTP  and IMAGE clone 40126376 (Source BioScience UK) containing the sequence of interest. A clone was grown in the presence of kanamycin, purified using a Qiagen maxi prep kit, and the resultant plasmid linearized with SpeI to prepare a template that made a 768 base antisense riboprobe that was used without hydrolysis. The region of sequence used to produce the riboprobe did not show significant homology to any other known mouse mRNA sequences in the RefSeq.Specific localization of et al, The methods for pre-treatment, hybridization, washing, and dipping of slides in Ilford K5 for autoradiography were performed as previously described  before developing in Kodak D19 and counterstaining by Giemsa's method. Sections were examined under conventional or reflected light dark-field conditions (Nikon Eclipse ME600 epi-illumination microscope with Q imaging MicroPublisher 5.0 camera) that allowed individual autoradiographic silver grains to be seen as bright objects on a dark background.et al, Monocytes/macrophages: Limbs were fixed in 4% paraformaldehyde overnight and decalcified for 6 weeks in 15% EDTA. The limbs were bisected longitudinally and paraffin-embedded, and 4-μm sections were cut. Immunohistochemistry (IHC) was performed on deparaffinized and rehydrated sections, with specific primary antibody against F4/80 (rat anti-mouse (Biolegends)), as previously described .et al, et al, et al, +, CD11b+ cells, while the monocytic cells were identified as Ly-6G−, CD11b+, and CD115+ cells as described previously  were used. Air pouch model procedures were approved by the institutional ethics committee and the United Kingdom Home Office (PLL 70/7335). Air pouches were produced as previously described  is added at 30 min. Cells and supernatants are harvested at 1 h. Protein levels of mCCL2 cytokine in the supernatants were analyzed as described above. Experiments were performed in triplicates.Neutrophils are enriched to 82.7% purity  or fracture supernatant + rhTNF at 1 ng/ml (treatment). After 4 h, cells were fixed with 4% paraformaldehyde in PBS for 15 min at 37°C, quenched with 50 mM NHBone factures are very common. While the majority heal uneventfully, certain sub-groups would benefit from strategies to accelerate healing. These include high-energy open fractures, which are limb-threatening injuries prone to delayed and non-union, and fragility fractures in osteoporotic bone. The rates of recovery and mobilization are limited by the fracture healing time in patients with fragility fractures, thus accounting for the excessive morbidity and mortality seen in this vulnerable group of patients. There is currently no approved therapy for accelerating healing of fragility fractures.in vivo and in vitro approach, we demonstrate the importance of the early innate inflammatory response in the healing of bone fractures. Local administration of TNF, a pro-inflammatory cytokine, within 24 h of injury accelerates fracture repair in vivo. Conversely, administration of either anti-TNF or rm-IL-10, an anti-inflammatory cytokine, impairs fracture healing. TNF is expressed by both neutrophils and monocytes/macrophages following fracture. Addition of recombinant TNF to the fracture environment up-regulates the innate immune response by promoting the recruitment of neutrophils followed by monocyte/macrophages, the latter through the up-regulation of the monocyte chemoattractant, CCL2. Depletion of neutrophils impairs fracture repair in vivo. While the production of CCL2 by neutrophils is controversial in the literature, we demonstrate enhanced expression of CCL2 in neutrophils exposed to fracture environment following addition of TNF using immunocytochemistry. Macrophages are known to play a key role in orchestrating endochondral bone repair. Inhibition of CCR2, the main receptor for CCL2, found on monocytes/macrophages, led to significant impairment of fracture repair in vivo. These data demonstrate the role of the innate immune response via the TNF/CCL2/CCR2 axis in fracture repair.Using a combined in vivo is of particular interest as clinical translation would address one of the major unmet medical needs of the 21st century.This is the first direct evidence on the importance of the innate immune response in fracture healing and how it may be targeted to accelerate fracture repair. Our observation that this strategy pertains to osteoporotic bone t-tests. Multiple comparisons were made using one-way analyses of variance (ANOVA) followed by Bonferroni or Dunnett post-test analysis for multiple groups. Exact P-values are provided for t-tests, and multiplicity-adjusted P-values are provided for multiple comparisons. P < 0.05 are considered statistically significant. Significant results are expressed using asterisks, where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Statistical analyses were performed using GraphPad Prism version 6 . For every figure, statistical tests are justified as appropriate. All data were expressed as mean ± standard error of the mean (SEM). The significance of the difference between two groups was determined with a one- or two-sided unpaired


The aim of this study was to investigate the potential predictors of switching tumor necrosis factor (TNF)-α inhibitors in Korean patients with ankylosing spondylitis (AS). The patients who had been treated with TNF-α inhibitors were divided into two groups depending on whether they had switched TNF-α inhibitors. Demographic, clinical, laboratory, and treatment data at the time of initiation of TNF-α inhibitor treatment were compared between switchers and non-switchers, and within switchers according to the reasons for switching. Of the 269 patients, 70 (23%) had switched TNF-α inhibitors once; of these, 11 switched again. The median follow-up time was 52.7 months. Three- and five-year drug survival rates were 52%/48% for infliximab, 62%/42% for etanercept, and 71%/51% for adalimumab, respectively. Switchers were more likely to be prescribed disease-modifying anti-rheumatic drugs than non-switchers. A history of joint surgery and complete ankylosis of the sacroiliac joint was more frequent in switchers. Multivariate Cox’s proportional hazard analysis showed that the use of adalimumab as the first TNF-α inhibitor was less likely to lead to switching and complete ankylosis of the sacroiliac joints was more likely to lead to switching. The principal reasons for switching were drug inefficacy and adverse events, but the differences in the clinical data of these two groups of switchers were not significant. In AS patients who are candidates for TNF-α inhibitor therapy, switching may improve the therapeutic outcome based on clinical information. Ankylosing spondylitis (AS) is an inflammatory rheumatic disorder mainly affecting the axial skeleton. Inflammation of the sacroiliac joint, spine, and entheses is the main characteristic of AS and it eventually leads to ankylosis of the affected joint. Tumor necrosis factor (TNF)-α inhibitors are a major advance in the treatment of AS and their efficacy has been proven in several randomized controlled trials –3. HowevIn Korea, infliximab, etanercept, and adalimumab are the commercially available TNF-α inhibitors. Etanercept for patients with AS was approved by the Korean Health Insurance Review & Assessment Service (HIRA) in May 2005, followed by infliximab in November 2006 and adalimumab in April 2007. Head-to-head trials comparing the safety and efficacy of these three TNF-α inhibitors are lacking but their efficacies are considered to be similar. Infliximab is an IgG1 chimeric monoclonal antibody with its Fab portion derived from mouse; it is administered by intravenous infusion. Both etanercept, a recombinant 75-kDa TNF receptor IgG1 fusion protein, and adalimumab, a humanized monoclonal antibody, are given by subcutaneous injection. The different molecular structures and routes of administration of these three drugs could influence both their efficacies and their association with adverse events in patients receiving them. Accordingly, patients who do not respond to a TNF-α inhibitor or suffer an adverse event during its use may benefit by switching to one of the other inhibitors.For ethical reasons, the switching rate of TNF-α inhibitors and its effects cannot be investigated through randomized placebo-controlled studies. However, the many nationwide drug registries, such as BIOBADASER , BSRBR (The British Society for Rheumatology Biologics Registers), DANBIO , and NOR-DMARD (Norwegian DMARD register) provide the basis for observational studies. To date, there is no well-organized registry of similar information in Asia. Therefore, in this work our primary objectives were: (1) to analyze the switching percentage, the order of switching, and the clinical characteristics of AS patients in Korea who had switched TNF-α inhibitors and 2) to identify the predictors for switching TNF-α inhibitors.This retrospective cohort study was conducted in a single tertiary center. Patients diagnosed with AS according to the modified New York Criteria  and in wPatients in whom standard treatment failed and who had active disease were eligible for TNF-α inhibitor therapy, according to the Korean HIRA guideline based on ASAS consensus statement for the use of an anti-TNF agent for AS . The faiThe term "inefficacy" defined as an improvement of less than 50% or as a score of more than 2 in BASDAI 3 months after the initiation of treatment and every 6 months thereafter. Inefficacy included a lack/loss of effect of a TNF-α inhibitor. "Switchers" denoted the group of patients who switched from the prescribed TNF-α inhibitor to one of the other two inhibitors. "Non-switchers" denoted the group of patients who either continued with the first TNF-α inhibitor or discontinued the first TNF-α inhibitor but did not receive any other biological agent.To identify predictors for switching, patients were divided into two groups according to whether they had switched TNF-α inhibitors. The reasons for switching considered in this study were occurrence of an adverse event and inefficacy of treatment. Medical records were reviewed for information on patient age, body mass index (BMI), familial history, surgical history, marital status, education level, smoking status, and alcohol consumption at the time of initiation of TNF-α inhibitor treatment. Physical examination, laboratory findings, and imaging findings were reviewed at the time of treatment initiation and 3 months thereafter. BMI was calculated as the body weight in kilograms divided by the square of the height in meters. Peripheral arthritis was based on the clinical finding of one or more swollen and tender joints. Sacroiliitis was graded using plain radiographs according to the New York criteria : grade 02 test or Fisher’s exact test. Univariate and multivariate Cox regression analyses with hazard ratios (HRs) were used to identify factors predicting TNF-α inhibitor switching. P < 0.05 was considered to indicate statistical significance.Data processing and statistical analyses were performed using SPSS version 21.0 . Continuous variables are presented as the medians and interquartile range and were compared using a Mann-Whitney U-test. Categorical variables were analyzed using a χAll 269 patients with AS who participated in the study had been administered at least one TNF-α inhibitor. The median age of the patients was 40 years (range: 19–73) and 218 (81%) were males. The median duration of follow-up was 57.2 months (range: 3–143 months). Three- and five-year drug survival rates were 52%/48% for infliximab, 62%/42% for etanercept, and 71%/51% for adalimumab, respectively .In the case of the first TNF-α inhibitors, 70 of 269 (26%) patients were switchers and 199 of 269 (74%) patients were non-switchers. In the case of second TNF-α inhibitors, 11 of 70 (15.7%) were switchers and 59 of 70 (84.3%) were non-switchers. In the case of third TNF-α inhibitors, no patient who switched their TNF-α inhibitors has been identified to date. Among the 199 (74%) patients who did not switch first TNF-α inhibitors (non-switchers), 150 continued with the first inhibitor and 49 completely discontinued their use. Among 49 patients who discontinued TNF-α inhibitors, 34 did so due to low disease activity, 4 due to pregnancy and breast-feeding, and 2 due to economic issues. In total, nine patients discontinued their TNF-α inhibitors due to adverse events: six experienced mild injection reaction, and they maintained low disease activity even after discontinuation, two patients were discontinued due to pulmonary tuberculosis, and one patient discontinued due to congestive heart failure.Infliximab was the first TNF-α inhibitor used in the majority of patients , followed by etanercept  and adalimumab . Of the switchers, 38 patients (54.2%) were initially on infliximab, 26 (37.1%) were on etanercept, and 6 (8.5%) were on adalimumab. For the second TNF-α inhibitor, 13 (18.5%) patients were administered infliximab, 12 (17.1%) received etanercept, and 45 (64.2%) were treated with adalimumab . The mosp = 0.010 and p = 0.013). In the baseline laboratory findings, median erythrocyte sedimentation rate and C-reactive protein levels were higher in switchers than in non-switchers (p = 0.038 and p = 0.002). Methotrexate and sulfasalazine as well as DMARDs were more frequently prescribed to switchers  and the median prescribed dose of prednisolone was higher (p = 0.022).Demographic, socioeconomic, clinical, laboratory, and treatment data at the time of TNF-α inhibitor initiation were compared between switchers and non-switchers . The firp = 0.044) (p = 0.005). The same was true for patients with uveitis. C-reactive protein levels tended to be lower in the group with adverse event. We also had attempted to divide the ‘inefficacy’ group into ‘lack of efficacy’ and ‘loss of efficacy.’ Lack of efficacy was defined as initially not responding to TNF-α inhibitors within 3–4 months. Loss of efficacy was defined as an initial response to TNF-α inhibitors and a secondary loss of efficacy over 6 months. According to this definition, only 4 patients were allocated to the lack of efficacy group and 41 to the loss of efficacy group. When we analyzed the data of only the loss of efficacy group, there was no difference from the original result.The principal reasons for switching were drug inefficacy  and the occurrence of adverse events , with infusion reaction (13 patients) being the most common among the latter. The median dosage of methotrexate at the time of TNF-α inhibitor initiation was higher in the group of patients who had switched inhibitors because of drug inefficacy than in the group reporting adverse events  . Among tMultivariate Cox’s proportional hazard models were used to assess the reliability of the switching predictors identified . The usep = 0.030 and 26.9% vs. 50.0%, p = 0.003). Median C-reactive protein levels were also higher in the inefficacy group (p = 0.001). Methotrexate and any DMARD were more frequently prescribed to patients in the inefficacy group than to those in the non-switchers group (p = 0.014 and p = 0.001). Complete ankylosis of the sacroiliac joint was more likely to lead to switching in the inefficacy group, based on an analysis of the data by multivariate Cox’s proportional hazard analysis . These results are similar to those between switchers and non-switchers.The group of switchers due to drug inefficacy was compared with the group of non-switchers. The percentages of patients who underwent joint surgery before starting their first TNF-α inhibitor and of patients with complete ankylosis of the sacroiliac joint were higher in the inefficacy group than in the non-switchers group  . French Infliximab was the most frequently used agent among the three TNF-α inhibitors considered in this study, followed by adalimumab and etanercept. The most common sequence of switching was infliximab to adalimumab. Infliximab was the first TNF-α inhibitor introduced for the treatment of RA in Korea. However, it had been used for the treatment of AS even before receiving the approval of the Korean HIRA for this purpose. The early off-label use of infliximab probably explains why patients with more severe AS were initially treated with infliximab than with the other two TNF-α inhibitors. Accordingly, preference and accessibility may influence the decision to switch from infliximab to another agent. We analyzed baseline characteristics according to TNF-α inhibitor, additionally. Age, ESR, and CRP, at the time of initiation of TNF-α inhibitors, were highest in the infliximab group. Disease duration was longest in infliximab group but there was no statistically significant difference among the three agents. Concomitant uses of DMARDs and MTX were lowest in the adalimumab group.In several reports, TNF-α inhibitors were discontinued because of the development of anti-drug antibodies, which occurs more often with infliximab than with adalimumab. This difference can be explained by the structures of these inhibitors. Infliximab, a chimeric monoclonal antibody, has a murine component and therefore a greater likelihood of inducing anti-drug antibodies than adalimumab, a humanized monoclonal antibody . BecausePatients treated with adalimumab as the first TNF-α inhibitor were less likely to switch to another inhibitor. In the BIOBADASER registry, the use of adalimumab was a protective factor for drug discontinuation related to an adverse event in patients with RA and spondyloarthropathy . AlthougIn the current study, patients who had complete ankylosis of the sacroiliac joint when TNF-α inhibitor treatment was initiated were likely to switch to another inhibitor. This association between grade of sacroiliitis and likelihood of switching is a novel finding of our study. The radiologic response of patients with AS to TNF-α inhibitors can be measured using the Ankylosing Spondylitis spine Magnetic Resonance Imaging-activity (ASspiMRI-a) system, the Berlin modification of ASspiMRI-a and of the Spondyloarthritis Research Consortium of Canada (SPARCC) scoring system. These scoring systems use magnetic resonance imaging to measure vertebral inflammation . In our There are several potential limitations to the interpretation of our data. The first is the study design. As ours was an observational study, the choice of TNF-α inhibitors was not randomized but was instead affected by factors such as physician preference, concerns associated with the introduction of a novel agent, and the route of drug administration. The second limitation is the nature of our patient series. Patients were selected from a single tertiary center located within the local community. Thus, the patients’ living standards and their ability to access the center may have biased enrollment to favor patients with more severe disease. Third, inefficacy could not be further distinguished as lack of effect vs. loss of effect. Fourth, we did not measure anti-drug antibody levels to predict therapeutic response in these patients. Finally, we did not measure functional status of patient such as Bath Ankylosing Spondylitis Functional Index.In conclusion, 70 (26%) of the 269 AS patients enrolled in our study had switched TNF-α inhibitors during the 12 years of follow-up. The use of adalimumab as the first TNF-α inhibitor was less likely to lead to switching and complete ankylosis of the sacroiliac joint at the time of TNF-α inhibitor initiation was more likely to lead to switching. In AS patients who are candidates for TNF-α inhibitor therapy, switching is considered to be an important treatment option, which highlights the different molecular structures and routes of administration of TNF-α inhibitors.S1 File2 test (Table G).Survival curves of the three TNF-α inhibitors . Survival rates of the three TNF-α inhibitors (Table A). Predictors of TNF-α inhibitor switching excluding the 9 patients who discontinued TNF-α inhibitors due to adverse events (Table B). Univariate logistic analysis used to evaluate the predictors of complete discontinuation of TNF-α inhibitors (Table C). Comorbid conditions of patients according to whether they subsequently switched or not (Table D). Cox proportional regression analysis used to evaluate predictors of TNF-α inhibitor switching after adjusted for age, gender, disease duration, and BASDAI using propensity score matching (Table E). Baseline characteristics and laboratory findings of patients starting their first TNF-α inhibitor according to TNF-α inhibitor using ANOVA test (Table F). Baseline characteristics and laboratory findings of patients starting their first TNF-α inhibitor according to TNF-α inhibitor using χ(PDF)Click here for additional data file.


We show that although never laminar, the tuning of DS-RGC axons targeting the mutant tectum is normal. Analysis of mutant tectal neurons at late developmental stages reveals that directional tuning is indistinguishable from wild-type larvae. Furthermore, we show that structural plasticity of tectal dendrites and RGC axons compensates for the loss of lamination, establishing connectivity between DS-RGCs and their normal tectal targets. However, tectal direction selectivity is severely perturbed at earlier developmental stages. Thus, the formation of synaptic laminae is ultimately dispensable for the correct wiring of direction-selective tectal circuits, but it is crucial for the rapid assembly of these networks.A common feature of the brain is the arrangement of synapses in layers. To examine the significance of this organizational feature, we studied the functional development of direction-selective (DS) circuits in the tectum of  •Robo2 directs lamination of direction-selective retinal axons and tectal dendrites•Tectal lamination is required for rapid assembly of direction-selective circuits•Functional direction-selective circuits eventually form when lamination is lost•Structural plasticity compensates for the loss of tectal lamination A feature of the brain is the arrangement of synapses in layers. The significance of this architecture is unknown. Nikolaou and Meyer show that lamination is ultimately dispensable for circuit function but that it is crucial for rapid circuit development. The majastray (astti272z) mutant zebrafish in which the laminar arrangement of retinal inputs to the tectum is severely disrupted. The astti272z allele is a functional null mutation of robo2—part of an evolutionarily conserved group of transmembrane proteins that, together with their Slit ligands, have been shown to be important for long-range axon guidance, growth, and targeting  connections between RGCs and tectal neurons affected when lamination of RGC axons is lost? To answer this, we took advantage of the argeting . Targetif Slit1a . The funastray (astti272z) mutant. Therefore, before determining how tectal circuits devoted to processing of motion were altered in animals with disrupted laminar input, we first determined whether direction-selective circuits are established correctly in the mutant retina. We did this by generating wild-type (WT) and astti272z larvae expressing a presynaptically targeted, genetically encoded reporter of neural activity specifically in RGCs ), and recording visually evoked activity in presynaptic terminals of RGC axons within the intact tectum  larvae while visually evoked responses were imaged in the contralateral tectum , which is approximately 24 hr after the first RGC axons enter the tectum, and 7 dpf when synapses between RGC axons and tectal dendrites and visually evoked behaviors are well established (astti272z larvae imaged at 4 and 7 dpf). These reveal distinct distributions of preferred angles that are strikingly similar in WT and mutant tecta , 43% (downward motion), and 38% (forward motion). At 7 dpf, the fraction is 44% (upward motion), 54% (downward motion), and 46% (forward motion)  A–S3F. Thastti272z mutants expressing GCaMP5G in the majority of neurons in the brain (Tg(elavl3:GCaMP5G) (R2 > 0.8) were applied to all visually responsive voxels followed by automatic aggregation of similarly tuned voxels into cell body-sized units (see astti272z larvae imaged at 4 and 7 dpf). Surprisingly, cumulative histograms reveal almost identical distributions in the preferred angles of tectal cells in WT and astti272z larvae at 7 dpf .  The reduction in DS tectal cell number is not due to delayed proliferation or differentiation, because the numbers of mitotic (PH-3+) and postmitotic (HuC/D+) cells in the developing astti272z tectum at 48 hpf are comparable to those found in WT animals   (Movies GCaMP5G)  and S4. GCaMP5G) A–S4F. DSnits see . The resat 7 dpf B and 3D,at 7 dpf  are presz larvae H.  a change in functional fate, in which tectal cells that are not fated to become DS maintain their morphology in the mutant tectum but form synapses indiscriminately with the misplaced DS-RGC axons and, in so doing, adopt directional tuning , and a higher proportion of them possess diffuse dendritic arbors (>16 μm extent in laminar axis) (astti272z: 84%). Thus, dendritic lamination is lost but tuning properties of tectal cells targeted by the FoxP2.A:Gal4FF are unaltered in the mutant. These data suggest a model of functional recovery in which structural plasticity in the mutant tectum establishes functional connections between DS-RGCs and their normal tectal targets. It is interesting to note that tectal cells tuned to posterior motion and some vertically tuned tectal neurons also have laminar dendrites and that this lamination appears to be lost in the mutant tectum. This suggests that structural plasticity perhaps ensures appropriate functional wiring of multiple tectal neuron subtypes when lamination cues are lost , allowing us to perform functional imaging of the tectal population receiving input from the transplanted eye at 4 dpf and then again at 7 dpf . These findings indicate that Robo2 expression is required in both RGCs and tectal neurons for the rapid (by 4 dpf) development of DS retinotectal connections. However, at 7 dpf, the number of DS tectal cells in the two experimental groups match those found in the control, WT-to-WT group . We show that Robo2-positive tectal cells receiving input from Robo2-negative RGC axons have laminar dendrites  and their synaptic partners—the intrinsically photosensitive RGCs (ipRGCs)  . However(ipRGCs) . Becausee neuron . The losobserved  is likelDrosophila, suggesting that an evolutionarily conserved role for Slit-Robo signaling as a global neuropilar organizer  of the retina have implicated homophilic and heterophilic cell-surface molecules on axons and dendrites in synaptic partner recognition. For example, members of the immunoglobulin-domain-containing superfamily  have been identified as being important elements of a cell-surface recognition code that ensures precise connectivity in the chick and mouse retina zc60, Tg(UAS:SyGCaMP3)kg1 and Tg(elavl3:GCaMP5G)a4598 have been previously described  together with GCaMP6F or tdTomato effector plasmids, as previously described . See also .Conceptualization, N.N. and M.P.M.; Methodology, N.N. and M.P.M.; Investigation, NN; Formal Analysis, N.N. and M.P.M.; Writing – Original Draft, N.N. and M.P.M.; Writing – Review & Editing, N.N. and M.P.M.; Funding Acquisition, M.P.M.