Background: Neuro-inflammatory circuits in the tissue regulate the complex pathophysiology of pain. Protective nociceptive pain serves as an early warning system against noxious environmental stimuli. Tissue-resident mast cells orchestrate the increased thermal sensitivity following injection of basic secretagogue compound 48/80 in the hind paw tissues of ND4 mice. Here we investigated the effects of pre-treatment with TNF-α neutralizing antibody on compound 48/80-provoked thermal hyperalgesia.Methods: We treated ND4 Swiss male mice with intravenous anti-TNF-α antibody or vehicle 30 minutes prior to bilateral, intra-plantar compound 48/80 administration and measured changes in the timing of hind paw withdrawal observed subsequent to mice being placed on a 51oC hotplate. We also assessed changes in tissue swelling, TNF-α gene expression and protein abundance, mast cell degranulation, and neutrophil influx in the hind paw tissue.Findings: We found that TNF-α neutralization significantly blocked thermal hyperalgesia, and reduced early tissue swelling. TNF-α neutralization had no significant effect on mast cell degranulation or neutrophil influx into the tissue, however. Moreover, no changes in TNF-α protein or mRNA levels were detected within 3 hours of administration of compound 48/80.Interpretation:  The neutralizing antibodies likely target pre-formed TNF-α including that stored in the granules of tissue-resident mast cells. Pre-formed TNF-α, released upon degranulation, has immediate effects on nociceptive signaling prior to the induction of neutrophil influx. These early effects on nociceptors are abrogated by TNF-α blockade, resulting in compromised nociceptive withdrawal responses to acute, harmful environmental stimuli. Compound 48/80 (c48/80)-provoked thermal hyperalgesic responses in the hind paw tissue of male ND4 Swiss mice are mediated by the degranulation of tissue-resident mast cells2. The increased sensitivity to thermal stimulus is dependent, in part, on neutrophil influx into the affected tissue, and on histamine signaling; the response is completely abrogated in mast cell-deficient mice, and is substantially reduced by blocking neutrophil influx or treatment with histamine receptor antagonists2. The importance of neutrophil influx in nociceptive cascades has been shown in different rodent models5 and the important regulatory roles of inflammatory cytokines including IL-1β, TNF-α, and IL-6 in pain signaling have also been demonstrated7. Here we show that c48/80-induced early thermal sensitivity in ND4 Swiss mice is markedly reduced by pre-treatment with a neutralizing antibody against TNF-α. Anti-TNF-α antibody administration does not significantly affect levels of mast cell degranulation or neutrophil influx into the affected tissue. We also found no increase in protein and mRNA levels of TNF-α in the tissue within the first 3 hours following c48/80 administration suggesting that this blockade targets pre-formed TNF-α including that stored in the granules of tissue-resident mast cells. Our data suggest that pre-formed TNF-α may act rapidly on nociceptors that are known to reside in close proximity to mast cells8 and modulate nociceptor sensitization thresholds9 and function10. TNF-α blockade therefore compromises the early, protective nociceptive withdrawal responses that normally act to protect tissues from further exposure to acute, injurious environmental stimuli11.Pain is one of the cardinal components of inflammation and tissue-resident immune cells are important players in the regulation of protective nociceptive responsesThree-six month old male ND4 Swiss mice  were housed in Macalester College’s research animal facility with a 12-hour light/dark cycle and free access to food and water. A total of 73 mice were used for the experiments shown here  to ensure that appropriate statistical analysis could be performed on data acquired from these experiments. Macalester College’s Institutional Animal Care and Use Committee approved all experimental procedures (Protocol B08Su1).2. Either 200μg/kg of anti-TNF-α neutralizing antibody  or 200μl vehicle was injected intravenously 30 minutes prior to c48/80 injection in a protocol adapted from Rochaet al.12. Rochaet al. administered anti-TNF-α antibodies i.v. 5 mins prior to carrageenan treatment in a mouse model of mechanical hyperalgesia12.All drugs were administered using 0.9% saline  vehicle or phosphate buffered saline . Mice received bilateral intra-plantar (i.pl.) treatments with c48/80 ) or saline alone as previously described2. Two baseline hotplate latencies were taken 24 and 48 hours before the experiment. Mice with >10 second differences between baselines or <15 second averages were excluded from the experiment. Nociceptive thermal sensitivity was quantified by subtracting the mean baseline thermal latency from the experimental thermal latency at each time point for each mouse.To assess thermal sensitivity, single mice treated with i.pl. c48/80 or vehicle were placed in a Plexiglas cylinder on a hotplate analgesia meter  maintained at 51.0 ± 0.5°C and removed when prolonged retraction, flipping/licking of the hind paw, or jumping with both hind paws off the hotplate were observed, but no later than 40 seconds, as previously described2.Change in hind paw width measured using digital calipers  was calculated as an average of the left and right paw widths. Baseline paw widths for each mouse were taken pre-treatment and subtracted from post-treatment paw widths to calculate tissue edema as previously described2 inhalation, weighed, frozen at -80°C in 5.6μl/mg tissue weight of 50mM K2HPO4 buffer (pH 6.0)  containing 0.05% hexadecyltrimethylammonium bromide (HTAB) , thawed, homogenized in 5x the storage volume of HTAB buffer, sonicated with a 550 Sonic Dismembrator , freeze-thawed, and centrifuged  as previously described13. Absorbance was recorded using a BioTek PowerWave XS plate reader  at 450nm after a 20-minute incubation in 50mM phosphate buffer (pH 6.0) with 0.025% hydrogen peroxide  and 0.167mg/mlo-dianisidine-dihydrochloride  at room temperature in the dark. Myeloperoxidase (MPO) levels were normalized to tissue weight and presented as OD/g of wet tissue.Footpads were extracted from hind paws of mice euthanized by CO2 inhalation, flash-frozen in liquid nitrogen and stored at -80°C.For protein and gene expression studies, hind paws were excised from mice euthanized by COFor protein studies, flash-frozen paws were homogenized in Cell Lysis Buffer  supplemented with protease inhibitor  using a Tissue-Tearor . Homogenates were incubated on ice for 20 minutes, centrifuged at 2000 rpm for 10 minutes at 4°C, and lysate supernatants stored at -80°C. We quantified TNF-α cytokine levels by ELISA according to the manufacturer’s instructions .14 using TaqMan Gene Expression Assay Primer/Probe Sets and TaqMan MasterMix  in a Bio-Rad iCycler  using β-2-microglobulin  and TNF-α  primer sets. Fold expression was normalized to β-2-microglobulin levels and calculated as previously described15.To measure cytokine gene expression, total RNA was extracted from flash-frozen plantar tissue , quantified with Nanodrop ND-1000 Spectrophotometer , and reverse-transcribed in a 2720 Thermal Cycler  using the Superscript III First-Strand Synthesis System  with 100ng of RNA per reaction. Relative transcript abundance was determined by quantitative comparative reverse transcriptase PCR (qRT-PCR)2 inhalation) were fixed in 10% buffered formalin  for 24 hours, transferred to 70% ethanol , decalcified for 1–2 weeks in 15% EDTA, hydrated, and embedded in paraffin. 4μm sagittal sections were stained with toluidine blue (Tb) at pH 2.3 for 3 minutes  or hematoxylin and eosin (H&E)  for the detection of mast cells or neutrophils, respectively, at 400x magnification with ≥3 biological replicates per treatment. Tb-stained mast cells were counted in 10 fields/section and degranulation was scored based on the number of granules observed outside the boundary of the cell: intact (0), mild (1–10), moderate (10–20), and extensive (20+) as previously described2. The same investigator performed all counts and was blinded to treatment groups. Neutrophils were imaged between the heel and the toes on the plantar side of the hind paw section using an Olympus CX21LED light microscope and camera .Excised hind paws  and graphed with PRISM 5.0 . All statistical analyses were performed using JMP 9.0 . All data are presented as the mean ±SEM. Data were analyzed using one-way ANOVA, and the Tukey-Kramer HSDMale ND4 Swiss mice bilaterally injected with 1500ng of c48/80 in the hind paws showed hyperalgesic withdrawal responses ~10 seconds sooner than their baseline withdrawal 30 minutes after treatment; these responses were resolved by 2.5 hours . Pre-tre2 and confirmed that the levels of mild, moderate and extensive degranulation were not altered in anti-TNF-α treated mice  in the hind paw tissue of mice injected bilaterally with 1500ng of c48/80 were not significantly reduced with anti-TNF-α pre-treatment  values of beta2m (reference) and TNF-alpha.Neutrophil counts: Neutrophils were counted in the plantar region from heel to the toes, in 10 separate sections (fields of view) at 1000x. Total number of neutrophils equals the sum of neutrophils in all 10 sections.Myeloperoxidase activity: The data show optical density (OD) readings at 450nm and "BA" values = OD - average of the blanks. Average "BA" values per gram of tissue weight = MPO activity as OD/gm of wet tissue.Mast cell degranulation: The data show numbers and percentages of intact and mildly, moderately, extensively degranulated mast cells in the hind paw tissue.Hyperalgesia: "BL Latency" signifies baseline latency values in seconds and "Delta latency" signifies change in thermal latency  at each time point for each mouse.Edema: "BL R" and "BL L" = baseline paw width in mm for the right and left hindpaw of each mouse respectively. Baseline values are subtracted from averages of left (L) and right (R) paw widths at each time point to give the delta paw width at each time point.ELISA: The data show the concentrations of TNF-alpha protein in hind paw tissue lysate assayed in duplicate and the average concentration in pg/ml of paw lysate. Click here for additional data file.11 and in the former instance, serves as an early warning system that prompts an organism to retreat from noxious environmental stimuli.Pain is a complex physiological and pathological phenomenon with intricate underlying neuro-immune circuitry. Pain can be either protective or maladaptive2. Here we show that pre-treatment with TNF-α neutralizing antibody just prior to c48/80 administration significantly reduces early thermal hyperalgesic responses. TNF-α neutralizing antibody treatment does not affect mast cell degranulation; levels of mild, modest and extensive degranulation following c48/80 treatment were similar to those previously described2. Mast cells contain pre-formed TNF-α in their cytoplasmic granules and this pro-inflammatory cytokine is one of the earliest mediators released by these cells upon activation16. We looked for evidence of changes in TNF-α protein and mRNA levels shortly after c48/80 treatment and found none, suggesting that the pre-formed TNF released by mast cells may be the main source of this cytokine in these experiments. As TNF-α is a known attractant of neutrophils17, we expected TNF-α blockade to result in a reduction of infiltrating neutrophils. We have previously shown that blockade of neutrophil influx can abrogate c48/80-provoked thermal sensitivity2. However, we found here that in the first 2.5 hours following c48/80 administration, TNF-α neutralizing antibody pre-treatment had little to no effect on recruitment of tissue neutrophils. The influx of neutrophils in the presence of TNF-α neutralizing antibodies is supported by several different kinds of evidence – myeloperoxidase activity in the tissue, visualization of neutrophils by H&E staining and neutrophil counts in H&E stained tissue sections. However, given our relatively small sample size for these assays, it is possible that a reduction in neutrophil influx can be seen with larger numbers of mice. Nevertheless, our observations are in keeping with earlier studies that show that mast cell mediators other than TNF-α can contribute to the induction of neutrophil influx18. In these experiments, pain was significantly abrogated as early as 30 minutes after c48/80 treatment despite the presence of neutrophils.We have previously shown that mast cells are required for enhanced sensitivity to heat stimulus following c48/80 treatment in the hind paw tissue of mice and blockade of neutrophil influx or histamine signaling can abrogate these responses7. Direct injection of TNF-α in the hind paw tissue produces thermal and mechanical hyperalgesia in rodents19; anti-TNF-α antibody administration shortly before challenge reduces the hyperalgesic responses to carrageenan12 in mice and blockade of TNF-α induced neutrophil influx impairs thermal carrageenan pain in rats3.TNF-α is one of the early inflammatory cytokines with a known role in various nociceptive cascades3 but rather the release of TNF-α and other cytokines such as IL-1β in the tissue following an inflammatory insult serves to attract infiltrating neutrophils that continue to fuel the nociceptive cascade3. In preliminary experiments, we have found that pre-treatment with antibodies against IL-1β reduces neutrophil influx into the tissue but does not abrogate c48/80-provoked thermal pain at early time points (data not shown). Therefore, the rapid anti-hyperalgesic effect that we observe here may be due to the blockade of earlier effects of TNF-α on nociceptor sensitization mediated by cyclooxygenase (COX) and p38 mitogen-activated protein (MAP) kinase activation20 or effects of TNF-α on nociceptor ion channels21. Our observations here suggest that pre-formed TNF-α, e.g. from mast cell granules in the skin mast cells, is the likely target of the TNF-α neutralizing antibody that produces an early reduction of thermal sensitivity. This is underscored by the documented proximity of nerves and mast cells in the tissue8 and the known effects of mast cell-derived TNF-α on nerve physiology - elongation of cutaneous nerves in oxazolone contact hypersensitivity22, and susceptibility of sensory neurons to agents such as capsaicin, in a model of tracheal vascular hyper-permeability23. These known interactions further support the idea that nociceptive neurons could be early downstream targets of the first release of pre-formed TNF-α immediately after mast cell degranulation. In this study, we show that early, protective, nociceptive withdrawal responses to heat stimulus are compromised by the blockade of TNF-α signaling. It has been documented that individuals who use TNF-targeted biological therapeutics to manage chronic autoimmune and inflammatory conditions can experience a higher risk of bacterial and viral infections24. Our findings here suggest that the use of TNF-α blockade therapies may also compromise the protective, nociceptive responses of such individuals leaving them more vulnerable to tissue damage from injurious environmental stimuli.Our findings here add nuance to the prevailing model of mast cell and neutrophil modulation of peripheral nociception with evidence that TNF-α blockade can also abrogate very early thermal pain responses without a proportional reduction in neutrophil influx. Hind paw thermal pain provoked by c48/80 is markedly abrogated despite the presence of infiltrating neutrophils in the tissue. In a model of carrageenan-induced pain in rats, Cunha and colleagues have shown that levels of TNF-α in the tissue are not affected by the absence of infiltrating neutrophils i.e. these cells are not the primary sources of TNF-α Thank you for addressing my comments.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The paper is now more clear, and has improved after addressing the referees' comments.Thank you and congratulations to the authors.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for addressing my previous comments. This paper makes an important contribution to the literature.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. show that TNF-alpha blockade reduced early tissue swelling in a mast cell-dependent thermal hyperalgesia model. The paper is well constructed and clear, and experiments have been well designed and conducted. There are minor questions that I mention below.Anti-TNF-a neutralizing antibody dose is mainly expressed in the article as ul/Kg, please write it as ug/Kg.Since toluidine blue staining is performed differently in different tissues please indicate pH and the time of incubation.In the Methods for TNF-a expression measurements it is said “relative transcript abundance was determined by quantitative RT-PCR” but the technique used is semi-quantitative.When explaining Figure 1A does not mention that at 30 minutes it is also significantly different when comparing the control and TNF treated groups.Please comment more on the mast cells degranulation status of figure 2. Is 60% mild degranulation for this model an expected value? Have you seen or would you expect different percentages using different C4880 doses?Pre-treatment with TNF-a antibody seems to have a small effect on MPO activity but not a significant one, probably because of the low n number (n = 3). This could be discussed as a possibility.ChatterjeaI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Dr. Torres,Thank you for reviewing our paper.  Our responses to your comments follow: 1. We have now corrected this throughout the manuscript.2. We have added this information to the relevant methods section.3. We have clarified that our approach uses comparative quantitative PCR analysis and added an additional reference for this nomenclature.  4. We have added this to the description of the report of the results from Figure 1A.5. We have added a sentence to the discussion indicating that the levels of degranulation seen here are similar to those we have previously observed.6. We have added some clarifying language indicating that these findings are based on a relatively small sample size but that multiple lines of evidence indicate that at least in these experiments, treatment with anti-TNF neutralizing antibodies did not completely abrogate neutrophil influx into the c48/80-treated hind paw tissue. et al. is well-designed; its conclusions are balanced and justified based on the results shown. I think that it could be relevant to know whether TNF-alpha injections can mimic the effects of c48/80 injections in the model used: hyperalgesic withdrawal responses.The study by Chatterjea DFigure 4a leads to the conclusion that neutrophil influx is not affected by treatment with anti-TNFalpha antibodies and c48/80. Data is based on n=3 according to figure legend, though there is a trend for neutrophil numbers to be reduced by treatment with anti-TNFalpha antibodies. I am wondering whether the authors have considered increasing the number of mice for this experiment. If it is definitely not the case that neutrophils numbers are reduced, it remains unexplained why there is a discrepancy with a previous study, in which the authors demonstrated that neutrophils are an important component of the hyperalgesic response induced by c48/80.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Dr. Piliponsky,Thank you for reviewing our paper. In our discussion of the finding in Figure 4A, we have now added some clarifying language indicating that these findings are based on a relatively small sample size but that multiple lines of evidence indicate that at least in these experiments, treatment with anti-TNF neutralizing antibodies did not completely abrogate neutrophil influx into the c48/80-treated hind paw tissue.  et al. (2011Zhang) (reference 9 in the paper) proposed that TNF-alpha sensitizes meningeal nociceptors indirectly via local COX activity and the p38 MAPK signalling cascade, rather than by direct actions on nociceptors. et al. Wang reported that TNF receptors in the sciatic nerve were expressed primarily on Schwann cells rather than neurons, leaving open the possibility that Schwann cells are involved in the inflammatory cascade triggered by TNF-alpha.This paper makes a strong case for the early involvement of mast cell-derived TNF-alpha in thermal hyperalgesia and oedema during inflammatory reactions. The only contentious issue is the suggestion that TNF-alpha acts directly on nociceptors, rather than via the cascade of inflammatory mediators triggered by TNF release. The references cited in support of this view either did not directly address this point, or did not provide the intended support. For example,One other minor point: I think this sentence in the first paragraph of the Results "Saline-treated control mice had no increase in thermal sensitivity and showed no tissue swelling after c48/80 treatment" should be "Saline-treated control mice had no increase in thermal sensitivity and showed no tissue swelling".I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Dr. Drummond,Thank you for reviewing our paper. Following your suggestion, we have rephrased the sentence describing the effects of TNF-a on nociceptors as possibly mediated by inflammatory intermediates. We have also corrected the sentence in the first paragraph of the results section.


As the epidermal growth factor receptor (EGFR) is a known transducer of proliferative signals and a potent activator of MAPKs, we hypothesized that the EGFR participates in TNF-dependent MAPK activation and IL-8 secretion by intestinal epithelial cells (IECs). We show that the EGFR is tyrosine-phosphorylated following treatment of IECs (HT-29 and IEC-6) with TNF-α. This requires EGFR autophosphorylation as it was blocked by the EGFR kinase inhibitor AG1478. Autophosphorylation was also inhibited by both a Src-kinase inhibitor and the metalloproteinase inhibitor batimastat. TNF treatment of IECs resulted in the accumulation of soluble TGF-α; treatment of IECs with batimastat suppressed TGF-α release and immunoneutralization of TGF-α resulted in decreased EGFR and ERK phosphorylations. TNF-α treatment of IECs resulted in an association between EGFR and HER2 and inhibition of HER2 using a specific inhibitor AG879 in combination with AG1478-suppressed TNF-α-dependent ERK phosphorylation and IL-8 release. Downregulation of HER2 via siRNA resulted in a significant decrease in ERK phosphorylation and a 50% reduction in IL-8 secretion.TNF- Inflammatory bowel diseases (IBDs), comprised of ulcerative colitis and Crohn's disease, are chronic, relapsing-remitting inflammatory diseases of unknown etiology. Current understanding suggests a critical role for the innate immune system in the context of a permissive genetic background and the intestinal microflora . Interes The mechanism(s) by which chronic inflammation contributes to carcinogenesis are poorly defined. Tumors, including CRC, are infiltrated by activated immune cells including T cells, neutrophils, macrophages, and dendritic cells which secrete various cytokines, chemokines, proteases, and growth factors. This results in the modification of the surrounding stroma creating an environment conducive to tumor growth, invasion, and eventual metastasis , 4–6.α) is a proinflammatory cytokine known to play a central role in the development of intestinal inflammation and IBD [α have been demonstrated in IBD patients [α was recently shown to play a critical role in the development of colorectal cancer in an animal model of chemically induced colitis-associated cancer [ Tumor necrosis factor-alpha  [α mediates these functional changes in IECs remains poorly understood. TNF-α is known to act through two distinct cell-surface receptors, a 55-KDa receptor and a 75-KDa receptor referred to as TNFR-I and TNFR-II, respectively, although most biological activities are attributed to the type I receptor [α receptor signaling was the EGF receptor (EGFR) [ TNF-s (IECs) –18. Howereceptor , 20. Hisr (EGFR) , a 170 kr (EGFR) . Howeverr (EGFR) , 23. Ther (EGFR) , 23. α receptors (TNFRs) do not possess any known catalytic activity and instead rely exclusively on adaptor molecules for the recruitment and transmission of extracellular signals [α-dependent cellular changes in a cell- and tissue-specific manner. TNF is a potent activator of MAPK signaling; however, the mechanisms whereby TNF-α activates the ERK MAPK pathway, remain poorly understood [α receptor, suggesting a direct link between TNFR-I and ERK [α [α-dependent EGFR transactivation and suggest that this event is required for ERK pathway activation in both hepatocytes and mammary epithelial cells [In contrast to EGFRs, TNF- signals . Work ovderstood , 20. GRB and ERK . In addid ERK [α , 27. Alsd ERK [α , 29. Othal cells , 31.α in the transformed intestinal epithelial cell line HT-29 leading to expression of the angiogenic and chemotactic cytokine interleukin 8 (IL-8) [α-mediated activation of the ERK pathway leading to the secretion of IL-8 in intestinal epithelial cells. We report that maximal ERK activation and IL-8 secretion in response to TNF-α requires the release of TGF-α and the activation of the EGFR family of receptors.We have previously described the activation of the ERK signaling pathway in response to TNF-8 (IL-8) . EGFR ge8 (IL-8) , 33. The Unless otherwise stated all chemicals were purchased from Sigma . μg/mL insulin. For experimental treatments, cells were grown in either 6 or 12 well tissue culture plates . HT-29 and IEC-6 cells were obtained from American Type Culture Collection . HT-29 cells were cultured in RPMI 1640 media  supplemented with 10% heat-inactivated fetal calf serum , 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 2% sodium bicarbonate, and 10 mmol/L HEPES. IEC-6 cells were cultured in DMEM supplemented with 5% fetal calf serum, 2 mmol/L glutamine, and 5 α  or epidermal growth factor  in the presence or absence of the ERK pathway inhibitor PD98059 , the platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitor AG1298, Src kinase inhibitor PP2 , the tyrosine kinase inhibitor genistein  the matrix metalloproteinase inhibitor batimastat (BB94) , the TNF-alpha converting enzyme (TACE) inhibitor TAPI-1 , the EGF receptor tyrosine kinase inhibitor AG1478 , and the HER2 receptor tyrosine kinase inhibitor AG879 or TGF-α neutralizing serum . Cells were treated with the inhibitors for 30 mins prior to treatment with TNF-α or EGF. Control monolayers were treated with an equal volume of vehicle . Prior to experiments designed to measure ERK activation or EGFR/HER2 transactivation, cells were incubated in serum-free media  overnight in order to reduce growth factor-mediated activation. All experiments were conducted in serum-free media. Confluent monolayers (passage 25–45) were incubated with human recombinant TNF-α or 50 ng/mL EGF for 3 hrs. IL-8 protein in supernatants was measured via ELISA as follows: 96 well Maxisorp ELISA plates  were coated with 4 μg/mL capture monoclonal anti-IL-8 antibody  in PBS (pH 7.4) overnight. Plates were then blocked overnight . Plates were washed 4 times between all steps with 0.05% Tween-20 PBS pH 7.4. 100 μL samples and standards  were incubated in the plates overnight. Biotinylated polyclonal anti-IL-8 antibody  was added (20 ng/mL in PBS pH 7.4) and plates incubated for 2 hrs. 100 μL Streptavidin-HRP  was added for 1 hr, followed by development with 100 μL TMBS . Reaction was stopped with acid (0.5 M H2SO4) and plates read immediately at 450 nm using an ELISA plate reader . All steps were carried out at room temperature. ELISA was sensitive to <30 pg/mL. TGF-α in cell culture supernatants was measured using a commercial TGF-α ELISA following manufacturer's instructions . For the purpose of measuring IL-8, HT-29 monolayers were stimulated with 10 ng/mL TNF-μL/well (500 uL/dish for IEC-6 cells) ice-cold modified RIPA buffer  and sonicated on ice for 30 secs. Lysates were centrifuged at 4000 RPM for 2 min and supernatants transferred to new tubes. Protein concentrations were determined using a commercial Lowry Assay, . Protein concentrations were adjusted to the same concentration (5 mg/mL) then, 5 μg of anti-EGFR, anti-her-2 antibody added , or antiphosphotyrosine  and incubated on a rotator overnight at 4°C. Antibody was precipitated by the addition of 50 μL of a 50% protein A/G-sepharose bead suspension for 2 hr at 4°C. Beads were washed 4 times with ice-cold modified RIPA buffer, supernatant was aspirated, then 60 μL 2X protein sample buffer was added per sample. Samples were boiled for 5 min, centrifuged at 10000 rpm for 1 min, and proteins separated by SDS-PAGE as described above. For TGF-α immunoneutralization studies, HT-29 cells were treated with 1–10 μg/mL anti-TGF-α or Ig control sera  for 30 min prior to stimulation with TNF-α for 15 min. Cells were then harvested and analyzed for phospho-ERK content as described in the following. Cells were grown in six well plates (100 mm dishes for IEC-6 cells) and treated in duplicate as described in figure legends (results). Cells were harvested in 200 α or 50 ng/mL EGF and harvested in Mono Q buffer  at different times. Following sonication for 30 secs, samples were centrifuged at 12000 rpm for 1 min to remove insoluble material and protein concentrations were determined using a commercial Lowry Assay  using BSA standards made in Mono Q buffer. Lysate concentrations were adjusted to ensure even protein loading, mixed with an equal volume of 2X protein sample buffer , 2 μM microcystin ), boiled for 2 mins, and separated via electrophoresis (10% acrylamide gels). Proteins were transferred for 1.5 hrs (2 hrs for EGFR/HER2 immunoprecipitation experiments) at 400 mA in transfer buffer  onto a PVDF membrane . Membranes were blocked for 1 hr using 3% skim milk (5% BSA for antphosphotyrosine blots) and incubated overnight in primary antibody. The antibodies used were as follows: anti-ERK-1 , anti-phospho-ERK 1/2 , anti-phosphotyrosine , anti-EGFR, and anti-HER2 . Secondary staining was conducted using HRP-conjugated goat sera specific for mouse or rabbit Ig as required  followed by chemiluminescent detection using a commercial reagent following manufacturer's instructions . Comparisons were made only among samples isolated and transferred together onto the same membrane. Multiple exposures were done to ensure that film was not overexposed. In order to confirm equal loading of protein, all western blots using phospho-specific antibodies were stripped and reprobed with antibody against the nonphosphorylated kinase.  Monolayers were stimulated with 10 ng/mL TNF-α for 15 mins. Cells were washed 2X with ice-cold PBS and harvested on ice. TACE activity was measured using a commercially available fluorimetric TACE assay kit  as per manufacturer's instructions. Fluorescence was measured every 5 mins for 3 hrs and plotted over time. Data represents fluorescence following 1 hr incubation with fluorescent substrate which is within the linear portion for all curves. HT-29 cells were incubated in serum-free media overnight, washed once with serum-free media, and stimulated with 10 ng/mL TNF-5 cells were transfected with 80 pmols siRNA reagent  using Lipofectamine 2000  following manufacturer's instructions. Cells were cultured for a further 48 hrs in serum-free media prior to treatment with TNF-α as described in results and figure legends. Single-cell suspensions of HT-29 cells were prepared by trypsinizing 100 mm confluent monolayers. 5 × 10t-test. P < 0.05 was considered statistically significant.Unless otherwise stated, data shown in figures are representative experiments. Comparable results were obtained in additional experiments. Bar graphs are expressed as mean ± SD from at least three separate experiments. Differences between mean values were analyzed using the Student's α rapidly stimulates the phosphorylation (activation) of multiple MAPK pathways in HT-29 cells, including the ERK pathway leading to IL-8 secretion [α signaling, some studies suggesting that the EGFR acts downstream of TNF receptors [ We have previously shown that TNF-ecretion . Previoueceptors , 34–38. eceptors , 15. As α stimulation in various cell types [α in intestinal epithelial cells [α in hepatocytes and mammary epithelial cells, respectively [α in intestinal epithelial cells. HT-29 cells were cultured in serum-free media overnight, stimulated with 10 ng/mL TNF-α for various times, and the EGF receptor immunoprecipitated. EGFR tyrosine phosphorylation was then assessed by western blotting using antiphospho-tyrosine sera. As shown in α. Previous studies have described changes in EGFR tyrosine phosphorylation in response to TNF-ll types , 34–38. al cells , 16. Argectively , 31. Theectively –41. We sα treatment requires the intrinsic kinase activity of the EGFR (transactivation), HT-29 cells were treated as above, except cells were incubated with the EGF receptor tyrosine kinase inhibitor AG1478 for 15 mins prior to TNF-α stimulation. As shown in μM AG1478. AG1278 (5 μM), a PDGF-receptor tyrosine kinase inhibitor which is structurally similar to AG1478, did not affect EGF receptor tyrosine phosphorylation . As shown in α, suggesting that EGFR tyrosine kinase activation in response to TNF-α requires MMP activity. We next examined whether MMP activity is required for EGFR transactivation in response to TNF-α-converting enzyme (TACE) is a metalloproteinase which derives its name from its ability to cleave membrane-bound TNF-α leading to TNF-α release, but it also cleaves multiple EGFR ligands including amphiregulin, HB-EGF, epiregulin, and TGF-α [α-stimulated TNF-α release [α treatment.  We next sought to identify the MMP responsible for TNF-dependent EGFR transactivation. TNF-nd TGF-α . TACE is release . We therα has previously been implicated in TNF-α-stimulated EGFR transactivation [α and measured TGF-α in the culture media. As shown in α resulted in a 60% increase in soluble TGF-α compared to unstimulated controls. Pretreatment of cells with BB94 completely blocked TNF-α-stimulated TGF-α release as well as basal TGF-α release in unstimulated cells. On the other hand, pretreatment of HT-29 cells with increasing concentrations of the TACE inhibitor TAPI-1 had a dose-dependant effect on TNF-stimulated TGF-α release but did not alter basal TGF-α production ; thus it is unlikely that TNF would act via Pyk, a calcium-dependent kinase suggested to play a role in other systems such as that of carbachol-stimulated EGFR transactivation in T84 intestinal epithelial cells [μM AG1478, 2 μM PP2 (a Src-kinase inhibitor), or 100 μM genistein (tyrosine kinase inhibitor) for 15 mins prior to treatment with TNF-α for 15 mins. The results of this experiment are shown in α treatment resulted in increased EGFR tyrosine phosphorylation and this was blocked by AG1478. Interestingly, PP2 also abrogated EGFR tyrosine phosphorylation as well as the phosphorylation on tyrosine residues on proteins that coprecipitate with the EGFR (data not shown). This was also true of genistein, a broad specificity tyrosine kinase inhibitor, although genistein had a smaller effect upon the phosphotyrosine content of coprecipitating proteins. These results suggest the participation of Src-like kinases in relaying the signal that links TNF-α to the EGFR. In parallel experiments, we looked at the effects of these inhibitors upon TNF-stimulated ERK phosphorylation , HER3 (ErbB3), and HER4 (ErbB4) . Heterodα for various times and immunoprecipitated the EGFR. These immunoprecipitates were then probed for the presence of HER2. As shown in α treatment resulted in the time-dependant recruitment of HER2 to EGFR with peak association at 15 mins. Interestingly, this association is transient as it is no longer evident by 30 mins. Next, we assessed whether HER2 becomes phosphorylated on tyrosine residues in response to TNF-α. For this purpose serum-starved HT-29 cells were stimulated as before and tyrosine-phosphorylated proteins immunoprecipitated using antiphosphotyrosine sera. Samples were then probed via western blotting using anti-HER2 sera. As shown in α and after 5 mins of stimulation with TGF-α. To answer this question, we first stimulated HT-29 cells with TNF-μM AG1478 and 2.5 μM of the HER2-specific inhibitor AG879 prior to stimulation with TNF-α. As shown in  We next asked whether inhibition of HER2 in combination with inhibition of the EGFR would result in greater inhibition of ERK activation than inhibition of the EGFR alone. HT-29 cells were incubated in the presence or absence of 5 α-stimulated IL-8 secretion by intestinal epithelial cells [α for 6 hrs. The amount of secreted IL-8 was then measured in the supernatants via ELISA. As shown in α-stimulated IL-8 release was inhibited only at 10 μM AG1478 . On the other hand, while 1 μM AG1478 was sufficient to completely block EGFR phosphorylation  to completely block TNF-stimulated EGFR phosphorylation and downstream signaling. Our data with AG1478, the EGFR inhibitor, was initially very difficult to interpret. We observed a complete blockade of EGFR phosphorylation with AG1478; however, we could at best only partially block TNF-dependant ERK activation and had almost no effect upon IL-8 secretion with this drug alone. In the study by Janes et al, they pretreated cells with IFN-γ before all their experiments in order to enhance apoptosis in response to TNF-α. IFN-γ pretreatment is a key difference between their experimental design and ours; however, we were unable to completely block ERK activation or IL-8 secretion with AG1478 with or without IFN-γ pretreatment (unpublished data). However, using combined EGFR and HER2 inhibition, we can achieve greater ERK and IL-8 inhibition than either inhibitor alone. Interestingly, inhibition of HER2 using AG879 alone had a profound effect upon IL-8 secretion (~50% reduction at 2.5 μM), but combined inhibition using both AG1478 (1 μM) and AG879 (2.5 μM) resulted in greater than 80% inhibition. This may represent a nonspecific effect on the part of our inhibitors or a greater role for the EGFR/HER2 receptor complex upon IL-8 secretion, which may involve the activation of pathways other than the MEK/ERK pathway. Recently, Sethi et al. have suggested that the EGFR can stimulate NFκB activation independent of IKK through the phosphorylation of IκB on tyrosine 42 [κB activation and IL-8 secretion in IECs, there is significant evidence pointing to the importance of IKK-dependent IκB phosphorylation and degradation leading to NFκB activation and proinflammatory gene expression in these cells [κB activation and IκB degradation. Previously, Janes et al. showed that TNF-se cells . Future α release, Src inhibition should result in reduced EGFR and Her-2 activities leading to decreased ERK activation. Perhaps in the absence of Src, TNF-stimulates ERK activity through a yet undetermined mechanism. Alternatively, there could exist two parallel pathways leading to EGFR transactivation downstream from TNF-α, one Src-dependent, and one MMP-dependent. TNF-α has been shown to activate both in other systems [α in TNF-dependent ERK activation using human HT-29 cells; perhaps there exist cell line/species-specific differences that underlie the noted discrepancies between these studies While this paper was in preparation, Hobbs and coworkers have shown TNF transactivation of the EGFR stimulates COX-2 expression in mouse intestinal cells. They provide evidence to suggest the participation of Src and p38, kinases in an MMP-independent manner. In our hands, a Src inhibitor (PP2) and a tyrosine kinase inhibitor (genistein) completely blocked EGFR phosphorylation and yet had no effect upon ERK activation . It woul systems , 37, 44.α release. The TACE inhibitor TAPI-1 inhibits TNF-stimulated TGF-α release but this is most evident at high concentrations. In addition, TNF failed to stimulate TACE activity. Interestingly, there is precedent for this as Myhre et al. have recently shown that TACE may be regulated by at the level of cellular localization as opposed to enzymatic activity [ TACE seemed like a good candidate to be the MMP activated by TNF leading to TGF-activity . In the present study we focused on the role of the EGFR/HER2 signaling pathway in TNF-stimulated IL-8 secretion. However, this pathway is likely to contribute to many aspects of TNF signaling in IECs. Both the EGFR and TNF are known to profoundly affect intestinal epithelial cell function. Of particular interest in this regard, Janes et al. have shown that this pathway may modulate IEC apoptosis which may have implications towards the development of cancer in the context of inflammation . Work by IL-8 and other cytokines such as IL-6 have been shown to play a critical role in tumor growth in multiple cancer models independent of inflammation such as in Ras-driven models of cancer . Interesin vivo to see the effect of EGFR/Her2 signaling inhibition in the context of a colitis-induced cancer model. Third, attempts at abrogating EGFR signaling in the context of TNF-α signaling must keep in mind the participation of other EGFR binding partners such as HER2.The fact that EGFR/HER2 participates in TNF signaling may have several important therapeutic implications. First, it suggests that therapies which target the EGFR/HER2 may potentially affect immune responses in the gut. Second, EGFR/HER2 activation by TNF may contribute to inflammation induced carcinogenesis. This possibility will have to await testing


HWY336 bound to both kinases reversibly via noncovalent interactions, and inhibited their activity by interfering with access of a protein substrate to its binding site. The binding affinity of HWY336 to MKK4 was measured by surface plasmon resonance to determine a dissociation constant (dK) of 3.2 µM. When mammalian cells were treated with HWY336, MKK4 and MKK7 were selectively inhibited, resulting in inhibition of c-Jun NH2-terminal protein kinases in vivo. The structural model of HWY336 bound to either MKK4 or MKK7 predicted that HWY336 was docked to the activation loop, which is adjacent to the substrate binding site. This model suggested the importance of the activation loop of MKKs in HWY336 selectivity. We verified this model by mutating three critical residues within this loop of MKK4 to the corresponding residues in MKK3. The mutant MKK4 displayed similar kinase activity as wild-type kinase, but its activity was not inhibited by HWY336 compared to wild-type MKK4. We propose that the specific association of HWY336 to the activation loop of MKK4/MKK7 is responsible for its selective inhibition.A protoberberine derivative library was used to search for selective inhibitors against kinases of the mitogen-activated protein kinase (MAPK) cascades in mammalian cells. Among kinases in mammalian MAPK pathways, we identified a compound (HWY336) that selectively inhibits kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7). The IC JNK/SAPKs are activated in response to chemical and environmental stresses through MKK4/MKK7. Studies have shown that inflammatory cytokines stimulate p38 MAPKs via MKK3/MKK6. Finally, ERKs are activated by mitogens and growth factors through sequential activation of MEKK Raf and MEK1/2 to control cell division and differentiation. Each MAPK pathway functions in parallel by mediating specific signals. Nevertheless, significant crosstalk exists between these pathways to affect the activation of a single MAPK and form a complex network The evolutionarily conserved mitogen-activated protein kinase (MAPK) signaling pathway is activated by diverse extracellular stimuli to regulate proliferation, differentiation, and cell death in eukaryotes. The well-characterized MAPK family can be subdivided into three groups: c-Jun NHcd]pyrazol-6(2H)-one] inhibits JNK activity Synthetic inhibitors against specific components of MAPK cascades have been excellent tools for studying the mechanisms behind this signaling pathway in mammalian cells. For example, PD98059 [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one] and U0126  inhibit MEK1 and MEK2 Protoberberine alkaloids have anti-tumor activity in mice and cytotoxic activities against human cancer cell lines HEK293T and HEK293 cells were grown in DMEM (GIBCO) supplemented with 10% (v/v) fetal bovine serum (GIBCO), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (GIBCO). CHO cells were cultured in F-12K nutrient mixture medium (GIBCO) containing 10% FBS, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 µg/ml of amphotericin B (GIBCO). Cells were treated with 600 mM D-sorbitol (Sigma-Aldrich) for 30 min to activate MAPKs prior to purification.Each of 80 protoberberine derivatives was dissolved in DMSO to a final concentration of 1 mg/ml. A stock solution containing 1 mg/ml of SP600125, SB203580, or U0126 (A.G. Scientific Inc.) was also prepared in DMSO.TCCACTGATGCC 3′ (reverse). The plasmid was treated with 10 U of DpnI  for 1 hr after completion of PCR. The Q253Y I258V R262M mutations in MKK4 and R283Y K288V R292M mutations in MKK7 were confirmed by sequencing. Expression of MKK4, MKK4-Q253Y I258V R262M, MKK7, and MKK7-R283Y K288V R292M was confirmed by western blot using anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) and HA probe (F-7) monoclonal antibody (Santa Cruz).The pCDNA3-Flag-MKK4-Q253Y I258V R262M vector and the pCDNA3-HA-MKK7-R283Y K288V R292M vector were created by site-directed mutagenesis with the pCDNA3-Flag-MKK4 vector  and the pCDNA3-HA-MKK7 vector  as a template. We mutated Gln to Tyr, Ile to Val, and Arg to Met in the MKK4. The following primers were used to amplify this mutant plasmid: 5′GGCATCAGTGGATACCTTGTGGACTCTGTGGCCAAGACAATGGATGCTGGCTGT3′ (forward),5′ACAGCCAGCATCCATTGTCTTGGCCACAGAGTCCACAAGGTATCCACTGATGCC3′ (reverse). Similarly, Arg to Tyr, Lys to Val, and Arg to Met in the MKK7 were mutated with the following primer: 5′GGCATCAGTGGATACCTTGTGGACTCTGTGGCCAAGACAATGTCAG CTGGCTGT3′(forward), 5′ACAGCCAGCTGACATTGTCTTGGCCACAGAGTCCACAAGGTA g for 5 min at 4°C. The cell pellets were washed with cold PBS and solubilized with ice-cold 1× lysis buffer . Cellular extracts were centrifuged for 20 min at 10,000×g to remove cellular debris. The supernatant was used for immunoprecipitation. Cell lysate  was incubated overnight at 4°C with antibody specific to each kinase. The following antibodies were used: anti-MEK1 , anti-MEK2 , and anti-JNK   at a 1∶200 dilution; anti-MKK3 , anti-MKK4 , anti-MKK6 , anti-MKK7 , anti-ERK1 , and anti-p38   at a 1∶150 dilution. After the incubation, 60 µl Sepharose A-conjugated protein A (Sigma-Aldrich) was added and mixed for 90 min at 4°C. Immunoprecipitation of each kinase was confirmed by silver staining or western blot analysis.Cells were cultured on 100 mm plates for 2 days, scraped into 1.5-ml tubes, and collected by centrifugation at 2,000×in vitro kinase assays, protein A bead-bound kinase was washed with cold kinase buffer , and incubated with HWY336, U0126, HWY289, berberine, or DMSO alone for 20 min at 30°C. The kinase reaction was conducted for 30 min at 30°C by addition of 0.5 µg JNK (Millipore) or 1 mg/ml myelin basic protein , 1 mM ATP, and 10 µCi 32P-ATP (Perkin Elmer).For To determine whether HWY336 binding to MKK4 and MKK7 was reversible, each purified kinase was treated with HWY336 and washed three times with kinase buffer containing varying concentrations of NaCl from 0 to 500 mM prior to the kinase assay. Purified MKK4 and MKK7 were pre-incubated with 1 mg/ml MBP or 1 mM ATP prior to the addition of HWY336 to determine whether HWY336 competed with protein substrate or ATP for binding to MKK4 and MKK7.After HWY336 treatments, total protein (50 µg) of cell lysate was separated by 10% SDS-PAGE and transferred to a PVDF membrane. Specific proteins were detected using the following primary antibodies: anti-JNK, anti-p-JNKs, anti-p38, anti-p-p38, anti-MKK4, and anti-p-MKK4 .The optical set-up for SPR detection consisted of two concentric dual motorized stages that were employed to implement angle-scanning capability. To measure binding constants, imaging SPR detection was performed by maintaining the angle of light incidence at 59°. Light source from a He-Ne laser  was p-polarized before incident on a SPR detection sample that was index-matched to an SF10 prism substrate. A p-i-n photodiode  assessed the signal, which was subsequently fed to a low noise lock-in amplifier. The whole procedure was computer controlled and fully automated. Each measurement was repeated multiple times for statistical analysis.SPR sample chips were fabricated by evaporating a 2 nm thick chromium adhesion layer and a 40 nm thick gold film on an SF10 cover glass substrate. Evaporated sample chips were cleaned in a plasma cleaner . For SPR measurements, streptavidin was first coated on the gold surface by soaking the sample in a 1 mM solution of 8-amino-1-octanethiol  for 8 hrs in a dark environment and incubating the gold chip with 4 mM SATP solution for 1.5 hrs in a humid chamber. SPR sample surface was deacetylated in an incubation process using de-acetylation buffer  for 25 min, followed by streptavidin attachment on the surface using 20 µl of streptavidin-maleimide solution incubated on the SPR samples for 1 hr. After PBS washing, 10 nM biotinylated MKK4 solution  was immobilized for 2 hrs. For binding of MKK4 with HWY336, HWY336 solution with PBS buffer was injected onto the SPR samples through a fluidic channel for 120 min using a micropump (KD Scientific).Homology modeling for MKK4 was performed using the Modeller9v4 software that calculated a model composed of non-hydrogen atoms, based on aligning the sequence to be modeled with known related structures. A three-dimensional model was obtained by optimization of molecular probability density function using a variable target function procedure in Cartesian space that employed conjugate gradients and molecular dynamics with simulated annealing.Molecular docking used a reported MKK7 structure and our homology model of MKK4 using the SYBYL 8.1 software package . Protein structure was prepared using a biopolymer module to prepare a structure that was suitable for docking assessments. Hydrogen atoms were added to the structure, atom types and charges were assigned using AMBER7 FF99, and side chain amides were modified. HWY336 was sketched using the SYBYL 8.1 sketch program and minimized using the Tripos force field and Powell method with a termination gradient set to 0.05 kcal/mol. The molecule was fully minimized with Gasteiger-Hückel charges. The docking study was performed using the Surflex-Dock module of SYBYL 8.1, which used an empirical scoring function to score ligand and protomol guided docking in vitro kinase assays were performed with MEK1, MEK2, MKK4, MKK7, MKK3, MKK6, and MAPKs  immunoprecipitated from either HEK293 or CHO cells, using MBP as a common substrate for kinases  had no effect . This se50 of HWY336 was 6 µM for MKK4  of ligand-protein binding was 3.2 µM for MKK4 and HWY336, which was comparable to those of MEK1 and its well-known selective inhibitors, U0126 (Kd = 2±0.5 µM) and PD0325901 (Kd = 5±1 µM) The results in When we assessed whether HWY336 competed with a protein substrate or ATP for binding to MKK4 and MKK7, the inhibitory effect of HWY336 was unaffected by pre-incubation with ATP , but washttp://www.ebi.ac.uk/Tools/clustalw2/index.html) To understand the basis of inhibitory selectivity of HWY336 to MKK4 and MKK7, we used molecular docking modeling of HWY336 and MKK4. As a first step in establishing a specific docking model for HWY336 binding to MKK4 and MKK7, the homology and phylogeny of these kinases were examined. In mammals, the MAPK cascades exist as two parallel pathways, MKK3/MKK6 and MKK4/MKK7 To further elucidate the inhibitory action of HWY336, we mapped its docking site by creating a three-dimensional model of MKK4 and MKK7. Since no crystal structure was currently available for MKK4, we used MKK7 (PDB code: 2DYL with resolution 2.45 Å) as a template for three-dimensional homology modeling for MKK4. MKK4 and MKK7 share 49% sequence identity. Therefore, the missing activation loop of MKK7 was first generated using the Modeller9v4 program Molecular docking generates and scores putative protein-ligand complexes. Given the crystal structure of a binding target, molecular docking automatically samples ligand conformation and protein-ligand interactions with a specified region of the protein surface. This method has been used successfully to identify active compounds by filtering out those that do not fit into the binding site. Because our data demonstrated that HWY336 treatment leads to complete inhibition of MKK4/MKK7 activity but not MKK3/MKK6 , we examThe gatekeeper hinge region, DFG motif, and Ser/Thr and Tyr residues were highly conserved between MKK3/MKK6 and MKK4/MKK7 . HoweverWe searched the possible ligand accessible cavities inside MKK4 as many as possible. 261 residue, which is important for the phosphorylation process (262 (Arg292 of MKK7) within the activation loops of MKK4 and MKK7, suggesting an important role of this amino acid residue. Hydrophobic interactions between HWY336 and MKK4/MKK7 were also important for its MKK inhibition. Hydrophobic interactions between HWY336 and MKK4  and MKK7 mutant (R283Y K288V R292M), maintained similar kinase activity as the wild-type proteins because these residues were also present within the activation loop of MKK3  had corresponding counterparts in S. pombe Wis1  for binding, further study on the mechanism underlying the effects of HWY336 would provide important information that could be used to overcome the specificity barrier in the development of MAPK inhibitors. In addition, the high selectivity of HWY336 for MKK4 and MKK7 suggests that further modification of these compounds may lead to the development of selective inhibitors as therapeutics.File S1Supporting information figures.Figure S1, HWY336 inhibits MKK4 and MKK7 selectively but does not inhibit other mammalian MEKs and MAPKs. MKK4 and MKK7 were immunoprecipitated from HEK293 or CHO cells following MAPK pathway activation as described in EXPERIMENTAL PROCEDURES. The same quantity of kinase was used in each assay. A) MKK4 and MKK7 activity assayed with MBP as a substrate after pre-treatment with 1 mM of DMSO (lane 1), 400 µM berberine (lane 2), 1 mM HWY289, another protoberberine derivative from the library (lane 4), or 1 mM HWY336 (lane 5). DMSO was used as a negative control (lane 1) and 3 mM U0126 was used as a positive control (lane 3). B, berberine; U, U0126; 289, HWY289; 336, HWY336. B) MEK1, MKK3/MKK6, JNK1/2, p38, and ERK1/2 were immunoprecipitated from activated HEK293 and CHO cells as described in Figure S2, The mechanism of HWY336-mediated inhibition of MKK4 and MKK7. Stress-activated MKK4 and MKK7 were immunoprecipitated from HEK293 cells, and the same amount of each kinase was used in the assays as confirmed by Coomassie staining or western blotting. A) Reversibility of HWY336 binding to MKK4 and MKK7. MKK4 or MKK7 was pre-incubated with 600 µM HWY336 for 10 min and washed with kinase buffer containing increasing concentrations of NaCl prior to kinase assays. B, C) The ability of ATP (B) or MBP substrate (C) to compete with HWY336 for binding to MKK4 and MKK7. Immunoprecipitated MKK4 and MKK7 were pre-incubated with different concentrations of either ATP (B) or MBP (C) and their kinase activities were assayed in the presence of 600 µM HWY336 or DMSO (lane 1). Figure S3, HWY336 interacts with MKK4 reversibly, and inhibits MKK4 by competing with JNK, a physiological protein substrate, and not with ATP. Stress-activated MKK4 were immunoprecipitated from HEK293 cells as described in Figure S4, Computer modeling predicts the conserved three-dimensional structures of MKK4 over MKK7. A) Three-dimensional structure of MKK7 (magenta) is superimposed onto the MKK4 (cyan) model structure. B) Three-dimensional modeling of HWY336 docking with MKK7. A proposed docked pose of ATP inside the MKK7 structure. The residues that vary  within the activation loop are indicated. HWY336 forms a hydrogen bond with Arg292 and the conserved Thr291, which is important in the phosphorylation process of MKK7 . C) Activation loop sequences and critical residues are highly conserved among MKK4/MKK7 and Wis1, which are all inhibited by HWY336. Figure S5, Prediction of a HWY336-binding site within the activation loop of MKK4. All possible solvent pockets were simulated and a HWY336-binding site was predicted within the activation loop of MKK4 using SITE ID module of SYBYL 8.1.(PDF)Click here for additional data file.


Plasmodium pre-erythrocytic stages.The liver stages of malaria parasites are inhibited by cytokines such as interferon-γ or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-α possibly because of differences in the models used. We have reassessed the role of TNF-α in the different cellular systems used to study the in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-α treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-α inhibition was mediated by a soluble factor present in the supernatant of TNF-α stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified.Human or mouse TNF-α were tested against human and rodent malaria parasites grown Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection. Plasmodium chabaudi in mice P. chabaudi adami blood infections less efficiently et al. reported that recombinant human TNF-α directly inhibited P. berghei grown in vitro in the HepG2 human hepatoma cell line, and in vivo in rats and mice P. yoelii parasites grown in cultured purified mouse primary hepatocytes were unaffected by TNF-α, whereas this cytokine inhibited the hepatic development of this parasite in vivoin vivo was actually mediated by the IL-6 secreted by non-parenchymal liver cells in response to TNF-α stimulation Plasmodium hepatic stages observed in in vitro assays of TNF-α activity.Tumour necrosis factor (TNF)-α is a cytokine with pleiotropic effects including anti-microbial activities All experiments and procedures involving mice were approved by the “Direction Départementale des Service Vétérinaires de Paris, France” (Authorisation No 75–129) and performed in compliance with regulations of the French Ministry of Agriculture for animal experimentation (1987). Human liver fragments used to prepare primary hepatocyte cultures were collected after written informed consent from patients undergoing a partial hepatectomy. The collection and use of these tissues were undertaken in accordance with French national ethical regulations and have been approved by the Ethic Committee of the Centre Hospitalo-Universitaire Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France.Different batches of human and murine TNF-α with similar specific activity were obtained from R&D systems. Endotoxin levels in the different batches used in this study were always below 1.0 EU per 1 µg as reported by the manufacturer. S-methyl-thiourea , a potent inhibitor of iNOS Plasmodium yoelii yoelii 265BY strain (Pyy265BY), of P. yoelii yoelii 17X strain clone 1.1 (Py17X), and of P. berghei ANKA cloned line transfected with GFP (PbA) Anopheles stephensi mosquitoes, 16 to 21 days after an infective mouse blood meal. After aseptic dissection, salivary glands were homogenized in a glass grinder and diluted in culture medium Plasmodium falciparum were obtained from infected salivary glands of A. gambiae mosquitoes which have been fed two weeks before on infected human blood cultures using a membrane-based feeder system BALB/cJ mice were purchased from Harlan Laboratories , and were housed in a pathogen-free rodent barrier facility. Sporozoites of the uncloned line of 5 per well), were cultured in DMEM (Invitrogen) supplemented with 10% FCS (Invitrogen), 2 mM glutamine, 1% penicillin-streptomycin of a 100 X stock solution (Invitrogen). Human hepatocarcinoma cells HepG2/CD81+ (HepG2 stably expressing CD81) (105 per well) 5 cells per well for primary murine hepatocytes, and of 2×105 cells per well for primary human hepatocytes cells, and cultured at 37°C, 5% CO2, in DMEM medium as above supplemented with 10−7 M dexamethasone (Sigma) after complete cell adherence (12–24 hours).Mouse hepatoma cells Hepa1–6 (ATCC CRL-1830) (103 cells/well) was evaluated using a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Briefly, cytokines were added in triplicate at decreasing concentrations. Medium was replaced 3 hours after sporozoite infection and every day thereafter up to day 4 (for rodent parasites) or 7  with fresh medium containing the cytokine at the same concentration. Twenty-four hours after the last medium change, 110 µl of a solution containing 10 µl of MTT (Sigma) solution (5 mg/ml) and 100 µl of medium were added and the cultures incubated for a further 4 hours. The formazan crystals that were formed were dissolved using 100 µl of a 1∶1 DMSO: ethanol solution. Optical density was read immediately at 570 nm with a reference wavelength at 630 nm Toxicity of the cytokine to primary culture of hepatocytes or hepatoma cell lines in flat-bottom 96 wells  of for 2 days. Plasmodium sporozoites (104 in a 50 µl) were added to this supernatant (50 µl) or to fresh medium (50 µl). The final solution (100 µl) containing the sporozoite was added to the culture. Medium was replaced after 3 h, 24 h, and then every day up to day 5 after sporozoite inoculation. Experimental determination of the number of liver stage parasites was performed in triplicate or quadruplicate. Cultures were stopped 48 h  or 5 days  after sporozoite infection, fixed with cold methanol and schizont numbers were assessed by immunofluorescence using antibodies recognizing Plasmodium liver stages as previously described After removal of medium from the culture chambers, ten thousands sporozoites were added in 100 µl of fresh supplemented medium with various quantities of TNF-α tested, and at different times during cultivation. Medium was replaced at −24, 0, 3, and 24 h after rodent malaria sporozoite inoculation, and at −24, 0, 3, 24 h and then every day up to day 5 after in vitroP. bergheiP. yoeliiet al. P. yoelii development, providing a good system to study P. yoelii liver stage biology, thus reducing the need for the more tedious primary murine hepatocyte cultures. We employed this cell line to assess the role of TNF-α against the hepatic stages of malaria parasites.HepG2 is a hepatoma cell line easily propagated P. yoelii sporozoites (1.29±0.0.05 A.U.) did not differ significantly.When human TNF-α was added to HepG2-CD81 cultures over a four-day period centred on the time of sporozoite inoculation, Pyy265BY hepatic parasite development was inhibited in a dose dependent manner . Signifiet al. where an inhibitory effect of human TNF-α against P. berghei NK65 strain was reported We next tested the effect of human TNF-α on PbA parasites, thus providing a reasonable comparison with the study of Schofield Plasmodium species that infect rodents In order to test whether the findings obtained using HepG2-CD81 cells can be extended to other host cell/parasite combinations, we repeated the TNF-α treatment (100 ng/ml initiated at 48 h before sporozoite inoculation and maintained for the subsequent 48 h) with mouse hepatoma cells Hepa 1–6 and primary cultures of purified mouse hepatocytes, both of which sustain the growth of in vivo treatment with this cytokine inhibited P. berghei NK65 liver stage development in situ, for e.g. by inducing the production of another anti-liver stage cytokine such as IL-6 by nonparenchymal liver cells. We wished to ascertain whether the inhibition noted above was due to direct interaction with hepatocytes P. yoelii sporozoites (0.41±0.05 A.U.) were not significant.We next tested the effect of human TNF-α on mouse cells since it was shown previously that P. falciparum in human primary hepatocytes, a host/parasite combination of direct clinical relevance. We used highly purified human hepatocytes to prevent indirect effect of TNF-α on contaminating nonparenchymal cells as shown previously P. falciparum liver stage development was inhibited when the cultures were treated with human TNF-α at 100 ng/ml, but not 1 ng/ml, 24 h or 48 h prior to sporozoite inoculation and until day 5 thereafter (P. yoelii sporozoites (0.793±0.03 A.U.) did not differ significantly.We then tested the effect of TNF-α on the development of ereafter . Inhibit did not reverse the effect of the cytokine against Pyy265BY HepG2/CD81 , a competitive inhibitor of the inducible nitric oxide synthase pG2/CD81  or againatocytes . We nextatocytes , or thatatocytes . We alsoP. falciparum sporozoites in normal medium were added to supernatant medium obtained from human hepatocytes treated for 48 h with TNF-α (1/1 volume). The mixture was then added to fresh human hepatocytes to initiate the infection. A strong significant inhibition of sporozoite development was observed, and it reached levels similar to those induced by direct TNF-α pre-treatment of the cultures  et al.The fact that the inhibitory effect of TNF-α was observed only when cultures were pre-incubated with the cytokine suggested that stimulated hepatocytes secrete an inhibitory factor and/or that the TNF-α treatment makes them refractory to infection. Host cell refractoriness is unlikely because addition of the supernatant from TNF-α-stimulated hepatocytes to the cultures was sufficient to obtain hepatic parasite inhibition. TNF-α alone or together with IL-6 and IL-1, is known to induce the synthesis of acute phase response proteins by hepatocytes. Although the acute phase response to inflammatory stimuli is evolutionary conserved, species-specific differences exist During malaria blood stage infection, the production of TNF-α is increased


In particular, the snoRNA U50, mediating the methylation of C2848 in 28S rRNA, has been suggested as a potential tumor suppressor-like gene playing a role in breast and prostate cancers and B-cell lymphoma. Indeed, we observed the downregulation of U50 in colon cancer cell lines as well as tumors. We then investigated the relationship between U50 and proliferation in lymphocytes stimulated by phytohemagglutinin (PHA) and observed a strong decrease in U50 levels associated with a reduced C2848 methylation. This reduction was due to an alteration of U50 stability and to an increase of its consumption. Indeed, the blockade of ribosome biogenesis induced only an early decrease in U50 followed by a stabilization of U50 levels when ribosome biogenesis was almost completely blocked. Similar results were found with other snoRNAs. Lastly, we observed that U50 modulation affects ribosome efficiency in IRES-mediated translation, demonstrating that changes in the methylation levels of a single specific site on 28S rRNA may alter ribosome function. In conclusion, our results link U50 to the cellular proliferation rate and ribosome biogenesis and these findings may explain why its levels are often greatly reduced in cancers. O-methylation of specific sites, respectively [et al. [Ribosome biogenesis is a highly coordinated process occurring in the nucleolus, where a polycistronic pre-ribosomal RNA (pre-rRNA) transcript is processed to generate the mature 18S, 5.8S, and 28S rRNA. During this processing, the rRNA sequences undergo extensive covalent nucleotide modification, largely directed by small nucleolar RNA (snoRNA)-protein complexes (snoRNP) [ectively ,4. In paectively  which of [et al. ); howeve [et al. . Further [et al. ,13). Thi [et al. . Indeed, [et al. –17 and t [et al. . Recent  [et al. –23. snoR [et al. ,24, whicIn this paper we investigated the relationship between snoU50 and cancer in colon cancer cell lines and tumors with particular regard to proliferation.p = 0.047) (p = 0.049) (p = 0.0004) (p = 0.0121)  , left. T= 0.047)  and we f= 0.049) , right. = 0.049) , left, wCompared to untransformed tissues, U50 was downregulated in tumors. Being that one of the major distinguishing characteristics between these tissues is their proliferation activity, we investigated whether there is a relationship between U50 and proliferation.i.e., the comparison of resting and stimulated primary human lymphocytes. Thus we isolated lymphocytes from healthy donors and performed lymphocyte proliferation assays by using phytohemoagglutinin (PHA). After PHA stimulation, we evaluated the expressions of both U50 and its host gene SNHG5 and observed a strong reduction of U50 levels in PHA stimulated cells, compared to controls  and SNHG5, whose genes were both transcribed by polymerase II. Results demonstrated that none were affected by Act-D 8nM treatment . The SNHPrompted by these observations, we investigated the possible effect of U50 modulation on mRNA translation. Indeed, U50 is responsible for the site-specific C2848 methylation on 28s rRNA, and It has recently been demonstrated that incorrect methylation of rRNA is associated with an impaired capacity to initiate translation through Internal Ribosome Entry Site (IRES) ,25 by inFurthermore, in order to investigate the effect of U50 overexpression, we used the pSrQ-U50 vector to stably transfect HCT116, SW620, and LoVo cell lines. In addition to HCT116, we chose SW620 and LoVo cells since they displayed the lowest U50 basal level in our cohort of colon cancer lines, as previously reported. We successfully obtained all U50 overexpressing lines, but an increase in C2848 methylation was achieved for the HCT116 line only  and S6. Both tumor and adjacent non-tumor  colon tissues selected from a series of thirty-four consecutive patients who had undergone surgical resection for primary carcinoma at the Surgical Department of the University of Bologna, on the sole basis of frozen tissue availability. For each patient, clinical information was recorded and the corresponding tissue was histologically characterized and processed following standard procedures to define bio-pathological features. Specimen collection and tissue analyses were approved by the Bologna University Ethical Committee on human tissue research. Tissues were preserved at −80 °C until use. A 60 mg piece for each sample was minced in liquid nitrogen and then lysed for total RNA extraction using Tri-reagent solution  according to the manufacturer’s instructions.2 according to the American Type Culture Collection instructions.All colon cancer cell lines were grown in their optimal culture medium and conditions at 37 °C in 5% COLymphocytes were recovered from healthy donors’ buffy coats collected by the transfusion center of Sant’Orsola-Malpighi Hospital in Bologna after ficoll gradient and monocyte depletion. Human lymphocytes were grown in RPMI-1640 medium , supplemented with 10% FBS, 1% of penicillin and streptomycin, 1% of glutamine, and 0.5% of non-essential amino acids . Lymphocyte proliferation assays were performed by adding 10 μg/mL of phytohemoagglutinin (PHA) (Sigma) to the medium.The inhibition of ribosome biogenesis and mRNA stability assay were performed by exposing cells to Actinomycin D (Act-D) (Sigma) at a concentration of 8 nM and 80 nM, respectively, and assessed at different time points . SW620 cells were seeded at 100,000 cells/well in 6-well plates and treated with Act-D, while human lymphocytes were seeded at 1,000,000 cells/mL, PHA stimulated for 72 h (where necessary), and then treated with Act-D. PHA stimulation was evaluated by cell counting and considered effective only when lymphocytes proliferation was increased more than 5 fold compared to control cells.^−Ct, assumed the values of untreated cells as reference (100%), and then calculated the relative percentage of investigated genes. For each sample, three replicates were analyzed. Analyses in cell lines and tumors were carried out by using aliquots of a single stock of cDNA obtained from HCT116 as internal calibrator. The analysis of U50 levels was performed by qPCR using a primer with a linker sequence attached to a U50-specific sequence for cDNA synthesis and reverse linker specific primer, and U50 forward specific primer for real-time PCR as described in Dong, 2008 [Total RNA was extracted from cell lines using Tri-reagent (Ambion) and 1 μg was reverse-transcribed using the High-Capacity cDNA Archive Kit . Real-time PCR analysis was carried out in a Gene Amp 7000 Sequence Detection System (Applied Biosystems) using the TaqMan approach for β-glucuronidase (GUS) (Applied Biosystems) and SYBR green approach for all other genes, using specific couples of primers. The housekeeping GUS gene was used to calculate the relative amounts of the studied target genes in all experiments except for those in which Act-D treatment was performed. In the latter case, for each sample we calculated the 2ng, 2008 . All priSince it has been shown that RNAi is an unsuitable tool for snoRNA KD , we usedFor U50 upregulation, we used a retroviral transduction system. We briefly transfected the Phoenix A cell line with 10 μg of pSIREN-RetroQ-U50 plasmid or the pSIRENRetroQ vector control (gifts of Prof. Dong)  using LiTo evaluate site-specific (C2848) methylation levels, we followed a modified previously described method . This moCapped mRNA was transcribed from linearized pR-CrPV-IRES-F (gift of Dr. D. Ruggero) , pF-EMCVt-test was used for patient data set when comparing normal and correspondent tumoral tissues. A two-sided Student’s t-test was used for the comparisons between two groups. p values <0.05 were regarded as statistically significant.All data were analyzed using Prism software, version 5.0a. A paired Recent reports have demonstrated that U50 was downregulated in prostate and breast cancers and in B-cell lymphomas ,21,23. I


TNF-α) gene polymorphisms and open angle glaucoma (OAG) risks, and the association between the TNF-α level in aqueous humor (AH) and the risks of glaucoma.This meta-analysis was performed to clarify the association between tumor necrosis factor alpha  and the 95% confidence interval (CI), while a pooled standardized mean difference with 95% CI was used for the comparison of TNF-α levels in AH between patients with OAG and controls. Publication bias was estimated using Begg’s funnel plots and Egger’s regression test.A computerized literature search was performed for the relevant available studies from three databases including PubMed, ISI Web of Science, and Embase. The fixed or random effect model was selected based on the heterogeneity test using the Q test and the TNF-α −308G/A, −857C/T, −863C/A, and −238G/A polymorphisms and the risks of OAG, respectively. The combined results showed that the TNF-α −308G/A gene polymorphism was significantly associated with risks of high-tension glaucoma , but not with normal tension glaucoma or exfoliation glaucoma. Ethnicity-stratified analysis revealed that a significant association also existed in Asians . None of the other polymorphisms was significantly associated with OAG risks. Furthermore, the pooled results of six studies showed that the TNF-α levels in the AH of patients with OAG was higher than that of the control subjects . Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger’s test.A total of 14 , five , three (645 cases and 666 controls), and three studies (404 cases and 625 controls) were finally included in the analyses for the associations between TNF-α −308G/A polymorphism is significantly associated with the risks of high-tension glaucoma. Since potential confounders could not be ruled out completely, further studies are needed to confirm these results.This meta-analysis suggests that patients with OAG may have higher TNF-α levels compared with the control subjects, and the  Glaucoma, characterized by optic neuropathy and a progressive loss of the retinal ganglion cells (RGCs), is the second leading cause of blindness worldwide . GlaucomTumor necrosis factor-alpha (TNF-α) is a cytokine that belongs to the TNF superfamily of 19 different protein ligands . TNF-α irs1800629), which could increase transcription six- to sevenfold [rs361525), −863C/A (rs645836), and −857C/T (rs1799724) have also been studied. However, the results have been highly inconsistent. Some studies suggested that the TNF-α −308 A allele was significantly associated with the increased risk of OAG [The human TNF-α gene is located within the highly polymorphic major histocompatibility complex class III region on chromosome 6p21.3 . Severalevenfold . Other pk of OAG -15, whilk of OAG -18, and k of OAG ,20. Furtk of OAG , which wk of OAG .TNF-α gene  polymorphisms and the risks of OAG. We also performed a meta-analysis to compare the TNF-α level in AH between patients with glaucoma and the control subjects.In the current study, we performed a meta-analysis of all eligible studies, to provide a more accurate estimate of the association of the A computerized literature search was performed for the relevant available studies from three databases including PubMed, ISI Web of Science, and Embase. The search strategy for identifying the relevant studies involved combinations of the following keywords: “tumor necrosis factor-α” or “TNF-α” and “glaucoma.” The reference lists of review articles, clinical trials, and meta-analyses were also hand-searched to collect other relevant studies. If more than one article was published using the same case series, only the study with the largest sample size was selected. The literature search was updated on August 30, 2012.TNF-α  polymorphisms and the risk of OAG or comparing the TNF-α level in AH of patients with glaucoma and that of the control subjects, (2) case-control design, and (3) gene polymorphisms studies must provide sufficient data for calculating the odds ratio (OR) and the corresponding 95% confidence interval (95% CI). All identified studies were carefully reviewed independently by two investigators to determine whether an individual study was eligible for inclusion in this meta-analysisThe inclusion criteria were as follows: (1) evaluating the associations between Data were extracted independently by two investigators who reached a consensus on all of the items. The following information was extracted from each study: (1) name of the first author, (2) year of publication, (3) country of origin, (4) ethnicity of the study population, (5) number of cases and controls, (6) gender and age of enrolled subjects, and (7) number of genotypes in cases and controls.TNF-α gene polymorphisms was tested for deviation from Hardy–Weinberg equilibrium (HWE) in the control subjects using the chi-square goodness of fit. The associations between the TNF-α  polymorphisms and OAG risks were estimated by calculating pooled ORs and 95% CI. The significance of the pooled effect size was determined with the Z test. Heterogeneity among studies was assessed using the Q test as well as the 2I statistic, which was documented for the percentage of the observed between-study variability due to heterogeneity rather than chance [Genotypic frequency for the n chance . A signin chance . Subgroun chance . Begg’s n chance .-α level in AH between the patients with OAG and the controls. The SMD was chosen because the TNF-α concentrations were measured using different methods in the included studies. The heterogeneity test was also performed by using the Q test and the 2I statistic as performed in other studies [A pooled standardized mean difference (SMD), together with 95% CI, was used to compare the TNF studies ,28. In cAll statistical analysis was performed using STATA version 11 . Two-sided p values less than 0.05 were considered statistically significant.TNF-α gene polymorphisms and OAG risks were retrieved. Among these studies, Fan et al. recruited three forms of patients with OAG [TNF-α (−308G/A) and TNF-α (−238G/A) [TNF-α (−308G/A) and TNF-α (−857C/T) [TNF-α (−308G/A), TNF-α (−857C/T), and TNF-α (−863C/A) [TNF-α −308G/A, −857C/T, −863C/A, and −238G/A polymorphisms and risks of OAG, respectively. The studies used blood samples for DNA extraction, while polymerase chain reaction-restriction fragments length polymorphism, TaqMan, or DNA sequencing methods were used for genotyping. Genotype distribution in control groups were in HWE in most of the studies except two studies for the TNF-α (−308G/A) polymorphism [Eleven studies investigating the association between with OAG , while Rwith OAG ,18. Thre−238G/A) ,17,29, o−857C/T) , and one−863C/A) ,15,20,30morphism ,19. DetaTNF-α −308G/A polymorphisms and OAG risks are shown in TNF-α −308G/A polymorphism and the risk of OAG . Subgroup analysis stratified by type of OAG and ethnicity further confirmed the null association except the homozygous genotype comparison for EXG. However, when the two studies in which the genotype distribution in the control groups deviated from HWE were excluded [TNF-α −308G/A polymorphisms and the risks of HTG , but not with NTG  or EXG . Furthermore, ethnicity-stratified analysis revealed that a significant association also existed in Asians   .TNF-α  polymorphisms and the OAG risks are shown in TNF-α  polymorphisms.The pooled results on the associations between the TNF-α  polymorphisms and the OAG risks . Egger’s test and Begg’s funnel plot were applied for comparison to assess the publication bias of the literature, and no possibility of publication bias for this test was observed (p=0.191).The results of the meta-analysis for the comparisons of TNF-α levels in AH between the patients with glaucoma and the controls are shown in TNF-α gene polymorphisms and OAG risks, in which several gene polymorphisms in the TNF-α gene promoter region including TNF-α  have been investigated. However, these epidemiological studies obtained contradictory results, which might be related to factors such as the sample size of the study, the ethnicity of the enrolled subjects, and the type of OAG. Considering the potential important roles of TNF-α in the etiology of OAG, a timely meta-analysis was conducted to provide an appropriate approach to obtain a more definitive conclusion.Glaucoma is the second leading cause of blindness worldwide. Growing evidence supports the important role of TNF-α as a mediator of neurodegeneration in OAG ,5. Some TNF-α −308G/A, −857C/T, −863C/A, and −238G/A polymorphisms and the risks of OAG, respectively. When all the studies were included, the combined results showed no significant association between the TNF-α −308G/A polymorphism and the risks of OAG. However, when the studies in which the genotype distribution in the control groups deviated from HWE were excluded [TNF-α −308G/A polymorphisms were significantly associated with the risks of HTG, but not with the risks of NTG or EXG. Furthermore, ethnicity-stratified subgroup analysis revealed that significant association also existed in Asians. Regarding the other genetic polymorphisms including −238G/A, −863C/A, and −857C/T, the results of the current meta-analysis demonstrated no significant association.In the current study, 14, five, three, and three studies were included in the analyses for the associations between excluded , the pooTNF-α −308G/A polymorphism and the risks of glaucoma [TNF-α −308G/A polymorphism and the risks of glaucoma were included; those were included in the current study, in which five additional newly published studies were included [TNF-α −308G/A polymorphisms and the NTG risks was investigated reported null association [TNF-α −308G/A gene polymorphism was significantly associated with HTG risk, but not with NTG risks. EXG is another type of OAG with quite different characteristics from POAG. EXG is the most frequently reported type of secondary glaucoma, which is caused by deposition of exfoliation material and liberated iris pigment in the trabecular meshwork leading to elevation of IOP [LOXL1) gene [In a recently published meta-analysis, Yu et al. investigated the association between the glaucoma . In thatincluded ,19,29. Iincluded . Furtherociation ,19. Thern of IOP . Thus, EL1) gene .TNF-α gene polymorphisms and the risks of OAG, we summarized the studies in which the TNF-α levels in the AH were detected in patients with OAG as well as in control subjects. Some studies provided the mean and standard deviation of TNF-α levels in patients with OAG and controls, while other studies reported the positive and negative AH samples of patients with OAG and controls  polymorphisms are not significantly associated with OAG risks. Furthermore, the pooled results of six individual studies indicate that patients with OAG may have higher TNF-α levels in AH than the control subjects, which supports the important roles of TNF-α in the development of OAG. Since potential confounders could not be ruled out completely, further studies are needed to confirm these results.In summary, the current meta-analysis suggests that the


The HIV-1 envelope spike, which consists of a compact, heterodimeric trimer of the glycoprotein gp120 and gp41, is the sole viral target of neutralizing antibodies. The gp120 component of the viral spike is known to be heavily glycosylated, and glycosylation can affect the conformation of envelope spikes. V1/V2 variable loops of gp120 are key target regions for a number of broadly neutralizing human antibodies, such as PG9 and PG16, CH01-CH04, and PGT141-145. Two glycosylation sites (N156 and N160) have been shown by mutagenesis studies to be important in forming the PG9 and PG16 epitopes. Recently, Peter Kwong and coworkers have resolved crystal structure of V1/V2 domain of HIV-1 gp120 from strains CAP45 and ZM109 complexes with antigen-binding fragment of PG9.We employ enhanced molecular dynamics sampling methods (eg. replica exchange molecular dynamics) to dissect the influence of disulfide bond and glycosylation on the conformational landscape of an indel free epitope region of V1/V2 loop. These methods are expected to capture the influence of glycosylation, solvent, rest of the gp120 protein and scaffold constructs on the conformation of V1/V2.We evaluate the backbone conformational preferences and solvent accessibility of each residue in the selected V1/V2 region and compare them to the antibody-bound conformation of this region. Both the disulfide bond that links V1 and V2 loops and the nearby glycosylations affect the beta sheet formation propensity of that region. Further characterization indicates that glycans predominantly influence the entropy of the V1/V2 loops.Our studies reveal how glycans can impact the electrostatic and hydrophobic surfaces of the V1/V2 regions that have been proposed to form the epitope for broadly neutralizing antibodies. Along with proximal disulfide bonds, glycans tend to affect the local beta-sheet propensities that can further contribute to the quaternary nature of the epitope.


Tumor necrosis factor (TNF), a pleiotropic cytokine mainly produced by activated macrophages, modulates a wide range of biological functions in multiple tissues and organs. Besides its effects on tumor cell death, TNF is a key mediator of both acute and chronic inflammation. Since the description of TNF in the 1970s and through consideration of structural homologies, a total of 19 TNF-related cytokines have now been regrouped into a large family called the TNF ligand superfamily (TNFSF), whose members interact with TNF receptor superfamily (TNFRSF) members. More than 150,000 scientific publications (!) concerning TNF and its family members are available, demonstrating the strong interest of the scientific community in this molecule. Numerous studies implicating TNF family members in the pathophysiology of human autoimmune/inflammatory diseases have supported the emergence of TNF blocking agents developed for treatment of human disease, particularly over the last decade. These biotherapies, in the form of (I) chimeric, humanized, or human anti-TNF monoclonal antibodies or (II) fusion proteins involving a soluble TNF receptor, have been very successful in ameliorating disease signs and symptoms, especially in patients suffering from rheumatoid arthritis (RA) and Crohn's disease. Nonetheless, several aspects of these beneficial effects remain enigmatic. Moreover, the modulatory factors influencing TNF production by macrophages are not all known. Nevertheless, it is expected that over the next few years we will witness an increasing number of diseases for which TNF-blockade therapy is indicated.In this special issue, eleven papers including research articles, review articles, and clinical studies provide new information and interesting discussion regarding current questions related to this hot topic.β and TNF secretion and described here a beneficial anti-inflammatory effect of gambogic acid in rat antigen-induced arthritis. Interestingly, F. R. Spinelli et al. show that blocking TNF biological effects during RA is associated with an increase of circulating endothelial progenitor cells, which might positively affect the endothelial function and may help in correcting the endothelial dysfunction observed in the disease.In the first group of articles, interesting data is presented about TNF-blocking therapies and modulation of TNF generation by macrophages. Y. Lv et al. studied the nonneuronal cholinergic system existing in macrophages and show in a murine monocyte/macrophage cell line that bacterial lipopolysaccharide (LPS) exposure enhances autocrine acetylcholine production associated with an attenuation of TNF release. In this same cell line, K. Borzęcka et al. determined that, during high dose LPS stimulation, CD14 together with scavenger receptors is required for the binding of LPS but has a limited and dispensable contribution to TNF production. R. Cascão et al. recently identified gambogic acid as a simultaneous blocker of IL-1In the second group of articles, possible off-label uses of anti-TNF therapy in various disorders are discussed. Due to their low prevalence, discussion of the use of anti-TNF modalities in Behçet's disease, sarcoidosis, and noninfectious uveitis in the review by D. Sánchez-Cano et al. was mainly based on case reports and case series. The clinical study of C. García-De-Vicuña et al. supports the usefulness of adalimumab in the treatment of refractory uveitis associated with juvenile idiopathic arthritis. P.-A. Jarrot and G. Kaplanski review the use of anti-TNF therapy in vasculitis and conclude that, except for Behçet's disease, this treatment has not shown significant efficacy. In their review article, A. Kumar et al. described how HIV infection is modulated by TNF and TNFR superfamily pathways and then discuss the emerging therapeutic options based on the modulation of TNF activity.The third group of articles tackle the topic of the management of anti-TNF therapy for its well-established indications. B. Mörck et al. envision being able to reduce the drug costs in active HLA-B27 positive ankylosing spondylitis over time by reducing the infliximab dose and by extending the interval between infliximab doses. On the other hand, R. Altwegg and T. Vincent discuss the usefulness of monitoring serum trough levels and anti-drug antibodies in the optimization of anti-TNF therapies during inflammatory bowel disease.Lastly, Y. Aiba and M. Nakamura open a debate concerning a putative therapeutic role of TL1A/DR3 inhibition. They suggest that the modulation of this particular TNF/TNFR superfamily member interaction may be a potential therapeutic target in several autoimmune diseases including inflammatory bowel disease, RA, ankylosing spondylitis, and primary biliary cirrhosis.We hope that readers of the journal will find in this special issue not only accurate data and updated reviews on the targeting of TNF and its family members in autoimmune/inflammatory disease treatment, but also relevant questions that remain to be resolved including the extension of current therapeutic indications and the optimization of the anti-TNF therapies to find a better balance between cost and effectiveness.Sophie  Desplat-JégoSophie  Desplat-JégoLinda  BurklyLinda  BurklyChaim  PuttermanChaim  Putterman