The induction of regulatory macrophages can be potentiated using the combination of anti-TNF and thiopurines, consistent with the enhanced efficacy of this combination therapy described in clinical trials. As thiopurines are unfortunately associated with significant side effects, we here aimed to identify alternatives for combination therapy with anti-TNF, using the macrophage induction model as a screening tool.Regulatory macrophages play a critical role in tissue repair, and we have previously shown that anti-tumour necrosis factor [TNF] antibodies induce these macrophages in vitro and in the T cell transfer model of colitis.Mixed lymphocyte reactions were treated with anti-TNF and a library of 1600 drug compounds. Induction of CD14+CD206+ macrophages was analysed by flow cytometry. Positive hits were validated In vivo, albendazole plus anti-TNF combination therapy was superior to monotherapy in a model of colitis, in terms of both induction of regulatory macrophages and improvement of clinical symptoms.Among the 98 compounds potentiating the induction of regulatory macrophages by anti-TNF were six benzimidazoles, including albendazole. Albendazole treatment in the presence of anti-TNF resulted in alterations in the tubulin skeleton and signalling though AMPK, which was required for the enhanced induction. Combination therapy also increased expression levels of the immunoregulatory cytokine IL-10. Albendazole enhances the induction of regulatory macrophages by anti-TNF and potentiates clinical efficacy in murine colitis. Given its favourable safety profile, these data indicate that the repurposing of albendazole may be a novel option for anti-TNF combination therapy in IBD. Currently, anti-tumour necrosis factor [TNF] monoclonal antibodies are the most effective treatment to achieve this therapeutic goal. However, despite its obvious success, anti-TNF monotherapy does not result in complete mucosal healing in all patients. In a landmark clinical trial, complete mucosal healing was observed in 30% of patients after 26 weeks of monotherapy with infliximab [IFX].2 Ablation of macrophages in the context of wounding results in significant delays in wound closure and increased inflammatory activity.3 We have previously shown that anti-TNF antibodies induce CD14+ CD163+ CD206+ M2-type regulatory macrophages which are highly suppressive of T lymphocyte proliferation and promote wound repair in vitro.4,5 Given the pivotal role of macrophages in wound healing, this is likely to be an important mechanism by which anti-TNF mediates mucosal healing. Indeed, we subsequently found that regulatory macrophages are induced in endoscopic responders to IFX but not in endoscopic non-responders.4 In line with the clinical additive effect of thiopurines and anti-TNF, we also observed that thiopurines enhance the induction of regulatory macrophages both in number and in immunosuppressive potential.Wound healing is an active process involving both immune suppression and repair of the damaged tissue. A key role in this process is played by regulatory macrophages, which inhibit the active immune response, remove apoptotic cells, and promote epithelial proliferation and wound contraction.6 Given this problematic safety profile, alternative drugs for use as anti-TNF co-medication would fill a currently unmet need. Here, we used our in vitro regulatory macrophage induction assay as a screening tool for the identification of potential alternative candidates to increase anti-TNF induced mucosal healing.Unfortunately, thiopurines often have to be discontinued due to either intolerance or serious side effects. In one large Dutch inception cohort of 363 patients, thiopurines had to be stopped in 39% of patients due to adverse events.Peripheral blood mononuclear cells [PBMC] were isolated from buffy coats obtained from healthy blood bank donors, using Ficoll density gradient centrifugation. PBMC of two individual donors were cultured in a 1:1 ratio for 48 h. Subsequently, anti-TNF [infliximab] or control IgG  was added, to a final concentration of 10 µg/ml. Where appropriate, additional compounds were added at the concentrations indicated. Albendazole [#A4673], mebendazole [#46404], docetaxel [#01885], paclitaxel [#T7402], budesonide [#PHR1178], fenbendazole [#35032], pyrythione zinc [#H6377], ciclopirox olamine [#C0415], pyrimethamine [#46706], quinacrine [#Q3251], mycophenolic acid [#M3536], adapalene [#A7486], amitryptiline [#A8404], duloxetine [#SML0474], triamterene [#T4143], and colchicine [#C9754] were all obtained from Sigma Aldrich. AMPK activator A769662 was obtained from SelleckChem  and Compound C from Calbiochem . Cultures were incubated for another 4–5 days and analysed by flow cytometry.For analysis of effects on isolated macrophages, mixed lymphocyte reactions (MLR) were cultured for 48 h. CD14-expressing cells were isolated using magnetic cell sorting  and cultured for an additional 72 h in the presence of anti-TNF and/or albendazole.For flow cytometry of cell cultures, cells were harvested using 5 mM EDTA and stained using αCD14-PE [clone MϕP90], αCD206-APC [clone 19.2], αCD163-PE [clone GHI/61], and αCD86-FITC , αCD14-PE–Cy7 [clone HCD14], and αHLA-DR-AF700 . Cells were analysed using a FACS Fortessa [BD] and analysed using FlowJo software .7 Briefly, KG1 or THP1 cells were incubated with compounds as indicated for 18 h, followed by fixation in warm 0.5% glutaraldehyde in microtubule stabilisation buffer . Subsequently samples were quenched using an equal volume of 0.25M glycine in PBS and pelleted. Cells were then resuspended in PBS containing 0.2% Triton-X100, 2% bovine serum albumin and 50 µg/ml RNAse A [Sigma], and incubated for 48 h. Tubulin was stained using mouse anti-tubulin-FITC [Sigma] and analysed by flow cytometry.Tubulin polymerisation was measured as previously described.ad libitum. The T cell transfer model has been described previously.8 Briefly, wild type CD4+CD45RBhigh cells are isolated from the spleen using magnetic depletion and flow cytometric cell sorting; 2.5 x 10E5 cells were transferred into C.B-17 SCID animals by intraperitoneal [i.p.] injection. Control animals received no T cell transfer. Three weeks after transfer, endoscopy was performed to ensure the onset of disease. Animals were randomised using an online randomiser [www.randomizer.org]. Treatment was allocated per cage because of possible faecal-oral transfer of albendazole. Treatment consisted of 1–100 µg/animal [titration experiment] or 25 µg/animal [combination treatment experiment] anti-murine TNF or IgG . For combination treatment, either 4 mg/animal albendazole [Sigma] in 0.5% carboxymethylcellulose or vehicle control was administred. This dose has been described previously for i.p. administration.9,10 All treatments were administered twice weekly by i.p. injection. Weight was monitored three times per week. In the combination treatment experiment, two animals were lost to follow-up  due to complications upon endoscopy and subsequent premature sacrifice, and were excluded from the analysis. Upon sacrifice, disease activity index [DAI] was determined by the level of diarrhoea [score 0–3], colonic inflammation/oedema [0–3], and weight loss [0–4] as previously described. All scoring was performed by a blinded observer. All animal studies were approved by the local animal ethical committee, and performed according to national guidelines.Female Balb/C and C.B-17 SCID animals were obtained from Harlan  and used between the ages of 8 and 20 weeks. Within each experiment, animals were within a 1 week age range. Animals were housed under SPF conditions in IVF cages in groups of 5. They were maintained on a 12-h light/dark cycle and provided with water and chow The primary outcome of the animal model is the histological scoring of colitis. Upon sacrifice, the colon was opened, washed, and cut longitudinally in half, and one part was processed for histology. Tissue was rolled into swiss rolls and routinely embedded in paraffin. Six-µm sections were cut and stained by haematoxylin and eosin [HE] staining. Sections were then blinded and randomised and scored for pathology, including the following parameters: immune infiltrate [0–3], goblet cell loss [0–3], crypt loss [0–3], crypt hyperplasia [0–3], and the presence of crypt abscesses ; total histological scoring was the combined score [0–15].Upon sacrifice, colon sections of approximately 10 mm were snapfrozen. RNA was isolated using the Isolate II RNA kit  and cDNA was generated using RevertAid reverse transcriptase  and random primers . Quantitative real-time polymerase chain reactions [RT-PCR] were carried out using Sensifast SybrGreen [GC Biotech]. For relative expression, all data were normalised against expression of the household gene cyclophilin, according to the 2^ddCt method.The MLR was performed in wells containing poly-L-lysine [Sigma] coated coverslips. Cultures were fixed using 4% PFA, permeabilised using 0.1% Triton X, and stained using αPhospho-AMPK , anti-CD14-FITC [BD Biosciences], and Goat-αRabbit-AF647 . Samples were embedded in SlowFade Gold containing DAPI [Invitrogen] and imaged using a Leica DM6000B microscope. Random images were taken by a blinded researcher, and analysed using ImageJ software [http://imagej.nih.gov/ij/],;pAMPK staining was normalised against DAPI.In situ hybridisation was performed using the RNAscope Reagent 2.5HD Red system , using probes Mm-Ifng, Mm-Il17a, and Mm-IL10. After development of the FastRed signalling, sections were washed once in tap water, and twice in PBS. Sections were then stained using anti-CD3  and Goat-αRabbit-AF488 [Invitrogen] and mounted in SlowFade Gold containing DAPI [Invitrogen] and imaged using a Leica DM6000B microscope. Images were analysed using ImageJ software [http://imagej.nih.gov/ij/]. CD3 positive staining was calculated relative to DAPI staining. T cell skewing was calculated as the number of CD3+ cells containing at least two positive spots, as per the manufacturers suggestion.Sections were cut freshly and dried overnight at 37°C. post hoc testing [Newman Keuls] and Kruskal-Wallis followed by Dunns post hoc analysis for quantitative PCR data. All bars represent means, with error bars indicating standard error of the mean [SEM]. Data were considered significant when p < 0.05.Data were analysed using GraphPad Prism . Statistical analysis included one-way analysis of variance [ANOVA] followed by All animal studies were approved by the institutional animal ethical committee and performed according to national guidelines.ECCO-JCC online]. Of all compounds tested, 98 compounds significantly enhanced anti-TNF mediated induction of regulatory macrophages at at least one concentration [ECCO-JCC online]. Notably, 6-thioguanin is one of the compounds present in the library and was one of the positive hits in the screen, validating the approach. Several of the positive compounds clustered in a few functional classes, such as steroids and cytostatic drugs . Remarkably, six out of nine benzimidazoles present in the library tested positive in the screen, whereas the closely related [nitro]imidazoles tested negative.Using a MLR with human peripheral blood mononuclear cells as previously described, we screened the Pharmakon 1600 library for drugs with the capacity to enhance the anti-TNF mediated induction of CD14+CD206+ regulatory macrophages. Each compound was tested at four different concentrations . As a positive control, 6-thioguanin, a metabolite of the thiopurine azathioprine, was included on each plate [ECCO-JCC online]. In view of the favourable safety profile and high tolerability of benzimidazoles and the novelty of their immunomodulatory potential, we further investigated the effects of benzimidazoles.To confirm the results obtained in the large-scale screen, 15 compounds from various drug classes were re-acquired from a second vendor and tested in a secondary assay. Compounds from various drug classes were selected based on clinical applicability. Of these 15 compounds, 13 indeed proved to enhance the macrophage-inducing capacity of anti-TNF, including the cytostatic agents docetaxel and paclitaxel, the steroid budesonide, and the anti-helminthics fenbendazole, albendazole, and mebendazole [5 Macrophages induced with anti-TNF/albendazole combination treatment inhibited T cell proliferation to a greater extent than equal numbers of macrophages induced with anti-TNF alone [ECCO-JCC online].Dose titration studies for albendazole and mebendazole revealed that, for both compounds, doses in the range 1–25 µM enhanced anti-TNF mediated macrophage induction . Due to NF alone . Thus, a11,12 Closer analysis of the data obtained in the screen revealed that two other tubulin-modifying drugs [vincristine and estramustine] also tended to increase anti-TNF mediated induction of regulatory macrophages, suggesting tubulin modulation as a potential mechanism . In contrast, 25 µg/animal twice weekly resulted in an intermediate response, both in body weight and in histological disease, and lower doses had no effect. We thus proceeded to test the efficacy of the combination therapy using an anti-TNF dose of 25 µg/animal twice weekly.Anti-TNF and albendazole combination therapy was further studied ECCO-JCC online], in line with previous data indicating that prevention of body weight loss is mediated mainly through neutralisation of systemic TNF, whereas intestinal healing requires additional effector mechanisms.14 Albendazole monotherapy did not improve colitis significantly. Supporting the hypothesis of a potentiating effect of albendazole on anti-TNF therapy rather than an independent immunosuppressive effect, levels of albendazole metabolites correlated significantly with disease activity in combination-treated animals , but not in the albendazole monotherapy group .As expected with this suboptimal dosing scheme, endoscopic and histological analysis clearly showed remaining inflammation in the animals on anti-TNF monotherapy -d. In coin situ hybridisation, T cell polarisation was investigated. Within the CD3+ present, a trend to decreased IFNγ expression was observed both with anti-TNF alone and with combination treatment, whereas IL-17 was only detected in a minority of CD3+ cells . These data suggest that the effects are mediated more though modulation of T cell numbers than through altered polarisation.Analysis of the T cell compartment confirmed a significantly decreased presence of CD3+ cells after anti-TNF plus albendazole combination treatment . Using cin vivo, colonic tissue was analysed for macrophage subtypes. Anti-TNF plus albendazole combination-treated animals showed decreased presence of total macrophages , consistent with data obtained in IBD patients upon response to anti-TNF.4 The macrophages present were further analysed for expression of Ly6C and CD206, which have been described to differentiate between immunosuppressive and inflammatory subsets.15 In placebo-treated animals, over half of macrophages were Ly6ChighCD206low, consistent with an inflammatory phenotype  and histological scoring .To investigate whether the therapeutic effect was associated with enhanced induction of regulatory macrophages henotype . In contin situ hybridisation. Cells expressing high levels of IL-10 were mainly found in the mesenchymal compartment in close proximity to CD3-expressing cells, but did not express CD3 themselves . These differences may be due to either the specific compound used [mebendazole versus albendazole], the use of a cell line versus primary cells, or the specific context of anti-TNF treatment in our experiments.Due to the large number of compounds included in the screen, the current data also provide clues regarding the biological mechanism of regulatory macrophage induction. Benzimidazoles enhanced this induction in the presence of anti-TNF, but the related families of imidazoles and nitroimidazoles did not. Benzimidazoles exert their anti-helminthic effect through modulation of helminthic tubulin, but also interfere with mammalian tubulin polymerisation.26 activated AMPK inhibits energy-consuming processes such as fatty acid and cholesterol synthesis, and activates catabolic mechanisms such as glucose and fatty acid uptake as well as fatty acid oxidation.27 This same shift in metabolic activity occurs during differentiation of M2-type macrophages. Conversely, inhibition of oxidative phosphorylation increases secretion of the pro-inflammatory cytokines IL6 and IL-12.28,29 In line with this, a role for AMPK in M2 polarisation has been described before. Macrophage-specific AMPK-/- animals fail to generate M2-type macrophages after muscle injury, and subsequently display delayed tissue regeneration.30 In microglia, a brain tissue-specific type of macrophage, AMPK signalling is critically important for expression of IL-10 after stimulation. Interestingly, deletion of AMPK signalling also inhibited expression of CD206 in these cells. We observed an increased expression of IL-10 and CD206 upon combination therapy, both in vivo and in vitro. The immunosuppressive effects of regulatory macrophages appeared mediated through general suppression of T cell responses, as decreased numbers of CD3+ cells were present in the colons after combination therapy, whereas T cell polarisation was not affected compared with anti-TNF therapy alone. These data strongly suggest the effect of anti-TNF+albendazole combination therapy is mediated through altered macrophage skewing, although we cannot exclude additional effects on other cell types in vivo.Downstream of these events, benzimidazoles induced AMPK activation, whereas inhibition of AMPK activity completely abrogated the effect of albendazole. AMPK is a highly conserved enzyme composed of three subunits, of which the alpha subunit contains the catalytic activity. AMPK mainly functions as an energy sensor, and is activated by phosphorylation upon decreasing levels of intracellular ATP. Through a multitude of intermediates,In summary, these data highlight the importance of regulatory macrophages as a therapeutic target in IBD, and show for the first time that benzimidazoles promote development of anti-TNF induced regulatory macrophages. This is an important step towards the repurposing of this well-established, safe, and widely available drug class for the treatment of IBD.This study was supported by the European Crohn’s and Colitis Organisation [ECCO grant 2015 to MW] and the Dutch Society for Gastroenterology [NVGE Gastrostart 2013 to GB].MEW reports non-financial support from Janssen, during the conduct of the study; and personal fees from Takeda, outside the submitted work. AC is currently an employee of Adaptimmune. PJK reports personal fees from Merck Sharp and Dohme, outside the submitted work. FMB reports grants from AbbVie, grants from Janssen Prevention Center of Janssen Vaccines & Prevention BV, Leiden, The Netherlands, part of the Janssen Pharmaceutical Companies of Johnson & Johnson, outside the submitted work. JFB reports personal fees from Abbvie, personal fees from Merck Sharp en Dohme, and personal fees from Takeda,outside the submitted work. GD’H reports grants from MSD, Abbot, GSK, personal fees from MSD, Abbot, Shire, personal fees from Ablynx, grants and personal fees from Abbvie, personal fees from Amakem, personal fees from AM Pharma, personal fees from BMS, personal fees from Boehringer Ingelheim, personal fees from Celgene, personal fees from Covidien, grants and personal fees from Ferring, grants from DrFALK Pharma, personal fees from Celltrion, grants and personal fees from Centocor/Jansen Biologics, personal fees from Engene, personal fees from Galapagos, personal fees from GSK, personal fees from Hospira, personal fees from Medimetrics, grants and personal fees from Millenium/Takeda, personal fees from Mitsubishi Pharma, grants and personal fees from MSD, grants and personal fees from Mundipharma, personal fees from Pfizer, grants from Photopill, grants and personal fees from Prometheus laboratories, personal fees from Receptos, personal fees from Robarts Clinical Trials, personal fees from Salix, personal fees from Sandox, grants and personal fees from Setpoint, personal fees from Shire, personal fees from TEVA, and personal fees from Tillots, outside the submitted work. GRB is currently an employee of GSK and reports grants from AbbVie, personal fees from AbbVie, personal fees from MSD, grants from Crucell BV, and from Takeda, outside the submitted work. All other authors report nothing to disclose.in vitro validation experiments. MW, FB, JB, and PK performed in vivo experiments. TH performed and analysed HPLC experiments. AS, GD, DE, LW, and GB supervised the study and provided clinical input. MW, LW, and GB wrote the manuscript, with input from all authors.MW, AD, AC, and DE designed, performed, and analysed the screen experiments. MW, AD, ZG, and PK performed Supplementary_informationClick here for additional data file.


Erythromonas ursincola, strain KR99 isolated from a freshwater thermal spring of Kamchatka Island in Russia, resists and reduces very high levels of toxic tellurite under aerobic conditions. Reduction is carried out by a constitutively expressed membrane associated enzyme, which was purified and characterized. The tellurite reductase has a molecular weight of 117 kDa, and is comprised of two subunits (62 and 55 kDa) in a 1:1 ratio. Optimal activity occurs at pH 7.0 and 28 °C. Tellurite reduction has a Vmax of 5.15 µmol/min/mg protein and a Km of 3.36 mM. The enzyme can also reduce tellurate with a Vmax and Km of 1.08 µmol/min/mg protein and 1.44 mM, respectively. This is the first purified membrane associated Te oxyanion reductase. This compound is lethal to most microorganisms at concentrations as low as 1 µg/mL . Ho. HoBlastE. ursincola, strain KR99, which is also capable of reducing tellurate. The slower rate for tellurate reduction could be attributed to the need for more electrons to transform it to Te° as compared to conversion of tellurite. More time is required to accommodate the transfer of the extra electrons, hence, there is a decreased rate. This is the first example of its kind, as well as the first from an AAP. Unlike the one isolated from Bacillus STG-83, this enzyme is not used for anaerobic respiration as KR99 does not grow anaerobically. Attempting to draw comparisons between the tellurite/tellurate reductase from strain KR99 and other known Te oxyanion reducing enzymes is challenging as this protein is unique. It was isolated from a Gram-negative bacterium, membrane associated, and specific to Te oxyanions. The previously published tellurite reductase from Bacillus STG-83 appears to have a similar Vmax and Km, however, it is much larger (197 kDa), comprised of three subunits, cytoplasmic in origin, and purified from a Gram-positive organism [In this work, we have isolated a membrane associated tellurite reductase from the AAP organism . In someorganism . Mass sporganism , but in organism , indicatorganism , attribuorganism ,14. Thisorganism , continu


Drosophila melanogaster, can be increased by reduced insulin-IGF-like signaling (IIS) or by changes in diet. Most studies testing whether IIS is involved in diet-mediated lifespan extension employ only a few diets, but recent data shows that a broad range of nutritional environments is required. Here, we present lifespan data of long-lived Drosophila, lacking three of the eight insulin-like peptides  on nine different diets that surround the optimum for lifespan. Their nutritional content was varied by manipulating sugar and yeast concentrations independently, and thus incorporated changes in both diet restriction and nutrient balance. The mutants were substantially longer-lived than controls on every diet, but the effects on the lifespan response to sugar and yeast differed. Our data illustrates how a greater coverage of diet balance (DB) and restriction can unify differing interpretations of how IIS might be involved in the response of lifespan to diet.Lifespan in many organisms, including  Drosophila melanogaster, can be increased by genetically reducing activity of the IIS pathway and also by a reduction of food intake without malnutrition .3Lifespan in many organisms, including the fruit fly 4 but two parameters are thought to be key, (1) does the mutation alter maximum life expectancy attained across all nutritional conditions, and, (2) is there a change in the shape of the response of lifespan to diet? A mutation that attains the first can be argued to extend life, at least in part, through a mechanism independent of DR since it builds on a state in which diet induced longevity is presumed already maximized. However, if a mutation shows the second type of change then it can be interpreted to mediate at least part of the DR response and thus that DR and the mutation are mechanistically linked.Typically, DR involves the use of two or more diets that represent increasing severity of restriction. The lifespan response to these treatments traces an inverted U-shape with relatively short life at high food concentrations, ascending to a peak at intermediate food levels, and decreasing again as nutrients become limiting and the organism increasingly suffers from starvation. When analyzing how a long-lived mutant modulates this response, several interacting effects are possible,7 Reduced IIS by knockout of Drosophila insulin-like peptides (dilps), the fly’s homologs for human insulin, has been proposed to mediate the benefits of DR because dietary yeast concentration affects the relative expression of dilp5.5 Moreover, knocking out three  of the eight dilps extends lifespan and reduces the magnitude of lifespan change in response to DR3—similar to what is observed when overexpressing a dominant negative form of insulin receptor (InR).6 However, other studies indicate that IIS and DR affect lifespan independently; flies without the IIS transcription factor dFOXO or lacking the insulin receptor substrate (chico) still demonstrate significant lifespan changes in response to DR and do not necessarily extend lifespan beyond that of controls on DR.7 Thus it is not yet clear to what extent IIS signaling and DR interact and through what mechanism.Reports vary on whether or not IIS and DR extend life through an overlapping or independent mechanism.8 and so DR in one laboratory is likely to be nutritionally different from that in another. Recent studies have revealed that not only the quantity but also the ratio between dietary protein and carbohydrates can account for the lifespan effects observed under DR.10 Thus, IIS-by-DR interaction studies could yield different outcomes because the mutation may alter the response of lifespan to some nutritional components, but not to others. If true, apparently contradictory outcomes of how a mutant affects the DR response can be resolved when they are viewed as distinct parts of a single lifespan response surface in multidimensional nutrient space.4Often, the specific set of ingredients and the practices adopted to impose DR are not identical between laboratoriesdilp2-3,5 mutants and control flies on nine food types, representing all combinations of three yeast and three sugar concentrations . These diets cover the range of foods used for past studies of IIS-by-DR interactions but extend them by incorporating changes in both DB and diet restriction (DR).To explore this potential explanation, we measured the lifespan of long-lived dilp2-3,5 mutants were longer-lived than controls by at least 21 days on each of our nine food types .11 Because each component of the canonical IIS pathway is embedded at a different point in this network they may modify the network’s overall response to diet in different ways. Further work to understand the mechanisms by which diet affects lifespan should incorporate these complex interactions between signaling networks and altered DR and DB.Both interactive and non-interactive effects of different IIS mutations on DR have been reportedSupplementary Figure S2Supplementary Figure S2.1Supplementary Figure S2.2Supplementary Table S1Supplementary Table S2Supplementary Data


This study reported the first assessment of carbon dots’ (CDots) antiviral activity to human norovirus virus-like-particles (VLPs), GI.1 and GII.4 VLPs. CDots with different surface passivation molecules, 2,2′-(ethylenedioxy)bis(ethylamine) (EDA)-CDots and 3-ethoxypropylamine (EPA)-CDots, were synthesized and evaluated. The results indicated both EDA- and EPA- CDots were highly effective to inhibit both strains of VLPs’ bindings to histo-blood group antigens (HBGA) receptors on human cells at CDots concentration of 5 µg/mL, with EDA-CDots achieving 100% inhibition and EPA CDots achieving 85–99% inhibition. At low CDots concentration (2 µg/mL), positively charged EDA-CDots exhibited higher inhibitory effect (~82%) than non-charged EPA-CDots (~60%), suggesting the surface charge status of CDots played a role in the interactions between CDots and the negatively charged VLPs. Both types of CDots also exhibited inhibitory effect on VLP’s binding to their respective antibodies, but much less effective than those to HBGA binding. After CDots treatments, VLPs remained intact, and no degradation was observed on VLPs’ capsid proteins. Taken together, the observed antiviral effects of CDots on noroviruses were mainly through the effective inhibition of VLPs’ binding to HBGA receptors and moderate inhibition of VLPs’ binding to their antibodies, without affecting the integrity of viral capsid protein and the viral particle. NoVs are a group of related non-enveloped, single stranded RNA viruses that have been classified in the Calicivirdae family. NoVs contain six genogroups (from GI to GIV), which can be further divided into different genetic clusters or genotypes based on their capsid sequence1. For example, GI includes nine genotypes and GII contains 22 genotypes3. Genogroups GI, GII, and GIV are responsible for disease in humans4.Human Norovirus (NoV) is the most common cause of nonbacterial, acute gastroenteritis outbreaks worldwide5, by breathing the air near an episode of vomiting, or by direct contact with an infected individual . NoV aerosols are formed during vomiting. A single episode of vomiting could release as many as 30 million virus particles6, while fewer than twenty virus particles can cause an infection7. NoV aerosols can also be formed by toilet flushing when vomit or diarrhea is present. The large amount of virus releasing from both fecal material and vomitus of infected individuals and the low infectious dose threshold are the factors that lead to the high number of human NoV annual outbreaks.NoV is extremely contagious and affects people of all ages. Human NoV transmission occurs by the fecal-oral route, usually through ingestion of contaminated food or water9. HBGAs are complex carbohydrates and represent terminal structures of glycan chains. They are highly polymorphic and include three major families: the ABO, secretor, and Lewis families. HBGAs are presented abundantly on the surface of mucosal epithelia of gastrointestinal track, where they may function as anchors for NoVs to initiate an infection10. Previous studies suggested that synthetic HBGAs or HBGA-expressing enteric bacteria could enhance NoV infection in B cells11.Studies have shown that NoVs recognize and interact with human histo-blood group antigens (HBGAs) in intestinal tissues as receptors or attachment factors in a strain-specific manner20. Most of these methods have been extensions of their antibacterial uses, whereas NoVs are known to be resistant to commonly used sanitizers and disinfectants21. Among the more recently developed alternative antiviral strategies, the use of nanoparticles has yielded promising results, including for example silver nanoparticles22, gold-copper core-shell nanoparticles23, and TiO2 nanoparticles coupled with illumination of low-pressure UV light24.The prevention and control of human NoVs infections have been challenging, despite the more significant effort in recent years based on different chemical and physical antiviral methodsin vitro, despite some progress very recently25, and to a lack of good animal model. Much of the research effort on NoVs has been based on the use of cultivatable surrogates such as murine norovirus, feline calicivirus, and poliovirus26 and more conveniently the virus-like-particles (VLPs). VLPs are self-assembled VP1 capsid proteins, which are expressed from open read frame 2 (ORF2) as a recombinant protein independent of other viral components. Each VLP is ~38 nm in diameter. While the VLPs do not contain the genomic RNA and are replication deficient, their structural and antigenic characteristics are indistinguishable from the native virion28. The NoV VLPs have been used as a promising vaccine platform for their ability to elicit a strong humoral and cellular immune response29. The characteristics of NoV VLPs and the easy production systems make them appropriate models for studying NoVs in biological assays and for understanding some specific questions about human NoVs. For example, VLPs were used successfully as a model in our previously reported study on the antiviral activity of gold-copper core-shell nanoparticles23. They have also been used as a model system for studying many other chemical and physical antiviral methods23. They are also useful in modeling virus-cell interactions28, and in identifying NoV binding receptors on human cells such as HBGAs30. In the work reported here, we used NoV VLPs as a model of human NoVs to explore the potential antiviral functions of the recently developed carbon dots.A major difficulty in the study of human NoVs in general has been due to challenges in the cultivation of the virus 31 are small carbon nanoparticles with surface passivation, for which more effective has been the chemical functionalization of organic molecules33. As a new class of quantum dot-like nanomaterials, CDots possess properties of bright fluorescence, no toxicity in vitro and in vivo, environmentally benign, simple synthetic routes, as well as photocatalytic functions resembling those found in conventional nanoscale semiconductors36. Many potential applications of CDots are being pursued across many fields such as chemical and biological sensing, bioimaging, nanomedicine, photocatalysis, and electrocatalysis34. Among the unique properties of CDots that are more relevant to the study reported here is the photo-activated antimicrobial function37. In fact, CDots with visible light illumination were highly effective in the inhibition of E. coli cell activities in several experimental settings, which has been attributed mechanistically to the photodynamic effect in CDots. Interestingly and surprisingly, we found in this study the significant antiviral activity of CDots toward NoV VLPs. More specifically, effects of the CDots on VLPs’ HBGA binding, antibody binding, and on the integrity of capsid protein and intergrity of VLPs particles were examined. Mechanistic implications of the results are discussed.Carbon dots (CDots)® 800CW, which was purchased from LI-COR Biosciences .Human NoV GI.1 VLPs and GII.4 VLPs, and their respective monoclonal antibodies (anti-GI.1 VLP antibody mAb3901 and anti-GII.4 VLP antibody NS14), were generously provided by Dr. Robert Atmar’s laboratory at Baylor College of Medicine . The secondary antibody used in ELISA tests was goat anti-mouse IgG H&L antibody conjugated to horseradish peroxidase (HRP), which was purchased from Abcam . The secondary antibody used in western blot assays was goat anti-mouse antibody labeled with IRDye38. In a typical experiment, a sample of carbon nano-powders (2 g) was refluxed in aqueous nitric acid  for 48 h. The reaction mixture was cooled back to room temperature, and centrifuged at 1,000 g to discard the supernatant. The residue was re-dispersed in deionized water, dialyzed in a membrane tubing (molecular weight cut-off ~500) against fresh water for 48 h, and then centrifuged at 1,000 g to retain the supernatant. Upon the removal of water, small carbon nanoparticles were recovered and used in the functionalization reaction with 2,2′-(ethylenedioxy)bis(ethylamine) 37 or 3-ethoxypropylamine 39 to yield EDA-CDots or EPA-CDots, respectively  in procedures similar to those reported previously39, the small carbon nanoparticles were refluxed in neat thionyl chloride for 12 h. Upon the removal of excess thionyl chloride, the treated sample (50 mg) was mixed well with carefully dried EDA liquid in a round-bottom flask, heated to 120 °C, and vigorously stirred under nitrogen protection for 3 days. The reaction mixture back at room temperature was dispersed in water and then centrifuged at 20,000 g to retain the supernatant. It was dialyzed in a membrane tubing (cutoff molecular weight ~500) against fresh water to remove unreacted EDA and other small molecular species to obtain EDA-CDots as an aqueous solution. The EPA-CDots were synthesized in the same procedures under similar experimental conditions, as reported recently39. Both EDA-CDots and EPA-CDots were characterized by using NMR, microscopy, and optical spectroscopy techniques, from which the results are consistent with the dot structure illustrated in Fig. 38.For the synthesis of EDA-CDots9. Briefly, saliva samples from healthy adult volunteers, including blood type A, B, and O, were collected. Saliva samples were immediately boiled for 5 min and centrifuged at 10,000 g for 5 min. The supernatant was collected and diluted to 1:2000 in PBS. For coating the plates with HBGAs, aliquot of 50 µL saliva dilution was used to coat 96-well plates at 4 °C overnight. Unbound saliva was removed and the wells were rinsed three times with PBS. The plates were then blocked with 100 µL Super-Block T20 (PBS) Blocking Buffer  for 1 h and rinsed with PBS twice.The effect of CDots treatment on VLPs was evaluated using a saliva-based HBGA binding assay according to a protocol reported previously with minor modificationsFor the HBGA binding assay, aliquots of 50 µL of 1.5 µg/mL VLPs, which were pretreated with 2 or 5 µg/mL EDA- or EPA- CDots at room temperature for 15 min, were added into the wells and incubated 1 h at room temperature. The unbound VLPs were removed and the wells were rinsed with PBST twice. The GI.1 or GII.4 VLPs binding to HBGAs in the wells were detected by using 1 µg/mL primary antibody mAb 3901 (to GI.1) or mAb NS14 (to GII.4), respectively, followed by the addition of 0.5 µg/mL secondary antibody–HRP-labeled goat anti-mouse IgG antibody. The antibody binding conditions included 1 h incubation at 37 °C and rinse with PBST twice. The final products were developed by adding 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate , and the absorbance reading at the wavelength of 450 nm was performed using a Spectra-Max M5 plate reader .#3591; Corning Incorporated, Corning, NY). 1 × PBS was added to reach the final volume of 50 µL in each reaction. The plates were constantly agitated on the shaker at the setting level of 2 at room temperature for 30 min, followed by 30 min incubation without agitation. The reaction solutions were discarded and the wells were washed with 100 µL 1 × PBS twice. The wells were then blocked with 100 µL Super-Block T20 (PBS) Blocking Buffer  for 1 h. After the blocking solution was discarded, each wells was washed with 100 µL PBST twice. Then, aliquots of 50 µL of 1 µg/mL anti-GI.1 VLP antibody mAb 3901 or anti-GII.4 VLP antibody mAb NS14 were added to each well to bind with the bound GI.1 or GII.4 VLPs, respectively, followed by 1 h incubation at 37 °C. After wash with PBST twice, aliquot of 50 µL of 1 µg/mL HRP-labeled goat anti-mouse IgG antibody solution was added to each well, and the plates were incubated at 37 °C for 1 h. After the incubation, the plates were washed with PBST, and the final products were developed using the TMB kits, and the absorbance at 450 nm in each well was measured.EDA- and EPA- CDots with various concentrations ranging from 0 to 60 µg/mL were used to treat 1 µg/mL GI.1 or GII.4 VLPs in medium-binding 96-well polystyrene plates (Costar2O). All the samples were incubated at 70–80 °C for 10 min and then loaded on 2 of precast 1.0 mm × 10-well NuPAGE® 4–12% Bis-Tris gels . The loading volume was 10 µL for each well. The gels were run in 1 × MOPS SDS running buffer  at 200 V for 1 h. One gel was used for staining, while the other was for western blotting. The gel for staining was prefixed with a 50% methanol and 7% acetic acid solution for 15 min and then washed with DI-H2O for 5 min, three times. The GelCode Blue stain  was used to stain the gel with constantly shaking for 1 h, followed by 1 h de-staining step in DI-H2O. The gel was then imaged using a LI-COR Odyssey Infra-red Imaging System .EDA- and EPA- Cdots at the concentration of 20 or 60 µg/mL were used to treat 33.3 µg/mL GI.1 or GII.4 VLPs in 1.5 mL centrifuge tubes. 1 × PBS was added to reach the final volume of 12 µL in each reaction. The tubes were constantly agitated on a shaker  at the setting level of 2 at the room temperature for 30 min. After the CDots treatments, each tube was added with 5 µL of 1 × NuPAGE LDS Sample buffer , 2 µL of 1 M DTT, and 3 µL deionized water  using 1 × NuPAGE® Transfer Buffer plus 10% MeOH and Hoefer Semi-Dry Transfer Apparatus  at 25 V for 1 h. The membrane was then blocked with 10 mL of 1:1 blocking buffer  and PBS at room temperature for 1 h. The primary antibody treatment was performed by soaking the membrane in 10 mL of 1:1 PBST and blocking buffer, to which 4.11 µg of mouse monoclonal anti-GI.1 VLP antibody mAb 3901 or anti-GII.4 VLP antibody mAb NS14 had been added, followed by incubation at 4 °C overnight with gently shaking. Then, the antibody solution was discarded, and the membrane was washed 5 times, each with PBS plus 0.05% Tween 20 (PBST) for 5 min, and then treated with 0.5 µg of goat anti-mouse IRDye® 800CW antibodies in 10 mL of 1:1 PBST and blocking buffer at room temperature for 1 h. After washed 5 × 5 min with PBST under shaking, the membrane was soaked in DI-HGI.1 and GII.4 VLP samples were treated with or without CDots, then 10 µL of each sample was placed on a formvar/carbon TEM grid  for 30 min. All grids were gently wicked to remove the fluid on the surfaces by the use of filter paper. The grids were stained with 2% uranyl acetate for 60 s and TEM images were acquired using a FEI Technai G2 Twin TEM  in the Shared Materials Instrumentation Facility (SMIF) at Duke University.41. The binding of norovirus to HBGAs has been found to be highly diverse but strain-specific. Several binding patterns have been identified and grouped into two major binding groups based on the binding of 14 novrovirus strains to HBGAs, and a model of norovirus/HBGA binding has been proposed9. A retrospective study showed that type O individuals had a significant higher infection rate than those with other blood types30, while other studies showed Norwalk VLPs lacked the binding to saliva samples collected from nonsecretors, and saliva from type B individuals did not bind or weakly bound to Norwalk virus42. Therefore, in this study, we examined the effect of CDots on GI.1 and GII.4 VLPs’ binding to saliva HBGAs from blood Type A, B, and O.NoVs recognize human HBGAs as receptors or attachment factors, such binding events play an important role in host susceptibility to NoV infectionFigure EPA-CDots were also highly effective in the same inhibition, though relatively somewhat weaker on the marching concentration basis. As also shown in Fig. 2) terminal groups tend to be positively changed at physiological pH (-NH3+), whereas EPA-CDots with surface methyl (-CH3) terminal groups are not charged. As VLPs are negatively charged, they should be more attractive to the positively charged EDA-CDots, resulting in a higher “local concentration” of the dots around the VLP particles, even though the mechanistic details on the interactions of the CDots with VLPs and the associated inhibition effects are likely very complex11. On possible mechanisms for the observed strong inhibitory effects of CDots, one is such that the CDots would bind to the surface of VLPs and physically block the active sites on the VLPs used for bindings to the HBGA receptors. Based on the X-ray crystal structure on the prototype GI.1 of NoV43, it contains two domains: the shell (S) and the protruding (P) domain, and the HBGA receptor binding interfaces are located at the top of the P domain, containing carbohydrate binding pockets. These pockets involve several scattered amino acid residues that form extensive hydrogen bond network with individual saccharides, thus stabilizing the binding of HBGAs to the virus capsid protein45. Although the binding of norovirus with human HBGA is a typical protein-carbohydrate interaction in which the protruding domain of the viral capsid protein serves an interface for the oligosaccharide side-chains of the HBGAs40, some of the complexities in the HBGA binding interactions have been discussed in the literature11, including capsid P domain loop movements, alternative HBGA conformations, and HBGA rotations. In fact, the blocking of NoV HBGA binding sites has been used as a surrogate for a NoV neutralization assay by using sera from immunized animals or infected humans47. It was found that the ability of sera to block VLP-HBGA interactions could be correlated with the protection against infection in NoV-vaccinated chimpanzees and against the illness among infected human volunteers49. According to these reported studies, the blocking of the HuNoV capsid from recognizing their binding sites on host cells represents a promising strategy in preventing HuNoV infection. Thus, the observed effective inhibition of the NoV VLPs by the CDots  can nonspecifically bind to proteins through complementary charges, π-π stacking, and/or hydrophobic interactions52. Analyses of the binding between C60  and lysozyme revealed that the primary driving force for the binding is van der Waals interaction, while polar solvation and entropy are detrimental to the binding53. More relevant to the blocking of receptor sites, it was demonstrated that C60 could inhibit the activity of HIV-proteases by integrating with proteins to form hybrid functional assemblies54. Therefore, a conceptually analogous explanation on the observed inhibition of NoV VLPs might be such that the CDots interact with VLPs’ capsid protein by a combination of several driving forces, resulting in the blocking of the active sites on NoV VLPs responsible for their binding to HBGA receptors.Interactions between various carbon nanomaterials and proteins in different mechanisms have been well-documented in the literatureWe further examined the effect of CDots treatment on VLPs’ binding to their antibodies. Figure EPA-CDots were somewhat less effective in the inhibitory effect on both GI.1 and GII.4 VLPs’ binding to their respective antibodies, as compared in Fig. 55, where the disruption of viral envelope and/or capsid structure, not the degradation of the viral protein or genome, was the primary mechanism of HPP55.In both EDA- and EPA-CDots treatments, GI.1 VLPs were more effectively inhibited in their binding to mAb3901 antibodies than GII.4 VLPs’ bindings to mAb NS14 across the different CDots concentrations Fig. . This miet al., 2013) suggested that the CDots treatment may be a potential strategy to prevent the initial access or spread of NoV to humans by effectively inhibiting NoV binding to HBGA receptors.The treatment with CDots was clearly more effective in inhibiting VLPs’ binding to HBGA than to their antibodies. In a comparison of the results in Figs To gain some mechanistic insights into the inhibitory effect of CDots on VLPs’ binding capacity to their antibodies and HBGA receptors, we further examined the possibility of VLPs’ capsid proteins being degraded by CDots treatments. Briefly, GI.1 and GII.4 VLPs were treated with EDA- and EPA- CDots at two different doses (20 µg/mL and 60 µg/mL). The treated and untreated VLPs were analyzed by SDS-PAGE gel, followed by GelCode Blue staining. As shown in Fig. 57, recognizing a continuous epitope on the C-terminal of the capsid protein57. The antibody mAb 3901 also recognizes a domain between amino acid 453 and amino acid 495, and the lower band in the western blot is likely a fragment that contains this sequence. Similarly for GII.4 VLPs, the mAb NS 14 binds to the capsid protein and other protein fragments that contain the recognized epitopes. Clearly, for both GI.1 and GII.4 VLPs, the protein band patterns detected in western blotting were essentially the same as those observed in SDS-PAGE detected by GelCode Blue staining. Therefore, the results demonstrated that CDots treatments did not degrade the viral protein, as the viral proteins still retained correct primary amino acid sequences and were able to react with their antibodies.We also examined the possibility of the proteins being antigenically changed after CDots treatments by western blotting using mAb 3901 against GI.1 VLPs and mAb NS14 against GII.4 VLPs. As shown in Fig. 58. HeLa cell viability was over 90% after being incubated with 500 µg/mL of CDots for 24 h59. The observation that the morphology and integrity of VLPs remained unchanged after CDots treatments is consistent with these and other results reported in the literature.Shown in Fig. This study explored the effects of EDA-CDots and EPA-CDots on NoV GI.1 and GII.4 VLPs. The results demonstrated that the treatment with CDots effectively inhibited VLPs’ binding to saliva HBGA receptors , without degrading VLP capsid proteins or affecting the integrity and morphology of the VLP particles. Between the two types of CDots, the positively charged EDA-CDots were much more effective than the non-charged EPA-CDots in inhibiting the binding of VLPs to HBGA receptors, due to more favorable binding between EDA-CDots and negatively charged VLPs. These CDots also showed inhibitory effect on VLPs’ binding to their respective antibodies, but much less effective compared to those inhibitions to HBGA receptors. Nevertheless, the results from this study showed the proof-of-concept on CDots’ antiviral function through the inhibition of virus binding to HGBA receptors, which could be a promising strategy in preventing HuNoV infection/spread by disabling HuNoV recognizing their binding sites on host cells. As there is no effective vaccine for NoV, effective hand washing and cleaning of contaminated sites are recommended practices for prevention of NoV infection/spread, examples of potential applications include CDots-containing antiviral sprays for sanitizing NoVs contaminated sites, such as surfaces or instruments in hospital, patient vomiting on carpet/floor, bath room toilet after patient diarrhea. CDots can also be incorporated into routine hand wash soaps for antiviral purpose. For cases involving aerosol NoVs, incorporating CDots agents into air filtration devices may be explored. Though the practical application for such purpose is likely more complex and requires additional investigations, the reported initial assessment of CDots’ antiviral function is highly valuable, and will be significantly contributing to the knowledge matrix for the broad implications and application potential of CDots.


Treg deficiency causes dramatic immunologic disease in both animal models and humans. As they are capable to suppress the function and the proliferation of conventional CD4+ and CD8+ T cells, Treg-based cell therapies are under evaluation for the treatment of various autoimmune diseases and are currently employed to prevent graft-versus-host disease (GvHD) in clinical trials of hematopoietic stem cell transplantation. Even though tumor necrosis factor-α (TNF-α) is well known for its pro-inflammatory role, recent studies show that it promotes Treg activation and suppressive function. In the present review, we discuss the role of TNF-α in Treg function and the possible implications on the actual treatments for immune-mediated diseases, with a particular attention to GvHD.FoxP3 They survival –3. Thesesurvival –3, 6. Resurvival , 8.+ T cells, their paucity and the complexity of their isolation limit further clinical applications. Thus, different strategies have been tested to expand Treg number and/or enhance Treg function in vitro and in vivo  and other immune cells, Treg-based cell therapies are under evaluation for the treatment of immune-mediated diseases. Recent studies showed that adoptive transfer of Tregs prevents graft-versus-host disease (GvHD), a life-threatening immune complication of allogeneic hematopoietic stem cell transplantation (HSCT). In this setting, donor Tcons mediate alloreactions that eradicate tumor cells [graft-versus-tumor (GvT)], but that are also directed against normal tissues , causing GvHD , 10. Stu in vivo .+ T cell mediated alloreactivity exacerbating immune destruction of GvHD target tissues (Tumor necrosis factor-α (TNF-α) is widely known for its pro-inflammatory activity –20. In t tissues , 29. Fol tissues .TNF-α is synthetized as a trimeric type II transmembrane protein, which can be cleaved to give rise to soluble extracellular TNF-α. Both membrane and soluble TNF-α are biologically active –20. TNF-In this review, we will describe how TNF-α impacts on Treg phenotype and function and how Treg immune responses can be modified by TNF-α exposure over time. We will report controversial studies on human Tregs where TNF-α role is not fully elucidated yet. We will also discuss the possible implications of these studies on the actual treatments for immune-mediated diseases mainly focusing on GvHD and we will propose future clinical directions.In vitro, TNF-α in the presence of IL-2 increases the expression of CD25 and FoxP3, enhances the proliferation of Tregs and the suppression they exert on effector T cell proliferation. Mouse Tregs express higher levels of TNFR2 than CD4+ CD25− T cells, while both subsets barely express TNFR1  and are more suppressive in vitro than CD4+ CD25+ TNFR2− cells  were preserved even after host–Treg expansion induced by the TNFR2 agonist. The beneficial effects of the TNFR2 agonist were abrogated in TNFR2-deficient mice  (+ CD25high FOXP3+ CTLA4+ CD127− CD62L+ Fas+ HLA-DR+ CD45RO+ CCR5− CCR6− CCR7− CXCR3− ICOS−), they were endowed with a greater suppressive function, and produced lower levels of IFN-γ and IL-10. In fact, such highly suppressive Tregs co-expressing TNFR2 could ameliorate the onset of autoimmune diseases. For example, in type 1 diabetic patients the same authors observed an increase in resting CD45RA+ Tregs and a decrease in activated CD45RO+ Tregs. In vitro treatment with TNF-α or TNFR2 agonist antibody corrected the activation defect of Tregs in these patients  . TNFR2 spatients . Thus, Tin vitro  patients and treatment with anti-TNF-α antibodies restores it –52. Tregin vitro , 53. Thefunction . In anotfunction .in vitro analyses of TNF-α effects on Tregs from healthy donors. Some studies showed that suppression of Tcon proliferation exerted by Tregs was impaired in the presence of TNF-α  made TNF-α an ideal therapeutic target for conditions where a reduction of inflammatory response was needed , 59, 60.Studies on GvHD after HSCT showed that TNF-α levels are increased in patients with acute GvHD and tend to correlate with disease onset and progression , 30, 62.in vivo suppressive function, the potential benefit of the treatment in inflammatory conditions could be limited. Anti-TNF-α treatments are not effective in some of the autoimmune diseases in which TNF-α is involved. Moreover, patients treated with anti-TNF-α drugs can develop other immune-mediated complications (The clinical effects of anti-TNF-α therapy should be reconsidered by virtue of the new insights on TNF-α/TNFR2 pathway in Treg function. As TNF-α inhibition can reduce Treg ications , 66. In ications , TNF-α bications .As Tregs are critical for GvHD protection and control over time, limiting Treg function could be a potential pitfall of TNF-α blocking therapy in the HSCT setting. TNF-α that is produced after conditioning regimens with radiotherapy and/or chemotherapy can bind TNFR2 and at the same time activate Tregs and alloreactive T cells , 69. The+ FoxP3+ Tregs, thanks to their high expression of TNFR2, are preferentially activated when the whole CD4+ T cell pool is exposed to TNF-α. In these conditions, they acquire a proliferative and functional advantage in comparison to the effector CD4+ FoxP3− T cells suggesting selectivity of the TNF-α/TNFR2 pathway in the CD4+ T cell subset (+ CD25high FOXP3+ Tregs in one subject (+ CD25− effector T cells upregulate TNFR2 expression after TCR stimulation and become more resistant to Treg-mediated suppression (+ T cells was required to induce full-fledged experimental colitis in one study (in vivo.Based on the data discussed above, stimulation of the TNF-α/TNFR2 pathway is expected to activate and expand Tregs , 44, 47.pression . In addine study . Thus, tin vitro (Compared with the available anti-TNF-α drugs, blocking antibodies that selectively inhibit TNFR1 or TNFR2 could be used for different clinical purposes . Anti-TNin vitro .ex vivo and possibly in vivo (in vitro manipulation required for their expansion while preserving function and purity. TNFR2 agonists may represent an alternative strategy to expand in vitro a Treg population endowed with higher purity and enhanced activity, thus improving results of current Treg-based clinical trials for GvHD prevention in HSCT. Moreover, the ability of TNFR2 agonists to expand highly suppressive Tregs in vivo should be carefully evaluated in preclinical models. Such studies could open the possibility of Treg-based immunotherapies for autoimmune diseases where regulation of T cell response is impaired or for tolerance induction to organ-transplantation (Figure As TNFR2 is highly expressed by a Treg subset with maximal suppressive function, it could be used as a marker for Treg selection for adoptive therapy purposes. At the same time, treatments that specifically stimulate TNFR2 could selectively boost Treg function. TNFR2 agonists can activate and expand Tregs  in vivo . The usevia TNFR2 could enhance yield, purity, and efficacy of Treg-based cell therapies. Such approach could have a potential for quick clinical translation in HSCT trials where Tregs are in use to prevent GvHD and boost immune reconstitution. The rising growth of studies on mouse and human Treg function strongly support a new role for TNF-α and TNFR2 as key players in the complex interplay between immune cells during immune regulation and tolerance.TNF-α has been widely known for its pro-inflammatory activity, but the effects that follow the stimulation of its two main receptors should be carefully taken in consideration when evaluating the pathogenesis and the treatment of immune-mediated diseases. In this context, new discoveries on the role of TNF-α/TNFR2 pathway may provide relevant tools for a correct use in the clinic of anti-TNF-α treatments and for improving Treg-based therapies. While the blockade of this pathway is under investigation for cancer treatment, TNFR2 stimulation could be used to induce and expand Tregs thus controlling detrimental immune responses. Further studies are needed to evaluate whether Treg activation AM, SP, and AP wrote the manuscript. AV provided overall guidance and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sedentary behaviour—essentially low energy sitting time in waking hours—has emerged as an important topic in public health over the past decade or so. Although Morris and colleagues In this special issue of the journal we encouraged papers that could demonstrate innovation and thus advance the field. We also wanted to see more papers on translation of evidence. The behavioural epidemiology framework has five main phases: (i) the study of health outcomes; (ii) measurement of the behaviour; (iii) correlates of behaviour; (iv) interventions to change behaviour; and (v) translation of findings In this special issue, we published 19 papers led by authors from five countries . An approximate allocation of the main topic of each paper to one of the five phases of the behavioural epidemiology framework shows that papers were more likely to address measurement, correlates and interventions see . It was Regarding innovation and advancing the field, papers addressed a number of novel topics and approaches, including health outcomes through muscle inactivity time assessed via electromyography (EMG) The measurement of sedentary behaviour remains a challenging field. The papers in this special issue used a multitude of approaches, including different wearable devices, self-report measures, experience sampling Interventions, whether in the workplace or schools, were still mainly focussed on the provision of sit-to-stand desks. Such an environmental change can be successful in reducing sitting time, but more is needed on the acceptability and feasibility (e.g. cost) of such an intervention and whether simple alternatives are also possible. Some people will not work at a desk but still have high levels of sitting. Moreover, health outcomes need investigating where standing is compared more with different levels of movement This special issue goes a small way to advancing the field, but there is much still to do across all phases of the behavioural epidemiology framework, but especially in documenting health outcomes of different sedentary and alternative behaviours, types and settings of interventions, and translation into practice. As Co-Editors of this issue we would like to thank the authors for submitting their papers and the reviewers for their efforts in undertaking peer review.


Research paper recommenders emerged over the last decade to ease finding publications relating to researchers’ area of interest. The challenge was not just to provide researchers with very rich publications at any time, any place and in any form but to also offer the right publication to the right researcher in the right way. Several approaches exist in handling paper recommender systems. However, these approaches assumed the availability of the whole contents of the recommending papers to be freely accessible, which is not always true due to factors such as copyright restrictions. This paper presents a collaborative approach for research paper recommender system. By leveraging the advantages of collaborative filtering approach, we utilize the publicly available contextual metadata to infer the hidden associations that exist between research papers in order to personalize recommendations. The novelty of our proposed approach is that it provides personalized recommendations regardless of the research field and regardless of the user’s expertise. Using a publicly available dataset, our proposed approach has recorded a significant improvement over other baseline methods in measuring both the overall performance and the ability to return relevant and useful publications at the top of the recommendation list. The overabundance of information that is available over the internet makes information seeking a difficult task. Researchers find it difficult to access and keep track of the most relevant and promising research papers of their interest . The easAnother classical approach used by most researchers is to follow the list of references from the documents they already possessed . Even thAn alternative approach that has been proposed in the literature is the use of research paper recommender systems , 5, to aTo provide more accurate and relevant recommendations, recommender systems incorporate the users’ contexts and the possible contextual information of the consumed contents . Differeet al. in ..3]. The In order to evaluate the quality of our approach, for each of the target papers, we performed 5-fold cross validation to its references and citations by selecting 20% as a test set. We then assess the general performance using the three most commonly used evaluation metrics in retrieval systems: precision, recall and F1 measures. Precision given by On the other hand, recall given by Moreover, F1 measure given by As users often scan only documents presented at the top ranked of the recommendation list, we feel imperative to also measure the system’s ability to provide useful recommendations at the top of the recommendation list using the two most widely used ranked information retrieval evaluation measures: Mean Average Precision (MAP) and Mean Reciprocal Rank (MRR).P(Rik) denotes the precision of returned papers from the top until paper k is reached, N represents the length of the recommendation list, ni is the number of relevant papers in the recommendation list and I is the set of papers.Average precision (AP) is the average of precision values at all ranks where relevant research papers are found and Mean Average Precision (MAP) given by rank(i) is the highest ranking where the first relevant research paper i appears, and Np represents the total number of target papers.Mean Reciprocal Rank (MRR) given by N recommendations values. This is because our approach is able to critically remove recommending papers that are less related to the target paper.To be specific, the results of each evaluation metric in this section represent the overall averages over all the 50 researchers of our dataset. We start the comparison by assessing the general performance of our proposed approach in returning relevant research papers with the baseline methods based on the three most commonly used information retrieval evaluation metrics. Figs N = 5 and when N = 20. However, our proposed approach began to show the significant difference as the number of N increases, specifically when N is above 20. The low performance based on recall of our proposed approach is as a result of strict rules in qualifying a candidate paper. Thus, our approach is only after the most significant related recommending papers to the target paper and therefore leaving a lot of other less related papers unrecalled. Furthermore, F1 measure), and from the figure, the performance difference between our proposed approach and CCF is also insignificant for values of N less than or equals to 20. However, our approach began to show significant improvement over CCF when N is greater than 20. In all the three measures, the Co-citation method performs very low compared to our proposed approach. This is because the Co-citation method does not infer the hidden associations between paper-citation relations rather applies direct relations between a target paper and its neighboring papers.N. On the other hand, our proposed approach performs worse than CCF in a recommendation list of 5 based on recall and F1 performance measures. However, the major reason behind the low performance of our proposed approach based on recall is the strict rules in qualifying a candidate paper.Conclusively, the general performance of our proposed approach has outstandingly outperformed the baseline methods based on precision for all values of F1 measures. Therefore, a very large N value is extremely important in order to recall as much qualitative and useful recommendations as possible.Our proposed approach is designed to favor precision which has more influence on user satisfaction than recall. This is because precision is the key element in the process of implementing a search solution . Poor prDue to the fact that users usually scan only the top of the recommendation list, we also make the comparison based on how our approach is able to return relevant research papers at the top of the recommendation list. Figs As we have pointed out earlier, all these improvements are largely due to the strictness in qualifying a candidate paper which removed less relevant papers to the target paper. This, therefore, increases the system’s ability to return relevant and useful recommendations at the top of the recommendation list.In this paper, we utilized the publicly available contextual metadata to leverage the advantages of collaborative filtering approach in recommending a set of related papers to a researcher based on paper-citation relations. The approach mined the hidden associations between a research paper and its references and citations using paper-citation relations. The rationale behind the approach is that, if two papers are significantly co-occurring with the same citing paper(s), then they should be similar to some extent.As demonstrated using a publicly available dataset, our proposed method outperforms the baseline methods in measuring both the overall performance and the ability to return relevant and useful research papers at the top of the recommendation list. Based on the three most commonly used information retrieval system metrics, our proposed approach have significantly improved the baseline methods based on precision, recall and F1 measures. Our proposed approach has also recorded significant improvements over the baseline methods in providing relevant and useful recommendations at the top of the recommendation list based on mean average precision (MAP) and mean reciprocal rank (MRR).In addition to considering the collaborative relations among research papers, our next line of research is to also put into cognizance the public contextual contents, such as titles and abstracts of the recommending papers for better performances.S1 Dataset(DOCX)Click here for additional data file.


Fyn tyrosine kinase has been implicated in the pathogenesis of Alzheimer’s disease (AD). We have previously reported that upregulation of the FynT isoform in AD brains was partly associated with astrocyte activation. In this study, we demonstrated selective FynT induction in murine cortex and primary astrocyte culture after prolonged exposure to inflammatory stimulants, suggesting that FynT may mediate persistent neuroinflammation. To delineate the functional role of astrocytic FynT in association with TNF-mediated inflammatory responses, immortalized normal human astrocytes (iNHA) stably expressing FynT kinase constitutively active (FynT-CA) or kinase dead (FynT-KD) mutants were treated with TNF and compared for inflammatory responses using high-throughput real-time RT-PCR and Luminex multi-analyte immunoassays. FynT-CA but not FynT-KD mutant exhibited drastic induction of proinflammatory cytokines and chemokines after prolonged exposure to TNF, which could be attenuated by treating with Fyn kinase inhibitor PP2 or silencing via FynT-specific DsiRNA. FynT kinase activity-dependent induction of PKCδ expression, PKCδ phosphorylation, as well as NFκB activation was detected at the late phase but not the early phase of TNF signaling. In conclusion, selective FynT induction by TNF may facilitate persistent inflammatory responses in astrocytes, which is highly relevant to chronic neuroinflammation in neurodegenerative diseases including but not limited to AD. Neuroinflammation driven by activation of brain-resident immune cells, namely microglia and astrocytes, is a hallmark associated with neurodegenerative conditions, including Alzheimer’s disease (AD). In AD, amyloid beta (Aβ) has been recognized to initiate an inflammatory response through microglia activation and astrocyte recruitment146The non-receptor tyrosine kinase, Fyn, has recently been implicated in the pathogenesis of AD, which is mainly based on its neuropathogenic role that links Aβ toxicity to tau pathology914Compelling evidence of Fyn being able to modulate the production of proinflammatory cytokines has been mainly reported in peripheral immune cells including but not limited to activated mast cells2123in vivo and in primary astrocyte culture and speculate that astrocytic FynT may mediate persistent neuroinflammation through TNF signaling pathway, and contribute to the progression of AD. Using recombinant approaches, we focused on studying the underlying mechanism of FynT kinase activity in association with TNF-mediated inflammatory responses in astrocytes.In this study, we identified that FynT being the specific isoform modulated by inflammatory stimulants We have reported that higher proportions of FynT expression was detected in primary astrocytes as compared to primary neuronsInterestingly, a corresponding increase in FynT expression was detected in mixed cultures maintained in FBS, with higher expression in 1% compared to 0.1% FBS, while FynB expression remained unchanged under all culture conditions . CorrelaIn accordance with our findings, triple-IF staining of FynT , green, A recent study demonstrated that prolonged treatment of TNF or LPS led to Fyn induction in microgliaPrimary astrocyte culture treated with 5 or 50 ng/ml TNF were monitored closely for the changes of FynT and FynB expression. Consistently, we detected significant increment of TNF-induced fold change of FynT at day 1 post-treatment which could be sustained till day 3 . In contTo determine the role of FynT kinase activity in modulating TNF-mediated proinflammatory responses, we generated iNHA clones with ectopic expression of FynT wild-type (FynT-WT) and FynT mutants (constitutively active FynT-CA or kinase dead FynT-KD), as well as empty vector control (EV). Immunoblot analysis confirmed ectopic expression of FynT as well as their corresponding kinase activities of each clone using anti-Fyn and anti-pTyr416 antibodies .Upon 50 ng/ml TNF treatment, all iNHA clones demonstrated rapid induction of IL1B and IL6 expression at the early phase, regardless of their ectopic FynT kinase activity . RemarkaWe further measured the induction fold change of cytokines in each iNHA clone after exposure to TNF for 3 days in seven independent treatments. FynT-CA clone exhibited the highest fold change induction for IL1B (mean ± SEM = 338.2 ± 121.3) and IL6 (46.9 ± 11.2) . AlthougTo reinforce the observation that FynT tyrosine kinase activity facilitated TNF-induced proinflammatory cytokines expression after prolonged incubation, EV control, FynT mutants of FynT-CA and FynT-KD as well as FynT-WT clones were treated with TNF or left untreated for 3 days in four independent experiments and subjected to expression profiling from a panel of 14 inflammatory associated markers using Biomark HD system’s high-throughput real-time RT-PCR. These markers could be broadly categorized into proinflammatory chemokines , proinflammatory cytokines , anti-inflammatory cytokines  and TNF receptor, TNFR1. Using Principal component analysis (PCA), the variability of TNF-induced inflammatory markers expression in each iNHA clone could be clearly visualized in a 3D scatter plot, allowing the clones with distinct expression profiles to be identified easily. In Consistent with the PCA results, unsupervised hierarchical clustering analysis of 14 inflammatory markers revealed that iNHA clones could be well segregated into two independent branches, as untreated (−) and TNF-treated (+) group on the left dendrogram . FynT-CA®). PCA 3D scatter plot displayed 81.8% of the variation in the dataset  inhibitor PP2 was able to inhibit ectopic FynT kinase activity in FynT-CA clone in a dose-dependent manner, as determined by reduction of pTyr416 immunoreactivities without affecting Fyn protein level . We demoUsing Dicer-substrate siRNAs (DsiRNA) randomized sequence duplex (DsiRNA_NC) as control, we demonstrated that treatment FynT-CA and FynT-KD clones with pooled DsiRNAs specific targeting FynT (DsiRNA_FynT) , suggesting that TNF-induced PKCδ expression may be relevant to FynT induction at the late phase. Likewise, after prolonged exposure to TNF, only FynT-CA clone exhibited PKCδ induction at the late phase  signaling axis has been shown to mediate proinflammatory responses under LPS and TNF treatment in microgliate phase . Furtherte phase .Consistently, we observed that PKCδ protein level was upregulated at the late phase in FynT-CA but not in FynT-KD clone, which was accompanied by the increased phospho-Tyr311, a marker of PKCδ activation . FurtherParacrine stimulation by TNF can lead to activation of a key inflammatory regulator, Nuclear factor-kappa B (NFκB) and result in the expression of multiple proinflammatory cytokines and chemokines31et al. demonstrated that microglial Fyn expression could be augmented by prolonged exposure to TNF and LPS, suggesting that Fyn is likely a late response geneThe biological relevance of astrocytes exposure to prolonged TNF treatment would be in the context of chronic inflammation where TNF constitutively activated NFκB and persistently induced inflammatory responses in neurodegenerative diseases, including but not limited to AD34Alternative splicing has been proposed to regulate and fine-tune the diversity and plasticity required for immune systemConsistent with the observation of increased astrocytic FynT expression after prolonged TNF treatment , we idenSFKs have been shown to modulate TNF-induced NFκB activation through phosphorylating certain signaling molecules. For example, c-src phosphorylates IkBα38Both FynT-CA and FynT-KD clones exhibited rapid phosphorylation of PKCδ-Tyr311  and NFκBIn addition to PKCδ phosphorylation at Tyr-311, FynT kinase activity may also be directly or indirectly involved in transcriptional regulation of PKCδ after prolonged exposure to TNF . This spIn summary, we have demonstrated that primary astrocytes exhibited selective FynT induction under prolonged treatment of TNF, which is similar to our previous observation under Aβ treatment6 cells were seeded onto wells coated with 0.1 mg/ml Poly-D-lysine hydrobromide (Sigma) and cultured at 37 °C in a humidified 5% CO2 incubator. Half-change of media was done at day 4 in vitro (DIV 4). Neuron-enriched cultures were maintained in 1 μM AraC (Sigma) without FBS from DIV 4 onwards. On DIV 7, culture medium was replaced with NB medium minus phenol red with the same supplements for each corresponding conditions and treated with TNF.Primary cortical mixed cultures and astrocyte cultures were established from embryonic (E18) and postnatal (P0 to P1) Sprague Dawley rat brains, respectively, after protocol approval from the SingHealth Institutional Animal Care and Use Committee (2014/SHS/950). All procedures were carried out in accordance with the approved guidelines and regulations. Briefly, cortices were dissected and meninges were removed in ice cold Hank’s balanced salt solution. To obtain primary cortical mixed culture, dissected cortices were digested using papain dissociation system , centrifuged and resuspended in Neurobasal (NB) medium supplemented with 2.5% B27, 0.25% GlutaMAX™, 1× penicillin-streptomycin, with or without 0.1 or 1% FBS. 1 × 107 cells per 75 cm2 flask and kept in 5% CO2 incubator at 37 °C with media changed every 2 days. The confluent glial culture (12–14 DIV) was agitated at 250 rpm for 24 h on an orbital shaker with media changed twice to remove microglia. The adherent astrocytes were trypsinized and re-plated at a density of 5 × 105 cells per well in 6-well plates in 0.5% FBS containing DMEM-F12 and treated with TNF.To obtain primary astrocyte culture, isolated cortices were digested with 0.05% trypsin/0.5 mM EDTA in 1× PBS, filtered through Falcon 40 mm nylon cell strainer, centrifuged and resuspended in DMEM-F12 supplemented with 10% FBS, 1 mM L-Glutamine and 1× Penicillin-Streptomycin. Cells were plated at 1 × 10The systemic LPS-induced murine neuroinflammation model that we adopted in current approach has been reported in a separate study by our groupiNHA were primary human astrocytes stably transfected with constructs encoding E6, E7 and human telomerase reverse transcriptase (hTERT)Primary cortical cells grown on coverslips were fixed in 3.7% formaldehyde in PBS for 10 min, rinsed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, rinsed with PBS, blocked with 10% normal goat serum in PBS at room temperature for 1 h. Cells were co-incubated overnight with two different species of primary antibodies at 4 °C, followed by washing and incubating with corresponding secondary antibodies at room temperature for 1 h. For triple-IF, cells were followed by staining with fluorophore-conjugated antibody. Coverslips were mounted in Vectashield mounting medium containing DAPI. Images were visualized and captured using the Nikon A1R confocal microscopy system. Antibodies in used are listed in ® RNA kit (Macherey-Nagel) according to manufacturer’s instruction. 1 μg of the isolated total RNA was reverse-transcribed (RT) using High-Capacity cDNA RT kit (Applied Biosystems) in accordance to the manufacturer’s protocol. Amplification of the 2-times diluted cDNA was carried out in GoTaq® qPCR Master Mix (Promega) containing 200 nM primer sets (RNA extraction was performed using TRIzol reagent (Invitrogen) coupled with column purification. The aqueous phase obtained after centrifugation of TRIzol-Chloroform mixture was mixed with same volume of 70% ethanol and purified using NucleoSpinmer sets  using thSpecific target amplification (STA) was carried out for cDNA in 1× TaqMan PreAmp Master Mix (Applied Biosystem) containing 0.05 μM pooled target primer pairs, under the following condition: 95 °C for 2 min; 95 °C, 15 sec for 10 cycles; 60 °C, 4 min. After a clean-up step by treating with Exonuclease I (ExoI), samples were diluted 10-fold in TE buffer. Preparation and loading of samples and assay mix onto 192.24 Dynamic Array integrated fluidic circuit (IFC) was performed according to the manufacturer’s instructions. Briefly, 1.8μl of each diluted ExoI-treated STA product was mixed with 2 μl of 2× SsoFast EvaGreen Supermix with low ROX (Bio-Rad) and 0.2 μl of Delta Gene Sample Reagent (Fluidigm) before applied to IFC. 0.4 μl of each primer set (50 μM) was mixed independently with 2 μl of 2× Assay loading reagent (Fluidigm) and 1.6 μl of 1× DNA suspension buffer  as assay mix before applied to IFC. After completion the mixing of samples and assays in IFC Controller RX, IFC was then transferred to BioMark HD system (Fluidigm) and ran with program setting: 95 °C, 1 min; followed by 30 cycles of 96 °C, 5 sec and 60 °C, 20 sec. The melting curve analysis consisted of 3 sec at 60 °C followed by heating up to 95 °C with a ramp rate of 1 °C/3 sec. Standard curves of each gene was generated by 10× serial dilution of a pooled template DNAs for calculating relative signal intensity of each sample. Data normalization was carried out similar to real-time RT-PCR as mentioned above.® Multiplex Assays with Luminex LX200 instrument (Millipore) according to the manufacturer’s instruction. Analysis was performed using MILLIPLEX® Analyst Software Version 3.4 (Millipore).Conditioned culture media were collected at day 3 post TNF-treatment, centrifuged for 10 min at 4 °C to remove cell debris and stored at −80 °C until use. Measurement of released cytokines and chemokines including CCL2, CCL3(MIP1α), CCL4(MIP1β), CCL5(RANTES), CCL7(MCP3), CXCL1,2,3(GRO), CXCL8(IL8), CXCL10(IP10), CXCL12(SDF1α+β), CSF2(GMCSF), CSF3(GCSF), FGF2, IL1RN(IL1ra), IL1B(IL1β), IL6, LIF were by MILLIPLEX5 cells per well and transfected the following day with DsiRNA using HilyMax transfection reagent (Dojindo Laboratories). Briefly, 9 μl of HilyMax reagent was added to 120 μl Opti-MEM® I Reduced-Serum Medium (GIBCO) containing 3 μl of either 20 μM pooled DsiRNA_FynT or DsiRNA_NC, mixed well and incubated for 15 min at room temperature. The mixture was then added directly into each well. A second transfection was required to be performed two day after the initial transfection to effectively knockdown the ectopic FynT expression in each iNHA clone.Three DsiRNAs specific targeting FynT (DsiRNA_FynT), as well as normal control duplex (DsiRNA_NC), were purchased from Integrated DNA Technologies (sequences found in ® Reporter Assay Kit (Promega) according to manufacturer’s instructions. NFκB transcriptional activity was determined by Firefly luciferase activities after normalizing for transfection efficiency against Renilla luciferase activities.iNHA clones were co-transfected with NFkB(1) luciferase Reporter Vector (pNFκB-luc) (Panomics) and Renilla luciferase control reporter vector (pRL-CMV) (Promega) at a ratio of 100:1. At 24 h post-transfection, cells were left untreated or treated with 50 ng/ml TNF for 6 hours or 3 days and harvested for luciferase activity measurement using Dual-Luciferase® Dry Blotting System (Invitrogen), the membranes were blocked with Bløk-PO Noise Cancelling Reagents (Millipore) for 1 h, and incubated overnight with primary antibodies (TM western blot stripping buffer (Pierce) at room temperature for 20 min and rinsed with TBST.Samples were lysed directly in 2× Laemmli Sample Buffer (Bio-Rad) with addition of 5% of β-mercaptoethanol and boiled for 5 min. Lysates were measured for protein amount using the 2-D Quant Kit  according to manufacturer’s instructions. After sample separation by 10% SDS-PAGE and transferred onto nitrocellulose membranes using the iBlottibodies  in block® Genomics Suite Version 6.6 software was used to perform Principal component analysis (PCA) and unsupervised hierarchical clustering analysis. All data were transformed to log2 base scale and checked for batch effects. Once a batch effect was identified, it will be removed using a proprietary function of Partek Genomics Suite. All other graphs were plotted and statistical analyses were performed using GraphPad PRISM® Version 5 software.PartekHow to cite this article: Lee, C. et al. Selective induction of alternatively spliced FynT isoform by TNF facilitates persistent inflammatory responses in astrocytes. Sci. Rep.7, 43651; doi: 10.1038/srep43651 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The aim of this study was to describe a clinical case of a patient withAlzheimer's disease (AD) in use of an anti-TNF-α agent for rheumatoidarthritis (RA). The patient reported is an 81-year-old Caucasian man and retiredteacher, diagnosed with RA in 2008 and AD in 2011. Treatment with donepezil wasstarted in 2011 and the use of etanercept introduced in 2012. He was previouslytreated with adalimumab in 2010 for 18 months. In 2013, the subject was engagedin a clinical trial to assess a complementary non-pharmacological approach forAD, presenting significant cognitive improvement during the follow-up period. Wepropose the hypothesis of a synergistic effect of anti-TNF-α medicationused for the treatment of RA as the cause of the improvement in cognitiveresponse observed. These findings could suggest a possible use of this drugclass in the therapeutic management of AD. Neurons, glial cells, macrophages and other cells of the immune systemproduce this factor under extremely variable stimuli. Its broad spectrum of action,not only restricted to inflammatory phenomena, makes the therapeutic approachesinvolving TNF-α highly relevant.12 Moreover, thereare genetic characteristics that can explain an improved response to the treatmentof RA with anti-TNF-α drugs,13 which raises the possibility of applying the same concept toAD.10Since the early 1990s, an antagonistic relationship between AD and RA has beensuggested, given that a significantly lower prevalence of AD was found in patientsunder long-term treatment with non-steroidal anti-inflammatory agents forRA.14 Despite theconflicting evidence on the theme, great attention has been dedicated to anti-TNFtherapies for neurodegenerative diseases over the past decade, especially forAD.The main anti-TNF-α agents available are infliximab, adalimumab andetanercept, developed after the discovery of the key role played by TNF-α inthe systemic inflammatory process, serving as useful alternative therapeuticapproaches for several chronic inflammatory conditions, with RA figuring as theprototype disease.The goal of this study was to assess the cognitive changes observed in a patient withAD and RA in use of an anti-TNF-α agent.15 demonstratedtemporospatial disorientation and inability to evoke two out of three words (scoreof 24/30). HIV, VDRL, vitamin B12, renal and hepatic function exams were all normal.Cranial computed tomography scan showed global cerebral volume loss, with no signsof atrophy in the medial temporal regions. Magnetic nuclear resonance was notperformed at the time of diagnosis. Based on the results obtained, the patient wasdiagnosed with AD in 2011, and appropriate treatment with donepezil was prescribed,initially at 5 mg per day, then 10 mg, with good tolerance. Later, sertraline 50 mgper day was added to the scheme, followed by 25 mg of quetiapine during the nightdue to periods of nocturnal agitation. To date, The worst decompensation episodeoccurred owing to urinary tract infections and refractory joint pain related toRA.The patient, an 81-year-old Caucasian married, holding a Bachelor's degree, and aretired teacher, started to present behavioral changes approximately five yearsearlier: he left the home several times without informing his destination and becameblunted, isolating himself in the bedroom for hours at a time. The subject hadalways had good memory, however, from this date on, he manifested progressive memorydeficits, becoming repetitive and also having major difficulty remembering morerecent facts, even those related to his daily activities and personal life.Neurologic exam disclosed no other deficits besides cognitive dysfunction. Overtime, the patient presented marked mood swings, alternating between periods ofsomnolence and agitation. Likewise, time and space disorientations emerged,associated with increased memory impairment. The Mini-Mental State Examination(MMSE)The subject was also diagnosed with RA in 2008. At the beginning of the disease, hewas under treatment with methotrexate, 15 mg a week, and prednisolone, 20 mg a day.Methotrexate was later substituted by leflunomide, 20 mg per day, and thecorticosteroid was gradually withdrawn. The patient presented significant clinicalimprovement, persisting for approximately four months. The pain episodes became morefrequent and intense, leading to the use of adalimumab  initially at 40 mg a week with dosage reduction after two months to 40 mgevery fifteen days. With anti-TNF-α treatment, the patient reportedsubstantial improvement in the symptoms. Appropriate disease control was observedfor eleven months, when the subject again presented severe morning stiffness anddiffuse pain. Corticosteroid therapy was reintroduced, with administration of 5 mg aday of prednisolone. Six months later, the patient was still reporting joint painand persistent morning stiffness. Thus, it was decided to modify theanti-TNF-α strategy through the use of etanercept (Enbrel™ - July2012) at 50 mg a week. After therapeutic modification, there was significantimprovement in the symptoms related to RA, currently maintained for fifteenmonths.16 In this study, the subject wasselected as part of the group submitted to the complementary treatment,participating in weekly individual sessions of RO for six months. The Consortium toEstablish a Registry for Alzheimer's Disease (CERAD) Neuropsychologicbattery17 the Mini-MentalStatus Examination (MMSE)15 andthe Clock Drawing Test (CDT)18were used as outcome assessment tools. The patient showed significant cognitiveimprovement during the study  therapy in the treatment of AD.he study .19TNF-α is also associated with the increased accumulation of β-amyloidpeptide aggregates in the central nervous system and the induction of neuronaldeath.11 Furthermore, theidentification of high levels of specific cytokines in the cerebrospinal fluid andperipheral blood of subjects with AD, as well as elevated production of thesecomponents in the central nervous system,20 has made the subject of anti-TNF strategy for the treatmentof neurodegenerative diseases a very attractive field in recent years. Nevertheless,certain findings contradict this tendency, suggesting that the absence of influenceof TNF in the central nervous system may actually aggravate the pathophysiologicalscenario of AD.22The amyloid plaques in AD are closely related to microglia cells and activatedastrocytes, cells involved in the liberation of pro-inflammatory factors andcytokines, such as TNF-α.23 apre-clinical trial observed, for the first time, a reduction in the toxic effectsinduced by β-amyloid aggregates associated with decreased levels ofTNF-α in the central nervous system after the administration of peripheraletanercept in animal models.24These results contradict the findings of earlier trials involving the use of thesame drug.25 Considering thepharmacologic characteristics of these agents and experience gained in the treatmentof neurologic disorders, several studies have been conducted since 2004 addressingthe perispinal injection of anti-TNF medications for the treatment of AD.26 This research model yielded promising outcomes, evidencedby quick and sustained cognitive improvements in the individuals treated.12 However, further trials need to beconducted to fill some gaps in knowledge, such as the physiologic repercussions offully inhibitingTNF-α effects in the central nervous system and thepossibility of selective inhibition of these cytokine functions.11In those conditions that have formal therapeutic indication of the drugs,TNF-α inhibitors are applied peripherally. Although it has been establishedthat these medications are unable to cross the blood-brain barrier under theseconditions,16 suggest that concomitant use of ananti- TNF-α agent could have contributed to the superior cognitiveperformance of the patient. Analyzing the MMSE score progression during the trial,the patient's score increased by 2 points, a significantly greater improvement thanthe average for the other participants in the group (1.43 point).16 In a similar study conducted byOnder et al.,27 during a 25-weekfollow-up period, the mean improvement in MMSE score in the group submitted toregular RO sessions was 0.2 point (SE 0.4), much lower than that observed in thiscase report. Another trial with similar characteristics also showed less markedimprovement compared to that found in the present patient, reporting a meanimprovement of 0.71 points after a 6-month follow-up.28In the case report, the favorable progression of the arthritic symptoms afterstarting etanercept therapy and the marked cognitive response of the subject incomparison to the other participants in the controlled trial reported,17 an innovative methodologicalaspect of the trial, used as part of the cognitive assessment during follow-up, alsoyielded interesting results.16 Bythe end of the follow-up, the improvement in CERAD total score observed for thesubject was +14 points, more than twice the average improvement of the otherparticipants in the treatment group (6.43 points).16 Although there are no similar studies againstwhich these results can be compared and no other significant disparities in specificitems of the battery were found between the patient and the rest of the group, thesefindings may corroborate the hypothesis of a positive contribution ofanti-TNF-α agents in this individual's clinical outcomes.The patient's performance on the CERAD neuropsychological battery,20 Likewise, inflammatory signals can propagate through theblood-brain barrier via communication between peripheral and central immunecells.30 Anotherrelevant aspect that corroborates this hypothesis is that the adequate response tothe anti-TNF-α therapy observed in the patient described can be derived froma favorable genotype to this kind of intervention.13 This could also translate to otherbenefits from the suppression of TNF-α activity, observed in this case as themarked cognitive response obtained after engagement in a cognitive stimulationstrategy. This is especially pertinent in view of recent findings observed in thepre-clinical trial using animal models, which suggested for the first time that theperipheral administration of anti-TNF-α agents has therapeutic potential incountering the deleterious effects of neuroinflammation in AD.24Despite the inability to cross the blood-brain barrier, there are several mechanismsby which the inflammatory response can provoke neurotoxicity without direct releaseof cytokines into the central nervous system.This case report illustrates the possible action of anti-TNF-α drugs in thecontrol of AD. It is hoped that the promising studies currently being conducted cantranslate to novel treatment approaches for AD, confirming the initial outcomes.