Eicosapentaenoic acid (EPA) is a ώ-3 polyunsaturated fatty acid with anti-inflammatory and anti-cachetic properties that may have potential benefits with regards to skeletal muscle atrophy conditions where inflammation is present. It is also reported that pathologic levels of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α are associated with muscle wasting, exerted through inhibition of myogenic differentiation and enhanced apoptosis. These findings led us to hypothesize that EPA may have a protective effect against skeletal muscle damage induced by the actions of TNF-α.p < 0.05) and myotube diameter (p < 0.05) indices back to control levels. EPA protective activity was associated with blocking cell death pathways as EPA completely attenuated TNF-mediated increases in caspase-8 activity (p < 0.05) and cellular necrosis (p < 0.05) back to their respective control levels. EPA alone significantly reduced spontaneous apoptosis and necrosis of differentiating myotubes . A 2 hour pre-treatment with EPA, prior to treatment with TNF alone, gave similar results.The deleterious effects of TNF-α on C2C12 myogenesis were completely inhibited by co-treatment with EPA. Thus, EPA prevented the TNF-mediated loss of MyHC expression and significantly increased myogenic fusion (In conclusion, EPA has a protective action against the damaging effects of TNF-α on C2C12 myogenesis. These findings support further investigations of EPA as a potential therapeutic agent during skeletal muscle regeneration following injury. Skeletal muscle can be affected by a number of potentially damaging conditions which lead to muscle atrophy. These conditions can include ageing and disease states. With ageing, muscle wasting occurs which is a chronic condition complicated by multitude of factors. A number of models have been suggested to explain the ageing related loss of muscle tissue, amongst which is the immunological theory. Here changes in DNA methylation and mutation of the somatic cells  could lead to increases in autocatalytic processes which ultimately leads to self destruction. Hence, a gradual cumulative effect here, such as an immune inflammatory response may lead to muscle damage and sarcopenia .in vitro and in vivo have shown that raised levels of TNF-α causes increased muscle loss [in vitro studies suggest the effects of TNF-α are specific to the stage of myotube differentiation at the time of administration. Thus, delivery of TNF-α to primary human myoblasts or murine C2C12 myoblasts inhibits myosin heavy chain (MyHC) expression and myogenic differentiation [Disease states can be both chronic and acute, but both ageing and disease can share some common factors. In particular, inflammation has been suggested to be present in a number of disease states associated with muscle atrophy, such as cancer, heart failure, rheumatoid arthritis, chronic obstructive pulmonary disease, HIV/AIDS, and also ageing related muscle wasting. Associated with this response are pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α that are commonly present at elevated levels 0.5–10 ng/ml) during disease-5. A bim–10 ng/mlcle loss -12. At lntiation -16 wherentiation ,16. Howentiation .The cellular action of TNF-α is complex and is exerted through a number of signalling pathways in skeletal muscle. Recent findings suggest that low levels of TNF-α are required for myoblast proliferation and this aspect of myogenesis is regulated by activation of p38 mitogen-activated protein kinase (MAPK) . In contA number of experimental and clinical studies have described potential health benefits for ώ-3 polyunsaturated fatty acids (PUFA), notably in reducing the incidence of cardiovascular disease . EicosapThese findings led us to hypothesize that EPA may have a protective effect against skeletal muscle damage induced by pro-inflammatory TNF-α. Hence, the aims of the present study were to utilise TNF-α in a damage model of murine C2C12 myogenic differentiation and to determine whether EPA treatment was able to reduce the deleterious effects of TNF-α on skeletal muscle cell differentiation. We evaluated the responses of differentiating cells to TNF-α and EPA treatments by morphological criteria and by expression of MyHC and quantified their effects on apoptosis by measuring caspase-8 activity.Under permissive conditions in a low-serum culture medium (DM), C2C12 myoblasts will differentiate to form myotubes. After 5 days following initiation of differentiation by incubation in DM, the morphological appearance of formed myotubes was examined figure . EPA alop < 0.05) MI after 48 hours and at 5 days, compared to control treatment in DM. EPA alone had no significant effect on the MI. EPA also had no effect on the rate of differentiation as indicated by a comparison between EPA and control after 48 hours of treatment. Treatment with EPA, either as a pre-treatment or as a co-treatment with TNF-α, improved the MI with additional benefit derived from a longer incubation with EPA. Following pre-incubation with EPA, the MI was partially restored with levels significantly higher than with TNF-α, reaching 40% (p < 0.05) and 50% (p < 0.05), compared to 5% and 30% for TNF-α, after 48 hours and 5 days respectively. When EPA was added as a co-treatment with TNF-α, the MI was partially restored after 48 hours but was fully restored to control levels of 70% by 5 days (NS v. control). In figure p < 0.001). EPA co-treatment completely prevented these effects of TNF-α and the size distribution of myotubes was not significantly different from that of controls. Here, most myotubes were large with 40% having >10 nuclei, compared to 0% with TNF-α treatment (p < 0.001). EPA pre-treatment was able to partially reverse the effects of TNF-α and here myotubes were more heterogeneous, with proportionately more smaller myotubes and fewer large myotubes compared to controls (p < 0.001). The results presented in figure p < 0.05), reducing mean diameter after 5 days from approximately 27 μm to 8 μm, compared to control. EPA alone did not significantly alter myotube size. However, treatment of differentiating myotubes with EPA, either as a pre-treatment or as a co-treatment, with TNF-α improved myotube size. Following pre-incubation with EPA, myotube size was significantly higher than with TNF-α (p < 0.05), reaching approximately 20 μm. When EPA was added as a co-treatment with TNF-α, myotube size was also significantly increased compared to TNF-α (p < 0.05) reaching approximately 24 μm, a size not significantly different from controls (NS v. control).These protective effects of EPA were further evidenced when we investigated morphological parameters of myogenesis; namely myoblast fusion Figure  and myotp < 0.05) cell death in DM, compared to controls. These data suggested EPA was protective against cell death during myotube formation. Using an alternative approach of dye exclusion (Trypan Blue) which will not detect cells in the earlier stages of apoptosis with intact cell membranes, we confirmed that EPA significantly reduced cellular necrosis (p < 0.05) at an earlier time-point (48 h) from initiation of differentiation (figure p < 0.05) cellular necrosis by 48 hours after initiation of differentiation, compared to control with almost 50% of cells assessed as non-viable. EPA treatment was found to have a protective effect against the TNF-α-induced increase in necrosis. Co-treatment with EPA completely abolished (p < 0.05) the effects of TNF-α, compared to control  the TNF-α-induced damage, compared to TNF-α alone  in the proportion of apoptotic cells from approximately 20% to 6% following treatment with EPA, compared to untreated controls (figure p < 0.05) the caspase-8 activity associated with spontaneous apoptosis, compared to untreated controls (figure p < 0.001) increase in caspase-8 activity by 24 or 48 hours in DM, compared to respective untreated controls. EPA treatment was found to block the TNF-α-induced increase in caspase-8 activation at both of these time-points. Co-treatment with EPA completely blocked (p < 0.05) the effects of TNF-α on caspase-8 activity. A similar significant (p < 0.05) protective effect of EPA was obtained when cells were instead pre-incubated for 2 hours with EPA, prior to treatment with TNF-α alone.Further investigation of the apparent protective effect of EPA against the inhibition of myogenesis in differentiating C2C12 myotubes by TNF-α was carried out to determine whether protection against apoptosis was conferred by EPA. We first examined whether EPA had an effect on baseline levels of apoptosis. When 50 μM EPA was administered to C2C12 cells in DM there was apparent suppression of apoptosis measured after 5 days indicated by Hoechst 33258 staining figure . A calcus figure . To confs figure . We nextp < 0.05) effect of EPA on the reduction of myogenesis caused by TNF-α, that was evident in both the continuous and pre-incubation conditions. Alone, TNF-α caused a 9-fold decrease in MI after 48 hours whereas by 5 days this was only 2-fold. These data are consistent with reports that more mature myotubes are less susceptible to the damaging effects of TNF-α on myogenesis [It has previously been suggested that muscle cells exposed to TNF-∝ during differentiation will be adversely affected. This study examined the potential for EPA to protect skeletal muscle cells during their differentiation from myoblasts to myotubes in an inflammatory damage model. The main findings of this study were that EPA has a significant and protective effect on myogenesis in a murine C2C12 model of muscle damage. Undifferentiated C2C12 myoblasts, the in vitro equivalent of satellite cells, can be induced to differentiate into non-proliferating, multinucleated, fully differentiated myotubes over a period of days in the presence of a reduced-serum growth medium. Using this model, or primary human myoblasts, it has been shown that TNF-α has differential effects according to the developmental stage of myogenesis and these are mediated through alternative mechanisms that inhibit myotube formation or that induce myoblast and myotube apoptosis. Thus, in cultured differentiating primary human or C2C12 myoblasts, addition of TNF-α completely inhibits MyHC expression and myogenic differentiation ,14,29,30ogenesis ,14. In cp < 0.05) the TNF-α-induced damage, compared to TNF-α alone. However, as discussed above, the pre-treatment was sufficient to block caspase-8 activity induced by TNF-α. This may reflect an increased susceptibility of pathways associated specifically with caspase activation, to EPA inhibition, although this will require further clarification. A 2 hour pre-treatment with EPA has been used previously and found to effectively inhibit apoptosis in fully differentiated myotubes [Following TNF-α treatment, there is an initial 24 h period of myoblast proliferation associated with transient NF-κB and Jun kinases (JNK) 1 and 2 activation, followed by induction of apoptosis . More remyotubes . Other rmyotubes  to 24 homyotubes ,33,34. 2myotubes . It is p2 (PLA2) plays a major role in this process [2 via the TNF-R1. Interestingly, TNF-R1 expression is also upregulated by AA and can be inhibited by ώ-3 PUFAs including EPA [A definitive explanation for the ability of EPA to protect differentiating myotubes from the inhibitory effects of TNF-α is currently lacking. The most widely accepted proposition for its anti-inflammatory efficacy is that EPA changes the membrane phospholipid pool, becoming readily incorporated into the cell membrane at the expense of AA ,36. A po process . TNF-α mding EPA . Signallding EPA . Since oding EPA ,6. PUFA ding EPA . Similarding EPA . The abiding EPA ,35. It hding EPA . Peroxisding EPA . Recentlding EPA . Interesding EPA .In summary, the present study demonstrates that the ώ-3 PUFA EPA exerts a protective effect on differentiating C2C12 myotubes, attenuating the inhibitory effects of TNF-α on indices of myogenesis and apoptosis. These findings are consistent with reports in the literature regarding potential mechanisms of action for EPA, but these now require detailed investigation to clarify the nature of the observed benefits on skeletal muscle cells. These observations suggest for the first time that EPA may have potential benefits in skeletal muscle regeneration.4 cells/cm2 onto uncoated tissue culture plastic flasks or multi-well plates  for 24 h at 37°C and 5%CO2, at which point they had reached approx. 70% confluency. At this time myoblasts were induced to differentiate by briefly rinsing cells with PBS and replacing GM with DMEM containing antibiotics, supplemented with 2% (v/v) heat-inactivated horse serum, referred to as differentiation media (DM). For proliferation experiments myoblasts were maintained in GM beyond 24 h, up to 5 days. To evaluate their effects on myogenic differentiation, murine recombinant TNF-α  or EPA  were added to cell cultures directly following induction of differentiation. EPA was first complexed with fatty-acid free bovine serum albumin (BSA) (Sigma) as described previously [The murine skeletal muscle cell line C2C12 was obtained from the European Centre for Animal Cell Culture . C2C12 is a well established murine model for studying skeletal muscle differentiation. C2C12 myoblasts are able to undergo differentiation into spontaneously contracting myotubes on withdrawal of growth factors . Thus, meviously . Briefly®-546 IgG, at a dilution of 1:2000 in PBS for 1 h in the dark. After washing with PBS, cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI). The stained cells were analysed under a Nikon TE2000 inverted fluorescence microscope and DAPI and MyHC images were captured with a Hamamatsu Orca camera and merged using Image-Pro Lab v3.7 image analysis software .To study the effect of TNF-α and EPA treatments on differentiation of myotubes, immunocytochemistry was performed. Cultures were briefly rinsed with PBS, then fixed with ice-cold methanol for 2 min. Cells were then washed three times with PBS for 3 min, blocked with 1% BSA in PBS for 30 min, then incubated with 38 μg/ml of MF-20 antibody  to detect myosin heavy chain (MyHC) protein, at a dilution of 1:250 in 0.5% BSA/PBS for 1 h. After washing with PBS, cells were incubated with 2 μg/ml goat anti-mouse Alexa FluorMyotube metrics were also quantified using Image-Pro software to determine myotube diameter and a myoblast fusion index. Average myotube diameter was evaluated as the mean of five approximately equi-distant measurements taken along the length of the myotube. For each treatment, 10 fields of view were chosen randomly and 10 myotubes were measured in each field. A myogenic index (MI) was also calculated to indicate myotube fusion and the mean number of nuclei per myotube was calculated to indicate heterogeneity of myotube size. Using 10 images from randomly chosen microscope fields of DAPI and MyHC stained cells for each treatment, the total number of nuclei and the number of nuclei incorporated into myotubes were counted. The MI was calculated as the percentage of nuclei incorporated into myotubes (defined as containing at least two nuclei) relative to the total number of nuclei. Using the same images, the myotube heterogeneity was calculated as the number of nuclei per myotube, within categories of small (2–5 nuclei), medium (6–9 nuclei) and large (>10 nuclei) myotubes, expressed as a proportion (%) of the total myotubes.To determine the contribution of apoptosis to cell death during myotube differentiation, and the effects of EPA treatment on this, cells in DM were stained with Hoechst 33342 to identify apoptotic nuclei and images captured, as described above. Briefly, a final concentration of 1 μM Hoechst 33342 stain  was added directly to cells in culture. After 2–10 minutes media was removed and cells were rinsed in 1× apoptosis wash buffer (Invitrogen) and mounted using one drop of wash buffer and a coverslip. Analysis was performed immediately under fluorescence microscopy. To quantify the extent of apoptosis, an apoptosis index (AI) was calculated as described in , from 6 p < 0.05.Data is expressed as the mean ± SEM from three independent experiments. Statistical significance between two groups/treatments was tested using unpaired Student's t test. Statistical significance between more than two groups was tested using one-way ANOVA. Statistical significance was set at PM carried out the experimental studies. SP and JA conceived of the study, and participated in its design and coordination and helped to draft the manuscript. SP performed the statistical analysis. All authors read and approved the final manuscript.


Analysis of conditioned media generated by weakly and highly tumorigenic SKv-1 keratinocyte lines harbouring integrated human papillomavirus type 16 (HPV16) DNA sequences revealed a factor inhibiting TNF-alpha and TNF-beta cytotoxic activity. This inhibitory activity was specifically blocked by htr-9 monoclonal antibody (MAb) recognising 55/60 kDa type I TNF receptor suggesting that it is related to a soluble form of this particular receptor (sTNF-RI). The presence of sTNF-RI was confirmed by Western blot analysis of SKv-1 cell-conditioned medium showing a band of 31.5 kDa as well as by the specific enzyme-linked immunobiological assay (ELIBA). Release of sTNF-RI was a result of shedding because Northern blot analysis showed that SKv-1 cells expressed a full-length TNF-RI mRNA, and radioimmunoprecipitation of TNF-RI from [32S]cysteine-labelled cell extracts demonstrated the presence of normal 55 kDa molecule. Evaluation by ELIBA showed that highly tumorigenic SKv-12 cells released significantly more sTNF-RI than their weakly tumorigenic SKv-11 parental cells. Furthermore, human recombinant as well as SKv cell-derived sTNF-RI stimulated proliferation of weakly tumorigenic SKv-11 cells. This suggests that a progressive growth of some neoplastic cells may be, at least partially, a result of an increased spontaneous release of sTNF-RI that enables the cells to escape from local TNF-alpha-mediated growth inhibition.


The effect of L-carnitine and some of its acyl derivatives on serum TNF production and lethality in a murine experimental endotoxin shock model was investigated. In some instances, serum IL-6 production was also evaluated. In this experimental model, C57BL/6 mice received 30 mg/kg LPS (E. cell 055:B5) injected intraperitoneally, while L-carnitine and its derivatives were administered according to different schedules. Serum levels of TNF and IL-6 were evaluated 1 h following LPS injection. The treated animals were also monitored daily for differences in body temperature, feeding, and survival for 10 days after LPS injection. Although some derivatives were able to significantly affect TNF production, the marked decrease in serum TNF levels of LPS-treated mice was not paralleled by a substantial increase in survival.


Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1.The JNK pathway is a mitogen-activated protein (MAP) kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK), Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF) stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation.In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation.In contrast to mammals, it seems that in  Mkk4 and Mkk7 single mutants are embryonic lethal The JNK pathway, one of the three major classes of mitogen-activated protein (MAP) kinase pathways , is induced by pro-inflammatory cytokines, such as Tumor Necrosis Factor (TNF) and Interleukin-1 (IL-1), and several forms of environmental stress  Drosophila orthologs of Mkk4 and Mkk7 have been identified hemipterous/Mkk7 (hep), have been isolated Drosophila JNK, Basket (Bsk) hep lead to a defect in dorsal closure, a well characterized process in the Drosophila embryo that entirely depends on JNK signaling Drosophila Mkk4 only activates JNK but not p38 in vitro Drosophila Mkk4 have been identified and its functional relevance towards JNK activation therefore remains elusive. Based on the embryonic lethality of hep mutants it is obvious that Mkk4, which is expressed during embryonic development, cannot substitute for Hep function in this process. Although it has been reported that in mammals Mkk4 and Mkk7 may synergistically activate JNK DrosophilaDrosophila Mkk4 gene. Our genetic and biochemical experiments now demonstrate a non-redundant role for Mkk4 as a MAPKK acting in parallel of Hep in dTAK1-mediated JNK activation during both Eiger and Imd signaling.In a dominant suppressor screen for new components of the Eiger-JNK-pathway in DrosophilaDf(3R)Exel6149) that also suppresses the Eiger-induced small eye phenotype Exel6149 or in heteroallelic combinations. In some cases homozygous lethality is observed which is most likely due to second mutations on the chromosome. The absence of embryonic lethality associated with Mkk4 loss of function demonstrates that unlike Hep/Mkk7, Mkk4 is not rate limiting for dorsal closure of the Drosophila embryo.Mkk4 leads to a potent suppression of the Eiger-induced small eye phenotype -mediated JNK activation.Removing a single copy of henotype . Removinpression . Therefos effect . It is iMkk4 does not suppress the small eye phenotype induced by expression of an activated version of hep (CAhep) in the Drosophila eye hep and Mkk4 reduces this activity  or both copies of activity . Howeveractivity . In S2 cactivity  [13], [1reatment  suggestireatment . This lareatment . These rCA (strong) and HepWT (weak) (CA (Asp), we introduced the Ser277→Asp and Thr281→Asp mutations, which corresponds to the mutations that were introduced to generate HepCAAsp is not constitutively active, neither in flies (not shown) nor in S2 cells (Asp suppresses GMR-egr (CAGMR-hep (Mut) where mutations Ser277→Ala and Thr281→Val were introduced (WT (not shown) suggesting that the kinase activity of Mkk4 is not associated with its dominant negative effect upon overexpression. This effect may rather relate to differential binding ability towards dTAK1 and Bsk of Mkk4WT and Mkk4Asp. Indeed in CoIP experiments Mkk4Asp is still able to bind dTAK1 but no longer Bsk (In contrast to the intrinsic activity of HepT (weak)  and 2B [T (weak)  or in flT (weak)  or HepCAeak)  Mkk4 deficiency leads to similar immune phenotypes like dTAK1 loss of function. We challenged Mkk4 mutants with Ecc15 and monitored their survival over time. Relish and dTAK1 mutants, Mkk4 mutants survive like wild-type flies to this challenge. This result therefore suggests that Mkk4 is dispensable for the activation of the Imd/IKK/Relish cascade by dTAK1. Therefore, the involvement of Mkk4 in the Imd cascade is restricted to the dTAK1-mediated activation of the JNK branch  as well as the Regg1 parental line initially used to generate 1egr and 3egr alleles Regg1 P-element insertion, 1cRegg1, to create an isogenic wild-type control line of the Regg1, 1egr and 3egr alleles. As expected, egr transcripts were detected by RT-qPCR in all control lines  but not in 1egr, 3egr and 66egr mutant lines  and tubulinα1-Mkk4 transgene were obtained by standard P-element-induced transformation. 1dTAK1, E20Relish, rm7spz, semlPGRP-SA, Regg1, 1egr, 3egr, 66egr and 1hep fly strains were described previously 1cRegg1 was generated by classical transposase-mediated precise excision of the Regg1 P-element. Df(3R)Exel6149 was obtained from Bloomington stock center,. UAS-CAhep, GMR-gal4 and UAS-egr were previously described Fly stocks were maintained on polenta-agar medium at 23°C. Df(3R)Exel6149 was selected as a dominant suppressor deficiency. This deficiency removes 26 genes including Mkk4 and maps to the cytological location 85A Df(3R)Exel6149 at position 85A, which was isolated as a dominant suppressor from the deficiency screen). Chromosomes carrying a suppressor mutation were allowed to recombine with the RFP-marked chromosome in females. These virgins were crossed back to GMR-egr/CyO males. The number of non-CyO RFP(+) progeny (sorted under a fluorescent binocular) with a suppressed eye phenotype in relation to the number of non-CyO RFP(+) progeny with a small eye reflects the relative genetic distance to the RFP insertion. In stocks carrying a suppressor mutation that mapped very close to the RFP(+) insertion at 85E (only 1-3% recombination frequency between suppressor mutation and RFP(+) at 85E), and therefore also close to Df(3R)Exel6149 at 85A, the Mkk4 gene was checked for point mutations by sequencing. The Mkk4 gene was chosen because it was the most evident candidate from the 26 genes deleted in Df(3R)Exel6149.From our dominant suppressor screen no lethal complementation group on the third chromosome could be identified (10). Based on this observation we decided to combine two strategies in order to map dominant suppressor mutations on the third chromosome. First, we screened the whole Exelixis deficiency kit Mkk4 full-length cDNA (RE70055) was cloned into pUAST tubulinα1 promoter AspUAS-Mkk4 and mutUAS-Mkk4 the corresponding mutations were introduced by classical site directed mutagenesis.The Mkk4 coding region. PCR products were analyzed by standard sequencing.Genomic DNA was amplified by PCR using evenly spaced primers in the Mkk4, pUAST-HA-Mkk4, pUAST-AspMkk4, pUAST-HA-AspMkk4, pUAST-dTAK1, pUAST-dTAK1-FLAG, pUAST-hep, pUAST-CAhep, pUAST-bsk-FLAG, ptub-Gal4, pUAST-GFP.Schneider (S2) cells were cultured in Schneider's Drosophila medium  supplemented with 10% fetal calf serum and 1% penicillin/streptomysin at 25°. Cells were transfected with expression vectors, using Cellfectin (Invitrogen) according to the manufacturer's protocol. Expression vectors: pUAST-6 cells/well) were seeded into a 12-well plate. One day after seeding cells were transfected with the indicated expression vectors. Forty-eight hours after transfection the cells were harvested and lysed in lysis buffer containing 150 mm NaCl, 50 mm Tris-HCl (pH 8.0), 1% Nonidet P-40, 0.5% deoxycholic acid, and protease inhibitors . Lysates were mixed with an anti-HA antibody and 25 µl of Protein-A sepharose beads and allowed to rotate at 4°C overnight. The beads were then collected and washed with the lysis buffer four times. Proteins were eluted from the beads and resolved on a 4–12% NUPAGE gel system (Invitrogen) and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated with anti-FLAG M2 antibody (Sigma) followed by appropriate secondary antibodies conjugated with horseradish peroxidase (HRP). Signals were detected with ECL reagents .S2 cells (0.75×106 cells) and split into two halfs. One half was left untreated and the other half was treated with lipopolysaccharide (LPS) (Sigma) at a concentration of 50 µg/ml for 10 min  were seeded into a 24-well plate. One day after seeding cells were transfected with an AP1-luciferase reporter plasmid along with the indicated expression vector. The total DNA concentration (1 µg) was kept constant by supplementing with empty vector. Forty-eight hours after transfection, cells were harvested, lysed in passive lysis buffer, and luciferase activity was measured using the dual luciferase assay system (Promega). The values shown reflect the relative luciferase activity: the ratio of firefly (AP1 luciferase) and tub-renilla luciferase activity of one representative experiment in which each transfection was made in duplicate.S2 cells (0.4×10Double-stranded RNA (dsRNA) was prepared as described by the Dixon lab Mkk4: sense 5′-caatcccccggatcagctaag-3′; antisense 5′-cacatcccgatggataatctttagc-3′hep: sense 5′-gcaagtacattgtcaagtgcc-3′; antisense 5′-tggagcgttggatcgccattgg-3′Bsk: sense 5′-cgccgcaaaggaacttgg-3′; antisense 5′-tcagcatcataccacacg-3′dTAK1: sense 5′-gatgaccaacaatcgcgg-3′; antisense 5′-ggcgctgagtggcctcagc-3′GFP: sense 5′-gaacttttcactggagttgtcc-3′; antisense 5′-gccatgtgtaatcccagcagc-3′egr  and RpL32  were used to detect target gene transcripts. The amount of egr mRNA detected was normalized to control RpL32 mRNA values. Normalized data was used to quantify the relative levels of a given mRNA according to cycling threshold analysis (ΔCt). Relative ΔCtegr/ΔCtRpL32 ratios of WT controls were anchored in 1 to indicate fold-induction. Graphs represent the mean and S.D of relative ratios detected in 3 biological repetition of a pool of 15 males.SYBR Green quantitative real-time PCR analysis was performed as previously described Erwinia carotovora carotovora 15  170, 29°C); Enterococcus faecalis  or Staphylococcus aureus . Flies were incubated at the indicated temperatures and their survival was monitored twice every day.Systemic infections were performed by pricking 60 adult males of 4 to 7 days old in the lateral thoracic region with a thin needle previously dipped into a concentrated pellet of the following bacteria.


Mycobacterium tuberculosis (Mtb) infection. Formation and maintenance of granulomas are essential for control of Mtb infection and are regulated in part by a pro-inflammatory cytokine, tumor necrosis factor-α (TNF). To characterize mechanisms that control TNF availability within a TB granuloma, we developed a multi-scale two compartment partial differential equation model that describes a granuloma as a collection of immune cells forming concentric layers and includes TNF/TNF receptor binding and trafficking processes. We used the results of sensitivity analysis as a tool to identify experiments to measure critical model parameters in an artificial experimental model of a TB granuloma induced in the lungs of mice following injection of mycobacterial antigen-coated beads. Using our model, we then demonstrated that the organization of immune cells within a TB granuloma as well as TNF/TNF receptor binding and intracellular trafficking are two important factors that control TNF availability and may spatially coordinate TNF-induced immunological functions within a granuloma. Further, we showed that the neutralization power of TNF-neutralizing drugs depends on their TNF binding characteristics, including TNF binding kinetics, ability to bind to membrane-bound TNF and TNF binding stoichiometry. To further elucidate the role of TNF in the process of granuloma development, our modeling and experimental findings on TNF-associated molecular scale aspects of the granuloma can be incorporated into larger scale models describing the immune response to TB infection. Ultimately, these modeling and experimental results can help identify new strategies for TB disease control/therapy.Tuberculosis (TB) granulomas are organized collections of immune cells comprised of macrophages, lymphocytes and other cells that form in the lung as a result of immune response to  Mycobacterium tuberculosis (Mtb). Multiple host immune factors control the formation of a self-organizing aggregate of immune cells termed a granuloma in the lungs after inhalation of Mtb. One such factor, tumor necrosis factor-α (TNF), is a protein that regulates inflammatory immune responses. Availability of TNF within a TB granuloma has been proposed to have a critical role in the protective immunity against TB. However, direct measurement of the level of TNF in a granuloma is not experimentally feasible. Therefore, we develop a mathematical model based on an experimental model of granuloma developed in mice to predict TNF availability in a granuloma. We measure values of critical model parameters and explore mechanisms that influence TNF availability in the granuloma. We find that cellular organization in a granuloma and intracellular trafficking of TNF control TNF availability in a granuloma. Further, our model analysis also highlights anti-TNF drug properties that determine their TNF neutralization power. Our findings complement and extend those of recent studies on the role of TNF in the immune response against TB.Tuberculosis is a common and deadly infectious disease caused by a highly successful bacterium,  Mycobacterium tuberculosis (Mtb), and is responsible for three million deaths per year Tuberculosis (TB) is caused by a highly successful bacterium, Tumor necrosis factor-α (TNF) is a well-studied inflammatory cytokine that is produced by immune cells, especially activated macrophages and monocytes. TNF is expressed as a 26 kDa membrane-bound precursor protein  that can be cleaved by proteolytic activity of a metalloproteinase TNF-α converting enzyme (TACE) and released as a 17 kDa subunit  into extracellular spaces To elucidate the mechanisms by which availability of TNF in a granuloma is controlled, we focus on TNF interactions with immune cells that comprise a granuloma. We are interested particularly in TNF receptor dynamics. Receptor/ligand interactions at the cell membrane are responsible for initiating intracellular signaling pathways and ultimately the cell response to the external stimulus. However, trafficking events  have been demonstrated to take place under normal physiological conditions and can influence the availability of ligand, the number of ligand-bound receptors and thus receptor-mediated cell responses Mycobacterium purified protein derivative (PPD) antigen. This model is an appropriate choice for our study as it provides cytokine and cellular patterns that closely match those in an active mycobacterial infection The influence of the dynamics of TNF/TNFR trafficking processes on the availability of TNF in a TB granuloma has never been studied. Thus, we develop a reaction/diffusion-based partial differential equation (PDE) model that describes a TB granuloma as a continuous collection of immune cells forming concentric layers and includes TNF/TNFR binding and trafficking processes. Our multi-scale model is focused on TNF/TNFR-level reactions and interactions, while using a coarse-grain description of the cellular-level details representing a snapshot in time of a granuloma comprised of a static number of immune cells. To analyze the model, we use estimations for TNF/TNFR-associated parameter values from literature and then employ an artificial experimental mouse model of TB granuloma  to quantAnimal studies were conducted according to University of Michigan Committee on Use and Care of Animal (UCUCA)-approved protocol (No. 8307).The binding interactions and reactions controlling TNF/TNFR dynamics at the single-cell level regardless of the cell type are illustrated in Several TNF-neutralizing drugs have been developed and they work to interfere with TNF activity and thus are used to control inflammation in human diseases such as rheumatoid arthritis and Crohn's disease. These drugs are composed of either monoclonal antibodies (e.g. infliximab) or receptor fusion molecules (e.g. etanercept) that specifically bind TNF, acting as a competitive inhibitor for TNF binding to cell surface TNFRs and eventually neutralizing its functions To study the effect of TNF-neutralizing drugs of various properties on TNF/TNFR dynamics, we modeled a hypothetical drug as an agent that binds to sTNF or both sTNF and mTNF molecules and also inhibits sTNF binding to both TNFRs. We captured TNF neutralization-associated reactions  with MATLAB 7.5 . Simulations were run until a steady state was reached . Because TNF-associated molecular level processes studied here occur significantly faster than cellular level events that may change the structure of a granuloma , we assumed that the structure of granuloma is not changed during the 12-hour time course of simulations.separation index (s), to present the level of separation between different cell types in a granuloma, defined as:ol, gl are the lymphocyte (T cells and B cells) fractions in the outer compartment and in the whole granuloma, respectively. Thus a separation index of 0 is equivalent to a totally mixed cellular organization, whereas a separation index of 1 represents a separate cellular organization approximately as observed in human and non-human primate models of TB in which DCs and macrophages reside in the inner compartment (core) and lymphocytes compose the outer compartment (mantle). A schematic representation of the effect of changing s on cellular organization of the bead granuloma is shown in Supplementary inρ and outρ) are computed and replace general cell density ρ in diffusion equations of To study the influence that specific cellular organizations may have on the availability of TNF within a TB granuloma, we explicitly incorporated major granuloma-comprising cell types (determined from experiments performed herein), including macrophages, DCs, T cells and B cells into our mathematical model of a granuloma. We defined a metric, We assumed that some TNF/TNFR kinetic parameters, including the rate constants for TNF release by TACE activity, TNF/TNFR association and dissociation as well as TNFR internalization, shedding, degradation and recycling have the same values for different cell types. This assumption is based on consistency of experimental data on measurement or estimation of values of these parameters for a variety of cell types including various cell lines expressing TNF and/or TNF receptors with one another as well as other data on similar mammalian cell surface receptors The protective role of TNF in immunity to Mtb infection has been shown to depend primarily on the soluble form (sTNF) and its interactions with TNFR1 To identify parameters that significantly influence the outcomes of the two-compartment model of a granuloma, we used Latin hypercube sampling (LHS) f defined as the fraction of granuloma in the outer compartment. The parameter f directly determines the thickness of the inner and outer compartments as indicated in Supplementary synth_ink, synth_outk, 1_inR, 1_outR, 2_inR, 2_outR). Thus, these parameters together with f determine the overall rate of TNF and TNFR expression in the granuloma. Note that the separation index (s) defined above is not used in the absence of distinct cell types . Simulations sampled each parameter 1000 times, producing 1000 solutions to the model equations. To determine the correlation between parameter values and each of the model outputs, partial rank correlation coefficient (PRCC) values were calculated p-values derived from Student's t test. The choice of number of simulations (N) is determined by the desired significance level for the PRCC N = 1000 implies that PRCC values above +0.09 or below −0.09 are significantly different from zero (p<0.001). Model parameters then were categorized for their significance in affecting the model outputs based on their PRCC values.To reduce the number of parameters in LHS simulations, we replaced distinct cell type fractions with a general parameter 0C in the surrounding tissue with a flux into a granuloma that is dependent on drug permeability ck and the drug gradient at granuloma radius r = gr:0C was considered constant within the time course of simulation that is significantly shorter than the decay time reported for TNF-neutralizing drugs E, was defined as a function of the ratio of the spatially averaged steady-state concentration of sTNF before drug addition to the spatially averaged concentration of sTNF when drug exerts its steady state maximum effect, i.e. approximately 6 hours after drug addition.t = 0 stands for the steady state condition at which drug was added.To study the effect of TNF-neutralizing drugs of various properties on TNF availability in a granuloma, the model was run in the absence of drug until a steady state was reached and then the drug was added. We modeled the drug source as a concentration Mycobacterium purified protein derivative (PPD) as previously described We use an artificial mouse model of TB granuloma that has been demonstrated to provide a well-circumscribed lung granuloma typified by a type 1 cytokine phenotype characterized in TB 5 events were counted using a BD-LSRII system flow cytometer (BD Biosciences). F4/80+ CD11b+ macrophage, B220+ CD11c+ lymphoid dendritic cell (pDC), CD11b+ CD11c+ myeloid dendritic cell (mDC), B220+ B cell, CD4+ T cell and CD8+ T cell populations were gated following compensation for fluorochrome spectral overlaps. Cell fractions in granulomas were identified following analysis by FlowJo software .To identify the cellular composition of PPD bead granulomas, we used multi-color flow cytometry with fluorescing antibodies specific for immune cell markers, including macrophages, DCs, T cells and B cells. Other immune cells such as neutrophils and eosinophils were not quantified as they have been shown to constitute only a tiny fraction of PPD bead granulomas 6 cells per volume of 200 µl were stained with saturating concentrations of antibodies that were identified to be 2 mg/l for anti-TNFR1 and 1 mg/l for anti-TNFR2 antibodies. We generated a calibration plot from the fluorescence intensity measurements on Quanti-BRITE PE-conjugated standard micro-beads (BD Biosciences). This plot was used  to quantify TNFR1 and TNFR2 densities on the membrane of granuloma macrophages, mDCs, pDCs, B cells, CD4 and CD8 T cells based on the PE mean fluorescence intensities.To quantify the number of TNFR1 and TNFR2 molecules on the membrane of each cell type, we used quantitative flow cytometry with Phycoerythrin (PE)-conjugated anti-TNFR1 or -TNFR2 antibodies (BioLegend), together with staining of cell-specific markers as described above. Thus, cell suspensions at a concentration of 1×102 humidified environment for 4–5 hours. TNF-α proteinase inhibitor-1  at a concentration of 100 µM was added to inhibit TNF release from cell membranes 6 cells at a sequence of time-points within a 3 hour period of TACE inhibition at 37°C.Because TNF is initially synthesized as a membrane-bound molecule (mTNF), we can also use quantitative flow cytometry to quantify the rate of TNF synthesis by granuloma-comprising cells, including macrophages, DCs, B cells and T cells. Live granuloma cells were first isolated (from the lungs of a group of 10 mice) by using a Dead Cell Removal Kit (Miltenyi Biotec) and incubated at 37°C in a 5% COy = btae + c as the general form of Equation 10 (which is derived from Equation 3) using MATLAB. Parameters a, b, c then were used to calculate TACEk' and synthk.mTNF]0 is the steady-state initial number of mTNF on the cell membrane and TACEk' is the TNF release rate constant in the presence of TAPI-1 (TACEk'≤TACEk). Knowing synthk and the steady-state initial number of mTNF [mTNF]0, the value of TACEk can be calculated from:The rate of mTNF synthesis for distinct TNF-producing cell types was derived by nonlinear regression of the experimental data to an equation of the form To understand the extent to which granuloma properties  impact the availability of TNF, simulations were run for different values of model parameters within ranges given in synth_ink and synth_outk), the average TNFR1 density in the inner and outer compartments , the outer compartment fraction of granuloma (f) as well as both TNF receptor affinities for sTNF (d1K and d2K) and the rate constant for TNF-induced internalization of TNFR1 (int1k). Indeed, using different values of these particular parameters as inputs leads to outputs similar to those shown in To identify parameters that significantly influence the availability of TNF within a granuloma, sensitivity of the outputs of the model describing TNF trafficking in a granuloma to changes of input parameters was explored. d2K) as a competitor of TNFR1 for binding to sTNF. Conversely, TNFR1 internalization rate constant int1k, TNFR1 density in both compartments, equilibrium dissociation constant of TNFR1 (d1K), and the outer compartment fraction of granuloma f negatively correlate with this same output. Although greater affinity of TNFR1 for sTNF enhances the level of sTNF binding to TNFR1 in the core of granuloma (output 2) as the major TNF-producing compartment, it reduces the access of TNFR1 on the membrane of cells in the outer compartment to diffusing sTNF (output 3). Thus, increasing the effective diffusion coefficient of sTNF in the granuloma increases the sTNF-bound fraction of receptors in the outer compartment. Diffusion of shed sTNF-bound TNFR2 complex from the inner compartment to the outer compartment of granuloma can also explain the positive correlation of TNFR2 density in the core with the sTNF-bound fraction of TNFR1 in the outer compartment (output 3), while it is negatively correlated with the same response in the inner compartment (output 2) due to competition between receptors for binding to sTNF. Significant correlations of model parameters with the level of free sTNF concentration in the granuloma (output 4) are qualitatively similar to their correlations with output 1, except for d1K and sTNF/TNFR1 association rate constant on1k that are, respectively, positively and negatively correlated with output 4.Parameters that positively correlate with the sTNF-bound fraction of cell surface TNFR1 in the whole granuloma  in the percentage of both CD4 and CD8 T cells which represent the adaptive immune response was observed in day 4 granulomas compared with day 2 granulomas. On the other hand, macrophages and DCs were shown to form a slightly smaller portion of day 4 granuloma cell population. The percentage of B cells in granulomas did not significantly change from day 2 to day 4. Cellular composition of the granuloma and the increase in the level of T cell recruitment with time are qualitatively consistent with the experimental data on the infiltration of immune cells into the lungs of mice infected with Mtb as well as data on granulomas induced in lungs of Mtb-infected monkeys, although T cell recruitment occurs in a shorter time scale for PPD bead granulomas We used an artificial model of TB granuloma developed in mice following injection of PPD-coated beads to measure model parameters of interest. To identify the cellular composition of PPD bead granulomas, multi-color flow cytometry with fluorescing antibodies for specific immune cell surface markers was used as described in 3 molecules per cell and a lower level of expression for TNFR2 (The average numbers of TNFR1 and TNFR2 molecules on the membrane of day 2 and day 4 granuloma-comprising immune cells were quantified by flow cytometry using standard PE-conjugated beads as described in or TNFR2 . Furthersynthk by different types of immune cells in granulomas isolated at 2 and 4 days were measured by flow cytometry as described in TACEk' was not zero. A higher concentration of TAPI-1 (200 µM) has been shown to have stronger inhibitory effects on the release of TNF from the membrane of human peripheral blood T cells synthk, TACEk and TACEk' from three experiments are averaged and reported in Using TAPI-1 as a TACE inhibitor to suppress the release of TNF from the membrane of TNF-expressing cells over a 3 hour time course, the rates of mTNF synthesis In general, the differences between experimental data on day 2 and 4 granulomas, although significant, are fairly small. Thus, using our data on cellular composition, cell-specific rates of mTNF synthesis and TNFR densities from day 4 PPD-bead granulomas as well as literature data on TNF/TNFR kinetic parameter estimates as inputs to our model, we studied mechanisms that control steady state TNF availability within a TB granuloma. Here we illustrate the role of two important factors, (i) molecular level processes governing TNF/TNFR interactions and intracellular dynamics and (ii) cellular organization within the granuloma, in regulating TNF availability within a granuloma.To study the influence of TNF-associated molecular level processes on the availability of TNF, and thus TNF signaling within a TB granuloma, the distribution of sTNF in a granuloma was calculated by comparing modeling results in the presence of TNF intracellular trafficking with results of the model in the absence of TNF/TNFR internalization and shedding or TNF binding to TNF receptors. s) between populations of macrophages/DCs and lymphocytes within the granuloma  by anti-TNF drugs is influenced by drug properties, including drug/TNF association and dissociation kinetics, drug ability to bind to mTNF, and drug/TNF binding stoichiometry. Three classes of hypothetical drugs with defined properties were modeled loosely based on properties of human TNF-neutralizing drugs (e.g. infliximab and etanercept) and their efficiencies of TNF neutralization were compared. Since the general behavior of all classes of drugs was shown to be independent of cellular organization in the granuloma (data not shown), model results for a separation index of one are discussed below.on_sTNF/drugk and off_sTNF/drugk) for sTNF and drug are shown in on_sTNF/drugk without changing drug affinity leads to an increase in the drug neutralization efficiency .et al on the role of TNF in host defense against TB et al describes the temporal dynamics of the immune response to Mtb infection in active and latent phases within a time course of 500 days by inclusion of TNF immunological functions on macrophages and T cells. However, we focus in this study on a snapshot in time of a granuloma to study the steady state spatial distribution of available TNF. We used results of our model sensitivity analysis as a novel tool to lead experiments to measure critical model parameters in artificial granulomas induced in the lungs of mice following injection of mycobacterial PPD-coated beads. Finally, whereas TNF neutralization has been simulated by Marino et al via removing fractions of available sTNF and/or mTNF, we studied the effects of TNF-neutralizing drugs by incorporation of their mTNF and/or sTNF binding kinetics and stoichiometry.The detailed consideration of synthesis, diffusion, receptor binding and intracellular trafficking of TNF within the heterogeneous three-dimensional structure of a granuloma distinguishes our model from a previous study by Marino Model analysis helped us characterize two mechanisms for controlling the availability of TNF within a granuloma. These mechanisms include intracellular trafficking of TNF via internalization of recyclable TNFRs, and specific cellular organization within the granuloma, i.e. the level of separation between different classes of cells. Further, we demonstrated that for the resulting effect of cellular organization on spatial distribution of available TNF in the granuloma to be significant, intracellular trafficking of TNF is essential . Hence, −11 M) that can activate only a limited number of cell surface TNFR1 molecules are not capable of inducing apoptosis in the cells −13 M based on both modeling For sufficiently large values of the separation index in the granuloma, the model predicts significantly greater levels of sTNF binding to TNFR1 on the membrane of macrophages/DCs in the core compared with lymphocytes in the mantle , which mThe hypothesis of differential induction of TNF-mediated signaling pathways for classical granulomas such as ones observed particularly in human, nonhuman primate and guinea pig models of TB −7–10−6 M Finally, we used the model to predict and analyze the effects of TNF-neutralizing drugs with different properties on the availability of TNF within a developed granuloma. Average serum concentration of two murine analogs of human TNF blockers, infliximab and etanercept, after a single dose of drug, has been reported to be on the order of 10on_TNF/drugk = 5.7×104 M−1s−1, off_TNF/drugk = 1.1×10−4 s−1 and etanercept: on_TNF/drugk = 2.6×105 M−1s−1, off_TNF/drugk = 1.3×10−3 s−1) ck) by up to two order of magnitude (data not shown). Thus, the difference in binding properties of infliximab and etanercept must be considered when explaining the higher rate of TB reactivation induced from infliximab treatments in comparison with etanercept, although we anticipate that differential functional properties such as induction of apoptosis in TNF-producing cells by infliximab but not etanercept further influence the outcome of anti-TNF treatments We can use these modeling results to generate a hypothesis regarding experimentally observed differences in the neutralizing power of the two major human TNF blockers, infliximab and etanercept, based on their TNF binding specificities. Infliximab is a chimeric monoclonal TNF antibody that binds potently to both sTNF and mTNF. Up to three infliximab molecules can bind to one trimeric TNF molecule. Etanercept is a TNF receptor p75-IgG fusion protein that can also bind to both trimeric sTNF and mTNF. However, two receptor arms of etanercept contact two of the three receptor binding sites on different faces of the trimeric TNF, leading to a binding molar ratio of 1∶1 for TNF/etanercept binding. Further, etanercept has greater TNF association and dissociation rate constants in comparison with infliximab Although we have focused this study primarily on molecular and cellular scale processes within a snapshot of time in a granuloma, it will be necessary to consider multiple time and length scales (including dynamics in the lymphatic system) to further examine the role of TNF and anti-TNF therapies in the process of granuloma development and maintenance. We are currently working on this multi-scale approach Text S1Simulation results for the spatial profiles of different forms of TNF in the model(0.03 MB DOC)Click here for additional data file.Table S1Parameters defined or modified based on incorporation of different cell types in the granuloma model(0.07 MB DOC)Click here for additional data file.Table S2Parameter sets used to generate curves on (0.04 MB DOC)Click here for additional data file.Figure S1s and f used in the two-compartment model of PPD-bead granuloma. (A) Parameter s (separation index) is defined as indicated in Equation (5) to present the level of separation between different cell types in the granuloma model  when all cell types are present. A separation index (s) of 0 is equivalent to a totally mixed cellular organization. Increasing s leads to an increase in the level of separation in the cellular organization as s = 1 represents a cellular organization in which macrophages and DCs are separate from but surrounded by lymphocytes. (B) Parameter f is defined as the fraction of cellular granuloma in the outer compartment and is only used when distinct cell types are not considered in the model . Increasing f results in a decrease in corer while beadr and gr are maintained constant.A schematic representation of parameters (2.14 MB TIF)Click here for additional data file.Figure S2shed, sTNF-bound and internalized TNFRs in a granuloma for two sets of parameter values: (A), (B) synth_ink = 1 #/cell.s, synth_outk = 0.01 #/cell.s, 1_inR = 2_inR = 1_outR = 2_outR = 2000 #/cell. (C), (D) synth_ink = synth_outk = 0.1 #/cell.s, 1_inR = 2_inR = 1_outR = 500 #/cell , 2_outR = 5000 #/cell. For both simulations, s = 1 and f = 0.5. Other parameter values are as listed in beadr) and radius at which the two compartments are separated (corer).Simulation results for the steady-state concentration profiles of the model species, including sTNF, sTNF/TNFR2(2.22 MB TIF)Click here for additional data file.


The investigative anti-tumour agent 5,6-dimethylxanthenonone-4-acetic acid , developed in this laboratory as an improved analogue of flavone acetic acid , is currently in clinical trial. The ability of DMXAA to up-regulate tumour necrosis factor (TNF) mRNA and protein synthesis in cultured human peripheral blood leucocytes (HPBLs) has been investigated and compared with that of flavone acetic acid (FAA) and of bacterial lipopolysaccharide (LPS). Human peripheral blood leucocytes were isolated from buffy coats obtained from a blood transfusion centre and also from blood samples from laboratory volunteers. At a concentration of 400 microg ml(-1) and an incubation time of 2 h, DMXAA up-regulated mRNA synthesis in six of eight individuals tested, as measured by Northern blotting. The degree of up-regulation varied in different individuals from one to nine times that of control levels. In contrast, FAA caused no induction above that of control levels and in some cases suppressed expression relative to controls, extending previous data that DMXAA but not FAA up-regulates TNF mRNA in the human HL-60 tumour cell line. At the same concentration but with longer incubation times (6-12 h), DMXAA induced increases in TNF protein in 11 of 15 samples of HPBLs from buffy coats and also in 11 of 15 samples of HPBLs from volunteers, as measured by cytotoxicity assays with L929 cells. FAA caused no increase in TNF protein, while LPS induced TNF to approximately 20-fold higher levels than did DMXAA. Considerable heterogeneity of response was observed with both sources of HPBLs, and there was little or no correlation between the extent of TNF induction by DMXAA and LPS in individual samples. In vitro analysis of the response of human peripheral blood leucocytes to DMXAA may be a useful test in clinical trials of agents such as DMXAA.


The role of microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase) in the mutagenicity of benzo(a)pyrene-3,6-quinone  was studied using supF tRNA gene as the mutational target. pUB3 carrying the supF tRNA gene upon transformation into the Escherichia coli ES87 cells exhibited a spontaneous mutation frequency of 0.62 x 10(-6). Chemical modification of the pUB3 DNA with BP-3,6-Q caused a fourfold increase in the mutation frequency, compared with the spontaneous mutations. P450 reductase catalysed metabolic activation of BP-3,6-Q into reactive products (semiquinone and reactive oxygen species), which caused a further increase in the mutation frequency to eightfold over spontaneous mutations. Oxygen radical scavengers  blocked the P450 reductase-activated BP-3,6-Q-induced stimulation of mutations. This indicates that redox cycling of the semiquinone leading to the generation of reactive oxygen species (ROS) was directly responsible for the increased mutation frequency of P450 reductase-activated BP-3,6-Q. Analysis of the mutation spectra revealed that P450 reductase-activated BP-3,6-Q showed a significantly higher preference for frameshift mutations, particularly deletions, compared with the spontaneous mutations and the mutations generated by benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). The single most frequently observed mutation by P450 reductase-activated quinone (semiquinone + ROS) was deletion of a single guanosine. Among the base substitutions, G:C --> T:A, G:C --> A:T and G:C --> C:G were also noticed. Interestingly, NQO1 competed with P450 reductase and specifically prevented the P450 reductase-activated BP-3,6-Q-induced mutations. However, BP-hydroquinone  generated during the metabolic reduction of BP-3,6-Q catalysed by NQO1 caused specific mutations involving the deletion of a single cytosine from the DNA sequence 5'-CCCCC-3' in supF tRNA gene at a significantly high frequency. A similar cytosine deletion was also observed with benzoquinone hydroquinone (HQ), indicating that the deletion of cytosine is associated with a hydroquinone class of compounds. These results suggest that: (1) quinones and P450 reductase-activated products of quinones (semiquinones and ROS) are mutagenic compounds; (2) the mutational spectra of quinones, semiquinones and hydroquinones differ from each other with respect to their mutational frequency and specificity; (3) NQO1 competes with P450 reductase and protects the cells from quinone mutagenicity; and (4) the NQO1 -metabolized quinones (hydroquinones), if not eliminated, cause specific mutations that are not observed with quinones and P450 reductase-activated quinones (semiquinones and ROS).


In this report we demonstrate that the herpes simplex virus type 1 reiteration element 1 (RE1) (nt: 117158–117353) in concert with its flanking sequences is both a cell specific and stimulus inducible regulatory domain. This region of the virus genome and specifically the RE1 supports differential reporter gene expression in both baby hamster kidney cells and disassociated rat trigeminal ganglia and is present within a region that is implicated in regulating latency of the virus in neuronal cells. Further we demonstrate that this locus is a transcriptional regulatory domain and a target for the transcription factor CCCTC binding protein. We therefore set out to address the hypothesis that the RE1, URI and UI, which is similar to URI but has the RE1 region removed  can support differential cell and stimulus inducible expression in vivo and in vitro  removed , can act22.1The RE1, UI and URI regions were cloned upstream of the minimum SV40 promoter in the pGL3 promoter vector . The CTC2.2Baby hamster kidney (BHK) fibroblasts were transfected with Exgen 500 in vitro transfection reagent (Fermentas) using the standard protocol a transfection efficiency of 80–90% was achieved. BHK fibroblasts were maintained in Glasgow’s medium (Invitrogen) supplemented with 10% FCS and 1% (v/v) 100× penicillin/streptomycin .renillia luciferase  was added as an internal control in all experiments. Reporter gene expression was analyzed 48 h after transfection using the Dual Luciferase Reporter Assay System (Promega) and assayed for both luciferase and renillin activity using a luminometer (GLOMAX 96 microplate luminometer). The mean normalized luciferase values were calculated together with the SEM (luciferase/renillin). To normalise for transfection efficiency the total DNA concentration was standardized by co-transfection of reporter constructs with 1 μg of CTCF or 1 μg of pGL3b (Promega). The CTCF data was normalised to transgene expression from pGL3p co-transfected with CTCF.Disassociated TG were prepared from Wistar rats (2–5 days old) and cultured based on a previously published protocol 3In this communication we demonstrate using transient transfections that the URI domain, UI and RE1 components  have difThe RE1 in the context of URI is a repressor in fibroblasts B, as it One of the cellular factors that could affect virus reactivation is the transcription factor CTCF, as it is responsive to cellular challenges including UV light In genome wide analyses in Drosophila, CTCF is found to bind between closely spaced genes, which is an atypical distribution compared to other insulator proteins and suggest that it plays an active role in regulating gene activity In both fibroblasts and TG the RE1 domain acts as a repressor of an active enhancer when it was cloned into the pGL3c vector B. The pGCTCF has been implicated as a major determinant controlling HSV-1 latency. A chromatin insulator-like element in the HSV-1 LAT intron, CTCF repeat long 2 (CTRL2), has homology to the RE1 and contains nine copies of the CTCCC CTCF consensus. CTRL2 has been shown to bind CTCF and demonstrated enhancer blocking activity in vitro Taken together our data suggest this locus is involved in cell specific expression, and that the RE1 is key in the regulation of gene expression. Furthermore, the RE1 can act as an enhancer blocker, therefore it appears the locus may have different functions during the HSV-1 lifecycle. The data illustrates mechanisms by which tandem repeats can synergistically interact with their surrounding sequences to modulate enhancer function in a cell specific manner. An activity for CTCF at the UI and URI domains expands the repertoire of this transcription factor as a regulator of HSV-1 latency


Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci) in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-α. Our study demonstrates distinct profiles of TNF-α-activated gene expression in Thy-1 positive (Thy-1+) and negative (Thy-1−) subsets of mouse embryonic fibroblasts (MEF). TNF-α induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1− MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1− MEFs significantly attenuated TNF-α-activated gene expression. Mechanistically, TNF-α activated Src family kinase (SFK) only in Thy-1− MEFs. Blockade of SFK activation abrogated TNF-α-activated gene expression in Thy-1− MEFs, whereas restoration of SFK activation rescued the TNF-α response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-α-activated gene expression via interfering with SFK- and NF-κB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation. Thy-1 is a glycosyl phosphatidylinositol-anchored glycoprotein that is expressed in a variety of cell types, including T cells, thymocytes, neurons, endothelial cells, and fibroblasts Accumulating evidence indicates that fibroblasts are important modulators of inflammation and immunity Immunohistochemical analyses of human lung sections reveal that fibroblasts within fibroblastic foci of idiopathic pulmonary fibrosis do not express Thy-1, whereas most fibroblasts from normal lungs are Thy-1+ TNF-α is a major pro-inflammatory cytokine. Upon binding to its receptors on the cell membrane, TNF-α initiates an inflammatory cascade consisting of increases in COX-2, iNOS, and endothelial adhesion molecule expression, recruitment of inflammatory cells and eventual tissue destruction The current study investigates whether Thy-1 expression regulates gene activation by TNF-α in fibroblasts. Our results indicate that Thy-1 expression in mouse embryonic fibroblasts (MEF) substantially attenuates gene activation by TNF-α, which includes MMP-9, ICAM-1, and a reporter gene controlled by the human IL-8 promoter. Moreover, SFK activation is required for TNF-α-activated gene expression in MEFs. Our findings further suggest that Thy-1 interferes with SFK activation by TNF-α and thereby inhibits TNF-α-activated gene expression in MEFs.vs. a 6-fold increase over control for Thy-1+ MEF (data not shown). A similar profile was observed in ICAM-1 with a 18-fold increase in the mRNA levels of ICAM-1 in TNF-α stimulated Thy-1− MEF vs. a 4-fold increase in Thy-1+ MEF  were serum-starved and exposed to TNF-α (5 ng/ml) for 24 hours. Expression of MMP-9 and ICAM-1, two TNF-α-activated genes, were determined by quantitative RT-PCR. TNF-α induced a robust induction of MMP-9, a 12-fold increase over the control group in Thy-1− MEF, in contrast to a modest 4-fold increase in Thy-1+ MEF . The dify-1+ MEF . To confy-1+ MEF . In conty-1+ MEF . The TGFy-1+ MEF . More imy-1+ MEF . The higy-1+ MEF . Moreovey-1+ MEF . Since Ty-1+ MEF . Thy-1-my-1+ MEF  and gelay-1+ MEF . Similary-1+ MEF , D & E. in vitro using a Src Assay Kit as described in in vitro kinase assays confirmed that PP2, at least in MEFs under the current experimental conditions, activated rather than inhibited SFK activity. Consequently, PP2 rescued MMP-9 and ICAM-1 induction by TNF-α in Thy-1+ MEF to a level comparable to that in Thy-1− MEF  have led to an unexpected finding in that a commonly used SFK selective inhibitor PP2 displays minimal effect on SFK activation as well as TNF-α-activated gene expression in Thy-1− MEFs  & 4. In In summary, the current study reveals greater SFK activation and gene expression in response to TNF-α in Thy-1− MEFs compared with their Thy-1+ counterparts. Our results further suggest that Thy-1 down-regulates TNF-α-activated gene expression by interfering with SFK activation and NF-κB-mediated transactivation. These findings provide a novel mechanistic insight to the distinct roles of Thy-1 subsets of fibroblasts in inflammation and warrant further investigation of Thy-1-mediated regulation of the TNF-α-SFK-NF-κB network in fibroblasts residing within various tissues upon inflammation.Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2 were purchased from Sigma . The SFK selective inhibitors PP2 and SU6656 were purchased from Calbiochem . Recombinant mouse TNF-α and human TGF-β1 were obtained from Peprotech . The reporter constructs that express firefly luciferase regulated by the human IL-8 promoter either inact (IL-8-LUCwt) or with the NF-κB or AP-1 binding elements mutated (IL-8-LUCmtNF-κB and IL-8-LUCmtAP-1) were kindly provided by Dr. Gary Wu at the University of Pennsylvania School of Medicine Mouse embryonic fibroblasts (MEF) from C57 mice were isolated and sorted with FACS using FITC-labeled Thy-1-specific antibodies as previously described 5′-CGACCTGGAAGTCCAACTAC-3′, and reverse primer (nucleotides 177–160) 5′-ATCTGCTGCATCTGCTTG-3′; mouse ICAM-1 (GenBank NM_010493), forward primer (nucleotides 981–1000), 5′-GACGCAGAGGACCTTAACAG-3′, and reverse primer (nucleotides 1119–1102), 5′-GACGCCGCTCAGAAGAAC-3′; mouse MMP-9 (GenBank BC046991), forward primer (nucleotides 2033–2052), 5′-CCTGGAACTCACACGACATC-3′, and reverse primer (nucleotides 2261–2244), 5′-GGTGGTGGTGGTGGTCTC-3′. The ratio of GOI/36B4 was compared among samples and the fold change of GOI expression was obtained by setting the values from the untreated cells to one.Quantitative RT-PCR was performed to determine mRNA levels of Genes Of Interest (GOI) in MEFs subject to the indicated treatments as previously described Serum-starved fibroblasts were exposed to the indicated doses of TNF-α or TGF-β1 with or without pretreatment with PP2 (10 µM) or SU6656 (20 µM) for 30 minutes. The conditioned medium was collected at 48 hours after the specified treatments and processed for gelatin zymography as described elsewhere 4 cells per well. DMEM containing 5% FBS was added into the bottom chamber as a chemoattractant. TNF-α (5 ng/ml) was added at 16 hours after seeding and left in the medium for another 24 hours. The non-invading cells on the top surface of the membrane were removed by scrubbing twice with a swab. The invading cells on the bottom surface of the membrane were stained using Hema 3 system . Invading cells were enumerated under light microscopy. The invasion assays were performed in triplicates for each treatment group. Results shown were the average from three triplicates.The invasive potential of TNF-α-stimulated MEFs was assessed using the BD Biocoat Invasion System  following provided instructions. Briefly, serum-starved MEF cells were seeded into the top chamber at a density of 1×10in vitro SFK kinase assay. The total SFK-associated FAK was evaluated by immunoblot with a FAK-specific antibody after stripping the blot.The immunoblots for signaling molecules were carried out as previously described SFK catalytic activity in treated MEFs was analyzed using the Src Assay Kit  as per the provider's instructions. The cell extracts were prepared from the MEFs subject to the indicated experimental conditions and immunoprecipitated with an SFK-specific antibody as described previously 4 cells per well. On the next day, the two luciferase reporters IL-8-Luc and pRL-TK were co-transfected into MEFs along with either the backbone vectors or the vectors express mouse Thy-1 or dnFyn using Lipofectamine 2000 . The cells were serum-starved for 24 hours after transfection and then exposed to TNF-α (5 ng/ml) for 6 hours. The treated cells were harvested and assayed for luciferase activity using the Lumat LB 9507 Luminometer  along with the Dual-Luciferase Reporter Assay System (Promega) per the provider's instructions. Variation in transfection efficiency was monitored and corrected with the co-transfected pRL-TK. The ratio of Fire Fly luciferase activity to Renilla luciferase activity in each sample served as a measurement of normalized luciferase activity. The fold change of normalized luciferase activity was obtained by setting the values from the untreated cells at one. To determine the effects of Thy-1 and dominant-negative Fyn on TNF-α-activated gene expression, Thy-1− MEFs were transfected with either the backbone vectors or Thy-1 or dn-Fyn vectors followed by exposure to TNF-α. Total cell RNA (24 hr post-exposure to TNF-α) or the conditioned medium (48 hrs post-exposure to TNF-α) was collected to determine the expression of MMP-9 and ICAM-1. To determine effects of PP2 on activity of the human IL-8 promoter, IL-8-LUC and its variants with mutational inactivation of the NF-κB and AP-1 binding elements were transfected into MEF Thy-1 subsets in the presence of TNF-α (5 ng/ml) ± PP2 (10 µM). The transcriptional activity of NF-κB and AP-1 was assessed by transfecting Thy-1+ MEFs with NF-κB-LUC and AP-1-LUC in the presence or absence of TNF-α (5 ng/ml) ± PP2 (10 µM).Transient transfection and luciferase reporter assays were carried out as previously described The results that were obtained from at least three independent experiments were presented as mean±standard deviation. Statistical evaluation was performed between individual treatment groups by unpaired two-tailed Student's T-test using GraphPad Prism . A P value of less than 0.05 was considered significant.


Numerous studies have been performed aimed at limiting the extent of retinal injury after ischemia, but there is still no effective pharmacological treatment available. The aim of the present study was to examine the role of tumor necrosis factor (TNF)α and its receptors (TNF-R1 and TNF-R2), especially considering the neuroretina and the retinal vasculature since the retinal blood vessels are key organs in circulatory failure.Retinal ischemia was induced in pigs by elevating the intraocular pressure to 80 mmHg in one eye, while the other eye served as a control (sham-operated). One hour of ischemia was followed by 5 or 12 h of reperfusion. Retinal circulation was examined in vivo by fundus imaging and fluorescein angiography. TNF-α levels were measured in the vitreous using an angiogenesis antibody array test. The presence and amounts of TNF-α, TNF-R1, and TNF-R2 were investigated in the neuroretina and in the retinal blood vessels, using immunofluorescence staining and real-time PCR techniques.TNF-α mRNA in the neuroretina. TNF-α immunofluorescence staining was localized to the Müller cells and the outer plexiform layer of the neuroretina. The expression of TNF-R1 and TNF-R2 mRNA was increased in both the neuroretina and retinal arteries following ischemia-reperfusion. Immunofluorescence double staining for TNF-R1 and either smooth muscle actin or 4',6-diamidino-2-phenylindole (DAPI) indicated expression in the cell membranes of the vascular smooth muscle cells. Double staining with TNF-R1 and calbindin showed localization to the horizontal cells in the outer plexiform layer of the neuroretina.Fundus imaging showed obstructed blood flow when ischemia was induced, and reperfusion was clearly visualized using fluorescein angiography. Ischemia resulted in elevated levels of TNF-α protein in the vitreous and Retinal ischemia results in increased expression of TNF-α and its receptors (TNF-R1 and TNF-R2). Cellular signaling pathways involving TNF may be important in the development of retinal injury following ischemia and thus an interesting target for future development of pharmacological therapeutics. Diabetes, vein thrombosis, and arterial occlusion are the most common causes of retinal ischemia and a major cause of sight-threatening complications and blindness . It is i2-terminal kinase [Retinal ischemia is most probably multifactorial and probably has numerous overlapping signaling pathways that may contribute to the injury. Tumor necrosis factor α (TNF-α) is interesting in the context of retinal circulatory failure in that it is involved in mediating the harmful processes that are initiated following stroke -4 and isl kinase . These pl kinase . In contl kinase ,16. Bothl kinase . The funThe aim of the present study was to examine the role of TNF-α and its receptors in retinal ischemia-reperfusion injury in detail, with regard to both the neuroretina and the retinal vasculature, by using the porcine model of retinal ischemia-reperfusion induced by elevating the intraocular pressure (IOP) -20. To a™; Farmaceutici Gellini S.p.A., Aprilia, Italy) was used in combination with 2 mg/kg bodyweight xylazine  for premedication. After induction of anesthesia with thiopental, 12.5 mg/kg bodyweight , the animals were orally intubated with cuffed endotracheal tubes. Anesthesia was maintained by continuous intravenous infusion of 20 mg/ml propofol , at a dosage of 0.1–0.2 mg/kg/min, in combination with intermittent fentanyl administration  at approximately 3.5 μg/kg/h. Mechanical ventilation was established with a Siemens-Elema 900B ventilator  in the volume-controlled mode and adjusted to obtain normocapnia. The animals were ventilated with a mixture of oxygen (70%) and dinitrous oxide (30%). During the procedure, the mean arterial blood pressure of the pigs was 92±7 mmHg. After completion of the experiments, the animals were euthanized by a lethal injection of potassium, 2 mmol/kg .A total of 15 healthy landrace pigs  with a mean bodyweight of 70 kg were used for this study . Before surgery the animals were fasted overnight with free access to water. An intramuscular injection of 15 mg/kg bodyweight ketamine , and the Association for Research in Vision and Ophthalmology statement of Use of Animals in Ophthalmic and Vision Research.2*2H2O, 0.75 mg KCl, 0.30 mg MgCl2*6H2O, 3.90 mg Na-Acetate*3H2O, 1.70 mg Na-Citrate*2H2O 6.40 mg NaCl, citric acid to adjust pH and water for injection; AmoTM EndosolTM; AMO Groningen BV, Groningen, the Netherlands). The IOP was verified using a Tono-Pen®XL tonometer . The other eye served as a control, and the same surgical procedure was performed except for elevation of the IOP (this is referred to as the “sham-operated” eye in the text and figures). After 1 h, the cannulation needles were removed, and reperfusion of the retinal vasculature was allowed to take place for 5 or 12 h during anesthesia. Ischemia was confirmed by indirect ophthalmoscopic examination and fundus imaging as described below.The posterior chamber in one eye was cannulated with a 30-gauge needle, and the IOP was increased to 80 mmHg by continuous infusion of a balanced salt solution for ophthalmic irrigation  to a pupil diameter of 8–10 mm. The IOP was increased gradually from the normal level at10–20 to 40±5, 60±5, 70±5, and 80±5 mmHg, and the fundus was imaged continuously at each pressure level. Reperfusion was monitored using fluorescein angiography. Fluorescein  was injected when the IOP was stable at 80 mmHg. The IOP was then allowed to normalize, and the fundus was imaged during pressure normalization. The retinal angiographic images were acquired with a RetCam 3 fundus camera . Standard angiograms were recorded with a blue filter after the injection of fluorescein dye. A wire ophthalmologic speculum was required to separate the pigs’ eyelids for angiography. Video images were recorded at a rate of 30 frames per second, until full arteriovenous filling was observed.After 5 or 12 h of reperfusion, both eyes of each pig were enucleated under anesthesia (including the optic nerve). The eyes were dissected, the anterior segment was removed, and the vitreous humor collected. The vitreous was homogenized by aspiration through a Pasteur pipette and then centrifuged for 30 min at 16,000× g at 4 °C to precipitate insoluble tissue. The vitreous homogenate was snap frozen in liquid nitrogen and stored at −80 °C until further analysis.One limitation of the present study was that the retinal arteries and blood vessels of the eyes exposed to 5 h and 12 h of ischemia-reperfusion were dissected in two different ways and that the retina used for real-time PCR (see below) did not consist of whole retinas but pieces. However, since both eyes from a particular pig were dissected in the same way, it is unlikely that the dissection technique would have affected the results and conclusions drawn from the comparison of the ischemia-reperfused eye and the control eye from each animal. Dissection was performed as follows: A slice along the superior–inferior axis containing the optic nerve head of the eye was first removed. This was later prepared for immunofluorescence and histological investigation (see below). The retina was then dissected free from the sclera and retinal pigment epithelium in the remainder of the eye. Regarding the eyes exposed to 5 h of ischemia-reperfusion, the large retinal arteries were isolated from the neuroretina by careful dissection in a balanced salt solution for ophthalmic irrigation at 4 °C. Pieces of each neuroretina and the retinal arteries were frozen separately. Regarding the eyes exposed to 12 h of ischemia-reperfusion, the neuroretina was isolated by dissecting pieces lacking major blood vessels, in the same balanced salt solution, at 4 °C and frozen as above. After removing pieces of neuroretina, the rest of the retina was placed in distilled water on ice for 1 h. During this time, the tissue was gently and repeatedly aspirated through a Pasteur pipette under magnification, taking care not to damage the blood vessels. The blood vessels were collected and then frozen. The neuroretina and retinal arteries or blood vessels were collected, frozen, and stored at −80 °C until used for real-time PCR experiments.2PO4 and 72 mM Na2HPO4; pH 7.4; Sigma-Aldrich, St. Louis, MO) and then washed in the same solution with increasing concentrations of sucrose . The specimens were embedded in 30% egg albumin and 3% gelatin (Sigma-Aldrich) and were stored at −80 °C until sectioning. The specimens were sectioned at 12 µm in a cryostat  and placed on microscope slides , three sections on each slide. The slides were allowed to dry at room temperature for 30–60 min and were then stored at −20 °C until used.Slices from both eyes were fixed in 4% paraformaldehyde for 4 h. After fixation the tissue was rinsed in 0.1 M Sørensen’s phosphate buffer  plus 0.25% Triton X-100 (2×5 min). Blocking solution, containing PBS+0.25% Triton X-100+1% BSA (BSA) and an additional 5% normal serum was added to the slides to avoid nonspecific binding of antibodies. The slides were incubated for 1 h at room temperature. Specimens were incubated overnight at 4 °C with the same blocking solution but with 2% normal serum instead and the primary antibody of interest: 1:400 rabbit polyclonal TNF-R1 antibody , 1:400 goat polyclonal TNF-α (M-18) antibody , 1:200 mouse monoclonal calbindin antibody , and/or 1:200 mouse monoclonal anti-smooth muscle actin .The slides were dried at room temperature for 15 min and permeabilized in PBS , incubated in blocking solution with the appropriate secondary antibody—1:100 fluorescein isothiocyanate (FITC) swine antirabbit , 1:400 Alexa 488 donkey antigoat , 1:300 Texas red donkey antimouse —and incubated at room temperature for 1 h. The slides were then washed in PBS (3×15 min) and mounted in antifading mounting medium with or without 4´,6-diamidino-2-phenylindole . Slides destined for use with the primary antibody against TNF-α were placed in citric acid (pH 6.0) in a microwave oven at full effect for 7 min before the blocking step to open up the epitopes. Sections incubated without primary or secondary antibody were used as negative controls to verify the lack of autofluorescence and nonspecific secondary antibody staining. The staining location was analyzed using a light microscope equipped for fluorescence microscopy . Photographs were taken with a digital camera  mounted on the microscope.260/A280 ratio) using a spectrophotometer . All samples were stored at -80 °C until further analysis.An Allprep DNA/RNA/Protein Mini Kit  was used for the extraction of RNA. Briefly, the tissue was homogenized in buffer RLT, including β-mercaptoethanol, using a stainless steel bead and a Qiagen TissueLyser. The stainless steel bead was removed and the samples were centrifuged at 8,000× g. The supernatant was then transferred to an Allprep DNA spin column and centrifuged at 8,000× g. Ethanol (99.5%) was added to the flow through, and the samples were transferred to an RNeasy (Qiagen) spin column and centrifuged. On-column DNase digestion was performed according to the manufacturer’s instructions . The column was washed with RW1 buffer, and RPE (Qiagen) buffer, and the RNA was eluted in RNase-free water: 50 μl for neuroretina specimens and 30 µl for the retinal arteries/blood vessels. Following extraction, the RNA concentration and purity was measured by the absorbance at 260 nm . The cDNA obtained as described above was used as a template in a 25-µl reaction. A control without template was included in all experiments. Forward and reverse primers were designed using the Primer3 Input, version 0.4.0 [Actb) and elongation factor-1α (EF-1α) since these are believed to be continuously expressed at constant amounts in cells [Reverse transcription of total RNA to cDNA was performed using the Taqman GeneAmp RNA PCR kit  in a Perkin-Elmer DNA Thermal Cycler (Applied Biosystems). cDNA was synthesized from 1 µg total RNA in a 50-µl reaction with a master mix containing a reverse transcriptase and random hexamers, which act as primers. The reaction was run at 25 °C for 10 min, 37 °C for 60 min, and 95 °C for 5 min. Following reverse transcription, 50 µl double distilled Hon 0.4.0  (Table 1in cells . The pri® Green (Applied Biosystems) and the chosen forward and reverse primers  was added to the wells. One microliter cDNA from the specimens containing neuroretina (sham-operated or ischemia-reperfusion eyes) or retinal arteries/blood vessels (sham-operated or ischemia-reperfusion eyes) was added to the wells. The real-time PCR reaction was performed with the following profile: 1 cycle of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. This was followed by a dissociation step: 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. One standard curve was obtained for each pair of primers to confirm that the primers were optimal and that the cDNA had been amplified with the same efficiency during the real-time PCR process. The cycle threshold (CT) can be expressed in terms of the amplification efficiency (E), and the cDNA concentration:The forward and reverse primers were dissolved in RNase free water and a mixture of forward and reverse primers was made. A master mix containing RNase-free water, Power SYBRTNF-α, TNF-R1, and TNF-R2 mRNA in the specimens was calculated relative to the amount of β-actin and EF-1α mRNA in the same sample using the relation:where the amplification efficiency has the optimal value of 1. The amount of 0=original amount of TNF-α, TNF-R1, or TNF-R2 mRNA; R0=original amount of Actb mRNA; CTR is the CT value for β-actin; and CTX is the CT value for TNF-α, TNF-R1, or TNF-R2.where XTNF-α protein levels were determined in vitreous extracts from sham-operated and ischemia-reperfusion eyes following 12 h of reperfusion (n=6) using angiogenesis antibody array membranes . The assay was performed according to the according to the manufacturer’s instructions. In brief, the array membranes were blocked and rinsed before incubation with vitreous extracts for 1 h at room temperature. Membranes were subsequently rinsed and incubated with Biotin-Conjugated Anti-Cytokine/Anti-Angiogenesis Mix and then developed with Streptavidin-HRP Conjugate (both supplied in the kit). Protein spots on membranes were visualized using a Fujifilm LAS-1000 Luminescent Image Analyzer . The spot intensity was quantified using Image J software .t test with the Bonferroni correction for multiple comparisons. Calculations and statistics were performed using GraphPad Prism 5.0 software . Differences were considered significant when p<0.05. Results are presented as means±the standard error of the mean.Statistical analysis was performed using the paired Student ratio The pigs in this study were anesthetized throughout the study, and the interaction between ischemia and anesthesia is not known but may have impacted the results. To limit the impact on the results and conclusions drawn from the study, the results from the ischemia-reperfusion eye were always compared with the results from the sham-operated eye in the same animal. The other option would be to wake the animals and re-anesthetize them again after the reperfusion; this would, however, subject the animals to increased stress, which is also known to induce metabolic changes. Taken together, we believe that the conclusions drawn from the study have not been affected.A limitation to the present study is that satisfactory results could not be achieved for immunofluorescence with TNF-R2. Our real-time PCR shows an effect of ischemia-reperfusion on TNF-R2, and TNF-R2 has previously been suggested to be neuroprotective . This isThe retinal blood vessels were visualized by fundus imaging before the IOP was elevated and at increasing levels of IOP. Before the IOP was elevated , the pigs had a normal fundus with filled blood vessels and venous pulsations were seen. When the pressure was increased to 40 mmHg, the veins were slightly less filled and there were no venous pulsations. At 60 mmHg, blood perfusion was still maintained via both arteries and veins, although these blood vessels appeared narrower. Arterial pulsations could be observed, and the fundus was paler. At a pressure of 70 mmHg, the blood vessels were thin and the blood flow was slow and limited, with weak arterial pulsations. At this pressure, the fundus was clearly pale. At the highest IOP, 80 mmHg, blood flow was completely obstructed. Boxcaring (break up of blood flow into clumps of red blood cells giving an appearance of boxcars in the vessels) phenomena were seen in both veins and arteries, and a retrograde flow was seen in superior arteries. Fluorescein angiography was performed at the highest IOP (80 mmHg) when no perfusion of blood vessels could be seen. The IOP was then gradually reduced and the following events were observed. First, there was hyperfluorescence in the optic nerve head and filling of the large blood vessel of the choroidal circulation. This was followed by distinct pulsations and slight filling of the retinal arteries. At this stage medium-sized choroidal vessels, but not choriocapillaries, were perfused. Blood flow through the arteries was then restored, but there was still no venous filling. In the next stage, micro-emboli were seen to move slowly out of the veins to allow reperfusion. See TNF-α, TNF-R1, and TNF-R2 mRNA expression in the real-time PCR experiments were seen in the neuroretina for pigs subjected to 12 h of ischemia-reperfusion when using Actb and EF-1α as reference genes. Examples of comparisons of Actb and EF-1α as reference gene are as follows:Similar patterns of TNF-α was 1.72±0.93 with Actb and 2.29±0.44 with EF-1α as reference gene (p>0.3). TNF-R1 was 2.47±0.20 with Actb and 2.34±0.29 with EF-1α as reference gene (p>0.3). TNF-R2 was 1.19±0.20 with Actb and 1.07±0.14 with EF-1α as reference gene (p=0.2171). The results suggest that both Actb and EF-1α were trustworthy as references genes and from here on the results in figures and text are given in relation to Actb. The standard curves for each primer pair had similar slopes: 3.3 for TNF-α, 3.4 for TNF-R1, 3.1 for TNF-R2, and 3.3 for Actb, indicating that they were amplified with similar efficiency.TNF-α mRNA levels were elevated in the neuroretina following ischemia-reperfusion for 5 h but were not significantly altered after 12 h of reperfusion , but after the IOP was increased to 80 cm H2O (59 mmHg), the optic nerve oxygen tension fell sharply, and further increases in IOP above 120 cm H2O (88 mmHg) did not influence the oxygen tension of the optic nerve. This clearly indicates that a pressure of 80 mmHg, which has been used in this and previous studies [In the present study, the retinal vasculature was monitored in vivo using fundus imaging and fluorescein angiography after elevating the IOP to induce ischemia and lowering of the IOP to allow reperfusion. Fundus imaging showed that the retinal blood vessels were thinner than in normal eyes at an IOP of 60 mmHg, and at 80 mmHg there was no visible blood flow through the retinal blood vessels. Fluorescein angiography confirmed that there was no perfusion of retinal blood vessels at an IOP of 80 mmHg, which is a pressure used in this and other studies to create retinal ischemia -20. La C studies -20, is s studies , and mulIn the present study, retinal reperfusion following ischemia was visualized with fluorescein angiography. There is increasing evidence that reperfusion generates detrimental intracellular signaling cascades and contributes to enlargement of the ischemic injury , and sucTNF-α mRNA level in the neuroretina was significantly increased after ischemia and 5 h reperfusion, while after 12 h of reperfusion no significant increase was observed when compared to the sham-operated eyes. Similar results have been seen in a mouse model of central retinal artery occlusion, where TNF-α mRNA levels were increased early but then almost reached control levels again after 7 days [TNF-α mRNA has also been seen in a rat model of pressure-induced ischemia-reperfusion [TNF-α is one of many factors that may contribute to the development of ischemic injury. TNF-α is known to affect the growth, differentiation, survival, and physiologic function of a variety of cells, including those in the immune system, astrocytes, microglia, and smooth muscle cells . In the r 7 days . Furthererfusion . These rTNF-α has been suggested to be an inducer of apoptotic cell death via TNF-R1 occupancy in a caspase-mediated pathway, which includes the activation of caspase-8 . The binTNF-R1 and TNF-R2 mRNA was increased in both the neuroretina and retinal arteries following ischemia-reperfusion. Interestingly, TNF-R1 and TNF-R2 mRNA levels have previously been shown to be increased in the brain following ischemia [TNF-R1 mRNA levels have been shown to be increased in the retina in patients with glaucoma [In the present study, the expression of ischemia ,33, and glaucoma . Such obglaucoma . Delayedglaucoma . TNF-R1 glaucoma , and shoInhibitors of TNF-α are now being constructed and include a soluble TNF-α receptor antagonist (etanercept), three anti-TNF-α antibodies , and a humanized Fab fragment combined with polyethylene glycol (certolizumab) ,37. In tTNF-α mRNA in the neuroretina; the expression of TNF-R1 and TNF-R2 mRNA was also increased in both the neuroretina and retinal arteries. These results indicate that the TNF signaling pathways may be interesting targets for the development of pharmacological therapeutics aimed at preventing the development of retinal injury following ischemia.In conclusion, ischemia was produced by increasing the IOP to 80 mmHg. At this pressure blood flow to the retina was obstructed, as shown by fundus imaging. After 1 h of ischemia, the IOP was allowed to return to normal levels and the retinal vasculature was reperfused, as shown by fluorescein angiography. Retinal ischemia-reperfusion resulted in elevated levels of TNF-α protein in the vitreous and