In this study, we sought to determine the cellular source of inducible nitric oxide synthase (iNOS) induced in lymphatic endothelial cells (LECs) in response to tumor necrosis factor (TNF), the effects of iNOS on lymphatic smooth muscle cell (LSMC) function and on the development of arthritis in TNF-transgenic (TNF-Tg) mice, and whether iNOS inhibitors improve lymphatic function and reduce joint destruction in inflammatory erosive arthritis.We used quantitative polymerase chain reactions, immunohistochemistry, histology, and near-infrared imaging to examine (1) iNOS expression in podoplanin + LECs and lymphatic vessels from wild-type (WT) and TNF-Tg mice, (2) iNOS induction by TNF in WT LECs, (3) the effects of iNOS inhibitors on expression of functional muscle genes in LSMCs, and (4) the effects of iNOS inhibitors on lymphatic vessel contraction and drainage, as well as the severity of arthritis, in TNF-Tg mice.iNOS messenger RNA levels than WT cells, and iNOS expression was confirmed immunohistochemically in podoplanin + LECs in lymphatic vessels from inflamed joints. TNF (0.1 ng/ml) increased iNOS levels 40-fold in LECs. LSMCs cocultured with LECs pretreated with TNF had reduced expression of functional muscle genes. This reduction was prevented by ferulic acid, which blocked nitric oxide production. Local injection of L-N6-(1-iminoethyl)lysine 5-tetrazole-amide into inflamed paws of TNF-Tg mice resulted in recovery of lymphatic vessel contractions and drainage. Treatment of TNF-Tg mice with ferulic acid reduced synovial inflammation as well as cartilage and bone erosion, and it also restored lymphatic contraction and drainage.LECs from TNF-Tg mice had eight fold higher iNOS is produced primarily by LECs in lymphatic vessel efferent from inflamed joints of TNF-Tg mice in response to TNF and inhibits LSMC contraction and lymph drainage. Ferulic acid represents a potential new therapy to restore lymphatic function and thus improve inflammatory arthritis by inhibiting local production of nitric oxide by LSMCs.The online version of this article (doi:10.1186/s13075-016-0963-8) contains supplementary material, which is available to authorized users. The lymphatic system plays a critical role in maintenance of fluid homeostasis and normal immune responses . It is aThe lymphatic vasculature is composed of capillaries and collecting vessels that have different morphology, structural composition, and function . For exa+Gr-1+ myeloid cells [Nω-nitro-l-arginine methyl ester [l-NAME] and l-N6-(1-iminoethyl)lysine 5-tetrazole-amide [l-NIL]), as well as ferulic acid  is a primary regulator of lymphatic vessel contraction via its vasodilatory signal in LSMCs , 21. In id cells . Most reid cells . Severalid cells , 25, as id cells –29. In aid cells , NOS inhid cells , and incid cells . Howevery for RA , 34), onl-NIL or saline was injected into footpads during the NIR-ICG imaging session. The lymphatic pulse and clearance were monitored by NIR-ICG imaging after treatment. The effects of long-term NO inhibition were assessed by adding 100 ng/ml of L-NIL or 100 ng/ml of the general NOS inhibitor L-NAME  to the drinking water, which was changed every day for 6 weeks. Ferulic acid  was dissolved in saline. Mice were gavaged daily with ferulic acid solution (20 mg/kg/day) or saline for 12 weeks. We determined that the purity of ferulic acid was more than 98 % by high-performance liquid chromatography  . The TNFImmunohistochemistry (IHC) was performed on frozen sections of fresh popliteal lymph nodes (PLNs) and limb tissues containing lymphatic vessels obtained from the mice, as described previously . Tissues+ cells were collected from the LS column by flushing with 0.5 ml of MACS buffer. About 20,000 cells were obtained per mouse leg. To characterize PDPN+ cells as LECs, cells were stained with goat antimouse vascular endothelial growth factor receptor 3 antibody  followed by Alexa Fluor 488–conjugated antigoat immunoglobulin G secondary antibody , or with biotin-conjugated mouse LYVE-1  followed by allophycocyanin-conjugated streptavidin , or PE-conjugated mouse PDPN antibody, and subjected to flow cytometry . The data were analyzed with FlowJo software .Soft tissue and muscles were removed from entire legs and digested with 2 ml of 1 mg/ml collagenase I  for 1 h at 37 °C. Digestion was terminated by adding 8 ml of 2 % fetal bovine serum (FBS) in PBS and filtered with a 40-μm Falcon cell strainer . Centrifuged cells were washed twice with MACS buffer , recentrifuged, and resuspended with 95 μl of MACS buffer plus 5 μl of PE-conjugated anti-PDPN antibody  for 30 minutes at 4 °C, and then with 95 μl of MACS buffer plus 5 μl of anti-PE microbeads  for another 30 minutes at 4 °C. Cells were resuspended with 0.5 ml of MACS buffer and passed through an LS separation column . After extensive washing, PDPNt) value of each sample for an individual gene from the corresponding Ct value of β-actin (ΔCt). The ΔΔCt value was obtained by subtracting the ΔCt of the test samples from the ΔCt of control samples. These values were then raised to the power of 2 (2−ΔΔCt) to yield the fold expression relative to the reference level. The expression levels of functional muscle genes were examined using sequence-specific primers, which are listed in Additional file RNA was extracted from LECs and LSMCs using TRIzol reagent (Life Technologies), and complementary DNA was prepared from total RNA using the GeneAmp RNA PCR Core Kit . Quantitative polymerase chain reaction (PCR) amplification was performed in triplicate with gene-specific primers and iQ SYBR Green Supermix  in an iCycler real-time PCR machine (Bio-Rad Laboratories). The relative abundance of each gene was calculated by subtracting the threshold cycle (CWhole-cell lysate samples were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (20 μg of protein/lane). Immunoblotting was carried out using antibodies to smooth muscle myosin heavy chain 2 , h1-calponin , SMα22) , nuclear factor κB2  at 1:1000 dilution, iNOS  at 1:800 dilution, and β-actin  at 1:5000 dilution. Bands were visualized using enhanced chemiluminescence . Signal intensity of bands was measured by densitometry.6/well in six-well plates overnight, pretreated with 1 μM aminoguanidine hemisulfate salt (Ami) and FLA (250 μM) for 3 h, and then with 1 ng/ml TNF for 24 h. Supernatants were collected, and nitrite levels were assessed using an NO assay kit . This kit transforms nitrate to nitrite via nitrate reductase first, and then uses Griess reagent to detect the total amount of nitrite, from which the total NO levels are calculated. It does not detect reactive oxygen species [LECs were seeded at 10 species .l-glutamine, and antibiotics. The culture medium was changed every 3 days. Smooth muscle cells covering lymphatic vessels migrated from the vessels after 3–4 days, and vessel segments were removed aseptically. The cells were trypsinized after 7–10 days with 0.25 % trypsin in 0.02 % EDTA and transferred to a new gel-coated dish (passage 0). After 1–2 weeks, the cells reached confluence and were split into two dishes (passage 1). The cells were fixed with 10 % formalin for 10 minutes, blocked with 0.2 % Triton-100 and 1 % BSA in PBS for 30 minutes at RT, stained with FITC anti-αSMA antibody (1:400) at 4 °C overnight, and observed under a fluorescence microscope . Whole-cell lysates were examined by Western blot analysis using a 1:1000 dilution of an anti-sMYH antibody  and a 1:5000 dilution of anti-β-actin antibody .We followed a published protocol for isolating LSMCs . Mesente6 cells/coverslip in a 10-cm dish. After 2 days, LECs were treated with 1 ng/ml TNF  with or without a selective iNOS inhibitor  for 24 h. Coverslips with LECs were then transferred into another 10-cm dish, which had already been coated with 2 × 105 LSMCs for 4 days. After 24 h of coculture, coverslips with LECs were removed and LSMCs were harvested for analysis.A murine LEC cell line established from Freund’s adjuvant–induced benign lymphangiomas   activity to identify osteoclasts. Sections of the entire ankle joint, including the distal tibia and proximal metatarsal bones, were digitized using an Olympus VS120-S5-E whole-slide imaging system (Olympus America). All images were analyzed using Olympus OlyVIA software (Olympus America). We outlined ROIs, which included distal tibia, talus, calcaneus, proximal metatarsal bone, synovial capsules, ligaments, and adjacent soft tissues. The total tissue area of the ROI was determined automatically with Olympus VS120 software and ranged from 3.77 mmt test. One-way analysis of variance test followed by a Bonferroni-corrected posttest was used for comparisons of more than two groups. p Values <0.05 were considered statistically significant.Data are presented as means ± standard deviations. Statistical analyses were performed with SPSS 16.0 software . Differences between two groups were compared using Student’s + myeloid cells within LYVE-1+ lymphatic vessels in the PLNs , but not in their WT littermates . In contrast, saline or L-NAME did not change the lymphatic pulse in either TNF-Tg or WT mice. Similarly, L-NIL significantly increased lymph drainage in TNF-Tg mice compared with saline , while L-NAME decreased lymphatic drainage in both WT and TNF-Tg mice. These data indicate that only selective iNOS inhibition by L-NIL significantly increased lymphatic vessel contractions and lymphatic flow in TNF-Tg mice, but not in WT littermates. The nonspecific NOS inhibition by L-NAME significantly decreased ICG clearance in both WT and TNF-Tg mice, consistent with a critical role for endothelial nitric oxide synthase (eNOS) signaling in basal lymphatic function.To confirm that lymphatic function recovery was due to iNOS inhibition and to assess drug effects on normal lymphatic vessel function, we treated cohorts of TNF-Tg mice with severe ankle arthritis and their WT littermates with saline, L-NIL, and the nonspecific NOS inhibitor L-NAME for 6 weeks Fig. . UnfortuAngelica sinensis extract [Given the toxicity challenges associated with selective iNOS inhibitors and our interest in understanding the mechanism of action of traditional Chinese medicines (TCMs) that have been used successfully to treat patients with RA who do not respond to disease-modifying antirheumatic drugs –46, we p extract  used to  extract , 48, 49,p < 0.05). In contrast, ferulic acid therapy restored lymph flow and lymphatic vessel contraction frequency in TNF-Tg mice to WT levels  induced a high level of expression of iNOS in LECs, indicating that LECs are very sensitive to iNOS induced locally by inflammatory cytokines. We reported previously that the circulating human TNF levels in TNF-Tg mice are about 0.1–0.2 ng/ml , 52. ThuLECs may be exposed to TNF in vivo by a number of mechanisms. For example, TNF levels will be increased in the interstitial fluid draining from inflamed joints when it passes into collecting lymphatic vessels as well as in soft tissues generally because of high serum TNF levels in these mice . Anotherl-NIL [p = 0.072) and synovial vascularity  vs. placebo [l-NAME and l-NIL. Because the primary cause of early death (6–12 months of age) of TNF-Tg mice is pulmonary hypertension, which is also the primary toxicity associated with NO inhibitors [Our study clearly indicates the contribution of NO from LECs to inhibition of LSMC function in the setting of TNF overexpression, which provides experimental evidence for the use of NOS inhibitors in the treatment of RA. It is important to note that iNOS is a well-established target for RA therapy, as the efficacy of two selective inhibitors—l-NIL  and GW27l-NIL —has alre placebo . Howeverhibitors , we spechibitors , 48, 49,We have demonstrated that dysfunction of efferent lymphatic vessels from inflamed hind limb joints in TNF-Tg mice involves TNF-induced iNOS expression and NO production by LECs, which affects LSMCs and results in decreased lymphatic vessel contractions and lymph drainage. iNOS inhibitors, and especially ferulic acid therapy, ameliorate TNF-induced arthritis, which is associated with restoration of lymphatic vessel contractions and drainage. Thus, inhibition of NO production and improvement of lymphatic vessel draining function together represent a potential therapeutic strategy for chronic inflammatory arthritis. It also represents a new mechanism of action for some established drugs that have been used to treat patients with inflammatory arthritis.


Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine and elevated in the regions of tissue injury and inflammatory diseases. The deleterious effects of TNF-α on fibroblasts may aggravate heart inflammation mediated through the up-regulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1). However, the mechanisms underlying TNF-α-induced VCAM-1 expression in cardiac fibroblasts remain unknown. This study aimed to investigate the roles of TNF-α in VCAM-1 expression and its effects on human cardiac fibroblasts (HCFs).The primary culture HCFs were used in this study. The results obtained with Western blotting, real time-quantitative PCR, and promoter activity analyses showed that TNF-α-induced VCAM-1 expression was mediated through TNF receptor (TNFR) 1-dependent gene up-regulation. Activation of TNFR1 by TNF-α transactivated c-Src-dependent EGF receptor (EGFR) linking to PI3K/Akt cascade, and then led to transcriptional activity of NF-κB. Moreover, the results of promoter reporter assay demonstrated that the phosphorylated p65 NF-κB turned on VCAM-1 gene expression. Subsequently, up-regulation of VCAM-1 promoted monocytes adhesion to HCFs challenged with TNF-α determined by cell adhesion assay.Taken together, these results indicate that in HCFs, activation of NF-κB by c-Src-mediated transactivation of EGFR/PI3K/Akt cascade is required for TNF-α-induced VCAM-1 expression. Finally, increased VCAM-1 enhances monocytes adhering to HCFs challenged with TNF-α. Understanding the mechanisms of VCAM-1 up-regulated by TNF-α on HCFs may provide rationally therapeutic interventions for heart injury or inflammatory diseases. Heart failure, the first cause of death in the world, is generally defined as the inability of heart supplying sufficient blood volume and is a progressive and complex pathological condition . The mosTNF-α is a major cytokine in the pathogenesis of cardiac injury, promoting inflammation, apoptosis, and production of ECM , 14 and In the serum of patients with non-ischemic heart failure, the levels of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 are often elevated. And the cellular infiltration is due to up-regulation of adhesion molecules including ICAM-1 and VCAM-1 to control cell adhesion and migration –26. ThesIn this study, we found that TNF-α induces c-Src-mediated signal activation in HCFs via TNFR1, in turn initiates the activation of EGFR, PI3K/Akt, and NF-κB. Activated NF-κB could accelerate VCAM-1 promoter activity to induce VCAM-1 expression in HCFs. Moreover, up-regulation of VCAM-1 promoted adhesion of monocytes to HCFs challenged with TNF-α. These results provide new insights into the TNF-α action, to up-regulate the VCAM-1 expression and then amplify the inflammatory responses in HCFs.1068) (#2236), Akt (Ser473) (#9271), and p65 NF-κB (Ser536) (#3031) antibody kits were from Cell Signaling . Human polyclonal antibody VCAM-1 (sc-8304), TNFR1 (sc-52739), phospho-c-Src (Tyr139) (sc-12928-R), c-Src (sc-18), p110 (PI3K subunit) (sc-7189), Akt (sc-8312), and p65 (sc-7151) antibodies were from Santa Cruz . Vimentin (MS-129-P) was from Thermo Fisher Scientific . GAPDH was from Biogenesis . Peroxidase AffiniPure goat anti-rabbit IgG (111-035-003) and anti-mouse IgG (115-035-003) were from Jackson ImmunoResearch . PP1, AG1478, LY294002, SH-5, and Bay11-7082 were from Biomol . TNF-α was from R&D Systems . Bicinchoninic acid (BCA) protein assay kit was from Pierce . Actinomycin D (Act. D), cycloheximide (CHI), enzymes, and other chemicals were from Sigma .DMEM/F-12 medium, fetal bovine serum (FBS), TRIZOL, Lipofectamine-PLUS reagent and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester  were from Invitrogen . The siRNAs for TNFR1 , c-Src , EGFR , p110 , Akt , and p65  were from Sigma . Hybond C membrane, enhanced chemiluminescence (ECL) and Western blotting detection system were from GE Healthcare Biosciences . PhosphoPlus EGFR  were isolated from human heart obtained from ScienCell Research Lab  and then characterized by immunofluorescent staining with an antibody specific to fibronectin . These cescribed , 37, andescribed . When thGrowth-arrested HCFs (incubation in serum-free DMEM/F-12 for 24 h) were incubated with TNF-α at 37 °C for the indicated time intervals. The cells were washed with ice-cold PBS, scraped, and collected by centrifugation at 45,000 × g for 1 h at 4 °C to yield the whole cell extract, as previously described . SamplesTotal RNA was isolated from HCFs treated with TNF-α for the indicated time in 10-cm culture dishes with Trizol according to the protocol of the manufacturer. The cDNA obtained from total RNA previously described . RT  was performed with the 7500 Real-Time PCR System  using primers and probe mixes for VCAM-1  and endogenous GAPDH  served as an internal control gene . Relative gene expression was determined by the ΔΔCt method, where Ct meant threshold cycle . All exp5 cells/ml (1 ml per well) in 12-well culture plates for 24 h, reaching about 80 ~ 90 % confluence. Cells were washed once with PBS and 0.4 ml of serum-free DMEM/F-12 medium was added to each well. The 0.8 μg plasmid DNA (or 20 pmol siRNA) and 1 μl PLUS reagent complex were prepared with serum- and antibiotics-free DMEM/F-12 for 15 min at room temperature. Two μl Lipofectamine™ was mixed gently with serum- and antibiotics-free DMEM/F-12. The mixtures were incubated for 30 min at room temperature. The mixtures (100 μl) were added into the wells. After transfection for 5 h, the cells were washed once with PBS and 0.5 ml of containing 10 % FBS was added and incubated for an additional 19 h. VCAM-1-Luc and κB-Luc  activities in HCFs were determined as previously described [For construction of the VCAM-1-Luc plasmid, human VCAM-1 promoter, a region spanning −1716 to −119 bp  was inserted between MluI and XhoI sites of pGL3-basic vector . All plasmids were prepared by using QIAGEN plasmid DNA preparation kit. Plasmid (or siRNA) transient transfection of HCFs was performed according to the protocol of Lipofectamine and PLUS reagent  with minor modifications. Briefly, HCFs were plated at 3 × 10escribed  using a 2 atmosphere. Before the adhesion assay, the HCFs were washed with PBS and then incubated in serum-free DMEM/F-12. THP-1 cells  were maintained in suspension in RPMI-1640 medium supplemented with 10 % FBS, 100 U penicillin, and 100 U streptomycin. Before labeling, THP-1 cells were washed and resuspended in serum-free medium. BCECF/AM (10 μM), a membrane-permeable fluorescent indicator with an Ex/Em = 503/520 nm, was used to label the THP-1 cells in serum free medium for 1 h at 37 °C. After labeling, cells were washed and resuspended in serum free medium at least three times and kept in the dark at room temperature and then the labeled THP-1 cell (2 × 106 cells/ml) were added to HCFs challenged with TNF-α, and cultures were incubated in a CO2 incubator for 1 h. Non-adherent THP-1 cells were removed and plates were gently washed twice with PBS. The numbers of fluorescently labeled adherent THP-1 cells were determined by counting four fields per 200× high-power field well using a fluorescence microscope .HCFs were placed on 6-well culture plates with cover slips and pretreated with the pharmacological inhibitors for 1 h before treatment with TNF-α for 16 h at 37 °C in a humidified 5 % COp < 0.05 level of significance.All data were estimated using GraphPad Prism Program . Data were expressed as the mean ± SEM and analyzed with a one-way ANOVA followed with Wilcoxon/Kruskal-Wallis test at To evaluate the effect of TNF-α on VCAM-1 protein and mRNA expression, HCFs were incubated with various concentrations of TNF-α for the indicated time intervals. As shown in Fig. To determine whether the effect of TNF-α on VCAM-1 expression is dependent on de novo protein synthesis, a transcription inhibitor Act. D and a translation inhibitor CHI were used. As shown in Figs. TNF-α has been shown to induce expression of adhesion molecules such as VCAM-1 in various cell types . In addiSeveral growth factor receptors such as EGFR activation have been shown to be regulated by c-Src leading to induction of various genes . Thus, wThe PI3K/Akt signaling cascade, the direct downstream molecules of EGFR, plays a key role in various physiological and pathological processes. Previous studies have indicated that PI3K/Akt pathway is involved in VCAM-1 induction by TNF-α in various cell types , 41. In a subunit of NF-κB) phosphorylation which was attenuated by pretreatment with Bay11-7082 during the period of observation  are higher than normal (2.6 ± 0.4 pg/ml) . HoweverIn this study, up-regulation of VCAM-1 by TNF-α is mediated through transactivation of c-Src/EGFR which were attenuated by their respective inhibitors (PP1 and AG1478) and siRNAs transfection, suggesting the involvement of c-Src/EGFR in these responses Figs.  and 4. IThe PI3K/Akt cascade is the downstream components of EGFR activation, which is implicated in the pathogenesis of inflammatory responses , 50 throInflammatory responses following exposure to cytokines are highly dependent on activation of NF-κB which plays an important role in expression of several inflammatory genes , 18, 53.The infiltration of immune cells is due to up-regulation of adhesion molecules including ICAM-1 and VCAM-1 , 24, 26 In summary, we reported here that TNF-α induced the expression of VCAM-1 gene in HCFs. The TNFR1, c-Src, EGFR, PI3K/Akt, and NF-κB cooperatively mediated these effects of TNF-α. Based on the observations from literatures and our findings, Fig.


Odontomachus brunneus during their interactions with a common ant predator: pit-building antlions. We observed that while trap-jaw ant workers escaped from antlion pits by running in about half of interactions, in 15% of interactions they escaped by mandible-powered jumping. To test whether escape jumps improved individual survival, we experimentally prevented workers from jumping and measured their escape rate. Workers with unrestrained mandibles escaped from antlion pits significantly more frequently than workers with restrained mandibles. Our results indicate that some trap-jaw ant species can use mandible-powered jumps to escape from common predators. These results also provide a charismatic example of evolutionary co-option, where a trait that evolved for one function (predation) has been co-opted for another (defense).Animals use a variety of escape mechanisms to increase the probability of surviving predatory attacks. Antipredator defenses can be elaborate, making their evolutionary origin unclear. Trap-jaw ants are known for their rapid and powerful predatory mandible strikes, and some species have been observed to direct those strikes at the substrate, thereby launching themselves into the air away from a potential threat. This potential escape mechanism has never been examined in a natural context. We studied the use of mandible-powered jumping in  The significance of predation in animal evolution is reflected in the diversity of strategies that prey employ to avoid it. Antipredator adaptations, such as crypsis, mimicry, chemical defense, or escape behaviors, each derail a specific stage of the predation sequence , 2. EscaOdontomachus, for example, are capable of snapping their mandibles shut as fast as 60 m/s and generate forces over 300 times their body weight [Trap-jaw ants have specialized spring-loaded mandibles that snap shut at some of the fastest speeds ever recorded for an animal movement. Members of the genus y weight , 8. The y weight , 10 and y weight ).Orectognathus versicolor and Odontomachus ruginodis direct their strikes against invading ants, flinging the attackers away from the nest entrance [Odontomachus might also be an adaptive escape behavior. They described vertical “escape jumps” following mandible strikes that were targeted against the substrate and were accompanied by a characteristic body and leg posture. Spagna et al. [Odontomachus can perform these escape jumps, and that the behavior is context-dependent, occurring more frequently in the presence of other predatory ant species.Although all trap-jaw ants use their mandible strike for predation, in at least two lineages, this behavior has been co-opted for defense. Nest-guarding workers of entrance , 13. Durentrance . More reentrance  suggestea et al.  demonstrOdontomachus trap-jaw ants are an effective antipredator escape behaviors by examining: (1) the frequency that trap-jaw ants use escape jumps during encounters with predators that co-occur in their foraging habitat, and (2) if escape jumps increase the probability of a trap-jaw ant surviving a predatory encounter.Together, this evidence is suggestive that spring-loaded mandibles have been evolutionary co-opted as an antipredator strategy in some trap-jaw ants, but several fundamental questions about escape jumps remain unanswered. No ecologically relevant predators or competitors have been identified against which trap-jaw ants use their mandibles for escape, and it is unclear if escape jumps actually improve individual survival. While collecting trap-jaw ants in central Florida, we observed ants foraging in areas inhabited by a common sit-and-wait predator: pit-building antlions (Neuroptera: Myrmeleontidae) . EncountOdontomachus brunneus were excavated from scrub habitat and pastures at ABS and the MacArthur Agro-ecology Research Center (MAERC), both located at the southern end of the Lake Wales Ridge in central Florida. Colonies ranged in size from approximately 15 to 75 workers, and four contained queens. In the laboratory, artificial nests were constructed from 32 cm X 20 cm plastic boxes with Fluon-coated walls. One or two plastic test tubes containing water trapped behind cotton plugs were provided for nest chambers. Ants were provided with water and 20% sugar solution ad libitum, and 4–10 termites or frozen crickets each day.All field research was conducted at Archbold Biological Station (ABS) with their permission over three summers (2011 to 2013). Twenty nests of Pit-building antlions (Neuroptera: Myrmeleontidae) are ubiquitous sit-and-wait predators of terrestrial insects, and are commonly found in trap-jaw ant foraging areas at ABS and MAERC  Fig 1 a. The larMyrmeleon carolinus, M. crudelis, or M. mobilis were collected along fire lanes and under shelters at ABS. Each antlion was transferred to an arena made from 11.5 cm diameter plastic cups coated with Fluon (Northern Products) and filled with 4 cm of sand collected from the sites where the antlions were found. Sand was sifted with a #20 US standard sieve  to remove large particles. After digging a pit in an arena , antlions were fed one ant (Dorymyrmex bureni) each day. Both ants and antlions were maintained at approximately 24° C with natural lighting cycles. Vouchers have been deposited in the invertebrate collection at ABS.Ultimate (third) instar larvae of We conducted 117 trials, each consisting of a single encounter between an individual trap-jaw ant and an antlion. The antlions were starved for 48 hours prior to observation and any larvae that had abandoned their initial pit or had not dug a pit at all were excluded from study. Ant workers were allowed to rest individually in Fluon-coated cups for approximately one minute and then tipped into the arena away from the antlion pit. Ants that fell directly into a pit or snapped their mandibles before entering a pit on their own were immediately returned to their nest for later use. Each trial was recorded with either standard (Panasomic TM80 high-definition video camera) or high-speed video .caught—the ant was grabbed by the antlion and held under the sand for at least 60 seconds, escape by running—the ant ultimately escaped by running out of the pit, and escape jump—the ant jumped completely out of the pit as a result of a mandible strike. We only scored outcomes if the antlion attacked (either threw sand at or attempted to grab the ant), and if no interaction occurred within 10 minutes, the trial was terminated and the individuals omitted from the analysis. Pit diameter, which is correlated to antlion size [A trial started after introducing the ant into the antlion arena and concluded after 10 minutes or when one of the following events was observed: ion size , was meaion size .To test the hypothesis that escape jumps improve the survival of individual ant workers, we experimentally prevented workers from jaw-jumping by restraining their mandibles and then measuring their escape frequency from antlion pits. Individual foragers were chilled on ice and the distal tips of the mandibles were glued together with cyanoacrylate glue (Scotch Super Glue Gel) .. After Odontomachus brunneus workers fell into antlion pits, they were captured approximately one-third of the time  or overall escape frequency . The duration of each trial ranged between 0.5 and 65 seconds and did not vary among the three outcomes . Individuals that escaped by jumping responded to antlion attacks by snapping their mandibles against the side or bottom of the pit, resulting in an explosion of sandy substrate and launching them completely out of the pit . It is unlikely that O. relictus workers are incapable of jumping due to physical constraints; the mean maximal force of O. relictus mandible snaps is similar to those of other Odontomachus species that also perform escape jumps [O. relictus, or if they require a specific context in which the behavior would be released.Preliminary data with other species of pe jumps , 15. It O. brunneus interacts. Several other predators have been described that may be specialists on trap-jaw ants, including workers of Dorymyrmex bureni, Formica archboldi [Pachycondyla spp. [O. brunneus and O. relictus were rare in stomach content surveys of the narrow-mouthed toad, Gastrophryne carolinensis, an ant specialist whose foraging areas and daily activity overlaps with Odontomachus at ABS [Antlions are only one of numerous predators and competitors with which rchboldi , and Pacyla spp. . Workerss at ABS . The absOdontomachus, however, trap jaws appear to have been evolutionary coopted and play a role on both sides of predator-prey evolutionary arms races. Many questions remain about the evolutionary history of trap jaws. There are more than 160 species in the sister trap-jaw ant genera Odontomachus and Anochetus, and only a small fraction have been studied in any detail with respect to their defensive use of the mandible snap. A full understanding of the evolutionary dynamics trap-jaw mandibles will require a more comprehensive documentation of the natural history of trap-jaw ants.Spring-loaded mandibles may represent a “key morphological innovation”, allowing trap-jaw ants to access an otherwise inaccessible resource  . In at lS1 VideoThis high-speed video (700 fps) depicts a successful antlion predation event. After flinging sand at prey, the antlion grabs an ant and drags it under the sand at the bottom of the pit.(MP4)Click here for additional data file.S2 VideoOdontomachus brunneus during interactions with an antlion, Myrmeleon carolinus. After striking the substrate, the ant jumps completely out of the pit.This high-speed video (500 fps) depicts an escape jump of (MP4)Click here for additional data file.S3 VideoOdontomachus brunneus jumping out of an experimental antlion pit.A standard speed (32 fps) video showing the trap-jaw ant (MP4)Click here for additional data file.S4 VideoThis high-speed (500 fps) video shows a trap-jaw ant snapping its jaws after being caught by an antlion, then being released, and an unsuccessful escape jump.(MP4)Click here for additional data file.


Assays for VLPs and total antigen together with animal immunisation trials confirmed preservation of antigenicity and immunogenicity of S-HBsAg in freeze-dried powder. Long-term stability tests revealed that the stored freeze-dried product was stable at 4°C for one year, but degraded at elevated temperatures. As a result, a basis for an efficient freeze-drying process has been established and a suitable semiproduct for oral plant-derived vaccine against HBV was obtained.The aim of this study was to develop a freeze-drying protocol facilitating successful processing of plant material containing the small surface antigen of hepatitis B virus (S-HBsAg) while preserving its VLP structure and immunogenicity. Freeze-drying of the antigen in lettuce leaf tissue, without any isolation or purification step, was investigated. Each process step was consecutively evaluated and the best parameters were applied. Several drying profiles and excipients were tested. The profile of 20°C for 20 h for primary and 22°C for 2 h for secondary drying as well as sucrose expressed efficient stabilisation of S-HBsAg during freeze-drying. Freezing rate and postprocess residual moisture were also analysed as important factors affecting S-HBsAg preservation. The process was reproducible and provided a product with VLP content up to 200  Effective subunit vaccines against hepatitis B virus (HBV), based on the virus surface antigen (HBsAg), were introduced over 25 years ago enabling implementation of hepatitis B mass vaccination programmes. Still, the number of chronic carriers and postdisease mortality around the world, particularly in developing countries, have been steadily growing , 2. The Research focused on the development of an oral plant-based vaccine against HBV has been conducted for over 15 years. Although initial experiments provided positive results as orally delivered raw plant tissue bearing HBsAg induced a specific humoral response , it was Freeze-drying, although being a well-established method commonly used for pharmaceuticals, is a process still subjected to empirical practice , 9. ThisThere are two main hypotheses explaining protein stabilisation during drying and storage. One is the “glass dynamics hypothesis”  which stThe aim of this study was to develop a stable dry-powder formulation containing S-HBsAg VLPs expressed in previously obtained lettuce , with thμg/g FW were harvested for lyophilisation experiments.Plant material used in the studies came from previously obtained lettuce expressing S-HBsAg . Only leTwo freezing methods, “fast” and “slow”, were tested on leaf blades. “Fast” freezing was performed by submerging the material into liquid nitrogen and immediately placing on a freeze-dryer shelf precooled to −35°C. The material subjected to “slow” freezing was put into a freeze-dryer chamber at ambient temperature and frozen at 2°C/min rate until −35°C. After an hour frozen samples were divided and subjected to different thawing protocols, “slow” in air of 4°C, or “fast” by immersing in 4°C water. The experiment was conducted in three replications.Leaf blades for lyophilisation were infiltrated with an excipient by placing the material in a vacuum chamber and lowering pressure to 50–200 mbar maintained for 2–15 min. Then a solution of a given excipient see  was intrP ≤ 0.05) using Statistica 8 software (StatSoft). Since many variables were investigated, the studies were conducted in a mode, in which only the most efficient variant of a given parameter tested in the current round of experiments was used for the next round.Since the absolute content of total or VLP-assembled S-HBsAg in the freeze-dried tissue depended on its original level in the plants, lyophilisation effectiveness was expressed as relative antigen preservation calculated as a ratio of the antigen content in processed tissue to the fresh one multiplied by the degree of weight loss. Each experimental variant was tested in at least three replications and analysed statistically  PVP40000, and 0.2% (w/v) BSA and supplemented with 0.05% (v/v) Triton X-100. Crude extracts were centrifuged for 10 min at 10.000 rpm at 4°C. The S-HBsAg content, both total and assembled into VLPs, was assayed using a MaxiSorp microplate (NUNC) coated with 0.5 μg/mL anti-SHBsAg IgG2a monoclonal antibody (mAb)  in carbonate buffer at pH 9.6. After washing with PBS containing 0.05% (v/v) Tween 20, plates were blocked with 5% (w/v) PBS-fat-free milk at 25°C for 1 h. Following washing samples, extracts were added to PBS-filled wells at 1 : 1 ratio and serially diluted. After incubation at 25°C for 45 min and washing, wells were incubated with primary and secondary antibodies, specific for a given test. For the VLPs assay, 0.5 μg/mL mAb IgG2b anti-SHBsAg  and polyclonal goat anti-mouse IgG2b (Sigma), and finally 1 : 10,000 diluted rabbit anti-goat AP-conjugated antibody (Sigma) were used, all with washing between steps. Total antigen content was assayed after samples incubation with rabbit anti-SHBsAg serum , followed by biotin-conjugated anti-rabbit goat polyclonal antibody (Sigma), and followed by AP-conjugated Extravidine (Sigma). The reaction with pNPP substrate (Sigma) was developed at 25°C for 1 h and absorbance at 405 nm was measured using the microplate reader, Model 680 (Bio-Rad). The S-HBsAg concentration was calculated in micrograms per gram of fresh weight (FW) or lyophilised dry weight (DW).The content of S-HBsAg, both total and VLP-formed, was assayed in lettuce leaves and in derived lyophilised tissue using quantitative sandwich ELISA tests performed according to typical guidelines. Samples were ground with extraction buffer, added at a ratio of 25 : 1 for leaves and 62.5 : 1 for lyophilised tissue and consisting of PBS at pH 7.4, 10 mM NaμL of PBS with 0.5% (v/v) Tween 20, then Laemmli buffer with 100 mM DTT was added and samples were incubated at 65°C for 15 min. Samples were run on 12% polyacrylamide gel with 0.1% (w/v) SDS and then blotted onto a nitrocellulose membrane (Roche). The blot was blocked with 3% (w/v) BSA in TBS and then incubated with the TBS-diluted rabbit polyclonal anti-SHBsAg antibody . The AP-conjugated goat polyclonal anti-rabbit whole-molecule antibody (Sigma) followed. The molecular weight of protein bands, visualized after a reaction with the BCIP/NBT substrate (Sigma), were estimated using a protein size marker (MBI Fermentas).Leaf fragment or lyophilised tissue, approx. 50–60 mg in weight, were ground in 300 i.m.) injection of Engerix B in a dose 500 ng of S-HBsAg or with PBS for control groups. Boosting was performed on the 42nd day after priming by oral administration (p.o.) of PBS-suspended powdered lyophilised tissue containing 50 ng of S-HBsAg VLPs, the control lyophilised tissue, or i.m. delivered PBS. Blood was collected 3 days before (preimmune) and 63 days after prime immunisation. Sera were collected from blood by centrifugation at 3 000 rpm at 4°C for 15 min. Anti-HBs antibodies (mIU/mL) were assayed twice using the Monolisa anti-HBs PLUS kit (Bio-Rad) and analysed statistically  using Statistica 8 software (StatSoft).Separate groups of 10–12-week-old BALB/c mice (5 per group) were primed by an intramuscular . After the profile with a 20°C shelf temperature, this protectant made it possible to obtain several times higher lyophilisation efficiency, in comparison to unsoaked tissue (124%). Increased VLPs level beyond 100% could be caused by partial disintegration of actual VLPs into smaller particles. Moreover, the total antigen level remained practically unchanged during that freeze-drying profile, providing a conclusion that VLPs degradation occurred only to a limited extent, oppositely to the cycle with primary drying at 5°C, where in connection with VLPs degradation the total antigen level increased to around 200%. A relatively good VLPs preservation (70%) was also observed for leaves soaked with 500 mM mannitol. However, at the same time the level of the total antigen remained unchanged, indicating that some degradation of S-HBsAg polypeptides released or aggregates released from VLPs might occur. In the other variants, low VLPs preservation together with high fluctuations of the total S-HBsAg level was observed, in most cases above the theoretical 100%. In addition to effectiveness values, some tendency was observed for VLPs preservation to increase in correlation to the increasing concentrations of excipients. This phenomenon partially coincided, especially for the variants with sucrose and mannitol, with less increased contents of total antigen. Regarding the abovementioned assumption that the higher the total antigen level the larger the pool of defect S-HBsAg forms, that effect is advantageous. Summarising this step of research, sucrose provided the best preservation of S-HBsAg in the form of VLPs and the simultaneous desirable, unchanged level of the total antigen; hence it was selected for next tests.The results of the experiments  proved tμg/g DW in the variant with 500 mM sucrose, than 11-12 μg/g DW obtained previously [In addition to the improved relative effectiveness of lyophilisation, absolute VLPs contents in processed tissues were substantially higher, up to 131.5 eviously . This caeviously . In consAlthough freeze-drying was the crucial stage, associated handling steps as tissue infiltration with a lyoprotectant and milling of the product could also affect the final efficiency of whole process. Literature data as well as our results showed that the preservation effect was associated with a high concentration of a protective excipient, here 500 mM sucrose . TherefoAfter freeze-drying, lyophilised tissue was milled to obtain the final semiproduct. Tests proved that within a period of 5–90 seconds, no significant differences occurred regarding both VLPs and total S-HBsAg content (data not shown). Yet, since approximately 20 seconds were enough to mill the tissue completely, this time was adopted.The successive step was focused on determining shelf temperature and duration of freeze-drying, which are of great importance for process economy and scaling up. Optimisation of freeze-drying is still a trial-based process. A higher drying temperature allows shortening cycle duration and consequently provides a more cost-effective process. Usually, each 1°C increase in product temperature decreases time of primary drying by about 13% , 44. In Starting point for our studies was temperature 5°C or 20°C for primary drying. The latter variant resulted in better S-HBsAg preservation, probably due to lower residual moisture of the product. Then, we decided to test higher drying temperatures to find upper limit, that is, collapse temperature, but also to prolong the process duration since it could not be excluded that low RM and S-HBsAg preservation could be achieved in that way. Tests  revealedApart from the temperature profile and duration, other parameters as chamber pressure and type and nature of drying container can influence process performance. Pressure should be maintained well below the ice vapour point at the product temperature to allow high sublimation rate. But it also must be kept over certain level to avoid large heterogeneity in heat transfer and product contamination. Therefore, the optimum chamber pressure is a compromise with the moderate values of 0.15–0.2 mbar giving optimal results and with little influence on process effect within this range , 49. AccIn our study we devised primary parameters for efficient preservation of S-HBsAg during freeze-drying on a laboratory scale. For a successful scale-up of this process, it is important to convert the developed small-scale cycle parameters to a full-scale production. When transferring to commercial scale equipment, it is likely that the dryer size would be 10–100 times larger. As the heat and mass transfer coefficients will change, respectively, shelf temperature will have to be adjusted accordingly in order not to exceed the maximum product temperature. In addition, an appropriate endpoint of drying should be established for a respective equipment to secure adequate final relative moisture level , 51. Anoμg VLPs/g DW was achieved as plant material with ≥10 μg VLPs/g FW was processed. The highest level in lyophilised material was 192.4 μg/g DW when plant material with 15.9 μg VLPs/g FW was used. The improved freeze-drying procedure resulted in considerable increase in the S-HBsAg VLPs content in comparison to the previous trial, where only approximately 10 μg VLPs/g DW were obtained after lyophilisation of untreated material coming from plants expressing the antigen at similar level [The lyophilisation process is a turn-based method; hence a batch-to-batch variation does occur , 42. Owiμg/g DW [μg/g DW, but some batches resulted in values as low as 192.4 μg/g DW, with the mean value for all batches at 664.2 μg/g DW. Summarising, the elaborated freeze-drying process expressed satisfactory reproducibility regarding S-HBsAg preservation. Furthermore, obtained material was characterised by a high VLPs content along with the total S antigen level being not excessive, which is beneficial in terms of oral vaccination.In contrast the situation regarding the total S-HBsAg level was quite different. The after-process values differed dramatically, showing no clear connection with VLPs preservation or degradation. Complete VLPs retention exhibited both an increase in the total antigen content up to 556% (batch V-2) or at an unchanged level. A possible explanation of its variation during freeze-drying might be the partial destruction of VLPs, resulting in an increase of the total antigen content, similarly as in freeze-thawing see . Even thμg/g DW . In this i.m. priming with the Engerix B vaccine received an oral booster with powdered lyophilised tissue (see μg/g DW and 199% or 538 μg/g DW for the total antigen. Hence, the dose of 50 ng S-HBsAg was delivered in 1.72 mg of freeze-dried tissue per individual subject. Results of mouse vaccination are summarised in To confirm the retained immunogenicity of S-HBsAg in freeze-dried plant tissue, animal immunisation trials were performed. Mice afterssue see  based onμg/g DW for 8 months. These values were close to those recorded in the previous report [μg/g of S-HBsAg VLPs in lyophilised plant material may be a natural upper limit that can be stable in the dried state at higher temperatures. Sucrose, while effective during freeze-drying itself, fails to serve as a protectant for S-HBsAg during storage at room or higher temperatures. Improvement in long-term storage at elevated temperatures, important in the aspect of vaccine distribution economy and common availability, would require further tests with a probable use of additional excipients. The obtained freeze-dried formula was stable when stored at 4°C, with the S-HBsAg VLPs content of over 200 μg/g DW, several times higher than the value reported previously [Apart from immunogenicity preservation of plant-associated S-HBsAg, long-term stability of the antigen is crucial for vaccine design. Respective tests were conducted with sucrose soaked, freeze-dried, and powdered plant material, which was subsequently placed in sealed containers and stored for one year at three temperatures, 4°C, 22°C, and 37°C . The VLPTo a large extent we have improved processing via freeze-drying of plant material containing the HBV small surface antigen for the purpose of a potential oral vaccine. The procedure included excipient infiltration, freeze-drying, and powdering. The obtained formulation resulted in approx. 100% preservation of S-HBsAg assembled into immunogenic VLP structures, as well as retaining the total antigen content at the desired, not excessive, level.μg/g DW. Finally, postprocessing preservation of native and immunogenic S-HBsAg in plant-derived preparation was confirmed during mouse immunisation trials when the orally administered preparation used as boosting elicited an immune response comparable to routine injection vaccination.Sucrose demonstrated appropriate properties as a lyoprotectant and screening of physical parameters of freeze-drying resulted in selecting appropriate shelf temperatures and drying times with regard to maintaining S-HBsAg structure and antigenicity. In addition, certain boundaries of freeze-drying parameters of shelf temperature and residual moisture were identified that cannot be exceeded to ensure the S antigen preservation. Consequently, the improved plant material processing was reproducible and made it possible to obtain the final product with a high content of S-HBsAg VLPs, up to 200 Long-term stability tests revealed that storage of the plant-derived preparation at 4°C made it possible to retain practically unchanged S-HBsAg VLPs level. However, at elevated temperatures VLPs were gradually degraded. Those results may be further expanded on studies of long-term stability at elevated temperatures, an important aspect for an easily distributed and commonly available vaccine. As a result of this research, a basis for an efficient freeze-drying process has been established and a suitable semiproduct for an oral plant derived vaccine against HBV was obtained.To assess excipient potency for stabilisation of S-HBsAg during the freeze-drying process series of substances were tested with regard to their reported lyoprotective properties and requirements of oral vaccine formulation. For the preliminary test, fresh lettuce leaves were infiltrated with sucrose, glucose, mannitol, glycine and glycerol at concentrations of 100, 250 and 500 mM under 100 mbar vacuum for 5 min and lyophilised at 5°C and 20°C for primary drying and 22°C for secondary drying.Basing on absolute antigen values and relative preservation, it could be stated that across all tested variants sucrose and then mannitol at highest concentrations appeared as the most suitable for lyoprotection of S-HBsAg. Statistical analysis revealed that sucrose 500 mM was the most effective variant regarding relative content of VLPs in both drying profiles. As a consequence this protectant was chosen for further testing as expressing the best potential of S-HBsAg preservation, even when used in the unfavourable drying profile.


AAV vectors encoding cDNA for dominant-negative tumor necrosis factor (DN-TNF) or GFP (control) were injected into the striatum of young adult wild type WT and YAC128 TG mice and achieved 30–50% target coverage. Expression of dominant negative TNF protein was confirmed immunohistologically and biochemically and was maintained as mice aged to one year, but declined significantly over time. However, the extent of striatal DN-TNF gene transfer achieved in our studies was not sufficient to achieve robust effects on neuroinflammation, rescue degenerating MSNs or improve motor function in treated mice. Our findings suggest that alternative drug delivery strategies should be explored to determine whether greater target coverage by DN-TNF protein might afford some level of neuroprotection against HD-like pathology and/or that soluble TNF signaling may not be the primary driver of striatal neuroinflammation and MSN loss in YAC128 TG mice.CNS inflammation is a hallmark of neurodegenerative disease, and recent studies suggest that the inflammatory response may contribute to neuronal demise. In particular, increased tumor necrosis factor (TNF) signaling is implicated in the pathology of both Parkinson's disease (PD) and Alzheimer's disease (AD). We have previously shown that localized gene delivery of dominant negative TNF to the degenerating brain region can limit pathology in animal models of PD and AD. TNF is upregulated in Huntington's disease (HD), like in PD and AD, but it is unknown whether TNF signaling contributes to neuronal degeneration in HD. We used  Mutant htt causes degeneration of GABAergic medium spiny neurons (MSN), predominantly in the caudate putamen and cortex, leading to uncontrolled movements, emotional disturbances, dementia and death. A number of cell-autonomous and non-cell-autonomous effects of mutant htt have been documented, suggesting that the mechanisms underlying HD pathology are complex Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG repeat expansions in the Chronic neuroinflammation is a characteristic feature of neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) in vitroWhile pathological effects of inflammation have been established in other models of neurodegenerative disease The YAC128 mouse genome contains a yeast artificial chromosome (YAC) that carries the entire human htt gene, including a 120 CAG repeat mutation The human full-length cDNA sequence required for generating DN-TNF protein (TNF variant A145R/I97T) in mammalian cells 2. The cells were plated at 30–40% confluence in CellSTACKs (Corning) 24 h before transfection (70–80% confluence when transfection). 1.8 mg helper plasmid pXYZ1 (AAV helper plasmid for serotype 1) and 0.6 mg rAAV transfer plasmid were co-transfected into HEK293 cells in a 10-CellSTACK culture chamber (Corning) using the calcium phosphate precipitation procedure 12–6×1012 vg (vector genomes)/mL and AAV-GFP virus titers were 6.4×1011–7.3×1012 vg/mL.AAV2 serotype 1 viruses were produced using Human Embryonic Kidney (HEK) 293 cells for packaging. Cells were maintained in Dulbecco's modified Eagles medium (DMEM), supplemented with 5% fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/ml streptomycin in 37°C incubator with 5% COCytoplasmic-to-nuclear translocation of the p65RelA subunit of the NFkB complex was used as a measure of TNF signaling activation. HEK293 cells were plated at the density of 0.02 million cells/well in a 12-well plate. Once cells attached, 2 µL AAV-GFP or AAV-DN-TNF were added to the 1 mL culture medium. After incubation for 3 days, the cells were treated with 10 ng/mL mouse TNF (R&D Systems)  for 30 minutes, then washed with PBS and fixed with 4% paraformaldehyde. Immunocytochemical staining was performed using anti-p65 primary antibody  and Alexa Fluor 594 secondary antibody (Invitrogen A11012).Generation and breeding of YAC128 HD transgenic mice (FVBN/NJ background strain) has been described previously 2 month old animals Table 1 Mice underwent behavioral testing on balance beam and rotarod apparatus  at 3, 5, 7, 9, 11 and 12 months of age. At each time point, mice were trained for 3 days on each task followed by testing on the fourth day. Training and testing were done at the same time each day. Beamwalk training consisted of 4 traverses across a 17-mm round beam into a home cage. On the test day, mice were required to traverse 3 different beams  into a home cage with 3 trials on each beam. The 3 trials were done consecutively with 30 min. of rest between different beam sizes. Average time to traverse the beam was calculated and used to compare groups. Rotarod training consisted of 3 runs in which mice were placed on an accelerating rotarod (0.2 rpm/s) until they fell from the rod. The test day consisted of 3 trials running on the accelerating rotarod with an inter-trial interval of 30 min. Average time spent running on the rod was measured and used to compare between groups.A human TNF-specific ELISA can be used to detect expression of DN-TNF without cross-reactivity with endogenous murine TNF. Mice were given a lethal dose of Euthasol (Burns Vet supply), decapitated and the left and right striata were dissected, weighed and immediately flash frozen in liquid nitrogen. Right samples were sonicated in 20 µL/mg of cold lysis buffer containing a protease inhibitor cocktail , centrifuged at 4°C and used for huTNF-specific ELISA according to manufacturer's instructions .Power SYBR green PCR Master Mix (Applied Biosystems) and a concentration of 0.15 µM of each PCR primer. All reactions were performed in triplicate and average values were used. mRNA expression levels were normalized to those of the mouse housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was found to be unchanged by genotype or treatment, and evaluated by the comparative CT method . Values were normalized to 3 month old WT animals and compared by 2-way ANOVA  followed by Bonferroni posthoc tests.Left striatum samples  were collected and flash frozen as described above and homogenized in ice-cold RLT buffer  containing 2-mercaptoethanol using a rotor-stator homogenizer followed by centrifugation in QIAshredder spin columns (Qiagen). RNA was isolated using the Qiagen RNeasy kit  according to manufacturer's instructions, treated with DNase I , and reverse transcribed using Superscript II RNase H-reverse transcriptase (Invitrogen). RNA concentration was determined by absorbance at 260 nm using a Nanodrop . Oligonucleotide primers for gene amplification of murine TNF, interleukin-6 (IL-6), chemokine (C-C motif) ligand 2 , chemokine (C-C motif) ligand 5 , protein tyrosine phosphatase receptor type C , glial fibrillary acidic protein (GFAP), CD68, chemokine (C-C motif) ligand 3 , inducible nitric oxide synthase (iNOS), chemokine (C-X-C motif) ligand 2 (MIP2), interferon gamma (IFNγ), interleukin-4 (IL-4), and interleukin-10 (IL-10) were obtained from Integrated DNA Technologies and validated by analysis of template titration and dissociation curves (primer sequences available on request). Quantitative real-time PCR was performed using an ABI Prism 7900HT Fast Detection System (Applied Biosystems). Each 10 µL reaction was performed in 384-well format with 25 ng of cDNA, 5 µL of Levels of murine interferon-gamma (IFNγ), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), chemokine (C-X-C motif) ligand 1  and tumor necrosis factor (TNF) in serum were analyzed using a multiplex assay according to manufacturer's instructions . For serum collection, animals were deeply anesthetized with ketamine/xylazine cocktail (described above), then decapitated. Blood was collected in vacutainers  and spun at 13,000 rpm for 10 min. The serum fraction was removed and frozen at −80°C. Serum samples from individual animals were used in the multiplexed immunoassay.Elite ABC kit (Vector Laboratories) and detected with diaminobenzidine (DAB) (Vector Laboratories). Labeled sections were mounted on slides and coverslipped using Glycergel Mounting Medium . Stereo Investigator analysis software  was used for carrying out unbiased stereological counts of NeuN-positive neuronal nuclei in the striatum, employing the optical fractionator method Animals used for quantification of MSNs were the same animals used for behavioral testing. After transcardial perfusion with 12.5 mL ice cold PBS supplemented with 0.1% glucose and 0.1% heparin followed by 50 mL 4% paraformaldehyde (PFA) in PBS, dissected brains were post-fixed in 4% PFA for 24 h at 4°C, equilibrated in 20–30% sucrose in PBS for 24–48 h at 4°C and stored in PBS plus 0.01% sodium azide at 4°C. The brains were then sliced into 30 µm-thick coronal sections using a SM2010R sliding microtome . Coronal sections spaced 360 µm apart throughout the striatum (+1.70 mm to −2.30 mm relative to Bregma) were collected. For quantification of neuronal loss, sections were stained with anti-NeuN monoclonal antibody  using a free-floating protocol. Biotinylated anti-mouse IgG reagent was used as the secondary antibody . Signal was amplified with a Vectastain Tissue was prepared for labeling as described for stereological quantification of NeuN positive neurons above. Coronal sections spaced 360 µm apart throughout the striatum (+1.70 mm to −2.30 mm relative to Bregma) were collected and stained with goat polyclonal anti-Iba1 antibody  using a free-floating protocol. Alexa Fluoro 594 goat anti-rabbit IgG  was used as the secondary antibody. Cover slips were applied to mounted brain sections using Fluoro-Gel anti-fade (Electron Microscopy Sciences). Serial fluorescent images were captured and the number of striatal microglia per field  was quantified by using Nikon NIS-Element software.Coronal sections spaced 360 µm apart throughout the striatum (+1.70 mm to −2.30 mm relative to Bregma) were collected from 5 randomly chosen wild type animals that received AAV-GFP vector injections and mounted onto slides. The volume of the striatum that contained GFP-labeled cells was estimated using the Cavalieri Estimator Probe of the Stereo Investigator analysis software. The grid size was set to 100 µm.T-tests, one-way, and two-way ANOVA's were performed using GraphPad Prism software version 6.0 for Mac, GraphPad Software, La Jolla CA. Two-way repeated measures ANOVA's were performed using SPSS , Greenhouse-Geissen test was used (Epsilon <0.75) to calculate the appropriate adjustment to degrees of freedom of the F-statistic. Statistics are reported in the figure legends. Asterisks indicate the results of the post hoc tests following a significant interaction.All animal studies were approved by the Institutional Animal Care and Use Committee at The University of Texas Southwestern Medical Center.To provide rationale for an anti-inflammatory intervention to rescue MSNs, we first assessed the inflammatory profile of YAC128 transgenic (TG) compared to wild type (WT) mice using three independent approaches. First, we measured mRNA levels of inflammation-related genes in striata of naive mice as they aged over 12 months Table 2.To obtain a second measure of inflammation in YAC128 TG mice, we quantified the levels of inflammation-related proteins in serum from 12-month-old YAC128 TG and WT animals using multiplexed ELISA Fig. 1B.As a third measure of the inflammatory profile of YAC128 TG mice, we quantified activated microglia in the striatum using Iba1 immunohistochemistry Fig. 1C.Fig. S1A). To confirm that the AAV-derived DN-TNF was bioactive and capable of blocking soluble TNF signal transduction, we infected HEK293 cells and measured TNF-induced cytoplasmic-to-nuclear translocation of the p65RelA subunit of NFkB in vitro using immunocytochemistry. GFP fluorescence confirmed high infectivity (data not shown) and cells infected with AAV-DN-TNF displayed minimal nuclear enrichment of p65, whereas AAV-GFP infected cells displayed strong nuclear enrichment of p65 .We generated AAV vectors that expressed either GFP alone (negative control) or dominant negative TNF DN-TNF  and GFP,, in vivo transduction with AAV-GFP achieved coverage of ∼30–50% of striatal volume in aged animals, indicating that not all striatal neurons were maintained in an environment of increased DN-TNF expression throughout the study. To directly quantify DN-TNF levels, we used a human TNF-specific ELISA that specifically recognizes the DN-TNF human protein sequence but not endogenous mouse TNF . Locomotor performance was not improved in YAC128 TG mice following AAV-DN-TNF injection  than those measured in the substantia nigra after delivery of DN-TNF via lentivirus, a treatment that was effective in reducing dopaminergic neuron death in a striatal 6-hydroxydopamine model of PD Few studies have addressed whether inflammation contributes to neuronal death in HD, but several reports in rodent models of HD-like pathology have provided evidence that it may indeed play a role. For example, one recent report showed that microglial CB(2) cannabinoid receptor signaling reduced microglial activation and neuroinflammation and prevented neuron loss in a mouse model of intrastriatal quinolinic acid-induced excitotoxicity One emerging hypothesis is that mutant htt expression causes immune cell dysfunction that contributes to CNS pathology in HD. Kwan et al. in vitro, suggesting that the signaling machinery for TNF death receptor function is in place in vivo MSN in the striatum display a different arsenal of inflammatory responses (or are less susceptible to soluble TNF) relative to other regions where we have successfully neutralized soluble TNF, such as the substantia nigra pars compacta (SNpc) and achieved neuroprotection. In support of this idea, chronic infusion of DN-TNF protein into SNpc, but not into striatum, attenuated neuroinflammation and loss of DA neurons in SNpc induced by a striatal 6-hydroxydopamine lesion We hypothesized that TNF could be directly toxic to CNS neurons via the TNF death receptor signaling pathway–as observed in PD models–or could contribute to an enhanced inflammatory environment in the striatum that would cause activation of MG and release of other cytotoxic molecules. In support of this idea, MSN-like striatally-derived neuronal cell lines are susceptible to TNF-induced apoptosis What other factors in addition to, or independent of, poor target coverage and transgene silencing might account for the inability of AAV-DN-TNF to rescue MSN in striatum in the YAC128 model of HD? First, TNF levels may not be critical for neuronal death. Post-mortem studies In summary, we conclude that the YAC128 mouse model displays age-dependent neuroinflammation in the striatum and is therefore an appropriate model in which to explore the role of inflammation in MSN degeneration and HD-like phenotypes. However, our ability to test the hypothesis that soluble TNF plays a critical role in mediating these effects was limited by the inherent drawbacks associated with viral-mediated gene transfer. Specifically, our single bilateral injections of AAV-DN-TNF yielded at best 50% coverage of the target structure (i.e. the striatum) and expression of DN-TNF declined over time. Together, this level of neutralization of soluble TNF could have been insufficient to attenuate age-dependent neuroinflammation, MSN degeneration, and locomotor deficits in the YAC128 mice. On the other hand, these results could indicate that TNF signaling in the brain has different consequences in the context of mutant htt expression than in other neurodegenerative diseases.Given the technical limitations of our study, we believe these data should not be interpreted to mean that TNF and neuroinflammation in general do not play a role in HD pathophysiology. Instead, our results may highlight the importance of deciphering which inflammatory signals contribute to neuron death and precisely where these signals are important. Several studies have documented increased inflammation in HD by comparing levels of cytokines and chemokines in brain. Quantitative PCR using postmortem HD patient brains revealed a significant increase in IL-10, IL-6, IL-8, MMP9 and CCL2 and a trend toward an increase in TNF in the striatum of HD patients compared to control patients in vitro resulted in inhibition of TNF-dependent NFκB signal transduction, limited AAV-DN-TNF in vivo spread after single bilateral striatal injections and decline in DN-TNF protein levels over time may have contributed to the lack of effects on age-related neuroinflammation, MSN loss, and locomotor deficits in YAC128 mice. Given these unfortunate but not uncommon technical limitations of gene transfer, alternative delivery approaches for DN-TNF should be explored to ensure greater coverage of large anatomical structures like the striatum to unequivocally establish whether neutralization of soluble TNF affords therapeutic benefit in this mouse model of HD.YAC128 mice display moderate age-related increases in inflammatory markers and thus represent a good model in which to test the role of neuroinflammation in progression of HD-like pathology. While high transduction efficiency of AAV-DN-TNF Figure S1AAV vectors for the expression of dominant negative TNF. (A) AAV2 construct for expression of DN-TNF and enhanced GFP , with eGFP expressed from an internal ribosome entry site. AAV2 control construct (bottom) for expression of eGFP alone . (B) DN-TNF protein produced by AAV2/1 serotype virus is biologically active. In unstimulated HEK293 cells, p65RelA displays a cytoplasmic distribution regardless of whether they are transduced with AAV-GFP or AAV-DN-TNF (left panels); in cells treated with TNF (right panels), AAV-GFP-transduced cells display translocation of the p65RelA subunit of NFkB to the nucleus (top right), demonstrating that TNF receptors have been activated by TNF binding. In cells transduced with AAV-DN-TNF (bottom right), p65 does not enter the nucleus because soluble TNF has been sequestered away from TNF receptors by dominant negative TNF. Scale bar, 100 µm.(EPS)Click here for additional data file.Figure S2Aged YAC128 transgenic animals exhibit locomotor deficits in several functional tests. YAC128 TG and WT male and female mice were compared in tests of motor function including (A) time running on the accelerating rotarod and (B) time to cross a 17 mm, 11 mm and 5 mm diameter at 7, 9 and 12 months. Statistical data for rotarod (A): interaction p = 0.003, genotype p = 0.032, time p = 0.358. Statistical data for beamwalk (B): interaction p = 0.304, p = 0.072, p = 0.007 for 17, 11 and 5 mm beams, respectively; time p = 0.049, p = 0.002, p<0.0001 for 17, 11 and 5 mm beams, respectively; genotype p = 0.048, p = 0.063, p = 0.005 for 17, 11, 5 mm beams, respectively . *p<0.05, **p<0.01, ***p<0.001.(EPS)Click here for additional data file.


Eleven new human polyomaviruses (HPyVs) have been identified in the last decade. Serological studies show that these novel HPyVs sub-clinically infect humans at an early age. The routes of infection, entry pathways, and cell tropism of new HPyVs remain unknown. VP1 proteins of polyomaviruses can assembly into virus-like particles (VLPs). As cell culturing systems for HPyV are currently not available, VP1-derived VLPs may be useful tools in basic research and biotechnological applications.Recombinant VP1-derived VLPs from 11 newly identified HPyVs were efficiently expressed in yeast. VP1 proteins derived from Merkel cell polyomavirus (MCPyV), trichodysplasia spinulosa-associated polyomavirus (TSPyV), and New Jersey polyomavirus (NJPyV) self-assembled into homogeneous similarly-sized VLPs. Karolinska Institutet polyomavirus (KIPyV), HPyV7, HPyV9, HPyV10, and St. Louis polyomavirus (STLPyV) VP1 proteins formed VLPs that varied in size with diameters ranging from 20 to 60 nm. Smaller-sized VLPs (25–35 nm in diameter) predominated in preparations from Washington University polyomavirus (WUPyV) and HPyV6. Attempts to express recombinant HPyV12 VP1-derived VLPs in yeast indicate that translation of VP1 might start at the second of two potential translation initiation sites in the VP1-encoding open reading frame (ORF). This translation resulted in a 364-amino acid-long VP1 protein, which efficiently self-assembled into typical PyV VLPs. MCPyV-, KIPyV-, TSPyV-, HPyV9-, HPyV10-, and HPyV12-derived VLPs showed hemagglutination (HA) assay activity in guinea pig erythrocytes, whereas WUPyV-, HPyV6-, HPyV7-, STLPyV- and NJPyV-derived VP1 VLPs did not.The yeast expression system was successfully utilized for high-throughput production of recombinant VP1-derived VLPs from 11 newly identified HPyVs. HPyV12 VP1-derived VLPs were generated from the second of two potential translation initiation sites in the VP1-encoding ORF. Recombinant VLPs produced in yeast originated from different HPyVs demonstrated distinct HA activities and may be useful in virus diagnostics, capsid structure studies, or investigation of entry pathways and cell tropism of HPyVs until cell culture systems for new HPyVs are developed. Orthopolyomavirus and Wukipolyomavirus) and avian (Avipolyomavirus) species , according to the native virus gene sequence with one nucleotide changed to remove a restriction endonuclease XbaI recognition site, and 2) HPyV12 VP1s380 with a S. cerevisiae codon-optimized gene sequence [GenBank: KP293751]. Shorter 364-aa-long versions of the HPyV12 VP1 protein with native (HPyV12 VP1n364) and codon-optimized sequences (HPyV12 VP1s364) were amplified from HPyV12 VP1n380 and HPyV12 VP1s380 using Phusion High-Fidelity DNA Polymerase and the primers: HPy12-VP1ATG2-NX: ctctagaatggcacccaagaggaaaaccacctg; HPy12-VP1stop-NX: gtctagattatggaactggtgttatttcttgtc; HPy12-VP1-ATG2-SX: ctctagaatggcacctaaaaggaagaccacatg; HPy12-VP1stopSX: gtctagattatggaactggagtaatttc.Two versions of the HPyV12 VP1 protein, consisting of a 380-aa chain, were commercially synthesized by GenScript: 1) HPyV12 VP1nEscherichia coli DH5α cells.All VP1-encoding DNA fragments were sub-cloned into the XbaI site of the yeast expression vector pFX7 for target protein expression . Recombi380, pFX7-HPyV12-VP1s380, pFX7-HPyV12-VP1n364, pFX7-HPyV12-VP1s364, and pFX7-NJPyV-VP1 were transformed into the S. cerevisiae strain, AH22-214 . The growing conditions for yeast transformants harboring plasmids with VP1-encoding genes from different HPyVs and conditions for induction of VP1 synthesis were similar to that previously described [2, 0.25 M L-Arginine, 0.001 % Triton X-100, 10 mM Tris/HCl, pH 7.2), lysed by vortexing with an equal volume of glass beads for 7 min, and then cooled on ice for 1 min between each vortexing step. A 15–30-μL aliquot of each yeast lysate and the supernatant obtained after centrifugation of each yeast lysate were prepared for analysis by mixing with the SDS-PAGE sample buffer  and then boiling for 5 min. Prepared samples were then subjected to SDS-PAGE and run in SDS-Tris-glycine buffer. The resultant gel was stained with Coomassie brilliant blue  for protein band visualization.The vector pFX7, without any insert or constructed plasmids, pFX7-KIPyV-VP1, pFX7-WUPyV-VP1, pFX7-MCPyV-VP1, pFX7-HPyV6-VP1, pFX7-HPyV7-VP1, pFX7-TSPyV-VP1, pFX7-HPyV9-VP1, pFX7-HPyV10-VP1, pFX7-STPyV-VP1, pFX7-HPyV12-VP1nescribed . Briefly2, 0.001 % 0.25 M L-Arginine, and Trition X-100, in 10 mM Tris/HCl-buffer, pH 7.2) with 2 mM PMSF and EDTA-free Complete Protease Inhibitor Cocktail tablets , and then were homogenized with glass beads using Bead-Beater GB26 . The supernatant collected after centrifugation of yeast lysate was loaded onto a 20–69 % sucrose gradient in DB150 buffer and then centrifuged at 100,000 × g  overnight at 4 °C. Thereafter, 2-mL fractions were collected and samples from each fraction were analyzed using Coomassie brilliant blue-stained SDS-PAGE. Fractions containing VP1 were pooled, diluted in DB150 buffer, and after a 0.5 h incubation step with DNAse-free RNAse , VP1-derived VLPs were concentrated by ultracentrifugation at 100,000 × g for 4 h at 4 °C. Afterwards, pellets containing VP1 were dissolved in DB150 buffer and subjected to ultracentrifugation on a CsCl gradient with densities ranging from 1.23 to 1.46 g mL−1 at 100,000 × g overnight at 4 °C. Fractions were collected and analyzed, as described above. After pooling and diluting purified VP1-containing fractions in DB150 buffer, VP1-derived VLPs were precipitated by ultracentrifugation for 4 h at 100,000 × g. Pellets with VP1-derived VLPs were dissolved in PBS, dialyzed against PBS overnight, aliquoted, and then lyophilized or stored in PBS in 50 % glycerol at −20 °C. JCPyV, BKPyV, SV40, and HaPyV VP1-derived VLPs were prepared as previously described [Recombinant VP1 protein purification was carried out as previously described . Brieflyescribed .To confirm VLP assembly of recombinant VP1 proteins generated in yeast, a sample of VP1 purified by ultracentrifugation was placed on 300-mesh carbon-coated palladium grid. VP1-derived VLPs adhering to the grid were negatively stained with 2 % aqueous uranyl acetate solution and examined with a Morgagni 268 electron microscope .2O2 .Samples of purified recombinant VP1 proteins derived from all HPyVs and HaPyV were mixed with the SDS-PAGE sample buffer , boiled for 5 min, applied to a SDS-PAGE and run in SDS-Tris-glycine buffer. Protein bands in SDS-PAGE were electro-transferred to Immobilon P membrane  or visualized by staining with Coomassie brilliant blue . The membranes were blocked with 5 % milk in TTBS  and incubated overnight in the relevant antibody solutions at room temperature. Anti-JCPyV, −MCPyV, and -WUPyV VP1 mouse sera (raised against the respective VP1 protein) and anti-HaPyV VP1 rabbit serum  were dil−1 to 0.025 μg mL−1) in 25 μL PBS and mixed with 25 μL of a 1 % (v/v) guinea pig erythrocyte suspension in PBS. HA activity was recorded after a 3 h incubation at 4 °C. The HA titer was defined as the reciprocal of the highest antigen dilution clearly showing HA.VLPs were subjected to a HA assay using guinea pig erythrocytes in a U-shaped microtiter plate. VLPs of each purified VP1 protein were serially diluted (50 μg mL


To date, immune-modulatory treatments in sepsis have proved unsuccessful. Better understanding of inflammatory processes might help tailor such therapies more effectively and improve their efficacy. It is now recognised that some patients may require immune stimulating therapies.1. TNF converting enzyme (TACE), activated via p38-mitogen activated protein kinase (MAPK) mediated pathway2, is essential for the shedding of TNF and TNF receptors (TNFRs)-1&2. Altered TACE behaviour may be mechanistically important in deactivation.Monocytes display altered behaviour in response to systemic inflammation; this has been called “deactivation” and is characterised by reduced HLA-DR expression and attenuated tumour necrosis factor-α (TNF) release in response to lipopolysaccharide (LPS) stimulusTo determine whether monocyte TACE activity is affected by sepsis and is reflective of illness severity.3.Sixteen patients with sepsis, 15 healthy volunteers and 8 patients with non-infectious systemic inflammatory response syndrome (NI-SIRS) were recruited and data collected at baseline (D0), day 2 (D2) and day 4 (D4). Peripheral blood mononuclear cells were separated by density gradient centrifugation with HLA-DR, TACE and TNFR expression  determined by flow cytometry. LPS-induced TNF release was measured using ELISA. Monocytes were isolated using magnetic bead selection and TACE catalytic activity  was measured using fluorescence resonance energy transfer assayHLA-DR expression and LPS-induced monocyte TNF release were attenuated in sepsis and NI-SIRS (p< 0.01), consistent with deactivation. Sepsis, but not NI-SIRS, produced elevated basal monocyte TACE activity (p< 0.01) while LPS-induced increases in TACE activity were attenuated. Elevated D0 basal TACE activity positively correlated with APACHE II score in sepsis (p< 0.01), but not NI-SIRS.TACE expression was unaltered across all groups, but LPS-induced TNFR shedding appeared reduced by sepsis. Sepsis patients displayed an attenuated p38-MAPK response to LPS.Our data indicate that sepsis induces specific changes in monocyte TACE catalytic activity profiles that may reflect illness severity, whilst such changes were not seen in NI-SIRS. These changes in TACE activity attenuated LPS-induced release of TNFR and TNF from monocytes, and appear to be mediated through altered upstream p38-MAPK signalling.


Chemotherapy-induced cognitive impairment (CICI) is a common sequelae of cancer therapy. Recent preclinical observations have suggested that CICI can be mediated by chemotherapy-induced plasma protein oxidation, which triggers TNF-α mediated CNS damage. This study evaluated sodium-2-mercaptoethane sulfonate (Mesna) co-administration with doxorubicin to reduce doxorubicin-induced plasma protein oxidation and resultant cascade of TNF-α, soluble TNF receptor levels and related cytokines.2) or saline administration occurred 15 minutes prior and three hours post doxorubicin. Pre-treatment and post-treatment measurements of oxidative stress, TNF-α and related cytokines were evaluated during the two experimental cycles of chemotherapy.Thirty-two evaluable patients were randomized using a crossover design to receive mesna or saline in either the first or second cycle of doxorubicin in the context of a standard chemotherapy regimen for either non-Hodgkin lymphoma or breast cancer. Mesna (360 mg/mCo-administration of mesna with chemotherapy reduced post-treatment levels of TNF-related cytokines and TNF-receptor 1 (TNFR1) and TNF-receptor 2 (TNFR2) . Patients with the highest pre-treatment levels of each cytokine and its receptors were the most likely to benefit from mesna co-administration.The extracellular anti-oxidant mesna, when co-administered during a single cycle of doxorubicin, reduced levels of TNF-α and its receptors after that cycle of therapy, demonstrating for the first time a clinical interaction between mesna and doxorubicin, drugs often coincidentally co-administered in multi-agent regimens. These findings support further investigation to determine whether rationally-timed mesna co-administration with redox active chemotherapy may prevent or reduce the cascade of events that lead to CICI.NCT01205503.clinicaltrials.gov  Symptoms of chemotherapy-induced cognitive impairment (CICI), or ‘chemobrain’, are common complaints of cancer survivors. While incidence estimates of CICI vary widely , CICI isin vivo mesna co-administration with Dox prevents Dox-induced plasma oxidative stress in mice . Al. Al13]. During cycles one and two of each participant’s planned treatment, blood samples were obtained immediately prior to test-article administration and six hours after the infusion of Dox. The blood samples were placed on ice at the bedside and the plasma was prepared within 30 minutes of being drawn to ensure as little modification of plasma proteins as possible. Once the plasma samples were prepared, they were snap frozen and stored at -80°C until analyses.Oxidative Stress Assays: The slot-blot method was used to determine levels of protein oxidation [indexed by protein carbonyl (PC) and 3-nitrotyrosine (3-NT)] and lipid peroxidation [indexed by protein-bound 4-hydroxy-2-nonenal (HNE) as previously described .Enzyme-linked Immunosorbent assay (ELISA): Plasma samples were used to measure TNF, TNFR1, TNFR2 and IL-18 levels, according to the human ELISA following the manufacturer’s instructions . The TNF, TNFR1, TNFR2 and IL-18 concentration in the sample was calculated from the recombinant human TNF and its receptors as well as IL-18 standard curves. The means of minimum detectable dose of the assays were 0.106 pg/ml, 0.77 pg/ml, 0.6 pg/ml and 12.5 pg/ml for TNF-α, TNFR1, TNFR2, and IL-18, respectively.Demographics and clinical characteristics of the 32 patients enrolled in the study are summarized in The co-administration of mesna with Dox reduced plasma levels of TNF-α, TNFR1 and TNFR2 compared to saline for patients with high baseline values receiving Dox for breast cancer and NHL. There were significant baseline differences between lymphoma versus breast cancer patients for TNF-α, TNFR1, TNFR2 and IL-18. To control for these differences, a linear mixed model found statistically significant differences in these measures over time as well as a baseline-by-treatment interactions. The baseline value was a significant covariate in these models . FurtherTo further analyze the baseline by treatment interaction, Bonferonni adjustments for post-hoc comparisons at various baseline levels were considered significant for any p<0.017. As shown in In addition, Unlike the findings with cytokine levels, there were no significant baseline differences between lymphoma versus breast cancer patients with PC , plasma protein-bound HNE  and 3-NT . With regards to PC, protein-bound HNE, and 3-NT, no statistically significant differences were observed between the mesna and saline groups at six hours post Dox administration, though a non-statistically significant reduction in PC was observed with mesna (p = 0.20).Grade 3 and 4 toxicities are reported in This study has demonstrated that mesna co-administration decreases TNF-α, TNFR1 and TNFR2 levels after Dox-containing chemotherapy as compared to receiving Dox-containing chemotherapy alone, indicating a potential role for mesna in mediating toxicities resulting from TNF-α signaling during cancer treatment. Furthermore, the finding that this effect is most pronounced in patients with higher baseline levels of TNF pathway-related cytokines suggests that these baseline levels may be biomarkers that can identify patients most likely to benefit from the strategy of rationally administering mesna to patients receiving redox active chemotherapy.We embarked on the preclinical and clinical evaluation of mesna interaction with Dox in relationship to TNF-α in part because of our initial findings that patients who were receiving chemotherapy who coincidentally received mesna with their chemotherapy had significantly less PC, a marker of oxidative stress (p<0.05) . HoweverIn a previous observational clinical study, paired samples from patients receiving Dox found that TNF-α plasma levels are significantly increased at six hours after treatment with Dox . This clMesna is approved by the U.S. FDA for the prevention of hemorrhagic cystitis after treatment with ifosfamide chemotherapy. Upon intravenous infusion, mesna is rapidly oxidized to mesna disulfide (dimesna). Mesna and dimesna are rapidly filtered and excreted by the kidneys. In the nephron dimesna is reduced back to mesna and in the bladder mesna detoxifies acrolein and other toxic ifosfamide metabolites. Mesna is rapidly filtered by the kidneys and has a very limited volume of distribution . Mesna iIn our pre-clinical murine studies, Dox administration resulted in increased brain oxidative stress, even though neither Dox nor its major metabolite cross the blood brain barrier and enter the CNS , suggestIn the current study, the amount of inflammatory cytokine reduction achieved by co-administration of mesna with chemotherapy positively correlates with the patient’s pre-treatment cytokine levels. As illustrated in Figs Our study has important limitations. First, though the rationale of the study was based on our preclinical data that link Dox-mediated plasma protein oxidation, TNF-α and cognitive dysfunction, we did not measure any cognitive outcomes in the patients enrolled in the study. Our rationale was that we first wanted to demonstrate that mesna had the postulated biochemical effect on plasma protein oxidation and inflammatory cytokines before exposing a cohort of patients to rationally administered mesna during all cycles of therapy. Also, we did not measure the levels of anti-inflammatory cytokines, which also could play a role in our model of pathophysiology of CICI.In summary, we have shown that co-administration of mesna during one cycle of Dox-based chemotherapy reduced the post-treatment levels of TNF-α, TNFR1 and TNFR2. Subset analyses further support the additional hypothesis that those patients who present with highest pre-treatment levels of TNF-α, TNFR1 and TNFR2 are the most likely to benefit from mesna co-administration and should be prospectively tested in future studies. Although this study did not directly address the potential clinical role for mesna in the prevention of CICI, it did demonstrate a clinical interaction between mesna and Dox on cytokine levels associated with CICI, and therefore provides a clinical basis for a future mechanistically-based prospective evaluation of mesna for the prevention of CICI.S1 CONSORT Checklist(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


EMBO Molecular Medicine, Puimège et al (Mus musculus), vs. resistant (Mus spretus) mice, where TNFR1 expression is low. They reported that this expression is under the control of a microRNA (miR-511), which itself is induced by glucocorticoids.Tumor necrosis factor (TNF) is a fascinating anti-tumoral cytokine, which plays a key role in orchestrating the fight of innate immunity against infection. Concomitantly, TNF is a major player of the inflammatory response, as illustrated by successful therapeutic strategies targeting TNF in patho logies such as Crohn’s diseases, rheumatoid arthritis or psoriasis. In mice, TNF is able to induce tissue injuries and lethal shock. In this issue of ge et al  elegantl Although TNF was discovered for its anti-tumoral activity, its use in cancer has been rather limited to rare cases of soft tissue sarcoma in limbs that allow isolated limb perfusion . In this experimental setting, the process becomes fully TNF dependent. Noteworthy, the LD50 of LPS was similar in wild-type and TNF KO mice, whereas, as compared to wild-type mice, TNF-deficient mice were far more resistant when LPS was administrated together with D-galactosamine  or damage-associated molecular patterns (DAMPs) activated monocytes/macrophages, TNF is a homotrimer molecule released from the cell surface by a specific enzyme . It acts through two different TNF receptors . The binding of TNF to the extracellular domains of TNFR1 bridges the receptors, thus gathering the death domains of the cytoplasmic moiety and initiating the signaling cascade ending either in apoptosis or in the production of many inflammatory mediators . TNF also induces the release of other cytokines, which can act in synergy with TNF within an amplificatory feedback loop (e.g. interferon-γ (IFNγ) and interleukin-1β (IL-1β)), or which drive TNF-induced lethal shock  independently of any natural shedding; indeed, soluble TNFRI levels in plasma were also lower in M. spretus as compared to that in M. musculus. They established that the 10 amino acid difference of TNFRI in both mouse strains was not associated with any functional changes. Since similar levels of TNFRI mRNA were found in both types of mice, they focused on TNFRI translational regulation. They discovered that a microRNA (miR-511) could down-regulate TNFRI translation, and observed a higher expression of miR-511 in M. spretus mice. The most fascinating were the experiments in which the authors were able to protect sensitive C57BL/6 mice against TNF-induced lethality with hydrodynamic injection of plasmids expressing miR-511 and to sensitize resistant SPRET/Ei mice with plasmids expressing anti-miR-511. These approaches are said by the authors to only target the liver. To further complete the beauty of their demonstration, using glucocorticoid receptor antagonist (RU486), adrenalectomized mice and dexamethasone and measuring circulating corticosterone, they convincingly established that miR-511 is under the control of glucocorticoids.In their recent work, Puimège et al  decipherThis work enlightens the complexity of the regulatory loops involved in inflammation. TNF can activate the hypothalamic–pituitary–adrenal axis that results in the release of glucocorticoids, cytoplasmic receptors being themselves regulated by miRNAs. Once complexed to their receptors, glucocorticoids bind to glucocorticoid response elements (GRE), present in the promoters of numerous genes, favoring or inhibiting the transcription. Anti-inflammatory properties of glucocorticoids reflect their capacity to regulate the transcription of more than 900 GRE-element-containing genes, supporting the production of annexin-A1 and IL-10, and inducing the release of certain miRNAs. The demonstration of the induction of miR-511 by glucocorticoids and its capacity to limit the expression of TNFRI is a new clue that allows a better understanding of the complex control of inflammation by glucocorticoids.