Limitless cell proliferation, evasion from apoptosis, dedifferentiation, metastatic spread and therapy resistance: all these properties of a cancer cell contribute to its malignant phenotype and affect patient outcome. MYBL2  is a transcription factor of the MYB transcription factor family and a physiological regulator of cell cycle progression, cell survival and cell differentiation. When deregulated in cancer cells, MYBL2 mediates the deregulation of these properties. In fact, MYBL2 is overexpressed and associated with poor patient outcome in numerous cancer entities. MYBL2 and players of its downstream transcriptional network can be used as prognostic and/or predictive biomarkers as well as potential therapeutic targets to offer less toxic and more specific anti-cancer therapies in future. In this review, we summarize current knowledge on the physiological roles of MYBL2 and highlight the impact of its deregulation on cancer initiation and progression. MYBL2 is a highly conserved member of the MYB family of transcription factors.MYBL2 is an important physiological regulator of cell cycle progression, cell survival and cell differentiation.Deregulation of MYBL2 expression is involved in cancer initiation and progression.High MYBL2 expression is significantly correlated with poor patient outcome in numerous cancer entities.What are further players of the MYBL2 downstream transcriptional network mediating its cancer-promoting properties?How can MYBL2 and players of its downstream transcriptional network be exploited as therapeutic targets to improve patient outcome?Which additional cancer entities are also affected by MYBL2 deregulation and which patients could specifically benefit from using MYBL2 as a biomarker or therapeutic target?1 characterize the malignant phenotype of a cancer cell. MYBL2 , a transcription factor of the MYB family of transcription factors, contributes to these properties of a cancer cell. MYBL2 is a physiological regulator of cell cycle progression, cell survival and cell differentiation, but due to its frequently found deregulation in cancer, it significantly drives cancer initiation and/or progression.Limitless replicative potential, evading apoptosis, tissue invasion and metastasis: these classical hallmarks of cancer, as originally proposed by Hanahan and Weinberg,MYB was the first discovered family member and is the mammalian homolog of the retroviral v-Myb oncogene that causes acute leukemia in birds and can transform hematopoietic cells.3MYBL1 and MYBL2 have been cloned based on the homology to MYB.4 In mammals, MYB expression is mainly restricted to hematopoietic cells, colonic crypts and brain,6 whereas MYBL1 is expressed in several regions of the developing central nervous system, germinal B-lymphocytes and reproductive systems of both genders.8 In contrast, MYBL2 is expressed in basically all proliferating cells,3 which is a possible explanation for the lethal phenotype of MYBL2 knockout mice showing early embryonal death as a result of impaired inner cell mass formation,9 whereas MYBL1 deletion results in viable mice and MYB deletion leads to late embryonal death by cause of lacking erythropoiesis.10The MYB family of transcription factors comprises three members: MYB (c-Myb), MYBL1 (A-Myb) and MYBL2 (B-Myb). 11 whereas MYBL1 has been associated with Burkitt’s lymphoma and several types of leukemia.12 In contrast, MYBL2 deregulations occur in a broad spectrum of cancer entities as it is a central regulator of cell cycle progression, cell survival and cell differentiation in many tissue types (see ‘MYBL2 in cancer’ section). In this review, we summarize the physiological roles of MYBL2 in cell cycle regulation, cell survival and cell differentiation, and describe its deregulation as well as the resulting functional and clinical implications in cancer.According to their tissue-specific expression, MYB and MYBL1 deregulations have been associated with certain specific cancer entities: MYB was shown to be involved in several types of leukemia, colon and breast cancer,MYBL2 is a cell cycle regulated and a cell cycle regulating gene. Its expression is controlled by the DREAM multiprotein complex , which is crucial in coordinating cell cycle-dependent gene expression and represses most cell cycle genes during cellular quiescence.13 This complex consists of the dimerization partner , the RB-like proteins p130 or p107, E2F (E2F4 or E2F5) and the multi-vulval class B core .13 Upon cell cycle entry, p130 or p107 dissociate from the MuvB core and from repressor E2Fs  due to loss of DYRK1A-dependent phosphorylation of LIN52, allowing activator E2Fs (E2F1 or E2F2 or E2F3) to transactivate early G1/S cell cycle genes, including MYBL2.13 Accordingly, MYBL2 is repressed by the DREAM complex during cellular quiescence and becomes subsequently expressed in late G1 and in S phase.13 Additionally, at a post-transcriptional level, MYBL2 expression can be suppressed by microRNAs.1922 Interestingly, Cyclin A-dependent phosphorylation simultaneously reduces MYBL2 protein expression by facilitating ubiquitin-mediated proteolysis of MYBL2, probably to regulate the proper alternation of events during cell cycle progression.23 p300, a transcriptional co-activating protein, is also able to stimulate MYBL2 activity by MYBL2 acetylation, depending on its phosphorylation by Cyclin A.24 However, contrary to Cyclin A, Cyclin D1 strongly inhibits transactivation activity of MYBL2 through direct interaction.25 Mechanistically, it was proposed that Cyclin D1 abolishes the activating function of p300.24 Several other co-activators, such as PARP1, ZPR9, TAF(II)250, or co-repressors, such as p107, p57 or CDK9, were further shown to modulate transactivation properties of MYBL2.33Apart from MYBL2 expression, the transcriptional activity of MYBL2 is regulated by posttranslational modifications and protein–protein interactions. During late G1 and S phase, MYBL2 is phosphorylated by Cyclin A/E-CDK2, which enhances its transactivation activity, probably by releasing it from the nuclear receptor co-repressors N-CoR and SMRT, which maintain MYBL2 in an inhibited state when non-phosphorylated.Cyclin B1 (CCNB1),34CDK1,34Cyclin A2 (CCNA2)34 , cell cycle genes homology region  elements and FOXM1 binding motifs co-occur in the promoters of these genes.36 A model emerged over the years: When DREAM dissociates upon cell cycle entry, MYBL2 becomes increasingly expressed and activated, and forms a complex with MuvB in early and mid S phase. RNAi experiments showed that knockdown of either MYBL2 or components of the MuvB core complex inhibits target gene promoter binding of the other.36 Consistently, depletion of either MBS or CHR motifs in the promoters of these target genes independently prohibits target gene promoter binding.54 These results clearly indicate a dependency of both factors to one another in transactivating late cell cycle genes in early and mid S phase. Afterwards, the MYBL2–MuvB complex recruits FOXM1 in late S phase, forming a MYBL2–MuvB–FOXM1 complex.13 MYBL2 increasingly undergoes phosphorylation-dependent proteasomal degradation in late S phase,23 leading to predominant MuvB–FOXM1 complex in G2/M phase, whereby FOXM1 is increasingly activated by phosphorylation.55 Depletion of FOXM1 did not affect MYBL2 target gene promoter binding, but conversely, MYBL2 or LIN9 depletion reduced FOXM1 target gene promoter binding, indicating that the MYBL2–MuvB complex is required for FOXM1 target gene promoter binding, but not vice versa.36 As the FOXM1 DNA-binding domain has an extraordinarily low binding affinity to its target sequence and MBS and CHR motifs are necessary for FOXM1 to bind the target promoters, it was proposed that the MYBL2–MuvB complex is needed to increase target specificity for FOXM1 binding.13 The residual MuvB–FOXM1 complex dissociates due to increasing APC/C-CDH1-dependent FOXM1 degradation during M phase56  MYBL2 depletion leads to stalling of replication forks, disorganization of the replication program and an increase in double-strand breaks.41 It has been shown that these effects are, at least in part, mediated by deregulation of MYC and FOXM1 transcription, which are important for normal S phase progression, indicating that MYBL2 protects cells from genomic damage during S phase by promoting proper cell cycle progression.41 Chromosomal fragmentation, shorter and thicker chromatids, end-to-end fusion and chromatid loss upon MYBL2 knockdown indicates that reduced activity of MYBL2 is also associated with structural chromosomal instability.69The association between MYBL2 and cell proliferation has already been described early by Arsura An association between MYBL2 and cell survival has already been reported in early studies. However, over the years, the role of MYBL2 in the regulation of cell survival became increasingly clear and is mainly mediated via transcriptional regulation of specific target genes, but can also be mediated by direct protein–protein interaction. The transcriptional regulation by MYBL2 seems to depend on the cell type: In most cell types MYBL2 appears to have pro-survival functions, whereas it mainly has anti-survival functions in cells of neural origin when exposed to apoptotic stimuli .et al.47 showed that MYBL2 overexpression in interleukin 2-dependent murine T cells is associated with enhanced transactivation of the anti-apoptotic Bcl-2, and hence diminished cytokine dependence and enhanced resistance to apoptosis induced by doxorubicin, ceramide and dexamethasone. Consistently, the transfection of a Bcl-2-non-expressing human B-cell line with a MYBL2 expression vector induced the expression of Bcl-2 and vice versa, antisense depletion of MYBL2 decreases Bcl-2 levels and enhances apoptosis.70 Furthermore, results of Cervellera et al.50 indicate that ApolipoproteinJ/Clusterin is a MYBL2 target gene, whose expression mediates resistance to apoptosis induced by the chemotherapeutic drug doxorubicin in neuroblastoma. Santilli et al.62 further confirm these results: under conditions of thermal stress, MYBL2-dependent ApolipoproteinJ/Clusterin expression is enhanced due to redox modification of MYBL2 and constitutes a protective response mechanism to thermal injury in MEFs. MYBL2 has also been shown to suppress autophagy and to promote cell survival of ovarian oocytes by binding the promoter and directly activating the transcription of VDAC2.51Grassilli 71 This indicates a required role for MYBL2 in neuronal apoptosis after E2F de-repression due to apoptotic stimuli.72 A model has been proposed by which E2F4–p130 protein complexes protect neurons from cell death by occupying the MYBL2 promoter under basal conditions, whereas under conditions of cell stress these complexes are lost and replaced by E2F1 transactivating MYBL2 and thus promoting cell death.73 In neurons, MYBL2 is able to bind the promoter and to transactivate the pro-apoptotic gene BCL2L11 (Bim).48 The following interaction of Bim with the cellular apoptotic machinery leads to caspase activation and apoptotic cell death.48 MYBL2 was also shown to be required for beta-amyloid-dependent induction of Bim and cell death, relevant in Alzheimer’s disease.74 Also in Drosophila, dMyb promotes the programmed cell death of specified sensory organ precursor daughter cells.75 However, not only in neural cells, but also in TGF-β1-treated M1 myeloid leukemia cell lines overexpressing MYBL2, TGF-β1-induced apoptosis was found to be accelerated.76However, in contrast to these findings, MYBL2 seems to have a contrary role concerning cell survival predominantly in neural cells. MYBL2 knockdown protects pheochromocytoma cells, sympathetic neurons and cortical neurons against cell death elicited by NGF withdrawal or DNA damage.77 On the one hand, MYBL2 can enhance STRAP-mediated inhibition of TGF-β signaling pathways, such as apoptosis and growth inhibition, by inhibiting TGF-β receptor association with SMAD3 and enhancing TGF-β receptor association with SMAD7, and thereby prevent translocation of SMAD3 in the nucleus in response to TGF-β1 .77 On the other hand, co-expression of MYBL2 results in increased STRAP-mediated stimulation of p53 nuclear translocation, p53-induced apoptosis and cell cycle arrest via reduction of p53–MDM2 association .77Independent of the transactivation capabilities of MYBL2, it is further able to regulate cell survival by direct interaction with the serine–threonine kinase receptor-associated protein (STRAP), for which MYBL2 can serve as a positive regulator.78Several lines of evidence indicate that MYBL2 contributes to the maintenance of an undifferentiated and/or stem cell-like phenotype of a cell. Especially in stem cells, the balance between cellular quiescence on the one hand, and cell division in order to generate more stem cells  or to give rise to mature cells (differentiation) on the other hand, is important for the maintenance of the stem cell pool.79 Later on, in neuroblastoma cells, MYBL2 expression was found to be downregulated during retinoic acid-induced neural and glial differentiation and conversely, constitutive expression of MYBL2 prevents retinoic acid-induced neural differentiation.80 Compatible with this, levels of p130, a member of the DREAM complex (see ‘MYBL2 in cell cycle regulation’ section) that is able to suppress the MYBL2 promoter upon transfection, was shown to be strongly upregulated during mid/late differentiation stages, whereas MYBL2 levels decrease.81 Comparable results indicating a role for MYBL2 to maintain cells in an undifferentiated state have also been shown for several different cell types, such as leukemic cell lines,82 male gonocytes,83 intestinal epithelial cells38 and keratinocytes.84Early results showed that MYBL2 protein levels decrease upon differentiation of human myeloid cell lines.52 Especially for the maintenance of pluripotency, MYBL2 directly regulates the expression of POU5F1, SOX2 and NANOG, which are critical mediators of differentiation and pluripotency in ESC.85 Similarly, MYBL2 was shown to control self-renewal and differentiation of hematopoietic stem cells, possibly by downregulating ID1 and CEBPα, which promote cellular differentiation, while upregulating GATA2, a transcription factor shown to promote proliferation at the expense of differentiation86. Additionally, MYBL2 expression can be deregulated at a post-transcriptional level via deregulation of microRNAs, a class of small non-coding RNAs often found to be deregulated in cancer97 and of which some were shown to suppress MYBL2 mRNA expression.19On the one hand, altered 98 Physiologically, p53 directly activates p21, which prevents following p130 phosphorylation by cyclin-dependent kinases, leading to a switch from MYBL2–MuvB to DREAM complex by shifting MuvB-associated proteins from MYBL2 to E2F4/DP1/p130.99 It has been described that the p53-p21-DREAM pathway represses MYBL2 expression, especially under conditions of cellular stress, such as DNA damage, which mechanistically explains MYBL2 deregulation, and thus deregulation of cell cycle progression and cell survival in p53-mutated cancers.101 This is in accordance with results from Parikh et al.102 showing that MYBL2 is disproportionately upregulated in many p53 mutant cancers. MYBL2 has even been shown to overcome DNA damage-induced G2 checkpoint arrest in p53 mutant cells103 and constitutive expression of MYBL2 has been shown to overcome p53-induced G1 checkpoint arrest.59 Furthermore, the oncoviral HPV16 E7 protein is able to deregulate DREAM complex assembly and to thereby drive MYBL2 expression.104 Consistent to this, HPV16-immortalized cells show upregulated expression of MYBL2.105 Mechanistically, the HPV16 E7 oncogene can bind to p130, promote its proteasomal degradation and thereby disassemble the DREAM complex.106 E7 in addition directly binds to the MYBL2–MuvB–FoxM1 complex, leading to cooperative transcriptional activation of mitotic genes.106 MYBL2 moreover mediates abrogation of DNA damage-induced G1 checkpoint arrest, via regulation of CDK1 expression in E7 transformed cells107 and was shown to rescue oncogene-induced cellular senescence,108 a permanent cell cycle arrest that cells must bypass during cancer development,109 probably by suppressing p16INK4A expression.110p53 signaling is frequently altered in cancers.111 cervical cancer,112 colorectal cancer,89 hepatocellular carcinoma,91 leukemic cells,15 lung adenocarcinoma42 and neuroblastoma (in MYCN-amplified cell lines).113As described for non-malignant cells (see ‘MYBL2 in cell cycle regulation’ section), MYBL2 has also been shown to drive cell proliferation and/or cell cycle progression in cancer cells, such as breast cancer,47 These results from Grassilli et al. are in accordance with results from Levenson et al.,114 showing that MYBL2 is upregulated upon genetic suppressor element-induced drug resistance to DNA-interactive agents, such as aphidicolin, hydroxyurea, cytarabine, etoposide, doxorubicin and mafosfamide in fibrosarcoma cells. In neuroblastoma, MYBL2 directly regulates expression of ApolipoproteinJ/Clusterin and thereby mediates resistance to apoptosis induced by doxorubicin.50Resistance to chemo- and radiotherapy is one of the main properties of a cancer that determines cancer progression and patient outcome. MYBL2 overexpression in interleukin 2-dependent murine T cells was shown to be associated with enhanced resistance to drug-induced apoptosis by doxorubicin, ceramide and dexamethasone, due to increased transactivation of the anti-apoptotic Bcl-2 by MYBL2.101 Also, upon ionizing radiation, Cyclin F suppresses the MYBL2-regulated transcriptional program by directly interacting with MYBL2 and thereby suppressing Cyclin A-mediated phosphorylation of MYBL2.115 On the contrary, under non-physiological conditions, such as in p53 mutant cells, MYBL2 fails to dissociate from MuvB, which contributes to increased G2/M gene expression in response to DNA damage.103 In accordance, DT40 chicken B cells lacking MYBL2 show increased sensitivity to DNA damage elicited by UV irradiation and alkylation.116 Consistently, in Ewing sarcoma cells, MYBL2 can be destroyed quickly upon UV irradiation, leading to induction of apoptosis,117 whereas this is not the case in neuroblastoma, where MYBL2 levels do not change upon irradiation, making the cells resistant to UV-induced apoptosis.117 Interestingly, in neuroblastoma cells MYBL2 is found to be hypophosphorylated and overexpression of a non-phosphorylatable MYBL2 mutant in HEK 293 cells can protect cells from UV-induced apoptotic cell death, suggesting that decreased Cyclin A-dependent phosphorylation, accompanied by decreased activation but also decreased proteasomal degradation, can facilitate the survival promoting activity of MYBL2.117However, MYBL2 was not only shown to mediate resistance to chemotherapeutic agents, but also resistance to DNA damage, as, for example, caused by radiation. Under physiological conditions, such as in p53 wild-type cells, DNA damage results in p53-dependent binding of p130 and E2F4 to MuvB and the dissociation of the MYBL2–MuvB complex.89 hepatocellular carcinoma91 and leukemia cells.118Consistent with these results, a pro-survival role for MYBL2 has also been shown in several cancer cell lines, such as colorectal cancer,et al.58 have shown that the introduction of an inducible dominant interfering Myb protein into ESC lead to dissociation of ESC colonies into dispersed single cells and to reduced adhesion of the ESC to the culture dish. Cell adhesion analyses have shown that MYBL2 suppression decreased the adhesion with extracellular matrix proteins, such as Laminin, Collagen and Fibronectin, probably due to reduced cell surface expression of Beta1 Integrin.58Early results of Iwai 119 has been proposed: In breast cancer cells, MYBL2 knockdown is able to restore the expression of the epithelial marker E-Cadherin, the formation of cell–cell junctions and to suppress cell invasion, anchorage-independent growth and tumor formation.120 Conversely, MYBL2 overexpression decreased the expression of the E-Cadherin, but increased expression of mesenchymal markers.120 Mechanistically, it was proposed that MYBL2 upregulates the expression of the major EMT regulator SNAIL, thereby mediating activation of EMT and cancer cell invasion.120However, in contrast to these early findings, a role for MYBL2 in epithelial-to-mesenchymal transition (EMT), a process in which epithelial cells lose their polarity and gain migratory and invasive properties,111 Similar results were shown for prostate cancer and renal cell carcinoma: MYBL2 is overexpressed in prostate cancer (xenograft) metastases,121 whereas in renal cell carcinoma MYBL2 was found to be expressed in metastases from primary tumors being MYBL2 negative.93In accordance with this, MYBL2 protein levels have been shown to be significantly upregulated in matched breast cancer metastases compared to the primary tumor.123 Also, several inhibitors interfering with players of the downstream transcriptional network of MYBL2, such as inhibitors against Aurora kinases,124 FGF receptors,125 Kinesins,126 Bcl-2127 and BIRC5 (Survivin)128 have already been in clinical trials and may serve as an effective, more specific and less toxic future anti-cancer therapy in cancers highly expressing MYBL2.MYBL2 is a central regulator of cell cycle progression, cell survival and cell differentiation. Deregulation of MYBL2 expression can contribute significantly to cancer progression by promoting cancer cell proliferation, therapy resistance, metastatic spread and is correlated with poor patient outcome in several cancer entities. Therefore, MYBL2 and/or players of its downstream transcriptional network could serve as effective targets for cancer treatment. Although no direct MYBL2 inhibitor is available yet, CDK2 inhibition could be used to reduce MYBL2 activity in MYBL2 high-expressing cancers. Yet, highly specific CDK2 inhibitors are lacking, but several more or less specific CDK inhibitors have already been in clinical trials for cancer treatment.


Do highly productive researchers have significantly higher probability to produce top cited papers? Or do high productive researchers mainly produce a sea of irrelevant papers—in other words do we find a diminishing marginal result from productivity? The answer on these questions is important, as it may help to answer the question of whether the increased competition and increased use of indicators for research evaluation and accountability focus has perverse effects or not. We use a Swedish author disambiguated dataset consisting of 48.000 researchers and their WoS-publications during the period of 2008–2011 with citations until 2014 to investigate the relation between productivity and production of highly cited papers. As the analysis shows, quantity does make a difference. Highly Cited Researchers database, where they are listed and recognized per area. Using another database, Scopus, Klavans & Boyack phrase them ‘superstars’ in a large-scale study of publication behaviour, thereby showing that superstars publishes less in isolated areas (retrieved using a clustering procedure), in dying areas, or in areas without an inherent dynamics [One astonishing feature of the scientific enterprise is the role of a few extremely prolific researchers . Thomsondynamics . Highly Within bibliometrics there is a discussion on how to identify and measure “superstars”, and many papers discuss the correlation between the various indicators developed for performance measurement. One of the stable outcomes is that there is a high correlation between the numbers of papers a researcher has published and the number of citations received . From thnormal science, Kuhn [If one agrees that in science it is all about top (cited) publications, the question comes up what an efficient publication strategy would look like. Is publishing a lot the best way–or does that generally lead to ce, Kuhn , with once, Kuhn  and is ace, Kuhn  was ablece, Kuhn ), and thce, Kuhn ).However, others seem to see this differently. Firstly, recent empirical research suggests that on the long run, average output per researcher (corrected for the number of co-authors) has not increased at all . This suThis brings us to the question what the relation is between overall output (number of publications) and the number of high impact papers of researchers. Several possibilities exist, and only the last two would be evidence that more is not better:Increasing returns from productivity: The number of top papers increases faster than the total output . This was done as the academic labour market to quite a large extent is Scandinavian.All articles by each researcher are ranked, based on received citations and according to the about 260 subject categories as specified in the Web of Science, and the articles are divided into CSS  classes . While measures based on percentile groups (e.g. PPtop1% etc.) are arbitrarily constructed, CSS have some advantages concerning the identification of outstanding citation rates . The CSSIn the following we will investigate the relation between quality and quantity in several different ways. We do this, as from a methodological perspective different options are open, without a convincing argument which one would be the better. By using a variety of methods, we avoid to produce results that are only artefacts of a specific method.number of CSS3 papers increases with productivity  publications of an author as the independent variable, and the also integer counted number of top cited publications of the same author. We do this for each of the definitions of ‘top cited’ that were discussed above, using field-normalized citations . This is done for the total population of (publishing) researchers, without normalizing for field-based productivity differences. As the total set of researchers is dominated by life and medical sciences and by natural sciences, and as these groups have comparable average publications and citations, we assume that this does not influence the results. However, under point (iv) below, we introduce a way of taking field differences in productivity into account.We do the same analysis as under (ii), but use fractional instead of integer counting of publications and top-cited publications. This helps to investigate the effect of different ways of counting on the relations under study.http://science-metrix.com/en/classification>).We introduce field-normalized  productivity, and calculate the relation between in this way defined productivity and having at the number of fractionally counted publication in CSS1 and CSS3. As we produced field normalized output counts, we can provide an integrated analysis of all researchers across all fields. This is done with a method–Field Adjusted Production (FAP)—based on Waring estimations. This method was initially developed by Glänzel and his colleagues , 27, durWe calculated for each of the seven productivity classes the average number of CSS3 papers . So, in In number of CSS3 publications differ between the classes for all fields  more often non-significant. The estimated means of the number and share of CSS3 papers for the individual fields, as well as the 95% confidence levels are in Supporting information. The main ‘deviant’ patterns are for computer science, for the humanities and for the social sciences, where the highest productivity classes actually have a lower share of CSS3 papers than the one but highest class–as was also visible in We have done a regression analysis with high-cited papers as dependent variable, and productivity as independent variable. We did the analysis for the various definitions of top cited papers. For papers in the top 1% cited papers  the corrThe lower the citation threshold the higher the correlation. Why this is the case needs further investigation. One may conclude that the higher the level of citations, the more randomness in the data. However, a probably more obvious explanation is that the small number of high productive researchers with many top papers always have co-authors to these high cited papers who themselves are not highly productive, and may be in the science system only for a few years. This could be PhD students, temporary research staff, or post docs who e.g. have not pursued an academic career and stopped publishing. In that sense one also expects top cited authors in the lower productivity segments, reducing the explained variance. To control for this effect, one may include only PIs in the analysis, something we will address in a subsequent study.Why would this effect be stronger for the higher citation threshold? This is related to the distribution of productivity. As generally is the case in bibliometric distributions , also inThis effect is also stronger if there are more co-authors per paper. This is easily understood, as the more co-authors a top paper has, the more not productive co-authors exist that do have co-authored top-cited papersWe may conclude that co-authoring equalizes and is not beneficial to the small elite. Depending on the field, between 92% and 98% of the researchers does not have a single paper in the CSS3 class in the four years period under consideration. The number of co-authors differs between fields, and indeed this is related to the steepness of the curves in We did the above type of analysis also using fractional counting of productivity. The patterns are the same, with correlations of .37 and .79 Figs . But theAs discussed above, productivity figures differ between fields, and it may therefore be useful to normalize productivity data to field specific averages. The relation between the number of CSS1 papers and the total field adjusted output is strong: the correlation is high with r = 0.80 , and notWe do the same for CCS3 papers as dependent variable, the much narrower defined top, and field-adjusted productivity as independent variable. If we only include natural and health/life sciences, the correlation is considerable (r = 0.58), and about equally large as without applying FAP . HoweverAs the above results show, there is not only a strong correlation between productivity (number of papers) and impact (number of citations), that also holds for the production of high impact papers: the more papers, the more high impact papers. More specifically, for most fields there are constant or increasing marginal returns. In that sense, increased productivity of the research system is not a perverse effect of output oriented evaluation systems, but a positive development. It strongly increases the occurrence of breakthroughs and important inventions , as woulThe analysis also gives an indication of the output levels that one may strive at when selecting researchers for grants or jobs. To produce high impact papers, certain output levels seem to be required–of course at the same time dependent on which field is under study.Future work in this research line will cover various extensions: Firstly, we plan to extend the analysis to some other countries, which of course requires large-scale disambiguation of author names. Secondly, we will in a next version control for number of co-authors, and for gender . The forS1 File(PDF)Click here for additional data file.


The corresponding figures for rigidity were 43 vs. 15% (p < 0.001), for postural instability 71 vs. 22% (p < 0.001), and for tremor at rest 5 vs. 6% (not significant). The total UPDRS-m score was significantly higher among individuals with NPH (median = 12) than without (median = 1) and correlated significantly with the degree of NPH symptoms (r = −0.72) and ventriculomegaly (r = 0.31). In this study, parkinsonian symptoms, except resting tremor, were frequent in individuals with NPH and correlated with the severity of NPH symptoms. Asymmetric symptoms were uncommon. We recommend a liberal use of neuroradiological imaging when investigating a patient with parkinsonian features.It may be challenging to differentiate normal pressure hydrocephalus (NPH) from neurodegenerative disorders such as Parkinson’s disease. In this population-based study, we wanted to describe the frequency of parkinsonian symptoms among individuals with and without NPH, and whether the motor examination part of the Unified Parkinson’s Disease Rating Scale (UPDRS-m) score differs between these groups. Furthermore, we wanted to find out whether there was a relationship between UPDRS-m score, NPH symptoms, and radiological signs of NPH. A sample of 168 individuals over the age of 65 with and without self-reported symptoms of NPH underwent a computerized tomography of the brain and clinical examination, including UPDRS-m to grade parkinsonian symptoms. According to diagnostic guidelines, 38 fulfilled criteria for NPH, whereas 130 had unlikely NPH. Bradykinesia was significantly more common among those with NPH (79%) compared to those with unlikely NPH (32%) ( Parkinsonian symptomatology exists in addition to the classical triad of gait, cognitive, and urinary symptoms in normal pressure hydrocephalus (NPH) –4, and mThe Unified Parkinson’s Disease Rating Scale (UPDRS)  is a widAs parkinsonian features are important in the differential diagnosis of neurological disorders, we wanted to describe the frequency of such symptoms and compare the UPDRS-m score between individuals from the general population with and without signs of NPH. Furthermore, we wanted to find out whether there was a relationship between UPDRS-m score, NPH symptoms, and radiological signs of NPH.This study is part of an ongoing epidemiological study on the prevalence of NPH. Out of the total population of 28,000 individuals aged 65 years or older living in Jämtland County, 1000 were randomly selected from the Swedish population register and received a questionnaire on NPH symptoms. The questionnaire is based on an on-line screening tool for NPH , and con2O, or less. However, when CSF pressure measurements are not performed, as in the present study, the diagnosis “possible NPH with neuroradiological support” can be used instead. It requires an NPH-specific radiological picture of the brain with narrowing of the sulci and subarachnoid spaces over the high convexity/midline surface [To diagnose NPH, we used the guidelines by Mori et al. , which a surface . FinallyRadiological evaluation was made with a CT scan of the brain. Previously described radiological markers were analyzed, i.e., Evans’ index, callosal angle, signs of narrow medial sulci, focally enlarged sulci, dilated fissure Sylvii, and size of temporal horns  Fig. 2)2).Fig. 2To investigate the degree of parkinsonian motor symptoms, UPDRS-m  was usedWe used a scale developed by Hellström et al.  to measuThe neurologist was blinded to radiological data and, accordingly, the radiologist was blinded to clinical data.Descriptive statistics were used to present the frequency of different parkinsonian symptoms among individuals with and without signs of NPH.Differences in the level of parkinsonian symptoms and UPDRS score between the three groups, “unlikely”, “possible”, and “probable” NPH, were tested with the Fisher–Freeman–Halton exact test and Kruskal–Wallis statistical test, respectively.p < 0.05.Spearman correlation analyses were used to measure the association between UPDRS-m score and NPH score and the UPDRS-m score and continuous radiological variables , respectively. The level of significance was set to All analyses were performed using SPSS .The Regional Ethical Review Board in Umeå approved the study (Dnr 2014/180-31) and all participants gave written, informed consent.The sample consisted of 168 individuals, 75 men and 93 women, mean 75 (66–92) years. According to diagnostic criteria, 11 individuals had “probable NPH”, 27 individuals had “possible NPH”, and 130 had “unlikely NPH”. Table The frequency of specific parkinsonian symptoms according to NPH diagnosis is shown in Table Except for facial expression and tremor, the UPDRS-m scores differed significantly between the groups of “unlikely”, “possible”, and “probable” NPH Table . As exper = −0.72, p < 0.001) , whereas the radiological marker callosal angle (r = −0.11) did not.The score of NPH symptoms  correlat01) Fig. . FurtherIn this population-based study, bradykinesia and rigidity were more than twice, and postural instability more than three times as frequent in individuals with NPH than in those without NPH. In contrast, tremor at rest was unusual and similar between the two groups. The total UPDRS-m score was significantly higher among individuals with NPH and correlated to the severity of NPH symptoms. Our findings confirm the results from the previous studies on hospital-based material that parkinsonism can be a part of the clinical syndrome of NPH , 12–15.Of the 38 individuals with NPH, 71% showed parkinsonism according to the UK Brain Bank Criteria . This eqParkinsonian signs were also present in the group with “unlikely NPH” where we found bradykinesia in one-third of the individuals evaluated, rigidity in one out of seven, and postural instability in one out of five. These results are similar to those found in the previous population-based studies , 18.The pathophysiology of NPH is not fully understood. Theories involve a high resistance to CSF outflow contributing to ventricular enlargement, mechanical pressure of the brain parenchyma, disturbance of cerebral blood flow (CBF), and increased water content in periventricular areas , 25. In The reversibility of parkinsonian signs that has been reported after shunt surgery or CSF removal supports that these symptoms are caused by hydrocephalus and not just a result of comorbidity –15. A reThe findings in the present study highlight some of the diagnostic challenges when meeting a patient with parkinsonian features. However, there are some clinical hallmarks that can be useful. In contrast to PD , tremor Although it may complicate the diagnostic procedure, parkinsonian symptomatology in NPH should not exclude the patient from shunt surgery, as the symptoms diminish postoperatively –14.The strength of this study is the relatively large, unselected sample with individuals from the general population. Only three individuals had been under investigation for NPH: one had received a shunt a few years prior to the study, one received a shunt afterwards (June 2016), and one declined operation. The shunt operated patients improved postoperatively; they were both wheelchair bound before the operation and regained walking ability.2O in “probable NPH” [The study has some limitations, as well. We did not exclude individuals with common comorbidities such as arthrosis and vascular disease, to minimize the risk of excluding those who suffered from NPH as well. Although this might have influenced the UPDRS-m score, we believe that it increases the generalizability of the results. This is supported by the fact that the group without NPH did not show more parkinsonian symptoms than what has been previously reported in the general population of elderly , 18. We ble NPH” . HoweverFinally, we do not yet know how many of those with NPH in this study will be offered and respond to shunt surgery, i.e., fulfill the diagnosis of “definitive NPH”.In this study, parkinsonian motor symptoms, except resting tremor, were frequent among individuals with NPH and correlated with the severity of NPH symptoms. Asymmetric distribution was rare. We recommend a liberal use of neuroradiological imaging when investigating a patient with parkinsonian features, in particular when the symptoms do not response to dopaminergic treatment.


Mice with prior hippocampal neurodegeneration are more susceptible to systemic TNF-α, displaying:•Acute working memory deficits not present in normal animals similarly challenged.•Exaggerated sickness behavior with respect to normal TNF-challenged animals.•Heightened brain expression of TNF-α mRNA and IL-1β mRNA and protein. de novo neuronal death, synaptic loss or tau hyperphosphorylation that was distinguishable from that arising from ME7 alone. The data indicate that acutely elevated TNF-α has robust acute effects on brain function, selectively in the degenerating brain, but more sustained levels may be required to significantly impact on underlying neurodegeneration.Inflammation influences chronic neurodegeneration but its precise roles are not yet clear. Systemic inflammation caused by infection, trauma or co-morbidity can alter the brain’s inflammatory status, produce acute cognitive impairments, such as delirium, and drive new pathology and accelerated decline. Consistent with this, elevated systemic TNF-α is associated with more rapid cognitive decline over 6 months in Alzheimer’s disease patients. In the current study we challenged normal animals and those with existing progressive neurodegeneration (ME7 prion disease) with TNF-α (i.p.) to test the hypothesis that this cytokine has differential effects on cognitive function, sickness behavior and features of underlying pathology contingent on the animals’ baseline condition. TNF-α (50 μg/kg) had no impact on performance of normal animals  on working memory (T-maze) but produced acute impairments in ME7 animals similarly challenged. Plasma TNF-α and CCL2 levels were equivalent in NBH and ME7 TNF-challenged animals but hippocampal and hypothalamic transcription of IL-1β, TNF-α and CCL2 and translation of IL-1β were higher in ME7 + TNF-α than NBH + TNF-α animals. TNF-α produced an exaggerated sickness behavior response  in ME7 animals compared to that in NBH animals. However a single challenge with this dose was not sufficient to produce  Analyside novo neuronal pathology after a single (i.p.) challenge with TNF-α was explored using quantification of apoptotic cell death, synaptic loss and tau hyperphosphorylation post-TNF-α in ME7 and NBH animals.We sought to specifically investigate whether exogenously added systemic TNF-α has deleterious effects on cognitive function, inflammation, sickness behavior and neurodegeneration on a background of robust chronic neurodegeneration. In this study we used the ME7 model of prion disease, which shows robust synaptic loss, extracellular amyloidosis and a sequence of behavioral and cognitive impairments  and whic22.1ad libitum. Mice were anaesthetized intraperitoneally (i.p.) with Avertin  and positioned in a stereotaxic frame (Kopf instruments). Holes were drilled at −2.0 mm (anteroposterior) and ±1.6 mm  from Bregma and 1 μl 10% w/v ME7-infected C57BL/6 brain homogenate was injected into the hippocampus using a Hamilton microsyringe  to a depth of −1.7 mm. The needle was left in situ for 2 min before slow withdrawal. Control animals were administered 1 μl 10% w/v normal brain homogenate (NBH). Mice were placed in an incubator at ∼25 °C for recovery. When returned to their home cage they were administered Sucrose (5% w/v) and Carprofen  in their drinking water for 2 days post-surgery. Animals were monitored for recovery from surgery. All animal experimentation was performed under a license granted by the Minister for Health and Children, Ireland, with approval from the TCD Animal Research Ethics Committee and in compliance with the Cruelty to Animals Act, 1876 and the European Community Directive, 86/609/EEC. Every effort was made to minimize stress to the animals.Female C57BL/6 mice  were housed in cages of 5 at 21 °C with a 12:12 light, dark cycle. Food and water access was de novo pathology that more intense systemic inflammation was required to produce damage  at doses of 50 μg/kg or 250 μg/kg prepared in sterile saline . Lower doses were used for the T-maze experiments and higher doses for the pathology experiment. Our previous studies using these 2 TNF doses  suggeste/kg LPS; ) versus /kg LPS; ). This fde novo neuropathology induced by systemic inflammation in ME7 animals has been exclusively at 18–19 weeks, which is the period when neuronal cell soma loss is determined to be occurring in this prion model . However our prior demonstrations of on model .2.2Hippocampal-dependent working memory was assessed in mice by observing alternation behavior in a novel water T-maze task . The T-m2.3An ‘open field’ consisted of a plastic base (58 cm × 33 cm) surrounded by walls of 19 cm. The floor of the box is divided into a grid of equal sized squares. Measurement was made of distance travelled (grid squares crossed) and total number of rears (frequency with which the mice stood on their hind legs in the maze). Each animal’s baseline activity was measured prior to experimentation. Animals were scored for 2 min in the Open Field.A thermocouple rectal probe  was used to measure core body temperature. The mice were habituated to measurement of rectal temperature for 2 days prior to experimentation to minimize/standardise stress effects. Baseline temperature was taken at the time of challenge, followed by the timepoints indicated. Body weight was likewise taken immediately before challenge and at a number of times up to 18 h .2.4g for 15 min to remove cells and the plasma was then aliquoted and stored at −20 °C. Three tissue punches were taken from the brain: the left hippocampus, right hippocampus and the hypothalamus at the appropriate anterior-posterior coordinates from Bregma. Tissue was snap-frozen in liquid nitrogen and was stored at −80 °C until use.Animals for mRNA and protein analysis were terminally anaesthetised with sodium pentobarbital at 2 or 4 h post-challenge . The thoracic cavity was opened and blood collected in heparinised tubes directly from the right atrium of the heart. The animal was then rapidly transcardially perfused with heparinised saline to wash out the blood. Whole blood was spun at 1500xAnimals for immunohistochemical examination were terminally anaesthetised with sodium pentobarbital  at 9 or 18 h post-challenge and transcardially perfused with heparinised saline for approximately 3 min followed by 10% neutral buffered formalin  for approximately 15 min. Brains were post-fixed in formalin and then embedded in paraffin wax. Coronal sections (10 μm) were cut on a Leica RM2235 Rotary Microtome  at the level of the hippocampus and floated onto electrostatically charged slides  and dried at 37 °C overnight.2.52O2 were used as substrate and the reaction was stopped with 1 M H2SO4 before optical density was read at 450 nm with correction at 570 nm. Standard curves were constructed and samples were quantified only if the absorbance fell on the linear portion of the standard curve. IL-1β protein in the hippocampus was quantified using a Quantikine mouse IL-1β kit  according to manufacturer’s instructions.Plasma samples were diluted appropriately and then analysed for TNF-α and CCL2. Mouse TNF-α and CCL2 were quantified by sandwich-type ELISA, using R&D systems duo set kits . A standard protocol was followed. The capture anti-TNF-α and CCL2 antibodies were diluted 1/180 (approximately 1 μg/ml) in PBS, and used to coat a 96-well maxisorb microplate overnight  with 100 μl per well. Plates were then washed with PBS + 0.05% Tween and blocked with PBS + 1% BSA before addition of 100 μl samples and standards for 2 h at room temperature. Detection antibodies were used at a dilution of 1/180 and incubated on the plates for 1.5 h. Streptavidin poly-horseradish peroxidase  was diluted in 1:10,000 and incubated in the dark at room temperature for 20 min. Samples, standards, detection antibodies and streptavidin poly-horseradish peroxidase were diluted in high performance ELISA buffer . TMB and H2.6Total RNA was isolated using the RNeasy Plus Mini method  as per the manufacturer’s instructions. To ensure complete DNA elimination from the column-bound RNA, an on-column DNase step was performed. The RNA yield and quality of each sample was quantified based on Optical Density (OD) using the NanoDrop’ND-1000 UV–vis spectrophotometer . cDNA synthesis was carried out using a High Capacity cDNA Reverse Transcriptase Kit . cDNA was stored at −20 °C until use in RT-PCR. All primer and probe sets were designed using Applied Biosystems Primer Express software and amplified a single sequence of the correct amplicon size, as verified by SDS-PAGE. Where no probe sequence is shown, the DNA binding dye SYBR green was used in its place. Primer pair/probe sequences are shown in 2.72O2/methanol (20 min). Sections were then pre-treated with citrate buffer (pH 6) for 2 × 5 min in the microwave (except SY38). IBA-1 sections were then pre-treated with 0.04% pepsin in 0.1 M HCl for 20 min prior to blocking. Slides were blocked with the appropriate serum. Primary antibodies were incubated overnight at 4 °C. Thereafter the ABC method was used as previously described . The DeadEnd Fluorometric TUNEL system  was used in conjunction with a biotinylated anti-Fluorescein secondary to visualise apoptotic cells in accordance with manufacturer’s instructions.Sections were labelled with IBA-1, 1/2000, ab5076 , SY38, 1/2000 , AT8, 1/200, MN1020 , Caspase 3, 1/50, ab3623 ; SY38 sections were treated with 0.2 M boric acid, pH 9 at 65 °C for 30 min then cooled to room temperature prior to quenching. All sections were quenched with 1% Hescribed  with per2.8TUNEL-positive and Caspase-3-positive cells were counted by two experimentors, blinded to experimental treatment. Tau was quantified in the CA3 region by determining the area fraction labelled by the AT8 antibody. The density of synaptophysin staining was determined by monochrome pixel density analysis of digitally captured images using Leica QWIN image analysis software  as described in . The tra2) was automatically determined by counting all the objects between 200 and 6000 pixels2.IBA-1 quantification: The number of Iba-1 positive cells was determined, in 10 μm sections, in the dorsal part of the hippocampus  in high-quality microphotographs taken under 20X objective. At least four counting frames per animal were analysed  with Image J software . Files were converted to 8 bit binary images and the intensity threshold set at 150. The number of cells per field  followed by Bonferroni 33.16,328 = 3.09). An a priori prediction from previous studies in the laboratory suggested that the effect of TNF-α on working memory would be a transient one (The effect of TNF-α (50 μg/kg) on working memory was examined using the alternation T-maze. All animals were confirmed to have acceptable baseline performance ⩾70% for three consecutive days pre-challenge. On the days of saline or TNF-α treatments, animals underwent 3 blocks of 5 trials  and 2 further blocks of 5 trials at 24-h post-challenge to confirm recovery. Systemically administered TNF-α induced a transient deficit in working memory in the T-maze task in ME7 animals, while there were no deficits in NBH animals similarly challenged . Using 3ient one  and at t3.2Transcripts for selected inflammatory cytokines were examined in the hippocampus and hypothalamus following systemic TNF-α administration. IL-1β, TNF-α and the chemokine CCL2 all showed acute elevation after systemic TNF-α that was greater in ME7 animals than in NBH. = 24.66, P < 0.001]. Post-hoc Bonferroni comparisons revealed a significant difference between NBH + TNF-α and ME7 + TNF-α at 2 h (P < 0.01) and between NBH + saline and ME7 + saline (P < 0.05)  = 72.99, P < 0.001], and TNF-α administration  both significantly increased hippocampal TNF-α expression. Post-hoc comparisons showed that disease was associated with significantly (p < 0.01) higher hippocampal TNF-α mRNA expression across all treatment groups (P < 0.001)  = 49.15, P < 0.001], and of TNF-α . Post-hoc Bonferroni comparisons revealed significant differences between NBH + TNF-α at 2 h and ME7 TNF-α at 2 h (P < 0.001) but also between NBH + saline and ME7 + saline (P < 0.001)  a. Diseas< 0.001) b. CCL2 m< 0.001) c. = 32.53, P < 0.0001], and of TNF-α treatment , but no significant disease × treatment interaction. Post-hoc Bonferroni comparison revealed a significant increase in IL-1β mRNA expression in the hypothalamus in all ME7 groups compared to all NBH groups  = 28.47, P < 0.0001] and of TNF-α treatment [F = 19.5 P < 0.0001] but there was no disease × treatment interaction  and 4 h (P < 0.01) in comparison to the same treatments in NBH animals. CCL2 mRNA in the hypothalamus revealed a significant effect of disease  and TNF-α treatment . In the case of CCL2, post-hoc Bonferroni comparison revealed no significant difference between NBH + TNF-α and ME7 + TNF-α groups in the hypothalamus at 2 h post-challenge  = 45.67777, P < 0.0001]. CCL2 also showed a significant increase in plasma following TNF-α treatment  but levels of both of these cytokines were equivalent in TNF-α-challenged ME7 and NBH animals.Systemic levels of TNF-α and CCL2 proteins were examined in the plasma using ELISA g,h. TNF- = 19.8, P < 0.001] and by TNF-α treatment . However, there was also a significant disease × treatment interaction  and post-hoc Bonferroni comparison revealed a significant increase in hippocampal IL-1β protein in ME7 animals compared to NBH animals at both 2 (P < 0.001) and 4 (P < 0.05) hours post-TNF-α.Hippocampal expression of IL-1β protein was examined using ELISA following systemic TNF-α administration i. IL-1β  = 3.624, P < 0.05]; To further explore the brain inflammatory milieu, the hypothalamic mRNA expression of other genes implicated in sickness behavior and thermoregulation were assessed . COX-2 m = 60.76, P < 0.0001] and post-hoc Bonferroni comparisons showed a significant difference between NBH + saline and ME7 + saline (P < 0.05) and between NBH + TNF-α and ME7 + TNF-α at 2 h (P < 0.001) and at 4 h (P < 0.01)  b. = 11.10, P < 0.005] and treatment  showed significant effects, albeit with a small effect size.Receptor activator of nuclear factor κB (RANK) expression was slightly increased by TNF-α treatment and slightly increased by disease per se. Both disease  and treatment . Although the interaction was not quite significant [F = 2.75] Bonferroni post-hoc test revealed a significant increase in ME7 + TNF-α at 2 h when compared with NBH + TNF-α at 2 h (P < 0.01) (Regarding RANK ligand (RANKL), this was robustly induced by TNF-α, particularly in ME7 animals. Two way ANOVA showed a significant effect of disease [F < 0.01) d.3.3 = 19.91) and an interaction between disease, treatment and time  = 15.94). TNF-α caused modest weight loss, presumably by inducing acute hypophagia following treatment  = 34.36) and this was also somewhat greater in ME7 + TNF-α animals, when measured at 18 h  = 17.63).The data on CNS cytokines and RANKL predicted differential effects of systemic TNF-α (250 μg/kg) on sickness parameters in NBH and ME7 animals. Therefore core body temperature, body weight, locomotor activity and rears in the open field were examined at the indicated times post-challenge. TNF-α induced acute hypothermia peaking at 2 h and returning gradually to baseline at 18 h a. The re at 18 h b. There  = 8.58) and an interaction between disease, treatment and time  = 4.67). There was no main effect of treatment on rears however, there was a robust interaction between disease, treatment and time  = 11.06). The ME7 + TNF-α group once again showed a prolonged decrease in rearing activity following systemic TNF-α treatment  on ME7 pathology was examined using markers for microglial activation and neuronal and synaptic pathology . Synaptophysin was significantly decreased in the hippocampal layers by ongoing disease pathology in ME7 in comparison to NBH a vs. b. Both caspase 3 and TUNEL were used to determine whether systemic TNF-α could increase ongoing apoptosis in the ME7 model  = 48.23) of ME7 animals  was also used to visualise apoptosis since it labels fragmentation of nuclear DNA. TUNEL-positive cells appeared throughout coronal sections of ME7 brains, appearing as dense labelling within a condensed nucleus. In the hippocampus the ME7 + Saline group significantly greater levels of apoptosis were apparent compared to the NBH animals  has significant effects on muscle cachexia, it is more likely that short-term body weight changes are mediated via decreased motivation to consume food which, in LPS and IL-1-induced sickness behavior, is also largely mediated by prostaglandins. Similarly, although the neuroanatomy and molecular mechanisms of sickness-induced hypoactivity remain poorly understood both indomethacin  and the 4.3de novo pathology. It is important to acknowledge a weakness of the current experimental design in that the ME7 animals models used here showed very significant synaptic and neuronal pathology prior to TNF-α challenge. If one assumes a relatively modest de novo injury, even if occuring selectively in the already degenerating brain, it would be predicted to be relatively difficult to detect on a background of very significant pathology, with the associated variability between different animals. We observed inter-individual differences in severity of pathology, evident in apparently increased Caspase-3 and IBA1 in ME7 + TNFα at 9 h, these were generally within the range of this inter-individual variability. Nonetheless, we have previously shown that acute exacerbation of neuropathology is detectable after systemic LPS (at 500 μg/kg; de novo cell death in the hippocampus of these animals. Thus, it appears that acutely elevated TNF-α is not sufficient to induce de novo hippocampal cell death and neither is systemic TNF-α essential for hippocampal cell death induced by LPS.Systemically applied LPS exacerbates existing pathology and accelerates functional decline in multiple models of neurodegeneration including prion disease, amyotrophic lateral sclerosis, Parkinson’s disease and Alzheimer’s disease  and therIt may be significant that elevated baseline TNF-α was a stronger predictor of decline than acute SIE in AD patients  and one 5The data indicate that acutely elevated TNF-α has robust acute effects on brain function in the degenerating brain, which may be highly relevant for illness-induced exacerbations of brain dysfunction including depression, delirium and post-operative cognitive dysfunction but levels of this pro-inflammatory cytokine may need to be more sustained to significantly impact on underlying neurodegeneration.


Infliximab (IFX) did not bind directly to soluble or membrane‐bound mouse TNF‐α nor did it have any effect on TNF‐α‐induced nuclear factor kappa B (NF‐κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF‐α‐independent Trichuris muris‐induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post‐infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in‐vitro data, in‐vivo treatment of T. muris‐infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF‐α, observed anti‐inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti‐inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)‐α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF‐α was investigated  Infliximab (IFX) is a neutralizing monoclonal chimeric antibody first developed against human tumour necrosis factor (TNF)‐α in BALB/c mice Preclinical models add value to understanding the complex mechanisms of cytokine neutralization, and since 2012 more than 25 studies have demonstrated an anti‐inflammatory effect of IFX treatment in mice, with an inferred mechanism of TNF‐α neutralization. Several studies have shown an overall reduction in TNF‐α after treatment in‐vitro and in‐vivo approaches to study the specific effects of IFX in mice. We used a TNF‐α‐independent infection‐driven mouse model of chronic colitis Trichuris muris infection presents with transmural colonic inflammation, a dominant T helper type 1 (Th1) response and inflammatory cell influx. However, it has been shown that the lack of receptors for both TNF‐α We were interested to examine the specificity of IFX for mouse TNF‐α in more detail to understand the value of IFX in preclinical mouse models. Here we used both Six‐week‐old male AKR mice  were housed under specific pathogen‐free conditions with free access to food and water. TNF‐α‐deficient mice  were maintained and bred on a C57BL/6 background at the University of Manchester. All experiments were conducted under the UK Home Office Animals Scientific Procedures Act 1986 (revised January 2013) at the University of Manchester.Binding of IFX to recombinant (r)TNF‐α was studied using standard denaturing Western blot techniques. Briefly, 2‐mercaptoethanol and heat‐treated rTNF‐α was loaded onto a preprepared iBlot sodium dodecyl sulphate (SDS) denaturing gel  in denaturing SDS running buffer . After transferring to a nitrocellulose membrane, the gel was blocked , washed [1% phosphate‐buffered saline (PBS)‐Tween20 ×4] and primary antibodies added . Membranes were incubated for 16 h at 4°C, washed and secondary antibodies  were added (1 h with shaking). Membranes were then washed and treated with ECL  before imaging using Bio‐Rad Chemidoc . Data were processed using Image Lab Software (Bio‐rad).5 cells/well. Cells were stimulated with lipopolysaccharide (LPS) (50 μg/ml) in triplicate, with or without IFX (20 μg/ml). Cells were incubated at 37°C/5% CO2 for 24 h. Transmembrane tmTNF‐α was detected by flow cytometry . Recommended isotype control and TNF‐α‐deficient mice were used as a negative control. Cells were stained separately for cell death markers propidium iodide [PI‐phycoerythrin (PE)] and annexin V [AV‐fluorescein isothiocyanate (FITC)], according to the manufacturer's instructions .Spleen cells were isolated using standard techniques. After red blood cell lysis cells were washed and plated in RPMI‐1640 complete media  at 5 × 10pac1 and nhe1. Lentivirus production and transduction was carried out as described previously 2. Wells were measured every 10 min during a 12‐h period. Data were normalized to an untreated control.Mouse embryonic fibroblasts stably expressing a luciferase‐labelled consensus nuclear factor kappa B (NF‐κB) promoter  was constructed. Lentiviral luciferase reporter for NF‐κB transcriptional activity was made by insertion of a multiple cloning site into pRE1x plasmid (a kind gift from U. Shibler) Trichuris muris parasites were harvested and maintained as described previously T. muris eggs in distilled water by oral gavage. A single injection of 5 mg/kg (paediatric dose) of infliximab , was given intraperitoneally (carrier 0·9% sodium chloride) 35 days post‐infection. Control antibodies were given at the same concentration; anti‐mouse TNF‐α , rat IgG1 or human IgG .®  for 24 h (4°C) before RNA extraction. For histology, 0·5 cm of whole colonic segments from the proximal ascending colon were taken and fixed in neutral buffered formalin. The remaining large bowel was assessed for worm burden.Serum samples, mesenteric lymph node (MLN) and intestines were taken at autopsy. The colon was measured from ileo‐caecal valve to rectal margin; 0·5 cm of whole colonic tissue immediately distal to the ileo‐caecal valve was isolated into 1·0 ml AllProtecthttp://rsbweb.nih.gov/ij) following image capture using SPOT™ Imaging Solutions camera and Advanced SPOT software . Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining was performed on 5 μm paraffin sections, according to the manufacturer's instructions  Sigma Aldrich).5 μm paraffin‐embedded sections were stained with Harris haematoxylin and eosin using standard protocols. Slides were randomized and blinded. Colonic crypt length and muscle thickness (μm) were measured using Image J software , with one‐way analysis of variance (anova) and unpaired Student's t‐test post‐hoc analysis. Data are expressed as mean ± standard error of the mean (s.e.m.).Statistical testing was performed using GraphPad PrismWe set out initially to determine the binding capacity of IFX to both soluble (sTNF‐α) and membrane‐bound (tmTNF‐α) mouse TNF‐α. Although IFX was able to block binding of anti‐human TNF‐α antibody to recombinant human TNF‐α by ELISA, it had no equivalent effect in a mouse TNF‐α ELISA Fig. a. This wT. murisAs IFX has been shown to induce anti‐inflammatory effects in mice, we chose to measure efficacy in a TNF‐α‐independent model of colitis, induced by a natural pathogen of mice + or inducible nitric oxide synthase (iNOS)+ cells after treatment, suggesting that IFX did not affect macrophage survival or influx in the short 10‐day window post‐treatment  in the proximal colon at the site of infection, primarily at mucosal and submucosal sites, clearly indicating a systemic effect of the human antibody administration , suggesting that IFX mediated its effect on cell survival independently of TNF‐α, a conclusion reinforced by the similar effect of human IgG. Very little cell apoptosis was observed at the epithelial interface; the most abundant cell death was observed in the base of the crypts for all treatments.ion Fig. . A highlin vitro that IFX can inhibit the TNF‐R1 : TNF‐α reverse signalling pathway between T cells and macrophages, affecting cell survival in‐vivo observations by culturing whole spleen cells with or without IFX, human IgG1 and/or anti‐human Fc antibody. IFX and human IgG control treatment induced significant cell death in whole spleen cell populations over a 24‐h period; both apoptotic and necrotic cells could be seen (Fig. It has been shown Since 2012 many peer‐reviewed papers have studied the effect of IFX in mouse models of inflammation, from diabetes et al. compared both IFX and TNF‐α kinoid treatment in arthritic human TNF‐α transgenic mice, showing that both treatments had the same immunological outcome The mechanism of action of IFX outside TNF‐α binding has been the subject of many studies over the years, concluding that its efficacy in humans cannot be accounted for by TNF‐α neutralization alone In summary, it is clear that in many murine models of inflammation a consequence of IFX treatment is reduction in TNF‐α S. E. L., J. T. M., P. P., K. J. E. and J. L. P.: study concept and design. M. B. A., S. E. L., M. L., H. E., L. B. and J. B.: acquisition of data. S. L., M. B. A., M. L., H. E., K. J. E. and J. L. P.: analysis and interpretation of data. M. B. A., K. J. E. and J. L. P. drafting of the manuscript.The authors declare no disclosures, financial or otherwise in relation to this work.


Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes. Membrane-bound and soluble TNF strongly interact with two receptors, TNFR1 and TNFR2, but the two forms of TNF are differentially effective in receptor activation.1 Whereas membrane-bound TNF activates TNFR1 and TNFR2 efficiently, soluble TNF is sufficient for TNFR1 activation but largely inactive upon binding to TNFR2. TNFR1 belongs to the death receptor subgroup of the TNF receptor family and can trigger apoptosis and necroptosis.4 However, cell death induction by TNFR1 is typically efficiently antagonized by concomitant activation of the cytoprotective classical NFκB pathway and/or ubiquitous expression of anti-apoptotic proteins.2 The latter involve FLIP proteins which generally inhibit death receptor-induced caspase-8 activation but also complexes containing TRAF2, cIAP1 and cIAP2 which specifically interfere with caspase-8 activation in context of TNFR1 signaling.4 Worth mentioning, TRAF2-cIAP1/2 complexes also mediate K63-linked ubiquitination of RIP1 in the TNFR1 signaling complex, thereby facilitating TNFR1-mediated activation of the classical NFκB pathway. Indeed, TNFR1 signaling is predominantly pro-inflammatory as TNFR1-induced cell death is blocked as long as the aforementioned protective mechanisms are not impaired.Tumor necrosis factor (TNF) is a pleiotropic cytokine that occurs as a type II transmembrane protein but can be released from the plasma membrane by proteolytic processing.5 TNFR2 activation has been linked to a variety of immune regulatory functions, which, in contrast to the activities of TNFR1, often result in anti-inflammatory effects.6In contrast to TNFR1, TNFR2 contains no cytoplasmic death domain. Upon ligand binding, TNFR2 recruits TRAF2 and various TRAF2-associated proteins, such as TRAF1, cIAP1 and cIAP2, but also interacts with other signaling proteins independently of TRAF2.in vivo, demonstrating additive, synergistic or even antagonistic effects. At the cellular level, several mechanisms for the cross-talk between TNFR1 and TNFR2 have been identified.1 Besides the obvious competition for ligand binding, TNFR1 and TNFR2 can induce, for example, autocrine TNF production in a cell type-specific manner.1 In context of TNFR1 activation by soluble TNF, subsequent induction of membrane-bound TNF results in costimulation of TNFR2, thereby converting the initially transient activation into sustained autocrine signaling. In addition, TNFR1 and TNFR2 compete for the cytoplasmic pool of TRAF2–cIAP1/2 complexes. By depletion and/or degradation of TRAF2, TNFR2 is capable to modulate TNFR1 signaling.1 Moreover, TNFR2 but not TNFR1, stimulates the alternative NFκB pathway by triggering proteolytic processing of the inactive p100/RelB dimers into active p52/RelB NFκB complexes.7 Notably, TNFR2-induced alternative NFκB signaling can be enhanced by TNFR1-mediated induction of p100 and RelB expression via the classical NFκB pathway.7Murine models shed light on the complex interplay of the TNFR1–TNFR2 system 17 However, the molecular mechanisms of TNF-induced cell death in macrophages are incompletely understood and were, therefore addressed in our study. Using macrophages genetically deficient for TNFR1, TNFR2 or TNF together with TNFR1- and TNFR2-specific TNF variants, we show that TNFR2 activation sensitizes macrophages for TNFR1-mediated necroptosis triggered by autocrine produced TNF and provide evidence that this is related to TNFR2-induced depletion/degradation of TRAF2-cIAP1/2 complexes.In macrophages, the complexity of the TNF-TNFR1/2 system is especially relevant. Macrophages on one hand co-express TNFR1 and TNFR2 and are on the other hand a pathophysiologically important source of TNF, for example, in response to a variety of pathogen-associated molecular patterns (PAMPs). TNF not only acts as a macrophage-derived effector molecule, but in an autocrine fashion also controls macrophage activation and survival, as seen for example during infection with mycobacteria.18 and consequently selectively stimulates TNFR1 in murine cells, revealed robust TNFR1 responsiveness as seen by activation of the classical NFκB pathway and upregulation of the NFκB target genes TRAF1 and p100 . Typical murine macrophage surface markers such as F4/80 and CD11b were detectable 5 days after differentiation into macrophages was initiated . Flow cyand p100 . To deterophages . In macrrophages . In accorophages , TNC-scTNF80. For this purpose, macrophages derived from Hoxb8-immortalized murine MPCs of TNF- and TNFR1-deficient mice  showed no cytotoxic activity in macrophages differentiated from HoxB8-immortalized MPCs, TLR4 stimulation with LPS in the presence zVAD-fmk resulted in robust cell death .31TLR3 and TLR4 are not only major inducers of TNF production in macrophages but have also the potential to trigger necroptosis in this cell type.ll death . This cy33 Macrophages activated by PAMPs, for example, LPS, are an integral part of the host's first line of defense against invading pathogens. Some bacteria evade macrophage control by inducing cell death of infected macrophages. This occurs by various mechanisms reaching from caspase-dependent apoptosis over IL-1β-induced pyroptosis to RIP1/RIP3-mediated necroptosis.34 Bacteria-triggered necroptosis in macrophages has been demonstrated for various bacteria producing pore forming toxins such as Staphylococcus aureus, Listeria monocytogenes, uropathogenic strains of Eschericha coli and Salmonella enterica serovar Typhimurium (S. Typhimurium).36 Although the pore forming toxin producing bacteria trigger necroptosis by membrane disruption and ion dysregulation, S. Typhimurium-induced macrophage necroptosis was attributed to IFNβ induction and TNFR1/R2-independent RIP1/RIP3-mediated necroptosis.27 The latter is in line with studies demonstrating TLR3- and TLR4-induced necroptosis of macrophages.37 Despite the prototypic nature of TNF-induced necroptosis and the overwhelming role of the TNF-TNFR system in macrophage biology, the potential of macrophages to undergo TNF-induced necroptosis has not been evaluated yet in detail. Especially, the specific role of the two TNF receptors in TNF-induced necroptosis in macrophages remained unclear. We, therefore, elucidated in our study the interplay of endogenous TNF with TNFR1 and TNFR2 as well as the individual contribution of the two TNF receptors to TNF-induced necroptosis in murine macrophages.Macrophages are perhaps the best studied primary cell type capable to undergo necroptotic cell death. Indeed, macrophages are strongly responsive to the majority of natural inducers of necroptosis including TNF, interferons and various viral and bacterial PAMPs.39 We, therefore, analyzed the involvement of endogenous TNF and TNFR1 in macrophage necroptosis following TNFR2 activation. Indeed, it turned out that (i) TNFR2 induces TNF under necroptotic conditions in macrophages rophages , indicatmulation .50 The effects of SMAC mimetics in macrophages resemble those described for TNFR2 activation in several ways as (i) both trigger degradation of cIAP1 and cIAP2, thereby sensitizing cells for TNFR1-induced cell death and (ii) both activate transcription factors of the NFκB family enabling (in a cell-type dependent manner) autocrine TNF production. In line with this notion, we found that a mixture of zVAD-fmk and BV6, but not BV6 alone, triggered TNF- and TNFR1-dependent necroptosis  rabbit mAb IgG, clone 14D4, # 2859; anti-TRAF2, rabbit polyclonal IgG, # 4724), Miltenyi Biotec, Bergisch Gladbach, Germany , Santa Cruz Biotechnology Santa Cruz, CA, USA , Enzo Life Sciences, NY, USA  and Affymetrix eBioscience . zVAD-fmk was obtained from Thermo Fisher Scientific Waltham, MA, USA (Bachem # N-1510.0025) and necrostatin-1 from Biomol, Hamburg, Germany (# AP-309). For stimulation of TLR3 and TLR4, high- and low-molecular weight poly(I:C) and LPS form the mouse TLR1-9 agonist kit (# tlrl-kit1mw) from InvivoGen  have been used. Human TNF was a kind gift from Prof. Daniela Männel . The strongly agonistic murine TNFR2-specific nonameric murine TNF variant TNC-sc(mu)TNF80 was produced and characterized as described elsewhere (under revision elsewhere). To control TNC-sc(mu)TNF80 for its LPS content, the latter was determined with the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific) as described in the manual of the manufacturer. If necessary LPS has been removed using the Pierce High Capacity Endotoxin Removal Resin packed in columns according to the recommendations of the supplier. Afterwards samples were controlled for successful LPS removal.The SMAC mimetic BV6 was synthesized as described in ref. 52 In brief, C57Bl/6 mice  were sacrificed in accordance with animal welfare. Femurs were cut and flushed with icecold PBS. After filtration (100 μm pores) on ice, cells were pelleted  and resuspended in 5 ml ammonium sulfate solution  to lyse red blood cells. Non-lysed cells were separated by additional centrifugation , washed tree times with ice-cold PBS and cultivated for 2 h in 10 ml RPMI 1640 medium supplemented with 10% (v/v) FCS and 30 μM ß-mercaptoethanol to allow adherence of fibroblasts. The supernatant with non-adherent cells was then transferred to a new cell culture disc and supplemented with 10 ng/ml murine IL-3 (Miltenyi Biotec), 10 ng/ml murine IL-6 (Miltenyi Biotec), 4% (v/v) conditioned cell culture supernatant of SCF producing CHO cells and additional 5% (v/v) FCS. After 2 days cells were used for transduction  with 3 ml HoxB8 encoding virus supernatant (DMEM-Glutamaxx (Thermo Fisher Scientific), 10% (v/v) FCS). A plasmid for estrogen-regulated HoxB8 expression (3HAERHBH-HoxB8) was kindly provided by Hans Haecker . After 3 h, 3 ml Optimem, 10% (v/v) FCS, 1% (v/v) L-glutamine, 30 μM ß-mercaptoethanol with 1 μM estradiol and 4% (v/v) SCF containing cell culture supernatant were added. Three days later, 2 ml supernatant were exchanged with RPMI 1640 medium with 10% (v/v) FCS, 1% (v/v) L-glutamine and 30 μM ß-mercaptoethanol. After additional 3 days, 3 ml of cell culture were transferred into a new well and supplemented with 3 ml of fresh medium. Medium was changed three times per week and every time freshly supplemented with 1 μM estradiol  and 4% (v/v) conditioned cell culture supernatant of SCF producing CHO cells. To induce differentiation into macrophages, the progenitor cells were washed three times with warm PBS supplemented with 10% (v/v) FCS and seeded on uncoated cell culture plates or flasks in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1% (v/v) L-glutamine, 30 μM ß-mercaptoethanol and 10% (v/v) conditioned cell culture supernatant of M-CSF producing L929 cells. Cell culture media was replaced after 3 days. Five days after initiating the differentiation, cells were harvested. An aliquot was analyzed using flow cytometry to control the successful differentiation into macrophages. The remaining cells were seeded for the experiments into 96-well (45 × 103 per well) or 6-well plates (1 × 106 per well).Hoxb8 immortalized murine MPCs were generated essentially as described elsewhere.To detect cell-surface expressed proteins, cells were harvested and resuspended in PBS. PE-labeled antibodies of the specificity of interest and corresponding isotype control antibodies were added at the dilution recommended by the supplier for 0.5 h on ice. After removal of unbound antibodies (3 × wash with PBS) cells were analyzed with a FACSCalibur  following standard procedures.MPCs were seeded in 6-well plates and were differentiated to macrophages. The latter were then subjected to stimulation with the reagents of interest and whole cell lysates were prepared. For this purpose, the cells were scraped into ice-cold PBS with a rubber policeman, pelleted by centrifugation and after resuspension in 4 × Laemmli sample buffer (8% (w/v) SDS, 0.1 M dithiothreitol, 40% (v/v) glycerol, 0.2 M Tris, pH 8.0) supplemented with phosphatase inhibitor cocktail II (Sigma) lysed by sonification (ten pulses) and heating for 5 min at 96 °C. Remaining insoluble debris was removed by centrifugation and the resulting whole cell lysates were subjected to SDS–PAGE. Separated proteins were transferred from the gel to nitrocellulose and after blocking of nonspecific binding sites by incubation in Tris-buffered saline with 0.1% (v/v) Tween 20 and 5% (w/v) dry milk, immunoblotting was performed with the primary antibodies of interest, horseradish peroxidase-conjugated secondary antibodies  and the ECL western blotting detection reagents and analysis system (Thermo Fisher Scientific).Tnf mRNA levels were quantified using the TaqMan mouse Tnf (Mm00443258_m1) gene expression assay (Applied Biosystems) and an ABI Prism 7900 sequence detector (Applied Biosystems). qRT-PCR reactions were performed in triplicates for each sample of an experiment and normalized to the housekeeping gene Hprt1 (Mm00446968_m1). mRNA levels were calculated using the SDS 2.1 software (Applied Biosystems).Total RNA was isolated using the RNeasy mini kit from Qiagen  according to the protocol of the manufacturer. Two micrograms of total RNA were transcribed into complementary DNA using the high-capacity cDNA reverse transcription kit . 4/well) were cultivated overnight in 100 μl culture medium in 96-well plates and stimulated the next day in triplicates with the reagents of interest. After 36 h cell viability was determined using the MTT assay or crystal violet staining. For normalization of viability each experiment not only included untreated cells, but also cells that had been treated with a cytotoxic mixture  sodium azide) triggering maximal cell death. The average of the triplicate of cells treated with the cytotoxic mixture was defined as 0% viability and the average of the triplicate of the untreated cells was set to 100% viability. Each measurement was finally normalized according to these values and the averages of the various triplicates were considered as a data point for further analyses using the One-way ANOVA Bonferroni's multiple comparison test function of the GraphPad Prism5 software .MPCs or macrophages derived thereof (2 × 10


Anti-TNF agents have revolutionised rheumatoid arthritis (RA) treatment; however, a third of patients fail to achieve therapeutic responses. Unexpectedly, studies in murine and human arthritis have indicated that anti-TNF treatment can increase circulating T helper 17 (Th17) cells, but the relationship to treatment response is unclear. To identify immune correlates of anti-TNF treatment response, we conducted a longitudinal study using clinical, ultrasound and T cell assessments.n = 25) were studied at protocol visits during the initial 12 weeks of anti-TNF treatment. Improvement in the disease activity score of 28 joints (DAS28) >1.2 defined treatment responders (n = 16) and non-responders (n = 9). Changes in synovial thickening and vascularity of 10 metacarpophalangeal joints were quantitatively assessed by grey scale and power Doppler ultrasound. The frequency of circulating Th17 cells was determined by IL17 enzyme-linked immunospot assay (Elispot) and flow cytometry (fluorescence-activated cell sorting (FACS)).Patients with RA (6 360 (280–645) vs 632 (367 − 1167), p = 0.003). The increase in CD4 + IL17+ cells at 12 weeks was confirmed by FACS (median (range) %, 0.7 (0.5–0.9) vs 1.05 (0.6–1.3); p = 0.01). The increase in circulating Th17 cells inversely correlated with reduction in synovial vascularity  and thickening . Higher frequencies of circulating Th17 cells at baseline were associated with poorer anti-TNF treatment response defined by ultrasonographic measures.The frequency of circulating IL17-producing cells increased significantly 12 weeks after anti-TNF initiation (Elispot median (range) specific spot forming cells (spSFC)/10These results demonstrate a link between changes in circulating Th17 cells with resolution of ultrasonographic features of synovial inflammation and vascularity during anti-TNF treatment. The findings may reflect redistribution of Th17 cells from inflamed joints or TNF-driven regulation of Th17 cell production.NCT01060098. Registered 29 January 2010.ClinicalTrials.gov: The online version of this article (doi:10.1186/s13075-016-1197-5) contains supplementary material, which is available to authorized users. Th17 cells are a highly pro-inflammatory T helper (Th) cell subset, which have been shown to contribute to arthritis pathogenesis . Their sAnti-TNF treatment has revolutionised the management of RA, leading to improvement in signs and symptoms, and slowing progression identified on radiography . HoweverIn collagen-induced arthritis (CIA), a mouse model of RA, amelioration of arthritis during anti-TNF treatment has intriguingly been shown to increase the numbers of Th17 cells in draining lymph nodes, while reducing the numbers of these cells in the inflamed paws . SubsequIn the present study, we extend these observations to longitudinally investigate the relationship between the frequency of circulating Th17 cells during anti-TNF therapy in patients with RA with clinical and morphological changes, in particular synovial thickening and vascularity as assessed by ultrasonography in response to treatment.n = 25) with an established diagnosis according to the 1987 American College of Rheumatology criteria [Patients with RA (criteria  were recn = 16) or non-responders (n = 9) to anti-TNF based on whether they achieved improvement in the DAS28 score >1.2 from baseline to 12 weeks on treatment, based on European League Against Rheumatism (EULAR) response criteria [Patients were defined as responders n = 6 or non-6/ml in heat-inactivated fetal bovine serum (FBS)  containing 10% dimethyl sulfoxide , placed overnight at -80 °C in a cryogenic vessel containing isopentane and subsequently transferred to liquid nitrogen. To minimise intra-assay variability, all measurements at all time points were analysed simultaneously for each patient.Peripheral venous blood was collected in glass tubes containing sodium heparin and PBMC were isolated by density gradient centrifugation using Lympholyte . PBMC were cryopreserved at a density of 5 − 10/106/ml in RPMI containing 10% human AB serum (Sigma). Cell viability by trypan blue was consistently >95%: 2 × 105 cells were cultured in triplicate in RPMI/10%AB serum containing 1 μg/ml anti-CD3  for 20 hours. PHA (1 μg/ml) or medium alone were used as positive and negative controls respectively. Sterile multiscreen 96-well plates  were coated with IL17 capture antibody , used according to manufacturer’s instructions, and incubated at 4 °C for 16 hours. The stimulated cells were transferred to the coated plates for a further 24 hours. The plates were washed and biotinylated anti-IL17 antibody (R&D Systems), used according to manufacturer’s instructions, was added for 2 hours. Streptavidin-AP (R&D Systems), used according to manufacturer’s instructions, was added to the plates for 2 hours at room temperature in the dark. BCIP/NBT solution (R&D Systems) was added for 30 minutes to allow visualisation of coloured spots. The spots were counted using an automated Elispot reader . The number of specific spot forming cells (spSFC) was determined as the mean number of spots in the presence of stimulation agent minus mean number of spots in wells containing medium only.PBMC from the baseline visit and weeks 4 and 12 on treatment were thawed, washed and resuspended at 2 × 106/ml were placed in culture medium  and stimulated for 5 hours with phorbol myristate acetate  and Ionomycin (500 ng/ml)  in the presence of 10 μg/ml Brefeldin A (Sigma). Cells were incubated with Aqua-Live/dead fixable dead cell kit , used according to manufacturer’s instructions, for 30 minutes at 4 °C and subsequently stained with anti-CD4-FITC and anti-CD8-PerCP/Cy5.5  for 30 minutes at 4 °C before fixation with Cytofix (BD Biosciences). Cells were permeabilised with phosphate-buffered saline containing 1% bovine serum albumin and 0.05% saponin (Sigma) and stained with anti-IL17-PE (BD Biosciences) for 30 minutes. Cells were acquired and analysed on the FACS CantoII using FACS DIVA software (BD Biosciences). Live cells were identified by FSC-A/FSC-W profile and negative staining for Aqua-live/dead kit. Lymphocytes were identified by back-gating from CD8 and using the forward and side scatter profile.Thawed PBMC at a concentration 15 × 10At each study visit, patients underwent high-frequency grey scale and power Doppler ultrasound over the dorsum of 10 MCP joints in the transverse plane using the GE Logiq9 machine with a two-dimensional M12L transducer  performed by the same sonographer (DH) to ensure consistency. Settings on the machine were kept constant: grey scale frequency 14 MHz, power Doppler frequency 7.5 MHz, Gain 50, PRF 1.4 KHz, wall filter 127 Hz. To standardise image acquisition, the hands were maintained in a position of rest using a splint. The timing of scanning at each visit was within 1 hour of the baseline visit. Images were anonymised and stored for future analysis.Anonymised clips and images were analysed by the same assessor (DH) for synovial thickening and vascularity in the transverse plane, by a quantitative scoring method previously described by us using a computerised image analysis system  , 29. Synp values less than 0.05 were considered significant.The analysed parameters obtained at protocol time points on treatment were compared to baseline using the Wilcoxon signed rank matched-pairs test. Correlation was analysed using Spearman rank. Data were analysed using Prism version 5 ; n = 25, 18 female and 7 male; mean age ± SD 57.4 ± 11.7 years; mean disease duration ± SD 10.6 ± 9.2 years) with active disease (mean DAS28 ± SD 5.72 ± 0.84) were followed longitudinally during the initial 12 weeks of treatment with anti-TNF . Anti-TNF treatment led to a significant reduction in DAS28, as early as 1 week after anti-TNF initiation in the whole cohort, with further reduction at 4 and 12 weeks on treatment  for Trans STA (ICC = 0.99) and Trans PDA (ICC = 0.99) indicated excellent agreement between the two scans confirming the imaging technique was consistent and reproducible. To assess within-scan intra-reader reproducibility in image analysis, 10 anonymised scans were randomly selected and the grey scale and PDUS were re-read in a blinded fashion and the intra-class correlation coefficient for Trans STA (ICC = 0.91) and Trans PDA (ICC = 0.99) indicated excellent agreement.r = 0.59, p = 0.01). The kinetics of change of both quantitative measures of synovial thickening and synovial vascularity did not differ significantly between those patients treated with etanercept and those with adalimumab and both measures showed improvement with anti-TNF treatment, irrespective of type (data not shown).At baseline, quantitative measures of synovial vascularity correlated positively with disease activity  increased significantly 12 weeks after anti-TNF initiation (median (range) spSFC/106 360 (280–645) vs 632 (367–1167), p = 0.003) compared to baseline   %, 0.7 (0.5–0.9) vs 1.05 (0.6–1.3), 01) Fig.  and d.r = -0.39, p = 0.04) and the change in Trans PDA score  from baseline to 12 weeks on treatment  from baseline to week 12 on treatment and the change in Trans STA score  , from baseline to week 4  and from baseline to week 12 on treatment   , from baseline to week 4  and from baseline to week 12  and the change in synovial vascularity by PDUS from baseline to week 1 on treatment 02) Fig. . There w01) Fig. . These r Fig. . T6, 538 (280–725) vs 405 (310–540), p value not significant) and FACS (EULAR non-responders vs good responders median (range) %, 0.9 (0.7–1.2) vs 0.7 (0.48–0.9), p value not significant).In view of the relationships observed between a higher frequency of Th17 cells at baseline and a smaller improvement, or worsening, in synovial thickening and vascularity during treatment, we compared the frequency of circulating Th17 cells prior to anti-TNF initiation between EULAR good responders and non-responders to therapy. Anti-TNF non-responders showed a trend towards having a higher frequency of circulating Th17 cells at baseline compared to good responders, both by Elispot (EULAR non-responders vs good responders median (range) spSFC/10We conducted a longitudinal investigation of patients with RA during the initial 12 weeks of anti-TNF treatment using clinical, ultrasonographic and T cell assessments to gain an understanding of immune correlates of treatment response. This longitudinal evaluation allowed us to identify a link between changes in circulating Th17 cells, evaluated by cellular assays, and resolving synovial inflammation and vascularity during anti-TNF treatment.Anti-TNF treatment led to a significant and sustained improvement in clinical measures of disease activity and morphological improvement in synovial thickening and vascularity determined by grey scale and PDUS during 12 weeks of treatment. We observed strong positive correlations between DAS28, a composite measure of disease activity, and synovial vascularity score by PDUS, a more objective and quantitative measure of synovitis in the limited set of joints assessed. These findings are in agreement with previous studies , 30–33. Synovial thickness and vascularity scores improved during anti-TNF treatment in EULAR good responders, but interestingly they exhibited different kinetics of change, with synovial vascularity showing earlier and more marked improvement compared with synovial thickening scores. PDUS signal has been shown to reflect vascularisation of pannus in RA and to correlate with histological changes of synovitis and synovial membrane microvascular density , 33. OneUsing Elispot and intra-cellular cytokine staining, we demonstrated an increase in circulating Th17 cells during anti-TNF treatment in patients with RA. These results were obtained using two different but complementary techniques for assessing cellular immune responses and were consistent, thus strengthening our findings. The increase in circulating Th17 cells during anti-TNF treatment has been indicated in two small studies but these have evaluated Th17 cells using flow cytometry only, or by measurement of IL17 production by stimulated PBMC using ELISA at one time point on treatment , 25.We found significant negative correlation between the change in numbers of Th17 cells from baseline to 12 weeks on treatment and the change in ultrasound scores for synovial thickening and vascularity from baseline to 12 weeks. Thus, as the frequency of Th17 cells increases in peripheral blood during anti-TNF treatment, there is a corresponding improvement in synovial thickening and vascularity. Our results suggest that the increase in Th17 cells in peripheral blood during treatment is associated with improvement in synovial thickening and vascularity.This is the first study to link changes in T cell immunopathology assessed by cellular assays with the morphological changes in inflamed joints assessed by PDUS during anti-TNF treatment. These correlations are consistent with the mechanism of action of anti-TNF agents. One of the key mechanisms of action through which anti-TNF has been shown to lead to improvement in arthritis is through reduction in trafficking of inflammatory cells to joints through reduced synovial expression of chemokines and adhesion molecules and also reduced angiogenesis and synovial VEGF expression , 37, 38.Yersinia-induced reactive arthritis in mice lacking TNFp55 receptor was associated with more severe disease. Increased levels of IL17, IL23 and IL12p70 were found in the arthritic joints of these mice and antibody blockade of IL17 was shown to reduce arthritis severity. The increase in Th17 responses in the TNFRp55-/- mice was shown to be mediated by an increase in IL12/23p40 [Another possible mechanism through which anti-TNF may cause an increase in circulating Th17 cells is through an increase in the p40 subunit shared between IL12 and IL23, the key cytokines involved in differentiation of Th1 and Th17 cells, respectively. In a study by our group using the CIA mouse model of RA, anti-TNF therapy ameliorated arthritis by decreasing numbers of Th1 and Th17 cells in arthritic joints, but also caused an increase in Th1 and Th17 cells in draining lymph nodes [12/23p40 . Taken tAnother interesting finding to emerge from this study is that anti-TNF non-responders showed a trend towards a higher baseline frequency of Th17 cells compared to responders and this trend was observed using results from both Elispot and intra-cellular cytokine staining. Two other studies also point to an association between higher baseline levels of IL17 or a higher frequency of Th17 cells and poor anti-TNF treatment response in RA; although in these studies this relationship has been investigated using clinical measures of disease activity only and at a single time point on treatment, rather than longitudinally , 41. We We have used two different but complementary techniques (Elispot and intracellular cytokine staining) to assess the frequency of Th17 cells prior to anti-TNF initiation and using both methods, we found a significant relationship between a higher baseline frequency of Th17 cells and poor treatment response assessed by ultrasonographic measures. If these associations are also confirmed in larger patient cohorts and in patients with other types of inflammatory arthritis aside from RA, the characterisation of Th17 cells as a marker of anti-TNF non-response raises the possibility of being able to tailor biologic therapy of inflammatory arthritis according to individual patient immunological profiles. Further investigation is warranted into whether patients with higher baseline frequencies of circulating Th17 cells may have more IL17-driven disease and whether these patients may derive greater benefit from treatment with a combination of anti-IL17 and anti-TNF agents.The present study shows that anti-TNF treatment induces an increase in circulating Th17 cells in patients with RA and this increase correlates with reduction in synovial thickening and vascularity. This is the first study to link increases in circulating Th17 cell numbers and IL17 production with resolution of ultrasonographic features of synovitis during anti-TNF treatment. We also showed that higher baseline frequencies of Th17 cells were associated with poorer anti-TNF treatment response. These observations raise the possibility that dual inhibition of IL17 and TNF may be a means to further advance therapeutic outcomes . Fischer