Tumor necrosis factor-alpha (TNF-α) is elevated early in injured brain after traumatic brain injury (TBI), in humans and in animals. Etanercept (a TNF-α antagonist with anti-inflammatory effects) attenuates TBI in rats by reducing both microglial and astrocytic activation and increased serum levels of TNF-α. However, it is not known whether etanercept improves outcomes of TBI by attenuating microglia-associated, astrocytes-associated, and/or neurons-associated TNF-α expression in ischemic brain. A well clinically relevant rat model, where a lateral fluid percussion is combined with systemic administration of etanercept immediately after TBI, was used. The neurological severity score and motor function was measured on all rats preinjury and on day 3 after etanercept administration. At the same time, the neuronal and glial production of TNF-α was measured by Immunofluorescence staining. In addition, TNFα contents of ischemic cerebral homogenates was measured using commercial enzyme-linked immunosorbent assay kits.In addition to inducing brain ischemia as well as neurological and motor deficits, TBI caused significantly higher numbers of microglia-TNF-α double positive cells, but not neurons-TNF-α or astrocytes-TNF-α double positive cells in the injured brain areas than did the sham operated controls, when evaluated 3 days after TBI. The TBI-induced cerebral ischemia, neurological motor deficits, and increased numbers of microglia-TNF-α double positive cells and increased TNF-α levels in the injured brain were all significantly attenuated by etanercept therapy.This finding indicates that early microglia overproduction of TNF-α in the injured brain region after TBI contributes to cerebral ischemia and neurological motor deficits, which can be attenuated by etanercept therapy. Studies in this model could provide insight into the mechanisms underlying neurological motor disturbance in brain-injured patients. Tumor necrosis factor-alpha (TNF-α) is elevated in both the serum and cerebrospinal fluid of humans after traumatic brain injury (TBI) ,2. It isEtanercept is a TNF antagonist with anti-inflammatory effects. Systemic administration at the dosage of 5 mg/kg of body weight improves outcomes of TBI in rats by reducing microglial and astrocytic activation and activated inflammation  . Again, We explored these two questions by studying the interrelationship between the activation of microglia-TNF-α, astrocyte-TNF-α, and neuron-TNF-α double labeling cells in the ischemic brain areas and the development of cerebral ischemia and neurological and motor dysfunction as well as the attenuating of cerebral ischemia and neurological and motor dysfunction by etanercept therapy after TBI.ad libitum. All protocols, designed to minimize discomfort in the animals during surgery and in the recovery period, were approved by the Institutional Animal Care and Use Committee of Southern Taiwan University of Science and Technology.Male Sprague–Dawley rats (280–300 g), obtained from the Animal Resource Center of the Taiwan National Science Council, were housed four per group at an ambient temperature of 22 ± 1°C with a 12-h light–dark cycle. Pelleted rat chow and tap water were available The rats were anesthetized with sodium pentobarbital  and a mixture containing ketamine , atropine , and xylazine  . Both the femoral artery and vein on the right side were cannulated with PE50 polyethylene tubing to monitor blood pressure and analyze blood gas respectively. After cannulation, the wound was sutured and the rats were placed in a stereotaxic frame. Each rat’s scalp was sagittally incised, and then the rat was subjected to a FPI ,12. PrioThe rats (n=96) were randomly allocated into the following groups: 1) FPI+saline control group (n=32): rats were subjected to FPI plus a dose of normal saline (1 ml/kg body weight [i.p.]) once every 12 h for 3 consecutive days ; 2) FPI+In experiment 1, etanercept (5 mg/kg [i.p.]) (n=8) or saline (1 ml/kg [i.p.]) (n=8) was randomly administrated immediately after FPI, and their effects on neurological motor performance were assessed preinjury and on 4 days post-FPI. Another 8 untreated sham-operated rats were used as controls. Etanercept  was reconstituted with normal saline according to the manufacturer’s instructions.In experiment 2, three days after FPI, immediately following the neurological motor tests, all the rats used in experimental 1 were killed 3 days after FPI for brain contusion assessment.In experiment 3, etanercept (5 mg/kg [i.p.] (n=8) or saline (1 ml/kg [i.p.] (n=8) was randomly given to each rat immediately after FPI once every 12 h for 3 consecutive days, and the effect on the number of the co-localization of TNF-α and microglia specific marker cells, TNF-α and neurons specific marker cells, and TNF-α and astrocyte specific marker cells in the cortex, white matter, hippocampus, or hypothalamus was determined. Untreated sham group, FPI+saline group, and FPI-etanercept group were killed 72 h post-FPI (n=8 for each group) for immunohistological evaluation.In experiment 4, untreated sham group (n=8), FPI+saline group (n=8), and FPI+etanercept group (n=8) animals were killed 72 h post-FPI for determination of TNF-α protein level in ischemic brains.Acute neurological injury was assessed in all rats on the day prior to and on 3 days after surgery using a neurological severity score (NSS) . NSS is The inclined plane was used to measure limb strength . The rat3 25; EDTA 0.026; and CaCl2 2.5) containing 5 mM glucose. Segments of cerebral cortex  were weighted, cut into small pieces, dispersed by aspiration into a pipette and suspended in 1 ml of physiological salt solution in a test tube. Samples were kept on wet ice for 20 min before use. The homogenates were centrifuged at 7,500 rpm  for 20 min. The supernatants were used for measuring TNF-α concentrations. TNF-α concentrations were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits  and following the manufacturer’s instructions. The minimum detectable concentrations of TNF-α were 1.1 pg/ml. There was no cross-reactivity reported with other cytokines. All samples were assayed is duplicate.Cerebral hemispheres were quickly dissected free and kept on ice in physiological salt solution  staining procedures are described elsewhere . Three dSerial 50-μm sections corresponding to coronal coordinates 0.8 mm to 5.3 mm posterior to the bregma were incubated in 2 mol/L HCL for 30 min, rinsed in 0.1 mol/L boric acid (pH 8.5) for 3 min at room temperature and then incubated with primary antibodies in phosphate-buffered saline (PBS) containing 0.5% normal bovine serum at 4°C overnight; secondary antibodies-incubated for 1 h at room temperature. The antibodies therein were, sequentially, mouse monoclonal anti-NeuN , mouse monoclonal anti-GFAP antibody , mouse monoclonal anti-Iba-1 antibody , goat polyclonal anti-TNF-α antibody , DyLight 488-conjugated donkey-anti-goat IgG antibody , and Alexa Fluro 568-conjugated donkey anti-mouse IgG antibody . The sections were coverslipped with the mounting medium . The labeled cells were calculated in 5 coronal sections from each rat and expressed as the mean number of cells per section. For negative controls sections, all the procedures were without the primary antibody. Primary and secondary antibodies for multiple-staining are listed in Table Images of the fluorescent immunohistochemistry for immune cells were captured at 100x magnification using a fluorescence microscope system , and images from bregma −0.8, -1.5, -2.5, -3.0, and −3.5 mm from each animal were evaluated. In each image, immune-positive cells showing staining with a cellular morphology and above background level were manually and exhaustively counted using the Axiovision image analysis software (Zeiss Gmbh), All cell counts were performed by an investigator (C. P.C.) blinded to the treatment status of each animal.post hoc multiple comparisons among means. Analyses for all behavioral variables used Student’s unpaired t-test to compare variables between groups. Bonferroni’s analysis was then performed when appropriate, to determine post-hoc significance at individual time point. Data was analyzed using Statistica, Software® and, in all cases, statistical significance was set at P<0.05.The data are presented as mean ± standard deviation (SD). A repeated measures analysis of variance was used to test the treatment-by-time interactions and the effect of treatment over time on each score. Duncan’s multiple range test was used for The average intensity of the fluid pulse delivered to animals in the injured group was 2.24±0.05 atm (mean±SEM). Immediately following this impact, all rats experienced a period of apnea (lasting approximately 25 sec), hypertension (approximately up to ~140 mmHg and lasting ~25 sec), and tachycardia (~390 beats/min and lasting more than 120 minutes). Sham-injured animals showed no apnea, hypertension, or tachycardia. There was no difference between 2 treatment groups.Three days after the rats had been subjected to FPI, behavioral tests revealed that the NSS of both the  group and the (FPI+etanercept) group were significantly (P<0.05) higher than those of the untreated sham-FPI group  . The cerebral ischemia areas were significantly (P<0.01) smaller in the FPI+etanercept group than in the FPI+saline group   group had significantly P<0.001) larger areas of brain ischemia than did the sham-FPI group  higher for the FPI+saline and the FPI+etanercept groups than for the sham group 72 h after FPI or sham operation Figure , but sigThe major findings of our present study are: (1) etanercept injected systemically reduces motor and neurological deficits caused by FPI by day 3 after FPI; (2) the increased numbers of the co-localization of TNF-α and microglia specific marker cells are significantly and selectively higher in the ischemic cortex, white matter, hippocampus, and hypothalamus during FPI which can be attenuated by etanercept therapy; (3) overproduction of cerebral TNF-α in the ischemic cortex caused by FPI can be attenuated by etanercept; (4) by day 3 after FPI, neither the co-localization of TNF-α and astrocyte specific marker cells nor the TNF-α and neuron specific marker cells can be seem in the ischemic brain regions. Our data suggest that systemic administration of etanercept may improve outcome of FPI by attenuating the activation of microglial TNF-α in the ischemic brain. The hypothesis is in part supported by many investigations. For example, Li and Colleagues  has repoTNF-α transduces death- and survival-signaling through its cognate receptors TNFR1 and TNFR2 and is involved in the inflammatory response following TBI -25. IncrAccumulated evidence shows that TNF-α and its receptor play an important role in the pathophysiology of TBI ,24,25. IEtanercept, when administered systemically at the dosage approved for its licensed indications (~50 mg/week in human), would not be expected to achieved therapeutic levels in the cerebrospinal fluid because of its high molecular weight . It shouThe present study focuses mainly the effects of etanercept on microglial overproduction of TNF-α in the rat brain effected by TBI. Actually, in addition to TNF-α, TBI-induced increased levels of both interleukin-1β and interleukin-6 were all significantly reduced by etanercept treatment . AdditioIt should be mentioned that in the present study, there appears to be quite a few instances of Iba1 and TNF-α labeling that do not coincide and the staining morphology is quite different in some instances (mor elongated Iba1 staining versus round/amoeboid TNF-α staining). Furthermore, Iba1 is not a particularly distinguishing cell-specific marker for microglia in the injured brain as infiltrating macrophages also express this antigen. In addition, although our present study showed that the sham-group displayed no evidence of damage 3 days post-FPI, Jones et al.  did dispFinally, it should be mentioned that given that the sham animals in the present study did not receive injections on any treatment the authors might comment on the possible confound that the stress associated with the injections may have had on the TBI group versus sham.The present study demonstrates that TBI, in addition to inducing cerebral contusion and neurological motor deficits, cause the microglial overproduction of TNF-α in the injured cortex, hippocampus, white matter, and hypothalamus. The TBI-induced neurological motor deficits and microglial overproduction of TNF-α can be significantly attenuated by the post-TBI application of etanercept. Our data identify a role for the microglial production of TNF-α in the outcomes of TBI in rats. Etanercept may attenuate cerebral contusion and neurological motor deficits during TBI by inhibiting the activation of the microglia-TNF-α double positive cells in the ischemic brain region.The authors declare that they have no competing interests.CCC and CPC designed and conducted most of the experiments. CHC, CCW, CUC, CMC, BCC, CZY designed and conducted some of the experiments. CCC and CPC wrote most of the manuscript. All authors analyzed the data, revised the manuscript and gave final approval for publication.


Our visual system can robustly detect an important feature in a scene, such as the identity of an object, even when its retinal image can vary substantially. Neural basis for such invariant recognition is thought to be the common property in visual cortex where a cell retains its activity even with large changes in stimuli along a certain dimension. For example, a face-selective cell may keep firing when the viewing angle of the face is changed; a V1 complex cell may remain active when the phase of an oriented grating is changed. A modern approach to identify such invariant dimensions is a semi-automatic analysis of responses to a large number of randomly chosen stimuli; a well-known method is spike-triggered covariance analysis (STC), which has successfully estimated receptive field elements with distinct phases for V1 complex cells [This work aims at generalizing the previous semi-automatic method to reveal invariant dimensions in V2. STC is not suitable for direct applications to V2 cells since these typically have a much higher-degree of nonlinearity compared to V1 cells and since a large number of parameters are needed to characterize V2 cells and therefore STC requires an unrealistic amount of data. Instead, our approach is to assume a population model of V1 cells and analyze their outputs with respect to the responses of a V2 cell, where the latter part uses a Bayesian version of STC ,4.We applied our method to a publicly available dataset of 127 V2 cells responding to a large number of natural images . Our ana


Increased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin  signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.Cells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.TNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.Here, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration. We have previously analyzed Wnt-β-catenin  signaling in nucleus pulposus cells and reported that activation of Wnt signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of IVDs -3. Incre pathway ,6. As th pathway . Wnt sig pathway -11. HoweLow back pain is strongly associated with IVD degeneration, which in turn is associated with sciatica and disc herniation ,13. The Animal experiments were performed according to a protocol approved by the Animal Experimentation Committee of our institution .To determine the β-catenin-TCF/LEF transcriptional activity, nucleus pulposus cells and annulus fibrosus cells were transiently transfected with the TCF/LEF reporter gene Topflash  or Fopflash (mutated TCF binding site) . The Fopflash construct is identical to the Topflash construct, with the exception that it contains mutated copies of TCF/LEF binding sites and is used as a control to measure nonspecific activation of the reporter construct. K3-luc  and the SOST expression plasmid (number 10842) were purchased from Addgene . The wild-type (WT)-β-catenin expression plasmid and the backbone plasmid were provided by Dr Raymond Poon . The β-catenin small interfering RNA (siRNA) (number sc-29209) and control siRNA duplexes were purchased from Santa Cruz Biotechnology . The Dickkopf (DKK)-1, -2, -3, and -4 expression plasmids and the backbone (pMY-IRES-EGFP) plasmids were provided by Dr Siegfried Janz  .Renilla reniformis luciferase gene as an internal transfection control. Recombinant TNF-α was purchased from . We used 6-bromoindirubin-3′-oxime (BIO)  to examine Wnt signaling activity. BIO is a cell-permeable, highly potent, selective, reversible, and ATP-competitive specific inhibitor of glycogen synthase kinase (GSK)-3a/b activity [We used the vector pGL4.74  containing the activity .et al. [2. Confluent nucleus pulposus and annulus fibrosus cells were harvested and subcultured in 10-cm dishes. Low-passage (<3) cells cultured in monolayers were used for all experiments, because cells obtained from the rat IVD tissues exhibited variable morphology until passages 2 or 3.Nucleus pulposus cells and annulus fibrosus cells were isolated from the lumbar discs of 11-week-old Sprague Dawley rats n = 32) using methods reported by Hiyama 2 using m3 cells/well) and incubated for 24 h. The cells were treated with 10 ng/mL TNF-α or 1.0 μM BIO, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (vol/vol) in PBS, blocked with PBS containing 10% FBS, and incubated overnight at 4°C with antibodies against TNF-α  or β-catenin . The cells were washed and incubated with anti-rabbit Alexa Fluor 488 secondary (green) antibodies  at a dilution of 1:200 and 10 μM 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature, for nuclear staining. The samples were observed under a fluorescence microscope connected to a digital imaging system. Negative controls without the primary antibody were prepared.Nucleus pulposus cells were plated in flat-bottom 96-well plates (3 × 105 cells/plate) with or without TNF-α for 24 h, and the total RNA was extracted from the cells using the TRIzol RNA isolation protocol (Invitrogen). RNA was treated with RNase-free DNAse I. Total RNA (100 ng) was used as a template for the real-time PCR analyses. The cDNA was synthesized via the reverse transcription of mRNA, as described previously [Wnt1, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt10a, Wnt10b, Wnt16, LRP5, LRP6, β-catenin, LEF1, TCF4, TNF-α, TNFR1 and TNFR2 were added. The primers were synthesized by  or  and are shown in Table glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used to normalize each sample, and the arbitrary intensity threshold (Ct) of amplification was computed. The expression scores were obtained using the ΔΔCt calculation method.Nucleus pulposus cells were cultured in 10-cm plates . Nuclear or total cellular proteins were separated on a sodium dodecyl sulfate (SDS) polyacrylamide gel and were electrotransferred onto nitrocellulose membranes . The membranes were blocked with 5% BSA in Tris-buffered saline and Tween 20 (TBST)  and incubated overnight at 4°C in 5% BSA in TBST with anti-TNF-α  or anti-β-catenin  antibodies. Immunolabeling was detected with an ECL reagent . The Western blot data were quantified using Image J pixel analysis (NIH Image software). Data from the western blots were presented as band density normalized to that of the loading control (actin).Treated nucleus pulposus cells were placed immediately on ice and washed with cold PBS. Proteins were prepared using the CellLytic NuCLEAR extraction kit . All of the wash buffers and the final resuspension buffer included 1 × protease inhibitor cocktail , NaF (5 mM), and Na4 cells/well 1 day before transfection. Cells were co-transfected with 100 to 500 ng of expression plasmids or the backbone vector together with the reporter plasmids. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent. Cells were cultured for 24 h and treated with a specific concentration of TNF-α or BIO. The cells were harvested 24 h after treatment and a Dual-Luciferase™ reporter assay system (Promega) was used for the sequential measurements of firefly and Renilla luciferase activities. The results were normalized regarding transfection efficiency and were expressed as a relative ratio of luciferase to pGL4.74 activities (denoted as relative activity). Nucleus pulposus cells were transfected with a plasmid encoding green fluorescent protein, to check transfection efficiency, which was 60 to 70% in nucleus pulposus cells. The luciferase activities and relative ratios were quantified using a Turner Designs Luminometer Model TD-20/20 instrument (Promega).Nucleus pulposus cells and annulus fibrosus cells were transferred to 24-well plates at a density of 3 × 10t-test or analysis of variance, to assess variance. Significance was accepted at P <0.05 and is denoted with an asterisk in the figures.Typically, data were compiled from at least three independent triplicate experiments, each performed using separate cultures and on separate occasions. The responses were presented as the fold change relative to the untreated control. The data were presented as the mean ± SD. Data were compared between groups using Student’s P = 0.2026; 0.5 μM, 1.81 ± 0.37, P <0.001; 1.0 μM, 1.81 ± 0.59, P <0.001) . To validate these findings, we performed loss-of-function experiments using an siRNA for β-catenin. Suppression of gene expression was confirmed by real-time RT-PCR (data not shown). TNF-α promoter activity was inhibited in nucleus pulposus cells that were co-transfected with the β-catenin siRNA  (P <0.05) . The expression of TNF-α mRNA was significantly higher (by 1.5-fold) in the annulus fibrosus than in the nucleus pulposus  (P <0.05). To determine whether a concomitant elevation in TNF-α protein expression was associated with Wnt signaling, the cells were evaluated using western blotting  by western blotting. The results demonstrate that soluble TNF-α protein was significantly elevated after the stimulation of TNF-α compared to the control cells  for 6 and 24 h. Real-time PCR analysis demonstrated that TNF-α treatment for 24 h increased the expression of the et al. reported that TNF-α reduced the synthesis of matrix molecules and upregulated the mRNA expression of MMP-1, -3, and -13 and ADAM-TS4 and ADAM-TS5 [2, the regulation of IL-6, and MMP-1 is mediated exclusively by TNF-R1.The canonical Wnt signaling and the proinflammatory cytokine TNF-α play critical roles in development, homeostasis, and cancer -25. HoweADAM-TS5 . StudiesADAM-TS5  on synovet al. have shown that both IL-1 and TNF-α are expressed in IVDs and are upregulated in the presence of degeneration [et al. have also reported that TNF-R2 is not expressed by IVD cells either in normal or degenerate IVDs [in vivo. However, no increase in TNF-R1 synthesis was seen during IVD degeneration.Furthermore, Le Maitre neration . TNF-α cate IVDs . In addiThe complexity of the Wnt signaling cascade should enable the activation and/or repression of many specific signals and targets. Remarkably, we found that Wnt signaling, which suppresses the proliferation of nucleus pulposus cells and induces cell senescence, activated the expression of TNF-α. Direct evidence for the activation of TNF-α by Wnt signaling was obtained from experiments such as a reporter assay, real-time PCR, western blotting, et cetera. Furthermore, Wnt signaling was modulated by several different families of secreted negative regulators. The results of this study suggest that inhibition of Wnt signaling may be able to contribute to the suppression of disc degeneration, because Wnt signaling increased the expression of TNF-α and TNF-α activated Wnt signaling. Wnt antagonists can be divided into two functional classes. These two major classes function in quite different ways: the secreted Frizzled-related protein (sFRP) class binds to Wnt ligands, whereas the DKK or sclerostin class binds to a component of the Wnt receptor. Thus, in theory, the outcome appears to be that sFRPs inhibit canonical (β-catenin-dependent) and noncanonical (β-catenin-independent) pathways, whereas DKKs or sclerostin inhibit only the canonical pathway. The DKK family comprises four members (DKK-1 to DKK-4) in mammals ,34. DKK et al. reported that TNF-α increased the expression of β-catenin and MMP-13, and significantly inhibited matrix synthesis, which resulted in the degeneration of rabbit IVD cells. These authors also showed that blocking Wnt/β-catenin signaling using DKK-1 protected the normal metabolism of IVD tissues in rabbits [The most-studied member of the DKK family is DKK-1. The binding of DKK-1 to the LPR5/6 receptor and a cell-surface coreceptor, Kremen-1/2, promotes internalization of the receptor complex and dampens the Wnt signal. Both DKK-1 and DKK-2 bind to the LRP5/6 receptor, which is expressed in cells and has higher affinity compared with Fz and Wnt. The characteristic developmental function of DKK-1 is its head-inducing activity. Previously, Ye  rabbits . HoweverSOST gene. Similar to DKK, sclerostin binds to Lrp5/6 and antagonizes canonical Wnt signaling [SOST gene. As a result, it might have resulted in the activation of Wnt signaling in nucleus pulposus cells.The Wnt-antagonizing activity of DKK-4 appears to be indistinguishable from that of DKK-1, whereas DKK-3 has distinct roles in regulating the Wnt pathway, depending on the cell types examined. For example, DKK-3 potentiates Wnt signaling in human Müller glia MIO-M1 and HEK293 cell lines , but inhignaling -46. Low Overall, our results indicate that inhibition of Wnt signaling suppresses a catabolic response via the inhibitory action of TNF-α in nucleus pulposus cells. However, it remains to be determined which of the proposed receptors for DKKs or sclerostin, including LRP-4, -5, and -6, are important for the responses observed in our study and whether the effect is dependent on canonical or noncanonical Wnt signaling, or on other types of signaling. In addition to the limitation inherent in the use of rats, because of the ambiguity of the notochordal cells in this animal, the functions of the rat disc compared with the functions of the human IVD need to be considered in the interpretation of the findings of this study. Regarding this issue, additional studies using different species are required to evaluate and conclude whether the mechanism involving the expression of these molecules is specific to nucleus pulposus cells, in particular regarding the human situation.in vitro evidence that the activation of Wnt signaling upregulates the proinflammatory cytokine TNF-α and might cause the degeneration of nucleus pulposus cells. We speculated that blocking the Wnt signaling might protect nucleus pulposus cells against degeneration. The DKK or sclerostin families of proteins are natural regulators of Wnt signaling and can specifically block this pathway. These findings suggest that overexpression of DKK-3, DKK-4, or sclerostin inhibit TNF-α expression by specifically blocking the Wnt channel. Inhibition of Wnt signaling using DKKs or sclerostin exerts a protective and reversing effect in the TNF-α-induced degeneration of IVD cells.Here, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide 2; RT-PCR: Reverse transcription polymerase chain reaction; sFRP: Frizzled-related protein; SOST: Sclerostin; TBST: Tris-buffered saline and Tween 20; TCF: T-cell factor; TNF-α: Tumor necrosis factor-α; WT: Wild-type.ADAMTS: A disintegrin and metalloproteinase with thrombospondin motifs; BIO: 6-bromoindirubin-3′-oxime; BSA: Bovine serum albumin; Ct: Intensity threshold; DAPI: 4′,6-diamidino-2-phenylindole; DKK: Dickkopf; DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; GSK: Glycogen synthase kinase; IL-1β: Interleukin-1 β; IVD: Intervertebral disc; LEF: Lymphoid enhancer factor; MMP: Matrix metalloproteinase; PBS: Phosphate-buffered saline; PCP: Planar cell polarity; PGE2: Prostaglandin EThe authors declare that they have no competing interests.AH participated in study design, performed experiments, analyzed data and wrote the paper. KY prepared the isolation of cells and performed experiments and the statistical analysis. TN performed experiments and analyzed data. DS participated in study design, performed experiments and performed the statistical analysis. JM participated in study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The soluble TNF-α protein levels after the stimulation of TNF-α  by western blotting. The results demonstrate that soluble TNF-α protein was significantly elevated after the stimulation of TNF-α compared to the control cells.Click here for file


By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG1 was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-α neutralization bioassay. Furthermore, h357 IgG1 possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-α. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 µg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use.A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-α) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified  Tumor necrosis factor (TNF-α) is a pro-inflammatory cytokine produced primarily by cells of the immune system, including macrophages and monocytes. TNF-α is present as a homotrimeric protein in which each subunit is initially translated as a 26 kDa transmembrane precursor protein. After being cleaved at a site proximal to the transmembrane domain of TNF-α by TNF-α converting enzyme, a soluble trimeric form of TNF-α is released and exerts its activity by binding to two structurally distinct type I and type II TNF receptors (TNFRI and TNFRII) on effector cells. The transmembrane form of TNF-α is also known as its unique biologic functions, such as cytotoxic activity and polyclonal B cell activation, in a cell-to-cell contact manner via variable domain resurfacing is another approach that can maintain the specificity and binding affinity of parental antibody, which can reduce the immunogenicity of antibodies through the replacement of surface exposed residues in the murine FRs with those usually found in human antibodies The major impediment of the murine monoclonal antibody in clinical practice is that it may elicit human anti-murine antibody (HAMA) response in patients via computer-aided molecular modeling followed by functional characterization of the humanized antibody. The results indicated that the humanized antibody, h357, retained in vitro bioactivities similar to the parental antibody. Moreover, the humanized h357 IgG is capable of binding to the cells bearing the transmembrane TNF-α and triggering ADCC with effector cells and CDC. The humanized 357 IgG at a dose level of 50 µg/mouse/day significantly inhibited disease progression in a mouse model of collagen antibody-induced arthritis when compared with saline treated mice. These results provided a basis for the development of h357 IgG as therapeutic use.This study is in an attempt to humanize a murine anti-human TNF-α monoclonal antibody, m357, raised by immunization of mice with recombinant human TNF-α. In order to keep the parental binding affinity, we resurfaced m357 in the variable regions of both the heavy and light chains H) and light chain variable region (VL) of the murine anti-human TNF-α monoclonal antibody (designated as m357) were first obtained by RT-PCR . The three-dimensional structure of the deduced amino acid sequences of the VH and VL fragments of m357 was constructed individually by homology modeling (program MODELLER) as described under “H and VL domains of m357 were created using the crystal structures of the VH domain of the anti-breast tumour antibody SM3  L domain of the anti-Thermus aquaticus DNA polymerase I antibody  H and VL domains of m357 were obtained through Discovery Studio modeling 2.1 program as shown in The murine monoclonal antibody derived from the hybridoma cell line, 357-101-4 (ECACC No. 92030603), is of therapeutic interest for its reported strong neutralizing biological activity against human TNF-α H and VL domains of m357 were constructed individually; second, we used AREAIMOL program to calculate the solvent accessible residues for identifying the non-human like framework surface residues, and finally we mutated these surface residues to the human counterparts according the results of the sequence alignment of human framework. In order to replace the non-human like framework surface residues of the murine m357 on the variable domain, a set of highly homologous surface residues from a human target sequence were selected. We searched the IMGT database (http://imgt.cines.fr/) to identify the human VH and VL sequence pairs, which are most homologous to the corresponding variable regions of m357, while eliminating the sequences of phage-display or humanized antibodies from our search results. The most identical surface residues found from human sequences were the variable regions of PPS4  (Genbank DQ187629 and DQ187550)  Figure 2H and VL of m357, comprising the signal peptide at the N-terminus, were in-frame fused to the human IgG γ1 heavy chain and kappa light chain constant regions, respectively. For the expression of an intact humanized 357 (h357) IgG1 molecule, two different mammalian expression vectors, pSecTag2/Hygro and pcDNA3.3, were used to adopt the humanized heavy chain and light chain of h357 IgG, respectively. After co-transfection and antibiotic selection, one of the clones showing the highest expression level was chosen for further studies. The expression level of the recombinant h357 IgG was ∼14 mg/L. Both m357 and h357 IgGs were purified by protein A chromatography individually and the protein purities were determined by SDS-PAGE. As shown in The amino acid sequences of the humanized V50 of 3.07 nM and 2.30 nM, respectively. The results indicated that the humanized 357 IgG retained the TNF-α neutralization activity at concentrations similar to that of the murine IgG.To examine the functional activity of the anti-TNF-α antibodies, the ability of the antibody to inhibit soluble TNF-α activity was performed. TNF-α causes cell cytotoxicity to murine L929 cells. Both m357 and h357 IgGs were evaluated in L929 assays by co-incubation of antibodies with recombinant human TNF-α. As shown in D values of approximately 7.8 nM, 5.5 nM, 2.2 nM and 2.9 nM, respectively. The similar KD values in h357 and m357 IgGs indicated that the humanization process did not alter the TNF-α binding strength of the parental mouse antibody.A higher binding affinity between antagonists and soluble TNF-α can help maintain the stability of neutralizing complex and become a critical factor for neutralizing activity of anti-TNF-α antibodies D of 12.0 nM and 16.8 nM, respectively. The similar KD values in m357 and h357 indicated that the humanization process did not alter the transmembrane TNF-α binding affinity. The binding affinity in the nanomolar range suggested h357 IgG could potentially trigger more effector functions or apoptosis mechanism through binding to transmembrane TNF-α.TNF-α exists as a membrane associated precursor (transmembrane TNF-α) from which the mature soluble form is released by proteolytic cleavage mediated by TNF-α converting enzyme. Many studies have investigated that TNF-α antagonists can cause cell lysis by apoptotic, ADCC, CDC and outside-to-inside signaling mechanisms given that it has the binding ability to transmembrane TNF-α Previous studies have been reported that the binding affinity of infliximab and adalimumab toward the membrane form of TNF-α were better than that of etanercept, which can affect the cell killing of cell surface-expressed TNF-α by ADCC, CDC or apoptosis mechanisms and this may be one of reasons that cause different effects on clinical diseases To assess the therapeutic effects of h357 on rheumatoid arthritis development, the collagen antibody-induced arthritis mouse model was adopted, in which the disease was induced by the systemic administration of a cocktail of monoclonal antibodies that target various regions of collagen type II, which is one of the major constituents of articular cartilage matrix proteins, followed by lipopolysaccharides TNF-α antagonists, including antibodies, have been developed for binding to TNF-α as a mean to inhibit its biological activities, including TNF-α-induced cell activation and cytotoxicity. Anti-TNF-α therapies have been applied to a variety of autoimmune disorders, such as rheumatoid arthritis in silico primary structure-based algorisms which mainly focused on MHC class II peptide binding predictions. Meanwhile, recently developed T-cell based procedures are showing promise for predicting potential immunogenicity with some biologics including monoclonal antibodies. Further experimental work focusing on both in silico and T-cell assays of the h357 IgG will be assessed to further minimize the immunogenicity issues for drug development.Protein therapeutics from exogenous sources can potentially elicit immune response when administered in humans. The anti-drug antibodies could lead to partially or completely loss of drug efficacy. Preclinical immunogenicity assessment has drawn a lot of attention to FDA and is highly encouraged in the preclinical development stages. Many techniques, such as humanization, deimmunization and deaggrgation have been adopted to alter the structure of a protein in order to reduce the potential risk of immugenecity. Antibody humanization by CDR-grafting can often result in decrease or completely loss of antigen binding affinity because the murine frame-work regions encompassing the variable regions often contribute the integrity of antibody structure for the formation of correct CDR-epitope complex. The deimmunization approach we have adopted is a three dimensional structure resurfacing method based on the fact that most antigenic response to the variable region is directed to surface residues only. By replacing the amino acids that are potentially antigenic whereas preserving the most murine primary structure in the variable regions should in theory minimize the conformational change of the parental murine antibody and thus retain the antigen binding affinity. So far, there is little information available about the effects of antibody deimmunization by resurfacing on the antigen binding affinities. In this study, we demonstrated that this approach is feasible in retaining the antibody-antigen affinity. The next question is whether this resurfacing approach is truly a way to generate non-immunogenic proteins. Since antibody drugs are highly species specific, it is difficult to assess the immune response in humans when conducting preclinical animal experiments even with non-human primates. The adopted three dimensional structure resurfacing method currently we used is distinct from the http://www.jirb.org.tw/), case number 10-S-004, a committee for allowing all blood centers in Taiwan to provided blood for research purposes from those donors with written consent. All animal work was reviewed and approved by the Institutional Animal Care and Use Committee of Industrial Technology Research Institute .Human whole blood was collected from healthy donors at Hsinchui Blood Center, Taiwan, under a protocol approved by the Institutional Review Board of Industrial Technology Research Institute (IRB number 9901008) and reviewed by the Joint Institutional Review Board  (http://www.rcsb.org/pdb/home/home.do). The three-dimensional (3D) structure of the m357 Fv fragment was constructed by homology modeling based on the structures of the VH domain of the murine anti-breast tumor antibody Fab fragment SM-3[PDB entry: 1SM3] L domain structure of an anti-thermus aquaticus DNA polymerase I monoclonal antibody Fab fragment [PDB entry: 1AY1] The homology modeling process of m357 was performed using Discovery Studio Modeling 2.1 . Two separate BLASTP searches were performed for V7 cells of the hybridoma 357-101-4 (ECACC No. 92030603) which secrete anti-human TNF-α IgG (m357) was isolated and the first-strand cDNA was generated using oligo (dT) primers with Superscript III reverse transcriptase . The cDNA fragments encoding the heavy chain and light chain variable regions of m357 were obtained by PCR with the Heavy Primers  and the Light Primer Mix , respectively. The cDNA fragments encoding the humanized heavy chain and light chain variable regions of m357 were obtained by over-lapping PCR with primers covering the humanized regions. For the construction of the heavy chain of the humanized 357 (h357) antibody, the cDNA construct consisting the murine Ig kappa-chain V-J2-C signal peptide derived from the mammalian expression vector pSecTag2/Hygro , the VH region of h357, and the human IgG1 constant region  which derived from the cloning vector pFUSE-CHIg-hG1  were obtained by over-lapping PCR, followed by sub-cloning into the vector pSecTag2/Hygro  at Nhe I and Not I sites. For the construction of the light chain of the humanized 357 (h357), the cDNA construct consisting the above murine Ig kappa-chain V-J2-C signal peptide, the VL region of h357, and the human kappa light chain constant region which derived from the cloning vector pFUSE2-CLIg-hk  were obtained by over-lapping PCR, followed by sub-cloning into the mammalian expression vectors pcDNA3.3-TOPO TA . Plasmids containing the heavy and light chain cDNAs of h357 IgG1 were co-transfected into mouse myeloma NS0 cells  using Effectene (Qiagen). After selection with hygromycin B (400 µg/ml) and G418 (800 µg/ml) for 4 weeks, culture media from several stable clones were screened by ELISA with human TNF-α as coating and anti-human IgG Fcγ-HRP as detection reagents. An h357 IgG1 high-expressing clone was picked and cultured in shaker flask at an initial seeding density of 5×105 cells/ml in a chemically-defined medium HyQNS0 (Hyclone) without serum addition. Media were harvested 5 days later and h357 IgG1 was purified from the supernatant by Protein A  chromatography.The total RNA of around 105 cells/well into a 96-well plate and cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum for 16 hours. Then, several dilutions of the antibodies were prepared in medium containing actinomycin D (2 µg/ml) and TNF-α (100 ng/ml) and incubated at 37°C for 16 hours. After the supernatant were removed, 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) (Sigma-Aldrich) was added and incubated in 37°C for 4 hours. SDS solution (10%) was then added to the well. After 24 hours of incubation at room temperature, color in each well was recorded by colorimeter at 570 nm. Blank control , TNF-α control , and antibody control  were also designed in the experiment. The ED50 value was calculated by complex sigmoid non-linear regression analysis using SigmaPlot software .Neutralizing activities of m357 and h357 against human TNF-α were measured on the murine fibroblast L929 cells (ATCC Cat. No. CCL-1) treated with actinomycin D according to the method described previouslyA mutant form of transmembrane TNF-α, which is resistant to TNF-α converting enzyme-mediated cleavage, was generated by site-directed mutagenesis as described previously D) of each antagonist was calculated as the concentration of TNF-α required to achieve half-saturation of the total binding sites (maximum absorbance).The binding activities of h357 IgG, m357 IgG, etanercept and adalimumab to soluble TNF-α were determined by ELISA. For capturing h357 IgG, etanercept (Wyeth) and adalimumab (Abbott), the microtiter plate was coated with 2 µg/ml of goat anti-hunam IgG Fcγ fragment  in PBS overnight at 4°C. For capturing m357 IgG, the microtiter plate was coated with goat anti-mouse IgG Fcγ fragment . After blocking the wells with StartingBlock™ blocking buffer (Thermo Scientific), 2 µg/ml each of the TNF-α antagonists, as mentioned above, were added and incubated for 1 hr at 37°C. After washing, varing concentrations of human recombinant TNF-α  in PBS were incubated for 1 hr at 37°C. After washing, the bound soluble TNF-α was detected by incubation with horseradish peroxidase-conjuagted mouse monoclonal anti-TNF-α antibody  for 1 hr at 37°C and using 3,3′,5,5′,-tetramethylbenzidine as substrate. Absorbance was read at 450 nm on a microplate reader. The dissociation constant  containing 2% fetal bovine serum (fluorescence-activated cell sorting [FACS] buffer). Cells were washed with a FACS buffer for 3 times and were then stained with Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L)  for m357 IgG and Alexa Fluor 647 conjugated goat anti-human IgG (H+L)  for h357 IgG, respectively for 1 hr at 4°C. Fluorescence intensities were measured using a FACSCalibur flow cytometer .2 incubator at 37°C, followed by the addition of either human peripheral blood mononuclear cells (PBMC) as effector cells (effector to target ratio  = 20∶1 for ADCC assay) or human complement-rich serum (Quidel) . After an additional incubation for 16 hrs for ADCC assay and 5 hrs for CDC assay at 37°C, respectively, 100 µl of supernatant from each well were transferred into a clean flat-bottom 96-well plate. LDH substrate (100 µl) was added to each well and incubated for 30 min at room temperature protected from light. The absorbance of the samples was measure at 490 nm with an ELISA reader. Maximum LDH release was determined by incubating cells with lysis buffer. Percentage of specific lysis was calculated according to the following formula: % cytotoxicity  = [experimental release – spontaneous release]/[maximum release – spontaneous release]×100.Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. ADCC and CDC activities of h357 and m357 IgGs were measured by LDH Cytotoxicity Detection Kit (Clontech), which measures lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells. Briefly, NS0 cells stably expressing the transmembrane TNF-α were incubated with different concentration of h357 or m357 IgG for 1 hr in assay medium (DMEM +1% FBS) in a 5% COFor collagen antibody-induced arthritis experiments, 8- to10-week old male DBA/1J mice were purchased from Jackson laboratories . Mice were maintained under a climate controlled environment in a 12-h light/dark cycle. Arthritic mice were induced by intra-peritoneal (i.p.) injection of 3 mg/mouse of type II collagen specific antibodies (ModiQuest Research). Mice were further boosted with 25 µg lipopolysaccharides (LPS) (Sigma) by i.p. injection on day 6. Clinical arthritis scores were evaluated using a scale of 0–2 for each paw for a total score of 8. Paws were assigned a clinical score based on the index (ModiQuest Research scoring method): 0 =  normal; 0.25 =  one or two swollen toes; 0.5 =  three and four swollen toes; 0.75 =  slightly swollen footpad or ankle; 1 =  swollen footpad or ankle; 1.25 =  one or two swollen toes and swollen footpad or ankle; 2.0 =  swollen toes and swollen footpad and swollen ankle. At the time of disease onset, mice were administered by i.p. injection of 50 µg per does for 9 consecutive days. Scoring results were expressed as mean ± standard deviation (SD). Statistical differences between the control  and experimental groups were analyzed by Mann-Whitney U test for the severity of arthritis.Figure S1RT-PCR results showing the variable fragments and schematic representation of the strategy to assemble the heavy and light chains. (A) Amplification of the variable fragment cDNAs from the mouse hybridoma 357-101-4 secreting m357 IgG by RT-PCR with the Heavy Primers  and the Light Primer Mix , respectively. Lane M: Molecular weight marker. Lane 1: VH, ∼340 bp. Lane 2: VL, ∼325 bp. (B) Assembly of the cDNAs encoding the open reading frames of the heavy and light chains of the humanized 357 (h357) IgG1 by over-lapping PCR, respectively. The cDNA construct consisting the murine signal peptide (SP) derived from the cloning vector pSecTag2/Hygro, the VH region of h357, and the human IgG1 constant region  derived from the cloning vector pFUSE-CHIg-hG1 were obtained by over-lapping PCR, followed by sub-cloning into the vector pSecTag2/Hygro at Nhe I and Not I sites. The cDNA construct consisting the above signal peptide, the VL region of h357, and the human kappa light chain constant region derived from the cloning vector pFUSE2-CLIg-hk were obtained by over-lapping PCR, followed by sub-cloning into the mammalian expression vectors pcDNA3.3-TOPO TA . The locations of the primers and the restriction sites are shown in the diagram. SP, murine Ig kappa-chain V-J2-C signal peptide.(EPS)Click here for additional data file.


The somatically related antibodies, PG9 and PG16, neutralize 70-80% of HIV-1 isolates and bind a glycosylated epitope in the V1/V2 domain of HIV-1 gp120. Mutations in V1/V2, and sometimes V3 depending on the HIV-1 strain, affect neutralization and a glycan on Asn160 is required for neutralization. Both antibodies also preferentially bind the native trimer over monomeric gp120, especially PG16. The structure of PG9 in complex with its epitope, a scaffolded V1/V2 from HIV-1 strain ZM109, was recently solved and showed that PG9 targets a site of vulnerability comprising 2 glycans and a β-strand.To understand the differences in binding properties from these two somatically related antibodies, we first assessed their binding to monomeric gp120 and scaffolded V1/V2 proteins with different glycan types . In order for PG16 to bind the scaffolded V1/V2, the protein had to be expressed in mammalian cells in the presence of swainsonine, which inhibits glycan maturation past the hybrid state. A stable complex could be obtained between PG16 and a scaffolded V1/V2 domain from ZM109, and this complex was crystallized.Although the structure of PG16 bound to scaffolded V1/V2 resembled that of PG9, some differences were seen: 1) PG16 binding to the β-strand is weaker than PG9 with fewer charged interactions, 2) PG16 interacts with a hybrid glycan at position N173. The difference in binding recognition of PG9 and PG16 to monomeric gp120 depends on the type of glycans present. PG16 binds the protein portion of V1/V2 weaker than PG9 and this might explain the higher affinity of PG9 for the monomer. PG16 has evolved a second glycan site to compensate for weaker peptide interaction.The results show the importance of polyclonal response in infected individual to combat HIV-1, and in this case, to differential glycosylation.


Our collectible studies were searched from PUBMED, EMBASE, and the Cochrane Library between March 1994 and August 2013. Accordingly, 347 studies were collected totally, in which 15 articles and 23 trials were selected to study the NS in our meta-analysis. The TNF-α test showed moderate accuracy of the diagnosis of NS both in early-onset neonatal sepsis  and in late-onset neonatal sepsis . We also found the northern hemisphere group in the test has higher sensitivity (0.84) and specificity (0.83). A diagnostic OR analysis found that the study population may be the major reason for the heterogeneity. Accordingly, we suggest that TNF-α is also a valuable marker in the diagnosis of NS.Neonatal sepsis (NS) is an important cause of mortality in newborns and life-threatening disorder in infants. The meta-analysis was performed to investigate the diagnosis value of tumor necrosis factor- Neonatal sepsis (NS), as one of the major causes, leads to neonatal mortality and morbidity, especially in neonates born preterm . Early dα (TNF-α), by considering it as a useful marker. TNF-α is a cytokine involved in systemic inflammation, which belongs to a member of a group of cytokines that stimulate the acute phase reaction [Procalcitonin (PCT), a 116-amino acid peptide considered as a precursor in calcium homeostasis, has been proved to be a valuable marker for distinguishing sepsis from other noninfectious disease , 5. C-rereaction .α for detecting NS. Although a lot of works indicate that both PCT and CRP are two superior markers for diagnosis of sepsis and infection, we suggest here the TNF-α is also a promising marker in NS. A deeper meta-analysis of these studies is thus currently needed.Thus, the objective of this meta-analysis was to investigate the value of TNF-α in NS [α,” “neonatal,” “neonate,” “sepsis,” “infant,” “newborn,” and “tumor necrosis factors-α”. The reference lists of all known primary and review articles were also searched. No language restriction was used, so that we have examined the references of known articles to fully retrieve the data.In order to perform a systematic analysis of the available evidence on the efficacy of TNF-α in NS , the comIf an article does not include enough data for calculating sensitivity and specificity (2 × 2 table), we asked the corresponding author to provide us with necessary data. If there was no response from the corresponding author, a reminder was sent after one week. If we still cannot achieve the data after this process, the study was excluded from meta-analysis. The selection of articles was performed by two investigators independently to ensure the high accuracy.Data collected from the studies included the first author, publication year, diagnostic cut-off point and time, test methods, and sensitivity and specificity data. So the numbers of true-positive, false-positive, false-negative, and true-negative results were extracted for each study. Accuracy data was extracted to construct 2 × 2 table at a specific time. We have requested the information from the authors, if no enough data on the criteria was found in the studies.α test for diagnosis of NS. One group is the time points of TNF-α measurement for the diagnosis of early-onset neonatal sepsis (EONS), which were 0–72 h of age; the other group is the time points of TNF-α measurement for the diagnosis of late-onset neonatal sepsis (LONS), in which the age of neonates is older than 72 h. We calculated the sensitivity, specificity, diagnostic odds ratio (OR), and corresponding 95% confidence intervals (CI) from each study. We also gained the pooled sensitivity, specificity, and diagnosis OR from each group.We used Meta-Disc 1.4 software and Review Manager 5.0 to perform the statistical analysis . DiagnosQ statistic. Normally, I2 lies between 0% and 100%. If I2 < 50%, then there is a lot of homogeneity among studies in meta-analysis; whereas I2 > 50% shows there is more heterogeneity among studies. A value of 0% indicates no observed heterogeneity, and larger values show increasing heterogeneity [The diagnostic OR expresses how much greater the odds of having sepsis are for newborns who have a positive test result, relatively to newborns who have a negative result . For theogeneity . We explQ* values was received from the SROC curves. Meanwhile, the area under the (SROC) curves was also calculated from the SROC curves, which have been proposed as a way to assess diagnostic data in the context of a meta-analysis [In order to summarize these results, we constructed summary receiver operator characteristic (SROC) curves, which showed the relationship between sensitivity and the false positives (1-specificity). analysis .The literature search was completed in August 2013. We found 347 potentially relevant restudies, but only 15 articles met our inclusion criteria. α test in the diagnosis of proven early-onset neonatal sepsis (EONS). EONS was defined as the clinical sepsis in the 0–72 h after delivery, met the inclusion criteria in [Eleven articles and twelve trials were included to estimate the use of the TNF-teria in –27.I2 = 87.5%; specificity, I2 = 88.7%); it indicated that patient selection or other relevant factors might be responsible for heterogeneity.In these trials, we can get the TP, TN, FP, FN, sensitivity, specificity, DOR, PPV, and NPV from the articles. The sensitivity ranged from 20.8% to 100% and pooled sensitivity is 66.1% (95% CI 60.7%–70.1%), specificity ranged from 43.1% to 100% and pooled specificity is 75.6% (95% CI 72.2%–78.9%), and the detailed forest map is shown in α was 7.43 (95%CI 3.47–15.90), as shown in I2 = 77.9%). The SROC curve for TNF-α markers was plotted in Q*) of 0.7430 with the standard error was 0.04.The value of DOR of TNF-α test in the diagnosis of proven late-onset neonatal sepsis (LONS). LONS was defined as the clinical sepsis 72 h after birth, met the inclusion criteria in [Six articles and eleven trials were included to estimate the use of the TNF-teria in , 28–30.I2 = 91.9%; specificity, I2 = 87.5%), which indicated that patient selection or other relevant factors might be responsible for heterogeneity.In these trials, we can also get much information from the articles. The sensitivity ranged from 23.1% to 100% and pooled sensitivity is 68.0% (95% CI 62.8%–72.8%), specificity ranged from 73.2% to 100% and pooled specificity is 88.5% (95% CI 85.9%–90.7%), and the detailed forest map is shown in α was 37.44 (95% CI 19.07–73.48), as shown in I2 = 41.6%). The SROC curves for TNF-α markers were plotted in Q*) of 0.8696 with the standard error was 0.02.The value of DOR of TNF-In the LONS study, study populations come from different countries, but in general they can be further divided into two regions: the northern hemisphere and the southern hemisphere.α test in the northern hemisphere at the diagnosis of proven late-onset neonatal sepsis [I2 = 77.4%; specificity, I2 = 76.3%), which indicated that patient selection or other relevant factors might be responsible for heterogeneity.Eight trials were included to estimate the use of the TNF-l sepsis , 29, 30.α test in the northern hemisphere at the diagnosis of proven late-onset neonatal sepsis was 44.94 (95% CI 20.71–97.50), as shown in I2 = 47.1%). The SROC curves for TNF-α markers were plotted in Q*) of 0.8710 with the standard error was 0.02.The value of DOR of TNF-α test in the southern hemisphere at the diagnosis of proven late-onset neonatal sepsis [I2 = 0%; specificity, I2 = 56.0%), which indicated that patient selection or other relevant factors might be responsible for heterogeneity.Three trials were included to estimate the use of the TNF-l sepsis , 28. In α test in the northern hemisphere at the diagnosis of proven late-onset neonatal sepsis was 20.88 (95% CI 3.84–113.49), as shown in I2 = 35.5%). The SROC curves for TNF-α markers were plotted in Q*) of 0.1052 with the standard error was 0.309.The value of DOR of TNF-α test for NS in LONS, we compared TNF-α with PCT and CRP in LONS. Six articles and eleven trials were used to evaluate the diagnosis of TNF-α. Compared with 55% (95% CI 45%–65%) for the CRP test and 72% (95% CI 63%–81%) for the PCT test [α test was 68% (95% CI 63%–73%).In LONS, PCT and CRP have been proven to be useful markers for the diagnosis of NS , 9, 31. PCT test , the pooα was slightly higher than for the CRP and PCT test (88.5% (95% CI 86%–91%) versus 85% (95% CI 81%–88%) versus 77% (95% CI 72%–81%)). Furthermore, the pooled diagnostic OR for the TNF-α was higher than CRP and PCT (37.4 (95% CI 19.1–73.5) versus 8.6 (95% CI 3.5–21.0) versus 11.6 (95% CI 5.2–26.0)). The Q* value was slightly higher for the TNF-α than CRP and PCT (0.87 versus 0.75 versus 0.77). In SROC curve the TNF-α's AUC is almost equal to CRP (0.93 versus 0.96). As many articles reported that PCT and CRP are good markers, the TNF-α is also a good marker for the diagnosis of NS in LONS.The pooled specificity for the TNF-Heterogeneity is very critical in a meta-analysis, so we should try to explore the reason for the heterogeneity. Generally speaking, variations include several influence factors, for instance the cut-off value, study population, inject antibodies, and so forth.I2 (heterogeneity test) almost does not change (41.7% versus 41.6%). So the cut-off value is not the reason for the heterogeneity. Secondly, we consider the study population. The I2 is reduced from 41.6% to 0% when two studies of [Firstly, we consider the cut-off value. In our study, many data are so large, so we suppose that this is the possible reason for the heterogeneity. We excluded two studies of , 28 whosudies of  were excThe publication bias is difficult to avoid in meta-analysis . In eachNS is one of the most common diseases and life-threating disorder in neonate, and thus it can bring the high mortality and morbidity in infants. So the identification of biomarkers is very important to improve the diagnosis of NS. The clinical signs are nonspecific and laboratory indicators including blood culture are not reliable . The senα tests' sensitivity is 0.66 for the diagnosis of early-onset neonatal sepsis, and the specificity is 0.46 and the Q* is 0.74. At the late-onset neonatal sepsis, the TNF-α test's sensitivity is 0.68, whereas the specificity is 0.89 and the Q* is 0.87. In particular, TNF-α shows a higher accuracy for the diagnosis of NS in LONS. Therefore, we have further analyzed the regional issues in LONS. To this end, we have classified studies into two groups, that is, northern hemisphere group [α has appropriate accuracy for the diagnosis of NS and thus is a good biomarker for the diagnosis of NS.It is well known that an excellent marker should have high sensitivity and specificity. In our meta-analysis, the TNF-re group , 29, 30 re group , 28. Ourα' sensitivity ranged from 23.1% to 100%, and specificity ranged from 73.2% to 100%. The study from TNF-α is similar to CRP, so the TNF-α is a useful marker in the diagnosis of NS. In addition, PCT is more excellent marker which has better accuracy than CRP for the diagnosis of NS [α is slightly lower than that of the PCT test in EONS (66.1% versus 74%), the pooled specificity also in such (76% versus 86%). But in LONS, the pooled specificity of TNF-α is higher than that of the PCT test (89% versus 77%), although the pooled sensitivity is slightly lower than PCT (68% versus 72%). Generally speaking, the data of TNF-α and PCT is greater than CRP. This result again shows that TNF-α is good marker in the diagnosis of NS.CRP is an excellent marker and has been applied in clinic . The senα shows the moderate accuracy in the diagnosis of NS, both in EONS and LONS. If we test the accuracy of TNF-α by further dividing data into two regions, the study in northern hemisphere shows a better result. Because of the relatively few testing data, the experiments results need to be further studied and the clinical validation is also needed.In conclusion, TNF-


Tumor necrosis factor (TNF) is an important cytokine in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF antibodies have been successfully implemented in IBD therapy, however their efficacies differ among IBD patients. Here we investigate the influence of CD64 Fc receptor on the inhibitory activity of anti-TNFs in cells of intestinal wall.Intestinal cell lines, monocytes/macrophages and peripheral blood mononuclear cells (PBMCs) were used as models. The efficacies of adalimumab, infliximab and certolizumab-pegol were assessed by RT-PCR for target genes. Protein levels and localizations were examined by Western blotting and immunofluorescence. Antibody fragments were obtained by proteolytic digestion, immunoprecipitation and protein chip analysis. Knock-down of specific gene expression was performed using siRNAs.Infliximab had limited efficacy towards soluble TNF in cell types expressing Fc gamma receptor CD64. Both adalimumab and infliximab had lower efficacies in PBMCs of IBD patients, which express elevated levels of CD64. Infliximab-TNF complexes were more potent in activating CD64 in THP-1 cells than adalimumab, which was accompanied by distinct phospho-tyrosine signals. Blocking Fc parts and isolation of Fab fragments of infliximab improved its efficacy. IFN-γ-induced expression of CD64 correlated with a loss of efficacy of infliximab, whereas reduction of CD64 expression by either siRNA or PMA treatment improved inhibitory activity of this drug. Colonic mRNA expression levels of CD64 and other Fc gamma receptors were significantly increased in the inflamed tissues of infliximab non-responders.in vitro and ex vivo, whereas the presence of this receptor has no impact on the inhibitory activity of certolizumab-pegol, which lacks Fc fragment. These data could be helpful in both predicting and evaluating the outcome of anti-TNF therapy in IBD patients with elevated systemic and local levels of Fc receptors.CD64 modulates the efficacy of infliximab both  Inflammatory bowel disease (IBD) is a chronic, relapsing/remitting inflammatory condition of the gastrointestinal tract, which can be subdivided into two types: Crohn's disease (CD) and ulcerative colitis (UC). Although many pro-inflammatory cytokines have been implicated in the pathology of IBD, one of them, namely tumor necrosis factor (TNF), plays a pivotal role in the course of the disease 1 antibody , humanized monoclonal anti-TNF IgG1 antibody  and pegylated humanized anti-TNF Fab′ fragment . Until now, numerous in vitro models have been employed in order to study the efficacy of these drugs. Most of those studies focus on the comparison between different anti-TNFs using single type of assays or overexpression systems. However, what is lacking so far is the comparison between different cell types potentially targeted by TNF at the site of inflammation. In addition to the classical TNF neutralizing effect, anti-TNF agents are also capable of inducing mTNF-dependent signaling Based on the crucial role of TNF in inflammation, it was assumed that anti-TNF therapy would be potentially beneficial in chronic inflammatory conditions such as IBD Interestingly, it has been reported that anti-TNF therapeutics bind to Fc receptors in an Fc fragment-dependent manner Here, we report that binding of infliximab to CD64 modulates its inhibitory activity in different cell types of intestinal wall and that this has consequences for the infliximab therapy outcome in IBD patients.BBE , which could contribute to the observed differences between ADA and IFX, we next investigated the activation of Fc receptors in a setting where anti-TNF IgGs and TNF were pre-incubated to form immune complexes prior addition to the cell cultures. Interestingly, we observed that ADA, either alone or in complex with TNF, was more potent in inducing phospho-tyrosine signals than IFX , suggestIn order to investigate whether binding to Fc receptors can indeed influence the inhibitory efficacy of infliximab, we pre-incubated the cells with human serum containing high amounts of IgGs prior to stimulation with anti-TNF therapeutics. As expected, human serum improved the efficacy of infliximab in case of both fibroblasts and THP-1 cells , but theIn order to verify the role of Fc part of infliximab in limitation of its efficacy, we blocked the Fc fragments using protein A sepharose beads. To test the binding of anti-TNF antibodies to protein A sepharose beads, we employed standard SDS-PAGE/Coomassie approach ,A. Once In order to confirm that Fc fragments of ADA and IFX indeed modulate the efficacies of these drugs, we elevated the expression of high affinity IgG receptor CD64 prior to treatments. To this end, cells were pre-treated with IFN-γ for 48 h, followed by short-term incubation with anti-TNFs and treatment with TNF. Consistently with the existing data for both monocytes To investigate the role of Fc receptors in the responsiveness towards anti-TNF therapy we screened colonic biopsies of IFX responders and non-responders together with the healthy control subjects for the mRNA expression levels of various inflammatory targets and Fc receptors . EndoscoIn the present study we propose a new mechanism that modulates the inhibitory efficacy of infliximab, which is dependent on both its Fc fragment and the presence of high affinity Fc gamma receptor CD64. We have demonstrated that IFX was inefficient in cell types expressing CD64 including fibroblasts, monocytic THP-1 cells and different subsets of immune cells (PBMCs). We demonstrated that IFX was more potent in activating CD64 by inducing the transcription of pro-inflammatory genes, such as IL-8 and MCP-1. This was in striking contrast to CZP, which blocks TNF, but lacks Fc region. Fab fragments of IFX have statistically higher inhibitory efficacy, compared to the whole IgG molecules and both blocking Fc parts of this drug and changes in expression of CD64 correlate with the changes in its ability to block TNF-induced responses. Perhaps most importantly, we have shown that IFX non-responders express higher mRNA levels of CD64 in their inflamed colonic tissues.In agreement with our findings, it has been shown that ADA and IFX bind to Fc receptor-expressing THP-1 cells in a manner that is dependent on the addition of the human recombinant Fc fragment Our results imply that there are differences between tested anti-TNF IgGs in their Fc fragments, which could be directly responsible for the observed phenomena. Different glycosylation patterns within Fc fragment might be responsible for the observed differences in the efficacies between ADA and GOL . It is kRecently, it has been shown that certolizumab-pegol does not show significant therapeutic efficacy as compared to placebo in a large cohort of IBD patients indicating the importance of Fc fragment in providing desired therapeutic effect Recently, CD64 has been implicated as a modulator of responses against microbial challenge in the context of mucosal inflammation in the intestine in vitro , including 14 CD and 8 UC patients. Colonic biopsies were taken from the sigmoid colon in all patients except one CD patient in whom the sigmoid had been removed earlier after perforation. Specimens were taken from either inflamed or non-inflamed regions. Most IBD patients were immunosuppressed at the time of colonoscopy See . As contHuman recombinant IFN-γ (I3265), mouse monoclonal anti-human β-actin antibody (A5441), DAPI (D9564) and PMA (P1585) were purchased from Sigma-Aldrich . Recombinant TNF (C-63720) was from PromoCell/PromoKine . Human recombinant IL-1β (407615) was purchased at Calbiochem-Merck Chemicals (Germany). Adalimumab was from Abbott , infliximab from Essex Chemie AG , certolizumab-pegol from UCB Pharma , and golimumab from Centocor Ortho Biotech Inc. . Mouse monoclonal anti-α-smooth muscle actin (SMA) antibody (ab40865) was from Abcam , anti-villin antibody (MAB1639) from Chemicon (USA), and anti-CD16 (sc-70548), anti-CD32 (sc-53663), anti-CD64 (sc-1184), anti-TNFR1 (sc-7895) and anti-fibronectin (sc-9068) antibodies were from Santa Cruz Biotechnology . Anti-CD64 antibody (3860-1) used for detection of glycosylated band was obtained from Epitomics . Human IgG1 isotype control (ALX-804-133) was purchased at Enzo Life Sciences . CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems . For CMV detection we used primers specific for viral DNA polymerase UL54 BBE, CCD-18Co, and THP-1 cell lines were cultivated at 37°C in a 5% CO2 incubator. Both Caco2BBE and CCD-18Co cell lines were cultivated in high-glucose DMEM medium (Sigma-Aldrich) containing L-glutamine, sodium bicarbonate, 10% FCS, penicillin (100 U/ml) and streptomycin (100 µg/ml). THP-1 cells were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FCS, penicillin (100 U/ml) and streptomycin (100 µg/ml). All cell lines were purchased from ATCC-LGC Promochem . Ko77, F141, F148, and F188 fibroblasts were isolated and cultivated as described elsewhere Caco2Cell lines were incubated with pro-inflammatory cytokines TNF, IL-1β, and IFN-γ at the concentration of 50 ng/ml. Concentrations of the anti-TNF IgG1s used in the experiments were 10 µg/ml  and 24 µg/ml for certolizumab-pegol. For inhibition experiments cells were pre-incubated with anti-TNF therapeutics for 60 min at 37°C prior to treatment with TNF (50 ng/ml) for another 60 min.RNA was isolated with QIAcube  using the protocol for animal cell isolation with DNAse digest, or alternatively using TRIzol reagent . The amount and quality of RNAs were determined using ND-1000 spectrophotometer . Complementary DNAs were produced using High Capacity cDNA Reverse Transcription Kit . Real-time PCR reactions containing 10 µl of Fast Universal Master Mix (Applied Biosystems) and 1 µl of a TaqMan gene expression assay (Applied Biosystems) in 20 µl of total volume. The cycle conditions were: stage 1 ; stage 2 ; stage 3: 40 repeats . The expression levels of the target genes were normalized to the expression levels of β-actin using the comparative threshold cycle method.Protein concentrations were determined using the BCA Protein Assay Reagent .Protein electrophoresis was performed according to the standard protocol. Briefly, proteins were separated in 10% polyacrylamide gels (Tris/glycine) and transferred onto nitrocellulose membranes. For Western blotting, membranes were blocked with 5% solution of non-fat dried milk buffer in TBS-T (TBS+0.1%Tween) for 1 h at RT with gentle shaking. Primary antibodies were diluted in TBS-T containing 5% BSA and incubated with the membranes overnight at 4C°. Corresponding secondary antibodies were diluted in TBS-T buffer containing 5% blocking milk for 1 h at RT. Bands were detected by using ECL detection kit  and autoradiography. Densitometry analysis of the protein bands was performed using OptiQuant software.CCD-18Co cells were plated onto sterile glass coverslips and cultured until full confluence. Prior to their fixation cells were washed once with PBS and fixed with 3.7% solution of formaldehyde for 30 min at RT. After washing three times with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min at RT prior to incubation with blocking solution  in PBS) for 1 h at RT, and subsequently with anti-fibronectin and anti-CD64 antibodies overnight at 4°C. Coverslips were incubated with corresponding secondary anti-rabbit Cy2 and anti-mouse Cy3 antibodies diluted in blocking solution. Samples were mounted using anti-fade mounting medium  and analyzed by fluorescence microscopy .Fresh human peripheral blood (40 ml) was divided into eight parts for treatment with anti-TNFs and soluble TNF according to the protocol used for cell lines (see above). PBMCs were isolated in according to standard Ficoll  protocol. Briefly, diluted blood was overlayed onto Ficoll and centrifuged at 1700 rpm for 40 min. PBMCs from interphase were collected, washed once with PBS+0.1% BSA and harvested for RNA extraction. Detail description of blood samples from both healthy donors and IBD patients used for this set-up is listed in Complexes of ADA or IFX and TNF were formed by incubation at 37°C for 30 min in PBS prior to addition to cells. The ratio of anti-TNFs (100 µg/ml) vs soluble TNF (500 ng/ml) was the same as for stimulation experiments (see above).Adalimumab and infliximab were incubated with protein A sepharose beads  for 30 min at RT prior to incubation with fibroblasts for 30 min at 37°C. Afterwards, the cells were treated with TNF for 1 h, harvested and processed for RNA isolation.Adalimumab and infliximab were digested by IdeS endopeptidase as described elsewhere 6 cells per condition were suspended in 100 µl of Nucleofector® solution and 6 µl of 50 µM siRNA mix containing three different siRNA sequences: s5069, s5068, and s230564  were added for 10 min at RT. Next, the samples were subjected to nucleofection using high viability protocol (V001) and grown in standard medium for 3 days.Transient downregulation of CD64 expression in THP-1 cells was achieved using Nucleofector® kit V . Specifically, 10Statistical significance was determined by performing either ANOVA analysis with Tukey's multicomparison post-test or two-tailed paired t-test using Graph Pad Prism 5 software .Figure S1Ko77 and CCD-18Co fibroblast are CMV-positive. (A) Real-time PCR amplification curves show the expression of viral UL54 mRNA in both fibroblastic cell lines used in the study. The specificity of the PCR products was confirmed by the kinetics of dissociation curves for β-actin (B) and UL54 (C).(TIF)Click here for additional data file.Figure S2Intestinal epithelial cells, intestinal myofibroblasts and monocyte-macrophage cells can be efficiently treated with inflammatory cytokines to induce inflammatory responses. Caco2BBE (A–C), CCD-18Co (D–F) and THP-1 (G–I) cell lines were treated with three different cytokines to induce pro-inflammatory responses. Cells were harvested at different time points to monitor the kinetics of mRNA production of different genes. Values on Y-axis represent mRNA expression levels relative to ß-actin. Columns represent the mean values of three measurements within a single, representative experiment. Error bars represent SD.(TIF)Click here for additional data file.Figure S3Pro-inflammatory cytokines trigger specific signaling pathways in the cell lines used in the study. (A) IFN-γ, but neither TNF nor IL-1β, induces phosphorylation of STAT1 in intestinal epithelial Caco2BBE cells. (B) TNF induces phosphorylation of CREB, ATF-1 and p38 MAPK proteins in intestine-derived fibroblasts CCD-18Co. (C) TNF, but not IFN-γ, activates NF-κB in Caco2BBE cells as measured by electrophoretic mobility shift assay. Cells were treated with two different pro-inflammatory cytokines to test the specificity of the binding to the NF-κB-specific radiolabelled probe. Maximum activation was observed after 60 min. Addition of anti-p65 antibodies shifts the size of the protein-DNA complexes towards higher molecular weight, showing the specificity of the protein binding to the probe. (D) IL-1β activates NF-κB in Caco2BBE cells as measured by EMSA. Maximum activation was observed after 30 min. All cytokines were used at the concentration of 50 ng/ml.(TIF)Click here for additional data file.Figure S4Infliximab has limited efficacy in fibroblasts isolated from CD patients. (A) Fibroblasts isolated from CD patient (MC153) and (B) isolated from fistulizing CD patient (F188) were incubated with either adalimumab or infliximab before treatment with TNF. Columns represent the mean values of three measurements within a single, representative experiment relative to ß-actin. Error bars represent SD. Caco2BBE cells, intestinal fibroblasts and THP-1 cells express Fc receptors (C), but not mTNF (D). Recombinant TNF was used as a positive control (17 kDa). M: Molecular weight marker.(TIF)Click here for additional data file.Figure S5Golimumab displays reduced inhibitory efficacies in intestinal fibroblasts and THP-1 cells, but not in intestinal epithelial Caco2BBE cell line. (A) THP-1 cells (B) Caco2BBE, (C) Ko77, and (D) CCD-18Co cells were pre-incubated with golimumab and subsequently treated with TNF. The graphs show the results of a single experiment, measured in triplicates. Error bars represent SD.(TIF)Click here for additional data file.Figure S6Human serum changes the efficacy of IFX on fibroblasts and monocytes. (A) CCD-18Co fibroblasts were pre-incubated with human serum for 30 min prior to treatment with anti-TNF therapeutics and TNF. Graph shows result of a single experiment measured in triplicate. Error bars indicate SD of three measurements. (B) Quantification of TNF inhibition by infliximab in CCD-18Co fibroblasts expressed as percentage. (C) THP-1 cells were pre-incubated with human serum for 30 min prior to treatment with anti-TNF therapeutics and TNF. Graph shows result of single experiment measured in triplicates. Error bars indicate SD of three measurements. (D) Quantification of TNF-induced response inhibition by infliximab in THP-1 cells expressed as percentage. Graphs show the results of single experiment measured in triplicates.(TIF)Click here for additional data file.Figure S7Blocking of Fc and isolation of Fab fragments can be successfully applied for testing the efficacies of anti-TNFs. (A) Both adalimumab and infliximab, but not certolizumab-pegol bind to protein A sepharose beads. (B) Fab and Fc fragments can be produced by incubation with recombinant immunoglobulin-degrading enzyme of Streptococcus . (C) After proteolytic digestion Fab fragments of ADA and IFX were removed from the reaction mixture by immunoprecipitation using protein A sepharose beads. (D) Ko77 fibroblasts were pre-treated with either intact IgG1 molecules or Fc-purified digestion mixture containing Fab′ fragments of ADA or IFX and subsequently stimulated with TNF. The percentage of TNF inhibition was assessed based on the production of TNF mRNA by RT-PCR.(TIF)Click here for additional data file.Figure S8IFN-γ does not induce CD64 mRNA expression and does not influence inhibitory efficacy of IFX in Ko77 fibroblasts. mRNA expression levels of CD64 in Ko77 fibroblasts (A) and THP-1 cells (B) upon IFN-γ treatment. IFN-γ-induced progressive elevation of ICAM-1 mRNA levels in Ko77 (C), which was similar to the effect observed for CCD-18Co fibroblasts  m(TIF)Click here for additional data file.Figure S9The expression levels of CD64 can be decreased by either siRNA or PMA treatment in THP-1 cells. (A) Western blot analysis of CD64 in pGFP- and siRNA-transfected cells. The mRNA expression levels of CD64 (B), macrophage-specific marker CD68 (C) and CD16 (D) analyzed 48 h after the treatment with different amounts of PMA. The graphs show results from a single representative experiment measured in triplicates. Error bars indicate SD of triplicates.(TIF)Click here for additional data file.Figure S10Glycosylation at Asn297 does not influence the inhibitory efficacy of anti-TNF therapeutic antibodies. (A) Both adalimumab and infliximab were deglycosylated at Asn297 as indicated by the shift in molecular mass in SDS-PAGE and Coomassie staining. This modification did not result in significant change in the inhibitory efficacy of anti-TNF antibodies in either Ko77 fibroblasts (B) or in monocytic THP-1 cells (C). The graphs show results from a single experiment measured in triplicate. Error bars indicate SD of triplicates. ADA(d): de-glycosylated adalimumab; IFX(d): de-glycosylated infliximab.(TIF)Click here for additional data file.Table S1Basic characteristics of peripheral blood donors used in the study. 5-ASA: 5-aminosalicylic acid, IFX: infliximab.(TIF)Click here for additional data file.Table S2Basic description of human intestinal specimens used in the study. 5-ASA: 5-aminosalicylic acid; IFX: infliximab; ADA: adalimumab; ABA: abatacept; VED: vedolizumab; MTX: methotrexate; CZP: certolizumab-pegol; nd: not determined; N/A: not applicable.(TIF)Click here for additional data file.