Previous studies with gene-deficient mice (ADAMTS5-P) revealed that ADAMTS5  plays a functional role in adiposity and metabolic health. To confirm these observations, we have performed similar studies with an independently generated strain of ADAMTS5 deficient mice (ADAMTS5-J). Upon cold exposure as well as after high-fat diet feeding (diet-induced obesity or DIO model), these knockout (KO) mice developed less subcutaneous and gonadal white adipose tissue (WAT) as compared to their wild-type (WT) littermates (reduction was more pronounced in ADAMTS5-P mice). Enhanced browning of WAT, as monitored by expression of UCP-1 was seen in the ADAMTS5-J KO mice upon cold exposure but not in the DIO model (seen in both conditions with the ADAMTS5-P mice). Brown adipose tissue (BAT) mass was not different between KO and WT ADAMTS5-J mice, either upon cold exposure or in the DIO model (in contrast to the enhanced BAT mass with the ADAMTS5-P mice). Energy expenditure and thermogenesis were not significantly different between KO and WT ADAMTS5-J mice (in contrast to somewhat enhanced levels in ADAMTS5-P mice). Insulin sensitivity was improved in the ADAMTS5-J KO mice, and they were protected against non-alcoholic steatohepatitis in the DIO model (as the ADAMTS5-P mice). These data are thus similar for both strains of KO mice, confirming specificity of the phenotype, but some quantitative and qualitative differences are also observed. Hepatic triglyceride content was quantified with the FS* kit , as described [http://dx.doi.org/10.17504/protocols.io.katcsen) were stained with haematoxylin-eosin (H&E) to detect the presence of brown adipocytes in these sections (http://dx.doi.org/10.17504/protocols.io.j9qcr5w). This was confirmed by staining AT sections for UCP-1 using a polyclonal rabbit anti-mouse UCP-1 antibody , followed by biotin-coupled goat anti-rabbit secondary antibody  and a TSA biotinyl tyramide amplication kit  (http://dx.doi.org/10.17504/protocols.io.j9scr6e). Images were taken of the H&E and UCP-1 stained sWAT sections using a Axiovert 200M microscope  and the Axiovision Rel. 4.8.2. software (Zeiss) at a magnification of 400x. Liver sections (http://dx.doi.org/10.17504/protocols.io.katcsen) were stained with H&E or Sirius Red (http://dx.doi.org/10.17504/protocols.io.j9rcr56) to assess steatosis and fibrosis, respectively [et al [Metabolic parameters were determined using standard clinical laboratory assays . Two-sided values of p < 0.05 are considered statistically significant.versus 52% and 48% for ADAMTST5-J mice. For 295 ADAMTS5-P and 306 ADAMTS5-J mice that were genotyped, the genotype distribution followed the expected inheritance pattern for both males and females (Heterozygous breeding pairs of ADAMTS5-J mice (26 couples monitored) produced on average 5.5 ± 0.5 pups per litter with a mean time of 33 ± 1.7 days between litters; corresponding values for ADAMTS5-P mice (14 couples) were 6.4 ± 0.6 pups and 31 ± 2.0 days. Gender distribution was equal, with 51% male and 49% female for ADAMTS5-P  females . The somAt 8 weeks of age, body weights of male ADAMTS5-J WT and KO mice  were indCidea or Ucp1 was, however, not different between WT and Adamts5-/--J strains (Adamts5-/--J samples (in contrast to Adamts5-/--P mice) (ADAMTS5-J homozygous deficient and WT mice showed comparable body weights before and after (p = 0.08) 15 weeks of HFD feeding. Food intake was comparable for both genotypes . After 1 strains . UCP-1 p-P mice) .Analysis of plasma metabolic parameters did not reveal significant differences in glucose, cholesterol or triglyceride levels between both strains. Insulin levels were lower, resulting in a reduction of the HOMA-IR by 50% .2, VCO2 and calculated RER were not different between genotypes . Western blotting for UCP-1 revealed slightly elevated protein levels in sWAT of both KO strains (Adamts5-/--J versus Adamts5-/--P sWAT protein extracts showed reduced UCP-1 protein levels (p = 0.02) when levels were expressed relative to Adamts5+/+-P levels . Interestingly, Adamts5 gene expression was significantly lower in sWAT and GON AT of WT mice kept at 4°C as compared to 24°C , was slightly enhanced for KO mice  a significantly higher BAT mass, (ii) loss of body weight, and (iii) markedly higher expression of UCP-1 in sWAT. Therefore, we have also exposed WT and BAT mass . Express KO mice . Western WT mice . Fig 5E Adamts5-/--J mice  and Fasn (Fatty acid synthase) and the fibrosis markers Timp1 , Col1a1 (Collagen type 1) and Tgfβ1 (Transforming growth factor β1) was significantly lower for Adamts5-/--J as compared to WT mice (F4/80 and Csf1r (Colony stimulating factor 1 receptor) in liver tissues as compared to WT control littermates (Tnf-α (Tumour necrosis factor-α); Il-1β (Interleukin-1β) and Mcp-1 (Monocyte chemoattractant protein-1)) and M2 macrophage markers (Arg1 (Arginase 1) and Mrc1 (Mannose receptor c type 1)) did not show significant differences in gene expression between both groups  [Previous studies comparing ts5-/--P  and WT mts5-/--P , 12, 14.s5-/--J) .Adamts5-/--J mice of the present study with that of the Adamts5-/--P mice reveals some similarities supporting specificity of the phenotype, but also some quantitative and qualitative differences: (i) with respect to WAT mass: in Adamts5-/--J mice lower sWAT and GON fat mass was found, which was significant after 2 weeks of cold exposure, but only showed a trend in the DIO model; (ii) with respect to BAT mass: in Adamts5-/--J mice no significant difference with WT mice was observed after 2 weeks of cold exposure nor in the DIO model; (iii) with respect to browning of WAT: in Adamts5-/--J mice higher expression of UCP-1 mRNA and protein was found after 2 weeks of cold exposure (mRNA: 1.5- to 2-fold as compared to 5-fold for Adamts5-/--P mice). Furthermore, cold exposure for 72h led to lower UCP-1 protein levels (relative to ADAMTS5-P WT) in Adamts5-/--J versus Adamts5-/--P sWAT extracts. However, in the DIO model UCP-1 or Cidea expression levels in sWAT were not different between WT and Adamts5-/- -J mice (as compared to 30-fold enhancement for Ucp1 or 5-fold for Cidea for Adamts5-/--P versus WT) [2, VCO2 or RER (same as for Adamts5-/--P mice), and no effect of genotype was observed on energy expenditure or heat production, in contrast to Adamts5-/--P mice [-/--P and not in ADAMTS5-/--J mice as compared to their respective control littermates. In addition, (iv) with respect to insulin sensitivity: for Adamts5-/--J mice a 50% lower HOMA-IR was found in the DIO model as compared to WT mice, as also seen in the Adamts5-/--P mice (which was confirmed by insulin tolerance tests) [Adamts5-/--J mice had lower liver weight and hepatic triglyceride content and developed less steatosis or fibrosis, as shown by histological stainings and expression of marker genes (as observed for the Adamts5-/--P mice). Furthermore, with respect to the hepatic inflammatory profile: both KO strains showed reduced mRNA levels of the pan-macrophage markers F4/80 and Csf1r.Comparison of the phenotype of the rsus WT) . Indirec--P mice . These oe tests) , and (v)Adamts5-/--P mice. Browning of WAT is observed in both strains upon cold exposure, but only for the ADAMTS5-/--P strain in the DIO model (less stringent condition). The activated thermogenic profile in Adamts5-/--P mice was due to an increased activation of the β3-AR signaling pathway via protein kinase A and CREB [ADAMTS5-/--J mice may be a lower activation of the β3-AR pathway. Insulin sensitivity is improved in both KO strains, and both strains are protected against NASH.Thus, ADAMTS5 deficiency is associated with reduced WAT mass in both KO strains, and with enhanced BAT mass only in and CREB . Therefoin vitro differentiation into mature adipocytes of 3T3-F442A preadipocytes with stable Adamts5 gene silencing, or of embryonic fibroblasts derived from Adamts5-/--P mice, was significantly impaired as compared to control cells [De novo fat pad formation following injection of 3T3-F442A cells with Adamts5 knockdown in NUDE mice was also significantly reduced as compared to controls [Adamts5-/--P mice were performed in total in 60 WT and 54 KO mice on HFD, in several independent studies conducted over a 3 year period. In addition, similar data were obtained using a different diet composition [Specificity of the ADAMTS5-P phenotype is further supported by our finding that ol cells . De novocontrols . Furtherposition .Adamts5-/--P versus 100% C57Bl/6J for Adamts5-/--J [Adamts5-/--P mice, exon 2 (encoding part of the proteolytic domain) was flanked by loxP sites, in combination with Cre-recombinase under control of the Ella promoter [Adamts5-/--J mice, the targeting construct containing an IRES-lacZ-neomycin cassette, substituted 134 nucleotides of exon 2, including the catalytic site [et al. reported the presence of a truncated ADAMTS5 species (34–40 kDa) in muscle extracts of WT mice that was undetectable in equivalent KO samples (both Adamts5-/--P and Adamts5-/--J) [There may be several explanations for the quantitative differences observed between both ADAMTS5 KO strains. For both sets of studies true littermates were used, all males of comparable age and body weight and all correctly genotyped. Housing conditions and diets were also comparable. Although we have thus done every effort to perform the studies with both strains under comparable external and experimental conditions, we can not exclude differences in microbiota. It is indeed known that the bacterial composition of the gut can significantly affect the metabolic state , 20. Thets5-/--J , 15, 21)ts5-/--J , 23. Alsts5-/--J , 25. Furpromoter . For thetic site ) that ars5-/--J) . Some fus5-/--J) , 27. A ns5-/--J) .in vivo gene silencing of the target may help to confirm the phenotype.In any case, our observations strongly suggest that data obtained in a single strain of KO mice should be interpreted with great care. Ideally, a phenotype rescue by (tissue)specific (over)expression of the missing component should confirm specificity. In addition, a tissue-specific KO or S1 FigTnf-α (Tumour necrosis factor-α); Il-1β (Interleukin-1β) and Mcp-1 (Monocyte chemoattractant protein-1)) and M2 macrophage markers (Arg1 (Arginase 1) and Mrc1 (Mannose receptor c type 1)) in livers of obese WT (white bars) and Adamts5-/--J (black bars) mice were normalized to the housekeeping gene β-actin and shown relative to WT. Data are means ± SEM of 6 (WT) or 7 (Adamts5-/--J) experiments. ADAMTS5, a disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5; WT, wild-type; HFD, high-fat diet.Gene expression levels of M1 macrophage markers ((TIF)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.


Few studies have examined skeletal muscle O2 delivery/utilization, and no previous study has evaluated the differences between HF patients with reduced LVEF (HFREF) versus those with preserved LVEF (HFPEF). We used novel MRI-based techniques to non-invasively measure quadriceps (leg) blood flow, O2 extraction and VO2 recovery kinetics in clinically stable patients diagnosed with HFREF or HFPEF.Delayed oxygen uptake (VO2 saturation (%SaO2) were measured in the femoral vein post-exercise (knee-extension) using MRI , hemoglobin (Hgb) and hematocrit (from blood sampled prior to exercise) are used to calculate leg VO2, from the Fick equation . However, HFREF patients had severe impairment of VO2 recovery kinetics (increased MRT), while HFPEF had a moderate impairment, as compared to HC , however, the HFREF group had marked impairment in leg blood flow recovery dynamics, compared to both HFPEF and control groups  and HFPEF  patients were recruited from the Alberta HEART study. Quadriceps muscle mass, peak leg flow, A-VO2 recovery kinetics are related to the degree of functional impairment and are strongly predictive of mortality. We show for the first time that muscle-specific VO2 recovery kinetics are significantly more delayed in HFREF compared to HFPEF (reflecting a larger oxygen debt for a similar amount of work). These findings suggest distinct mechanisms may underlie the reduced exercise capacity in HFREF vs HFPEF, with potentially distinct diagnostic metrics and therapeutic approaches.Whole body VO


These mice exhibited impaired innate immunity and high susceptibility to cerebral Mycobacterium tuberculosis infection, a similar phenotype to complete TNF-deficient mice. Further, MT-TNF−/− mice were not able to control T cell responses and cytokine/chemokine production. Thus, our data suggested that collective TNF production by both myeloid and T cells are required to provide overall protective immunity against CNS-TB infection.Tuberculosis of the central nervous system (CNS-TB) is a devastating complication of tuberculosis, and tumor necrosis factor (TNF) is crucial for innate immunity and controlling the infection. TNF is produced by many cell types upon activation, in particularly the myeloid and T cells during neuroinflammation. Here we used mice with TNF ablation targeted to myeloid and T cell (MT-TNF Tumor necrosis factor (TNF) is a pleiotropic cytokine involved in protective cell-mediated immunity against mycobacterial infection. In humans, primates and mice, TNF is shown to be essential for the maintenance of granuloma structures and for effective control of pulmonary mycobacterial replication –4. MutatMycobacterium tuberculosis (M. tuberculosis) after primary lung infection  is a severe form of extrapulmonary tuberculosis, generally caused by hematogenous dissemination of nfection , 8. Earlnfection –11. In anfection , 13, andnfection , 15.Homeostasis is crucial for neural activities; therefore, the interactions between the CNS and immune systems must be tightly regulated to maintain balance between immune and neural functions. This is supported by the blood–brain barrier which separates the CNS from the regular systemic blood circulation that protects the CNS from the milieu of the periphery. However, research findings have demonstrated that both resident and infiltrating immune cells participate actively in immune responses in the CNS, which upon activation may secrete cytokines including TNF –20. AlsoM. tuberculosis infection. We used mice deficient for both myeloid- and T cell-derived TNF (MT-TNF−/−) and investigated outcomes after intracerebral M. tuberculosis infection in comparative studies with TNF floxed wild-type (TNFf/f) and complete TNF-deficient (TNF−/−) mice. We demonstrate the significance of TNF production by myeloid and T cells for controlling CNS-TB infection.In the brain, TNF is synthesized by both immune (including infiltrating cells) and non-immune cells (including neurons). Microglial cells, as the resident immune cells of the CNS, are thought to be the first line of defense and main source of TNF during CNS-TB , 22–24. f/f), myeloid TNF deficient (MN-TNF−/−), T-cell TNF deficient (T-TNF−/−), myeloid and T-cell TNF deficient (MT-TNF−/−), and complete TNF deficient (TNF−/−) were previously described  of M. tuberculosis H37Rv using Hamilton syringe . The burr hole was sealed with bone wax and the skin sutured.M. tuberculosis. Organs were weighed and homogenized in 0.04% Tween 80 saline. Also, 10-fold serial dilutions of organ homogenates were plated in duplicates on Middlebrook 7H10  agar containing 10% OADC , and incubated at 37°C for 19–21 days. The concentration of M. tuberculosis was then determined by counting the CFUs.Bacterial burdens in the brains, lungs, and spleens of infected mice were determined at specific time points after infection with Mouse brains were collected to generate single cell suspensions. Non-specific binding to cells was blocked through incubation with αFcγRIII (1 mg/ml of rat α-mouse CD32/16c). The following antibodies were used to stain the surface markers: CD11b-PerCP-Cy5-5 (Clone M1/70), CD11c-Alexa 700 (Clone HL3), CD45-APC (Clone 30- F11), CD80-FITC (16–10 A1), CD86-V450 (Clone GL1), MHCII/(I-A/I-E)-PE (M5/114.15.2), CD3?-Pacific Blue, CD4-Alexa 700, and CD8-PerCP-Cy5.5. All antibodies were from BD Pharmingen™ and BD Horizon™. After staining, cells were washed and fixed, then analyzed on BD LSR Fortessa (Beckton Dickinson) flow cytometer using Cell Quest software.M. tuberculosis infection. The chemokines MCP-1, MIP-1α, and RANTES; and the cytokines IFN-γ, IL-2, IL-6, IL-12p70, and TNF  were measured using commercially available ELISA reagents according to the manufacturer’s instructions. Chemokine and cytokine concentrations were measured by absorbance using a Versamax Microplate Reader  with SoftMax software.Supernatants from brain homogenates were prepared for cytokine and chemokine measurement by enzyme-linked immunosorbent assay (ELISA) after 3 weeks subsequent to intracerebral t-test. For all tests, a p-value of ≤0.05 was considered significant.The data are presented as the mean ± SEM. Statistical analysis was performed by two-way ANOVA and one-tailed M. tuberculosis and compared disease progression in various cell-specific TNF-deficient mice, including myeloid specific (MN-TNF−/−), T-cell specific (T-TNF−/−), and TNF deficient in both myeloid and T-cells (MT-TNF−/−). We found that MN-TNF−/− and T-TNF−/− mice survived the infection similar to wild-type TNFf/f mice  and day 21 (p < 0.01) postinfection. We next assessed the extent of dissemination of bacilli from the brain by measuring the levels of pulmonary and splenic bacterial burden  and macrophages (CD11b+CD45high) in the brain and evaluated activation status of these innate immune cells subsequent to 3 weeks cerebral M. tuberculosis infection. The flowcytometric analysis revealed significantly higher (p < 0.05) frequencies and absolute cell numbers of microglia and macrophages in both TNF−/− and MT-TNF−/− mice  expression of CD80 and MHCII in MT-TNF−/− compared to TNFf/f mice . At 3 weeks postinfection, the frequency of CD3+CD4+ cells in the MT-TNF−/− and TNF−/− mouse brains was more than twofold higher compared to TNFf/f mice  than in TNFf/f mice at week 3 postinfection. Therefore, these data suggest that in the absence of myeloid and T cell-derived TNF, as well as in the complete ablation of TNF, the overall T cell response was deregulated by means of T cell influx and activation.To explore whether the myeloid- and T-cell-derived TNF alter influx of T cells into the brain, we quantified CD3M. tuberculosis infection. We measured concentrations of the cytokines, TNF, IFN-γ, IL-2, IL-6, and IL-12p70, as well as the chemokines, MIP-1α, MCP-1, and RANTES, in the brain homogenates of TNFf/f, MT-TNF−/−, and TNF−/− mice after 3 weeks intracerebral infection  than the TNFf/f-infected brains.As expected, no TNF production was detected in TNFM. tuberculosis infection. These results also illustrate that deletion of TNF from myeloid and T cells rendered an uncontrolled increase of IFN-γ and IL-6 protein and reduced IL-2 and IL-12p70 production. The results further demonstrate that ablation of TNF in myeloid and T cells resulted in an impaired chemoattractant response during cerebral M. tuberculosis infection.Therefore, data showed that myeloid and T cell-derived TNF have differential effects on cytokine and chemokine production during cerebral −/− mice were highly susceptible to cerebral tuberculosis infection. Therefore, we have demonstrated ablation of TNF from myeloid and T cells resembled complete ablation of TNF, suggesting that myeloid and T cells are major sources of TNF in mediating immune protection against CNS-TB. It also provides important evidence that synergistic activity of TNF derived from myeloid cells and T cells is required for optimum protection.It has been demonstrated that elevated levels of TNF are associated with TB meningitis and tuberculoma , 13. MorM. tuberculosis induces a TNF-dependent host immune response orchestrated from various cell types  to study the role of neuron-derived TNF in CNS-TB (−/− mouse by intracellular TNF staining that shows TNF production in the TNFf/f neurons, but not in the Ns-TNF−/− and TNF−/− neurons. While neuron-derived TNF is dispensable in protective immunity against CNS-TB, neurons contribute to TNF production during CNS-TB (M. tuberculosis bacilli (M. tuberculosis infection are only beginning to be elucidated.During pulmonary nfection , 30. In nfection , 35. Somnfection –38; howeg CNS-TB , 22–24.  effects , 20. To n CNS-TB . In our g CNS-TB . Astrocyg CNS-TB –41. Alth bacilli , the rol−/− mice, uncontrolled cellular recruitment and poor immune activation in MT-TNF−/− mice have provided conclusive evidence that collective TNF production by myeloid and T cells is critical in immune regulation during CNS-TB infection. Taken together the survival of MN-TNF−/− and T-TNF−/− mice in CNS-TB, we also speculate the synergistic effect of myeloid and T cell-derived TNF in chronic infection. It has been demonstrated in a chimeric transfer study that both macrophage and T cell-derived TNF are required for sufficient and long-term protection against M. tuberculosis infection (of M. tuberculosis infection (Unlike the MN-TNFnfection . The connfection , 42, 43.nfection , 45. As −/− mice clearly demonstrated the important role of IFN-γ in the protective immune response to M. tuberculosis infection, while other studies using transgenic mice have shown that IFN-γ overexpression in the CNS increases disease progression (Tumor necrosis factor is involved in both immune and immunomodulatory responses and acts in synergy with T cell-derived IFN-γ to enhance the macrophages antimycobactericidal activity of nitric oxide and other RNI by iNOS. IFN-γ is mainly produced by T cells and natural killer cells in response to dying bacterial-infected macrophages , 47; thegression , 49. Our−/− and TNF−/− brains, yet, the infiltration of effector T cells was higher than in the TNFf/f mice. This coincides with the previous findings of dysregulated T cell trafficking to the brains of IL-2-deficient mice attributes to CNS autoimmunity (−/− and TNF−/− mice may be a direct result of TNF deficiency. Although the proinflammatory effects of TNF signaling through TNFRp55 and TNFRp75 are well documented, increasing evidence indicates the immunoregulatory effects of TNF–TNFRs on Treg cell function (M. tuberculosis-specific T cell activation (−/− mice and comparable to TNF−/− mice, which correlated with increased recruitment of infiltrating leukocytes into the brain. Therefore, it is possible in our study that the absence of TNF disrupts Treg function and causes uncontrolled T cell responses during CNS-TB infection. Taken together, TNF production by myeloid cell and T-cell is pivotal in regulating T cell immunity.Besides increase production levels of IFN-γ, the cytokine IL-2 was found to be lower in MT-TNFimmunity –52. Moreimmunity –55. Origimmunity –58. Tregimmunity , 60. Anofunction , 61, 62.function , 64. Morfunction , 65. We tivation . Recentltivation , 67. HerIn conclusion, our data demonstrate that ablation of TNF in myeloid and lymphoid T cells renders the protective immunity ineffective, leading to increase susceptibility. In conclusion, the findings in this study corroborate earlier reports of the importance of TNF in immune-mediated responses against tuberculosis. We further showed that the TNF production by both myeloid cell and T-cell is essential for this process and the future development of improved diagnostic and therapeutic strategies.N-JH, NMF, RK, and NA were involved in experiments and data analysis. VQ and BR contributed to the experimental design. N-JH wrote the manuscript. MJ oversaw the project. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of the present study was to investigate the levels of circulating CD14 in relation to the expression of tumor necrosis factor alpha (TNF-α) in monocytes, and serum levels of TNF-α and macrophage inflammatory protein-1 (MIP-1) in migraine patients. Numerous studies revealed controversial changes in the components of the immune system during attacks and the interictal period in migraine patients. Our study included 40 migraineurs and 39 controls. The levels of TNF-α, MIP-1 and CD14 were measured in peripheral monocytes and in sera with the Enzyme-Linked Immunosorbent Assay (ELISA) method, and the monocyte expression of TNF-α was also analysed by immunostaining. Serum CD14 concentrations were higher and the expression of TNF-α in monocytes was decreased in migraineurs. The serum MIP-1 level correlated with Verbal Rating Scale (VRS); the MIP-1:CD14 ratio in monocytes correlated with Visual Analogue Scale (VAS); the MIP-1:CD14 ratio correlated with Migraine Severity (MIGSEV)-Pain scores; and serum CD14 concentration correlated with migraine duration in years. Increased serum CD14 and depletion of TNF-α in monocytes can orchestrate other components of the immune system during the interictal period. The immune system and inflammatory mediators have been implicated in migraine pathophysiology, although migraine is not recognized as a classically inflammatory disorder. The results of numerous clinical and experimental studies support the neurogenic inflammation theory of migraine pathophysiology. Research in this area was inspired by clinical observation of comorbidity of migraine and atopic or inflammatory diseases ,2,3,4. CAttacks of migraine have been associated with a variety of changes in the immune system, including complement components, immunoglobulin levels, cytokine concentrations and lymphocyte subtypes count ,13,14,15The interictal phase was characterized by various changes in immunoglobulin levels ,17 and lThe presence of neurogenic inflammation in the trigeminovascular system during migraine attack  emphasizIncreased expression of IL-1β in the dura mater, further mast cell degranulation and IL-6 production stimulates inducible nitric oxide (NO) synthase (iNOS) in meningeal macrophages ,25.Mast cells, localized predominantly in the meninges and activated by the substance P-depending process, were also suggested to play a role in migraine pathophysiology . MigrainMonocytes are immune cells that are suggested to play a role in migraine pathophysiology. Increased NF-κB activity in monocytes from internal jugular blood of migraine patients peaked 2 h after attack onset and was accompanied by a transient reduction in IκBα expression. Up-regulation of monocytic iNOS was observed at 4 h, maintained at 6 h and reduced at the end of the attack .The purpose of the present study was to investigate the role of CD14 in relation to the expression of TNF-α in monocytes, serum levels of TNF-α and MIP-1 Macrophage Inflammatory Protein-1 (MIP-1) in migraine patients during the interictal period.The clinimetric evaluation of migraineurs included in the study revealed moderate to severe intensity of migraine attacks. This was demonstrated by mean results of MIDAS scale (43 ± 18), MIGSEV: MIGSEV-Pain (3.48 ± 0.51), MIGSEV-Nausea (3.24 ± 0.66), MIGSEV-Activity (3.40 ± 0.58), MIGSEV-Tolerability (2.72 ± 0.46), VAS scale (8 ± 2) and VRS (3.0 ± 1.5).p = 0.04) and a higher level of anticardiolipin IgG compared to the controls (p = 0.04), otherwise no significant differences in the metabolic and inflammatory profile were found, see We have found lower white blood cells (WBC) count in migraine patients (p = 0.009). TNF-α expression in monocytes from migraine patients was decreased compared to controls (p < 0.001). There were no statistically significant differences in serum concentrations of MIP-1 and TNF-α as well as in MIP-1:CD14 and TNF-α:CD14 ratios in monocytes between migraine patients and controls, see We have found a significantly higher concentration of soluble CD14 in migraineurs’ sera (p = 0.27), however, in both groups of migraineurs, macrophage TNF-α expression was significantly (p < 0.001) lower than in controls.The expression of TNF-α in monocytes did not differ between migraineurs with and without aura . None of the studied parameters were influenced by NSAID treatment, see TNF-α expression in monocytes did not differ between migraineurs using NSAIDs as abortive treatment and those not using NSAIDs , which binds lipopolysaccharide (LPS) ,34. SeruFurthermore, serum TNF concentrations correlated positively with MIP-1, which is a CC chemokine involved in the early stages of inflammation, wound healing and hematopoiesis . It has Forty migraine patients  were included in the study. All patients fulfilled International Headache Society 2004 criteria for migraine diagnosis . The stuhttp://imagej.nih.gov/ij).).Based on medical history, physical examination, and routine laboratory tests see , none oft-Student test. Variables with non-Gaussian distribution were presented as median and interquartile range, and tested with a non-parametric Mann–Whitney test. The correlations between variables were tested with univariate analysis and expressed as Spearman coefficient (rS).Statistical analysis was performed with the use of the licensed MedCalc software version 16.8.4—64 bit. First, we have tested the distribution of results with the d’Agostino–Pearson test. Then, the results with Gaussian distribution were expressed as mean ± standard deviation (SD) and analyzed with theConcluding, the abnormalities of TNF-α expression in monocytes, correlations of serum TNF-α and MIP-1 levels with clinimetric scores and increased circulating CD14 in migraine patients observed in the present study suggest the involvement of these factors that orchestrate components of the immune system during the interictal period. Understanding the role played by mediators of inflammation evaluated in our study may be promising for future therapeutic development.


Accurate Monte Carlo simulations for high-energy events at CERN’s Large Hadron Collider, are very expensive, both from the computing and storage points of view. We describe a method that allows to consistently re-use parton-level samples accurate up to NLO in QCD under different theoretical hypotheses. We implement it in MadGraph5_aMC@NLO and show its validation by applying it to several cases of practical interest for the search of new physics at the LHC. The search of new physics is one of the main priorities of the LHC. The recent observation of an anomaly in the di-photon spectra , 2 givesR2 and UV counter-terms) required by loop computations.In the recent years, efforts have focussed on providing accurate theoretical predictions for a large number of BSM models at Leading Order (LO), in the form of event generators. First, various programs such as FeynRules , LanHep It is now possible to generate Monte Carlo sample for a large class of BSM theories at LO and for an increasing number at NLO accuracy. Even though technically possible, producing samples for many models and benchmark points down to full detector level at the high luminosity expected at the LHC would require an unmanageable number of computing and storage resources. However, the stages of a simulation  are independent and factorise. Therefore changes in local probabilities happening at very short distance, i.e. from BSM physics, decouple from the rest of the simulation stages. This is particularly interesting since the slowest part of the simulation is the full simulation of the detector.A logical possibility therefore arises: one can generate large samples under a SM or basic BSM hypothesis and then continuously and locally deform the probability functions associated to the distributions of parton-level events in the phase space by changing the “weight” of each event in a sample to account for an alternative theory or benchmark point. Under a not-too-restrictive set of hypotheses which are easy to list, such an event-by-event re-weighting can be shown to be exactly equivalent (at least in the infinite statistic limit) to a direct generation in the BSM. Note that such an event-by-event re-weighting is conceptually different from the very common yet very crude method where events are re-weighted using a pivotal one-dimensional distribution. Event-by-event re-weighting is a common practice in MC simulations, yet currently it has been only publicly available at LO , 14 or aThe plan of this paper is as follows. Before introducing the NLO re-weighting method, we will focus on the LO case in order to explain the intrinsic limitations of such types of methods Sect. . In SectAs stated in the introduction, the re-weighting method consists in attaching a new weight to every parton-level event as corresponding to a different scenario. The new weights allow to predict accurately  all the LO differential distributions at the parton level, leading also to the possibility of performing a single shower and detector simulation for all the models under consideration. At LO accuracy the new weight |Mnew|2) , 14:1\do , 14:1\dStd(w) are respectively the mean and the standard deviation of the ratio of the weights and A few remarks are in order regarding the range of validity of this method. First, even if the method returns the correct weight, it requires that the event sampling related to Helicity  The helicity state of the external states of a parton-level event is optional in the LHEF convention, yet some programs  use thiColor-flow  A second piece of information presented in the LHEF is the color assignment in the large Internal resonances  In presence of on-shell propagators, the associated internal particle is written in the LHEF. This is used by the parton-shower program to guarantee that the associated invariant mass is preserved during the re-shuffling procedure intrinsic to the showering process. Consequently, modifying the mass/width of internal propagator should be done with caution since it can impact the parton-shower behaviour. This information can not be corrected via a re-weighting formula, as it links in a non-trivial way short-distance with long-distance physics.Selected results obtained with this re-weighting are presented in Sect. Second, the parton-level configuration feeder to parton-shower programs not only depends of the four-momenta but also of additional information, which is commonly encoded in the LesHouches Event File (LHEF) , 20. ConIn this section, we will present three re-weighting methods for NLO samples. First we will present a LO type of re-weighting that we dubbed “Naive LO-like” re-weighting introduced in VBFNLO (i.e. REPOLO ) and MadR, S, C, SC, MC correspond respectively to the contributions of the fully-resolved configuration , of its soft (including the Born matrix-element), collinear, soft-collinear limits (the counter-events) and the Monte Carlo (MC) counter-term. The Following the MC@NLO method , 25, theorig or new label). The Naive LO-like re-weighting computes the weights based on the multiplicity of the events before parton shower. i.e.,As this method does not consider the dependence of the virtual contributions, it fails to be NLO accurate. To ensure NLO accuracy, it requires that the effect of the new theory factorises out, i.e., whenR, S, C, SC, MC (see previous section). Q is the Ellis-Sexton scale and In order to have an accurate NLO re-weighting method, one should explicitly factorise out the dependence in the (various) matrix elements (i.e. in the Born squared matrix element – ribed in 4 introduR or F. The The expression of the endix of  and are 3.28) of ) and the(D.1) of ). This aendix of ). One caendix of . The infMore generally, the possible drawbacks and limitations on the statistical precision of the method are the same as for the LO case. However, for NLO calculations in MG5_aMC we face one additional source of statistical uncertainty due to the method used to integrate the virtual contribution. This method reduces the number of computations of the virtual by using an approximate of the virtual contribution based on the Born amplitudes times a fitted parameter sampling ), reduciTo reduce this effect, we propose to use a slightly more advanced re-weighting technique. We split the contribution proportional to the Born  are kept to a minimum. Unless otherwise stated, the settings used correspond to the default value of MG5_aMC (version 2.4.0). In particular the minimal transverse momentum on jet is of 20 GeV at LO and of 10 GeV at NLO.W and Z boson for the following dimension six operator:SU(2).For the first validation, we will use the effective field theory (EFT) in the Electro-Weak sector . We willZ boson at LO accuracy. Starting from a sample of Standard Model events (black solid curve), we have re-weighted our sample to get the SM plus the interference term with the dimension six operator for two values of the associated coupling: In Fig. The ratios between the differential curves obtained with each method are presented in the second inset. This inset contains also the statistical uncertainty (yellow band) for the ratio of two independent SM samples. The compatibility of those two ratio plots with the expected statistical fluctuation validates our approach/code implementation. The first inset presents the ratio between the EFT and SM predictions. It shows that the method works correctly for quite small and quite large modifications of the differential distributions.One can note that in the context of EFTs, the weight is linear in the dim-6 couplingZ and H boson in the EFT as implemented in the Higgs Characterisation framework/model [H is the Higgs doublet field and For our first NLO validation, we consider the associated production of a rk/model . We use rk/model : HD and In Fig. From the comparison of the two methods for the HD curve in the plot of the transverse momenta (top plot), we can observe that the statistical fluctuations are more pronounced for the curve obtained by re-weighting. This is an example of enhancement of statistical uncertainty due to the re-weighting as discussed around Eq. Q is the third generation left-handed quark doublet, t are respectively the Higgs and top quark fields, SU(3) generator.In this second NLO example, we will use the EFT framework in the context of the top-quark  and focuZ boson in the associated production of this boson with a top/anti-top quark pair. We present the result for both the full matrix element squared  and via the NLO accurate one (RWGT-NLO), The ratio to the SM curves are presented in the first inset while the ratio between our prediction and the direct computation in MG5_aMC for In this last example, we will present results for the associated production of a SM Higgs with one jet. In Fig. The first inset presents the ratio at LO and NLO of the infinite top mass limit over the full theory. For the NLO case, the full theory is approximated by the loop-improved method. The two ratios are very similar showing that the loop-improved method re-introduces the top-mass effects in a sensible way. The second inset presents the ratio between the re-weighting and the direct approach in the LO case, the statistical uncertainty of the ratio of two independent SM sample is presented by the yellow band. Its bumpy shape is due to the use of multiple samples with different cuts to decrease the statistical uncertainty. This ratio plot fully validates the re-weighting in the case of the LO curves.We have presented the implementation of several methods that can be used for re-weighting LO and NLO samples and discuss the associated intrinsic limitations. We have released a new version of MG5_aMC that allows the users to employe the various re-weighting methods presented in this paper in a fully automatic and user-friendly way. In particular we have introduced for the first time an NLO accurate re-weighting method and compared it with the approximate methods available in the literature. Other re-weighting methods like the Naive LO-like and the loop-improved are for the first time available in a public code.The comparison between the various methods shows that the approximate method (the Naive LO-like re-weighting) performs a satisfactory job. This indicates that the non locality of the MC counter terms is often more a theoretical problem than a contribution spoiling the NLO accuracy of the Naive LO-like re-weighting. Therefore the Naive LO-like re-weighting should be a good approximation in a quite large class of model/observable either when the virtual contribution is sub-dominant and/or when the effect of the BSM physics factorises. Consequently, we recommend phenomenologist to first test the Naive LO-like re-weighting and in case of loss of accuracy move forward to the slower NLO method. On the other hand for mass production at the LHC, where the samples are often used for more than one study, we recommend to always use the NLO accurate method.The framework introduced here is flexible enough to accommodate different types of re-weighting approaches. In the near future we plan to capitalise on this to allow different type of functionalities. First we plan to implement a standard systematics uncertainty computation module as it is done in , 38–42.


Over the last few decades, the institutionalisation of quantitative research evaluations has created incentives for scholars to publish as many papers as possible. This paper assesses the effects of such incentives on individual researchers’ scientific impact, by analysing the relationship between their number of articles and their proportion of highly cited papers. In other words, does the share of an author’s top 1% most cited papers increase, remain stable, or decrease as his/her total number of papers increase? Using a large dataset of disambiguated researchers  over the 1980–2013 period, this paper shows that, on average, the higher the number of papers a researcher publishes, the higher the proportion of these papers are amongst the most cited. This relationship is stronger for older cohorts of researchers, while decreasing returns to scale are observed for recent cohorts. On the whole, these results suggest that for established researchers, the strategy of publishing as many papers as possible did not yield lower shares of highly cited publications, but such a pattern is not always observed for younger scholars. Despitevar Bush , that scvar Bush , when anvar Bush  or the Rvar Bush . Variousvar Bush .publish as many papers as possible. For instance, in Australia, where publication counts were used without any emphasis on publication venue or citations, researchers have been found to increase the numbers of publications in journals with high acceptance rates and lower impact [The institutionalisation of these evaluations led many researchers to put large emphasis on the number of papers they published. This has led to adverse effects –10. Inder impact .In this research evaluation culture of quantity, researchers may have adopted different publication strategies. For example, some researchers might focus on publishing few, high-quality papers—e.g. being more ‘selective’  or ‘perfThis paper expands on such previous work, with a larger dataset and over a longer time period. Indeed, using a large dataset of distinct disambiguated researchers  who published at least one paper during the 1980–2013 period, this paper aims to better understand the relationship between publication activity and scholarly impact. More specifically, it aims to answer the following research question: what is the relationship between research productivity and scholarly impact? Is the share of scholars’ top papers increasing, stable, or declining, as their research productivity increases? In other words, can scholars be too productive? A good analogy for this is archery: if an archer throws one arrow, what is the probability that it hits the center of the target? Does an increase in the number of arrows thrown leads to an increase in the proportion arrows hitting the center of the target? Our working hypothesis is that authors with higher number of papers would also publish a higher proportion of top cited papers. Such hypothesis would be in agreement with Merton’s theory of cumulative advantage , and supThis paper uses Thomson Reuters’ Web of Science (WoS) database for the period 1980–2013. Only journal articles are included. Given that the unit analysed in this paper are individual researchers, we used the author disambiguation algorithm developed by Caron & van Eck  to identThree methods were tested to take into account the fact that researchers might publish in more than one of the four broad disciplinary domains. A first method was to consider author-domain combinations  as distiAs our aim is to assess researchers’ contribution to papers that have the highest impact, we isolated the top 1% most cited papers published each year for each discipline . Citations were counted until the end of 2013, and any overlap between the groups of citing and of cited authors (self-citations) were excluded. Hence, the paper only focuses on ‘external citations’ as these are the most relevant for evaluative purposes .de facto “new” scholars). For disciplines of the natural and medical sciences (Panel A), we observe a steep increase of the number of new scholars from the mid-1980s onwards. For the social and behavioral sciences (Panel B), the early 1980s decrease is followed by a relatively stable number of new authors, until the early 2000s when the number of new authors increase from about 40,000 to 70,000 annually. For law, arts, and humanities, the number of new authors is relatively stable, if not slightly decreasing, throughout the period.2 = 0.83) followed by natural sciences (R2 = 0.73), social and behavioral sciences (R2 = 0.68) and then by the social sciences and law, arts and humanities (R2 = 0.57).punch above their weight, and between 1.5% and 4% papers from researchers who published between 40 and 60 papers were in the top 1% most cited papers. Similar trends can be found in law, arts and humanities, with scholars who have published more than one paper having a share of top papers above average, and scholars with 10–20 papers obtaining a share of top papers between 1.3% and 2.6%.More specifically, medical and life sciences scholars with less than 10 papers generally publish less than 1% of their papers in the top 1% most cited groups of papers, the share of top papers increases with productivity, and reaches values between 2% and 3% for scholars who published more than 200 papers. In natural sciences, the increase is not as fast, with scholars who, roughly, publish less than 45 papers still being less likely to publish a high share of top papers. Along these lines, the results are also more scattered, with the group of scholars publishing 150 papers or more obtaining shares of top 1% most cited papers between 1% and 2.5%. In the social and behavioral sciences, very fast increase is seen in terms of shares of top cited papers: researchers with 4 papers or more already When the subset of younger researchers who have published their first paper between 2009 and 2013 is considered, different patterns are observed . For medAn important characteristic of this cohort is that it got socialized to research recently—when the evaluation culture was more present—which might make them prone to publish as much as possible. However, the drop in the share of top papers observed in each domain except natural sciences suggests that these academically-younger scholars struggle to keep impact high once a certain threshold is met. This might be due to the fact that these scholars have not yet secured permanent or tenure positions and, thus, might feel that they cannot be as selective as older scholarsIn order to take into account scholars’ various career paths, we compiled the proportion of top 1% most cited papers as a function of career length , panel Ato publish as much as possible. Our results have shown that, for older researchers, the higher the number of papers published, the more likely those ended up being amongst the most cited papers of their discipline. For younger scholars, however, such relationship could only be observed in the natural sciences and, to a certain extent, in the social and behavioral sciences. For the two other broad domains, higher scientific output was associated with decreasing shares of highly cited publications.Previous research has shown that, in many contexts, the focus on quantitative indicators for research evaluation has had adverse effects . This paSeveral factors can contribute to the explanation of this pattern. For instance, authorship criteria  and co-astratify [Leiden Manifesto [From a theoretical point of view, these results conform to the Mertonian theory of cumulative advantages : the higstratify  itself istratify , difficustratify , as wellanifesto  that indS1 Table(DOCX)Click here for additional data file.


Tissue necrosis factor-α (TNF-α) and complement component 3 (C3) are two well-known pro-inflammatory molecules. When TNF-α is upregulated, it contributes to changes in coagulation and causes C3 induction. They both interact with receptors on platelets and erythrocytes (RBCs). Here, we look at the individual effects of C3 and TNF-α, by adding low levels of the molecules to whole blood and platelet poor plasma. We used thromboelastography, wide-field microscopy and scanning electron microscopy to study blood clot formation, as well as structural changes to RBCs and platelets. Clot formation was significantly different from the naïve sample for both the molecules. Furthermore, TNF-α exposure to whole blood resulted in platelet clumping and activation and we noted spontaneous plasma protein dense matted deposits. C3 exposure did not cause platelet aggregation, and only slight pseudopodia formation was noted. Therefore, although C3 presence has an important function to cause TNF-α release, it does not necessarily by itself cause platelet activation or RBC damage at these low concentrations. We conclude by suggesting that our laboratory results can be translated into clinical practice by incorporating C3 and TNF-α measurements into broad spectrum analysis assays, like multiplex technology, as a step closer to a patient-orientated, precision medicine approach. They are not only upregulated in most inflammatory conditions, but their activities are closely linked. When TNF-α is upregulated, it contributes to changes in coagulation and C3 induction2. TNF-α plays a pivotal role in the disruption of macrovascular and microvascular circulation both in vivo and in vitro4, and is an important cytokine that can induce both apoptosis and inflammation5. In the presence of ROS, there is an increased production of TNF-α and, in turn, TNF-α signaling accentuates oxidative stress3. TNF-α upregulation is also associated with a changed coagulation propensity7. In short, TNF-α participates in vasodilatation and oedema formation, as well as leukocyte adhesion to the epithelium through expression of adhesion molecules. Furthermore, it regulates blood coagulation, contributes to oxidative stress at sites of inflammation, and indirectly induces fever6. TNF-α also plays a central role in the pathogenesis of insulin-resistant metabolic derangements.Tissue necrosis factor-α (TNF-α) and complement component 3 (C3) are two well-known pro-inflammatory molecules8. The relationship between upregulation of C3, inflammation, coagulation and hypofibrinolysis has been known for a long time10. Furthermore, pathology in the complement system is associated with an increased risk of pathological thrombotic processes11 e.g. stroke12 and in the pathogenesis of thromboembolism13.C3 is a major protein of innate immunity and the complement system, with crucial roles in microbial killing, apoptotic cell clearance, immune complex handling and modulation of adaptive immune responses2, atherosclerosis-induced inflammation14, rheumatoid arthritis15, human umbilical cord vein (HUVEC) endothelial cells16, and in insulin resistance17. It was also found that TNF-α synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid18. TNF-α may also induce epithelial to mesenchymal transition in injured renal tubular epithelial cells through inducing C3 expression19. Table C3 and TNF-α activity are interlinked, and examples of complement-dependent TNF release has been found in viral fulminant hepatitis20, with TNFR1, also known as tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) or CD120a, being the chief receptor. While RBCs do not express these receptors21, TNF can induce platelet consumption, and platelets do express TNFR1 and TNFR223. TNFR1 expressed on other cells also causes the release of factors with agonist activity for platelets24; and TNF-α is able to activate platelets through stimulation of the arachidonic acid pathway23.These two circulating pro-inflammatory molecules affect the coagulation and haematological system, as they interact with receptors on both platelets and erythrocytes (RBCs). Tumor necrosis factor receptors 1 and 2  are the primary TNF-α receptors25. Immune complexes, which have reacted with complement and bear C3b fragments also bind to the CR1 on human RBCs, and CR1 on RBCs serves as a transport system for immune complexes in the circulation to prevent immune complex deposition outside the fixed macrophage system28. Complement also interacts with the surface of activated platelets as well as with other components of the complement system including, C1q, C4, C3, and C9, which bind to activated platelets29. Furthermore, thrombin-activated platelets can actually initiate the complement cascade30, and C3a and its derivative C3a-des-Arg, induce platelet activation and aggregation in vitro31. Platelets express complement receptors C3aR, CR4, as well as a receptor for iC3b and C5a, and the C1q receptors gC1qR and cC1qR on their membranes. cC1qR, in particular, was shown to mediate platelet aggregating and activating effects29. Of importance is that platelets may also interact with the complement system via proteins that are not considered classical complement receptors, such as P-selectin32 or GP1bα33.Turning to complement, RBCs carry the complement receptor 1 (CR1), also known as C3b/C4b receptor or CD35, on its membrane35. During inflammation, various circulating inflammatory molecules are upregulated, and a crucial result of this combination of molecules is a pathological haematological system that ultimately translates to hypercoagulation, RBC dysfunction and platelet hyperactivation – all hallmarks of inflammation and cytokine upregulation. However, for clinical intervention, it is essential to know what the individual effects on these pro-inflammatory molecules are, to pinpoint possible biochemical interventions. Here we look at the individual effects of C3 and TNF-α, by adding low levels of the molecules to blood.In a series of papers, we have investigated the individual effects of inflammatory molecules on coagulationThe ethics committees of the University of Pretoria and Stellenbosch University (South Africa) approved this study (ethics clearance number 298/2016). A written form of informed consent was obtained from all healthy donors (available on request). The methods were carried out in accordance with the approved guidelines. Blood was collected and methods were carried out in accordance with the relevant guidelines of the ethics committee. We adhered strictly to the Declaration of Helsinki.Blood was collected from 14 healthy individuals who voluntarily enrolled for this study. Exclusion criteria for the healthy population were: known (chronic and acute) inflammatory conditions such as asthma, human immunodeficiency virus (HIV) or tuberculosis; risk factors associated with metabolic syndrome; smoking; and, if female, being on contraceptive or hormone replacement treatment. This population did not take any anti-inflammatory medication. Based on satisfying the exclusion criteria, the control donors were classified as ostensibly healthy. We therefore assumed that the TNF-α and C3 levels in our chosen sample population were in the ranges previously reported by researchers  and also to a higher final exposure concentration of 0.2 mg.mL−1 C3. These higher concentrations did not allow a clot to be formed on the TEG, as we could not obtain a clot R-time, suggesting that the added high concentrations were causing the clot to form too fast. However, we do believe that we simulated low-grade chronic inflammation with our low TNF-α and C3 final exposure concentration; but we do recognise that the physiological ranges during disease can be much higher.We exposed whole blood and plasma separately to either TNF-α (Sigma T6674) or C3 (Sigma C2910) at levels that represent low-grade chronic inflammation. Our final TNF-α exposure concentration in blood and plasma was 1 pg·mL2 in a disposable TEG cup. Recalcification of blood is necessary to reverse the effect of the sodium citrate collecting tube anticoagulation method and consequently initiate coagulation. The samples were then placed in the computer-controlled Thromboelastograph® 5000 Hemostasis Analyzer System  for analysis at 37 °C. Table 38.Viscoelastic assessment of clot kinetics was performed using thromboelastography (TEG). Whole blood (WB) and platelet poor plasma (PPP) from healthy donors was incubated with TNF-α or C3 for 10 minutes prior to assessment. WB was left at room temperature for 15 minutes following collection before being incubated. PPP was first thawed to room temperature from storage at −80 °C before incubation. 340 μL of naïve (untreated) or product-exposed WB or PPP was mixed with 20 μl of 0.2 M CaClRBC autofluorescence was used to follow the effects of the products added to WB. Images were acquired with the Zeiss CellDiscoverer 7 using a combination of three LED modules simultaneously: 385, 470 and 567 nm wavelengths. The following bandpass emission filters were used: 412–438; 501–527 and 583–601. A Plan-Apochromat 50×/1.2NA objective with a 2× tube lens and an Axiocam 506 mono-camera were used for image acquisition. The live cell imaging procedure included adding 99 μL of WB in a plate and imaging the naïve sample. Then, 1 μL of either TNF-α or C3 was added, and following stabilisation of the sample, images were captured at 3 minutes after exposure.38). Micrographs of WB smears were taken using a Zeiss crossbeam electron microscope and Merlin (Gemini II) FE SEM to study the ultrastructure of the both platelets and RBCs, with specific focus on their membranes. Image acquisition was performed independently at both the Universities of Stellenbosch and Pretoria. The images acquired at both these institutions are stored as raw data (see link below).Blood smears were prepared for SEM analysis using WB after exposure to TNF-α and C3 for 10 minutes. In short, 10 μL of unexposed naïve and product-exposed WB were placed on glass cover slips and smears were washed in PBS, fixed in 4% formaldehyde and 1% osmium tetraoxide before being dehydrated in increasing grades of ethanol and HMDS . This type of analysis allows us to compare each product exposure with the control (GraphPad Prism 7), with statistical significance taken as p ≤ 0.05.https://1drv.ms/f/s!AgoCOmY3bkKHuh5Av5hYQU5UQgdG. Raw data can also be access at the corresponding author’s researchgate link: https://www.researchgate.net/profile/Etheresia_Pretorius.Raw data, extensive SOPs for TEG and SEM, including original images without color and micrographs can be accessed at: 38; while PPP clot results reflect only the effect of the plasma proteins, chiefly fibrin(ogen). Table WB TEG parameters reflect clot properties due to both the cellular components (viz. platelets and RBC), as well as the plasma protein componentsFigure Figure 35.During inflammation, various circulating inflammatory molecules are upregulated, and a crucial result of this combination of molecules is a pathological haematological system that ultimately translates to hypercoagulation, RBC dysfunction and platelet hyperactivation – all hallmarks of inflammation and cytokine upregulation. However, for clinical intervention, it is essential to know what the individual effects on these pro-inflammatory molecules are, to pinpoint possible biochemical interventions. Here we look at the individual effects of C3 and TNF-α, by adding low levels of the molecules to blood. In a series of papers, we have investigated the individual effects of inflammatory molecules on coagulation39. In the current investigation, only TNF-α exposure, resulted in platelet clumping and activation . When C3 was added to whole blood, the R-time shorted significantly. Overall these two products seem to promote a faster clot initiation. There were significant differences in the TEG results when platelet poor plasma (PPP) was analysed. It is known that the diagnosis of hypercoagulability are based on changes in variables of hemostatic monitors such as a decrease in the time to clot initiation , an increase in the speed of clot propagation (angle), or an increase in clot strength . This indicates an enhancement of hemostasis or hypercoagulability as defined with TEG45. We conclude by suggesting that our laboratory results can be translated into clinical practice by incorporating C3 and TNF-α measurements into broad spectrum analysis assays, like multiplex technology, that measures various cytokines (e.g. interleukins). Developments in inflammatory marker quantification technology, like multiplex assays and arrays, allow for an improved evaluation and understanding of the dynamic nature of inflammatory responses47. Furthermore, multiplexed protein array assays provide high sensitivity and specificity using low sample volumes in a high throughput analysis and, in addition, can be used during therapeutic drug monitoring. With such approaches, therapy outcomes can also be followed with more accuracy, and in the long run, reduce treatment costs, as well as expedite wellness outcomes.Defining and understanding the role of cytokines and other inflammatory mediators, like TNF-α and C3 in (inflammatory) diseases, is becoming increasingly important, especially in patient-orientated and precision-based therapy initiativesConsidering that our results show that these two molecules have a pro-coagulant effect on blood, in addition to a place in diagnostics, it might be of importance to evaluate further the biochemical mechanisms by which C3 and TNF-α modulate pro-coagulant pathways. This might reveal a therapeutic potential for lowering the pro-coagulant state due to C3 and TNF-α, present in inflammatory diseases.


Fab, scFv) have not only reduced resulted immunogenicity, but also proved several benefits including better tumor penetration and more rapid blood clearance. Using affinity selection procedures in this study, a scFv antibody clone was isolated from naïve Tomlinson I phage display library that specifically recognizes and binds to TNF-α. The TNF-α recombinant protein was expressed in genetically engineered Escherichia coli SHuffle® T7 Express, for the first time, which is able to express disulfide-bonded recombinant proteins into their correctly folded states. ELISA-based affinity characterization results indicated that the isolated novel 29.2 kDa scFv binds TNF-α with suitable affinity. In-silico homology modeling study using ‘ModWeb’ as well as molecular docking study using Hex program confirmed the scFv and TNF-α interactions with a scFv-TNF- α binding energy of around -593 kj/mol which is well in agreement with our ELSIA results. The cloned scFv antibody may be potentially useful for research and therapeutic applications in the future.Tumor necrosis factor alpha (TNF-α) expression amplifies to excess amounts in several disorders such as rheumatoid arthritis and psoriasis. Although, Anti-TNF biologics have revolutionized the treatment of these autoimmune diseases, formation of anti-drug antibodies (ADA) has dramatically affected their use. The next generation antibodies ( Rheumatoid Arthritis is one of the most commonly long term inflammatory diseases. Although it generally involves the joints primarily, itis recommended as a syndrome which includes extra articular manifestations, such as rheumatoid nodules, pulmonary vasculitis or involvement, and systemic comorbidities . The infLately, the therapeutic management of patients with RA has seen a major evolution . A numbeIn the past decade, innovations in recombinant antibody technology have greatly facilitated the genetic manipulation of antibody fragments , 9. ScFvIn recent years, phage display of non-immune, human naïve scFv antibody repertoires have proven to be an important tool for the generation of highly specific antibodies -14. In tLarge scale expression and purification of TNF-α proteinE. coli cell expression, and cloned into a pET28a vector  containing an expression cassette with an N-terminal polyhistidine tag. Protein expression was conducted under native conditions  for nditions  and purinditions .Panning of phage display library13 cfu) at room temperature. Phage particles with affinity for the recombinant TNF-α were eluted using 100 mM triethylamine, and amplified in exponentially growing E. coli TG1 cells . The panning procedure was repeated for three rounds and the total eluted phage titer was determined after each round.Tomlinson I naïve scFv phage library was used for the selection of specific binders against recombinant TNF-α protein. The panning procedure was carried out by immobilizing recombinant TNF-α protein (100 µg/mL) overnight onto immunotubes , which had been washed with 1X PBS buffer, blocked with 2% skim milk in 1X PBS (w/v), and incubated with phage suspension  glucose and 100 µg/mL ampicillin. In order to induce scFv production, 96 colonies of TG1 and HB2151 containing the scFv phagemids were randomly picked up and inoculated in 2xTY medium supplemented with 100 µg/mL ampicillin and 0.1% (w/v) glucose in a microtiter plate. The cultures were grown while shaking (250 rpm) at 37 °C until the OD600 reached about 0.9. The transcription of scFv cassette was then induced with isopropyl β-D-thiogalactopyranoside (IPTG), which was added to the culture at a final concentration of 1 mM. Shaking was continued at 200 rpm in 30 °C environment overnight. The cells were isolated by centrifugation and the supernatants were used for subsequent assays.After three rounds of panning, Binding activity of scFv against TNF-α proteinThe binding activities of selected clones were investigated by Enzyme-Linked Immunosorbent Assay (ELISA). Briefly, About 100 µg/mL recombinant TNF-α in 1X PBS buffer was directly coated on high-binding Maxisorp microtiter plates . The wells were subsequently blocked with 2% BSA, in 1X PBS. 100 µL of scFv solutions (as described above) were then applied to the plates and incubated at 37 °C for 2 h. Bound scFvs were detected using anti c-Myc monoclonal antibody 9E10 followed by horseradish peroxidase conjugated to goat anti-mouse polyclonal antibodies. The plates were then evaluated using Biotek ELISA reader . Positive clones were further characterized by western blotting. Blotting analysisIn Western blotting, electrophoretically separated TNF-α protein was transferred from SDS-PAGE gel to nitrocellulose membrane (0.45 µm). The membrane was blocked with PBS buffer containing 2% (w/v) skim milk. The scFv proteins were detected by the anti c-Myc monoclonal antibody 9E10, followed by Goat anti-mouse IgG, coupled to alkaline phosphatase . Blots were developed using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT) as substrate.Homology modelingModWeb version r182 web server (https://modbase.compbio.ucsf.edu/modweb). Briefly, the heavy and light chain sequences were searched against antibody structure database followed by the identification of the template for framework (FRH/FRL) region.The 3D structure of the anti-TNF-α scFv was built by homology modeling based on the template crystal structure. To do so, the Anti-TNF-α scFv complex models were predicted using the structures determined by X-Ray crystallography of the different partners. The calculations were performed using the Molecular dockinghttp://hex.loria.fr/) was used to investigate the mode of interaction between the TNF-α and BSA (as negative control) with scFv. All water molecules were removed from the scFv model structure, and hydrogen atoms were added followed by minimal minimization in the presence of bound ligand using swis-pdb Viewer. The active site for docking was defined for all atoms within the 10 Å radius of the co-crystallized ligand. The correlation type was set to shape with electrostatic and subset parameters set at 0. The possible interactions were analyzed using Pymol software version 1.5.0.1 (http://pymol.findmysoft.com). The final model was selected based on the cluster size as described in the results session. Hex program , the translation of the DNA sequences into amino acid sequences and subsequent protein alignment were accomplished. The results confirmed that all positive clones obtained in the panning process contained the same sequence, namely TNF-α-scFv1  (GenBank accession number KY072973). Alignment analysis with the available sequences in the IMGT database (http://www.imgt.org/IMGT_vquest/share/textes/) showed that the VH and Vκ fragments of TNF-α-scFv1 were members of different groups as is shown in Figure 4.The scFv encoding regions of positive clones were PCR-amplified (834 bp) and subjected to fingerprinting analysis . To confBinding characteristics of the selected scFvTo investigate on the cross reactivity of scFv with other proteins, TNF-α-scFv1 solution (as described above) was used to detect the recombinant TNF-α protein as well as BSA, Glutathione-S-transferase (GST), Immunodominant Membrane Protein (IMP), and Fig Mosaic Virus Nucleic Capsid Protein (FMV NCP) using ELISA. According to Figure 5A, no cross reactivity of the prepared scFv with other negative control proteins was detected. Complementary Western blot analysis was performed to further confirm the specificity of the TNF-α-scFv1 against recombinant TNF-α as is shown in Homology model building and validation ModWeb version r182 web server. The PDB file 5B3N with the lowest DOPE and the highest GA341 assessment scores was selected (4Serine3) linker region of the model, which had no appropriate crystal structure in the homology model template. These results showed that the scFv model was suitable for the molecular docking study.Anti-TNF-α scFv was applied to the selected . QualityDocking of TNF-α and BSA with scFv To visually evaluate the interaction of the scFv with TNF-α, the TNF-α and BSA molecules were docked onto scFv using Hex program. The models with the lowest energy docking were selected from the resulting docking models. According to the docking results, the binding energy of scFv was estimated to be −593 kj/mol when fused to TNF-α. This is approximately 3.5 fold stronger than that of scFv-BSA (−165 kj/mol). These results are in agreement of the ELISA experiments. The final configuration of the docking complex of scFv-TNF-α is shown in e.g. Fab, scFv) not only have reduced the immunogenicity, but also have several benefits including better tumor penetration, more rapid blood clearance and lower retention times in non-target tissues (The existing evidence suggests that TNF-α is involved in the pathways of inflammatory responses . It has  tissues .10), robust and well-established E. coli expression system, scalability, short panning cycles and cost-effectiveness  were expressed in HB2151 and the other 8 clones (Average OD~3.1) in TG-1 which is in agreement with Tomlinson library protocol. Based on this protocol, expression of soluble scFv in TG-1 strain, which is able to suppress the termination and to introduce a glutamate residue at these positions may result in more positive clones. Co-expression of the scFv-pIII fusion, as a consequence of this suppressed termination, tends to lower the overall levels of scFv expression even in clones where there are no TAG stop codons in the scFv itself. To solve this problem, non-suppressor HB2151 strain was infected with selected phages. ScFv genes that did not contain TAG stop codons yielded higher levels of soluble scFv than those in TG-1. On the other hand, those that contained TAG stop codons would not produce any soluble scFv. In the present study, we described the use of Tomlinson I human single fold synthetic naïve phage display single chain antibody fragment library in phagemid/scFv format-fused to the pIII minor coat protein of M13 bacteriophage to identify small, fully humanized antibodies against TNF-α. For the evaluation of our results, the efficiencies of binder phages were measured as the fraction of input phages that bound to the TNF-α. The quantification of phages was performed by tittering them after each round of the bio-panning, with the input phage concentration being kept high during the rounds of selection enrichment , 21. Theet al. developed scFv specific antibodies against TNF-α using Tomlinson I library expressed in BL21 E. coli strain . At the same time a version of the disulphide band isomerase DsbC which lacks its signal sequence is expressed in the cytoplasm. DsbC isomerizes mis-oxidized substrates into their correctly folded state and greatly enhances the fidelity of disulphide bond formation . Furthermore, the results showed that the binding domain was mainly formed by K78, C69, Y82, S73, I72, T71, Q84, S19 and G17 amino acid residues. Homology modelling and molecular docking are two appropriate computational methods to study protein–ligand interactions. Here, Web based server ‘e.g. pET series) and then be transformed to the expression strains (e.g. SHuffle® T7 Express). We have, therefore, successfully isolated novel scFv clone variants from a naïve or single-pot library with specific binding affinity to TNF-α. In order to achieve high yield expression (due to low level periplasmic expression of this sequence), the sequence which encodes this identified anti-TNF-α scFv antibody can be sub-cloned into an expression vector under the control of a strong T7 promoter (


T肽是Tuftsin的衍生物，其基本功能是刺激巨噬细胞，提高巨噬细胞吞噬及分泌能力，通过增强效应细胞的细胞杀伤作用而被广泛用于多种恶性肿瘤术后抗肿瘤治疗。本研究旨在探讨T肽与化疗药物顺铂的联合能否提高铂类药物的抗肿瘤疗效及机制。应用酶联免疫吸附剂测定（enzyme-linked immunosorbent assay, ELISA）T肽和/或顺铂处理人巨噬细胞株U937后肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）和干扰素-γ（interferon-γ, IFN-γ）分泌水平的变化；应用裸鼠荷瘤动物模型，分析T肽和/或顺铂处理小鼠肿瘤变化情况以及小鼠用药期间外周血中巨噬细胞变化情况。①应用ELISA检测发现在T肽组和T肽联合顺铂组中，U937细胞培养液中TNF-α水平明显高于对照组和顺铂组；T肽联合顺铂组中IFN-γ水平明显高于对照组、顺铂组和T肽组；②在小鼠荷瘤模型中，T肽联合顺铂组的肿瘤体积明显小于对照组、顺铂组和T肽组，并且小鼠没有明显的体重下降；③在小鼠外周血的检测分析中发现，T肽联合顺铂组中活化巨噬细胞数呈现增长趋势。免疫组化分析各组中肿瘤组织Ki67显示T肽联合顺铂组抑制肿瘤细胞增殖。T肽能够促进巨噬细胞增殖和促进其分泌肿瘤细胞杀伤因子（TNF-α、IFN-γ）；在体内动物模型中，T肽能够提高化疗药物如顺铂的疗效，使联合组具有更强的肿瘤抑制作用，同时能够减轻化疗药物的毒副作用。


Pomacentrus moluccensis, and the Ambon damselfish Pomacentrus amboinensis, by the repeated use of a drop stimulus. We show that the length of thermal exposure of juveniles to elevated temperature significantly affects this escape responses. Short-term (4d) exposure to warmer temperature affected directionality and responsiveness for both species. After long-term (90d) exposure, only P. moluccensis showed beneficial plasticity, with directionality returning to control levels. Responsiveness also decreased in both species, possibly to compensate for higher temperatures. There was no effect of temperature or length of exposure on latency to react, maximum swimming speed, or escape distance suggesting that the physical ability to escape was maintained. Evidence suggests that elevated temperature may impact some fish species through its effect on the behavioural responses while under threat rather than having a direct influence on their physical ability to perform an effective escape response.The threat of predation, and the prey’s response, are important drivers of community dynamics. Yet environmental temperature can have a significant effect on predation avoidance techniques such as fast-start performance observed in marine fishes. While it is known that temperature increases can influence performance and behaviour in the short-term, little is known about how species respond to extended exposure during development. We produced a startle response in two species of damselfish, the lemon damsel  Predator–prey interactions are one of the key drivers structuring communities . PredatiRising environmental temperatures can substantially impact the behaviour and locomotor performance components of escape responses in fish . PreviouThe interaction between a predator and its prey is an intricate sequence that, at its end, leads to a predator striking at a prey and the prey being captured or escaping . Many amThe present study examines whether short-term or long-term exposure to elevated temperature regimes affect the fast-start escape response in two tropical damselfishes. Using a repeatable drop stimulus , we compared escape responses of juveniles from current-day controls (29 °C) to individuals exposed to 50 and 100 year projections , the Great Barrier Reef Marine Park Authority (G10/33239.1), and Queensland Fisheries (170251).Pomacentrus moluccensis, and the Ambon damselfish, Pomacentrus amboinensis). These species have been used in past studies on escape performance , James Cook University, Townsville. Fish were housed in densities of three of varying sizes  in 40 litre replicate tanks. This sizing order helped formed a natural hierarchy within tanks and reduced within tank fighting. Tanks were randomly allocated into one of three temperatures: 29.0 °C . Thermal sensitivity to elevated temperature (aim 1) was measured by exposing individuals to either 30 or 31 °C for a 4d period. To explore the extent of thermal plasticity (aim 2), separate treatments were held at either 30 or 31 °C for 90d. The control treatment remained at 29 °C for the entirety of the experiment. In total, there were 10 treatments: two short-term exposures, two long-term, one control, and repeated for each species. Holding tank temperatures were raised 1 °C/day to reduce the effect of heat shock . To contP. amboinensis n = 58, 30.57 ± 5.79 mm, P. moluccensis n = 83, 28.08 ± 3.59 mm) and tested for escape performance the following day. Treatments of P. amboinensis were significantly larger than P. moluccensis , however there were no differences across treatments for each species. Escape performance was tested in a circular arena (30 cm diameter) filled to a depth of 10 cm to reduce movement in the vertical plane and illuminated with fluorescent lighting strips. Water temperature of the arena reflected the treatment temperature that fish were held in. Water temperature was monitored with a C20 Comark thermometer . Temperature was maintained with a glass bar heater  and air stone for aeration. Complete water exchanges were conducted every 10 min or after 3 fish. The walls and top had opaque covers to prevent outside disturbance. To elicit an escape response, a startling stimulus consisting of a weight with a tapered end was released through a tube suspended above the centre of the arena. The suspended tube stopped ∼1 cm above the water’s surface concealing the stimulus weight as it fell thus creating a more sudden disturbance. A monofilament line was used to raise the weight and was long enough that the end touched just the water’s surface when released, preventing collision with the individual. To start a trial, fish were placed into a central habituation ring (10 cm diameter) via a water filled sample jar and given a one-minute habituation period. Following the habituation period, the central ring was gently raised and the stimulus weight was released, striking the water in an attempt to elicit an escape response. If a C-start was performed, no further attempts were conducted and the fish was returned to its holding tank. If no reaction was observed, fish were replaced into the habituation ring for another minute and the second attempt was conducted. Fish were given three attempts to perform a C-start before being recorded as “no reaction”. The escape response was recorded using a high-speed camera  directed at a 45° angled mirror placed beneath the testing tank.After the exposure period, individuals were measured Responsiveness: proportions of fish that responded with a “C-start” or “no C-start”. No C-start was subdivided into “avoid” to the stimulus by swimming away but not performing a desired C-start and “no reaction” to the stimulus. (b)Directionality: left and right side were split anteroposteriorly from directly above the fish. Directionality was determined by whether the head turned “away” or “toward” the side the stimulus occurred during stage 1 of the response. (c)Response latency (ms): time between the stimulus onset and first movement of the individual. (d)Escape distance (mm): distance travelled during the response to the end of stage 2. (e)−1): maximum velocity achieved at any time during the escape response.Maximum swim speed . Maximum swim speed (m s−1) was converted to body length s−1 to control for any effect of size. Control temperature did not consist of two exposure combinations like the elevated temperatures, therefore a full-factorial 3 × 2 design was not possible. Instead, the five treatments  were categorised into one “temperature treatment” factor. A binomial logistic regression was used to ascertain the effects of temperature treatment on the likelihood of an individual to elicit a C-start after the stimulus and the likelihood of that individual to turn away from the stimulus. Response latency, maximum swim speed, and escape distance were used as dependent variables analysed separately with one-way ANOVAs (α = 0.05) and temperature treatment as the fixed factor. These analyses were repeated for both species (replicates ranged from 7–12 for P. amboinensis and 9–19 for P. moluccensis). Preliminary analysis of response latency, maximum swim speed, and response distance used a linear mixed effects model with holding tank as a random factor to test for a tank effect. However, AIC values were lower for models not including random tank factor with ΔAIC <2 to models that did include random tank factor. Consequently, this term was dropped from the final model. We predicted that short-term exposure to elevated temperature would have a negative impact on escape responses. If beneficial plasticity was possible with long-term exposure, individuals exposed for 90d would exhibit improvement in performance that either approached or returned to present-day control levels.Statistical analyses were performed using IBM SPSS statistics (v23.0.0.2). Proximity of fish to the stimulus at the onset and body size can affect escape responses . The cenP. amboinensis (χ2(4) = 4.18, p = 0.38; P. amboinensis (χ2(4) = 11.23, p = 0.024; R2) of the variance in directionality of C-starts and correctly classified 67.60% of cases. Compared to controls, fish exposed to 30 °C for 4 days were 5 times more likely (33.33%) to turn towards the stimulus and 6.67 times more likely (40%) after 90 days. At 31 °C, fish were 25 times more likely (71.43%) to turn towards the stimulus, which was the same for both exposure durations. Temperature nor exposure duration had any significant effect on response latency  = 16.58, p = 0.002; R2) of the variance in proportion of individuals that elicited a C-start and correctly classified 80% of cases. At control, 82.61% of fish displayed a C-start. Of the fish that did not C-start, most showed avoidance behaviour (13.04%) and no reaction (4.35%) least of all. Responsiveness rose to absolute  after four days of exposure to either 30 or 31 °C. When 90 day exposure was compared to controls, individuals were 1.45 times less likely (76.47%) to perform a C-start at 30 °C and 3.55 times less likely (57.14%) at 31 °C. For individuals that did not display a C-start, there was an increase in no reaction up to 38% at 31 °C (from 4.35% at control). There was no significance in the regression model for directionality (χ2(4) = 5.98, p = 0.20; F4,60 = 1.23, p = 0.30, partial η2 = 0.052; F4,60 = 0.19, p = 0.94, partial η2 = 0.013; F4,60 = 0.85, p = 0.49, partial η2 = 0.054; P. moluccensis.For P. moluccensis followed our predictions by reducing the number of turns towards the stimulus and returning to control levels. For P. moluccensis, long-term exposure also altered responsiveness with fish responding less often with a C-start response at elevated temperatures. Such alterations in the C-start responsiveness to predators is likely to change prey survival, while the differential effects of temperature on the two closely related species suggests that future temperature elevation may affect community composition through differential survival.Studying how escape responses are affected by elevated temperature can be important when making predictions on the ability of prey to escape predators under climate change conditions . SupportP. moluccensis from 80% to 100%, and reduced avoidance reactions to 0% for both species. Generally, proportion of C-starts has been shown to increase with exposure to temperature both above  in this species has also been shown to occur for aerobic metabolism . Furthermore, precise measurement of escape success will require future study with only the prey, only the predator, and both exposed to elevated temperature as predators will also likely have specific responses to thermal conditions. Future studies may also consider a longitudinal approach with several populations from differing sites to consider adaptation to local thermal environments. Studies investigating the factors that influence predator–prey interactions, like this one, are crucial because these interactions determine population sizes and replenishment, leading to changes in community structure.To conclude, both species showed negative effects of increased temperature after short-term, though the impact was greater for ractions . Changes10.7717/peerj.3652/supp-1Supplemental Information 1−1), and response distance (cm).Percentages, means, and standard deviations for responsiveness (percent of reactions), directionality (percent of turns), response latency (ms), max speed (body lengths sClick here for additional data file.


Tumor necrosis factor-alpha (TNF-α) is a potent pro-inflammatory mediator and its expression is up-regulated in chronic obstructive pulmonary disease (COPD). Tristetraprolin (TTP) is implicated in regulation of TNF-α expression; however, whether TTP is involved in cigarette smoke-induced TNF-α expression has not been determined.TTP expression was examined by western blot analysis in murine alveolar macrophages and alveolar epithelial cells challenged without or with cigarette smoke extract (CSE). TNF-α mRNA stability, and the decay of TNF-α mRNA, were determined by real-time quantitative RT-PCR. TNF-α protein levels were examined at the same time in these cells. To identify the molecular mechanism involved, a construct expressing the human beta-globin reporter mRNA containing the TNF-α 3’-untranslated region was generated to characterize the TTP targeted site within TNF-α mRNA.CSE induced TTP down-regulation in alveolar macrophages and alveolar epithelial cells. Reduced TTP expression resulted in significantly increased TNF-α mRNA stability. Importantly, increased TNF-α mRNA stability due to impaired TTP function resulted in significantly increased TNF-α levels in these cells. Forced TTP expression abrogated the increased TNF-α mRNA stability and expression induced by CSE. By using the globin reporter construct containing TNF-α mRNA 3’-untranslated region, the data indicate that TTP directly targets the adenine- and uridine-rich region (ARE) of TNF-α mRNA and negatively regulates TNF-α expression at the post-transcriptional level.The data demonstrate that cigarette smoke exposure reduces TTP expression and impairs TTP function, resulting in significantly increased TNF-α mRNA stability and excessive TNF-α expression in alveolar macrophages and epithelial cells. The data suggest that TTP is a novel post-transcriptional regulator and limits excessive TNF-α expression and inflammatory response induced by cigarette smoke. Cigarette smoke exposure has been firmly associated with the development of chronic obstructive pulmonary disease (COPD) –4. COPD Persistent inflammation is thought as the main driving force of COPD pathogenesis ;7–10. InTumor necrosis factor alpha (TNF-α) is one of the potent pro-inflammation mediators and its expression is up-regulated in COPD lungs –17. Incrin vivo. Mice deficient in TTP developed a severe syndrome characterized by growth retardation and cachexia, arthritis, dermatitis, and autoimmunity [Tristetraprolin (TTP) is a widely distributed phospho-protein encoded by the immediate–early response gene –23. TTP immunity . Treatmeimmunity , demonstThis study aims to investigate whether cigarette smoke extract exposure dampens TTP function and whether impaired TTP function contributes to the increased TNF-α expression induced by cigarette smoke extract exposure. The results show that cigarette smoke extract exposure reduced TTP expression in alveolar macrophage and alveolar epithelial cells. Reduced TTP expression leads to significantly increased stability of TNF-α mRNA, and results in increased and prolonged expression of TNF-α in alveolar macrophage and alveolar epithelial cells. Exogenous TTP overexpression abrogated the increased stability of TNF-α mRNA in macrophages and epithelial cells exposed to cigarette smoke, resulting in significantly reduced TNF-α protein levels in these cells. Furthermore, the data revealed the molecular mechanism involved. TTP binds to and directly targets the adenine- and uridine-rich region (ARE) of TNF-α mRNA and negatively regulates TNF-α expression at the post-transcriptional level. Taken together, the data demonstrate that cigarette smoke exposure impairs TTP function resulting in prolonged TNF-α mRNA stability and persistent TNF-α expression. TTP is a novel post-transcriptional regulator and limits the TNF-α expression and pro-inflammatory response induced by cigarette smoke.Research cigarettes (3R4F) were purchased from the center for tobacco reference products at the University of Kentucky . The following purified antibodies were purchased: anti-TTP, anti- human influenza hemagglutinin (HA) tag, anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and anti- green fluorescent protein (GFP) , biotinylated rat anti-mouse anti-CD-45 , biotinylated rat anti-mouse anti-CD-16/32 . Enzyme-linked immunosorbent assay (ELISA) kits to determine the TNF-α protein level were purchased from the R&D System . All other reagents were purchased from Sigma  or Fisher Scientific .in vitro cigarette smoke exposure as described previously [CSE was used to study the effects of eviously –30. All eviously . The CSEeviously , these aPrimary alveolar macrophages were isolated from C57Bl/6 mice (Jackson Laboratory) and performed as described –32. The Generation, amplification, and utilization of the replication incompetent adenoviral vectors were described previously –36. Brie5’-AGATCTATCGATCTGCAGGATATCGCGGCCGCGTCGACAAGCTTGCATGC-3’) was inserted into a BglII site immediately downstream of the stop codon of the β-globin gene, which was previously subcloned into a tetracycline (Tet)-regulatory vector, pTRE (BD Biosciences). The TNF-α ARE  was then inserted between EcoRV and HindIII sites and infected cells as described previously by us [The construct expressing the human beta (β)-globin reporter mRNA containing the TNF-α ARE (pTRE-hGB-TNF), the pTRE empty control construct, and the pTet-Off construct were described previously by us . Brieflyly by us ;38.mRNA levels were determined by real-time quantitative RT-PCR and mRNA stability was evaluated through measurement of half-life as described by us previously . Half-liEnzyme-linked immunosorbent assay (ELISA) kits were used to determine the TNF-α protein levels per manufacture’s instructions.Western blot assays were performed as described by us previously . BrieflyAll data are summarized as mean ± S.E. For the statistical analysis, Sigma Plot  was used for this study. A Student t-test was used to compare two experimental groups. A p value of <0.05 was considered statistically significant.To understand the impact of cigarette smoke (CS) exposure on TTP function, we first examined TTP expression in alveolar macrophage and alveolar epithelial cells treated with CS extract (CSE). Primary alveolar macrophages and lung epithelial cells were obtained from C57Bl/6 mice. The rat alveolar epithelial cells (L2) and the mouse alveolar macrophages (MHS) were used for optimization of the test conditions and the experiments were repeated in primary alveolar macrophages and epithelial cells. Primary alveolar macrophages were treated with CSE (4% or 8% volume/volume) for 16 hours. Control primary alveolar macrophages were treated similarly with CSE control medium (cigarettes were not lit and not burnt). Western blot analysis of whole cell lysates showed that TTP level was significantly reduced in primary alveolar macrophages treated with CSE when compared to alveolar macrophages treated with CSE control medium , supportConsistently, CSE treatment had similar effects on TNF-α mRNA half-life and stability in rat alveolar epithelial cells , mouse aThe above data demonstrate that CSE induces TTP down-regulation , and CSEForced expression of TTP  significantly reduced TNF-α stability and half-life in alveolar macrophages , indicatSimilarly, forced expression of TTP significantly reduced TNF-α stability and half-life in rat alveolar epithelial cells (L2)  reporter gene was expressed when cells were cultured in medium without tetracycline (the Tet-Off system). The hGB expression was confirmed by detection of hGB mRNA in primary mouse alveolar macrophages  (nucleotides 1221–1310) into a tetracycline (Tet) regulated reporter vector  TTP expression on CSE-increased hGB mRNA stability, alveolar macrophages were infected with adenoviral TTP vectors and then treated with or without CSE. Forced exogenous expression of TTP significantly reduced hGB stability and half-life .are reporter and pTet-Off regulator vectors. The hGB reporter mRNA expression was confirmed in epithelial cells infected with both pTRE-hGB-TNFare reporter and pTet-Off regulator vectors  were infected with the pTRE-hGB-TNF vectors . Similar vectors . The fin vectors . Taken tTobacco smoke has been firmly associated with the development of chronic obstructive pulmonary disease (COPD) –4. COPD Tumor necrosis factor alpha (TNF-α) is one of the potent pro-inflammation mediators and its expression is up-regulated in COPD lungs –17. IncrTTP is a widely distributed phospho-protein encoded by the immediate–early response gene –23. TTP Although it is known that TTP negatively regulates TNF-α mRNA stability ;36;45–48in vivo [in vivo has not been examined in detail and is a focus of the future work. Given the evidence suggesting that TTP is one major regulator of the expression of TNF-α and other mediators [Furthermore, ectopic expression of TTP significantly reduced TNF-α mRNA stability and half-life in alveolar macrophages and epithelial cells . The datin vivo . Mice dein vivo . Treatmein vivo , demonstediators ;49–53 anediators –10, TTP are reporter), this study demonstrates that TTP negatively regulates TNF-α expression at the mRNA level by directly targeting the 3’-AU rich region (ARE) of TNF-α mRNA and degrading TNF-α mRNA (Regulation of the expression of pro-inflammation mediators is complicated and it is likely mediated by multiple pathways, either directly or indirectly ;54–58. IF-α mRNA . This meF-α mRNA . As persIn summary, we demonstrate in this study that cigarette smoke exposure induces TTP down-regulation in alveolar macrophage and alveolar epithelial cells. Reduced TTP expression leads to significantly increased stability of TNF-α mRNA and TNF-α expression in macrophages and epithelial cells exposed to cigarette smoke extract. Forced TTP expression abrogates the increased stability of TNF-α mRNA resulting in significantly reduced TNF-α expression in these cells. Importantly, the data demonstrate that TTP limits excessive TNF-α expression through a post-transcription mechanism by directly targeting the ARE regions of TNF-α mRNA and negatively regulates TNF-α expression at the post-transcriptional level. Taken together, the data demonstrate that TTP is a novel post-transcriptional regulator and limits the TNF-α expression and pro-inflammatory response induced by cigarette smoke. Cigarette smoke exposure impairs TTP function resulting in prolonged TNF-α mRNA stability and that subsequently leads to persistent TNF-α expression, which has been firmly associated with tissue injury and COPD pathogenesis.