We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6–GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are  Macrophages secrete cytokines as part of innate immune responses trans-membrane precursor of TNF is trafficked through the Golgi where its exit from the trans-Golgi network (TGN) occurs in tubular carriers for transport to recycling endosomes and thence to filopodia and phagocytic cups at the cell surface for cleavage and release The secretory pathway and trafficking machinery used by macrophages to traffic newly made cytokines to the cell surface for release are gradually being elucidated. The trans-Golgi cisterna/TGN where in their GTP-bound states they bind to a range of effectors. To date, more than 15 individual Rab6a and Rab6a' effectors have been identified Members of the Rab family of small GTPases mediate the specificity between donor and acceptor membranes for vesicle budding, docking, tethering and fusion in transport steps throughout eukaryotic cells Here, we investigated the role of Rab6 in regulating Golgi function for the trafficking and secretion of TNF in LPS-activated macrophages. Our findings serve to add Rab6 to the list of known molecular modulators of cytokine secretion and provide new insights about the role of Rab6 as a component of post-Golgi membrane carriers.cis- and trans-side) where it is involved in various trafficking steps  Rab6 (T27N). Rab6a(T27N)–GFP typically appeared as diffuse staining throughout the cell rather than being directed to the Golgi membranes , asterisTo independently test TNF trafficking and release we collected cell supernatants from LPS-activated (2 hours) transfected cells for measurement of secreted TNF . In conttrans-Golgi accumulation of COPI-coated vesicles In order to further examine roles for Rab6 in trafficking, as our next approach we depleted Rab6 in macrophages using siRNA. We have previously shown that similar depletion of Rab6 in HeLa cells was signified by a distinctive rearrangement of the Golgi complex with an increase in number of Golgi cisternae and a When RAW 264.7 cells were transiently transfected with siRNA Rab6, TNF was still present in the Golgi, despite the resulant and obvious changes in Golgi complex morphology in affected cells and significant reduction in TNF staining on the cell surface , ControlIn order to assess overall levels of TNF secretion we designed a small hairpin (sh) to deplete Rab6 in a more stable fashion. shRab6 tagged with mCherry (shRab6–mCherry) was produced as described in Materials and Methods, and the expressing cells were subsequently sorted by flow cytometry. Western blot analysis confirmed the efficiency of the shRab6 depletion of about 80% . In thestrans-golgin p230/golgin-245/GOLGA4 (hereafter p230) in vitro and in vivo impairs TNF trafficking out of the TGN, blocking its secretion We next sought to investigate the specific transport step affected by Rab6 disruption. We have previously shown that TNF exits the TGN in dynamic tubular carriers demarked by the In cells before and after LPS activation, Rab6a–GFP was partially co-localized on the Golgi complex with endogenous p230 , as we hFurthermore, we examined co-localization of p230 and Rab6 on Golgi membranes at the EM level in Rab6–GFP transfected macrophages using antibodies to immunogold label GFP and p230 . Prior tde novo within a defined time interval in single cells. This revealed that Rab6–positive tubules were predominant and even more prevalent after LPS. More tubules labeled for p230 appeared after LPS stimulation, as we have previously shown We next investigated the dynamic distribution of Rab6 and p230 in living cells transfected with either p230(GRIP)–mCherry (hereafter p230–mCherry) or Rab6–GFP or with both probes . When p2These observations suggest that Rab6 associates with vesicular and tubular carriers emerging constitutively from the TGN. Rab6 and p230 can appear on separate tubules or together on the same tubule. Interestingly, LPS promotes the formation of a class of Rab6/p230-positive vesicular/tubular carriers.The expression of inactive Rab6 isoforms, Rab6a(T27N)–GFP  and Rab6P4C–GFP (a Golgi membrane protein), galactose-1-phosphate uridylyl transferase –mCherry (a Golgi enzyme), golgin-97(GRIP)–mCherry (hereafter golgin-97–mCherry), p230–mCherry . BFA is an inhibitor of guanine nucleotide exchange factor for Arfs; treating cells with this drug causes dissociation of not only Arf itself but also effector coat proteins, such as COPI and AP-1 complexes, from Golgi membranes trans-Golgi and the TGN are well known sorting stations The intracellular transport of TNF and other cytokines through the constitutive secretory pathway in macrophages necessarily involves the Golgi complex as a major compartment for posttranslational modification and trafficking. The GDP-locked mutants of Rab6a and Rab6a' (T27N) demonstrably reduced or perturbed the accumulation of TNF at the cell surface and its short term release, whilst newly synthesized TNF was still present in the Golgi complex. This indicated a likely requirement for active Rab6a and/or Rab6a' for the trafficking of TNF between the Golgi and the cell surface. Indeed, it has been shown that Rab6(T27N) inhibits anterograde Golgi trafficking in other cells, including both intra-Golgi and post-Golgi trafficking steps Depletion of Rab6 protein from cells with siRNA or shRNA, which evidently included loss of both Rab6a and Rab6a' isoforms, had a dramatic effect on Golgi structure, causing the running together of Golgi ribbons previously visualized by tomography Upon Rab6 depletion, the secretion of TNF was reduced. Over a time course it was evident that Rab6 depletion most severely reduced the initial release of TNF, and then secretion levels recovered somewhat at later times. This recovery may be due to the LPS-induced expression of Rab6 identified here by Western blotting (since the siRNAs were not fully penetrable) or by some other compensatory effect. Nevertheless it appears that the Golgi cisternal dysmorphology resulting from Rab6 depletion does not itself prevent the transit of membrane-bound cargoes like TNF. The role of Rab6 in the regulation of membrane trafficking and the maintenance of Golgi organization has been recently reviewed i) vesicle/tubule fission from the Golgi orchestrating cargo exit from the Golgi/TGN through one of its effectors, myosin II ii) fission of one population of carriers iii) in docking and fusion of carriers at the cell surface Whereas the recycling endosomes dictate the final steps in the delivery of TNF and some other cytokines to the cell surface for release In macrophages we found Rab6 constitutively bound to many carrier tubules emerging from the TGN, consistent with its association on multiple types of carriers for post-Golgi transport. p230 was on a more specific subset of tubules, including those transporting TNF A priori this might also implicate Rab6 in selective, direct recruitment of p230 to the tubule membranes. p230 and golgin-97, both localizing at the TGN but in different membrane domains trans-golgins like GCC185 Depletion of Rab6 caused a significant cytosolic redistribution of p230, seemingly reducing the binding of p230, but not of golgin-97 to Golgi membranes. This accompanied the impairment of TNF transport and is thus a possible mechanism to explain this trafficking defect. In conclusion we have shown that Rab6 has essential roles in maintaining Golgi morphology and secretory trafficking in activated macrophages. One specific function for Rab6 is demonstrated by its requirement for the efficient delivery of TNF to the cell surface and for secretion in LPS-activated macrophages.Rab6 is part of a Rab cascade in the Golgi along with Rab33 and associated GEFs, Ric1p and Rgp1p to regulate both anterograde and retrograde transport through the Golgi 1–34), with an additional cysteine residue at the C-terminus . The sequence of peptide is: MFKKLKQKISEEQQQLQQALAPAQASSSSSTPTRC. The p2301–34 was directly injected subcutaneously into a rabbit and three additional boost injections were given 28, 56 and 91 days after the initial immunization followed by a terminal bleed at 103 days; the immunization as well as the bleeds were performed commercially by WEHI Antibody Facility . Antibodies were affinity-purified using a column of p2301–34 peptide conjugated to SulfoLink Coupling Gel (Pierce). The affinity-purified antibodies were demonstrated to be specific for p230 as (i) they detected perinuclear Golgi structures in mouse NIH3T3 cells, RAW 264.7 and bone marrow-derived macrophages by indirect immunofluorescence, whereas no reactivity was observed in cells depleted of endogenous p230 and (ii) they immunoprecitated a protein ∼230 kDa, corresponding to the size of endogenous p230.We used monoclonal antibodies to GM130 and golgin-97, and rat anti-TNF for intracellular staining (BD Biosciences), polyclonal anti-TNF for surface staining . The following were also used: nanogold-conjugated Fab fragments of anti-rabbit immunoglobulin G (IgG); protein A conjugated with colloidal gold, 10 or 15 nm ; anti-rabbit, anti-mouse and anti-rat antibodies conjugated with Alexa 488, Alexa 555 and Alexa 633 . To generate an anti-mouse p230 antibody, we used a synthetic peptide corresponding to the N-terminal 34 residues of mouse p230  Previously described GFP–Rab6a and GFP–Rab6a' Salmonella enterica serovar Minnesota Re595, BFA, and DMSO (Sigma-Aldrich), and TAPI (Santa Cruz Biotechnology).We used LPS from 2 at 37°C as previously described The RAW 264.7 murine macrophage cell line  was cultured in RPMI 1640 medium (Lonza) supplemented with 10% heat-inactivated FCS (Thermo Trace) and L-glutamine 2 mM (Invitrogen) in humidified 5% CO5'-TGATCACCCGATTCATGTA-3', corresponding to nucleotides 724–742 of the Rab6 coding sequence) separated by a short spacer (′-TTCAAGAGA-3′5) from the reverse complement of the same 19-nt sequence, and the transcriptional termination site for the RNA polymerase. This construct was used to generate a lentivirus expressing shRab6–mCherry according to a well established protocol A lentivirus-based shRNA system RAW 264.7 macrophages were transfected with the Rab6 dominant negative constructs and/or incubated in the presence of LPS 100 ng/ml, BFA 5 µg/ml for 2 h. Levels of secreted TNF in the culture medium were then quantified using a mouse TNF Elisa kit II (BD OptEIA) according to the manufacturer’s protocols.3, NH4Cl 50 mM in PBS) for 30 min, and then incubated with appropriate primary and secondary antibodies. Nuclei were counterstained with DAPI. Coverslips were mounted in ProLong Gold reagent (Invitrogen) prior to imaging. For live cell experiments, RAW 264.7 macrophages were cultured on 35 mm glass-bottom dishes (MatTek). Confocal microscopy on fixed cells was performed using a Leica TCS SP2 imaging system.Immunofluorescence staining was performed as previously described Live cell imaging was performed using a Personal DeltaVision Olympus IX71 inverted widefield deconvolution microscope equipped with an Olympus Plan apochromat 60× NA 1.35 oil objective and a 37°C incubator. Images were captured using a Roper CoolSNAP HQ2 monochrome camera. Imaging analysis of all other data was performed using ImageJ software .Pierce Chemical), and equal amounts of proteins were resolved on SDS-PAGE gels, transferred onto PVDF membrane  immunoblotted and detected with either enhanced chemiluminescence system (PerkinElmer Life Sciences) or Odyssey® infrared imaging detection (LI-COR® Biosciences). The ECL system utilized Biotrace- PVDF and HRP-conjugated secondary antibodies. Odyssey system required Immobilon-FL membrane and secondary antibodies coupled to IRDye® 800 or IRDye® 680 fluorophores.Cells were washed three times in ice-cold PBS and then lysed in sodium chloride 150 mM, Tris-HCl 50 mM pH 7.5, 1% Igepal . Protein concentrations were determined by a BCA assay-(bicinchoninic acid protein) . A value of p<0.05 was considered significant.Statistical analysis was performed using two-tailed Student’s Figure S1Rab6/p230 co-localization ratio on Golgi membranes is not affected by different fixatives. No difference in the co-localization level of Rab6–GFP with endogenous p230 was observed in RAW 264.7 macrophages fixed with cold methanol for 10 min at 4°C versus 4% PFA fixed macrophages. LPS incubation for 2 h induced a significantly increased co-localization of p230 on Rab6-positive Golgi membranes. Normalized co-localization levels are plotted in the graph. Original optical magnification 63X (A). Bar: 20 µm (A). ** = p<0.01 (pairwise comparisons).(TIF)Click here for additional data file.Figure S2Quantification of golgin redistributions in siRNA Rab6 cells. (A) Co-localization of p230–mCherry and golgin-97–mCherry in the experimental conditions summarized in P4P–GFP used as a Golgi marker. (B) Golgi localizing p230–mCherry and golgin-97–mCherry was normalized to the cytosolic redistribution. (C) Quantification of p230 redistribution in control and siRNA Rab6 LPS-activated cells as a ratio of Golgi complex and cytosolic area. * = p<0.05, *** = p<0.001 (pairwise comparisons).(TIF)Click here for additional data file.Figure S3Expression of Rab6a(T27N)–GFP and Rab6a'(T27N)–GFP inhibit Golgi-to-plasma membrane TNF trafficking, but not the Golgi localization of p230. Inactivation of single Rab6a or Rab6a' isoforms by over-expressing the GFP–tagged dominant negative (T27N) proteins resulted in an inefficient cytosolic redistribution of p230, which was for siRNA Rab6. Original optical magnification 63X. Bar: 10 µm.(TIF)Click here for additional data file.Movie S1RAW 264.7 macrophages were transfected with p230(GRIP)–mCherry. Images were acquired using a DeltaVision microscope for 5 min at 0.5 s intervals.(AVI)Click here for additional data file.Movie S2RAW 264.7 macrophages were transfected with Rab6a–GFP. Images were acquired using a DeltaVision microscope for 5 min at 0.5 s intervals.(AVI)Click here for additional data file.Movie S3RAW 264.7 macrophages were transfected with Rab6a–GFP and p230–mCherry. Images were acquired using a DeltaVision microscope for 5 min at 0.5 s intervals.(AVI)Click here for additional data file.Movie S4RAW 264.7 macrophages were transfected with Rab6a–GFP and p230–mCherry and stimulated for 2 h with LPS before recording. Images were acquired using a DeltaVision microscope for 5 min at 0.5 s intervals.(AVI)Click here for additional data file.Movie S5RAW 264.7 macrophages were transfected with Rab6a(T27N)–GFP. Images were acquired using a DeltaVision microscope for 5 min at 0.5 s intervals.(AVI)Click here for additional data file.Movie S6RAW 264.7 macrophages were transfected with Rab6a(T27N)–GFP and p230–mCherry, and stimulated for 2 h with LPS before recording. Images were acquired using a DeltaVision microscope for 5 min at 0.5 s intervals.(AVI)Click here for additional data file.


Inflammatory cytokines play a key role in the pathogenesis of joint diseases such as rheumatoid arthritis (RA). Current therapies target mainly tumor necrosis factor α (TNF-α) as this has proven benefits. However, a large number of patients do not respond to or become resistant to anti-TNF-α therapy. While the role of TNF-α in RA is quite evident, the role of TNF-β, also called lymphotoxin-α (LT-α), is unclear. In this study we investigated whether TNF-β and its receptor play a role in chondrocytes in the inflammatory environment.in vitro model of primary human chondrocytes was used to study TNF-β-mediated inflammatory signaling.An Cytokine-induced inflammation enhances TNF-β and TNF-β-receptor expression in primary human chondrocytes accompanied by the up-regulation of inflammatory (cyclooxygenase-2), matrix degrading  and apoptotic  signaling pathways, all known to be regulated by NF-κB. In contrast, anti-TNF-β, similar to the natural NF-κB inhibitor  or the knockdown of NF-κB by using antisense oligonucleotides (ASO), suppressed IL-1β-induced NF-κB activation and its translocation to the nucleus, and abolished the pro-inflammatory and apoptotic effects of IL-1β. This highlights, at least in part, the crucial role of NF-κB in TNF-β-induced-inflammation in cartilage, similar to that expected for TNF-α. Finally, the adhesiveness between TNF-β-expressing T-lymphocytes and the responding chondrocytes was significantly enhanced through a TNF-β-induced inflammatory microenvironment.These results suggest for the first time that TNF-β is involved in microenvironment inflammation in chondrocytes during RA parallel to TNF-α, resulting in the up-regulation of NF-κB signaling and activation of pro-inflammatory activity. Althougrotrimer ,7. RecenRA is a chronic, systemic inflammatory autoimmune disease characterized by inflammation of the synovial joints . BecauseIL-1β is a well-studied mediator of cartilage destruction in osteoarthritis (OA) and RA. This mediating effect occurs by reducing chondrocyte proteoglycan synthesis, increasing synthesis of matrix metalloproteinases (MMPs) and releasing nitric oxide ,12. IL-6Current treatment regimens for RA often target a specific cytokine to suppress inflammatory processes -17. Becain vivo collagen-induced arthritis mouse model, anti-TNF-β therapy dramatically improved the disease course comparably to anti-TNF-α therapy [Previous studies have indicated that TNF-β levels are elevated in the serum and synovial tissue of RA and OA patients -23. A re therapy . Recentl therapy .For safe and effective therapeutic use, however, more detailed information on the role of TNF-β in RA is needed. This is especially true regarding the interaction between chondrocytes, as the major architects of functional joint tissue, and TNF-β, which should be evaluated in more detail to better understand the inflammatory and degradative processes of RA.Curcuma longa, has strong anti-TNF-α activity and can further suppress TNF-α production through inhibition of nuclear factor κB (NF-κB) activation [In the search for effective and safe RA therapies, natural compounds have also shown ability to suppress inflammatory processes through inhibition of TNF-α. Curcumin (diferuloylmethane), when isolated from the rhizomes of tivation -28. Alsotivation . Antisentivation ,31. ASO-tivation ,33.in vitro model of RA and to investigate the underlying signaling pathways.Therefore, because an increasing body of evidence suggests that TNF-β may play a major role in an inflammatory joint environment, we decided to find out whether TNF-β and its receptor are expressed by primary human chondrocytes in an Antibodies to active caspase 3, MMP-9 and MMP-13 were purchased from R&D Systems . Anti-phospho-specific p65 (NF-κB/Ser536) was obtained from Cell Signaling Technology . Anti-TNF-β, anti-TNF-α and anti-TNF-β receptor (anti-TNF-β-R) antibodies were obtained from eBioscience . Monoclonal poly(ADP-ribose) polymerase (PARP) antibodies were purchased from Becton Dickinson . Cyclooxygenase 2 (Cox-2) antibody was obtained from Cayman Chemical . Monoclonal anti-β-actin antibody was purchased from Sigma-Aldrich Chemie . Antibodies against p53 were obtained from Santa Cruz Biotechnology . Alkaline phosphatase–linked sheep anti-mouse and sheep anti-rabbit secondary antibodies for immunoblotting were purchased from EMD Millipore . Secondary antibodies used for fluorescence labeling were purchased from Dianova . Gold particle–conjugated secondary antibodies were purchased from Amersham . All antibodies were used at concentrations recommended by the manufacturers. TNF-β and polyclonal rabbit anti-TNF-β antibodies were used as described previously  and wereCell culture growth medium consisting of Dulbecco’s modified Eagle’s medium/Ham’s F-12 (1:1), 10% fetal bovine serum (FBS), 1% partricin solution, 1% penicillin-streptomycin solution , 75 μg/ml ascorbic acid, 1% essential amino acids and 1% glutamine was obtained from Seromed . Epon was obtained from Plano . Curcumin with a purity greater than 95% was purchased from Indsaff  and dissolved in dimethyl sulfoxide, and a 5,000 μM stock was prepared and further diluted in cell culture medium. This commercial source of curcumin contains three major components: diferuloylmethane (the most abundant and active component of turmeric) (82%) and its derivatives demethoxycurcumin (15%) and bisdemethoxycurcumin (3%), together referred to as curcuminoids ,36. IL-1Primary human chondrocytes  were obtained from Provitro . Briefly, during monolayer expansion, chondrocytes were seeded at a density of 300,000 cells/T75 cell culture flask and grown until 70% confluence was reached in cell culture growth medium (10% FBS) . ChondroTo evaluate the effect of IL-1β on TNF-β expression in primary human chondrocytes, time- and dose-dependent experiments were carried out. Primary human chondrocytes in monolayer culture were either left untreated or treated with 10 ng/ml IL-1β for various durations  or with various concentrations of IL-1β  for 1 hour. Whole-cell extracts were prepared, and immunoblotting was carried out as described below.Transient transfection of primary human chondrocytes was performed as previously described . Phosphog. The supernatants (cytoplasmic extracts) were frozen at −80°C. Ice-cold nuclear extraction buffer (25 μl) was added to the pellets and incubated for 30 minutes with intermittent mixing. Extracts were centrifuged, and the supernatant (nuclear extracts) was transferred to the prechilled tubes for storage at −80°C. Total protein content was measured, samples were reduced with 2-mercaptoethanol and total protein concentrations were adjusted. After separation with SDS-PAGE under reducing conditions, the resultant was blotted onto a membrane using a Trans-Blot apparatus  and blocked for 2 hours in 5% (w/v) skimmed milk powder in PBS/0.1% Tween 20. Membranes were incubated overnight with the primary antibody directed against TNF-β, TNF-α, MMP-9 and −13, Cox-2, p53, cleaved caspase 3, NF-κB-p65, TNF-β-R, PARP and β-actin at a 1:1,000 dilution in blocking buffer at 4°C on a shaker, washed three times with blocking buffer, then incubated with the secondary antibody conjugated with alkaline phosphatase for 90 minutes at ambient temperature (AT). Membranes were washed three times in 0.1 M Tris (pH 9.5) containing 0.05 M MgCl2 and 0.1 M NaCl. Specific antigen–antibody complexes were detected using nitroblue tetrazolium and 5-bromo-4-chloro-3-indoylphosphate  as substrates for alkaline phosphatase.Whole-cell lysates or nuclear extracts used for Western blot analysis were prepared as previously described in detail ,41. BrieThe technique was performed as previously described in detail . Briefly4 solution. After dehydration in a series of ethanol washes, pellets were embedded in Epon and ultrathin cuts were made on a Reichert-Jung Ultracut E microtome (Leica Microsystems) and contrasted with a mixture of 2% uranyl acetate/lead citrate. Sections were examined under a Zeiss 10 transmission electron microscope .Detection of TNF-β-R expression on T lymphocytes (Jurkat cells) by immunoelectron microscopy was performed as previously described . Jurkat 6/ml) for 4 hours. Subsequently, nonadherent T cells were removed by gentle washing three times with PBS before paraformaldehyde fixation for evaluation by light microscopy.Primary human chondrocytes were grown to subconfluence in a monolayer culture and either left unstimulated or stimulated with 1 or 10 ng/ml TNF-β for 12 hours, washed and then cocultured with T lymphocytes (Jurkat cells)  in an Because it is unknown whether healthy or inflamed chondrocytes express TNF-β or TNF-β-R, we evaluated whether IL-1β stimulates chondrocytes to express TNF-β and TNF-β-R. Primary human chondrocytes in monolayer cultures were either left untreated  were cocultured with chondrocytes and incubated for 4 hours, and the number of T lymphocytes adherent to the surface of chondrocytes was quantified by light microscopy. Treatment with TNF-β did not morphologically change the chondrocytes. However, stimulation of chondrocytes with increased dosages of TNF-β resulted in an almost twofold increase in the adherence of T lymphocytes  activate the ubiquitous transcription factor NF-κB, which leads to further proinflammatory cytokine expression and thus to further tissue degradation. Activated NF-κB, also in chondrocytes, is known to be involved in the regulation of several genes, including by infection, adhesion, cell-cycle, apoptosis, survival and inflammatory processes, by activating IL-1β, TNF-α, IL-6, Cox-2 and MMPs ,46,54-58A possible mechanism underlying the inhibition of inducible NF-κB by curcumin  could be its capacity to inhibit the TNF-β signaling pathways in chondrocytes. Furthermore, our results demonstrate that anti-TNF-β inhibits IL-1β-induced NF-κB activation and its translocation to the nucleus in chondrocytes, demonstrating that TNF-β stimulates activation of NF-κB in chondrocytes upstream of IκBα dissociation, similarly to the signaling mechanism observed for TNF-α or lipopolysaccharide in chondrocytes ,60. ThesWe next investigated whether inhibition of NF-κB activation and its nuclear translocation influenced IL-1β-induced TNF-β production in chondrocytes. Previously, our group has shown that, in the human myelomonoblastic leukemic cell line ML-1a, both TNF-α and TNF-β induce activation of NF-κB by signaling through TNF receptor subunits P80 and P60 . In this1β2 and induce the production of proinflammatory chemokines [We observed that human chondrocytes express TNF-β receptors; therefore, further experiments were performed to determine their responsiveness to stimulation with TNF-β. In the present study, we have demonstrated that TNF-β treatment markedly induced its own expression in chondrocytes in a manner comparable to that of TNF-α . We haveemokines . These rin vitro. We have further shown that TNF-β treatment stimulated chondrocytes and made them attractive for adhesion of T lymphocytes, indicating a connection between TNF-β signaling in chondrocytes and the recruitment of T lymphocytes for the upkeep of microenvironment inflammation in the rheumatoid joint. These results demonstrate that, during inflammation in chondrocytes, TNF-β signals similarly to TNF-α. As many patients only weakly respond to anti-TNF-α therapy, or even become resistant to it [Our study demonstrates for the first time that TNF-β is an important regulator in activation of the NF-κB signaling pathway and its regulated proteins involved in inflammation, matrix degradation and apoptosis in human chondrocytes nt to it , blockinASO: Antisense oligonucleotide; AT: Ambient temperature; BSA: Bovine serum albumin; Cox-2: Cyclooxygenase 2; ECM: Extracellular matrix; FBS: Fetal bovine serum; IKK: IκB kinase; IL: Interleukin; LT-α: Lymphotoxin α; MMP: Matrix metalloproteinase; NF-κB: Nuclear factor κB; OA: Osteoarthritis; PARP: Poly(ADP-ribose) polymerase; PBS: Phosphate-buffered saline; RA: Rheumatoid arthritis; SO: Sense oligonucleotides; TNF: Tumor necrosis factor.The authors declare that they have no competing interests.CB, PS and MS carried out all of the experiments and drafted the manuscript. BBA and MS were responsible for the experiment design and data analysis. CB, PS and MS participated in histological and immunohistochemical analyses. BBA and MS participated in the evaluation of each experiment. BBA, CB and MS revised the paper, provided technical support and drafted the final version of the manuscript. All authors read and approved the final manuscript.


Endothelial dysfunction precedes atherosclerosis. Vasodilation induced by acetylcholine (ACh) is a specific test of endothelial function. Reproducibility of laser techniques such as laser-Doppler-flowmetry (LDF) and Laser-speckle-contrast-imaging (LSCI) to detect ACh vasodilation is debated and results expressions lack standardization. We aimed to study at a 7-day interval (i) the inter-subject reproducibility, (ii) the intra-subjects reproducibility, and (iii) the effect of the results expressions over variability.Using LDF and LSCI simultaneously, we performed two different ACh-iontophoresis protocols. The maximal ACh vasodilation (peak-ACh) was expressed as absolute or normalized flow or conductance values. Inter-subject reproducibility was expressed as coefficient of variation . Intra-subject reproducibility was expressed as within subject coefficients of variation , and intra-class correlation coefficients (ICC). Fifteen healthy subjects were included. The inter-subject reproducibility of peak-ACh depended upon the expression of the results and ranged from 55% to 162% for LDF and from 17% to 83% for LSCI. The intra-subject reproducibility (intra-CV/ICC) of peak-ACh was reduced when assessed with LSCI compared to LDF no matter how the results were expressed and whatever the protocol used. The highest intra-subject reproducibility was found using LSCI. It was 18.7%/0.87 for a single current stimulation (expressed as cutaneous vascular conductance) and 11.4%/0.61 for multiple current stimulations .ACh-iontophoresis coupled with LSCI is a promising test to assess endothelial function because it is reproducible, safe, and non-invasive. N°: NCT01664572. Atherosclerosis is a major health problem in Western countries In the clinical perspective, the development of an easy, non-invasive test to routinely assess endothelial function is still required. Different invasive and non-invasive techniques have been developed to study endothelial function and have been widely reviewed elsewhere An optimal tool for routine-use should, among others, be non-invasive, specific, and able to detect diseased patients Therefore, the goal of this study was to study in healthy subjects at a 7-day interval (i) the inter-subject reproducibility of ACh iontophoresis test, (ii) the intra-subjects reproducibility of ACh iontophoresis, and (iii) the effect of how the results of ACh iontophoresis test are expressed when comparing reproducibility during single and multiple stimulations using LDF and LSCI.Fifteen male or female volunteers (aged 18 years or older) without known cardiovascular disease were recruited in this study. Each subject gave his/her written informed consent prior to participation. This study received local institutional review board approval from the comité de protection des personnes (CPP) Ouest II, conformed to the Declaration of Helsinki, and was registered to the American National Institutes of Health database under reference N°: NCT01664572. The first subject was included July 2, 2012.All microvascular tests were performed with subjects resting supine in a temperature-controlled room (23±1°C). Tests were performed at least 2 hours after a meal. All tests were performed in the morning and at a same hour for each subject. All subjects had an acclimatization period longer than 15 min before the beginning of the cutaneous blood flow (CBF) acquisitions.The LDF relies on the Doppler effect. When photons of a laser light encounter moving particles (mainly red blood cells) in the studied tissues, the Doppler effect appears (modification of photon frequency). For LDF recordings, we used a laser Doppler flowmeter . Data of the CBF acquired by LDF were expressed as arbitrary units (a.u.) and recorded on a computer via an analogue to digital converter  with a sample rate of 18 Hz, on 16-bits.Recently, laser speckle contrast imaging (LSCI) has been commercialized Iontophoresis is a non-invasive method that drives a pharmacologically charged drug by electrorepulsion through the interstitium surrounding the blood vessels Blood pressure was recorded from the left middle finger  with mean arterial pressure (MAP) obtained from the continuous blood pressure signal. The MAP signal was recorded at a sampling rate of 18 Hz using an analog to digital converter  and analyzed offline using the Acknowledge ® software V3.5.4 .Local cutaneous temperature was measured on the right forearm using a surface thermocouple probe connected to an electronic thermometer .All the subjects participated in 2 protocols.In protocol 1, the endothelial test consisted of an iontophoresis of ACh with a single current application (0.1 mA and 30 s) as previously proposed This second protocol was performed on a different day than the first protocol. In protocol 2, the endothelial test consisted of an iontophoresis of ACh with multiple current applications  as previously proposed 2. Time of interest (TOI) is defined as the duration in seconds over which data  are averaged (absolute) corresponds to the CBF measured before the first current stimulation and is expressed in a.u. when LDF was used and in laser speckle perfusion units (LSPU) when LSCI was used. Baseline(CVC) corresponds to the CBF measured before the first current stimulation and expressed in Cutaneous Vascular Conductance (CVC). CVC is calculated as the laser signal value divided by the mean arterial pressure when the measurement is performed. CVC is expressed either in au/mmHg (for LDF) or in LSPU/mmHg (for LSCI). Baseline was measured with a ROI of 20 mm2 and a TOI of 10 s was chosen, when LSCI was used. For LDF, TOI was also of 10 s but no ROI can be defined with LDF. The following parameters of the maximal ACh vasodilation measurement obtained after current application are presented in The region of interest (ROI) is defined as the skin area of interest. The size of ROI was 20 mmResults are expressed as mean±standard deviation (SD).individual. The mean of cross-correlation coefficients  between D1 and D7 was calculated as the mean of individual cross-correlation coefficients and compared with the Wilcoxon test. Wilcoxon test was used because Rindividual distribution was not normally distributed when tested with Shapiro-Wilk test. Furthermore, the concordance of the average patterns  obtained with ACh iontophoresis between D1 and D7 was assessed using cross-correlation analysis (Raverage) for single current stimulation and multiple current stimulations.For each subject, the concordance of the vasodilation patterns between D1 and D7 was studied with cross-correlation analysis. For the cross-correlation analysis, the coefficient of correlation is a “r” value that, when reaching a maximum, is equal to the Pearson’s correlation coefficient. If the maximal “r” value is close to 1, the two signals are almost identical, whereas “r” is zero for two independent signals. The purpose of cross-correlation is to define whether signals have the same profile regardless of absolute values. Then for each subject we obtained a Rp value <0.05 was considered as statistically significant.The inter-subject reproducibility at a 7-day interval was evaluated by the coefficient of variations (inter-CV) calculated as the SD of CBF values of all subjects divide by the mean CBF of all subjects multiplied by 100. Comparisons of mean values of maximal vasodilation and time to peak between D1 and D7 obtained with LDF and LSCI were performed with the Wilcoxon test. A http://www.sportsci.org/resource/stats/) [(Standard deviation of the log-transformed paired differences/√2/100)]−1]. The lower the intra-CV, the better the reproducibility. Intra-subject reproducibility of measurements was also assessed by intra-class correlation coefficients (ICC) http://sportsci.org/ressource/stats/xrely.xls ) The intra-subject reproducibility at a 7-day interval was evaluated by the typical error of the estimate (TEE) of the peak of ACh as well as the coefficient of variations (intra-CV) according to the procedure proposed by Hopkins ω of 0.3 (to remain between the range of fair to good agreements), the estimated sample size was 13. Considering the risk of missing data, we included 15 subjects in this study.Number of subjects was calculated from ICCs by estimating the width of the 95% confidence interval for an expected correlation of 0.70 . Considering a two-sided interval, two measurements and a distance from correlation to limit General characteristics of the population are presented in The typical vasodilation patterns obtained by ACh iontophoresis either with a single current stimulation (protocol 1) or with multiple current stimulations (protocol 2) are presented in individual between D1 and D7 were 0.68 (0.24), and 0.74 (0.25) for LDF and LSCI, respectively. No statistical difference was found between Rindividual for LDF compared with Rindividual for LSCI. The Raverage between D1 and D7 were 0.82 using LDF whereas it was 0.98 using LSCI.The Rindividual between D1 and D7 were 0.65 (0.16) and 0.76 (0.20) for LDF and LSCI, respectively. This difference was statistically significant (p = 0.036). The Raverage between D1 and D7 was 0.94 using LDF whereas it was 0.98 using LSCI.The RMean values of the maximal ACh vasodilation measured at D1 compared to mean values of the maximal ACh vasodilation measured at D7 are presented in Intra-subject reproducibility of maximal ACh vasodilation at a 7-day interval using LDF and LSCI are presented in (multipleCVC) whereas the best CV was 18.7% [13.4–31.0] when results were expressed in peak ACh(CVC) with LSCI.In protocol 1, using LDF, the best CV was 80.0% [53.8–152.8] and was obtained when results were expressed in peak ACh(multiple CVC), whereas using LSCI, the best CV was 11.4% [8.2–18.5]; it was obtained when results were expressed in peak ACh(absolute).In protocol 2, using the LDF, the best CV was 50.1% [34.6–89.7]; it was obtained when results were expressed in peak ACh(absolute) or in peak ACh(CVC) or in peak ACh(multiple CVC). Which every way the results were expressed, the CVs obtained with LDF were higher than those obtained using LSCI. Only the intra-subject reproducibility measured with LSCI fulfilled both conditions  but this depended on the way of expressing the results.Using LDF, intra-subject reproducibility was always lower than using LSCI when results were expressed either in peak AChThis study provides original results about the inter-day reproducibility of laser techniques (LDF and LSCI) in order to assess endothelial function in clinical routine or clinical research. The major findings are: (i) the typical vasodilation patterns induced by ACh iontophoresis measured by LDF and LSCI are highly correlated at a 7-day interval; (ii) the inter-subject reproducibility is better when using LSCI; (iii) the intra-subject reproducibility is highly improved with LSCI and depends on the way of expressing the results.et al., by Debbabi et al. and Sauvet et al. using LDF 2) might induce non-specific vasodilation, which can be a confounding factor when intophoresis is used 3 and a late plateau, which involves muscarinic receptor and prostaglandins First, we confirmed the typical vasodilation patterns previously described by Durand http://www.itamar-medical.com/EndoPAT/FAQ.html) whereas with FMD the measurement lasts longer than 20 min Second, LDF and LSCI showed a high concordance between D1 and D7 with a statistically lower concordance with LDF compared with LSCI in protocol 2. Therefore, kinetic of the vasodilation involved in the ACh iontophoresis is similar at a 7 day-interval. Moreover we demonstrated that peak ACh is rapidly obtained in less than 10 minutes for both protocols showing the interest of such endothelial testing in clinical routine. The duration of endothelial function testing is a key point because this determines the applicability of a test in clinical routine.As compared with the most used non-invasive endothelial function tests ), ACh iontophoresis measured either with LDF or LSCI appears shorter in term of duration measurement without taking into account the time for set-up. Endothelial function assessment with peripheral arterial tonometry is performed in approximately 15 minutes  and the post-occlusive reactive hyperaemia a CV of 8% (when data were expressed as CVC) using LSCI whereas CV was of 40% (when data were expressed as CVC) and 30% (when data were expressed as CVC) when LDF was used, respectively Fourth, we found that the intra-subject reproducibility was improved with LSCI compared with LDF. This corroborates other results, which compared other microvascular tests measured with LSCI and LDF. Roustit (increase CVC) and peak ACh(increase absolute) result in a worse reproducibility as compared with the other ways of expressing the results. Further, as it has been suggested by several authors, expressing the results in CVC is better than expressing the results in absolute values in order to take into account differences and variations in blood pressure Last, this study showed that the way of expressing the results has an important effect over the variability of the peak ACh. For both protocols (1 & 2), expressing the results in peak AChThe main limitation of this study is that we did not show that the peak ACh is sensitive to health status changes or treatments. However, using laser flowmetry these points have been previously shown In order to assess endothelial function in clinical routine and human research, the use of LSCI, appears preferable to the use of LDF in term of reproducibility. The simplicity of the use of LSCI, and its excellent reproducibility should facilitate future studies dealing with cardiovascular risk factor management and patient outcomes.


Virus-like Particles (VLPs) display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV) that provides broad protection from diverse HPV types in a mouse pseudovirus infection model.Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types.We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine. It is estimated that nearly 500,000 individuals worldwide are affected with Human Papillomavirus (HPV)-associated cancer every year The current HPV vaccines (Gardasil® and Cervarix®) are derived from virus-like particles (VLPs) consisting of the HPV L1 major capsid protein. Gardasil® contains L1-VLPs derived from HPV16 and 18, as well as VLPs derived from two low-risk HPVs associated with genital warts (HPV6 and 11). Cervarix® only contains HPV16 and HPV18 L1-VLPs. Both vaccines induce high-titer protective antibodies to the HPV types included in the vaccine, but unfortunately these antibodies are type-specific, meaning that vaccination provides very little cross-protection to other “high-risk” HPV types Second-generation HPV vaccines providing broader protection against the spectrum of carcinogenic “high-risk” HPV types might permit further reductions in screening intensity in developed nations. Merck has indicated that a nonavalent L1-VLP vaccine is currently in clinical trials As an alternative tactic to broaden the efficacy of HPV vaccines, a number of groups have exploited the presence of highly-conserved cross-neutralizing epitopes in the L2 minor capsid protein of HPV to develop second-generation L2-based pan-HPV vaccines Although the sequence of the N-terminal domain of HPV L2 is relatively conserved amongst HPV types, there is some heterogeneity that can limit the breadth of reactivity of antibodies elicited against individual HPV L2 sequences. For example, a concatenated multimeric L2 fusion protein, which contains amino-terminal L2 peptides derived from 3 to 22 HPV types elicits broader neutralizing antibody responses than similar immunogens that contain L2 sequence from a single HPV type In our previous study L2 peptides were inserted into a constrained ß-hairpin structure (the AB-loop) on the surface of the PP7 coat protein. Because of these structural constraints, we were somewhat concerned that this context may not have been ideal for display of L2 peptides. Therefore, we explored a different display site at the N-terminus of the coat protein of an alternate VLP display platform based on the bacteriophage MS2. Somewhat surprisingly, immunization with MS2 VLPs displaying an HPV16 L2 peptide at the N-terminus of coat protein elicited much broader cross-reactive antibodies compared to the same epitope displayed on the AB-loop of PP7 VLPs. Moreover, mice immunized with MS2 VLPs displaying a HPV16 L2 peptide were strongly protected against vaginal and intradermal challenge with HPV pseudovirions representing diverse types. These data demonstrate that a single recombinant VLP displaying an L2 peptide can provide broad protection against HPV infection.The family of single-stranded RNA bacteriophage is a versatile platform for displaying heterologous epitopes. We have previously shown that insertions of diverse short peptides into a surface-exposed constrained loop (the AB-loop) on the coat protein of both bacteriophage MS2 and PP7 are compatible with VLP assembly We took advantage of an engineered version of the MS2 coat protein to make N-terminal recombinants. Genetic fusion of two copies of the MS2 coat protein forms a single-chain dimer version of the protein that, when expressed, spontaneously self-assembles into VLPs that are more stable than VLPs formed from coat protein monomers 3 after a single immunization and>104 after boosting (2) anti-L2 antibody titers after a single 250 ng dose of VLPs. Boosting with 500 ng of VLPs increased these titers to ∼104 . As shown in To investigate whether the broader cross-reactivity observed with 16L2(17–31) Nterm MS2 VLP sera is due to its ability to bind to linear or conformational epitopes, 8-mer peptides spanning the 16L2 epitope  were synthesized and used as target antigens in an ELISA. As shown in Papillomaviridae and form a disulfide bond in their native context The region of L2 that we are targeting contains two cysteine residues (C22 and C28) that are absolutely conserved throughout the nd immunization. As expected, mice immunized with either 16L2(17–31) Nterm MS2 VLPs or 16L2(17–31) AB-loop PP7 VLPs showed almost complete protection from high-dose vaginal challenge with homologous HPV16 PsV  Nterm MS2 VLPs] or at the AB-loop of PP7 coat protein [16L2(17–31) AB-loop PP7 VLPs] or control VLPs, and were then challenged with a panel of PsVs  three to five weeks after the 2PV16 PsV . ProtectPV16 PsV . ImmunizThe HPV minor capsid protein L2 has great potential as the basis of a second generation HPV vaccine. L2 immunogens can induce antibody responses that are much more broadly neutralizing than the L1-VLPs that make up the current HPV vaccines. However, L2-based vaccines have their own hurdles to overcome in order to be clinically effective. For one, L2 vaccines induce considerably lower neutralizing titers than L1-VLP based vaccines. This raises concerns that the protection provided upon vaccination with L2 will be short-lived. In addition, the sequence of L2 does vary amongst HPV types and, in general, immunization with single L2 peptides typically results in sera that only cross-neutralizes a subset of HPV types. For example, rabbit antisera induced upon immunization with synthetic linear peptides encoding short (15–20 amino acid) peptides spanning the N-terminal domain of HPV16 L2 effectively neutralize HPV16 PsV, but only display varying degrees of cross-neutralizing activity against HPV6, 18, 31, and 58 PsV We and others have shown that these limitations can be addressed by altering the way that L2 is displayed to the immune system. For example, the immunogenicity of L2 can be improved by the use of potent adjuvants or by increasing the multivalency of L2 through the use of concatemeric constructs The lack of broad cross-reactivity of antibodies induced by individual L2 peptides is exemplified by our previous studies using the PP7 VLP display platform. Display of L2 amino acids 17–31 from individual HPV types on bacteriophage PP7 VLPs induced high-titer type-specific antibodies to HPV peptides but generally low-titer cross-reactive antibodies to heterologous HPV types. These single L2 PP7 VLPs only induced broadly reactive and protective antibody responses when all eight VLPs were mixed together as a single immunogen In this regard, we explored whether display of L2 at an alternative site on the surface of a bacteriophage VLP could broaden the spectrum of protection provided by a single recombinant L2 VLP. We have identified three different sites on the bacteriophage VLP that allow for surface display of antigens; the N-terminus, the AB-loop, and the C-terminus. We have previously described the insertion of peptides on to the AB-loop of bacteriophage PP7 and MS2 In this study, we have shown that the N-terminus of the MS2 single-chain dimer coat protein can be modified to display HPV16 L2 peptides ranging from 10 to 27 amino acids (but not 52 amino acids) without affecting VLP assembly. These peptides are displayed on the surface of VLPs in a highly immunogenic format; all three L2-MS2 VLPs generated in this study induced high-titer anti-HPV16 L2 IgG antibody responses even at low (sub-microgram) doses without IFA. The different peptides elicited slightly different degrees of cross-reactive antibodies against heterologous HPV L2 peptides; the VLP displaying amino acids 17–31 induced the most broadly reactive antibody response. Strikingly, display at the N-terminus of MS2 elicited much more broadly reactive antibody responses than display of the same peptide in AB-loop of PP7 coat protein. It is likely that this configuration allows more conformational flexibility, perhaps, leading to a better exposure of conserved amino acids in this epitope thereby inducing a more broadly reactive antibody response against a conformational epitope. Correspondingly, mice immunized with 16L2(17–31) Nterm MS2 VLPs were more broadly protected upon vaginal and intradermal challenge with a diverse panel of HPV PsVs.Thus, in this manuscript we describe a single, potentially low-cost, bacterially-expressed VLP-based vaccine that elicits high-titer broadly neutralizing antibody responses against diverse HPV types at low doses. Bacteriophage VLPs can be expressed at high levels, are stable at room temperature CGGGCTTTGTTAGCAGCCGG 3′), which anneals downstream of a unique BamHI site in the pDSP62 plasmid was used for all PCR amplifications above. PCR fragments were cloned using NcoI/BamHI sites into the pDSP62 expression plasmid. For peptide 14–65 insertion, plasmid pDSP62-16L2(14–40), which contains the nucleotide sequence for L2 peptide 14–40, was used as a template for whole plasmid PCR amplification using phosphorylated primers. All constructs were sequenced to confirm the presence of the L2 peptide inserts.L2 sequences representing HPV16 L2 amino acids 20–29, 17–31, 14–40, and 14–65 were cloned onto the amino-terminus of a single-chain dimer version of the MS2 coat protein in plasmid pDSP62 C41 cells (Lucigen) were transformed with recombinant pDSP62-16L2 plasmids and recombinant 16L2-MS2 VLPs were expressed and purified as previously described To examine the native redox state of the two cysteines within the structural context of bacteriophage VLPs, L2-VLPs were biotinylated at 1∶50 molar ratio (VLPs:biotin) using 1-Biotinamido-4-[4′-cyclohexanecarboxamido]butane  for two hours at room temperature. Biotinylated VLPs were then purified from residual BMCC-biotin using the Amicon 100 K MWCO centrifugal filters (Millipore). In some cases, VLPs were reduced or denatured prior to the BMCC-biotin reaction. Reduction was performed by overnight incubation with 10 mM dithiothreitol (DTT) while denaturation was performed by incubating VLPs in 2% SDS for 1 hour at 37°C. Reduced or denatured VLPs were purified by 3X PBS buffer exchanges using Amicon 100 K MWCO centrifugal filters. For western blotting, non-reducing protein loading dye  was added to 500 ng of each VLP, heated for 5 minutes and resolved under denaturing conditions on a 12% SDS PAGE gel. VLPs were then transferred to a PVDF membrane and blocked as described above. Thio-biotinylated VLPs were probed with 1∶5,000 dilution of streptavidin-conjugated HRP for two hours and the membrane was developed as described above.All animal work was done in accordance with the National Institutes of Health and the University of New Mexico Institutional Animal Care and Use Committee (UNM IACUC) guidelines and was approved by the UNM IACUC (protocol 12-100827-HSC). Groups of Balb/c mice were immunized twice intramuscularly (i.m.) at a two-week interval with 5 µg of 16L2 Nterm MS2 VLPs , 16L2 AB-loop PP7 VLPs (displaying L2 amino acids 17–31), or, as negative controls, a mixture of wild-type MS2 and PP7 VLPs. Vaccine was prepared with incomplete Freund’s adjuvant (IFA). Another group of Balb/c mice was immunized i.m. with 250 ng of 16L2(17–31) Nterm MS2 VLPs without IFA and were boosted 4 weeks later with 500 ng without IFA. Two weeks after the second immunization, sera were collected (except for the mice immunized without IFA whereby sera was collected at one and three weeks after first immunization and then one week after second immunization) and anti-L2 IgG titers were determined by peptide-based ELISA using disulfide-constrained L2 peptides representing amino acids 14–40 from HPV1, 5, 6, 16, and 18 (as described http://home.ccr.cancer.gov/lco/packaging.htm) were generously provided by Chris Buck, Susana Pang, John Schiller, Martin Muller, and Tadahito Kanda. pVitro plasmids for HPV33, 35, 39, 51, and 53 L1/L2 expression were kindly provided by Richard Roden.HPV5, 6, 16, 31, 33, 35, 39, 45, 51, 53, and 58 PsVs with encapsidated reporter plasmid (pClucf) encoding both luciferase and green fluorescence protein (GFP) genes were produced in 293TT cells as previously described 4–3.0×106 PsV infectious units. Forty-eight hours post-PsV challenge, mice were vaginally instilled or injected intradermally with 0.4 mg of luciferin  and three minutes later their images were taken (with a five minute exposure) using a Caliper IVIS Lumina II . Average radiance (p/s/cm2/sr) was determined from the images by drawing equally sized regions of interests surrounding the site of PsV instillation. Statistical significance was determined by one-tailed unpaired t-test.Prior to challenge, groups of five female Balb/c mice were given two i.m. immunizations with 5 µg of 16L2(17–31) Nterm MS2 VLPs, 16L2(17–31) AB-loop PP7 VLPs, HPV16 L1L2 VLPs, or with equal mixture of wild-type MS2/PP7 VLPs with IFA. Three to five weeks after the last immunization, mice were treated with 3 mg of Depo-Provera (Pharmacia Corp) except the group for intradermal challenge. Five days post-Depo-Provera treatment, mice were challenged either cervicovaginally or intradermally with HPV PsVs as previously published


Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2  Laryngeal carcinoma (LC) is one of the most prevalent malignant tumors in the head and neck area. Approximately a global population of 190, 000 people are diagnosed this disease per year This study was approved by the Ethics Committee of First Affiliated Hospital of Sun Yat-Sen University. All efforts were made to minimize mice suffering. All mice were killed by cervical dislocation and decapitations and all surgeries were performed under anesthesia with urethane (2 mg/kg).EcoliDH-5α, plasmid pcDNA3.1 (+) and plasmid pIRES were preserved in the Biochemical Laboratory of Sun Yat-Sen University. Human laryngeal carcinoma cell line Hep-2 was purchased from Shanghai Cell Bank, Chinese Academy of Science. The human laryngeal carcinoma cell Hep-2 was incubated in DMEM medium containing 10% fetal bovine serum at 5% CO2 and 37°C.Escherichia coli JM109, transformed recipient bacteria Weighing 18–22 g, 56 SPF BALB/c nu/nu nude mice  aged 4–6 weeks were purchased from Animal Center (certificate number: SCK (Yue) 2003-0001 Yue regulatory certificate number 2007A015), Guangzhou University of Chinese Medicine. They were then kept and used for the experiments in the Laboratory Animal Center of Sun Yat-Sen University in Guangzhou .Molecular CloningAccording to the nucleotide sequence of TNF-α gene (access number: U77396) and CD gene (access number: AY331712) provided in GenBank, EcoR Ι and Not Ι restriction enzyme sites were inserted into up-stream and down-stream of TNF-α gene; Xho Ι and Apa Ι restriction enzyme sites were inserted into the 5′ terminal of the up-stream and down-stream primers of CD gene. Initiation codon and termination codon that agreed with the reading frame were also introduced. PCR primers were designed in accordance to the designing principles and were shown in Liposome-mediated gene transfection was performed as the protocol of Lipofectamine 2000. Hep-2 cells were transfected by the plasmids pcDNA 3.1(+)-TNF-α-CD, pcDNA3.1(+)-CD, pcDNA 3.1(+)-TNF-α and pcDNA3.1(+) (blank control), respectively. At 2 days after the transfection, cells were incubated with 600μg/ml G418 for additional two weeks. Positive clones were obtained, amplified and named as Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α and Hep-2/0, respectively.Total RNA was extracted from Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α and Hep-2/0 cells by guanidinum-phenol-chloroform method. Primers were designed according to the gene sequences published in GenBank Table 2 3 Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α, Hep-2/0 and Hep-2 parent cells were inoculated into 96-well plate, with 18 wells for each type of cells. The plate was then incubated at 37°C and 5%CO2 for 24 hours. 5-FC was then added at the concentration level of 0, 0.001, 0.01, 0.1, 1 and 10 mmol/L, with 3 wells for each concentration level. The cells were then incubated for another 96 hours and were added with MTT, in which the cells were cultivated for another 4 hours. Supernatant was then removed and Dimethyl Sulfoxide (DMSO) was added. OD value at 570 nm was detected by ELISA reader. Cell survival rate was calculated by: survival rate(%) = A/B×100% .A number of 5×10A total of three repeated experiments were performed. Survival curve was generated with mean survival rates as ordinate axis and concentration levels of 5-FC as abscissa axis.3 mixed cells with varied percentages of parent cells and each type of transfected cells  were inoculated onto 96-well plate, with 3 wells for each cell mixture. At 24 hours later, 1 mmol/L 5-FC was added. After being incubated for 96 hours, the cells were added with MTT. Another 4 hours later, supernatant was removed and DMSO was added. OD value at 570 nm was detected by ELISA reader. Cell survival rate was detected by 3 repeated measurements.The bystander effect refers to the induction of biological effects in cells that are not directly traversed by a charged particle 3 cells of each type were inoculated into 96-well plate and were incubated at 37°C and 5%CO2 for 24 hours. Then 1 mmol/L 5-FC was added and the cells were incubated for the next 96 hours. Then cells were prepared into single cell suspension. The cell concentration was maintained at 5×105–1×106 cells/ml, with 3 tubes for each type of cells. The cell suspension was then sent to the First Affiliated Hospital, Sun Yat-Sen University for flow cytometry.Apoptotic rates in Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α and Hep-2/0 cells when treated by 1 mmol/L 5-FC were detected by flow cytometer (Prodidium Iodide Staining). A total of 5×103 Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α and Hep-2/0 cells were inoculated into 96-well plate, which was then incubated at 37°C and 5%CO2 for 50 hours. Supernatant was obtained from each well and was divided, with 100 <mu>l in each portion. According to the protocol of TNF-α Elisa detection reagent kit , standard wells of 125 pg/ml, 62.50 pg/ml, 31.25 pg/ml, 15.65 pg/ml, 7.8 pg/ml and 0 pg/ml and blank well were prepared; experimental wells were also prepared using the supernatant of each cell culture, with 3 wells for each type of cells. OD value at 570 nm was detected by ELISA reader. Standard curve was generated and OD values of experiment samples were obtained using the standard curve.A total of 5×106 cells of each type into 0.2 mL PBS.Tumor cell suspension was inoculated into 53 BALB/c nu/nu nude mice. The nude mice were randomly assigned into 4 groups. Experiment groups: −1·d−1 was given by intraperitoneal injection everyday ; for Hep-2/0 group: the same amount of NS was injected. The treatment lasted for 12 lasted days. Every 3 days, vernier caliper was used to measure the size of the tumor (maximum major diameter and transverse diameter) and tumor volume was calculated by the formula: V = 1/2ab2. Tumor growth curve was plotted. Curve fitting equation was V = Aek t. Tumor doubling time was calculated by T = ln2/K (k: growth rate). Based on the mean tumor volume of each group, tumor growth curve was generated and tumor inhibition rate at day 29 was calculated. The formula of tumor inhibition rate: tumor inhibition rate(%) = [1−(baseline tumor volume of experiment group−tumor volume of experiment group at the end of experiment)/(baseline tumor volume of control group−tumor volume of control group at the end of experiment)]×100% Treatment started at 8 days after cell inoculation. For Hep-2/TIC, Hep-2/CD and Hep-2/TNF-α group, 5-FC at the dosage of 500 mg· kgSPSS12.0 statistical software was used. Data were shown as mean value ± standard deviation (A). Whereas electrophoresis analysis on PCR-amplified products of CD gene showed specific band between 1.2 kb and 1.5 kb, which is consistent with target gene in terms of gene length (B).Electrophoresis analysis was performed on the RT-PCR-amplified products of TNF-α gene and revealed 700 bp amplified segments Fig. 1AC).When cleavaging pcDNA3.1 (+)-CD with double enzymes of Xho Ι and Apa Ι, agarose gel electrophoresis analysis on cleavaged products revealed a band between 1.2 kb and 1.5 kb, which was consistent with target band Fig. 2A. A, band 1). While in transfected Hep-2/TIC and Hep-2/TNF-α cells, electrophoresis on RT-PCR products revealed specific bands of TNF-α mRNA . In Hep-2/0 cells which were not transfected with CD gene, no mRNA of CD gene was detected . While in transfected Hep-2/TIC and Hep-2/CD cells, electrophoresis on RT-PCR products revealed specific bands of mRNA of CD gene .For Hep-2/0 cells which were not transfected with TNF-α gene, no noticeable TNF-α mRNA can be detected ; differences between Hep-2/CD group and three other groups were also significant (P<0.05); however, difference was not statistically significant between Hep-2/TNF-α and Hep-2/0 groups (P>0.05). Cytocidal effect was seen in both Hep-2/TIC and Hep-2/CD group, more prominently in Hep-2/TIC group than in Hep-2/CD group (P<0.05). When Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α and Hep-2/0 cells accounted for 100% of all cells and no parent cells were added, significant difference in cell survival rate was seen between each two of the four groups when treated by 1 mmol/L 5-FC (P<0.05). In Hep-2/TNF-α and Hep-2/0 cells, no by-stander effect was shown. When Hep-2/CD and Hep-2/TIC cells accounted for 20% of the mixed cell, cytocidal effect was seen in 70% and 80% of the mixed cells, respectively. By-stander effect was strongest in Hep-2/TIC cells when treated by 5-FC. %, (30.07±0.95)%, (86.00±4.59)% and (88.99±2.96)%, respectively when treated by 1 mmol/L 5-FC; significant differences were seen when comparing Hep-2/TIC group to three other groups . Among all groups, Hep-2/TIC group showed the highest apoptotic rate. % in Hep-2/0 group, (28.53±1.25)% in Hep-2/TNF-α group, (38.13±2.32)% in Hep-2/CD group and (53.93±2.22)% in Hep-2/TIC group; statistically significant differences were observed when comparing each two groups . The difference of TNF-α protein concentrations in supernatant was not significantly different between Hep-2/TIC and Hep-2/TNF-α cells (P>0.05); also, no significant difference was observed between Hep-2/CD and Hep-2/0 cells (P>0.05). .In Hep-2/TIC and Hep-2/TNF-α cells with positive expression of TNF-α gene, expression of TNF-α protein was detected in the supernatant of the cell culture of Hep-2/TIC and Hep-2/TNF-α. Concentrations of TNF-α protein in the supernatant of both these cell culture were significantly different as compared to that in Hep-2/CD or Hep-2/0 cells (P>0.05). Fig. 7.A classic subcutaneous inoculation method was used to establish the nude mice models of human laryngeal carcinoma. In total, tumor developed in 44 mice, with a tumor formation rate of 83%. With regard to different groups, respectively 10 mice of Hep-2/TIC group, 11 mice of Hep-2/CD group, 11 mice of Hep-2/TNF-α group and 12 mice of Hep-2/0 group developed tumor. Averagely tumor formation took 6–8 days. At day 8 after cell inoculation, diameter of the grafted tumor was around 5 cm. During the experiment, respectively 1 mouse of Hep-2/TNF-α group and Hep-2/0 group showed significant weight loss, arched back, decreased activity and listlessness at day 8 and day 10 of drug administration, respectively. They died later and were excluded from the experiment. Respectively 1 nude mouse of Hep-2/CD group and Hep-2/0 group showed tumor necrosis at day 18 and day 20 after cell inoculation and were excluded from the experiment.3, 65.37±2.66 mm3, 64.84±5.41 mm3 and 64.02±4.78 mm3, respectively); no significant difference was seen between each two of them (P>0.05). At day 29 after cell inoculation, volume of grafted tumor in Hep-2/TIC group, Hep-2/CD group, Hep-2/TNF-α group and Hep-2/0 group was 80.47±33.74 mm3, 135.5±14.83 mm3, 525.8±40.70 mm3 and 678.3±52.34 mm3, respectively; significant difference was seen between each two of them (P<0.05). Tumor inhibition rates of Hep-2/TIC group, Hep-2/CD group and Hep-2/TNF-α group were 97.40%, 88.58% and 24.96%, respectively.Pictures of measured tumor tissues and growth curves of the grafted tumors in nude mice of varied groups were shown in At day 30 post the cell inoculation, pathological changes in Hep-2/TIC group, Hep-2/CD group, Hep-2/TNF-α group and Hep-2/0 group were shown accordingly in Surgery is still the main treatment of LSCC accompanied with radiotherapy and chemotherapy in the assistant position. In addition, the improvement space of normalized surgical procedures is also limited. LSCC's recurrence and metastasis often lead to the failure of treatment and 5-year survival rate is less than 60% TNF-α is a multifunctional cytokine which has been demonstrated in numerous preclinical models with potent anticancer property, importantly, the recombinant protein is in clinical trials phase in vivo and in vitro experiments confirmed the synergic anti-tumor effect of CD gene and TNF-α gene when using in combination. Based on the therapeutic rationale of CD suicide gene/pro-drug system and TNF-α, theoretically they manage to treat laryngeal carcinoma via multiple pathways while showing additive effect: TNF-α induces high permeability of tumor vasculature and thereby helps gather and concentrate 5-FU, a chemotherapeutic agent converted from 5-FC, in the tumor tissue. In vitro experiment has demonstrated that TNF-α could accelerate the gathering of chemotherapeutic agents in certain tumor cells and help reach the half inhibitory concentration in a short period of time In our study, both


Tumour necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. TNF-α acts by binding to its receptors, TNF-R1 (p55) and TNF-R2 (p75), on the cell surface. The aim of this study was first to investigate if there is altered expression of TNF-α and TNF-α receptors in cerebral artery walls following global or focal ischemia, and after organ culture. Secondly, we asked if the expression was regulated via activation of the MEK-ERK1/2 pathway.in vivo after subarachnoid hemorrhage (SAH) and middle cerebral artery occlusion (MCAO), and in vitro by organ culture of isolated cerebral arteries. The localization and amount of TNF-α, TNF-α receptor 1 and 2 proteins were analysed by immunohistochemistry and western blot after 24 and 48 h of organ culture and at 48 h following SAH or MCAO. In addition, cerebral arteries were incubated for 24 or 48 h in the absence or presence of a B-Raf inhibitor (SB386023-b), a MEK- inhibitor (U0126) or an NF-κB inhibitor (IMD-0354), and protein expression evaluated.The hypothesis was tested Immunohistochemistry revealed enhanced expression of TNF-α, TNF-R1 and TNF-R2 in the walls of cerebral arteries at 48 h after MCAO and SAH compared with control. Co-localization studies showed that TNF-α, TNF-R1 and TNF-R2 were primarily localized to the cell membrane and the cytoplasm of the smooth muscle cells (SMC). There was, in addition, some expression of TNF-R2 in the endothelial cells. Immunohistochemistry and western blot analysis showed that these proteins were upregulated after 24 and 48 h in culture, and this upregulation reached an apparent maximum at 48 h of organ culture. Treatment with U0126 significantly reduced the enhanced SMC expression of TNF-α, TNF-R1 and TNF-R2 immunoreactivities after 24 and 48 h of organ culture. The Raf and NF-κB inhibitors significantly reduced organ culture induced TNF-α expression while they had minor effects on the TNF-α receptors.The present study shows that cerebral ischemia and organ culture induce expression of TNF-α and its receptors in the walls of cerebral arteries and that upregulation is transcriptionally regulated via the MEK/ERK pathway. Stroke is a serious neurological disease and a leading cause of death and severe disability in the world . There aTNF-α is a pleiotropic cytokine produced by many cell types, and is involved in blood-brain barrier, inflammatory, thrombogenic, and vascular changes associated with brain injury . TNF-α hThe present study aimed to address two questions: First, is the expression of TNF-α, TNF-R1 and TNF-R2 altered in cerebrovascular smooth muscle cells (SMCs) following MCAO, SAH and organ culture? Second, what intracellular signaling events are involved in regulating the expression of these molecules? This was examined by in vitro application of signal transduction blockers, such as the MEK/ERK1/2 inhibitor U0126, the B-Raf inhibitor SB3860-b, and the NF-κB inhibitor IMD-0354 ,19. The 2O:O2 (70%:30%) and thereafter the animals were kept anaesthetized through a mask with 1.5% halothane during the operation. To confirm proper occlusion of the right MCA a laser-Doppler probe  was fixed on the skull (1 mm posterior to the bregma and 6 mm from the midline on the right side) to measure regional cortical blood flow. A polyethylene catheter was inserted into a tail artery to measure the mean arterial blood pressure, pH, pO2, pCO2, and plasma glucose. A rectal temperature probe connected to a homeothermal blanket was used to maintain body temperature at 37°C during the procedure. An intraluminal filament technique was used to induce transient MCAO, previously described in detail by Memezawa [Male Wistar-Hanover rats , weighing approximately 300-350 g (n = 6 per group), were obtained from Harlan Horst, the Netherlands. The animals were housed under controlled temperature and humidity with free access to water and food. The experimental procedures were approved by the Lund University Animal Ethics Committee (M43-07). Anaesthesia was induced using 4.5% halothane in NMemezawa . The resMemezawa ,22. TherMemezawa ,22.2O:O2 mixture (ratio 30:70). All animal experiments were performed following the national laws and guidelines and were approved by the Danish Animal Experimentation Inspectorate and the Ethics Committee for Laboratory Animal Experiments at the University of Lund. The rats were intubated and artificially ventilated with inhalation of 0.5-1.5% halothane in a N2O:O2 mixture (ratio 70:30) during the surgical procedure. The depth of anaesthesia was carefully monitored and the respiration checked by regularly withdrawing arterial blood samples for blood gas analysis . A temperature probe was inserted into the rectum of each rat to record the body temperature, which was maintained at 37°C by a heating pad. An arterial catheter to measure blood pressure was placed in the tail artery, and a catheter to monitor intracranial pressure (ICP) was placed in the subarachnoid space under the subocciptal membrane. At either side of the skull, two laser-Doppler flow probes were placed over either hemisphere to measure cortical cerebral blood flow (CBF). Finally, a 27 G blunt canula with side hole was introduced 6.5 mm anterior to bregma in the midline at an angle of 30° to the vertical using a stereotactic frame. After 30 min of equilibration of the animal, 250 μl blood was withdrawn from the tail catheter and injected intracranially at a pressure equal to the mean arterial blood pressure (MABP) (80-100 mmHg). Subsequently, the rat was kept under anaesthesia for another 60 minutes to allow recovery from the cerebral insult after which catheters were removed and incisions closed. For a more detailed description, see previous studies [Subarachnoid hemorrhage was induced by a model originally devised by Svendgaard et al  and desc2 and decapitated. The brains were removed and immersed in ice-cold bicarbonate buffer solution. The right and left MCAs, and the basilar artery (BA) were dissected out using a dissection microscope, snap frozen, and stored at -80°C for immunohistochemistery.After 48 hours of observation, MCAO, sham and SAH rats were anaesthetized using CO2 and decapitated. The brains were quickly removed and chilled in ice-cold bicarbonate buffer solution. The BA, the right and left MCAs, and the circle of Willis were removed and dissected free from the brain and surrounding tissue under a dissection microscope. The artery segments were placed individually into wells of a 12-well plate with 2 ml serum-free Dulbecco's modified Eagle's medium (DMEM) [2 in air for 24 or 48 h in the presence or absence of the intracellular signal inhibitors. The arteries were transferred into new wells containing fresh medium every 24 h. After 24 or 48 h, the vessels were snap frozen and stored at -80°C for immunohistochemistery and western blot.A total of 111 Male Wistar Hannover rats , weighing 350 to 420 g, were used for organ culture. The animals were anesthetized with COm (DMEM) . Incubat3, 4.6 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 1.5 CaCl2 and 5.6 glucose. Dulbecco's modified Eagle's medium (DMEM) contained L-glutamine (584 mg/L) and was supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) .The specific inhibitors used included: an IkB kinase 2 (IKK-2) inhibitor IMD-0354 -5-chloro-2-hydroxybenzamide) (30 nM) , a speci2-conjugated donkey anti-rabbit  diluted 1:200 in PBS containing 0.3% Triton X-100 and 1% BSA. Finally, the sections were washed with PBS and mounted with anti-fading Vectashield mounting medium . Immunoreactivity was visualized using an epifluorescence microscope  at the appropriate wavelengths and photographed with an attached Nikon DS-2Mv camera. The same procedure was used for the negative controls except that either the primary or the secondary antibody was omitted to evaluate autofluorescence and non-specific secondary antibody binding levels.Cerebral arteries from MCAO, SAH, sham, fresh, culture (24 and 48 h) and culture + inhibitors were placed into Tissue TEK , frozen on dry ice and sectioned into 10-μm thick slices in a cryostat . Three sections were collected and placed on each microscope slide  Mounted sections were fixed for 10 minutes in ice-cold acetone (-20°C) and then rehydrated in phosphate buffer solution (PBS) containing 0.3% Triton X-100 for 15 min. The sections were permeabilized and blocked for 1 h in blocking solution containing PBS, 0.3% TritonX-100, 1% bovine serum albumin (BSA) and 5% normal donkey serum, and then incubated over night at 4°C with the following primary antibodies: rabbit polyclonal to TNF-α  diluted 1:500, rabbit polyclonal to TNF-α receptor 1  diluted 1:1800, and rabbit polyclonal to TNF-α receptor 2  diluted 1:50. All primary antibodies were diluted in PBS containing 0.3% Triton X-100, 1% BSA, and 2% normal donkey serum. Sections were subsequently incubated for 1 h at room temperature with secondary Cy™2- conjugated donkey anti-rabbit  diluted 1:200 and Texas Red-labeled donkey anti-mouse  diluted 1:300 in PBS containing 0.3% Triton X-100 and 1% BSA. The sections were mounted with Vectashield mounting medium with 4', 6-diamidino-2-phenylindole (DAPI) . The antibodies were detected at the appropriate wavelengths using a Nikon confocal microscope .Double immunofluorescence labeling was performed for TNF-α, TNF-R1 or TNF-R2 and smooth muscle actin, a selective smooth muscle cell marker. For the latter, a mouse anti-rat smooth muscle actin antibody  was used at 1:200, diluted in PBS containing 0.3% Triton X-100, 1% BSA, and 2% normal donkey serum. The secondary antibodies were Cy™Cultured MCA, BA and circle of Willis (CW) vessels were homogenized in cell extract denaturing buffer  containing phosphatase inhibitor and protease inhibitor cocktails . Whole cell lysates were sonicated on ice for 2 min, centrifuged at 15 000 × g at 4°C for 30 min, and the supernatants were collected as protein samples. Protein concentrations were determined using standard protein assay reagents  and stored at -80°C awaiting immunoblot analysis. The protein homogenates were diluted 1:1 (v/v) with 2× sodium dodecyl sulfate (SDS) sample buffer (Bio-Rad). Protein samples  were boiled for 10 min in SDS sample buffer and separated on 4-15% SDS Ready Gel Precast Gels  for 120 min at 100 V and transferred to nitrocellulose membranes (Bio-Rad) at 100 V for 60 min. The membrane was then blocked for unspecific binding for 1 h at room temperature with PBS containing 0.1% Tween-20 (Sigma) and 5% non-fat dried milk, thereafter incubated overnight at 4°C with primary antibodies: rabbit polyclonal anti TNF-α, TNF-R1 and TNF-R2  or mouse polyclonal anti β-actin , followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibodies  for 1 h at room temperature. The labelled proteins were developed using the LumiSensor Chemiluminescent HRP Substrate kit . To detect multiple signals on a single membrane, the membrane was incubated in Restore Plus western blot stripping buffer for 5-15 min at room temperature  between the various labelling procedures.http://rsb.info.nih.gov/ij/. Measurements were made in 4 to 6 different areas  for each section and 6 sections from each rat were evaluated. Thereafter, the mean values of the intensity per measured area were used (from five different rats per group). The increased intensity for the SAH group was compared to the sham group and for the MCAO the two sides were compared. Results are expressed as mean values ± S.E.M (in arbitrary units of fluorescence intensity). For the arteries in culture, the mean values were presented as percentage fluorescence in the culture groups compared to the fresh group, where the fresh group was set to 100%. The investigator was blinded to the treatment group of each sample.Fluorescence intensity was measured using the ImageJ software For western blot, cerebrovascular protein lysates from the different groups were compared. Cerebral arteries from two rats were pooled for each measurement . Three independent experiments were performed in duplicate. The membranes were visualized using a Fujifilm LAS-4000 Luminescent Image Analyzer , and band intensity was quantified using Image Gauge Version 4.0 . The immunoblot optical density values were presented as percentage activity in the vehicle and treated groups compared to the fresh groups, where the fresh group was set to 100%.P-values less than 0.05 were considered significant; "n" refers to the number of rats.Data are expressed as the mean ± standard error of the mean (S.E.M). Statistical analyses were performed using the nonparametric Kruskal-Wallis with Dunn's post hoc test for comparison between more than two groups and Mann-Whitney test for comparison between two groups. The levels of TNF-α, TNF-R1 and TNF-R2 proteins expressed in the cerebral arteries after 48 h post MCAO for 2 h and reperfusion were investigated by immunofluorescence. Microscopic evaluation showed enhanced expression of TNF-α, TNF-R1 and TNF-R2 in smooth muscle cells in the MCAO group as compared with the control group  Figure . MeasureHere we tested the hypothesis that TNF-α and its receptors might be upregulated in the artery wall at 48 h following SAH. Protein levels of TNF-α, TNF-R1 and TNF-R2 were investigated by immunofluorescence. Microscopic evaluation and fluorescence intensity measurements revealed that SAH resulted in a more intense immunoreactive signal corresponding to TNF-α , TNF-R1  and TNF-R2  in smooth muscle cells of MCA and BA as compared to the sham group Figures  and 2b.The relative expression levels of TNF-α, TNF-R1 and TNF-R2 proteins were investigated in cerebral arteries following 24 or 48 h of culture in the presence of U0126, SB-386023b, IMD-0354 or control solvent, and in fresh vessels (non-cultured). Western blot analysis revealed a significant increase in levels of TNF-α at 24 and 48 h as compared to fresh vessels  and of TNF-R1  , a B-Raf inhibitor , the MEK1/2 inhibitor , or vehicle (same volume). Immunohistochemistry revealed enhanced expression of TNF-α, TNF-R1 and TNF-R2 in vehicle-treated samples, particularly at 48 h of culture . Additionally, a weak expression of TNF-R2 was observed in the endothelial cell layer  Figure .in vivo cerebral ischemic models and during in vitro organ culture (used as a stress model). The upregulation observed here was confirmed by western blot quantification. In support, quantitative real time-PCR has demonstrated enhanced expression of TNF-α mRNA in cerebral vessels at 24 h after SAH, MCAO, organ culture, and in a time study between 1 to 48 h following SAH [This study is the first to show that cerebral arteries show similar protein expression profiles with respect to TNF-α and its receptors in two wing SAH ,30. An uwing SAH .Several studies have recognized that inflammation is a key element in the pathophysiology and outcome after stroke . CytokinTNF-α, TNF-R1 and TNF-R2 mRNA levels have been shown to increase in the brain after both permanent and transient MCAO in rat and mouse ,36,37, aSeveral investigators have suggested a role of the MAPK-MEK-ERK pathway in the regulation of TNF-α following cerebral ischemia. Studies have shown that TNF-α can increase the permeability of the blood-brain barrier (BBB) via activation of the ERK1/2 pathway, increase the expression of TNF-R1 and TNF-R2, and that treatment with U0126 inactivates this signaling pathway and decreases the expression of the TNF receptors ,42. Our Binding of TNF-α to its cell surface receptors results in activation of mitogen activated protein kinase (MAPK) which may lead to activation of e.g. two transcription factors, Activation Protein-1 (AP-1) and NF-κB . NF-κB rin vitro organ culture of isolated cerebral arteries and in vivo ischemia models (MCAO and SAH) result in upregulation of TNF-α and TNF-α receptors after 48 h in cerebral vessels walls. Previous studies have revealed that organ culture of cerebral arteries results in upregulation of inflammatory factors such as cytokines and matrix metalloproteinases (MMPs) after 24 h in a way similar to that seen in ischemia models [In this study we have shown for the first time that a models . In addia models  and in ea models  there isin vivo in which we showed that (i) SB386023-b following experimental SAH [On the other hand the mechanical stress has been reported to activate MAPKs  and thisntal SAH  preventental SAH . The spental SAH , therebyIn the present study, by immunohistochemistry we report that all three inhibitors decreased expression of TNF-R1 protein at 48 h incubation. In contrast, the increased expression of TNF-R2 was significantly prevented only by U0126 treatment. The western blot experiments provided partial support for this by a tendency for inhibition of expression by U0126. One possible explanation for the difference might be that TNF-R1 contains a death domain in its cytoplasmic region whereas TNF-R2 lacks this . ActivatOur results show that cerebral ischemia  and organ culture are associated with enhanced TNF-α, TNF-R1 and TNF-R2 protein levels in the wall of cerebral arteries. In addition, treatment with inhibitors of the MEK/ERK signaling pathway and NF-κB transcription factor prevent these enhanced expressions to various extents. The results suggest that inhibition of TNF signaling pathways by inhibition of the intracellular MAPK ERK1/2 pathway may constitute interesting targets, whereby inflammatory processes elicited following stroke may be prevented or attenuated.The authors declare that they have no competing interests.AM carried out the main part of the experiments, participated in the design, statistical analysis and writing of the manuscript. QC and LSK performed the western blot experiments and participated in writing of the final manuscript. LE conceived the study, directed the work, and drafted the manuscript. All authors have read and approved the final manuscript


Tumor necrosis factor-α (TNF-α) has been discussed as a potential anticancer agent for many years, however initial enthusiasm about its clinical use as a systemic agent was curbed due to significant toxicities and lack of efficacy. Combination of TNF-α with chemotherapy in the setting of hyperthermic isolated limb perfusion (ILP), has provided new insights into a potential therapeutic role of this agent. The therapeutic benefit from TNF-α in ILP is thought to be not only due to its direct anti-proliferative effect, but also due to its ability to increase penetration of the chemotherapeutic agents into the tumor tissue. New concepts for the use of TNF-α as a facilitator rather than as a direct actor are currently being explored with the goal to exploit the ability of this agent to increase drug delivery and to simultaneously reduce systemic toxicity.This review article provides a comprehensive overview on the published previous experience with systemic TNF-α. Data from 18 phase I and 10 phase II single agent as well as 18 combination therapy studies illustrate previously used treatment and dose schedules, response data as well as the most prominently observed adverse effects. Also discussed, based on recent preclinical data, is a potential future role of systemic TNF-α in combination with liposomal chemotherapy to facilitate increased drug uptake into tumors. A 51 kDa soluble homotrimeric cytokine is derived from the transmembrane form via proteolytic cleavage by the metalloprotease TNF-α converting enzyme (TACE) . TNF-α iin vitro and in vivo studies. These studies highlighted the possible role of TNF-α as an anticancer agent and galvanized support for the numerous phase I and phase II studies that followed.After the initial observation that TNF-α induced hemorrhagic necrosis of tumors in mice treated with BCG and endotoxin , the potIn vitro studies demonstrated that TNF-α had a growth inhibitory effect on SV40-tranformed human mammary epithelia cells and a cytotoxic effect on breast cancer cell lines. Interestingly, there was no effect on normal human mammary epithelial cells [In vivo, TNF-α has shown activity against a wide variety of murine tumor types and human tumor xenografts [in vitro and in vivo data were suggestive that TNF-α had the potential to be highly specific anti-cancer therapy, with activity against a number of tumor types.al cells . Similaral cells . Intrigual cells . Comparial cells . In vivonografts -21. Takein vitro and in vivo. TNF-α has been shown to enhance the cytotoxicity of DNA topoisomerase inhibitors actinomycin D, adriamycin, and etoposide against murine bladder tumor cell line (MBT-2) in in vitro and in vivo models [Preclinical evidence also suggested a synergistic effect of TNF-α with a variety of chemotherapeutics o models . The enho models . Intereso models . Secondlo models .in vitro and in vivo studies using the TNF-α resistant melanoma cell line B16BL6 demonstrated that IFN-γ sensitizes cancer cells to the effects of TNF-α, inducing necrosis and tumor response, which were previously absent [et al. [TNF-α also demonstrated enhanced antitumor effects in vitro when used in combination with other cytokines . Inductiy absent . Later, y absent . TNF-α a [et al.  tested Tin vitro and in vivo. Concomitant TNF-α and IFN-α in a murine lung metastasis model significantly increased survival [Combinations of TNF-α with IFN-α and IL-2 also showed synergistic cytotoxic and cytostatic effects survival . TNF-α asurvival , 33.The combination of TNF-α and radiotherapy has been less extensively studied. Investigation of the interaction of TNF-α and radiation in 14 human tumor cells lines demonstrated synergistic or additive cytotoxicity with the maximum effect when TNF-α was given 4-12 hours before irradiation . The mecNumerous phase I and phase II studies have been conducted to ascertain the toxicity profile and efficacy of systemic TNF-α. Studies have encompassed a wide range of tumor types in both adult and pediatric patients. In the majority of phase I and phase II studies, TNF-α was administered as an intravenous bolus injection or infusion. However, a few phase I studies have evaluated TNF-α with subcutaneous or intramuscular administration.2, given as a 30 minute intravenous infusion was identified as MTD in several studies. Dose-limiting toxicities (DLT) as well as other side effects that were observed seemed to have been universal and in most cases reasonably well tolerated and reversible. Common DLTs included: hypotension, thrombocytopenia, leukopenia, neurotoxicity, fever, nausea/vomiting, as well as general symptoms of malaise and weakness , leukopenia (38%), thrombocytopenia (13%), fever / chills (69%), headache (25%), nausea / vomiting (69%) and hepatopathy (10%). However, compared to other phase II studies this regimen was fairly dose-dense which may have increased the significant toxicity observed.Phase II studies using systemically administered rhTNF-α are detailed in Table 2 and was similar to the dose used for rhTNF-α monotherapy. In one study of rhTNF-α and BCNU in advanced melanoma, a response rate of 20% was seen with BCNU alone compared to 10.5% with BCNU and rhTNF-α [Phase I and II studies that investigated the safety and efficacy of systemic rhTNF-α combined with carmustine , actinom rhTNF-α . Additio rhTNF-α . HoweverMany studies have combined systemic administration of rhTNF-α with other cytokines such as: IFN-γ -72, IL-2Three studies that combined rhTNF-α with external beam radiation have also been reported -79 and tTranslation of systemic TNF-α from research to clinic has been hampered by significant systemic toxicity and a lack of efficacy at MTD , 46. Futin vivo [2) and 4 of 15 dogs treated had a partial tumor response. DLT was similar to that observed with unconjugated TNF-α, with hypotension and coagulopathy being observed. This study suggests that PEG-rhTNF-α may limit some of the undesirable toxicity seen with unconjugated TNF-α and allow for greater antitumor responses.Polyethylene glycol (PEG) conjugated proteins have shown increased retention and decreased immunogenicity in vivo . Attemptin vivo -84. Thamin vivo . Comparain vivo . Interesin vivo but not in vitro with tumor cell lines, indicating that this synergism is due to an indirect effect of NGR-TNF-α on host vasculature [2 of NGR-TNF-α and 75mg/m2 of doxorubicin was recommended. The study provided hope for future development of TNF-α and doxorubicin combination therapy with 1 of 15 patients achieving a partial response and 10 of 15 patients with stable disease for a median duration of nearly 6 months.Asparagine-glycine-arginine conjugated to the N-terminus of TNF-α (NGR-TNF-α) specifically binds the aminopeptidase N (CD13) of tumor vasculature . CD13 isculature . A recenculature . A phasein vivo studies demonstrated that the uptake of radiolabeled liposomes in tumors was increased by approximately 6 fold in mice that were concomitantly treated with TNF-α [2PR) effect. In vivo experiments using the combination of TNF-α and liposomal doxorubicin showed a significantly increased survival benefit in tumor-bearing mice treated with the combination in comparison to mice treated with either TNF-α or liposomal doxorubicin alone. Although single-agent liposomal doxorubicin alone delayed tumor growth and led to improved survival, the tumors eventually grew back, whereas the combination treatment with TNF-α and liposomal doxorubicin led to a long-term survival in 80% of the treated animals. These findings are in accordance with previously published data showing improved treatment outcomes in rat osteosarcoma and murine melanoma tumor models that were treated with low-dose TNF-α plus liposomal doxorubicin in comparison to TNF-α plus free doxorubicin [An alternative concept for the use of TNF-α in the treatment of human cancers exists. Preclinical th TNF-α . The mecorubicin , 91. Thein vitro and in vivo preclinical studies. Sadly, the promise of systemic TNF-α has, as of yet, not translated to a patient therapy and enthusiasm has been curbed due to the toxicity profile and lack of efficacy at MTD. Combination with chemotherapy in the setting of hyperthermic isolated limb perfusion has proven quite successful, based not only on a direct anti-proliferative effect of TNF-α, but also due to its ability to increase drug penetration into tumor tissue. The future development of systemic TNF-α as an anticancer treatment will rely on exploring ways to reduced systemic toxicity and exploit novel mechanisms of action to deliver greater efficacy simultaneously with decreased toxicity. A number of avenues are currently being explored based on promising preclinical and early clinical data. The novel concept of using systemic TNF-α to facilitate increased tumor penetration of liposomal chemotherapy seems particularly promising and worth exploring clinically.TNF-α has been proven an effective anticancer agent in