During inflammation, immune cells accumulate in damaged areas and release pro-inflammatory cytokines and neurotrophins. Brain-derived neurotrophic factor (BDNF) plays a neuromodulatory role in spinal cord dorsal horn via the post-synaptic tyrosine protein kinase B (trkB) receptor to facilitate pain transmission. However, the precise role of BDNF and trkB receptor in the primary sensory neurons of dorsal root ganglia (DRG) during inflammation remains to be clarified. The aim of this study was to investigate whether and how BDNF-trkB signaling in the DRG is involved in the process of inflammatory pain.We used complete Freund's adjuvant- (CFA-) induced and tumor necrosis factor-α- (TNF-α-) induced inflammation in rat hindpaw as animal models of inflammatory pain. Quantification of protein and/or mRNA levels of pain mediators was performed in separate lumbar L3-L5 DRGs. The cellular mechanism of TNF-α-induced BDNF and/or trkB receptor expression was examined in primary DRG cultures collected from pooled L1-L6 DRGs. Calcitonin gene-related peptide (CGRP), BDNF and substance P release were also evaluated by enzyme immunoassay.CFA injection into rat hindpaw resulted in mechanical hyperalgesia and significant increases in levels of TNF-α in the inflamed tissues, along with enhancement of BDNF and trkB receptor as well as the pain mediators CGRP and transient receptor potential vanilloid receptor subtype 1 (TRPV1) in DRG. Direct injection of TNF-α into rat hindpaw resulted in similar effects with retrograde transport of TNF-α along the saphenous nerve to DRG during CFA-induced inflammation. Primary DRG cultures chronically treated with TNF-α showed significant enhancement of mRNA and protein levels of BDNF and trkB receptor, BDNF release and trkB-induced phospho-ERK1/2 signal. Moreover, CGRP and substance P release were enhanced in DRG cultures after chronic TNF-α treatment or acute BDNF stimulation. In addition, we found that BDNF up-regulated trkB expression in DRG cultures.Based on our current experimental results, we conclude that inflammation and TNF-α up-regulate the BDNF-trkB system in DRG. This phenomenon suggests that up-regulation of BDNF in DRG may, in addition to its post-synaptic effect in spinal dorsal horn, act as an autocrine and/or paracrine signal to activate the pre-synaptic trkB receptor and regulate synaptic excitability in pain transmission, thereby contributing to the development of hyperalgesia. Inflammation and pain largely share a common course of progression; patients with inflammation may suffer hyperalgesia and/or allodynia to various mechanical, thermal and chemical stimuli . InflammTNF-α is a potent pro-inflammatory cytokine that has been used frequently in laboratory studies to evoke inflammatory reactions. TNF-α activates the release of many cytokines, such as IL-1β, IL-6 and IL-8, and participates in the development of inflammatory hyperalgesia mainly through its receptor, TNFR1 and TNFR2 -11.Neurotrophins like NGF, neurotrophin 3/4 (NT-3/4) and brain-derived neurotrophic factor (BDNF) can be released from DRG, acting to either support neuronal development  or partiThe aim of this study was to investigate the role of BDNF-trkB within DRG during the progression of inflammatory pain, specifically focusing on whether and how the pro-inflammatory cytokine TNF-α influences trkB expression and signaling. To accomplish this aim, we used CFA-induced inflammation in rats as a chronic pain model to examine neurochemical changes in lumbar DRG. Levels of the pro-inflammatory cytokine TNF-α were first determined in the inflamed tissues. Consequently, a set of animals was injected with TNF-α directly into hindpaw to assess inflammatory pain responses. In addition, we used DRG primary cultures and treated with TNF-α for mechanistic and functional studies. We demonstrate that BDNF and trkB receptors are up-regulated in DRG of CFA- and TNF-α-induced inflammatory pain models. In addition, retrograde transport of TNF-α was observed in the CFA-induced inflammation model. Both trkB receptors and trkB-induced ERK signaling were increased after chronic TNF-α-treatment in DRG primary cultures. Furthermore, both CGRP and substance P release were enhanced after chronic TNF-α pre-treatment or acute BDNF administration in DRG cultures. We conclude that experimental arthritis and TNF-α initiate a BDNF-trkB feed-forward pathway in peripheral sensory neurons, and this up-regulation of BDNF-trkB system may participate in pain sensitization.ad libitum. The animal handling and drug treatments were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Animal Care Committee of Chang-Gung University.Male Sprague-Dawley (SD) rats  weighing 250-280 g  or 3-4 weeks of age (for primary culture) were used in the current study. After arrival, animals were acclimatized to the room with controlled temperature (22 ± 2°C), air humidity (50 ± 10%) and 12 hours day-night cycle (light on at 7:00 AM) for at least 7 days before experimentation. The animals were housed 2 or 3 per cage with food  and water 2O. NGF 2.5S  was reconstituted with PBS.CFA  was freshly prepared . Recombinant rat TNF-α protein  was reconstituted with phosphate buffered saline (PBS)/0.1% bovine serum albumin (BSA). Recombinant human BDNF protein  was reconstituted with ddHin vivo model of inflammatory pain, animals were anesthetized with isoflurane and locally injected with CFA, TNF-α or vehicle. In the CFA-treated group, animals were injected with 100 μl CFA into the right hindpaw with the contralateral side serving as an untreated control. Other animals received saline injection in the hindpaw to serve as a vehicle control. In the TNF-α-treated group, animals received 500 ng TNF-α (30 μl) in the hindpaw twice daily for three consecutive days, followed by daily mechanical von Frey tests. Lumbar L3~L5 DRGs or hindpaw tissues were collected 72 hours after CFA or TNF-α injection and samples of DRG were divided into following different groups: CFA , TNF-α , control  and saline groups.To produce an von Frey method ; TRPV1-forward, 5' GCG AGT TCA AAG ACC CAG AG 3'; TRPV1-reverse, 5' GGC ATT GAC AAA CTG CTT CA 3'[GenBank: NM_031982]; trkB.FL-forward, 5' AAG ATC CTG GTG GCC GTG AAG A 3'; trkB.FL-reverse, 5' CGG CTT CGC GAT GAA AGT CCT T 3'[GenBank: NM_012731]; BDNF-forward-1, 5' CAA GGC AAC TTG GCC TAC CC 3'; BDNF-reverse-1, 5' GAG CAT CAC CCG GGA AGT GT 3'; BDNF-forward-2, 5' TTA CCT GGA TGC CGC AAA CA 3'; BDNF-reverse-2, 5' TGG CCT TTT GAT ACC GGG AC 3' [GenBank: NM_012513]; CGRP-forward, 5' AAC CTT AGA AAG CAG CCC AGG CAT G 3'; CGRP-reverse, 5' GTG GGC ACA AAG TTG TCC TTC ACC A 3'[GenBank: M11597]. Threshold cycle, Ct, which correlates inversely with the target mRNA levels, was measured. ABI Prism Primer Express was used to design the specific gene primers. After normalization to 18S values, data were expressed as percentage of corresponding controls.Total RNA was isolated from cultured DRG neurons or tissues using TRIzol2PO4 3.3 mM, K2HPO4 21.9 mM, NaCl 154 mM) overnight at 4°C. IHC images were collected from 14 μm sections prepared using a freezing microtome .SD rats were sacrificed by decapitation; L3-L5 DRGs were removed and post-fixed in 4% (w/v) paraformaldehyde solution for 48 hours at 4°C, then cryo-protected with 20% (w/v) sucrose in potassium phosphate buffer saline  sodium tetrahydridoborate for 5 min. Afterward, the sections were incubated in primary antibody in 2% (w/v) normal goat serum/0.3% (v/v) TX-100/KPBS at 4°C for 48 hours. The antibodies used were anti-trkB antibody , anti-BDNF antibody , anti-CGRP antibody . Afterward, sections were incubated with 1:200 biotinylated goat anti-rabbit IgG secondary antibody for 1 hour. Immunoreactivity was visualized by the avidin-biotin complex method . After incubating with the avidin-biotin complex for 1 hour, sections were developed using 3, 3'-diaminobenzideine  and nickel ammonium sulfate in KPBS. Sections were mounted onto gelatin-coated slides, dehydrated through a serial alcohol gradient, degreased with xylene and then covered slipped with Entellan . Co-localization of BDNF and TNFR1 was evaluated by immunofluorescent double stain. Primary antibodies  were mixed and incubated at 4°C for 72 hours. After washed with KPBS, sections were incubated with secondary antibodies  for 1 hour at room temperature. Sections were then incubated with 1:1000 DAPI  for 5 min before mounting with glycerol. Signals of immunofluorescent stain were observed by fluorescence microscope.Tissue sections were rinsed twice with KPBS. Endogenous peroxidase was quenched by pre-treating the sections with 0.03% (v/v) H2O2 followed by pretreatment with 1% sodium tetrahydridoborate. After incubating with primary antibody and rinsing with PBS/0.3% TX-100, the cells were incubated in 1:200 biotinylated goat anti-rabbit IgG secondary antibody for 1 hour and rinsed with PBS. After incubation with avidin-biotin complex for 1 hour, ABC reaction was performed with a mixture of DAB and nickel ammonium sulfate. Cells were mounted on slides, dehydrated, and cover-slipped with mounting solution. The color of precipitate formed by DAB was monitored microscopically.Cultured DRG neurons were fixed with 2% (w/v) paraformaldehyde solution  for 30 min. Cells were rinsed with PBS/0.3% TX-100 following blocking with 2% normal goat serum in PBS/0.3% TX-100 for 30 min. Cells were then incubated in primary antibody in PBS/0.3% TX-100/2% goat serum at 4°C for 48 hours. The antibodies used were anti-trkB antibody (1:2000), anti-BDNF antibody (1:1000), anti-CGRP antibody (1:8000). TrkB staining was visualized via immunofluorescence. After rinsing three times, cells were incubated with FITC-conjugated goat anti-rabbit IgG secondary antibody  for 1 hour. Afterward, cells were then incubated with 1:1000 DAPI for 5 min before mounting with glycerol. Co-localization of TNFR1 and phospho-ERK were evaluated by immunofluorescent double stain. Primary antibodies  were mixed and incubated at 4°C for 72 hours. After wash with PBS, secondary antibodies, Rodamine-conjugated goat anti-mouse and Dylight 488-conjugated goat anti-rabbit , were also mixed together and added for 1 hour. Slides were then incubated with 1:1000 DAPI for 5 min before mounting with glycerol. Immunofluorescent stain signal was observed by fluorescence microscope. On the other hand, BDNF and CGRP staining were visualized via avidin-biotin complex method. Similar to immunohistochemistry, endogenous peroxidase was quenched by pre-treating the sections with 0.03% Hmax® ImmunoAssay system , CGRP EIA kit  or substance P EIA kit , respectively. For BDNF assay, after 5 nM TNF-α treatment for 48 hours, medium of DRG cultures were collected. The cells were lysised, sonicated and then centrifuged at 10,000 × g for 15 min. The supernatants were analyzed and represented as total BDNF content. For CGRP and substance P EIA assay, after 5 nM TNF-α treatment for 48 hours, culture medium were replaced by a fresh medium and allowed cells stable for 2 hours. Afterwards, 400 ng/ml BDNF was added for 30 or 60 min and the supernatant was collected and analyzed immediately according to the manufacturer's protocols. The microplate was read at 405 nm (yellow color) in an enzyme-linked immunosorbent assay (ELISA) plate reader .The release of BDNF, CGRP and substance P were determined using BDNF Eg, 4°C for 20 min and supernatants were collected. Blood samples were withdrawn from the animals' trunks into a blood collection tube containing 5.4 mg K2 EDTA and centrifuged at 1,000 × g for 10 min in the room temperature to collect the serum. Samples were analyzed immediately according to the manufacturer's protocols. The concentration of TNF-α was determined by the absorbance at 450 nm with reference filter at 540 nm.The amount of TNF-α in rat hindpaw or DRG was evaluated using a rat TNF-α EIA kit (R&D system). After CFA-injection for 72 hours, rat hindpaw or L3~L5 DRGs were isolated and analyzed immediately. Tissues were collected in PBS solution containing proteinase inhibitors leupeptin, aprotenin and PMSF (Sigma). After homogenization, tissues were centrifuged at 10,000 × Recombinant rat TNF-α protein (10 μg) was labeled with Alexa Fluor 488  according to the manufacturer's protocols. Animal received Alexa Fluor 488-TNF-α (25 μl) in CFA pre-treated hindpaw 24 hours before sacrifice (72 hours after CFA injection). Alexa Fluor 488 (25 μl) injection in CFA pre-treated hindpaw served as control. After injection, ipsilateral saphenous nerve was isolated immediately and monitored under fluorescence microscope. The fluorescent signals in the saphenous nerve were separately measured in three segments: distal (0.5 cm from the heel), middle (1.3 cm from the heel) and proximal (2 cm from the heel).in situ cell death detection kit , following the manufacturer's protocols, DRG primary cultures were fixed with 2% paraformaldehyde for 1 hour, then incubated with 0.1% Triton X-100/0.1% sodium citrate for 2 min on ice. Afterwards, the cultures were washed with PBS and then treated with 50 μl TUNEL reaction mixture (containing green Fluorescein) at 37°C for 60 min in the dark. After enzyme reaction, cultures were washed with PBS and double stained with DAPI  for 5 min. Numbers of apoptotic cells were evaluated by observation under a fluorescence microscope.Cell apoptosis was evaluated using an ImageJ  software was used to quantify the immunostaining images. A threshold was set by ImageJ to define the positive neurons among different experiments of the same study. For DRG immunostaining, three different sections (spacing 250~300 μm) were selected to calculate the total number of positive neurons and expressed as the percentage of positive neurons relative to the total number of neurons visualized in DRG sections. For the data in Table p < 0.05 was considered significant.All data were analyzed with GraphPad InStat . Results are expressed as mean ± SEM. The data were analyzed by one-way ANOVA followed by Tukey multiple comparisons test or un-paired Student's t-test. A value of SD rats receiving 100 μl CFA in the intraplantar surface developed mono-arthritis within 72 hours. CFA-injected hindpaw appeared swollen and enlarged by the third day after injection Figure . In contvon Frey testing. As shown in Figure To measure pain threshold, mechanical hyperalgesia was assessed in three experimental groups via daily To determine if the pro-inflammatory cytokine TNF-α was involved in CFA-induced hyperalgesia, tissue content of TNF-α was measured in rat hindpaw, DRG and serum 72 hours after CFA-injection by a TNF-α EIA kit. As shown in Figure von Frey. Similar to the CFA-injected animals, the hindpaw receiving TNF-α injection exhibited swelling  and medium (p = 0.0025) neurons after CFA injection, with the percent increase of trkB-positive neurons equally distributed in three different size neuronal populations. It was noted that trkB receptors were mainly expressed in small-sized neurons (77.66% of control and 76.84% of CFA treated), moderately expressed in medium-sized neurons (16.42% of control and 17.35% of CFA treated) but rarely expressed in large-sized (5.92% of control and 5.81% of CFA treated) DRG neurons.To explore whether CFA-induced trkB over-expression occurs in a particular type of DRG neuron, trkB-positive neurons were separately counted based on the diameter of the neuron  in L3 DRG. As shown in Table The changes in expression of BDNF, trkB and CGRP protein in L3-L5 DRGs were also evaluated 72 hours after TNF-α injection (twice daily for three days) in the hindpaw. Data are presented as percentage of positive neurons in DRG. L3-L5 DRGs were counted separately. As shown in Figure Previously, it was reported that under peripheral inflammatory conditions, TNF-α binds to TNFR1 and activates inflammatory signals by retrograde transport to the DRG . Double in vivo inflammatory condition. TNF-α (5 nM) was added to the culture medium at 3 days in vitro (DIV) and maintained for up to 48 hours. As shown in Figure In order to investigate the cellular mechanism of inflammatory responses, primary DRG cultures were used . Since we observed significantly increased TNF-α in rat hindpaw after CFA injection, and mechanical hyperalgesia develops in rats after direct treatment with TNF-α, the pro-inflammatory cytokine TNF-α was applied to mimic the To further confirm the impact of TNF-α on BDNF-trkB system in the DRG, TNF-α-induced BDNF release was evaluated by BDNF EIA. After treatment with TNF-α for 24 or 48 hours, BDNF release and expression were analyzed in supernatant medium and cultured cells, respectively. The results show that BDNF release was significantly enhanced after TNF-α treatment for 48 hours Figure . Total BIt has been reported that NGF increases the expression of BDNF in peptidergic small neurons in DRG and the release of BDNF was targeted to spinal cord lamina II ,18. In oThe results show that NGF induced a significant time-dependent increase in mRNA levels of TRPV1, BDNF and CGRP Figure . On the To test the functional significance of TNF-α-dependent trkB receptor up-regulation, trkB-mediated signaling was evaluated in DRG cultures. BDNF (400 ng/ml) was acutely applied  to DRG cultures after the cells were treated with 5 nM TNF-α for 48 hours, and then the downstream signals of phospho-ERK were measured by immunefluorescent stain. Further, to determine if TNF receptor co-localized with phospho-ERK signals, double fluorescent stain was employed. The results clearly show that phospho-ERK is enhanced after acute BDNF treatment for 10 min and is further increased by 48 hours of TNF-α pre-treatment Figure , 9E. MorTo characterize the functional role of TNF-α-enhanced BDNF-trkB signaling, CGRP and substance P were evaluated in DRG cultures. Protein levels of CGRP were significantly enhanced in DRG of either CFA-treated Figure  or TNF-αNext, we examined whether BDNF could trigger another nociception mediator, substance P release from DRG, after TNF-α treatment. After treatment with 5 nM TNF-α for 48 hours, an acute treatment with BDNF (400 ng/ml) was applied for 30 or 60 min and the amount of substance P in the culture medium was analyzed using an EIA kit. As shown in Figure in vivo) and in DRG cultures 48 hours after TNF-α treatment (in vitro). As shown in Figure Retrograde axonal transport of TNF-α has been reported in various pathological conditions ,28. For et al. (1999) reported that mRNA of truncated trkB receptor increases in DRG after CFA injection, but the full-length trkB receptor does not. Further, Chien et al. (2007) reported that BDNF, CGRP, trkA and p75NTR are increased in DRG after CFA treatment [Numerous studies have attempted to understand the cellular mechanism underlying hyperalgesia and allodynia using a CFA-induced inflammation pain model. Our study focused on the neurochemical changes in lumbar DRG and clearly shows that protein levels of trkB, BDNF and CGRP are significantly increased after animals develop hyperalgesia. Up-regulation of BDNF and CGRP in DRG during inflammation is a well-known phenomenon along with the consequent effect of post-synaptic trkB over-expression in the dorsal horn of spinal cord ,29. On treatment , while lreatment -33. Thesin vivo and in vitro.In the present study, we found that BDNF protein but not mRNA was significantly enhanced in DRG during inflammation. Previously, it has been reported that levels of BDNF mRNA increase significantly in DRG 24 hours after CFA-injection ,34, whilOur results reveal a statistically significant increase in protein levels of trkB, BDNF and CGRP as well as mRNA of trkB and TRPV1, mainly in L3, but CGRP protein in L4 and trkB and CGRP proteins in L5 DRG after CFA treatment. On the other hand, TNF-α treatment led to increased protein levels of trkB, BDNF and CGRP predominantly in L4, with relatively minor effects in L3 and L5 DRGs. This segregated effect was unexpected because rat hindpaw are innervated by primary afferents from L3, L4 and L5 DRGs. A previous report regarding the nociceptive dermatomes in Wistar rat hindpaw demonstrated that the L3 dermatome is contained in the inner part of the paw; the L4 dermatome is contained in the middle part of the paw and the L5 dermatome is contained in the outside part of the paw . AlthougPro-inflammatory cytokines play an important role in the process of inflammatory pain and their release results in pain sensitization ,3. TNF-αin situ BDNF-trkB up-regulation. In addition to the effect of TNF-α on BDNF-trkB, it is well known that TNF-α can induce IL-6, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion through activation of MEK1/2, p38 MAPK and NF-κB pathways during inflammation [in vivo.Similar to the CFA-induced inflammatory condition, we found that TNF-α injection into hindpaw and application to DRG cultures produced a clear enhancement of trkB and BDNF expression in DRG. In addition, we observed a release of BDNF from DRG cultures after TNF-α treatment. Other than targeting the post-synaptic trkB receptor in the dorsal horn, we speculate that enhanced BDNF would also have chance to activate local trkB receptors in DRG, leading to trkB dimerization and auto-phosphorylation, which would ultimately trigger pre-synaptic signaling pathways . In suppammation ,45. Sincin vivo [NGF is an important mediator of inflammatory pain . After rin vivo , althougin vivo . Our obsin vivo . Althougin vitro results demonstrate that an increase in trkB receptors is triggered by either BDNF or TNF-α. This effect may eventually form a sensitization loop to increase pain transmission at the level of the DRG.Our results show that BDNF treatment increases mRNA expression for trkB receptor in DRG cultures. BDNF is reported to co-express with trkA (20%) and trkB (10%) receptors in DRG ,21. In ain vivo and in vitro observations that total CGRP protein increases after CFA-induced inflammation [et al [-/- mice through the actions of NGF and TNF-α [The finding that basal CGRP release is enhanced in DRG after chronic TNF-α treatment supports a previous study which showed that acute TNF-α treatment increases CGRP release in rat trigeminal ganglia . In addiammation ,50. Simiammation ,31 that n [et al  reportednd TNF-α . In thisWe hypothesize that during chronic inflammation, cytokines such as TNF-α are released locally and act on TNF receptors in nociceptors. TNF-α then undergoes retrograde transport to the DRG to enhance synthesis of BDNF, trkB and CGRP. NGF may also be released from immune cells around nociceptors and bind to trkA receptor to further increase the expression of TRPV1, BDNF and CGRP. The enhanced BDNF in DRG might have chance to act as an autocrine and/or paracrine signal to activate pre-synaptic trkB receptors synergistically with cellular events mediated by TNF-α (or by other inflammatory mediators), and up-regulate synaptic excitability in pain transmission (see Figure The authors declare that they have no competing interests in conducting this study.in vivo and in vitro experiments in this study and prepared the first draft of this manuscript. JCC conceived of the study, participated in its design and coordination and helped to draft the manuscript. LSR and HLW participated in experimental design and consulted on the study. All authors read and approved the final manuscript.YTL carried out all the


Costimulation of T cells via costimulatory molecules such as B7 is important for eliciting cell-mediated antitumor immunity. Presenting costimulation molecules by immobilizing recombinant B7 on the surface of nanovectors is a novel strategy for complementary therapy. Polyhydroxyalkanoates (PHAs) are a family of biodegradable, non-toxic, biocompatible polyesters, which can be used as a nonspecific immobilizing matrix for protein presentation. Recombinant protein fusion with PHA granule binding protein phasin (PhaP) can be easily immobilized on the surface of PHA nanoparticles through hydrophobic interactions between PhaP and PHA, and therefore provides a low-cost protein presenting strategy.Escherichia coli strain BL21 (DE3). The purified B7-2-PhaP protein was immobilized on the surface of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)-based nanoparticles. Loading of 240 μg (3.2 pMol) of B7-2-PhaP protein per mg nanoparticles was achieved. Immobilized B7-2-PhaP on PHBHHx nanoparticles induced T cell activation and proliferation in vitro.In this study, the extracellular domain of the B7-2 molecule  was fused with PhaP at its N-terminal and heterogeneously expressed in recombinant in vitro. The B7-2-PhaP immobilized PHA nanosystem is a novel strategy for costimulation molecule presentation and may be used for costimulatory molecule complementary therapy.A PHA nanoparticle-based B7-2 costimulation molecule-presenting system was constructed. The PHA-based B7 presenting nanosystem provided costimulation signals to induce T cell activation and expansion  That is partly because some tumors can deliver antigen-specific signals to T cells, but do not deliver costimulatory signals necessary for full activation of T cells [It is generally accepted that the activation of naïve T cells requires two signals. One is provided by association of the T-cell receptor with the major histocompatibility complex–peptide complex. The other, known as costimulation, is necessary for optimal T-cell activation, which leads to clonal T-cell expansion, cytokine secretion and development of effector functions. Two main costimulatory molecules, B7-1 and B7-2, accomplish the coactivation function through binding with their ligand CD28 on the surface of T cells [in vivo. Based on the safety considerations of expressing B7 using a virus vector, we propose a novel strategy to present costimulatory molecules by immobilizing recombinant B7 on the surface of polyhydroxyalkanoate (PHAs)-based nanoparticles.Low levels of B7-1 and B7-2 have been detected on certain human cancers ,4 and inPHAs are a family of biodegradable, non-toxic, biocompatibility polyesters, which are synthesized by a wide range of microorganisms ,7, have co-3-hydroxyhexanoate) (PHBHHx)-based nanoparticles. The immunocompetence of the resulting fusion protein and functionality of the PHBHHx nanoparticles were analyzed.In this study, the extracellular domain of the B7-2 molecule was fused with PhaP at its N-terminal and the recombinant B7-2-PhaP protein was immobilized on the surface of poly(3-hydroxybutyrate-Escherichia coli BL21 (DE3), with a glutathione S-transferase (GST) tag at the N-terminal of the resulting fusion protein. A flexible peptide linker (GGGGSGGGSGGGS) was inserted between B7-2 and PhaP to ensure correct folding. The GST-B7-2-PhaP fusion protein was efficiently produced by recombinant E. coli BL21 (DE3)  column. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis showed that soluble GST-B7-2-PhaP fusion protein with a molecular mass of 73 kDa was successfully produced in the recombinant E. coli BL21 (DE3) and a single band of the purified GST-B7-2-PhaP fusion protein was detected from the eluted sample  for 16 h and then collected by centrifugation. After the immobilization process and removal of the nanoparticle proportion, no proteins were detected in the supernatant of samples with the initiation GST-B7-2-PhaP concentration of 1, 1.5 and 2 mg ml-1. In the sample to which 2.5 mg ml-1 GST-B7-2-PhaP protein was added, 0.236 mg ml-1 protein remained in the supernatant after removing the coated nanoparticles. This indicated that approximately 400 μg of GST-B7-2-PhaP fusion protein could be immobilized on the surface of 1 mg PHBHHx nanoparticles. Western blot analysis also demonstrated that GST-B7-2-PhaP fusion proteins could be detected on the samples eluted from the nanoparticles  stimulation, activated T cells proliferated. T cells treated with anti-CD3 antibody (5 ng ml-1) alone received only the primary signal for T cell activation, and thus did not become activated or proliferate. However, addition of either immobilized B7-2-PhaP (1 μg ml-1) or free B7-2-PhaP protein led to significant cell expansion. The B7-2-PhaP loaded PHBHHx nanoparticles showed a higher activity for stimulated cell expansion than non-immobilized free B7-2-PhaP protein. PHBHHx nanoparticle treatment also induced the stimulation of cell proliferation. Immobilized B7-2-PhaP on PHBHHx nanoparticles together with anti-CD3 antibody demonstrated the highest activity for the stimulation of cell proliferation. These results indicated that PHBHHx-based B7-2-PhaP presenting nanoparticles can be used to activate T cells.Following PHAs are linear biopolyesters produced as energy- and carbon-storage materials by many bacteria. The medical applications of PHA have been extensively explored in recent years for implant biomedical applications including sutures, nerve conduits, patches, slings, cardiovascular patches, stents, guided tissue repair/regeneration devices, bone marrow scaffolds and so on. PHA nanoparticles have also been developed as controlled drug-release vectors owing to their biocompatibility and biodegradability.The surface of PHA-based nanoparticles can be modified by many different types of signaling molecules and thus can be used for purposes other than targeted delivery. This can also be achieved by engineering proteins other than PhaP such as other PHA surface associated proteins including PhaC, PhaR, and PhaZ. These proteins provide targets for displaying various molecules on polymer nanoparticle surfaces for protein immobilization and display. The phasin PhaP is a small amphiphilic protein that can associate with most hydrophobic polymer granule surfaces via strong hydrophobic interactions. PhaP is abundant on the surface of natural PHA granules, and can reach approximately 5% (wt/wt) of the total cell protein in PHA-accumulating bacteria. Thus, multifunctional polymer nanoparticles can be obtained through protein engineering of PhaP and applied to a variety of versatile situations. The functional fusion proteins were able to produce easily by recombinant bacteria cultivation. This method provides a simple and convenient way to immobilize and present proteins on the surface of hydrophobic biomaterials without complicated procedures to modify both proteins and materials.-1 immobilized B7-2-PhaP proteins was applied to cultured cells, the amount of nanoparticles used was approximately 4.2 μg ml-1. Previously we demonstrated that 5 μg ml-1 PHBHHx nanoparticles could enhance the cell viability of rodent lymphocytes from spleen. Interestingly, the main degradation product of the PHBHHx polymer, 3-hydroxybutyrate, can promote interleukin-2 and interferon-γ secretion by rodent T lymphocytes (data not published). This may explain the slight T cell proliferation resulting from the non-coated PHBHHx nanoparticle treatment in this study.Human B7-2 is a 329-amino-acid (aa) protein containing a putative 23-aa signal peptide, a 224-aa extracellular domain, a 21-aa transmembrane domain, and a 61-aa cytoplasmic domain. The absence of the signal peptide, the transmembrane domain, and the cytoplasmic domain did not affect the costimulatory activity of B7 molecules. In this study, the 224-aa extracellular domain was subcloned and fused with PhaP and immobilized on the surface of PHBHHx nanoparticles through the hydrophobic interactions between PhaP and polymer. As measured by western blot, 400 μg of GST-B7-2-PhaP fusion proteins could be immobilized on the surface of 1 mg PHBHHx nanoparticles, which by extrapolation implies 240 μg (3.2 pMol) B7-2-PhaP fusion protein was present on the surface of 1 mg of nanoparticles. When 1 μg ml-1 phytohemagglutinin to increase cell numbers prior to the proliferation assay. Thus, the expanded cells were mainly T cells, although small numbers of B cells and other monocytes remained in the culture. This may explain why non-immobilized B7-2-PhaP fusion proteins also resulted in cell proliferation. PhaP is a bacterial protein that might be recognized by B cells, thus triggering an immune response resulting in lymphocyte proliferation. However, whether the B7-2-PhaP fusion protein was immobilized or not, the combined treatment of B7-2-PhaP protein and anti-CD3 antibody achieved the highest proliferation ratio and number of activated T cells. Compared with the non-immobilized free B7-2-PhaP protein, the B7-2-PhaP protein loaded nanoparticles showed a higher activity for cell proliferation stimulation. This can be explained by the advantage of the immobilization system, which can be used to provide several signal molecules that interact concurrently with ligands on cell surfaces.Phytohemagglutinin is a lectin found in plants, and is used as a mitogen to trigger T lymphocyte cell division. Isolated lymphocytes were treated with 5 μg mlE. coli BL21 (DE3). The purified B7-2-PhaP protein was immobilized on the surface of PHBHHx nanoparticles through the hydrophobic interaction between PhaP and polymer. The immunocompetence study showed that the PHA-based B7 presenting nanosystem could provide costimulatory signals for T cell activation, leading to T cell expansion in vitro. The B7-2-PhaP immobilized PHA nanosystem provides a novel strategy for the presentation of costimulatory molecules and may be a useful tool for costimulation molecule complementary therapy.In this study, a novel PHA nanoparticle-based B7 costimulation molecule-presenting system was developed. Both the vehicle and the presented functional protein were produced by microorganism fermentation and purified from the culture broth. The extracellular domain of the B7-2 molecule was fused with PhaP, and the B7-2-PhaP protein was hetero-expressed by recombinant Ralstonia eutropha H16 was subcloned from the plasmid pTwin2-EPhaP . The primers B1 (5’-GCGGATCCATGGCTCCTCTGAAGATTC-3’) (the double line marks the BamHI restriction site) and B2 (5’-ACCAGAGCCACCTCCTGAACCGCCTCCACCTGTAATCCAAGGAATG-3’) were used to amplify extracellular domain of B7-2 coding sequence. The primers P1 (5’-GGTTCAGGAGGTGGCTCTGGTGGATCGCTCACCCCGGAACAAGTT-3’) and P2 (5’-CGCTCGAGAGCCGTCGTCTTCTTTGCCGT-3’) (the double line marks the XhoI restriction site) were used to amplify the PhaP gene. The PCR products of B7-2 and PhaP were mixed at the ratio of 1:1 and fusion with each other by overlap extension PCR. Then BamHI and XhoI digested B7-2-phaP segment was inserted into the corresponding sites downstream of the GST tag coding sequence of pGEX-4T-3 expression vector to generate the final plasmid pGEX-B72P  was subcloned from the plasmid B7-2(IgV + C)/TEasy, which contains the cDNA sequence of B7-2. The gene encoding PhaP of 72bp was E. coli strain BL21 (DE3) was used to express GST-B7-2-PhaP fusion proteins. E. coli BL21 (DE3) transformants carrying the pGEX-B72P plasmid was cultured at 37°C in 500 ml shake flasks containing 200 ml Luria Bertani medium  supplemented with 100 mg ml-1 ampicillin and 3% (v/v) ethanol in a shaking incubator at 200 rpm. At an OD600 of 0.4-0.6, 0.1 mM isopropyl β-D-thiogalactopyranoside was added to induce the expression of the fusion gene and the recombinant E. coli BL21 (DE3) carrying pGEX-B72P was continued in culture at 20°C for another 6 hours.g for 10 min and re-suspended in 20 ml phosphate-buffered saline (PBS) buffer followed by sonication  for 10 min at 40% output. After centrifugation at 12000 × g for 30 min at 4°C, the clarified soluble protein fraction was collected. The target recombinant GST-B7-2-PhaP fusion protein was purified by GS4B affinity chromatography  according to the manufacturer’s protocol.Cells were harvested by centrifugation at 6000 × The eluted protein was purified and condensed by the dialysis bag method. Sample collected after purification was analyzed by 12% SDS-PAGE. The purified recombinant protein was stored at −20°C after lyophilization for further usage.The PHBHHx material was produced, isolated and characterized as described previously ,16. The -1. A concentration of 5 mg ml-1 of PHBHHx nanoparticles was mixed with various amounts of purified GST-B7-2-PhaP fusion proteins, followed by stirring and incubation at 4°C for 12-16 h. Subsequently, the colloidal system was collected by centrifugation at 12,000 × g for 30 min and washed twice with PBS. The immobilized GST-B7-2-PhaP fusion protein was calculated according to the difference between the amounts of protein in the initial sample and that in the supernatant after incubation, and was quantified using the bicinchoninic acid method using a protein assay kit Beyotime  according to the manufacturer’s protocol.Fusion protein GST-B7-2-PhaP was dissolved in PBS at a final concentration of 1-2.5 mg mlg centrifugation for 10 min, the supernatant was analyzed by SDS-PAGE and western blot to verify the immobilization of GST-B7-2-PhaP on the surface of PHBHHx nanoparticles. Briefly, proteins were transferred to poly (vinylidene fluoride) (PVDF) membranes after electrophoresis, and were then blocked for 1 h at room temperature with 5% bovine serum albumin in TBST buffer. PVDF membranes were incubated overnight with an anti-GST antibody . The PVDF membrane was subsequently hybridized with secondary horseradish peroxidase-conjugated goat anti-mouse IgG  and the recombinant protein was detected by incubating with a chemiluminescence solution purchased from Thermo Scientific  according to the manufacturer’s instructions.The assembled GST-B7-2-PhaP-PHBHHx nanoparticles were resuspended in protein loading buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 1% β-mercaptoethanol) and boiled for 2 min to allow detachment of denatured GST-B7-2-PhaP fusion protein from PHBHHx nanoparticles. After 12,000 × 2 atmosphere overnight. After disposal of adherent cells, the suspended cells were recovered and treated with 5 μg ml-1 phytohemagglutinin for 72 h to expand the T cell population. For the T cell proliferation assay, cells were passaged and seeded in triplicate at 1 × 104 cells per well in 96-well culture plates, followed by incubation with the corresponding treatment for another 72 h. The cell proliferation was analyzed by MTT assay. Cells treated with 5 μg ml-1 phytohemagglutinin served as the positive control. T-cell surface glycoprotein CD3 antibody (500 ng ml-1) was used to provide the first signal for T cell activation.Five milliliters of whole blood was donated by a healthy volunteer. Lymphocytes were separated from non-coagulated whole blood using a human lymphocyte separation medium according to the manufacturer’s instructions . The isolated cells were suspended in a culture bottle with high-glucose RPMI1640 medium  supplemented with 10% fetal bovine serum  and maintained at 37°C in a humidified 5% COt-test. Differences were considered to be statistically significant when P < 0.05.All results were expressed as mean ± standard deviation of experiments performed in triplicate. Statistical differences were evaluated using a one-way analysis of variance with a Student’s The authors declare that they have no competing interests.XYL designed and conducted all experiments and prepared the manuscript. QQL participated in the construction of plasmids, expression and purification of recombinant protein. MCL prepared the polymer nanoparticles and participated in the immobilization of recombinant protein. YLZ participated in the design of cell purification and cultivation experiments and manuscript preparation. LLW carried out the cell culture and proliferation assay. All authors read and approved the final manuscript.


Cell growth conditions and purification methods are important in determining biopharmaceutical activity. However, in studies aimed at manufacturing virus-like particles (VLPs) for the purpose of creating a prophylactic vaccine and antigen for human papillomavirus (HPV), the effects of the presence of a resin-bound ligand during purification have never been investigated. In this study, we compared the structural integrity and immunogenicity of two kinds of VLPs derived from HPV type 16 (HPV16 VLPs): one VLP was purified by heparin chromatography (hHPV16 VLP) and the other by cation-exchange chromatography (cHPV16 VLP). The reactivity of anti-HPV16 neutralizing monoclonal antibodies (H16.V5 and H16.E70) towards hHPV16 VLP were significantly higher than the observed cHPV16 VLP reactivities, implying that hHPV16 VLP possesses a greater number of neutralizing epitopes and has a greater potential to elicit anti-HPV16 neutralizing antibodies. After the application of heparin chromatography, HPV16 VLP has a higher affinity for H16.V5 and H16.E70. This result indicates that heparin chromatography is valuable in selecting functional HPV16 VLPs. In regard to VLP immunogenicity, the anti-HPV16 L1 IgG and neutralizing antibody levels elicited by immunizations of mice with hHPV16 VLPs were higher than those elicited by cHPV16 VLP with and without adjuvant. Therefore, the ability of hHPV16 VLP to elicit humoral immune responses was superior to that of cHPV16 VLP. We conclude that the specific chromatographic technique employed for the purification of HPV16 VLPs is an important factor in determining the structural characteristics and immunogenicity of column-purified VLPs. Saccharomyces cerevisiae (S. cerevisiae) expression system, and the other is Cervarix™ (GlaxoSmithKline), which uses an insect cell expression system Of all the types of human papillomavirus (HPV), type 16 is considered to be the most significant, as it is responsible for approximately 50% of cervical cancers in vitro because the production and assembly of native HPV virions are strictly controlled by the cell cycle HPV VLPs have been used as surrogates of the native HPV virion in studies of HPV structure, infection mechanisms and epitope display, as the continuous production of native HPV virion is practically impossible et al. have suggested that the reactivity of anti-HPV31 L1 monoclonal antibody (Mab) towards HPV31 VLPs prior to and after heparin binding are different Numerous viruses, including HPV, undergo conformational changes as they interact with cell surface receptors S. cerevisiaeIn the manufacture of recombinant HPV VLP, the interaction between the VLP and resin-bound ligand during purification has the potential to affect the structure and immunogenicity of the resulting VLP. However, the effect of the resin-bound ligand used in manufacturing HPV VLPs has not been studied until now due to the complexity of the purification process. Previous methods developed for purifying HPV VLPs are not only inefficient but also inconvenient. They require several chromatography steps or ultracentrifugation onto a sucrose cushion followed by size-exclusion chromatography In this study, we compared the structural integrity and immunogenicity of HPV16 VLPs purified by heparin chromatography (hHPV16 VLPs) and cation-exchange chromatography (cHPV16 VLPs) . Our resth International Papillomavirus Conference, C.B. Buck also suggested that treatment with a low concentration of ammonium sulfate (25 mM) in neutral pH increases the extent of disulfide bonding S. cerevisiae use successive ultracentrifugation with a sucrose cushion, size-exclusion chromatography and ion-exchange chromatography or microfiltration We prepared four types of HPV16 VLPs to investigate the effect of the purification method on VLP conformation, and we introduced an ammonium sulfate precipitation step to prepare hHPV16 VLP and cHPV16 VLP , which iThe HPV16 VLP binding residues for H16.V5 and H16.E70 antibodies are known to be critical for eliciting anti-HPV16 neutralizing antibodies To confirm the purity of L1 protein of each VLP preparation, SDS-PAGE was performed . There wTo investigate the effect of the heparin chromatography on the conformation of HPV16 VLPs, we compared the Mab reactivity of scHPV16 VLP before and after heparin chromatography . The L1 To further investigate the structural integrity of the hHPV16 VLP and cHPV16 VLP, we used density gradient ultracentrifugation and transmission electron microscopy (TEM) analysis, both of which have been previously used to analyze capsid structure We further investigated the particle size distributions of two kinds of HPV16 VLPs using dynamic light scattering (DLS). As shown in in vitro antigenicity in vitro antigenicity of the cHPV16 VLP was confirmed to be lower than that of the hHPV16 VLP  contents of hHPV16 and cHPV16 VLP prior to immunization. The hHPV16 and cHPV16 VLP concentrations were 0.49 and 0.32 EU per 1000 ng L1 protein, respectively . It has We investigated the immunogenicity of two kinds of HPV16 VLPs through three separate protocols referred to as protocol-1, 2 and 3 . In prot+ cells was scored. As shown in + T cells in the hHPV16 VLP-immunization group was significantly higher than the response observed in the cHPV16 VLP-immunization group. These results indicate that immunogenicity of hHPV16 VLP in eliciting humoral and cellular immune responses is superior to that of cHPV16 VLP.CD3 is a well-characterized marker of T lymphocytes In this study, we compared the structural integrity and immunogenicity of hHPV16 VLP and cHPV16 VLP. Our results indicate that the use of heparin chromatography is more advantageous in obtaining highly immunogenic HPV16 VLP when compared to cation-exchange chromatography. The epitopes of H16.V5 and H16.E70 are critical for inducing anti-HPV16 neutralizing antibodies In this study, we separated two forms of hHPV16 VLP by analytical ultracentrifugation: a fully assembled and a dissociated form . A TEM aPreviously, we confirmed that the final yields of L1 protein resulting from heparin and cation-exchange chromatography are similar The shielding of key epitopes within the three-dimensional structure of the native envelope trimer of human immunodeficiency virus (HIV) is a major problem in neutralizing vaccine development because the conformational masking offers the virus an opportunity to evade host defenses Previously, it has been suggested that the upstream and downstream processing of virus-like particles during production may affect to their structure and immunogenicity Five- to six-week-old female BALB/c mice were purchased from Orient Bio  and acclimatized for 1 week prior to immunization. All animal experiments were performed in accordance with the National Research Council's Guidelines for the Care and Use of Laboratory Animals and with the Chung-Ang University Guidelines for Animal Experiments. In addition, all experiments were approved by the University Committee for animal experiments .S. cerevisiaeHPV16 VLPs were expressed in HPV16 VLP-producing Protein concentration of each VLP preparation was determined with a Bio-Rad Bradford protein assay kit  with bovine serum albumin  as standard. The purities of L1 proteins were further determined by SDS-PAGE and Western blot analysis described below.ELISAs were performed as previously described SDS–PAGE was performed using the Laemmli method Optiprep density gradient analysis was performed as previously described For TEM analysis, purified hHPV16 VLP and cHPV16 VLP were prepared and analyzed as previously described The size distribution of each HPV16 VLP was analyzed using a DLS-700 system  at 23°C. Each HPV16 VLP was prepared in 25 mM MOPS containing 75 mM NaCl pH 7.0 . The preparations were measured using duplicate assays, and the representative result of each VLP preparation was presented.Three kinds of immunization protocols were used to investigating the immunogenicity of the HPV VLPs. The protocols are briefly summarized in 6 cells/well) for 5 day at 37°C with 10 µg/ml of HPV16 VLP (hHPV16 VLP and cHPV16 VLP mixed in a 1∶1 ratio). The cells were harvested, washed with staining buffer (1% FBS in PBS) and stained with anti-mouse CD3 peridinin chlorophyll protein-eFluor® 710 (PerCP-eFluor®) antibody . The cells were washed twice with the staining buffer, and the stained cells were examining with a FACSAria flow cytometer . Thirty thousand events were acquired with the live lymphocyte gates during the scoring of proliferated CD3+ CFSElow cells.Splenocytes from the PBS-, hHPV16 VLP- and cHPV16 VLP-immunized mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) using a CellTrace™ CFSE cell proliferation kit . The CFSE-labeled splenocytes were cultured in 96-well flat-bottom cell culture plates  activity of the culture supernatants was measured by a colorimetric SEAP assay. The neutralization activities of the mice sera obtained from immunization protocol-1 were determined as described previously with slight modification To investigate the neutralizing ability of anti-HPV16 L1 mouse sera, a pseudovirus (PsV)-based neutralization assay was performed as previously described The statistical significance of differences between groups was determined using two-tailed Student's t-tests. P<0.05 indicated a statistically significant difference.Figure S1Effect of ammonium sulfate precipitation on the extension of intermolecular disulfide bonding of HPV16 VLP. To compare the degrees of intermolecular disulfide bonding between HPV16 VLPs before and after ammonium sulfate precipitation, non-reducing Western blotting was performed as described at the website of National Cancer Institute (NCI) (http://home.ccr.cancer.gov/Lco/ImprovedMaturation.htm). To detect HPV16 L1 protein, rabbit anti-HPV16 L1 polyclonal antibody and HRP-conjugated goat anti-rabbit IgG was used.(TIF)Click here for additional data file.Figure S2Measurements of endotoxin levels contained in hHPV16 VLP and cHPV16 VLP. To measure the endotoxin levels, RAW264.7 (2×104 cells / well) cells were seeded in a 96-well cell culture plate 24 hours prior to stimulations. To block Toll-like receptor 4 on RAW264.7 cells, the cells were treated with 50 µg/ml of polymyxin B  for 30 min at 37°C prior to LPS and HPV VLPs treatments. The cells were treated with LPS, hHPV16 VLP and cHPV16 VLP at concentrations of 10 ng/ml, 10 µg/ml and 10 µg/ml, respectively. Four hours after the treatments, the levels of TNF-α in the culture supernatants was measured using an ELISA kit according to manufacturer's instructions . Values are presented as the means ± SD of duplicate assays.(TIF)Click here for additional data file.Figure S3Neutralization assay results. The mice were immunized three times with 1000 ng of hHPV16 VLP or cHPV16 VLP, in combination with aluminum hydroxide Click here for additional data file.Figure S4Anti-HPV16 L1 IgG titers and neutralization activities of mice sera following immunizations with schHPV16 and scHPV16 VLP. Mice were immunized subcutaneously three times with 8 ng of schHPV16 VLP or scHPV16 VLP without adjuvant. Ten days after the last immunization, the sera were obtained and analyzed as described in the Materials and Methods to determine the anti-HPV16 L1 IgG titers and neutralization activities. The horizontal bars are the median values .(TIF)Click here for additional data file.Table S1Determinations of LPS levels in Mock and HPV16 VLPs. Mock samples were prepared form the cell lysate of parent cell of HPV16 L1-producing S. cerevisiae. The concentrations of hHPV16 VLP and cHPV16 VLP were determined by SDS-PAGE and Western blotting prior to the assay. The LPS level of each sample was determined using the limulus amoebocyte lysate (LAL) based colorimetric system  according to the manufacture's instruction.(DOCX)Click here for additional data file.


A literature review was performed to assess whether massage benefits patients postoperatively following coronary bypass grafts (CABG) and or valve replacement/repair. A case study on a patient who had suffered a hypoxic brain post cardiac arrest was conducted.A review on MEDLINE and Cochrane using search terms massage, cardiac and ICU identified nine research papers on the benefits of massage postoperatively for the aforementioned patient group. Other papers were listed but unrelated to cardiac surgery. None of the nine papers identified for this review were ICU specific in the title but the ICU was mentioned in the main text body. For the purpose of this review the selected papers are researching the effects of massage on physiological parameters, anxiety, pain, calm and perceived stress indicators in the CABG and/or valve repair/replacement. Out of these nine papers, one is British (2002). Five are American (2006 to 2012), two are Brazilian (2010) and one is an Indian paper (2010). All papers are randomised control trials (RCTs). Papers written prior to 1999 were excluded from this literature review.P >0.05). Pain, anxiety, rest and a calm score perform better with P >4.71 overall. Another American study showed that the length of stay is reduced if a patient receives healing touch postoperatively. However, in one RCT pain and anxiety increases and in some case SpO2 decreases. A case study was chosen by the author and the results support the benefits of massage with the cardiac patient group.Research from 1999 states there are methodological errors in prior papers and very few large-scale studies prior to this date, destabilising the validity and reliability of research from the papers written before 1999. The later research suggests that any change in the measured physiological parameters of blood pressure, heart rate and respiratory rate are insignificant (There appears to be some benefits but larger-scale studies are required within this and other ICUs in Britain. The author will be inquiring at her workplace whether further studies can be performed and whether she can initiate the research. She will identify further case studies and trial massage on select patients. The author recommends training for nurses interested in massage therapy so a bigger caseload can be identified.


An intercellular communication network that controls intestinal homeostasis in animals treated acutely with the pro-inflammatory cytokine TNF-α is uncovered by multi-scale systems analysis. Intestinal epithelial cells exist within a complex environment that affects how they interpret and respond to stimuli. We have applied a multi-scale in vivo systems approach to understand how intestinal immune cells communicate with epithelial cells to regulate responses to inflammatory signals. Multivariate modeling analysis of a large dataset composed of phospho-signals, cytokines, and immune cell populations within the intestine revealed an intimate relationship between immune cells and the epithelial response to TNF-α. Ablation of lymphocytes in the intestine prompted a decrease in the expression of MCP-1, which in turn increased the steady state number of intestinal plasmacytoid dendritic cells (pDCs). This change in the immune compartment affected the intestinal cytokine milieu and subsequent epithelial cell signaling network, with cells becoming hypersensitive to TNF-α-induced apoptosis in a way that could be predicted by mathematical modeling. In summary, we have uncovered a novel cellular network that regulates the response of intestinal epithelial cells to inflammatory stimuli in an in vivo setting. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that operates through a pleiotropic signaling network to influence a diverse array of cellular behaviors, including the induction of apoptosis, survival, proliferation, cell cycle arrest, or differentiation Dysregulation of TNF-α signaling contributes to several distinct chronic inflammatory conditions in the gut, such as inflammatory bowel disease, celiac disease, and peptic ulcer disease Under both homeostatic and pathologic conditions, the activity of the innate and adaptive components of the intestine can strongly influence the behavior of the intestinal epithelium and vice versa. For example, T cell receptor (TCR)-γδ+ lymphocytes regulate the proliferation and differentiation of epithelial cells through extracellular factors such as keratinocyte growth factor-1 Applying a systems biology perspective to explore communication mechanisms within the complex intestinal microenvironment will undoubtedly lead to novel insights into TNF-α biology, inflammatory diseases, and, more generally, heterologous cell interactions that occur as part of biological phenomena in complex tissues. Previously, we have demonstrated that the complex signaling effects elicited by TNF-α in the intestinal epithelium can be mathematically integrated into a signaling network state to predict epithelial cell behavior We have previously used systems biology to characterize how cell intrinsic signaling regulates the response of the intestinal epithelium to TNF-α Mutation of Rag1 affects the development of both B and T cells. To determine whether loss of either of these types of lymphocytes is specifically responsible for the enhanced apoptotic response of the intestinal epithelium, we acutely treated animals that lacked either T cells or B cells with TNF-α. Interestingly, T cell receptor beta/delta (Tcrb/Tcrd) double null animals, which lack T cells, and Immunoglobulin heavy chain mu (Ighm) null animals, which lack B cells, both exhibited an enhanced apoptotic response to TNF-α relative to WT controls , suggestWhile WT and Rag1 null mice differ in the presence of resident T cells within the intestinal lamina propria, we noted that acute treatment of WT mice with TNF-α also led to recruitment of additional T cells to the intestine . To deteThe commensal flora constitutes another feature of the intestinal microenvironment that plays an important role in regulating epithelial homeostasis. As such, we sought to determine whether acute elimination of the endogenous microbiota would affect the response of the intestinal epithelium to TNF-α. Treatment of WT mice with broad-spectrum antibiotics eliminated the commensal flora population that could be cultured from fecal pellets , but didTo identify the mechanisms by which immune cells modulate the magnitude of TNF-α-induced apoptosis in epithelial cells, we first asked whether network-level signaling responses to TNF-α varied in the different genetic backgrounds. To sample the network state, we collected a time course dataset across central signaling pathways  from intThe observation that lymphocyte perturbation resulted in altered epithelial signaling and TNF-α response prompted us to investigate whether immune cells communicate with epithelial cells through cytokine networks. To examine whether cytokine expression is disrupted at a network level, we measured a panel of inflammatory cytokines in the intestine after TNF-α stimulation  . The cytTo test directly whether it singularly regulates the epithelial apoptotic response to TNF-α, we functionally neutralized MCP-1 in WT animals with anti-MCP-1 antibody prior to TNF-α administration. Next, we collected a signaling dataset representing a time course of TNF-α stimulation (5 µg) coincident with MCP-1 neutralization . Application of this signaling dataset to the original three-dimensional PLSDA model of signaling  demonstrMCP-1, like TNF-α, is a pleiotropic cytokine that can affect a diverse array of cell types in a variety of ways. As such, it was unclear exactly how MCP-1 was functioning within the intestine to regulate the epithelial response to TNF-α. We set out to test multiple hypotheses relating to the relationship between MCP-1 and TNF-α-induced epithelial apoptosis. Based on an abundance of published work, our initial hypothesis was that lymphocytes secrete MCP-1 to recruit macrophages to the lamina propria and that this protects against TNF-α-induced epithelial apoptosis. Based on this hypothesis, we predicted that MCP-1 expression in the intestine would be detectable in intravillus lymphocytes. Surprisingly, we found that immunohistochemistry (IHC) for MCP-1 detected expression primarily in secretory intestinal epithelial cells, namely in goblet cells in the villi and Paneth cells at the base of the crypt . This obWhile these IHC and RT-PCR studies support the hypothesis that MCP-1 detected in the intestine is primarily derived from epithelial cells, a more direct way to link MCP-1 to secretory cells is to modify the number of those cells within the epithelium. Treatment of mice with dibenzazepine (DBZ), an inhibitor of γ-secretase, has been shown to increase the number of secretory epithelial cells in the intestinal epithelium While our data suggested that MCP-1 comes from epithelial cells, the mechanism of action of MCP-1 remained a question. Published studies have shown that this cytokine can act directly on epithelial cells, which also express the MCP-1 receptor, CCR2 MCP-1 is mainly known as a chemo-attractant that recruits monocytes, macrophages, dendritic cells, and T cells to sites of inflammation. We next tested the hypothesis that epithelial cells secrete MCP-1 to recruit a protective immune cell type (possibly macrophages) to the intestine. Nevertheless, by performing FACS analysis on immune cell populations from the gut, we found no evidence that Rag1 null animals or WT animals treated with anti-MCP-1 had fewer intestinal macrophages than control WT animals .All of our initial hypotheses relating to the function of MCP-1 were developed under the assumption that WT animals are actively resistant to TNF-α-induced apoptosis. Because our experimental results failed to support these hypotheses, we explored the possibility that Rag1 null mice are actively sensitive to TNF-α-induced apoptosis. We tested the hypothesis that the low levels of MCP-1 in Rag1 null intestines led to an increased number of deleterious immune cells relative to WT animals. Indeed, we identified three distinct immune populations that were over-represented in Rag1 null intestines: CD11b- macrophages, natural killer (NK) cells, and plasmacytoid dendritic cells (pDCs) . To deteOur data implicate pDCs in actively sensitizing the intestinal epithelium to TNF-α-induced apoptosis. We next investigated whether the increased or decreased presence of pDCs is sensed through the epithelial signaling network to change the magnitude of TNF-α-induced apoptosis. Our original modeling analysis comparing WT and Rag1 null animals identified four early signals (0.5 h post-TNF-α) that combinatorially contribute to the magnitude of TNF-α-induced apoptosis: p-Rsk, p-Mek, p-IκBα, and p-Akt . We measThe major question that remained was: How does the change in the number of pDCs affect the epithelial signaling network? We posited that pDCs might communicate with epithelial cells, directly or indirectly, using cytokines. Based on our original PLSDA model, we identified MCP-1 as being protective because it correlated with late/low apoptosis . We specTo test whether IFN-γ plays a mechanistic role in the potentiation of TNF-α-induced apoptosis by pDCs, we first treated WT animals with exogenous IFN-γ concurrent with TNF-α. We found that a single injection of IFN-γ was sufficient to enhance TNF-α-induced apoptosis in the WT intestinal epithelium . ConsistTissue homeostasis is maintained by a complex biological network consisting of cell intrinsic and extrinsic factors. Data-driven computational modeling provides a powerful framework for understanding how complex networks are established and maintained in a heterogeneous system MCP-1 belongs to the CC chemokine group, whose members are characterized by two adjacent cysteine residues in their N-termini. MCP-1 is known to signal through the G protein coupled receptor CCR2 to induce a variety of chemo-attractant functions. CCR2 expression was initially established in monocyte and macrophage cell lines, and hence, most studies of MCP-1 function have focused on mononuclear cell recruitment. Nevertheless, CCR2 is also expressed by other immune and non-immune cell types Our major goal in this study was to understand how heterologous interactions within the intestinal microenvironment play a role in regulating the epithelial response to TNF-α. Our initial hypothesis connecting MCP-1 to the epithelial TNF-α response was that it is expressed by lymphocytes, and that its presence affected epithelial sensitivity to TNF-α. Our results painted a more complex picture, however. For example, we found that MCP-1 was produced primarily by secretory epithelial cells . It is nTwo pieces of evidence suggested that MCP-1 indirectly influences epithelial signaling by playing a proximal role in regulating the identity of the immune cell complexion of the intestinal lamina propria. First, the lack of lymphocytes in Rag1 null mice was associated with an enhanced presence of several other immune cell types , which cUnlike conventional dendritic cells, whose main function is to sense and present antigen, pDCs are a special class of dendritic cells that secrete cytokines. Although evidence supports the existence of a common progenitor for both types of dendritic cells, pDCs exhibit morphologies, expression profiles, and homing properties of secretory lymphocytes In this study, we have uncovered a novel interaction network within the intestinal environment that controls the epithelial response to TNF-α . In thisAll of the experimental animals used in this study were 8-wk-old mice on a C57BL/6J genetic background. For TNF-α time courses, mice were anesthetized with Avertin , and then administered 5 or 10 µg of recombinant mouse TNF-α (Abazyme) in phosphate-buffered saline  by retro-orbital injection. Mice from the same set were treated at the same time, but were sacrificed at various time points. Control animals were anesthetized, injected with PBS (50 µl) by retro-orbital injection, and then sacrificed 2 h later.For sample collection, animals were sacrificed and the small intestine was removed and washed in PBS supplemented with protease inhibitor. Duodenal and ileal tissues were lysed and homogenized immediately in Bio-Plex lysis buffer (Bio-Rad), or else fixed with formalin for subsequent immunohistochemical analysis. Duodenal samples were obtained from the 1 cm of small intestine immediately adjacent to the stomach. Ileal samples were obtained from the 3 cm of small intestine immediately adjacent to the cecum. All animal work performed in this study was undertaken according to approved protocols and animal welfare regulations as put forth by the Subcommittee on Research Animal Care (SRAC) at the Massachusetts General Hospital.For cytokine neutralization experiments, anti-MCP-1 antibody , anti-IFN-γ , or anti-MAdCAM1  was administered by retro-orbital injection 2 h prior to the TNF-α time course. Exogenous MCP-1  or IFN-γ  was administered by retro-orbital injection concurrent with TNF-α treatment. To increase the number of secretory epithelial cells, animals were treated daily for 5 d with DBZ  and then treated with TNF-α. DBZ was prepared as described previously To perturb the microbiota, mice were given drinking water supplemented with broad-spectrum antibiotics  for 10 d prior to the TNF-α time course. To confirm the effect of the antibiotic, one fecal pellet was freshly collected from each animal, homogenized in Ringer's solution (Fisher), and plated on Rose/MacConkey agar plates (Hardy Diagnostics) immediately in a 1∶5 dilution.To ablate immune cells, mice were administered anti-NK1.1 , anti-PDCA-1 , or clodronate liposomes  for 48 h prior to the TNF-α time course.Standard immunohistochemistry procedures were followed. Slides were stained with anti-cleaved caspase 3  with EDTA antigen retrieval. The immunohistochemistry procedure was altered for intracellular cytokine staining for MCP-1 such that all solutions were supplemented with 0.01% saponin (Sigma-Aldrich). Slides were stained with anti-MCP-1  with citrate antigen retrieval. Apoptosis was quantified by quantitative Western blotting analysis of cleaved caspase 3  in tissue lysates. Western blots were quantified on a LI-COR Odyssey infrared imaging system. Each measurement was normalized to CC3 values from a control TNF-α time course (wild-type mice) loaded on the same gel.Quantitative measurements of protein phosphorylation were collected from lysed tissue samples using Bio-Plex (Bio-Rad), a multiplexed, bead-based system for analyzing total and phospho-protein levels in suspension PLSDA modeling was done as described previously After dissection, the 3 cm of duodenum of each mouse was collected and washed in PBS with 0.5% BSA and 2 mM EDTA. The intestine was weighed, homogenized mechanically, and incubated in 0.2 mg/mL collagenase in DMEM for 1 h at 37 degrees to separate the epithelial cells, prior to single cell suspension preparation with a cell strainer.For FACS analysis of immune cells at a systems level, the single cell suspensions were divided into three aliquots for staining. The first aliquot was stained with fluorescently conjugated antibodies for CD90.2, NK-1.1, CD11b, F4/80, Ly-6c, CD103, B220, Ly-6G, CD49b, CD11c, and TER-119 for 1 h. The second aliquot was stained for B220, CD11c, NK-1.1, CD11b, CD49b, and PDCA1 for 1 h. The third aliquot was fixed with Cytofix/Cytoperm solution (BD Biosciences) and stained for CD3, CD8, and CD4 for 1 h, and washed and stained for Fox3p overnight. All antibodies were obtained from BD Biosciences. Stained samples were analyzed with a BD LSRII flow cytometer.For experiments in which RNA was isolated from sorted cells, the preparation of the single cell suspension was altered such that the homogenized sample was incubated in 1 mg/mL collagenase in DMEM for 30 min at 37 degrees. For sorting epithelial cells, the sample was stained with an antibody for cytokeratin 20 (Acris). T cells were sorted with CD8 or CD4, B cells with CD19 or B220, and macrophages and dendritic cells with CD11C or F4/80. Sorting was performed with a FACSAria III (BD) directly into RLT buffer with β-mercaptoethanol. RNA was isolated from each population using an RNAeasy microkit (Qiagen).Real-time RT-PCR for MCP-1 was performed on the RNA samples using standard procedures on an ABI 7900HT (Applied Biosystems). The RNA expression level of each sample was compared to a standard sample on the plate, and normalized using TBP, HPRT, and GAPDH house-keeping genes to ensure the reproducibility of the results across different cell types, with the formula:Figure S1Effects of lymphocyte perturbation on acute TNF-α-induced apoptosis. (A) An example of a Western blot for caspase 3 cleavage and β-Tubulin in the duodena of Rag1 null mice (left) and wild-type mice (right) as a time course following TNF-α (5 µg) stimulation. (B) Caspase 3 cleavage induced 2 h post-TNF-α (5 µg) administration within the duodena of wild-type control mice (cyan), Rag1 null mice (red), TCRβ/δ (Tcrb/Tcrd) null mice (dark red), and immunoglobulin heavy chain mu (Ighm) null mice (brown). Data are normalized to wild-type control mice and error bars represent SEM for three mice. (C) Quantification of CD8+ T cells in the duodena of wild-type mice by FACS in wild-type controls (clear) or mice pretreated with anti-MadCAM1 (2 mg/kg) for 2 h (shaded). Treatment with anti-MadCAM1 prevents TNF-α-induced recruitment of T cells to the intestine. Error bars represent the SEM for three mice. (D) Caspase 3 cleavage within the duodena of wild-type control mice (white), Rag1 null mice (green), and wild-type mice pretreated with anti-MadCAM1 (shaded). Data are normalized to wild-type control mice and error bars represent the SEM for three mice.(PDF)Click here for additional data file.Figure S2Acute TNF-α-induced apoptosis in the intestine is not affected by modulation of the microbiota. (A) Fecal flora of wild-type mice on normal drinking water or on drinking water supplemented with broad spectrum antibiotics  for 10 d. Fecal flora were plated on MacConkey agar (pink) and Rose agar (red). (B) Time course of caspase 3 cleavage induced by TNF-α as determined by quantitative Western blotting in the duodena of wild-type mice after antibiotic treatment above (broken line) or normal drinking water (solid line). Data are normalized to the peak signal of the wild-type control. Error bars represent the SEM for three mice.(PDF)Click here for additional data file.Figure S3Signaling network sampled in our analysis. Here, we present a manually curated network of inflammatory signaling according to canonical pathways. We depict components and connections that are included in our analysis . Note that additional connections between the nodes likely exist, and note the presence or absence of particular connections that are cell context dependent.(PDF)Click here for additional data file.Figure S4Time courses of protein phosphorylation signals activated following exposure to TNF-α in vivo. Data points are the means of the median fluorescent intensities resulting from the phospho-protein assays, normalized to a loading control dataset on each plate. The left side of each graph set contains the duodenal data, while the right side contains the ileal data. The color scheme is based on genotype , while the solid and broken lines represent low (5 µg) and high (10 µg) doses of TNF-α, respectively. Error bars represent the SEM for three mice.(PDF)Click here for additional data file.Figure S5y-axis quantifies the positive or negative contribution of a particular signal to LV3. Late signals correlate to an early apoptotic phenotype, consistent with our previous findings.Modeling signaling network shifts in modulating TNF-α-induced apoptosis. (A) A 3-D PLSDA model describes dosage, regional, and genotypic effects on TNF-α-induced apoptosis. Error of classification into class 1 , class 2 , and class 3  of calibration data, using models with increasing numbers of latent variables. The average error across all classes (black) includes the first three classes and the three ileum classes (no apoptosis). Classification error is defined as the probability of misclassifying a sample given the sample distribution and a model. (B) Error of classification into classes 1, 2, and 3 of data kept out of the cross-validation procedure, using models built with the rest of the data, with increasing numbers of latent variables. The average error across all classes (black) includes the first three classes and the three ileum classes (no apoptosis). Note that the average error is greater than the error for individual classes due to the fact that the ileum classes (no apoptosis) are very similar at the signaling and phenotypic level and cannot be distinguished from one another. (C) Receiver operating characteristic (ROC) curves depicting specificity  versus sensitivity (true positive rate) as the numerical threshold for classification is changed. The quality of a classification model can be evaluated by the degree by which the curves deviate from the center diagonal, with more deviation describing a better classification. ROC curves for the six classes using a PLSDA with three LVs. The red circle represents the discriminant threshold selected for a model. (D) Loadings on LV3, the latent variable for describing the timing of apoptosis. The (PDF)Click here for additional data file.Figure S6Time courses of cytokine protein expression after administration of TNF-α in vivo. Data points are the means of the median fluorescent intensities resulting from the cytokine assays performed on duodenal lysates, normalized to a loading control dataset on each plate. The color scheme is based on genotype , while the solid and broken lines represent low (5 µg) and high (10 µg) doses of TNF-α, respectively. Error bars represent the SEM for three mice.(PDF)Click here for additional data file.Figure S7y-axis is the numerical result calculated with the PLSDA function of being classified into the “late/low apoptosis”  phenotypic class. The red broken line is the threshold that defines classification. Note that the non-specific antibody control classified into class 1. (B) PLSDA model-based prediction of the classification into the “early/low apoptosis”  phenotypic class with signaling data from duodenal samples from animals treated with TNF-a after pre-treatment with anti-MCP-1 (magenta) or non-specific antibody (cyan). (C) PLSDA model-based prediction of the classification into the “no apoptosis”  phenotypic class with signaling data from duodenal samples from animals treated with TNF-α after pre-treatment with anti-MCP-1 (magenta) or non-specific antibody (cyan).Statistical modeling of TNF-α-induced apoptosis in the context of MCP-1 neutralization. (A) PLSDA model-based prediction of the apoptotic phenotype in the duodenum based on phospho-protein signaling data from samples derived from animals treated with TNF-α after pretreatment with anti-MCP-1 (magenta) or non-specific antibody (cyan). The (PDF)Click here for additional data file.Figure S8Evaluation of exogenous MCP-1 activity and confirmation of immune cell ablation. (A) Evaluation of MCP-1 restoration in Rag1 null animals. A single injection of 2.5 µg of recombinant murine MCP-1 restores of intestinal level to that of wild-type mice. (B) Effect of exogenous MCP-1 on TNF-α-induced apoptosis in Rag1 null animals. Exogenous MCP-1 fails to protect. (C) FACS plots demonstrating ablation of pDCs in Rag1 null animals by treating with anti-PDCA1 (20 mg/kg) for 2 d. (D) Validation of cell depletion experiments. Natural killer cells were depleted by pretreating mice with anti-NK1.1 (12 mg/kg) for 2 d. pDCs were depleted by treating mice with anti-PDCA1 (20 mg/kg) for 2 d. Macrophages were depleted by treating animals with clodronate liposomes (250 µl) for 2 d. In all experiments, error bars represent the SEM for three mice.(PDF)Click here for additional data file.Figure S9Early TNF-α-induced signaling is modulated by plasmacytoid dendritic cells. Early signaling (0.5 h) induced by TNF-α in the duodenum as measured by Bio-Plex phospho-protein signal assays for wild-type mice (cyan), wild-type mice pretreated with anti-MCP-1 for 2 h (magenta), Rag1 null mice (red), and Rag1 null mice pretreated with anti-PDCA-1 for 2 d (yellow). Data are normalized to a loading control dataset on each plate. Error bars represent the SEM for three mice.(PDF)Click here for additional data file.Figure S10Early cytokine induction by TNF-α is modulated by plasmacytoid dendritic cells. Early cytokine protein levels (0.5 h) induced by TNF-α (5 µg) in the duodenum as measured by Bio-Plex cytokine assays for wild-type mice (cyan), wild-type mice pretreated with anti-MCP-1 for 2 h (magenta), Rag1 null mice (red), and Rag1 null mice pretreated with anti-PDCA-1 for 2 d (yellow). Data are normalized to a loading control dataset on each plate. Error bars represent the SEM for three mice.(PDF)Click here for additional data file.


In addition, we tested its effects under normal conditions as well as under inflammatory conditions induced by tumour necrosis factor-α (TNF-α) administered either by cotreatment, pretreatment, or posttreatment. Immunocytochemistry, MTT, wound-healing, and flow cytometry assays were performed. The resveratrol treatment significantly enhanced BAEC proliferation and attenuated TNF-α-induced impairment of proliferation at the optimal doses of 1–50 µM. Resveratrol at a high dose (100 μM) and TNF-α impaired BAEC migration, while low doses of resveratrol (1–50 μM) attenuated TNF-α-induced impairment of BAEC migration. Moreover, resveratrol inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. Taken together, our results suggest that the resveratrol protects BAECs after inflammatory stimulation as well as ameliorates inflammatory effects at low concentrations. Consequently, resveratrol should be considered as a candidate drug for the prevention and treatment of inflammatory vascular diseases.Endothelial injury induced by inflammatory factors plays a critical role in the pathogenesis of cardiovascular disease. Endothelial cell (EC) apoptosis, proliferation, migration, and cellular adhesion molecule (CAM) expression contribute to the development of atherosclerosis. We investigated the effects of resveratrol (0.1–100  Nearly confluent cell monolayers were “wounded” in a cross-shaped pattern with a sterile 200 The expression of proportions of cell specific cellular adhesion molecules was quantified by standard immunofluorescence cell sorting techniques , 20. Thet-test. P < 0.05 was considered statistically significant. SPSS version 17.0  was used for statistical analyses. All quantitative variables were expressed as means ± SEM from at least three separate experiments. Comparisons between groups were made using one-way ANOVA followed by Dunnett's post hoc test. To evaluate the difference of two groups, we used two-tailed Student's Primary baboon arterial had a typical cobblestone shape Figures  and tookμM) on the growth of primary BAECs (α when administered as cotreatment (μM) highly significantly enhanced EC proliferation after 24 h incubation in the optimal range of 10–50 μM   for 24 h significantly decreased (P < 0.01) BAEC proliferation by comparison with controls . No effects were detected at 1 or 100 μM, suggesting a dose-dependent effect of resveratrol on baboon ECs subjected to inflammatory conditions. Pretreatment with resveratrol (0.1–100 μM) (α for 4 h (P < 0.05), especially at doses of 1–50 μM (P < 0.01). Additionally, posttreatment with resveratrol (0.1–100 μM) . Together, the data indicated that the resveratrol could enhance primary baboon EC proliferation and could prevent cytotoxicity induced by TNF-α.We first tested the effect of various concentrations of resveratrol  . TNF-α tcontrols . However–100 μM)  for 24 h–100 μM)  for 24 hα treatment. As shown in μM; P < 0.05) increased wounded BAEC migration by comparison with controls, but, at a high concentration , it decreased the BAEC migration during 15 h. No effect was observed at concentrations of 0.1, 1, and 50 μM. As shown in α (P < 0.01) dramatically impaired BAEC migration at 15 h compared with controls, while resveratrol (1–50 μM) in the presence of TNF-α significantly increased the BAEC migration rate by comparison with TNF-α treatment alone (P < 0.01). No protective effect of 100 μM resveratrol was observed.We investigated whether resveratrol could affect BAEC migration with or without TNF-μM) and TNF-α. E-selectin, ICAM-1, and VCAM-1 expression on BAECs were significantly increased by TNF-α (10 ng/mL) stimulation at 4 h by comparison with controls  and TNF-α (10 ng/mL) by comparison with TNF-α treatment alone  for 24 h (α stimulation (P < 0.01). BAECs that were treated with TNF-α for 4 h and then incubated with resveratrol  for 24 h (μM) and VCAM-1  expression compared with controls .To investigate whether resveratrol affected the level of inducible cell adhesion molecule expression of E-selectin (CD62E), ICAM-1 (CD54), and VCAM-1 (CD106), BAECs were analysed by flow cytometry. BAECs were cotreated Figures , pretreaα at 4 h , but thefor 24 h  inhibitefor 24 h  exhibiteRecently, resveratrol has gained considerable attention because of its anticancer , anti-inin vitro primate model. The advantages of this model are as follows. First, it is easier to obtain primary macrovascular ECs from baboons in comparison with humans. Second, baboons exhibit a high degree of genetic, biochemical, physiological, and anatomical similarity to humans, and more closely resemble humans in comparison with other species in the pathology of vascular diseases [α compared with venous ECs, and they may play a key role in macrovascular disease under adverse conditions [α and thus provide an effective model for translating therapies to human clinical trials [in vivo. We have established a protocol for primary BAEC culture and previously performed fundamental investigations on their morphology and functions [in vitro studies as a prelude to in vivo studies.In this study, we investigated the effect of resveratrol on primary baboon femoral artery as an diseases . Third, nditions . Fourth,l trials . BAECs cunctions , 26. Addunctions . Thus, wμM, and it attenuated cellular impairment caused by TNF-α treatment when administered to the cells as cotreatment, pretreatment, or post-treatment. Data concerning the influence of resveratrol on EC growth have not been completely consistent. Previous studies demonstrated that resveratrol induces increasing levels of apoptosis in with increasing concentration (25–100 μM) and induces apoptosis at 100 μM by cleavage of caspase 3 [μM) had no effect. Csiszar et al. demonstrated that 10 μM resveratrol prevented EC apoptosis induced by cigarette smoke extract [μM attenuated apoptosis mediated by oxidative stress  [Proliferation and migration of ECs may play a crucial role in vascular self-repair in normal physiological as well as pathological conditions. EC monolayer integrity is maintained via proliferation and migration of neighboring cells. We first investigated different experimental settings to verify the effects of resveratrol on BAEC proliferation under normal and inflammatory conditions. Resveratrol highly significantly enhanced BAEC proliferation at the optimal concentrations of 10–100 aspase 3 , 27, whi extract . Our resnitrite) , 30. Witin vitro and in vivo findings from experiments of animals and humans is thought to depend largely on parent resveratrol plasma concentration and bioavailability. The greatest plasma concentrations of resveratrol have been reported to be 2.36 μM by Boocock et al. [μM by Brown et al. [in vitro (~32 nM–100 μM) in research with human cells. However, tissue resveratrol levels may be higher than what is suggested based on plasma levels because resveratrol is lipophilic. Moreover, resveratrol has shown efficacy at very low concentrations [in vitro effects of resveratrol on inflammation reflect in vivo effects that can be achieved by oral ingestion of resveratrol and if oral ingestion is cardioprotective. The translation of k et al.  and 4.2 n et al.  when 5 gtrations  in studitrations . Our preα (0.1–50 μM). We found that a low concentration of resveratrol had no inhibitory effect on BAEC migration. However, a high concentration (100 μM) of resveratrol decreased BAEC migration. In et al. [μM in human and bovine brain EC because of its antiangiogenesis properties. Our results also demonstrated that low doses of resveratrol attenuated the impairment of BAEC migration caused by inflammatory conditions, suggesting a mechanism for its anti-inflammatory properties. Cicha et al. [μmol/L in a Rho-associated-kinase- (ROCK-) dependent manner. Resveratrol promoted proliferation and migration of cerebral by activation of phosphoinositide 3 kinase (PI3-K)/Akt and mitogen-activated protein kinase (MAPK)/ERK signaling pathways [EC migration is an essential process for a variety of vascular functions such as tumour growth, vascular remodelling, and vascular wound healing. Our results indicated that resveratrol at a low concentration ameliorates impairment of BAEC migration induced by TNF-n et al.  showed ta et al.  observedpathways . Howeverpathways . We showα in BAECs. We demonstrated for the first time that resveratrol inhibits TNF-α-induced expression of ICAM-1 and VCAM-1 during long-term incubation after TNF-α stimulation, as well as by pretreatment and cotreatment. There was no inhibitory effect of resveratrol on CAM expression during short-term incubation, and this characteristic may contribute to its time-dependent inhibition of NF-κB [E-selectin, which may be restricted by its long-term incubation. Inflammation is defined in part by the upregulation of cell adhesion molecules on the surface of in response to cytokines. Adhesion molecule expression is the molecular basis of leukocyte-endothelium interactions and an important characteristic of inflammatory reactions, which are critical in atherogenesis . We inveof NF-κB . E-selecα-induced ICAM-1 and VCAM-1 expression, which may protect cells from TNF-α-induced cytotoxicity. However, the underlying molecular mechanisms by which resveratrol exerts its physiological effects require further investigation. Together, our findings highlight the power of resveratrol to protect primary ECs in culture and suggest that resveratrol may offer an alternative therapy for the prevention and treatment of cardiovascular disease. Preclinical trials with baboons are planned to evaluate the protective effects of resveratrol.Resveratrol decreased TNF-α. Resveratrol may provide a pharmacological approach for suppressing injury under inflammatory conditions and for reducing risk of cardiovascular disease and diabetes.In summary, our data suggest that resveratrol may protect baboon ECs from cytotoxicity induced by TNF-


To the Editor, Budd-Chiari syndrome (BCS) is an uncommon condition induced by thrombotic or non-thrombotic obstruction of hepatic venous outflow. BCS most often occurs in patients with underlying thrombotic diathesis, including such myeloproliferative disorders (MPDs) as polycythemia vera and paroxysmal nocturnal hemoglobinuria, and pregnancy, oral contraceptives, tumors, chronic inflammatory diseases, clotting disorders, and infections . Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine produced primarily by macrophages and T-cells that has a range of inflammatory and immunomodulatory activity . TNF-α iRecently, Fleischman et al. reported that Jak-2 V617F activity is positively correlated with TNF-α mRNA expression, suggesting that Jak2 V617F directly up regulates TNF-α mRNA in myeloproliferative neoplasm (MPN) patients . As suchDNA was obtained from peripheral blood samples of BCS patients (n = 84) and healthy controls (n = 101). DNA was isolated using a MagnaPure LC 2.0 automatic isolation system . The TNF-α -308 promoter region was then amplified using specific primers. A 194-bp product was amplified using the primers, F: 5’-ATTGGAAATAGGTTTTGAGGGTCAT- 3’ and R:5’TCTCGGTTTCTTCTCCATCGC-3’ . These PCR products were digested using PagI  restriction enzyme. Re-stricted fragments were run on 3% agarose gel and viewed under UV light [Among the 84 BCS patients, TNF-α -308 G/A polymorphism was present in 8.3% (n = 7), whereas the frequency of TNF-α -308 G/A polymorphism in the healthy Egyptian controls was 7.9%. The distribution of TNF-α -308 G/A polymorphisms in the BCS patients and controls is shown in the Fleischman et al. reported that TNF-α plays a central role in promoting clonal dominance of Jak2 V617Fexpressing cells in MPN. They showed that Jak2 V617F kinase regulates TNF-α expression in cell lines and primary MPN cells, and that TNF-α expression is correlated with Jak2 V617F allele burden . GhaffarConflict of Interest StatementNone of the authors have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.