Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation  Currently, a major hurdle to achieving robust ex vivo HSC expansion is the inability to distinguish between the autocrine and paracrine signals that govern hematopoiesis. For example signaling from VEGF via an internal autocrine loop has been shown to regulate HSC survival9in vitro11Hematopoietic stem cells (HSCs) continuously generate mature blood cells to renew or maintain life-long hematopoiesis. The dynamic regulation of HSC number and progeny involves complex signaling mechanisms, which are strongly influenced by the local microenvironment. The urgent need for a better understanding of HSC regulation is motivated by their fundamental role in the life-long hematopoiesis and their marked regenerative potential after transplantation in clinical therapies of several diseases such as cancer or autoimmune disorders1ex vivo is mimicking the HSC microenvironment by the integration of bioengineering approaches with our constantly expanding knowledge of the soluble signals involved in hematopoiesis131516182023in vitro. These modular toolboxes along with in-depth in vitro analysis provide tools to facilitate the understanding of autocrine and paracrine signaling in HSC regulation.A promising strategy to increase HSC expansion et al.in vitro in the context of a multiplex immunoassay analysis of cell-secreted growth factors. Based on a mechanistic model of HSC signals, a partial least squares (PLS) algorithm allowed the identification of the key players in the regulation of HSPC fate in our setting26in vitro biological systems.In the present study, we develop and use a microcavity platform to contribute to the deciphering of autocrine and paracrine signals in HSC fate regulation. Motivated by previous work of Csaszar E in vitro. For human HSPCs a 15 μm diameter microcavity is very well suited to establish a single cell constraint, while 40 μm diameter microcavities can already harbor up to 12 HSPCs (see next section). The cell-sized depth of the microcavities of 10 μm allows to impose spatial constraints on the cells while still enable cells to migrate and proliferate over the microstructured surface2425The basic premise behind the development of our microcavity platform was that autocrine and paracrine signals, as well as juxtacrine cell-cell and cell-ECM signals, can be distinguished by comparing single-cell and multi-cell arrangements of HSPCs ex vivo HSC culture and we recently demonstrated it to maintain HSC functionality in mouse repopulation studies significantly better than standard cell culture24The microstructured PDMS system is well established for In contrast, the hydrogel microcavity system is based on end-functionalized sPEG crosslinked with heparin as a functional glycosaminoglycan31334 GCSF mobilized human CD34+ HSPCs, isolated from the peripheral blood of healthy donors (after obtained informed consent), were seeded on top of microcavity arrays made from either hydrogels (sPEG-HEP and sPEG-sPEG) or PDMS. Tissue culture plastic (TCP) substrates served as controls. The cultures were supplemented with SCF, TPO and FLT3L at 10 ng·mL−1, the minimum concentration known to maintain or stimulate mild proliferation of HSPC over 7 days24To examine the effects of spatial constraint and material properties on HSPC expansion, 3·10Over a 7 day culture period, HPSC number declined on all surfaces except for 40 μm diameter hydrogel microcavities . The low−1) did yield cytokine-driven HSPC proliferation, it did not appear to influence the differential impact of microcavity size and material properties on cell expansion (4 cells per surface did not influence microcavity-dependent cell expansion (5 for 15 μm microcavity arrays and 7.2·105 for 40 μm microcavity arrays).While increasing the concentration of supplementary cytokines  in the culture medium beyond physiological concentrations  to calculate the coefficient values of our mechanistic model and the impact of the x variables on their explanation of the y response, by calculating the variable importance (VIP) value of each variable274950The highly complex parameter set based on the experimental data and the introduced model was evaluated using a PLS algorithm, as such methods have been found useful to analyze high-dimensional data, especially if variables 2 = 0.95)−1 background cytokines  instead of 10 ng·mL−1. Although we obtained a linear relationship between the predicted and measured cell numbers again (2 = 0.61). The slope of the linear regression calculated by the 10 ng·mL−1 model was about 3-fold lower leading to a 3-fold underestimation of cell number. This indicates that the cytokine background of SCF, TPO and FLT3L scales proliferative activity of HSPC independent of specific parameters of the microcavity and material environment.As shown in To reveal the level and significance of stimulatory and inhibitory effects that each factor under investigation has on HSPC cell number, we plotted their model coefficients against their corresponding VIP values . From thThe latter finding motivated us to minimize the original model in order to reveal key parameters in HSPC regulation. We systematically rebuilt our original model using the PLS derived coefficients. We started with the three factors  that had the highest VIP scores (VIP > 1.6) and then in stepwise fashion added the 3 factors  with the next-highest VIP values (1.6 > VIP > 1.1) one factor at a time .By applying the model coefficients for heparin, autoHGF and autoRANTES, (VIP > 1.5) we observed that material characteristics, namely heparin content, not microcavity size, impacted cell number . The lowBy applying a platform of biofunctional microcavity arrays, we revealed key parameters in autocrine, paracrine and juxtacrine signaling of HSPCs. Our results reinforce the concept of using spatially defined niches, reflecting the environment inside the BM, to control stem cell quiescence and expansion over long time periods. We demonstrated that our microcavity array platform can be used to control microenvironmental signals that affect HSPC expansion. The HSPC expansion data were used to validate a newly established mechanistic model by a PLS approach to identify key parameters in the convoluted signaling system of HSPC regulation. By that we found autocrine signals in single-cell niches to be critical in HSPC quiescence. Finally, the sequestering of heparin-binding cytokines by the hydrogel scaffold was identified as a new tool to control HSPC maintenance.in vitro HSPC culture. Autocrine signals from RANTES and HGF were shown to have a small, but significant, effect on cell growth, while their paracrine signals were shown to be not relevant. The found model coefficients agree to reports on HSPC proliferation showing a supporting function of RANTES (in vitro exposure of HSPCs to RANTES yields increased levels of HSPC after transplantation in vivo) and hindering influence of HGF (diminished proliferation during in vitro HSPC expansion)2524541617404556A novel feature of our microcavity platform is that it enables the separation of autocrine and paracrine signaling mechanisms of certain mediators of in vitro exposure)740On the other hand, paracrine signaling cytokines IL12 and ANG-2, both not affected by adsorption to heparin in our measurements, contribute to a strong material independent microcavity effect. Again, the found model coefficients agree to reported effects in HSPC proliferation with a supporting function of IL12, mostly in a synergistic manner with other factors such as SCF, and a hindering influence of ANG-2  affected HSPC expansionIn summary, our findings suggest that micron-sized spatial constraints allow for creating HSC microenvironments with gradated functionality and impact on cell fate decisions. Functionalized microcavity arrays together with multiplex analysis and statistical analysis were proven useful to decipher specific signaling pathways of the dynamic regulation of HSPC. The introduced methodology can be applied to multiple related studies centering around the microenvironmental regulation of stem cells.Fabrication of PDMS microcavity arrays was performed as described previouslyAfter curing, PDMS replicas were carefully peeled off the silicon master and unreacted prepolymer and curing agent were removed by swelling in heptane  for 24 h. Afterwards heptane was entirely removed by storage for 24 h in a vacuum oven at 60 °C, PDMS microcavity arrays were punched with a 11 mm punch press and attached on top of cleaned glass slides (Ø 11 mm). Subsequently the PDMS surfaces were treated with low pressure oxygen plasma  for 60 s and incubated in a solution of 20 mMalt-maleic anhydride) (PEMA)  were produced by adding 40 μL of a 0.3wt% solution of PEMA dissolved in ethanol on the amino-functionalized surface. PEMA was covalently linked to the surface by annealing at 120 °C for 2 h. Afterwards excess PEMA was removed by autoclaving in MilliQ for 20 min at 120 °C .3-aminopropyl-triethoxy-silane (ABCR) dissolved in ethanol (Sigma-Aldrich) for 2 h. After intense washing in ethanol and subsequent annealing for 1 h at 120 °C films of poly dissolved in phosphate buffered saline (PBS) (Sigma-Aldrich) for 1 h. Finally, surfaces were washed in PBS and CellGro® SCGM medium  prior to cell culture.To covalently bind FN, the anhydride moieties on the surface of PEMA-coated PDMS microcavities were regenerated at 120 °C for 2 h and the microcavity arrays were incubated in 40 μg·mLAt first, sacrificial polystyrene (PS) microstructures were fabricated via SAMIMet al.alt-maleic anhydride) (PEMA)  were produced by spincoating  30 μL of a 0.3wt% solution of PEMA dissolved in acetone and tetrahydrofuran (THF) (1:2 (v/v)) (both Sigma-Aldrich). The coverslips were treated at 120 °C for 2 h to covalently link the polymer film to the carrier. Excess polymer was removed by washing in acetone. Prior to immobilization of sPEG-heparin hydrogels, reactive anhydride groups of PEMA coated glass slides were created by treating for 2 h at 120 °C and in the following incubated in a solution of 100 μg·mL-1 N-(2-Aminoethyl)maleimide (Sigma-Aldrich) dissolved in MilliQ. After 30 min of incubation the surfaces were rinsed with MilliQ and dried. Hydrogels were formed via maleimide-thiol Michael-type addition using a polymer content of 5 wt-% with a molar ratio of sPEG to heparin of approximately 2:1. Heparin  functionalized with 8 maleimide units per molecule  was dissolved in PBS and ultrasonicated for 1 min in ice-cooled water. At the same time thiol end-functionalized 4-arm sPEG was dissolved in PBS by ultrasonication and pH was adjusted to pH 5. In the case of pure sPEG hydrogels, maleimide end-functionalized 4-arm sPEG was used instead of heparin. A volume of 7.5 μL of each thiol functionalized sPEG and heparin or maleimide functionalized sPEG solution was intensively mixed for 10 s. A volume of 14 μL of the gel precursor solution was transferred to the surface of a maleimide functionalized PEMA-coated glass coverslips and the gel drop adhering to the coverslip was placed on top of a microstructured PS surface. Next, hydrogels were allowed to form in the presence of the PS microstructures for 2 h at 22 °C. The PS microstructure was dissolved by immersion in xylene . Solvent was exchanged three times after 1 h of incubation. Next the coverslip tethered hydrogels were washed with acetone, ethanol and PBS and left in PBS. Prior to cell culture, the gels were sterilized by washing with a solution of 0.02% ProClin® (Sigma-Aldrich) in PBS.Next glass coverslips were modified with a reactive maleic anhydride copolymer coating as described in detail by Pompe Granulocyte colony-stimulating factor (G-CSF) mobilized human HSPCs were immuno-magnetically isolated for CD34+ cells according to the manufacturer’s instructions . All leukapheresis products were provided by the University Hospital Carl Gustav Carus, Dresden. Leukapheresis and subsequent cell experiments were carried out in accordance to the approved guidelines of the ethics committee of the University Hospital Carl Gustav Carus Dresden including a written informed consent from healthy donors. Briefly, a sample of 2 mL leukapheresis blood was mixed with 4 mL ACK lysing buffer  and incubated for 5 min to lyse all red blood cells. Afterwards the sample was centrifuged at 300 g for 5 min  and the supernatant was discarded. The remaining cell pellet was washed with 12 mL of isolation buffer (2 mM EDTA and 0.2% HSA in PBS) (both Sigma-Aldrich) and suspended in the staining solution containing 400 μL FcR blocking agent, 400 μL anti-human CD34-antibody (conjugated to 50 nm sized magnetic beads) and 1200 μL isolation buffer. After incubation for 30 min at 4 °C, the samples were washed by the addition of 10 mL isolation buffer and centrifuged at 300 g for 5 min. Subsequently, cells were separated with a MACS® LS column (Miltenyi Biotec) attached to a MACS® separator (Miltenyi Biotec) retaining the magnetically labeled cells within the column. The flow through was discarded and the CD34+ cells were flushed in a 5 mL isolation buffer after removal of the column from the magnetic field of the separator. To increase the purity of CD34+ cells, the procedure was repeated with a MACS® MS column subsequent to the first separation, and the magnetically captured CD34+ cells were flushed in 1.5 mL isolation buffer. Cell numbers and surface marker expression were measured with a MACSQuant Analyzer (Miltenyi Biotec).® SCGM medium  supplemented with 10 or 30 ng·mL−1 of the growth factors SCF (Miltenyi Biotec), TPO and FLT3L . Prior to cell isolation, all scaffolds were pre-incubated in 48 suspension well plates (Fisher Scientific GmbH) for >12 h in cell culture media without supplemented cytokines at 37 °C. For cell experiments, 500 μL of cell suspensions containing either 30000 or 60000 freshly isolated cells per mL were added on top of the hydrogel scaffolds. Between 2–3 microcavity samples were used per condition. Cells grown on TCP under the same conditions served as the control. After 7 days in culture the supernatants, including non-adherent and adherent cells, were collected from the surfaces. Cells adhering to the FN functionalized microcavity scaffolds were detached using Accutase®  (10 min at 37 °C), washed and resuspended. A portion of the harvested cells was used for live/dead cell staining with propidium iodide (PI) (Miltenyi Biotec) and counted with a MACSQuant Analyzer. The remaining cells were stained in PBS/5% (vol/vol) fetal calf serum  and 2 mM EDTA using CD34-FITC (clone: AC136) and CD133-APC (clone: 293C2) antibodies and cytometrically measured with MACSQuant Analyzer. Dead cells were excluded using PI. Obtained data were analyzed and processed using FlowJo software .Cells were grown in serum-free CellGro® technology . Three different Bio-Plex Pro™ magnetic cytokine, chemokine, and growth factor assays were used: AngioPlex, 27-Plex and the TGF-β 3-Plex assay . After 7 days of cell culture, the medium including all cells, was collected and centrifuged at 300 g for 5 min. Equal volumes of cell culture supernatants were stored at −80 °C until analysis. After thawing 110–160 μL aliquots from at least two donors per condition were transferred into a 96 protein low binding plate supported with 0.5% FCS. Samples were transferred in 50 μL volumes into a Bio-Plex Pro™ 96 flat bottom well plate (BioRad) and the assay procedures were performed according to the manufacturer´s instructions. Technical replicates were collected in parallel. Automated washing was performed using a Bio-Plex Pro™ wash station equipped with a magnet (BioRad). Finally, samples were analyzed via cytometric imaging with a Bio-Plex 200 and analyzed with the Bio-Plex Manager™ Software, Standard Edition (both BioRad).HSPC-secreted factors were measured using multiplex immunoassays based on the xMAP® Pro (10.0.1.) and the software integrated NIPALS algorithm were usedy response and the measured growth factor levels, paracrine or autocrine signaling activities, adhesive as well as cell-cell interactions, and heparin content of the scaffolds were set as x variables. Values for the x variables were parameterized as shown in i, mostly caused by technical reasons, were completed by using the mean value of factor i calculated from all samples. Data were centered and scaled to have mean 0 and a standard deviation of 1. The optimum numbers of extracted factors were calculated applying the K-Fold cross validation method. The Van der Voet T2 test was applied to test whether models with different numbers of extracted factors differ significantly from the optimum model. Based on the results of the cross validation, the minimal numbers of latent factors were chosen to explain at least 90% of the variation of cumulative x and y variables and to not differ significantly from the optimum model by Van der Voet T2 tests. After determining the necessary number of latent factors, coefficients as well as the variable importance in the projection (VIP), a measure estimating the importance of a variable in a PLS model, for each x variable were calculated. Subsequently, variables having a smaller VIP score than 0.8 were deleted from the model2) were calculated to validate the prediction of the algorithm.For partial least square (PLS) analysis of the mechanistic model, the software JMP® Pro (10.0.1.). Analysis of the statistical significance between two samples was performed either by one-way ANOVA or two-way ANOVA (results obtained from at least two donors) and Tukey-Kramer’s post hoc test. Asterisk denote statistical significance as follows: *P < 0.05, **P < 0.01, ***P < 0.001. All data are presented as mean values ± standard deviation.All data were analyzed using GraphPad Prism 6  and JMPHow to cite this article: Müller, E. et al. Distinguishing autocrine and paracrine signals in hematopoietic stem cell culture using a biofunctional microcavity platform. Sci. Rep.6, 31951; doi: 10.1038/srep31951 (2016).


Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr) gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R) that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.Thyroid-stimulating hormone (TSH) is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R) that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional  Lhβ and Fshβ were generated next by the duplication of preGthβ during 2R. Tshβ derived from preTshβ but the presence of a 2R-derived Tshβ subunit sister gene has never been demonstrated Callorhinchus milii) interestingly revealed the presence of two copies of Tshβ subunit related genes. Whether they resulted from a specific, local duplication of the Tshβ gene or from the conservation in cartilaginous fish of the Tshβ subunit sister gene derived from the 2R, could not be determined at that time The vertebrate pituitary glycoprotein hormones, TSH and the two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are heterodimers composed of a common α subunit, and a β subunit that confers hormonal specificity Tshβ sister gene was however identified in some teleost genomes, that was shown to be derived from the 3R The scenario appears rather similar in extant teleosts The glycoprotein hormones exert their action by interacting with specific and evolutionarily related G protein-coupled receptors. The glycoprotein hormone receptors (GPHR) are characterized by a large extracellular hormone-binding domain composed of a leucine rich domain connected to a seven-transmembrane domain by a hinge region Tshβ subunit genes and Tshr genes were derived from 2R and 3R were questions we addressed in this study. We took advantage of the recently released genomic data from several species that have a phylogenetically relevant position among vertebrates: two representatives of cartilaginous fish (chondrichthyes), a group that preceded the divergence of ray-finned fish (actinopterygies) and lobe-finned fish (sarcopterygies), the elephant shark, an holocephalan for which a new version of the genomic assembly was recently released Leucoraja erinacea), an elasmobranch; the spotted gar (Lepisosteus oculatus) a ray-finned fish representative that took root before the teleost radiation and the 3R Latimeria chalumnae), a lobe-finned fish that appeared just prior to lungfish Anguilla anguilla and A. japonica), representatives of the basal group of Elopomorphs Tshβ related subunit and for a Tshr related gene. The tissue distribution of the two Tshβ and the two Tsh receptors was analysed in the European eel.Whether additional All aspects of animal care and experimentation were in accordance with the Ethic committee of the Museum National d'Histoire Naturelle and approved by the Institutional Animal Care and Use Committee of the Animal Protection and Health, Veterinary Services Direction, Paris, France.http://blast.ncbi.nlm.nih.gov/Blast.cgi), Ensembl (http://www.ensembl.org/Multi/blastview), DDBJ (http://blast.ddbj.nig.ac.jp/), as well as on web sites for little skate (Skatebase: http://skatebase.org/skateBLAST and elephant shark (http://esharkgenome.imcb.a-star.edu.sg/blast/). Eel sequences were identified from European and Japanese eel genomes available on the website Eel Genome of ZF-Genomics (http://www.zfgenomics.org/sub/eel) in addition to the assembly available in NCBI, using the CLC BIO software .Blast analyses Tshβ or Tshr genes (Protein sequences were predicted from retrieved genomic or Expressed Sequence Tag (EST) sequences by using consensus splice donor and acceptor site and by sequence identity comparison with related hr genes .http://www.cbs.dtu.dk/services/SignalP/). Receptor transmembrane domains were predicted using TOPCONS (http://topcons.cbr.su.se/).The signal peptide cleavage site was determined using SignalP . The phylogenetic reconstructions were performed on-line by using a maximum likelihood method with PhyMyL 3.0 software http://www.phylogeny.fr/) with HKY85 as substitution model for TSHβ-related nucleotide sequences and WAG for TSHR-related amino-acid sequences and default settings for the other parameters. The robustness of the reconstruction was estimated by the aLRT score and/or by bootstrapping over 500 replicates. Nucleotide sequences of the entire coding region  were used for Tshβ subunits with truncation in the 3′ end of the longest sequences. The amino acid sequences were used for the TSHR tree reconstruction.Alignments were fitted manually using Se-AL editor .Mapping the genomic neighborhoods of Tshβ and Tshr were identified and annotated in the eel from the eel genome databases, using CLC BIO software.Flanking genes of duplicated Tissue distribution analysis was performed on RNA samples previously prepared from female silver migrating eels caught in the River Loire, France Tshβ  for European eel Tshβ  were preMessenger RNA was quantified on LightCycler using the LightCycler FastStart Master plus Sybr green I kit  as recommended by the manufacturer. The final primer concentration used was 500 nM. Each sample was run in duplicate using a 1/5 cDNA dilution. The PCR conditions were 95°C for 10 min followed by 50 cycles at 95°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec. The specificity of amplified product was checked by melting curve analysis after the amplification reactions. Relative transcript abundance was calculated from standard curves prepared from pituitaries and thyroid follicles cDNA using LightCycler software. Transcript levels were normalized using total tissue RNA content as previously described in Tshβ subunit genes were characterized from the coelacanth genome  were demonstrated to belong to the fourth paralogous group of genes derived from the duplicated ancGphβ genomic region  previously characterized in elephant shark from a pituitary library Tshβ subunit paralogous gene set  through ostariophysian species    . The firo salar)  suggestic region , close tTshβ subunit sequences of vertebrate representatives were aligned for a phylogenetic analysis. As expected from the synteny analysis, a monophyletic group emerged that clusters the coelacanth Tshβ2 together with the elephant shark Tshβ-related subunit HQ174784  that took place early in the radiation of teleosts. The Tshβ3 sequence branch length from this phylogenetic tree was 1.6 longer in average  and 6 (20 amino acids apart) as compared to LHβ and FSHβ subunits ancGphβ was generated by a duplication of Gpb5 just prior to the emergence of vertebratespreTshβ evolutionary precursor likely acquired these two codon insertions at the time of the 1R. It can be inferred from the aligned sequences that the insertion/deletion event was not generated at the splice site  to an asparagine (N) at the first position, two amino acids upstream of a conserved threonine. Since glycosylation sites are more likely to be created by generating a serine or threonine residue downstream of an existing asparagine TSHβ3 sequences display particular signatures . The mosTshβ2 subunit cDNA Tshβ cDNAs from the elephant shark Only one receptor gene was identified in the coelacanth and elephant shark genomes, although they have two TSHβ subunits. It is thus likely that the additional TSH2 made up of TSHβ2 and the common α subunit, would act through binding to the same TSH receptor, as the classical TSH. Such a redundancy might have led to the loss of the second TSH in tetrapods and in ray-finned fish. The lungfish Tshr occurred early in the teleost radiation Tshra-type receptor as previously named Tshrb is maintained in a similar genomic region as Tshra, in agreement with it resulting from the 3R (strict double conserved synteny). In contrast, in more derived teleost species such as cod, medaka and stickleback, Tshrb was found in a new genomic region  domain and, the large 10th exon encodes the transmembrane domain together with the carboxy-terminal cytosolic tail  is present in the genome of tetraodontiformes . The loss of the duplicated Tshrb in this teleost group is in agreement with the fact that they possess only one TSH. In contrast, Tshrb was also absent in ostariophysians , while they possess two potential TSHs. This indicates at least two independent losses of Tshrb during the radiation of teleosts. As with coelacanth or elephant shark TSH and TSH2, ostariophysian TSH and TSH3 might act through a unique TSHR receptor.A single The TSHR sequence alignment showed high conservation features throughout the vertebrate evolution , Fig. S3In both types of TSHR, two potential glycosylation sites were conserved, one close to the LRR2 region  unlike the eel. In particular, Tshβ transcripts are found in the saccus vasculosus, at the base of the hypothalamus in salmon and may be involved as a photoperiodic signal transducer The tissue distribution analysis by qPCR of the two duplicated ituitary . A low eTshβ3 was identified among transcripts isolated from liver in the adult Antarctic toothfish (FE210400.1) and from the ovary in the yellow perch (GO658547.1) indicating that the expression of this gene in these species as well as in the eel, is not restricted to the pituitary gland.Tshβ subunits, the duplicated Tshr showed a differential tissue distribution pattern. The thyroid follicles showed a high expression of Tshra while a low level of Tshrb transcript could be detected  in early vertebrates , concurrvolution . A seconFigure S1TSHβ subunit-related sequences alignment.(PDF)Click here for additional data file.Figure S2Reconstructed eel genomic regions flanking TSHβ (A) and TSHR (B) genes.(PDF)Click here for additional data file.Figure S3TSHR-related sequence alignment.(PDF)Click here for additional data file.Figure S4Relative evolution rates between TSHβ and TSHβ3 and between TSHR-a and -b sequences.(PDF)Click here for additional data file.Table S1Database references for TSHβ subunit-related sequences.(PDF)Click here for additional data file.Table S2Database references for TSHR-related sequences.(PDF)Click here for additional data file.Table S3European eel primer sets for quantitative real-time PCR.(PDF)Click here for additional data file.Table S4Database references for the genes in TSHβ and TSHR genomic regions.(PDF)Click here for additional data file.


Chronic non-bacterial osteomyelitis (CNO) is an inflammatory, non-infectious disorder of the skeletal system with unknown aetiology. Therapeutic options are NSAIDs, steroids and DMARDs . However, a considerable number of patients have a severe disease course and bisphosphonates or TNF-α blockade might be a therapeutic option.We performed a multicentre, retrospective chart review of all patients diagnosed with CNO in two paediatric rheumatology centres in the last 10 years and treated with pamidronate and/or TNF-α blockade. 17 patients were treated with pamidronate and/or TNF-α blockade. Out of these 17 patients, 10 were treated with pamidronate alone and showed clinical improvement. Three of the 17 patients were initially treated with TNF-α blockade; two had a positive response, one patient stopped therapy due to minor side effects. Two or one patients were initially treated with pamidonate or TNF-α blockade, respectively, but did not show clinical improvement. Interestingly, switching from pamidronate to TNF-α blockade or vice versa was associated with clinical improvement in these patients. One patient was treated with a combination therapy using pamidronate and TNF-α blockade and showed a good clinical response.Pamidronate and TNF-α blockade showed a good response in therapy refractory CNO patients. Severe adverse effects were not observed. However, some patients seem to benefit from one of the two therapeutic options in particular. Future studies are needed aiming to identify clinical and/or radiological parameters that might guide the decision to the appropriate treatment in an individual patient.


Platelet-derived growth factors (PDGFs) bind to two receptors, PDGFRα and PDGFRβ to mediate cell proliferation, migration and survival. Although epithelial cells typically do not express high levels of PDGFRs, their expression has been reported to increase in breast cancer cells that have undergone epithelial to mesenchymal transition.PDGFR signaling was inhibited using Sunitinib malate, Imatinib mesylate or Regorafenib in murine and human luminal-like and claudin-low mammary tumor cell lines or Masitinib in only the human cell lines. A scratch wound assay was used to assess tumor cell migration while immunofluorescence for phosphorylated histone H3 or cleaved caspase 3 was used to determine tumor cell proliferation and apoptosis, respectively.Sunitinib and Regorafenib, but not Imatinib, were capable of significantly inhibiting the migration of both murine and human luminal-like and claudin-low breast cancer cells while Masitinib inhibited migration in both human breast cancer cell lines. Sunitinib but not Regorafenib or Imatinib also significantly suppressed tumor cell proliferation in all four cell lines tested while Masitinib had no significant effect on human breast cancer cell proliferation. None of the PDGFR inhibitors consistently regulated mammary tumor cell apoptosis.Sunitinib, Regorafenib and Masitinib may prove clinically useful in inhibiting breast cancer cell migration and metastasis while only Sunitinib (and possibly Regorafenib in some breast cancer subtypes) is effective at inhibiting both migration and proliferation of breast cancer cells. Platelet derived growth factors (PDGFs), as the name suggests, were originally purified from platelets –4. PDGFsPDGFRs are typically not expressed in normal epithelial cells but are expressed in fibroblasts and smooth muscle cells  where thWith respect to breast cancer, less is known about the importance of PDGFs or PDGFRs. Studies have shown that PDGF-D is upregulated in invasive breast cancer while PDGF-BB is found at higher levels in patients with breast cancer compared to those with benign breast disease and the expression PDGFRs is a predictor of poor prognosis –11. Addiwww.cancer.gov). Regorafenib is FDA approved for the treatment of colorectal cancer and gastrointestinal stromal tumors (www.cancer.gov). Regorafenib can bind to and inhibit PDGFRβ as well as VEGFR2, VEGFR3, Ret, Kit and Raf [www.cancer.gov). A more recently described inhibitor of PDGFRα and PDGFRβ is Masitinib. Masitinib inhibits Kit, PDGFRα, PDGFRβ and Lyn [A number of agents capable of inhibiting PDGFR signaling have been developed including Sunitinib malate (Sutent), BAY 73–4506 (Regorafenib) and Imatinib mesylate (Gleevec). Sunitinib malate inhibits PDGFRβ, VEGFR2, Kit and FLT-3 –20. This and Raf ,22. Imat and Lyn . Masitin and Lyn .We have previously shown that PDGFRs were elevated in a murine mammary tumor cell line that has mesenchymal characteristics and closely resembles the human breast cancer subtype, claudin-low –27. ClauIn the current manuscript we extended our previous work to determine whether inhibitors of PDGFR could suppress migration and survival of murine and human luminal and claudin-low mammary tumor cells. We found that Sunitinib malate and Regorafenib significantly inhibited migration of all 4 cell lines tested. Sunitinib was also capable of suppressing proliferation in all 4 cell lines while Regorafenib suppressed proliferation only in the human claudin-low breast cancer cell line. Imatinib only significantly inhibited migration in the human luminal breast cancer cell line and had no significant effect on proliferation in any of the cell lines. Masitinib inhibited migration of both human breast cancer cell lines but had no effect on proliferation or apoptosis.RJ345 and RJ348 cells were generated in our lab and have previously been described . RJ345 aRegorafenib (Bay 73–4506), Imatinib mesylate (Gleevec), Masitinib (AB1010), and Sunitinib malate (Sutent) were purchased from Selleck Chemicals (Houston TX). All inhibitors were re-suspended in DMSO.Cells were plated in 6-well plates in fully supplemented media such that they were 90-100% confluent at the time the scratch was performed. Once the scratch was made the media was removed and replaced with fully supplemented media containing different concentrations of Masitinib, Regorafenib, Sunitinib or Imatinib. Images of the scratch were captured immediately after the scratch was induced and 24 or 48 hrs after drug administration. Percentage of the wound closed was calculated using ImageJ software (NIH). All drugs were diluted such that each well contained 0.1% DMSO and the control well contained 0.1% DMSO.Cells were plated in fully supplemented media on sterile glass coverslips in 6 well dishes at 20-30% confluence. Cells were allowed to adhere overnight and then the medium was removed and replaced with fully supplemented media containing Masitinib, Regorafenib, Sunitinib, or Imatinib. All drugs were diluted such that each well contained 0.1% DMSO and the control well contained 0.1% DMSO. Twenty-four hours later the media was removed, the cells washed 3 times in ice cold PBS and then fixed in 10% neutral buffered formalin for 1 hour at room temperature. Cells were washed 3 times in PBS and blocked with 5% BSA in PBS containing 0.1% Tween 20. For proliferation, cells were incubated with a 1:2000 dilution of an antibody against phospho-histone H3 (S10)  overnight at 4°C. For apoptosis, cells were incubated with a 1:500 dilution of an antibody against cleaved caspase 3  overnight at 4°C. The cells were then washed twice in PBS and incubated with a 1:200 dilution of the appropriate fluorescently labeled secondary antibody for 1 hour at room temperature. Cells were washed, counterstained with DAPI and visualized using an Olympus BX61 fluorescent microscope  using Metamorph imaging software . The number of positive cells and total number of cells were counted from at least 3 independent experiments.An ANOVA followed by a Dunnett’s posthoc analysis was performed to determine statistically significant values. Values were considered significant when p < 0.05.We have previously shown that RJ348 cells have characteristics similar to human claudin-low breast cancer while RJ345 cells resemble human luminal breast cancer . We haveSince the most dramatic effect observed following PDGFR knockdown in the RJ348 cells was an inhibition of tumor cell migration  , tumor Next, cell proliferation in response to 5 μM of each inhibitor was evaluated. Figure When apoptosis was evaluated, only 5 μM of Sunitinib significantly induced apoptosis and this was only observed in the RJ345 cells. The graph in Figure A more recently identified inhibitor of PDGFRα and PDGFRβ, Masitinib, was evaluated at 5 μM and 10 μM in MCF-7 and MDA-MB-231 cells and was found to significantly inhibit migration in both MCF-7 and MDA-MB-231 cells at both 5 μM and 10 μM Figure  but did Our previous study showed that murine claudin-low mammary tumor cells expressed elevated levels of PDGFRα and PDGFRβ compared to murine luminal mammary tumor cells. In addition, knockdown of PDGFRα or PDGFRβ significantly reduced migration but not proliferation of the claudin-low mammary tumor cells . AlthougThere were a number of surprising findings in this study. The first surprising finding was that Regorafenib and Sunitinib were equally capable of suppressing mammary tumor cell migration and proliferation in both luminal-like and claudin-low cell types. Based on western blots from our previous study  we anticThe second surprising finding was that Sunitinib inhibited proliferation of the claudin-low cell lines. At least for the RJ348 cells, our previous study showed that knockdown of PDGFR either had no effect on proliferation or induced a slight increase in proliferation. In the current study Sunitinib inhibited proliferation of all four cell lines. This effect is likely results from inhibition of kinases other than PDGFRβ.50 for PDGFR inhibition (100 nM) compared to Sunitinib (PDGFRβ IC50 of 2 nM) and Regoragenib (PDGFRβ IC50 of 22 nM). It is also possible that a kinase targeted by both Sunitinib and Regorafenib, but not Imatinib, is critical for migration and proliferation in the tumor cells tested. However, the fact that Masitinib, which inhibits both PDGFRα and PDGFRβ as well as c-kit (similar to Imatinib), significantly inhibited migration of both human breast cancer cell lines, but had no effect on proliferation or apoptosis argues against the IC50 or other kinase targets limiting Imatinib’s efficacy. Masitinib’s IC50 for PDGFRα and PDGFRβ is reported to be higher than Imatinib’s and the kinases inhibited by Masitinib are also inhibited by Imatinib. Therefore, it appears that inhibition of PDGFRβ can suppress breast cancer cell migration but does not regulate breast cancer cell proliferation. This would be consistent with our observations using PDGFR RNAi in RJ345 and RJ348 cells in that inhibition of PDGFRs can suppress migration but not proliferation [The third surprising finding was that Imatinib was less effective at inhibiting migration than either Sunitinib or Regorafenib. Our previous study showed that concomitant knockdown of both PDGFRα and PDGFRβ was the most effective strategy for inhibiting RJ348 migration and knockdown of PDGFRα was more effective at suppressing migration than PDGFRβ knockdown . Since Iferation . The abi3-thymidine assay. Migration was not evaluated [50 of Imatinib in both MCF-7 and MDA-MB-231 cells was approximately 7 uM [A review of the literature revealed that Regorafenib has not been tested on MCF-7 or MDA-MB-231 cells in vitro. One manuscript did evaluated the effects of Regorafenib on MDA-MB-231 cells in vivo and found that Regorafenib at 3 mg/kg and higher significantly inhibited MDA-MB-231 xenograft growth . For Sunely 7 uM . At 5 μMely 7 uM . In the ely 7 uM . The finely 7 uM . TherefoPharmacokinetic studies in mice and rats have shown maximal plasma concentrations of Sunitinib that can be achieved are in the range of 3–3.5 μM . In humaAlthough clinical trials using Sunitinib in the treatment of breast cancer have been disappointing thus far –43 a rec


The Southern Ocean is among the least explored marine environments on Earth, and still little is known about regional and vertical variability in the diversity of Antarctic marine prokaryotes. In this study, the bacterioplankton community in both epipelagic and mesopelagic waters was assessed at two adjacent stations by high-throughput sequencing and quantitative PCR. Water temperature was significantly higher in the superficial photic zone, while higher salinity and dissolved oxygen were recorded in the deeper water layers. The highest abundance of the bacterioplankton was found at a depth of 75 m, corresponding to the deep chlorophyll maximum layer. Both Alphaproteobacteria and Gammaproteobacteria were the most abundant taxa throughout the water column, while more sequences affiliated to Cyanobacteria and unclassified bacteria were identified from surface and the deepest waters, respectively. Temperature was the most significant environmental variable affecting the bacterial community structure. The bacterial community composition displayed significant differences at the epipelagic layers between two stations, whereas those in the mesopelagic waters were more similar to each other. Our results indicated that the epipelagic bacterioplankton might be dominated by short-term environmental variable conditions, whereas the mesopelagic communities appeared to be structured by longer water-mass residence time and relative stable environmental factors. The Southern Ocean has unique oceanographic dynamics with major implications for the global ocean circulation and water stratification systemDespite recent improvements in molecular biological methods  which can help reveal the massive taxonomic microbial richness1579et al. reported that only 25% bacterioplankton taxa were common at the surface waters between the Southern and Arctic Oceans, and the coastal surface communities from both polar oceans exhibited more dissimilar from their respective open ocean communities due to local variability in environmental conditions; whereas the differences were not as pronounced among those microbial taxa at deep waters largely due to long water-mass residence time and connectivity through ocean circulationThere appears to be a subtle interaction between inflows, water column stability, periodic overturns and bacterioplankton community composition in the Southern Ocean, as well as in other aquatic systems12131516The aim of this study was to assess the overall variability in bacterial community structure along well-defined vertical gradients and between two adjacent locations of the Southern Ocean. This study will allow us a new perspective of how bacterioplankton communities change along the water column in terms of abundance and diversity, and how environmental gradients and historical processes control the overall variability of microbial communities in epipelagic and mesopelagic waters in the Southern Ocean.in situ with a XR-420 CTD profiler . CTD salinity was calibrated following an inter-comparison with high-precision salinity measurements made with shipboard CTD instruments. Different sampling depths were selected in order to explore the entire water column in the transition from epipelagic to mesopelagic areas at both stations. Water samples for bacterial community analysis were collected using a closing Kemmerer bottle at nine depths ranging from 0 to 700 m in station A and from 0 to 350 m in station B, respectively. About 3 liters per sample were immediately filtered in triplicate through 0.22 μm pore size polycarbonate membranes  for DNA extraction. The membranes were placed into sterile 5 mL centrifuge tubes, and stored in a −80 °C freezer until molecular analyses. Total DNA was extracted directly from the membranes using a FastDNA spin kit  according to the manufacturer’s instructions. Purified DNA was dissolved in 50 μl ddH2O and stored at −20 °C until use.Water samples were collected during the Antarctic summer from two sampling sites located around 150 km apart from each other . Depth, water temperature, salinity, dissolved oxygen, and electrical conductivity were measured Ex Taq DNA polymerase , 5 μl of Ex Taq buffer, 4 μl of deoxyribonucleotide triphosphate (dNTP) mixture (2.5 mM each), 100 ng of DNA template, and 33.5 μl of ddH2O. PCR was performed in the following thermal cycles: initial denaturation at 94 °C for 5 min; 25 cycles at 94 °C for 30 s, 51 °C for 30 s and 72 °C for 60 s; and a final extension at 72 °C for 10 min. The purified PCR products were ligated into the pMD18-vector  and transformed into Escherichia coli DH5α competent cells . The plasmids containing 16S rRNA gene fragments were sequenced using an automated ABI3730 DNA Sequencer . Successfully inserted plasmid DNA was then extracted using the MiniPrep kit  and plasmid concentrations were determined by spectrophotometry using a BioPhotometer . A standard curve was prepared from linearized plasmid serial dilutions containing 104 and 1010 16S rRNA genes copies calculated directly from the concentration of extracted plasmids. The curve was generated by plotting the threshold cycle values versus log10 of the gene copy numbers. The amplification efficiency (E) was estimated using the slope of the standard curve through the following formula: E = (10−1/slope) − 1. The reasonable efficiency of the PCR should be between 95% and 105%To assess bacterial 16S rRNA gene copy number as a proxy for bacterial abundance, we ran quantitative PCR on each sample of DNA in conjunction with primers 341F (5′- CCTACGGGNGGCWGCAG-3′) and 515R (5′-ATTCCGCGGCTGGCA-3′)Ex Taq™, 0.5 μM of each primer (341F and 515R), 2 μl of total DNA, and RNase-free water. The thermocycling steps of the quantitative PCR were according to the manufacturer’s instructions . All the measurements were performed in triplicate and no signal was detected in our no template control (NTC) samples. In this study, the R2 value for linear regression of threshold value and standard abundance was 0.992, indicating that the assays were quantitative across the range of 16S rDNA concentration tested. The amplified products were visualized on a Gel Documentation system  after running in 1% agarose gel.Quantitative PCR amplification was performed from known concentrations of template DNA to construct standard curves for the quantification of environmental samples. PCR amplification of 16S rRNA genes was performed in triplicate on an Applied Biosystems 7500 Real-Time PCR System . The quantitative PCR assay was carried out in a volume of 20 μl including 10 μl SYBR Premix Taq DNA polymerase , 10 μM of each primer, and 20 ng of target DNA. Sequences were processed by using MOTHUR v.1.20.1http://rdp.cme.msu.edu)www.ncbi.nlm.nih.gov/blast) were also performed to provide information on the OTUs that were not classified by RDP. Dominant OTUs were defined as those with a representation ≥5% within a sample, abundant OTUs were defined as those with a representation ≥1% within a sample, and rare OTUs were defined as having an abundance <0.01% within a sample, which corresponds to the definitions of Ghiglione et al.et al.et al.The hypervariable V4 region ~207 bp) of the 16S rRNA gene was amplified using the primers: 16S-F (5′-AYTGGGYDTAAAGNG-3′) and 16S-R (5′-TACNVGGGTATCTAATCC-3′)7 bp of tH’) was calculated for the 16S rRNA gene diversity. The H’ was determined with the following equation: H’ = −ΣPilnPi. The term Pi was calculated as follows: Pi = ni/N, where ni is the number of sequences of the ith OTU and N is the number of sequences of all OTUs in a communityThe Shannon-Wiener index  waters, the samples were clustered into three groups . Thus, the similarity percentage (SIMPER) procedure was used to identify those species (or OTUs) that contributed most to the similarity and dissimilarity among the three groupsThe normality of the environmental variables and bacterial abundance was checked using the Shapiro-Wilk test and variables were log(x + 1) transformed to improve normality and homoscedasticity for multivariate statistical analyses. The gradient length of the longest axis explored by detrended correspondence analysis (DCA) for microbial communities was longer than 3 SD (standard deviation) units, thus further analyses were conducted using a unimodal response model. Canonical correspondence analysis (CCA) was employed to evaluate the effect of environmental factors  on the bacterial community structure. To evaluate the significance of the conditional effects, Monte Carlo permutation of full models was applied with 999 unrestricted permutations. There exist the possibility of collinearity among salinity and electrical conductivity, therefore only salinity was reserved for the CCA analysis. The above analyses were performed using the R software package with the vegan library (R Development Core Team).11 L−1 and 6.18 × 109 L−1, respectively (8 copies L−1) at the 700 m layer. However, the lowest gene copy number value in station B was found at 25 m, with the copy number of 1.49 × 108 L−1. Overall, the average abundance of 16S rRNA genes in the surface epipelagic waters was higher than that in deeper mesopelagic waters in station A. However, both number of OTUs and Shannon-Wiener diversity showed an opposite trend at both stations because they increased with water depth and showed a significant difference between surface and deep waters (Water temperature was significantly higher in the epipelagic (Groups 1 and 2) than deeper waters (Group 3) in which higher values of salinity and dissolved oxygen were recorded . The copectively . The bacp waters .The high-throughput sequencing generated a total of 489,589 quality-filtered sequences, averaging 37,661 sequences per sample, which were classified into 8,977 distinct OTUs at 97% similarity level. The rarefaction analysis showed that bacterioplankton richness was satisfactorily captured within the whole set of OTUs from the 13 samples, although non-saturated curves from single samples would suggest that diversity was not entirely covered at a local scale . RegardlThe result of CCA ordination showed that temperature was the significant environmental variable affecting bacterial community . The firThe heat map clearly showed the distribution patterns of bacterioplankton communities between surface epipelagic and deep mesopelagic waters . There wP < 0.05). Cyanobacteria were among the dominant taxa especially at station A, and their abundance in upper layer waters was higher than in the deeper waters. Surprisingly, the OTU16, belonging to Actinobacteria, was only found in the surface sample of station B  as those OTUs accounting for ≥5% of the sequences in at least one sample . These ttation B .Few reports have compared bacterioplankton communities at different water depths in the Southern Ocean15et al. suggested that melt water stratification and the transition to varying conditions in the Southern Ocean surface waters may have an impact not only on the micro-eukaryotic community but also on bacterial community compositionDistinct vertical distribution patterns of the bacterial communities were identified in the transition from epipelagic to mesopelagic waters. The surface community was dominated by few taxa with a high abundance, while the deep communities consisted of many taxa with lower abundance. These differences suggest a direct effect of vertical gradients along the water column31Roseobacter and SAR11 clades) and Gammaproteobacteria (including SAR86 clade) in terms of relative abundance and taxonomic richness is consistent with community patterns observed throughout the global ocean33et al.343539The predominance of both Alphaproteobacteria .Accession codes: All sequence data from this study have been deposited in the public NCBI database (http://www.ncbi.nlm.nih.gov/) under the accession number SRX386611.


We describe our recent work demonstrating that classical monocytes constantly enter the intestinal mucosa and how the environment dictates their subsequent fate. We believe that understanding the factors that drive intestinal macrophage development in the steady state and how these may change in response to pathogens or inflammation could provide important insights into the treatment of IBD.The intestine contains the largest pool of macrophages in the body which are essential for maintaining mucosal homeostasis in the face of the microbiota and the constant need for epithelial renewal but are also important components of protective immunity and are involved in the pathology of inflammatory bowel disease (IBD). However, defining the biological roles of intestinal macrophages has been impeded by problems in defining the phenotype and origins of different populations of myeloid cells in the mucosa. Here, we discuss how multiple parameters can be used in combination to discriminate between functionally distinct myeloid cells and discuss the roles of macrophages during homeostasis and how these may change when inflammation ensues. We also discuss the evidence that intestinal macrophages do not fit the current paradigm that tissue-resident macrophages are derived from embryonic precursors that self-renew  Immunological Reviews.This article is part of a series of reviews covering Mucosal Immunity appearing in Volume 260 of As discussed elsewhere in this volume, the intestine is the largest mucosal surface of the body and is the single biggest compartment of the immune system. It is exposed constantly to an array of foreign antigens and must discriminate between harmful and harmless antigens to ensure that the appropriate response is mounted to each. Robust protective immune responses must be generated in response to pathogenic insult, but similar responses mounted against innocuous dietary proteins or commensal bacteria can lead to the development of chronic inflammatory disorders such as celiac disease and Crohn's disease, respectively.Mononuclear phagocytes (MPs) comprising both macrophages (mϕ) and dendritic cells (DCs) are central to this discrimination process and therefore hold promise as targets for potential novel therapies to treat IBD. In this article, we discuss the role of mϕ in gut homeostasis and describe how they change when inflammation or infection is present. Furthermore, we discuss the ontogeny of intestinal mϕ and highlight that unlike many other tissue mϕ, those resident in the gut wall must be replenished continually by blood monocytes, whose fate is ultimately dictated by the environment into which they arrive. These unusual mϕ are ideally adapted to their surroundings and raise many questions about current views of the MP system and its functions.E integrin, and CX3CR1, respectively +MPs are one of the most abundant populations of leukocytes in the healthy intestinal mucosa, and this is the largest population of mϕ in the body + MHCII+ MPs expressing CD64 and/or F4/80 are highly phagocytic cells with abundant foamy cytoplasm in vivo requires the CSF-1R + MPs as mϕ.A useful approach to this comes from recent work that has demonstrated that expression of CD64 (the high affinity FcRγ1), F4/80 and MerTK in situ and some cells of the mϕ lineage express lower levels of CX3CR1 When identified appropriately, murine intestinal mϕ can be shown to express a number of characteristic surface molecules in addition to F4/80 and CD64, including the hemoglobin scavenger receptor CD163 and the mannose receptor CD206) 6 in situ− F4/80− CD11c+ MHCII+ MPs display poor phagocytic activity, expand markedly in response to exogenous flt3L administration, and express the DC-specific transcription factor Zbtb46− F4/80− MPs also migrate constitutively to the MLNs in a CCR7-dependent manner and excel at priming naive T cells − DCs exist de facto marker of mucosal DCs. Importantly, CD103− CD11b+ DCs also express intermediate levels of CX3CR1, making them almost identical to mϕ in terms of their surface phenotype. This emphasizes the need to use multiple markers to distinguish intestinal DCs from mϕ, using markers such as CD64, as well as CD26 and CD272, recently identified by the Immunological Genome Project as DC-specific markers In contrast, CD64+ CD14hi cells may represent recently arrived monocytes from blood. Importantly, CX3CR1 has also been shown to be expressed by intestinal macrophages in humans Characterization of mϕ in the human gut can also prove difficult, with investigators using different panels of pan-myeloid cell markers, including CD33, CD14, and CD13 in vivo, those resident in the gut wall adapt to their local environment in complex and specific ways that may not be reflected by the rigid classification of the M1-M2 paradigm.It has become commonplace to classify mϕ into one of two major subtypes based on their surface receptor expression and which mediators they produce – M1 and M2 lamina propria (LP) in close proximity to the epithelial monolayer Mϕ are found in the mucosa throughout the entire GI tract and are located mostly in the Fig. Like most tissue mϕ, those resident in the normal gut wall play essential housekeeping functions, such as the clearance of apoptotic or senescent cells, and tissue remodeling Fig. However, it is in their role as innate immune effector cells that mϕ are best known. Resident intestinal mϕ are highly phagocytic and are actively bactericidal + T cells  across the epithelium to capture soluble antigen in the lumen Fig. . Origina+ DCs can present bacterial antigen to naive T cells in the MLN Salmonella organisms in the lumen + cells may transport Salmonella in lymph to the MLN was interpreted as showing a role for migratory mϕ in this process + MP belonged to the population of bona fide CX3CR1 expressing DC that we and others have observed in intestinal lymph Despite their phagocytic activity and morphological appearance, there is similar uncertainty about whether LP mϕ play a role in the capture of and induction of immunity or tolerance to local bacteria. Although it is well established that CD103Tissue mϕ are traditionally viewed as part of the mononuclear phagocyte system (MPS). First described in 1972 + (CD115) monocyte subsets can be distinguished by their expression of Ly6C/Gr1 and the chemokine receptors CCR2 and CX3CR1 hi monocytes). The entry of Ly6Chi monocytes into the circulation from the BM is dependent on the CCL2-CCR2 chemokine axis and as a result, mice with genetic deletion of either of these molecules have markedly fewer circulating monocytes hi monocytes are now classified as ‘classical’ monocytes, as they fulfill all the roles previously attributed to generic monocytes in the MPS. In human, an analogous population of classical monocytes is identified as CD14hi CD16− and these also express CCR2 It is now known that the monocyte pool is heterogeneous and, in mice, CSF1Rlo monocytes) were initially proposed to be the precursors of steady state tissue mϕ lo monocytes is to act as phagocytes in the bloodstream, patrolling and maintaining the vasculature lo monocytes in human are identified as CD14lo CD16+, and these express relatively high levels of MHCII A smaller population of murine monocytes expressing lower levels of Ly6C and CCR2, but higher amounts of CX3CR1  mice Why the gut mucosa should need continuous recruitment and differentiation of Ly6Chi monocytes appear to differentiate into mature mϕ through a series of intermediaries which involves downregulation of CD14 and CD11c, and upregulation of MHCII, CD209 and CD163 An analogous monocyte differentiation process seems to exist in human intestine. We recently showed that CD14Fig. −/− mixed BM chimeric mice in situ self-renewal Csf1op/op mice, which have a mutation in the gene encoding CSF1 hi to Ly6Clo monocytes in blood is dependent on CSF1-CSF1R interactions The environmental cues that shape monocyte differentiation in the intestinal mucosa remain to be identified with certainty, and it is likely that multiple factors are responsible for the imprinting of the phenotypic and functional characteristics adopted by maturing monocytes Fig. . These flo monocytes, which mature from Ly6Chi monocytes A distinctive feature of intestinal mϕ is their expression of MHCII at levels even greater than those seen on mucosal DCs. Indeed, acquisition of MHCII is one of the first phenotypic changes to occur during monocyte maturation in the intestine, although its functional significance remains unclear hi phenotype appears to be associated with the development of an anti-inflammatory signature in fully differentiated intestinal mϕ. Given that the majority of mϕ in other tissues lack CX3CR1, it is clear that the local environment of the gut must instruct developing monocytes to upregulate this molecule. The factor responsible has not been identified, although TGFβ is a clear candidate, as it is known to induce CX3CR1 expression by microglia in the brain + MPs to extend cellular protrusions into the intestinal lumen As noted above, acquisition of the CX3CR1+ cells appears not to lead to spontaneous IBD in mice + CD4+ T cells, as these are abundant in the normal intestine and IBD occurs after specific deletion of IL-10 in these cells −/− mice + T cells is also dependent on the microbiota The IL-10–IL-10R axis plays a crucial role in conditioning the behavior of mϕ in the mucosa. One such effect is the upregulation of scavenger receptors such as CD206 and CD163, which is reduced in IL-10-deficient mice Hyporesponsiveness to activation via TLRs and other stimuli is a cardinal feature of resident intestinal mϕ. Early work suggested that this was the result of a failure to express pattern recognition receptors. However, it is now clear that gut-resident mϕ in mice and human express a full range of TLR −/− mice or mice with LysM-mediated deletion of the IL-10R signaling molecule STAT3 display exaggerated pro-inflammatory responses to bacterial products + MP could also reflect defective TGFβR-dependent signaling in mϕ, as TRAF6 and its downstream partner TAK1 are critical components in this pathway At least some of these inhibitory processes may be driven by IL-10 The control of mϕ activity may also involve dedicated inhibitory receptors including SIRPα and CD200R1. Indeed, the CD200–CD200R1 axis appears to play an important role in regulating alveolar mϕ activity + CD11c− cells, which will include many cell types and exclude the majority of resident mϕ which express CD11c. Therefore, the full impact of the microbiota on the behavior of resident colonic mϕ remains to be elucidated.The development of the hyporesponsive state appears to involve recognition of microbial signals, as colonic MP from GF mice respond robustly to stimulation with the TLR4 agonist LPS hi monocytes continuously enter the steady state intestinal mucosa as a surveillance measure. In the absence of any threat, the monocytes adopt an anti-inflammatory phenotype which is imprinted by local factors in the mucosa. The nature and mechanisms of action of these mediators remain to be defined fully.Together, these findings demonstrate that fully responsive Ly6Clo/− population of resident mϕ It is well known that the composition of the human intestinal mϕ pool changes considerably when there is perturbation of homeostasis. For instance, in the mucosa of Crohn's disease and ulcerative colitis patients, there is an accumulation of pro-inflammatory macrophages. These can be identified by their high expression of CD14, in contrast to the CD14+ T cells into lymphopenic hosts Helicobacter hepaticus coupled with neutralization of IL-10 (our unpublished observations). In all these cases, the infiltrate is characterized by a reversal in the ratio of CX3CR1hi and CX3CR1int cells, caused by the accumulation of Ly6Chi monocytes and their CX3CR1int progeny  T. gondii-induced ileitis, elicited Ly6Chi monocytes may actually prevent immunopathology by inhibiting the ability of local neutrophils to produce tissue damaging TNFα and ROI. This PGE2-dependent process involves production of IL-10 by monocytes T. gondii infected CCR2-deficient animals hi monocytes are diminished in GF mice, underlining how the local microenvironment determines the fate of monocytes in the mucosa Contrasting with their role as inflammatory effector cells, recent studies suggest that Ly6City Fig. . For insTrichuris muris, Nippostrongylus brasiliensis, Heligmosomoides polygyrus, Brugia malayi, and Litomosiodes sigmodontisMϕ have also been implicated in Th2 cell-mediated resistance to intestinal helminth infections hi monocytes as in the models of inflammation discussed above, this has never been investigated directly. In addition, recent studies of macrophage expansion during helminth infection of the peritoneal and pleural cavities suggest that this may be driven by IL4- and CSF-1-dependent proliferation of the pre-existing resident population in situ expansion of mϕ and monocyte recruitment during intestinal helminth infection could generate important insights into the plasticity and functional properties of mucosal macrophages.The origin of the alternatively activated macrophages in helminth infection is also uncertain. Although the requirement for CCL2 in parasite expulsion could indicate they are derived from recruited Ly6Chi MP accumulate in the mucosa of IBD patients hi cells in human are the equivalent of Ly6Chi monocytes in the mouse. As in mice, the CD14hi cells that appear in human intestine display enhanced production of TNFα, IL-1β, and IL-6 hi cells in the inflamed mucosa derive from circulating monocytes + eosinophils through their production of CCL11 (eotaxin-1) + mϕ in the inflamed mucosa have enhanced expression of CD40 As discussed above, CD14+ T cells. By their ability to produce large amounts of cytokines, these monocyte-derived ‘DCs’ (mo-DCs) may complement and shape the generation of effector T cells by conventional, flt3L-dependent DCs hi monocytes entering the inflamed mucosa may behave in this manner, acquiring migratory capacity and transiting to the draining MLN et al. + CD11b+ Ly6C− DCs in the colonic mucosa was depleted by administration of anti-CCR2 antibody and concluded that these were derived from Ly6Chi CCR2+ monocytes + DCs can express CCR2 in vivo depletion of CCR2+ cells may not be specific to monocytes and their progeny. Importantly, in our hands, CCR2+ CD11b+ DCs are derived from committed DC precursors and Ly6Chi monocytes cannot repopulate any DC population in either steady state or inflamed intestine + APC do accumulate in the MLN during experimental colitis, the vast majority of these arrive via the bloodstream in a CCR7-independent manner An area of controversy in myeloid cell biology is whether monocytes can generate APCs that can contribute to the initiation of adaptive immune responses. Under the influence of IFNγ, monocytes in inflamed tissues acquire MHCII and are proposed to migrate to draining LN in a CCR7-dependent manner, where they may prime naive CD4int cells returns to normal size, paralleled by a reduction in granulocyte numbers int cells are cleared by apoptosis, as has been shown for the monocyte-derived cells recruited to the inflamed peritoneum hi monocytes replenish CX3CR1hi macrophages in the steady state, it is also conceivable that some of the elicited Ly6Chi cells may convert into anti-inflammatory ‘resident’ mϕ once the inflammation begins to resolve, although this has not been shown directly. Even if this does not occur, we have already noted that the resident population of CX3CR1hi mϕ appears to persist during mucosal inflammation and irrespective of their origin, these anti-inflammatory cells may play an active role in resolution of tissue damage (Fig. in vitroFollowing the clearance of an infectious or inflammatory agent, the intestine must restore homeostasis so that chronic inflammation does not ensue. This is accompanied by major changes in the mϕ pool. During the resolution of colitis in mice, the expanded population of CX3CR1in situ, does not apply to the intestine. Rather, the mucosal mϕ compartment requires continuous replenishment by blood monocytes that are Ly6Chi in mice and CD14hi in human and once considered to be inflammatory in nature. Under steady state conditions, these monocytes differentiate locally into anti-inflammatory mϕ that express scavenger receptors, MHCII, are highly phagocytic and hyporesponsive to pro-inflammatory stimuli, but produce large amounts of IL-10. This process is dictated by cues received from the immediate environment and ensures that mucosal mϕ can ingest and degrade dying tissue cells and any commensal bacteria that penetrate across the epithelial barrier without provoking inflammation. They can also help maintain epithelial integrity and contribute to physiological tissue remodeling. Interruption of the mechanisms that promote the development and/or function of anti-inflammatory mϕ leads to inflammation in response to luminal materials such as the microbiota. During inflammation, or in response to invading pathogens, the normal pattern of monocyte differentiation is disrupted, leading to the accumulation of potent pro-inflammatory effector monocyte/mϕ. Although these clearly play important roles in tissue pathology and protective immunity, they also may be needed for controlling harmful neutrophil activity and for generating the inert mϕ required for resolution of inflammation and tissue repair. These properties underline the plasticity of this population of ‘classical’ monocytes and understanding the local factors that determine their various fates after arrival in the mucosa under different conditions could hold promise as new therapeutic targets for the treatment of IBD.There have been significant advances recently in our understanding of how mϕ contribute to the maintenance of homeostasis in the intestine, largely due to improvements in methods for identifying them more precisely. Furthermore, we and others have established approaches which help explore the origins and developmental stages of intestinal mϕ. In particular, our findings show that the current paradigm that tissue-resident mϕ are derived from fetal precursors that self-renew


Fair intentions should prompt positive affect, like gratitude, triggering cooperative behaviours; while intended unfairness should trigger negative affect, like anger, fostering anti-social actions. It is, however, contended that when people lack information to infer others’ intention they may use ‘normative’ beliefs about fairness - what a typical fair individual ‘should’ do in these circumstances – to guide their behaviour. In this experiment we examined this assertion. We had 122 participants play a one-shot, double-anonymous game with half playing as potential helpers (P1s) and half as recipients (P2s). Whether a participant was a P1 or P2 was chance-determined and all participants knew this. P1s decided whether to help P2s and whether to make their help unconditional (no repayment needed) or conditional (full or ‘taxed’ repayment). P2s decided whether to accept the offer and whatever conditions attached but were blind to the list of helping options available to P1s. We anticipated that recipients would refer to the ‘injunctive norm’ that ‘fair people should help “for free” when it is only by chance that they are in a position to help’. Therefore, without knowing P1s’ different helping options, unconditional offers should be rated by recipients as fairer than conditional offers, and this should be linked to greater gratitude with greater gratitude linked to greater reciprocation. Path analyses confirmed this serial mediation. The results showed that recipients of unconditional offers, compared to conditional ones, interpreted the helpers’ motives as more helpful, experienced greater gratitude and were more eager to reciprocate. The behavioural data further revealed that, when given a latter option to default, 38% of recipients of conditional offers did so.It is well documented that people would remunerate fair behaviours and penalize unfair behaviours. It is argued that individuals’ reactions following the receipt of a gift depend on the perceived intentions of the donors.  The ‘reciprocity rule’ stipulates that ‘we should attempt to repay, in kind, what another person has provided us (pp.20)’ The above suggests that when an individual decides whether to penalize or reward a partner the attribution of the partners’ intentions is crucial Greenberg and Westcott While people attend to others’ intentions they ‘do not attribute good or bad intention in a vacuum (pp.112)’, as Bicchieri In the present paper we endeavoured to examine this idea using a one-shot variant of the trust game. Particularly, in this game 1) all players were anonymous to one another and the experimenters (thereby avoiding any incentives to build reputations); 2) players interacted in dyads with one being a potential helper (P1 hereafter) and the other playing a potential recipient (P2 hereafter), 3) who ended up as either P1 or P2 in every pair, was by pure chance thus removing any attributions about ability; and 4) P2s were completely unknowledgeable about the options P1s had available to them. P1s could offer unconditional help  or conditional help (full repayment of repayment with tax). Thus, under these conditions, P2s could only rely on their beliefs of what people ‘ought’ to do in this context – their injunctive norm Thus we examined whether the difference in the types of helping offered  would be sufficient to induce different attributions of fairness in recipients towards the helper. The null hypothesis would state that in the absence of any information about the options open to the helpers, all help, conditional or otherwise, would be perceived as equally fair. Alternatively, it is plausible to hypothesize that individuals make use injunctive ‘norms’ (i.e. how people ought to behave) of fairness. When somebody’s good fortune is acquired by chance, rather than the owner having to earn the ‘Property Rights’ As well as hypothesizing that an unconditional offer would trigger a favourable appraisal of the donor’s intentions, compared to a conditional one; we also believed that this would generate a greater level of gratitude One hundred and twenty-two students from University of Nottingham participated in the experiment . Each participant was paid an inconvenience allowance of £2 (US$ = 3.27) and was given an opportunity to earn up to £3.50 (US$ = 5.72) more.The experiment was a modified trust game conducted using Z-Tree at least 50 points more to reach the ‘Bonus Threshold’ (i.e. 200) to claim extra money (i.e. the bonus). We allocated the initial endowments this way to create a situation in which all players were, to begin with, equal in the sense that they were all entitled to no additional bonuses as their initial bonus points stood at 150. It was made clear to every participant that each bonus point would be worth one penny (US$ = 0.17 cents) but only when he/she made the 200 point threshold was he/she entitled to any bonus. The only way to reach that threshold, as we told our participants, was by rolling a fair, six-sided die once. The possible payoffs were a loss of –50 points, 0 points, and gains of 50, 100, 150, and 200 points depending on the number on the dice.Each player was initially endowed with 150 money-equivalent ‘Bonus Points’ and was told that he/she needed However, it was emphasized that a larger number on the dice did not guarantee a more favourable allocation of bonus points. The purpose of this die-rolling arrangement was to lead the participants to believe that what segregated the ‘winners’ (i.e. hitting the threshold) from the ‘losers’ (i.e. missing the threshold) was better luck instead of ability. Every participant then learned that in this game he/she had a ‘partner’, who was unidentifiable and anonymous. They were reminded that ‘neither joint effort nor competition with your partner is needed’.Nevertheless, the participants’ scores were pre-determined. Regardless of the actual die-roll, P1s always received a ‘Die-rolling’ score 200  that exceeded the 200 point threshold, while P2s would always fall short of the 200 point threshold. Half the P2s (N = 30) ended up with a final total of 100 bonus point  and the other P2s (N = 31) ended up with 150 points . This difference between the P2s was aimed to manipulate the potential cost of helping for the P1s. The P2s with 100 point were in the ‘High-Cost’ helping condition, and would require twice the magnitude of their partners’  minimum ‘donations’ (i.e. 100) compared to the ‘Low-Cost’ recipients with a 150 points (i.e. 50), to reach the 200 point threshold. Each player then learned about his/her payoff and that of his/her partner.good (bad) luck), and (2) Ability (‘To what extent you think that your partner’s High (Low) score is because of his/her capability (incompetence)’). Both items were measured using 7-point Likert type scales (1 = ‘Not At All’ and 7 = ‘Completely’). In so doing we checked if our participants perceived their partners’ (good or bad) outcomes as chance-determined rather than ‘earned’.At this point both P1s’ and P2s’ provided attributions regarding their partners’ Die-rolling scores. Specifically they indicated if they thought that their partners’ scores were due to (1) Luck (‘To what extent you think that your partner’s High (Low) score is attributable to his/her unconditionally (i.e. to give away the points as a gift and expect no repayment: repayment free offer), or conditionally by applying a ‘repayment clause’ for the amount donated. If a P1 chose to offer conditional help, he/she was asked to pick a level of repayment as the ‘repayment clause’ for his/her recipient . Donors could help either ecipient . All theAfter P1s made the decisions their P2 partners were shown, firstly, whether P1s decided to help; and if so, whether the offers were conditional or unconditional and if they were conditional what the repayment requirements were . P2s who received an offer were then asked to decide whether to accept it or not. It was made clear to them that the P2s would always be financially better off by any offer of help compared to no offer of help. We then assessed recipients’ perception of their donors decisions using the following constructs: (1) State Annoyance (‘I am annoyed by my partner’s decision.’), (2) State Gratitude , (3) State Indebtedness (‘I feel indebted to my partner’), (4) Obligation (‘I feel obliged to repay my partner’), (5) Perceived Genuine Helpfulness (‘My partner's transfer is motivated by his/her sincere desire to help me’), (6) Perceived Reasonableness (‘My partner’s decision is reasonable’), and (7) Reciprocating Tendency (‘I am eager to help my partner out if he/she is in need in the near future’). All were measured using 7-point Likert type scales (where 1 = ‘Not At All’ and 7 = ‘Completely’).We checked that the recipients of conditional offers understood the game and that they were always better off economically accepting (rather than declining) the offers, even if they fulfilled the repayment. P2s who accepted their conditional offers were then shown a reminder of their expected repayment. Immediately after that reminder P2s decide whether to repay and how much . ImportaIn this game P1s’ final payoffs were affected by their P2 partners’ actual acceptance and repayment decisions. That is, any cooperation or defaulting by P2s would directly impact on their respective P1 partners’ final payoffs. All of the P2s’ decisions  were fed back to their P1 partners. Afterwards, all participants were shown onscreen their respective compensations. We then asked all participants to complete the Marlowe-Crowne Social Desirability Scale MCSD, . In so dth Feb, 2013; Ref. Code: 267).This study was approved by the ethics committee of the School of Psychology at the University of Nottingham, and all participants were older than 17 and all gave written informed consent prior to participation as approved by the ethics committee (Approved 11p<.001), between participants’ average ‘Chance’ (M: 5.04) and ‘Ability’ attributions (M: 1.59). This suggested that participants in this game were generally more inclined to interpret their partners’ good (or mis-) fortune to chance rather than to ability.Both P1s and P2s agreed that their partners’ die-rolling outcomes were chance- instead of ability-governed. See S1a Table and S1b Table in There were 49 P2s (80%) who received an offer and of these 24 received a conditional  and 25 an unconditional offer. While there was one instance of rejection among the recipients of unconditional offers (rejection rate: 1/25 = 4%: 96% acceptance), three recipients of conditional offers declined theirs (rejection rate: 3/24 = 12.5%: 87.5% acceptance). Neither cost of helping nor gender significantly differentiated (1) the number of P1s who agreed to help , (2) the conditionality of offers P1s made, (3) helpers’ (N = 49) preferred repayment options and (4) P2s’ (N = 61) the post offer ratings. See S2a Table & S2b Table in Independent samples T-Tests were conducted to examine if the conditionality of offer alone, elicited differential emotions toward donors and interpretations of donors’ intent in recipients. As illustrated in 1 = 0.675, p<.05), and therefore reported more gratitude . This elevated gratitude predicts a greater eagerness to reciprocate . A bias-corrected bootstrap CI for this total indirect effect (a1d21b2 = 0.675*0.412*0.455 = 0.1266) based on 5,000 samples did not include zero (0.0119 to 0.3682), indicating a statistically significant indirect effect 1d21) did not include zero (0.0266 to 0.5867). Meanwhile, there was no evidence that (1) receiving an unconditional offer influenced reciprocity tendency directly , or that (2) receiving an unconditional offer influenced reciprocity indirectly through gratitude only, as the bootstrap CI for this indirect effect (a2b2) included zero (–0.1437 to 0.6326).A serial multiple mediator model was specified Associations of Marlowe-Crowne Social Desirability Scale (MCSD) scores with the main study variables revealed no significant correlations with most of the recipients’ ratings and donors’ helping decisions. See S4a Table and S4b Table in p = .012), between the defaulter’s ‘Expected Repayment’ (M: 86.6) and his/her ‘Actual Repayment’ (M: 38.9). This suggested that a significant level of defaulting occurred among these conditional recipients and that is attributable to those 8 defaulters. There was, however, no evidence that the level of conditionality influenced the number of P2s who (1) accepted (or declined) their conditional offers, and (2) repaid fully or defaulted  defaulted by either repaying partially or nothing, while the rest  all repaid fully as agreed. Interestingly, scrutiny of these eight defaulters response revealed one instance of zero repayment, while the rest (N = 7) on average repaid 58.4% of their expected repayment. A Wilcoxon Signed-ranked test demonstrated a significant discrepancy,  the greater the level of gratitude towards them, and resulting in a greater tendency to reciprocate. Furthermore, the behavioural data revealed that when recipients received a conditional offer, nearly 40% of them (8 out of 21) acted uncooperatively despite acceptance, either giving less than they agreed or defecting completely. This indicated that people, albeit lacking awareness of the options open to donors, still made differential judgments based solely on whether they received a conditional or unconditional offer. We interpret this as an example of the use of injunctive normative beliefs about fairness. That is, in the absence of all information about the donor, recipients could only infer their donors’ intent to be helpful with reference to the normative beliefs about what an average ‘fair’ individual Furthermore, analyses show that people’s tendency to want to reciprocate when treated fairly and be less cooperative when treated less fairly in a normative sense, is, in part, dependent on attributional and emotional processing. People who inferred that the donor’s intentions were good, –feel gratitude towards the donor - were more likely to be eager to help them back in the future. Thus intentions and gratitude are two key processes that help account for the link between conditionality of offers and willingness to reciprocate. Importantly, these results show that injunctive normative beliefs about fairness are important with respect to intentions While only a relatively small fraction of recipients received conditional offers a non-negligible percentage (38%) of them still showed uncooperative behaviour, and interestingly all but one of these defectors chose to be somewhat ‘altruistic’ by repaying the majority of their obligated repayment. It is, important to investigate the psychological or situational factors that motivate someone to engage in this kind of ‘impure’ free-riding or ‘cheap-riding’ There are a number of interesting observations about the unconditional offers. First, in the context of ‘free’ gift we observe that most recipients accepted (96% acceptance rate 24/25 for ‘free’ compared to 87.5% 21/24 for the ‘charged’ gifts) rather that declined the ‘free’ gift because of suspicion S1 Supporting InformationStatistical Analyses of Participants’ Attributions of Partners’ Desirable (or Poor) Die-rolling Outcomes.(DOCX)Click here for additional data file.S2 Supporting InformationEffecs of Cost of Helping.(DOCX)Click here for additional data file.S3 Supporting InformationEffects of Gender.(DOCX)Click here for additional data file.S4. Supporting InformationEffects of Social Desirability Responding.(DOCX)Click here for additional data file.S5 Supporting InformationEffect of Level of Conditionality on Recipients’  Decision Making.(DOCX)Click here for additional data file.S6 Supporting InformationMore Statistics for Player 2s’ Post-offer Ratings.(DOCX)Click here for additional data file.S7 Supporting InformationOn-screen instructions for the game.(DOCX)Click here for additional data file.S8 Supporting InformationCalculation of Participants’ Compensations.(DOCX)Click here for additional data file.