Rosa26stopMet knock-in context (Del-R26Met) reveals that most tissues are capable of buffering enhanced Met-RTK signalling thus avoiding perturbation of developmental programs. Nevertheless, this ubiquitous increase of Met does compromise selected programs such as myoblast migration. Using cell-type specific Cre drivers, we genetically showed that altered myoblast migration results from ectopic Met expression in limb mesenchyme rather than in migrating myoblasts themselves. qRT-PCR analyses show that ectopic Met in limbs causes molecular changes such as downregulation in the expression levels of Notum and Syndecan4, two known regulators of morphogen gradients. Molecular and functional studies revealed that ectopic Met expression in limb mesenchyme does not alter HGF expression patterns and levels, but impairs HGF bioavailability. Together, our findings show that myoblasts, in which Met is endogenously expressed, are capable of buffering increased RTK levels, and identify mesenchymal cells as a cell type vulnerable to ectopic Met-RTK signalling. These results illustrate that embryonic cells are sensitive to alterations in the spatial distribution of RTK action, yet resilient to fluctuations in signalling levels of an RTK when occurring in its endogenous domain of activity.The successive events that cells experience throughout development shape their intrinsic capacity to respond and integrate RTK inputs. Cellular responses to RTKs rely on different mechanisms of regulation that establish proper levels of RTK activation, define duration of RTK action, and exert quantitative/qualitative signalling outcomes. The extent to which cells are competent to deal with fluctuations in RTK signalling is incompletely understood. Here, we employ a genetic system to enhance RTK signalling in a tissue-specific manner. The chosen RTK is the hepatocyte growth factor (HGF) receptor Met, an appropriate model due to its pleiotropic requirement in distinct developmental events. Ubiquitously enhanced Met in Cre/loxP-based  The need to achieve precise control of RTK activation is highlighted by human pathologies such as congenital malformations and cancers caused by aberrant RTK signalling. Identifying strategies to restrain RTK activity in cancer and/or to reactivate RTKs for counteracting degenerative processes is the focus of intense research efforts. We designed a genetic system to enhance RTK signalling during mouse embryogenesis in order to examine the competence of cells to deal with changes in RTK inputs. Our data reveal that most embryonic cells are capable of: 1) handling moderate perturbations in Met-RTK expression levels, 2) imposing a threshold of intracellular signalling activation despite elevated Met-RTK inputs, and/or 3) integrating variable quantitative levels of Met-RTK signalling within biological responses. Our results also establish that certain cell types, such as limb mesenchyme, are particularly vulnerable to alterations of the spatial distribution of RTK expression. The vulnerability of limb mesenchyme to enhanced Met levels is illustrated by gene expression changes, by interference with HGF chemoattractant effects, and by loss of accessibility to incoming myoblasts, leading to limb muscle defects. These findings highlight how resilience versus vulnerability to RTK fluctuation is strictly linked to cell competence and to the robustness of the developmental programs they undergo. Signalling by receptor tyrosine kinases (RTKs) coordinates developmental processes and ensures tissue homeostasis. Upon ligand stimulation, RTKs activate a number of intracellular signalling cascades to influence in cells identity acquisition, movement, survival, and proliferation . CellulaThe vulnerability of cells to excess RTK signalling is conditioned by their competence to respond to an instructive RTK ligand by changing their behaviour. Such competence is influenced by the previous history of cells, during which their intrinsic capacity to respond to a given signal has been shaped through gene regulatory events leading to the acquisition or loss of molecular components necessary for the response . Cellular responses to a given RTK are also conditioned by the simultaneous activation of other signalling inputs and by their synergistic or antagonistic nature , 6. VulnAs highlighted by studies in cellular and animal models, constitutive activation of RTKs results in altered duration/intensity of intracellular signalling and/or participation of additional pathways not recruited under physiological conditions. Unsurprisingly, molecular pathways activated by aberrant RTKs are integrated within other signalling networks through crosstalk, causing signalling perturbations that in turn impact cell behaviour . HoweverIn order to provide insights into the capacity of cells to integrate enhanced RTK signalling above endogenous levels within developmental programs, we have engineered the first mouse model in which RTK signalling can be conditionally enhanced in a tissue-specific manner. Importantly, we opted for the wild-type version of the RTK to avoid perturbation of cellular mechanisms regulating signalling maintenance, extinction, and intracellular location. The chosen RTK is the hepatocyte growth factor (HGF) receptor Met, an appropriate model due to: a) its pleiotropic properties elicited in distinct cell types ; b) the Rosa26LacZ-stop-Met (R26stopMet) transgenic mice that permit, upon Cre-mediated excision, to: 1) increase Met expression above endogenous levels and/or 2) impose Met signalling in cells that do not normally express Met [R26stopMet transgenic mice, we previously demonstrated that enhancing Met signalling in the neural lineage delays the onset of motor neuron degeneration in a murine model of amyotrophic lateral sclerosis [R26stopMet transgenic mice with either ubiquitous or tissue-specific-Cre transgenic lines to determine which cells are vulnerable to moderately enhanced Met-RTK expression levels during development. These genetic studies revealed that exogenous Met phosphorylation is restricted to few territories in developing embryos, thus highlighting the existence of a map of competence to Met activation. Intriguingly, we found that most cells, whether relying on endogenous Met signalling or being normally devoid of Met expression, can buffer a moderate increase in RTK signalling. This correlates with a limited propagation of the enhanced Met-RTK input to its downstream signals. This demonstrates that the signalling network is resilient to intensity changes in Met-RTK input for normal development. However, one cell type, limb mesenchyme, is vulnerable to ectopic Met expression, as evidenced by a drastic impairment of muscle morphogenesis, and by molecular changes in the expression levels of some extracellular regulators of morphogens. Ectopic expression of Met in mesenchymal cells, although not interfering with early limb patterning events, prevents myoblast migration in a non-cell autonomous manner, leading to a drastic reduction of limb muscles. In contrast, enhanced Met signalling in myogenic cells themselves does not interfere with their migration or with limb muscle development. Vulnerability of limb mesenchyme is due to altered HGF bioavailability in the limbs, as assessed by functional studies. Together, our findings show that the accomplishment of developmental programs in a genetic setting with enhanced Met levels is ensured by the robustness of cell competence to buffer increased RTK activity and to conserve functional downstream signalling. These findings also indicate that developmental programs are sensitive to alterations in the spatial distribution of RTK action.To achieve conditional gain of wild-type Met-RTK signalling above an endogenous level, we employed the Cre-loxP strategy . In partress Met . By usinclerosis . Moreoveclerosis . In the Rosa26LacZ-stop-Met (R26stopMet) mice, which allow Met levels to be enhanced in a temporal and spatial regulated manner, and have used them to demonstrate that boosting neuronal RTK signalling delays disease onset in an amyotrophic lateral sclerosis animal model [R26stopMet mice to explore genetically the robustness of developmental programs following enhanced wild-type RTK expression. To enhance Met-RTK signalling levels in all developing tissues, we crossed R26stopMet mice with Tg(CMV-cre)1Cgn/J transgenics (namely Deleter-Cre) [Deleter-Cre;R26Met mice/embryos (referred to as Del-R26Met) in order to excise the LacZ-stop cassette in every cells and allow ubiquitous expression of the Met transgene  to cases with a mosaic and more pronounced staining  with a human Met probe, distinguishing the Mettg from the endogenous mouse Met (mMet), showed ubiquitous expression of Mettg in fully recombined Del-R26Met embryos compared to controls  is expressed in all skeletal muscles throughout development, permitting visualisation of muscle formation in fully recombined Del-R26Met embryos that no longer express the LacZ-stop cassette. Whole mount staining showed that β-galactosidase activity was drastically reduced in Del-R26Met developing limbs compared to controls  or enhanced (as in Del-R26Met mutants), the migration program may not occur. Alternatively, exogenous Met expression might act in limb mesenchymal cells by altering their permissiveness to invading myoblasts. To discriminate between these two possibilities, we selectively enhanced Met expression in either migrating myoblasts or in limb mesenchyme, using the Pax3-Cre knock-in or the Prx1-Cre transgenic lines, respectively [Prx1-R26Met and Pax3-R26Met mutants confirmed that expression of the Mettg is adequately targeted to Prx1 and Pax3 expression domains, respectively ), in which limbs are also devoid of migrating myoblasts  mutant limbs (Notum and Syndecan4 (changed in Del-R26Met limbs) were similar in MetLacZ/d(neo) and control limbs , glypicans –34 and s Hst3st1 –38. Amon Hst3st1 –41 or to Hst3st1 –44. We aigration . A totalettg see . We firscontrols . Second,et limbs . We nextand Sdf1 . In contol limbs . We nextyoblasts , but whent limbs . In contol limbs . AltogetPax3-R26Met mutants could reflect either the robustness of the migration response to increased activation levels of Met intracellular effectors or an intrinsic capability of cells to restrain Met-RTK input. By performing IHC on embryo sections, we followed Met signalling levels in control and mutant forelimbs at E10.5, when myoblast migration occurs. In myoblasts, levels of pTyr1234–1235-Met were enhanced in Del-R26Met (non-migrating cells blocked at the base of the limb) and Pax3-R26Met (migrating cells progressing within the limb) compared to controls, whereas no major changes were observed in Prx1-R26Met   Figs . By explyos Figs . This isyos Figs . This wayos Figs . These fMet signalling mutants [R26stopMet and Del-R26Met embryos and used to follow biochemical changes in the Met signalling cascade. As expected, Del-R26Met embryonic hepatocytes express the Mettg, but not the β-galactosidase . Quantification of the Met25H2 levels revealed an approximately 4-fold increase of total Met protein amount in Del-R26Met versus R26stopMet controls. Increased levels of Met proteins correlate with a concomitant increase in Met phosphorylation levels on Tyr1234–1235 , which are not further enhanced upon HGF stimulation  its phosphorylation levels in Del-R26Met cells was not further enhanced upon HGF stimulation  and on Tyr1349 , which were enhanced upon HGF stimulation (627), but not to propagate signalling activation to downstream pathways . Together, these findings indicate that the Mettg achieves its full signalling competence following a burst of HGF stimulation.To get insights into how enhanced Met signalling is restricted in embryonic cells, we analysed the phosphorylation levels of two other tyrosine residues implicated in receptor signalling and endocytosis. Intriguingly, we found basal levels of Met phosphorylation on Tyrmulation . Consistmulation . Thus, bDel-R26Met and control embryos , an enzyme involved in HGF processing , or because it is neutralized by the action of other signals acting on cells. This is not the case of migrating myoblasts that, although insensitive themselves to enhanced Met expression, failed to migrate towards the limb buds as a result of ectopic Met expression in mesenchymal cells. Ectopic Met in the mesenchyme perturbs transcriptional regulation of some genes and interferes with HGF bioavailability, thus rendering the mesenchyme non accessible to myoblasts . Our resDel-R26Met mice has enabled expression of the receptor at more physiological levels. A moderate increase in Met levels during development drastically affects limb muscle formation by perturbing myoblast migration. Although we cannot exclude subtle phenotypes in other developing organs, these results highlight the restricted sensitivity of the myoblast migration process to ectopic Met in limbs. We genetically demonstrate that myoblasts themselves are not susceptible to enhanced Met signalling levels for their migration, when gain of expression is achieved in the myogenic lineage with Pax3-Cre. This context contrasts with a genetic setting in which Pax3 gain-of-function triggers ectopic myoblast delamination and migration at non-limb somite levels, caused by Met over-expression [Del-R26Met, thus failing to elicit any qualitative change in myoblast behaviour, whereas Met signalling is enhanced above this threshold in Pax3 gain-of-function mutants, thus triggering ectopic delamination. However, it is also possible that the effects of Pax3 gain-of-function mutants involve missregulation of other Pax3 target genes. The Met specificity-switch mutants have previously revealed that myoblast migration is impaired by lowering qualitative and/or quantitative Met signalling levels [The strategy of using the wild-type Met-RTK, rather than constitutive active forms, to generate pression . It is tg levels , 28. ThuDel-R26Met limb mesenchymal cells, in spite of reduced scattering-induced activity, indicating that HGF secretion and release is not impaired by the occurrence of autocrine HGF/Met binding. Moreover, an event of HGF trapping by Met should recapitulate all developmental defects observed in Met loss-of-function mutants. Although the molecular mechanism remains to be identified, it is tempting to speculate that defects in HGF bioavailability could be linked to transcriptional and/or posttranscriptional changes caused by ectopic Met in limbs. This possibility is supported by our data showing signalling  and transcriptional (e.g. Notum and Syndecan4 downregulation) changes occurring in mutant limbs compared to controls. Thus, ectopic Met expression and activation in limb mesenchyme triggers a number of qualitative responses that cannot be buffered by cells. Our co-culture studies highlight a difference in HGF bioavailability, which could result from a change in expression levels of a modulator of HGF, such as either the downregulation of a HGF interactor acting as enhancer of HGF bioactivity, or the upregulation of a negative interactor that would interfere with the capacity of HGF to bind/activate Met  a putative HGF modulator could be altered at the level of transcription, post-transcription, and/or secretion; 2) modulators of chemoattractants do not exert an “on or off” action, but rather fine tune signalling mechanisms, and may act redundantly with one another; 3) distinct mechanisms could cooperate to cause this qualitative phenotype, each of them participating although with a variable quantitative extent.Our studies also uncover another intriguing aspect linked to myoblasts migration: their susceptibility to alterations in the spatial distribution of Met expression. The limb mesenchyme is a local source of HGF exerting a chemoattractant effect on myoblasts that migrate from the somites. In a wild-type context, migrating myoblasts experience a territory devoid of Met-positive cells along their path. Our results ruled out that ectopic Met would act by altering the HGF mRNA and protein expression levels in limb mesenchyme. Instead, they provide evidence that limb mesenchyme must be devoid of Met expressing cells to ensure normal HGF bioavailability. Although we cannot fully exclude that ectopic Met acts by titrating the HGF secreted by limb mesenchyme, a series of experimental arguments make this possibility unlikely. Comparable levels of HGF protein were found in conditioned media from control and vate Met . Among pyoblasts , Syndecayoblasts , 54, andyoblasts . AlternaBy using motor neurons as a cellular paradigm, we have provided examples of robustness versus sensitivity to RTK signalling in developmental outcomes , 23. We tg is fully phosphorylated on Tyr1234–1235 that are critical for its kinase activation, it only becomes fully signalling competent after HGF stimulation, as shown by HGF-induced increased phosphorylation levels on Tyr1003 and Tyr1349 implicated in receptor signalling and endocytosis and by activation of Akt, STAT3, and ERKs. Thus, in Del-R26Met cells, HGF switches Met from a subthreshold signalling status into a fully signalling competent form. These results also show that the R26stopMet mice represent a valuable genetic model to explore the consequences of moderate perturbations of Met as its full signalling competence still depends on ligand stimulation. A second point of restriction occurs at the levels of Gab1, which is phosphorylated at comparable levels in control and Del-R26Met cells upon HGF stimulation. Notably, in the absence of HGF stimulation Gab1 is phosphorylated in Del-R26Met hepatocytes , but not in Del-R26Met mesenchymal cells (not expressing endogenous Met), indicating that the competence of cells to restrict signalling of enhanced Met is different according to the cell type, and that this competence can be influenced by factors such as expression levels of the receptor. Another restriction occurs further downstream, as revealed by the comparable phosphorylation levels of Akt, STAT3, and ERKs observed in control and Del-R26Met hepatocytes  and myoblasts (by IHC in vivo). Such restrictions imply that different cell types possess distinct mechanisms that sense and calibrate the level of activation required for and compatible with biological programs. Overall, our studies provide additional insights into the existence of an exquisite monitoring of signalling levels within cells to attenuate enhanced RTK levels.The present study further expands our knowledge on robustness versus sensitiveness of cells in developmental processes with respect to subtle increases in RTK signalling. Besides the sensitivity of myoblasts to changes in Met signalling in limb mesenchyme, most other developmental processes appear robust and capable of buffering excess Met-RTK signalling . How canFor cells that do not express endogenous Met, the issue of buffering an ectopic Met is only relevant provided that cells possess the adequate co-factors and downstream effectors. When they do, buffering may occur through mechanisms similar to those in cells with endogenous Met. Whereas most territories/cell types with ectopic Met appear capable of resilience, the limb mesenchyme is vulnerable to this signalling perturbation, illustrated by changes in gene expression, by failure of HGF bioavailability, and by resulting myoblast migration defects.R26stopMet mouse model represents a valuable tool for future studies on whether and how enhanced Met signalling impacts muscle development at later time points and during regeneration [MetLacZ/+ embryos at E13.5  [Fat1 in mice causes muscle shape defects resulting from altered migration polarity of a selective group of muscles matching those affected in FSHD [Fat1 expression is also regulated by HGF/Met, by Pax3-FKHR, and by Lbx1 [Met gene amplification is frequently associated with sarcoma susceptibility in muscular dystrophy mouse models [R26stopMet mice can be instrumental for future evaluation of how Met signalling impacts on muscle physiology, regeneration, and/or pathologies.The conditional neration . This isat E13.5 . The migy (FSHD) . Loss of in FSHD . Fat1 ex by Lbx1 , 45, 57. by Lbx1  and tumoe models . TherefoR26stopMet mice modelling conditional gain of RTK signalling exemplify robustness versus sensitiveness of cells to signalling perturbations in order to ensure reproducible developmental outcomes. The genetic approach we employed permits subtle changes in RTK signalling, in contrast to others where constitutive RTK activation, either by point mutations or ligand overexpression, leads to dramatic biological consequences. It is therefore not surprising that the Del-R26Met mice do not recapitulate defects reported in other transgenics with an over-activation of the HGF/Met system. It is the case of mice in which HGF over-expression causes aberrant myoblast and neural crest migration, leading to ectopic muscle formation and melanosis in the central nervous system [R26stopMet genetic setting represents therefore a suitable system to explore the in vivo robustness of cells to subtle increase of RTKs signalling during tissue homeostasis as well as during development and may disclose unexpected switches in cell sensitivity from development to adulthood.The s system , 60. A nAll procedures involving the use of animals were performed in accordance with the European Community Council Directive of 24 November 1986 on the protection of animals used for experimental purposes (86/609/EEC). The experimental protocols were carried out in compliance with institutional Ethical Committee guidelines for animal research .R26stopMet mice 26Sortm1(Actb-Met)Fmai) carrying a conditional mouse–human chimeric Met transgene into the Rosa26 locus was previously reported [Prx1 [Pax3 [Deleter-Cre [Mlc3F2E-LacZ transgenic [Metd(neo/+) [MetLacZ/+ [The generation of the reported , 25. Theed [Prx1  and Pax3x1 [Pax3  promoteransgenic , the Metd(neo/+)  and MettLacZ/+  mouse liFor ISH, embryos were collected in phosphate buffered Saline (PBS) and fixed in 4% paraformaldehyde (PFA) overnight. Whole mount RNA ISH done by using the relevant digoxigenin-labeled RNA probes. X-Gal and Salmon-Gal staining were performed as previously described , 24, 61.2 in DMEM containing 10% (v/v) fetal bovine serum (FBS), 100U/mL penicillin, 100μg/mL streptomycin, 4mM L-glutamine, and 1mM of sodium pyruvate. Cells were first incubated for 24 h at 37°C, in 5% CO2 to allow attachment and colony formation. For forelimb co-cultures, plates were processed as followed. In the first group, the media was changed with fresh media containing increasing concentrations of recombinant human HGF  to estimate the bioactivity dose of HGF released by wild-type limbs (comparable to 5ng/ml of recombinant human HGF). In the second group, media was replaced and added together with freshly dissected 2 forelimbs derived from E10.5 embryos of different genotypes (done by PCR using remaining tissue). Twenty-four hours later, the MDCK cells were fixed with 4% PFA in PBS, then cells were stained with Crystal violet. For quantification, at least 10 images with several cell colonies were analysed. Three different categories were defined: category 1 corresponds to not-scattered colonies; category 2 corresponds to colonies in which cells start losing their contact; category 3 corresponds to colonies with visible scattered cells.Procedures were performed as described , 50. Bri2. For biochemical analysis of conditioned media, after 48 hrs cells were washed twice then cultured in DMEM complete media without serum for 24 hrs. For scattering assays, after 48 hrs a coverslip with previously plated MDCK cells was placed upside down in order to have MDCK cells in contact with media pre-conditioned by limb mesenchymal cells. In order to avoid MDCK and limb mesenchymal cell contact, a spacer was added between the plate and the coverslip. For HGF/Met inhibition, Met chemical inhibitors  or blocking anti-HGF antibody  where added prior to co-culture with MDCK cells on coverslips. After 24 hrs co-cultures, coverslips were fixed and processed as described above.Forelimbs from E10.5 embryos were dissected in HBSS supplemented with 100U/mL penicillin, 100μg/mL streptomycin, and 7mM Hepes (Life Technologies) and incubated for 5 min in 2% trypsin (Sigma). Limbs from individual embryos were processed separately and a piece of the body was used for genotyping. Then, trypsin was inactivated with DMEM supplemented with 10% (v/v) FBS, 100U/mL penicillin, 100μg/mL streptomycin, 4mM L-glutamine, 1mM of sodium pyruvate, and non-essential amino acids (defined as complete media). Cells were dissociated by pipetting, then spin down for 5 min, resuspended in DMEM complete media, and plated in 4 well plates (2 forelimbs per well). Cells were grown at 37°C in a humidified atmosphere of 5% COCulture of primary embryonic hepatocytes were performed as previously described , 46–48. 1234/1235-Met , anti-phospho-Tyr1003-Met (1:2000 for WB), anti-phospho-Tyr1349-Met (1:1000 for WB), anti-phospho-Tyr627-Gab1 (1:2000 for WB), anti-phospho-Ser473-Akt , anti-phospho-Ser727-Stat3 (1:2000 for WB), anti-phospho-Thr202-Tyr204-ERKs , anti-phospho-Thr202-Tyr204-ERKs , anti-ERKs (1:10000 for WB); from Santa-Cruz Biotechnology: anti-mouse Met (1:200 for WB), anti-human Met (1:1000 for WB), anti-HGF (1:500 for WB); from Abcam: anti-β-galactosidase (1:2000 for WB); from R&D: anti-mouseHGF (1:50 for IHC); from Sigma-Aldrich: anti-actin (1:12000 for WB); from Assay Designs: anti-human Met (1:500 for IHC); from hybridoma bank: Pax3 (1:10 for IHC), anti-myosin heavy chain II ; from Jackson: anti-rabbit IgG-peroxidase or anti-mouse IgG-peroxidase (1:4000 for WB), anti-mouse fluorescent-coupled secondary antibodies (1:400 for IHC), anti-mouse or rabbit biotin-coupled secondary antibodies (1:500 for IHC).Antibodies used were from Cell Signaling: anti-Met 25H2 (1:2000 for WB), anti-phospho-TyrTotal RNA was isolated from embryos using the RNeasy Mini Kit  according to manufacture instruction. cDNA was generated using the Reverse Transcription Kit (Biorad). cDNA (30ng) was amplified by real time PCR using 4 μL SYBR Green qPCR SuperMix-UDG with Rox (Biorad) and 2μL of forward and reverse primers (0.1 μM). The analysis was performed on each sample in triplicates using Applied Biosystems . Relative transcript levels were calculated using the comparative Ct method and normalized to the housekeeping gene GAPDH. Primer sequences are listed in New-born mice were scarified and placed in water overnight at 4°C, then eviscerated, and skin was removed. Samples were fixed in 96% ethanol overnight. Cartilage staining was performed using alcian blue solution for 24 hours . Samples were then rinsed in ethanol 96% for 1 hour and clearing was done for 6 hours in 2% KOH. Bone staining was performed using alizarin red solution for 24 hours . Samples were treated with 1% KOH/20% glycerol, then stored in glycerol/ethanol (1:1 volume).For whole mount ISH, quantification of signal intensity in limbs was performed using the ImageJ software. Briefly, images were first converted to a grey scale and then inverted to a negative scale . Signal intensity was measured along a horizontal line of a given pixel length (matching the forelimb). After background and threshold subtraction, the values were averaged between several samples of each genotype to generate an average signal distribution plot (considering left and right limbs separately). The total signal intensity was also calculated for each sample and plotted individually.25H2 and HGF protein levels, quantification of signal intensity was done on western blots using the image J software. Images were process as described above. Values were averaged between different samples.For MetResults were expressed as the mean ±SEM. Statistical significant differences were estimated by applying unpaired t-Student test for data showing normal distribution and by Mann-Whitney test otherwise. P values are indicated in Figs.S1 FigRosa26LacZ-stop-Met, namely R26stopMet) and after Cre-mediated recombination (tissue-specific-R26Met).  Whole mount (B) or transverse section (C) β-galactosidase staining of E10.5 R26stopMet and Del-R26Met embryos. Note that different degree of Cre-mediated recombination results in Del-R26Met mutants with high (right) or low (middle) β-galactosidase activity. hb: hindbrain; ov: optic vesicles; sc: spinal cord; di: diencephalon. (D) Genotype analysis of embryos showing the mutant allele before and after Cre recombination. Note that the efficiency of Cre-mediated recombination results into Del-R26Met embryos with total (right) or partial (middle) deletion of the LacZ-stop cassette. Scale: 500μm.(A) Schematic representation of transgenic mice carrying the LacZ-stop cassette followed by chimeric Met before ((TIF)Click here for additional data file.S2 FigMet (hMet) and mouse Met (mMet) probes in E10.5 control and Del-R26Met embryos. Scale: 500μm.Whole mount ISH with human (TIF)Click here for additional data file.S3 FigDel-R26Met embryos, whereas Mettg  is expressed by most cell types in all tissues, phospho-Met is present only in a restricted number of cell types. Middle and bottom panels show enlargement of top panels at the levels of different organs. (B) Schematic representation of E16.5 embryos showing the level of sections reported in panel A and C. (C) Immunohistochemical analysis of phospho-Met and human Met (hMet) in control embryos showing background levels. (D) Table summarizing organs positive or not for phospho-Met. Scale: 200μm.(A) In (TIF)Click here for additional data file.S4 FigDel-R26Met embryos compared to controls. Note residual cytoplasmic β-galactosidase staining in the R26stopMet line due to a small proportion of cells in which the LacZ-stop cassette was not completely deleted. (B) Schematic representation of transgenic mice carrying the MLC-LacZ transgene alone (controls) or together with the transgenic Met in Del-R26Met embryos (limbs are outlined in bottom panels). Scale: 500μm.(A) Whole mount β-galactosidase staining showing reduced developing appendicular muscles in E13.5 (TIF)Click here for additional data file.S5 FigPax3 probe showing reduced migrating myoblasts in the forelimbs of Prx1-R26Met (A), but not of Pax3-R26Met (B) embryos. As in Pax3-Cre line the Cre gene is inserted in the Pax3 locus resulting also into a loss-of-functional allele, the adequate control embryos in panel B correspond to Pax3-Cre heterozygous (Pax3).  Quantification analyses of Pax3 positive domain in forelimbs. Left panels: each plot represents the average signal distribution along the white line in forelimbs. Right panels: quantifications and statistical analyses of the sum of signal intensity based on intensity plots in left panels. Numbers of samples: control, n = 11; Prx1-R26Met, n = 6; Pax3, n = 4; Pax3-R26Met, n = 4. The sum of Pax3 signal intensity was calculated between point A and B: A indicating a fixed position between the somites and the limb whereas B being placed at a fixed distance from A. Note the reduced Pax3 level in Prx1-R26Met mutants. (E) Left panels: Whole mount ISH of E12.5 embryos with MyoD probe showing reduced developing appendicular muscles in Prx1-R26Met mutants (limbs are outlined in bottom panels). Right panel: Prx1-R26Met mice at birth show hyperflexed forelimbs. Scale: 500μm. Whole mount ISH of embryos with (TIF)Click here for additional data file.S6 FigDel-R26Met embryos with Shh (top) and Fgf8 (bottom) probes. Note that Shh and Fgf8 expression remain located in the posterior limb bud mesenchyme and in the apical ectodermal ridge, respectively. Scale: 500μm. (B) Skeletal staining of P0 control and Del-R26Met limbs showing no major patterning defects. Note that the deltoid tuberosity in the humerus is significantly reduced in Del-R26Met mutants (100%: n = 7). As the deltoid tuberosity requires muscle mechanical forces at later stages besides initiating signals such as BMP4 [(A) Whole mount ISH of E10.5 control and  as BMP4 , it is l(TIF)Click here for additional data file.S7 FighMet) and LacZ transcripts in E10.5 control (n = 11) and Del-R26Met (n = 11) limbs. Each dots corresponds to transcript levels in forelimbs of E10.5 individual embryos (done in triplicate). Columns correspond to the average value, expressed as mean. Note that upregulation of Mettg (hMet) is paralleled by downregulation of the LacZ transcripts in Del-R26Met embryos.  qRT-PCR analysis of candidate genes in E10.5 control and Del-R26Met mutant limbs. No significant changes are observed. Hs2st1: heparan sulphate 2-O sulfotransferase 1. Hs3st1: heparan sulphate 3-O sulfotransferase 1. Sdf1: stromal cell-derived factor 1. Columns correspond to the average value, expressed as mean ± s.e.m. Mann-Whitney and Student-t test.(A) qRT-PCR analysis of human Met ((TIF)Click here for additional data file.S8 FigMet distribution in developing muscles of E10.5 (A) and E13.5 (B) MetLacZ/+ and MetLacZ/d(neo) embryos. Note: a) the absence of muscles in limbs of E10.5 and E13.5 MetLacZ/d(neo) embryos; b) that Met expression is restricted to a subgroups of developing muscles at E13.5; c) that developing limb tendons express also Met at E13.5 and the pattern of expression is not altered in MetLacZ/d(neo) mutants. Scale: 500μm. Whole mount β-galactosidase staining showing (TIF)Click here for additional data file.S9 FigDel-R26Met limb mesenchymal cells after enrichment with heparin (left) or lectin (right) beads. Note no major differences in the level of uncleaved HGF between control and mutants. Numbers on the top of control and mutant lanes correspond to quantification analyses of uncleaved HGF. Quantification of the processed form of HGF was not performed because of the presence of a nonspecific band with the same molecular weight (ns). Lanes with human recombinant HGF (rec-hHGF: 5ng) and non-conditioned media (media) after purification. (B) Quantitative analysis of MDCK cell scattering using co-cultures with dissected forelimbs from control and Del-R26Met embryos with a recombination efficiency higher or lower than 60%. Quantification was performed according to categories defined in st category using forelimbs from highly recombined Del-R26Met mutants . Mann-Whitney and Student-t test.(A) Western blot analysis of conditioned media from control and (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


IgG4 reactions marked by infiltration of IgG4-positive plasma cells in affected organs occur in cancer patients and in patients with IgG4-related diseases. Extrahepatic cholangiocarcinomas including gall bladder cancer are often accompanied by significant IgG4 reactions; these reactions show a negative correlation with CD8-positive cytotoxic T cells, suggesting that the evasion of immune surveillance is associated with cytotoxic T cells. The regulatory cytokine IL-10 may induce IgG4-positive plasma cell differentiation or promote B cell switching to IgG4 in the presence of IL-4. Cholangiocarcinoma cells may function as nonprofessional antigen presenting cells that indirectly induce IgG4 reactions via the IL-10-producing cells and/or these may act as Foxp3-positive and IL-10-producing cells that directly induce IgG4 reactions. Moreover, IgG4-related disease is a high-risk factor for cancer development; IgG4-related sclerosing cholangitis (IgG4-SC) cases associated with cholangiocarcinoma or its precursor lesion biliary intraepithelial neoplasia (BilIN) have been reported. IgG4-positive cell infiltration is an important finding of IgG4-SC but is not a histological hallmark of IgG4-SC. For the diagnosis of IgG4-SC, its differentiation from cholangiocarcinoma remains important. Inflammatory biliary diseases with periductal fibrosis are categorized as sclerosing cholangitis. In addition to the prototype of sclerosing cholangitis, primary sclerosing cholangitis (PSC), IgG4-related sclerosing cholangitis (IgG4-SC) is categorized as sclerosing cholangitis. Although IgG4-SC is characterized by the infiltration of numerous IgG4-positive cells in the wall of bile ducts, this IgG4 reaction is also found in PSC, hepatolithiasis, and cholangiocarcinoma. In particular, the differentiation between IgG4-SC and cholangiocarcinoma is an important clinical issue. Moreover, carcinogenesis in IgG4-related diseases has been noted  and a feIgG4 is a minor immunoglobulin subtype that does not activate complement and comprises only 3–6% of all circulating IgG in adults , 8. MoreBiliary tract cancers can be anatomically divided into intrahepatic and extrahepatic cholangiocarcinomas; the latter includes hepatic hilar cancer, common bile duct cancer, gall bladder cancer, and cancer of the papilla of Vater. The biological behavior and carcinogenicity of each cancer differ, but the histology of most biliary tract cancers is the same as that of ordinary adenocarcinomas. In addition to neoplastic lesions, several types of cholangitis causing biliary stenosis are important in the differential diagnosis of biliary diseases. PSC and IgG4-SC clinicopathologically mimic extrahepatic cholangiocarcinomas. In particular, the clinicopathological differentiation of IgG4-related diseases from neoplasms is important because desmoplastic change and, rarely, mass formation as well as marked IgG4-positive cell infiltration are found. Moreover, the IgG4 reaction often occurs in malignant neoplasms including pancreatic cancer –12 and tThe IgG4 reaction is scattered within and around cancerous nests. IgG4-positive cells are particularly prominent around nests, invasive areas facing noncancerous biliary walls, and fibroadipose tissue and are interspersed with other inflammatory cells. Moreover, in biliary tract cancer with a marked IgG4 reaction, the surrounding nonneoplastic biliary mucosa and the carcinoma area are often accompanied with an IgG4 reaction Figures . One chaIn the advanced cholangiocarcinoma cases, the presence of the cholangiocarcinoma arising from IgG4-SC is speculated, but it is pathologically hard to differentiate between cholangiocarcinoma with IgG4 reaction and cholangiocarcinoma arising from IgG4-SC. However, the presence of IgG4-SC cases accompanying biliary precancerous lesion indicates the possibility of biliary carcinogenesis arising from IgG4-SC.β. High Treg cell frequency is speculated to reflect a poor prognosis in pancreatic and colon cancer patients [Some pancreatic cancer cases accompanied by the IgG4 reaction and/or elevated serum IgG4 levels –12 in adpatients , 20. In patients –25, suggpatients . Consequpatients . In otheβ and are involved in the IgG4 reaction [Th2-type cytokines, IL-4 and IL-10, are important in the pathogenesis of IgG4-related diseases including IgG4-SC. Treg cells are characterized by the production of IL-10 and TGF-reaction , 25, 28.reaction . Severalreaction –33. We dProfessional APCs such as dendritic cells expressing both MHC class II and the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) present antigens with costimulatory molecules to CD4-positive T cells and differentiate from CD4-positive cells into Th1, Th2, TH17, or Treg cells depending on cytokine milieu. However, MHC class II-positive cells lacking costimulatory molecules induce anergy in native T cells. T regulatory type 1 cells (Tr1 cells) are characterized by the production of IL-10 and are induced by immature dendritic cells . MoreoveTreg cells, Th2 cells, and IL-10-producing regulatory T cells mainly produce IL-10. Although Foxp3 is a master transcription factor for Treg cells, Foxp3 and IL-10 are expressed in several carcinoma tissues and cultured cancer cell lines, suggesting that cancer cells induce the Treg cell-like immunoregulatory milieu to evade immunosurveillance –33. To vAs mentioned above, cholangiocarcinoma cells are nonprofessional APCs and/or regulatory cells that directly induce IgG4 reactions in an IL-10-predominant cytokine milieu. Although the IgG4 reaction in biliary tract cancers and IgG4-SC are closely associated with the IL-10 regulatory cytokine milieu, it is possible that both mechanisms are specific for cancer tissues but different from IgG4-related pathogenesis.Patients with autoimmune pancreatitis occasionally have other types of cancer including pancreatic cancer –41 and b in situ carcinoma directly contributes to the progression of overt invasive cholangiocarcinomas. BilIN3 as well as BilIN1-2 is found in IgG4-SC as well as PSC, suggesting a risk for invasive cholangiocarcinoma progression in IgG4-SC. In the IgG4-SC case, moreover, the BilIN lesion expressed a mutated form of the p53 tumor suppressor protein [PSC and IgG4-SC target large bile ducts such as the hepatic hilar bile duct and extrahepatic bile ducts. Both diseases exhibit similar clinicopathological behaviors, that is, bile duct stenosis and biliary obstruction. PSC often develops into cholangiocarcinoma  , 45. The protein , suggestTwo mechanisms are possible regarding cooccurrence of cancer and autoimmunity: 1) sustained inflammation in the presence of an autoimmune disease is considered to create immunological environments favorable for cancer development and (2) cancers may induce autoimmune diseases as a paraneoplastic syndrome  sustaine, 29, andIgG4-SC complicated autoimmune pancreatitis can be differentiated by the same diagnostic criteria as autoimmune pancreatitis, such as serum IgG4 levels and lesion distribution. However, differentiating IgG4-SC cases without pancreatic and other organs involvement from conditions such as PSC and cholangiocarcinoma is challenging. Moreover, the mean IgG4 serum level is relatively lower in IgG4-SC cases without autoimmune pancreatitis than in cases with autoimmune pancreatitis. Furthermore, hilar hepatic lesions that resemble hepatic hilar cholangiocarcinoma frequently accompany IgG4-SC cases without autoimmune pancreatitis , 51. BilIn this review, we described the IgG4 reaction in cholangiocarcinoma. Correlations between IgG4 and malignant neoplasms are noted; however, their cause-and-effect relationship needs further clarification. Furthermore, a prospective study is needed to elucidate the role of IgG4-related diseases in carcinogenesis. At this moment, the most important procedure is ruling out cancer in the diagnosis of IgG4-SC. We, pathologists, have to know the IgG4 reaction in cholangiocarcinoma from the aspect of the presence of IgG4-positive cells, its density, and its distribution in order to avoid the rapid diagnosis that the presence of IgG4-positive cells in biliary mucosa is of help as evidence of IgG4-SC.


IgG4-related disease (IgG4-RD) is a recently recognized systemic inflammatory disorder that can affect most organs/tissues such as sarcoidosis. The kidney is a frequently affected organ with tubulointerstitial nephritis (TIN), the representative lesion of IgG4-RD. This review focuses on the latest knowledge of IgG4-related kidney disease (IgG4-RKD).A wide range of renal manifestations of IgG4-RD, that is TIN, membranous glomerulonephritis (MGN) and other glomerular lesions, and pyelitis, are collectively referred to as IgG4-RKD. Clinically, decreased renal function, or characteristic imaging findings such as multiple low-density lesions on contrast-enhanced computed tomography or diffuse thickening of the renal pelvic wall, are typical presenting features. Although a rapid response to corticosteroid therapy is a very important feature of IgG4-TIN, in cases in which renal function is moderately to severely decreased before therapy, only partial recovery of renal function is obtained.TIN with characteristic imaging findings is a typical manifestation of IgG4-RKD in the interstitium, while MGN is a representative manifestation of the glomerular lesions. Although IgG4 is a central feature of IgG4-RD, the recent discovery of IgG4-negative IgG4-RD raises questions about the causative role of the IgG4 molecule in this context. IgG4-related disease (IgG4-RD), a recently recognized systemic inflammatory disorder, generally presents as a mass-forming lesion, or lesions, or organ enlargement ,2. CliniThe kidney is a representative organ, and the various renal lesions are collectively referred to as IgG4-related kidney disease (IgG4-RKD) –18. TubuPatients with IgG4-RKD present at an average age of 65 years, and 73–87% are men –18. Two et al. followed serum complement levels in 14 patients with IgG4-RKD and found that three again showed a decrease at the time of relapse. Hyper IgG-aemia is also prominent in many cases, and serum IgG levels exceeded 3000 mg/dl in 50% of patients in our IgG4-RKD series .Elevated serum IgG4 levels are the most important serological finding in IgG4-RKD –3. Altho20Although neither proteinuria nor haematuria is usually detected in IgG4-TIN, many patients with coexistent glomerular lesions have proteinuria or haematuria, or both, and patients with membranous glomerulonephritis (MGN) may show even nephrotic levels of proteinuria. In contrast to drug-induced acute TIN, IgG4-TIN is usually not accompanied by urinary excretion of many white blood cells (WBCs) or WBC casts. This finding probably mirrors a very mild tubulitis, a histopathological feature of IgG4-TIN.Serum C-reactive protein levels are usually normal, with this being a useful marker to distinguish IgG4-RD from anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis or Castleman's disease.A distinguishing feature of IgG4-TIN is its characteristic imaging findings frequently observed on computed tomography (CT) ,22,23. CRecently, MRI has become a useful imaging method to detect IgG4-RKD from a very early stage ,25. A tyFDG-PET ,27 and gPlasma cell rich TIN with fibrosis and sometimes numerous infiltrating eosinophils are typical pathological findings of IgG4-RKD Fig.  4,30–32–3230–32.Similarly, Houghton and Troxell  reportedAnother important pathological feature is fibrosis –18,31,32The specific distribution of renal parenchyma lesions is another distinctive feature of this disease. First, the margin between affected and unaffected portions is very clearly demarcated, with this finding thought to correspond to the imaging feature of multiple low-density lesions ,18,32. SGranuloma formation and fibrinoid necrosis of the artery have never been reported in IgG4-RKD ,18, althet al..A good and rapid response to corticosteroid therapy is a very important feature of IgG4-RD ,2, and t60▪▪]. The reasons for this can be explained through longitudinal pathological and imaging studies.Although in the vast majority of IgG4-TIN cases prompt recovery of renal function is achieved within 1 month of corticosteroid administration, in cases in which the eGFR has already decreased to less than 60 before treatment, only partial recovery of renal function is obtained (mostly in the first month and plateauing thereafter) ▪▪. The rIn histopathological examination, a re-biopsy study revealed that localized severe fibrosis became obvious in some parts after long-term corticosteroid maintenance therapy, although only minor abnormalities were seen in other parts .▪▪]. In particular, some areas of the kidney showed complete recovery and disappearance of low-density lesions without atrophy even with long-term administration of the maintenance dose of steroid (Fig. In a longitudinal imaging study, contrast enhancement of the renal cortex recovered after therapy in almost all patients with multiple low-density lesions ▪▪. In paoid Fig. . In contoid Fig.  61]. Th. Th▪▪]. et al.[et al.[▪▪] recently showed that several single plasmablast-derived antibody clones established from patients with active IgG4-RD could react with autoantigens in the cytosole of Hep-2 cells. Therefore, further studies are needed to clarify whether disease-specific autoantibodies exist or not in IgG4-RD.There is controversy as to whether IgG4-RD is an autoimmune disease or not . IgG4-reet al., who proet al.. In contet al.. Next, met al.,63. Therl.[et al.▪▪ recentet al.[Recently, two cases of IgG4-negative IgG4-RD were reported ▪,67▪. Thet al. also shoper se plays a crucial role in inducing multiple systemic lesions or is only a bystander. Analysis of many more cases, including ones with IgG4-negative IgG4-RD, with longer-term follow-up will be needed to define more precisely the role played by IgG4 in this disease.TIN with characteristic imaging findings is a typical manifestation of IgG4-RD in the kidney. MGN can be a manifestation of IgG4-RD, although a variety of glomerular diseases are known to accompany IgG4-TIN. Although IgG4 is a key molecule and abundantly present in both the serum and tissues in this disease, it is unknown whether IgG4 We would like to thank Mr. John Gelblum for his critical reading of the manuscript.This work was supported by Health and Labour Sciences Research Grants for the Study of Intractable Diseases from the Ministry of Health, Labour and Welfare, Japan.None.▪ of special interest▪▪ of outstanding interestPapers of particular interest, published within the annual period of review, have been highlighted as:


Antibiotic use is a major driver for the emergence and spread of antibiotic resistance. Antimicrobial stewardship programmes aim to improve antibiotic prescribing with the objectives of optimizing clinical outcomes while at the same time minimizing unintended consequences such as adverse effects and the selection of antibiotic resistance. In 2013, a five-year national strategy for tackling antimicrobial resistance was published in the UK. The overarching goal of the strategy is to slow the development and spread of resistance and to this end it has three strategic aims, namely to improve knowledge and understanding of resistance, to conserve and steward the effectiveness of existing treatments and to stimulate the development of new antibiotics, diagnostics and novel therapies. This article reviews the antimicrobial stewardship activities included in the strategy and describes their implementation and evaluation. Staphylococcus aureus (S. aureus) or erythromycin resistance in Streptococcus pneumoniae from children), in other pathogens, particularly Gram-negative bacteria, resistance to key antibiotics is either rising or remains at unacceptably high levels [S. aureus (both methicillin-resistant and methicillin-susceptible) and Escherichia coli, and all cases of Clostridium difficile (C. difficile) infection [Antibiotic resistance poses a global threat to public health and clinical medicine. In the UK, surveillance of antibiotic resistance over several decades using a variety of data sources has provided insight into the prevalence of resistance in a range of pathogens. While in some instances resistance has declined in recent years  enfection ; (iv) upnfection ; and (v)nfection .The UK five-year national strategy for tackling antimicrobial resistance includes optimising antibiotic prescribing through stewardship , althougC. difficile) and the emergence and spread of resistance. Stewardship programmes should typically consider a range of issues including monitoring and evaluating antimicrobial prescribing and how this relates to local antimicrobial resistance patterns, together with the provision of regular feedback to prescribers. The National Institute for Health and Care Excellence, as the body responsible for providing evidence-based national guidance and advice to improve health and social care in England, recently issued guidance recommending the establishment of antimicrobial stewardship programmes [Although the epidemiology of antibiotic resistance is complex, it is recognized that use of antibiotics is a major driver for the emergence and spread of resistant bacteria. This has led to the development and implementation of antimicrobial stewardship programmes, which comprise a range of interventions to improve antibiotic use in order to optimize clinical outcomes, while at the same time minimizing unintended consequences including toxicity, the selection of certain pathogenic organisms  that provides healthcare free at the point of access, although medicines prescribed in primary care and hospital outpatient settings do have a fixed patient payment. Provision of medical care is overseen by NHS England and is organised through over 200 Clinical Commissioning Groups comprising groups of General Practices that work together to plan and design local health services . PrimaryWhile the data on temporal trends in antibiotic prescribing are informative, a major limitation is that information on the clinical indication for which antibiotics are prescribed is not currently available, making it unfeasible to determine from this dataset whether treatment guidelines are being followed. However, assessment of the quality of antibiotic prescribing in primary care (based on whether the antibiotic prescribed for a given indication is consistent with available guidelines) can be undertaken using additional datasets such as the Clinical Practice Research Datalink and the Health Improvement Network which collect electronic medical records (including diagnoses and prescriptions issued) from general practices covering approximately 6%–7% of the population. Two recent studies using the Clinical Practice Research Datalink and the Health Improvement Network, respectively, have independently shown that for many practices the level of antibiotic prescribing for upper respiratory tract infections is above what is clinically justified based on national guidance, with the latter study showing that current levels are greater than those seen before recommendations were made to reduce it ,18. Therhttp://www.imshealth.com/portal/site/imshealth). IMS Health reimburses hospitals for these data and provides data handling and analytical support. IMS Health now has an agreement that enables it to share hospital antibiotic prescribing data with ESPAUR as well as with the former Antimicrobial Stewardship Sub-Group of the Advisory Committee on Antimicrobial Resistance and Healthcare-Associated Infections [The routine collection of data on prescribing in hospitals in England has been challenging due to the majority of hospitals not having systems in place for electronic recording of patient-level prescribing records. However, English hospital pharmacies provide aggregate monthly data on all medicines issued to in-patients, wards and clinics to IMS Health, a company providing information and technology services to the healthcare industry   are responsible for providing prescribing advice to primary care prescribers, and this includes the development, implementation and audit of antimicrobial prescribing guidelines. National guidelines for the management of common infections in primary care that can be adopted for local use are published by Public Health England , but theTo support Clinical Commissioning Group-led stewardship activity, detailed primary care antibiotic prescribing data are available from both the NHS-Business Services Authority Information Services Portal , and theThe TARGET  toolkit is an on-line antimicrobial stewardship resource designed to help influence prescribers’ and patients’ personal attitudes, social norms and perceived barriers to optimal antibiotic prescribing ,29. BothInfection remains a significant cause of morbidity and mortality in critically ill patients. While the early recognition of infection and rapid initiation of broad-spectrum empirical antibiotic therapy is an essential component of the management of such patients, it is being increasingly emphasized that exposure of patients to antibiotics is a major driver for the emergence and spread of resistance and should be avoided where possible. Thus clinicians face the dilemma of trying to reconcile conflicting advice. To help address this issue, the Advisory Committee on Antimicrobial Resistance and Healthcare-Associated Infections has promulgated antimicrobial stewardship guidance for antibiotic prescribing in hospitals under the heading “Start Smart—Then Focus” ,31. ThisData for antibiotic consumption in hospitals and primary care in England was published for the first time in 2014, as part of the ESPAUR Report . The RepIn England, IMS Health and Rx-info are commercial organizations that specialize in the provision of information, services and technology for the healthcare industry including the NHS. The hospital prescribing data used in the ESPAUR report was provided by IMS Health. Whilst both IMS Health and Rx-info have in-house quality assurance processes, the datasets have not however been externally validated. In June 2015, Public Health England validated the antimicrobial usage data of acute care Trusts in England as part of a quality improvement programme. To encourage participation, there is a requirement for hospitals to undertake data validation as a component of the national antimicrobial Quality Premium  for Clinical Commissioning Groups .C. difficile.An evidence-based antimicrobial self-assessment toolkit (ASAT) for secondary care developed in collaboration with the Advisory Committee on Antimicrobial Resistance and Healthcare-Associated Infections and the Department of Health in 2012 has been updated and is now available as an on-line tool to enable individual secondary care organizations to benchmark themselves against the national average . The maiThe National Institute for Health and Care Excellence has recently published guidance on antimicrobial stewardship . The tarEducation and training is an important aspect of improving stewardship. A multi-professional group led by the Advisory Committee on Antimicrobial Resistance and Healthcare-Associated Infections and Public Health England published national antimicrobial prescribing and stewardship competencies in 2014 . The comPublic understanding of the issues around antimicrobial use and resistance is important for tackling antimicrobial resistance, as a patient’s expectations and demand can influence prescriber behaviour, particularly within primary and community care settings . The antIn light of the above, there is clearly an on-going need to increase awareness and knowledge among the public as to when antibiotics should or should not be taken. In 2015, Public Health England and the Department of Health published an analysis that identified key behaviours and drivers for antibiotic use in both the public and healthcare professionals ,38. SuchThe European Antibiotic Awareness Day organized by the European Centre for Disease Prevention and Control and held every year on 18 November is designed to encourage the responsible use of antibiotics . In the The next time I have a cold or flu I will talk to the pharmacist first about how I can treat my symptoms rather than making a general practitioner appointment”. At the time of writing, 13,772 individuals (including the three authors of this article) had signed up as Antibiotic Guardians. Pledges can be made on-line at http://antibioticguardian.com/.The importance of public engagement rather than just awareness is increasingly being recognized. As a result, in 2014, Public Health England extended the European Antibiotic Awareness Day campaign objective in the UK from raising awareness to supporting people to take concrete personal and collective action to use antibiotics prudently. This took the form of the Antibiotic Guardian campaign, initiated as part of a drive to change behaviour by encouraging the public and professionals to make on-line pledges about how they would make better use of antibiotics. Drawing on evidence from behavioural science, implementation intentions (or if-then plans) were used to develop pledges in relation to antibiotic use. An exemplar pledge for a member of the public is “The critical role of antimicrobial stewardship in tackling the problem of antibiotic resistance is reflected in its inclusion as a key action in the UK five-year antibiotic resistance strategy. Antimicrobial stewardship is also an integral part of national strategies formulated by other countries including Australia  and the


Mantle cell lymphoma (MCL) is a relatively uncommon type of non-Hodgkin lymphoma. It develops in the outer edge of a lymph node called the mantle zone. In contrast, IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) is characterized by elevated serum IgG4 and persistent bilateral enlargement of lacrimal glands (LGs) and salivary glands (SGs), with infiltration of IgG4-positive plasma cells. Recent studies indicated the importance of differentiation between IgG4-DS and malignant lymphoma.An 82-year-old man was suspected of IgG4-DS because of a high serum IgG level (2174 mg/dL) and bilateral swelling of LGs and SGs. Lip biopsy and fine needle biopsy of submandibular gland were performed, and subsequently, MCL was diagnosed through the histopathological findings.MCL most commonly occurs in the Waldeyer ring, but rarely in the stomach, spleen, skin, LG, and SG. We report an unusual case of MCL involving LGs and SGs mimicking IgG4-DS, which suggests that IgG4 testing may be useful in the differentiation of IgG4-DS in the presence of bilateral swelling of LGs or SGs. Mantle cell lymphoma (MCL) is a relatively rare type of non-Hodgkin lymphoma, accounting for ~3 % of malignant lymphoma in Japan. MCL typically occurs in middle-aged to older adults, with a marked male predilection, and has a poorer prognosis compared with other subtypes of non-Hodgkin lymphoma , 2. MCL IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS), also known as Mikulicz’s disease, is a unique condition characterized by enlargement of the lacrimal glands (LGs) and salivary glands (SGs) caused by infiltration of lymphocytes. IgG4-DS has been considered to be a subtype of Sjögren’s syndrome (SS) because of certain histopathological similarities, particularly lymphocytic infiltration . HoweverAn 82-year-old man was referred to our institution with bilateral swelling of the LGs and submandibular glands (SMGs) in November 2008. He had previously attended the ophthalmology department in our hospital with chief complaints of bilateral swelling of LGs, which were diagnosed as IgG4-DS by these clinical findings and high serum IgG (2174 mg/dL). LG biopsy was performed for definitive diagnosis and 2 days later he visited our institution on referral from our ophthalmology department for further evaluation of SMGs. He had several medical histories of colonic cancer, hypertension, and cerebral infarction.Physical findings showed no fever , blood pressure of 132/78 mmHg, no weight loss, and no nocturnal sweating. He was aware of dry mouth . Bilateral SMGs, sublingual glands (SLGs), and labial salivary glands (LSGs) were elastic, hard and swollen without pain Fig. . F-18 fl6/mm3, white blood cell count of 7150/mm3 , and platelet count of 179 × 103/mm3. His C-reactive protein level was 0.23 mg/dL. The serum soluble interluekin-2 receptor concentration was abnormally high (2251.4 U/mL). Immunological tests were negative for the anti-SS-A/B antibody and the anti-thyroid peroxidase antibody, but positive for rheumatoid factor. Serum levels of IgA, IgM, and IgE were within normal limits , but his serum IgG level was elevated (2174 mg/dL).He had a low hemoglobin level (13.4 g/dL), red blood cell count of 4.57 × 10IgG4-DS was suspected because of persistent symmetrical swelling of at least two pairs of LGs and major SGs for at least 3 months. We thus additionally examined serum IgG4 and it was in the normal range (43.7 mg/dL). We performed LSG biopsy and fine needle biopsy (FNB) of the left SMG in addition to LG biospy to check if LSG and SMG were the same disease. Histologically, all sections showed severe uniform infiltration of lymphoplasmacytes, without lymphoid follicular formation. The plasmacytoid cells showed nuclear pleomorphism. Immunohistochemical staining showed monotypic predominance of kappa-light chain and no infiltration of IgG4-positive plasma cells. The infiltrating lymphocytes were positive for B-cell markers (CD20 and CD79a), CD5, bcl-2, and cyclin D1, but negative for T-cell markers (CD3) and CD10  and chemotherapy . Although swelling of the LGs and SMGs diminished markedly, it became intractable to treatment at 1 year following therapy and finally terminated in patient death in November 2011.MCL is recognized as an aggressive lymphoma for which the median survival of patients is ~3 years . The cliImmunophenotypically, the neoplastic lymphocytes usually express CD5, CD20, CD43, and FMC-7, but not CD10 or Bcl-6 . The lymLarry et al.  reportedIgG4-DS is now recognized as a new emerging disorder, characterized by high serum IgG4, marked infiltration of IgG4-positive plasma cells, and severe fibrosis with hyperplastic ectopic germinal centers in LGs and SGs. We recently proposed “Comprehensive Diagnostic Criteria for IgG4-RD” . IgG4-RDThis study design was approved by the Ethics Committee of Kyushu University, Japan, and written informed consent was obtained from all of the patients and healthy controls .


The purpose of this study was to identify whether heroin relapse is associated with changes in the functional connectivity of the default mode network (DMN) during methadone maintenance treatment (MMT). Resting-state functional magnetic resonance imaging (fMRI) data of chronic heroin relapsers (HR)  and abstainers (HA)  were investigated with an independent component analysis to address the functional connectivity of their DMN. Group comparison was then performed between the relapsers and abstainers. Our study found that the left inferior temporal gyrus and the right superior occipital gyrus associated with DMN showed decreased functional connectivity in HR when compared with HA, while the left precuneus and the right middle cingulum had increased functional connectivity. Mean intensity signal, extracted from left inferior temporal gyrus of HR patients, showed a significant negative correlation corresponding to the degree of heroin relapse. These findings suggest that altered functional connectivity of DMN may contribute to the potential neurobiological mechanism(s) of heroin relapse and have a predictive value concerning heroin relapse under MMT. Among current health and social problems, substance abuse remains one of the most prominent, with stimulant use ranked the costliest and most dangerous4Previous studies have proved resting state functional connectivity (rs-FC) to be a useful tool in investigating brain networks through the analysis of synchronous low frequency blood oxygen level-dependent (BOLD) signal fluctuation6101112The use of ICA analysis of rs-FC was used previously in heroin relapse studies that sought to identify possible alteration of functional connectivity of the DMN: A set of brain regions that exhibit robust low frequency oscillations during the resting state, but deactivate during activityWhat current research efforts have not done, and what this study aims to do, is to compare the DMN of heroin relapse patients with that of heroin abstainers and to identify possible alterations which exist in the resting state functional connectivity of the DMN of a heroin relapser. The differences to be observed in the DMN between heroin relapsers and abstainers are supposed to give neurobiological explanation to the relapse condition, and can be referred to as evidence for predicting the treatment outcome. Basing on the findings of this study, an identification system, which can predict relapse, is proposed for heroin addiction patients. Accordingly, such a system would be helpful for better understanding the therapeutic effect of methadone treatment.Demographic information is presented in Corresponding ICA images were independently selected from the two groups according to the templates presented by GIFT. One-sample t-test result of DMN from HA and HR are shown in We used age and methadone dosage as covariates for the group comparison and the difference in results is shown in ROI signal abstracted from the left inferior temporal gyrus had a significant negative correlation with heroin relapse level, shown in The overarching goal of this study was to investigate the resting state functional connectivity in RSNs of patients who are susceptible to heroin relapse during MMT as compared to those who were able to sustain abstinence from heroin use. The main objective was to test the hypothesis that there exists significant relapse-related alteration of functional connectivity of the DMN between the two. Our study found that the left inferior temporal gyrus and right superior occipital gyrus were associated with a DMN decrease in functional connectivity for HR compared to HA. Additionally, the left precuneus and right middle cingulum show an increased functional connectivity in HR compared to HA. Intensity signals, extracted from the left inferior temporal gyrus of HR patients, showed a significant negative correlation with corresponding HRL.Based on the results obtained, it was concluded that the clusters, which exhibited between-group differences, disproportionately drive DMN functional connectivity in heroin relapse patients.As we hypothesized, significant differences in resting state functional connectivity were observed in the DMN of HR patients compared to HA. The DMN has proved pertinent to attention, self-monitoring and introspective thoughts and has a “sentinel” role within the resting state in which it enables the brain to broadly evaluate information from both external and internal environmentsThe left precuneus in DMN showed an increase connectivity in HR compared to HA in our study. The precuneus is implicated in memory retrieval, attentional tracking of stimuliThe cingulate region has been shown to play an important role in drug addiction. The literature comprehensively shows that its impairment remains as one of the most consistent findings in drug addiction disorders20212223242527Our results demonstrated a significant negative correlation between the signal intensity of left inferior temporal gyrus region and the HRL. This shows that heroin relapse reduces the resting state functional connectivity of the DMN by deactivating it as indicated in 30In our study, the minimum description lengths (MDL) criterion was used to estimate the number of extracted components from the data. Basing on these estimates, 30 components were extracted, among which DMN was divided into posterior and anterior 2 components. Within DMN, some brain regions behave differently than others. Some previous resting-state fMRI study processed similar decompositionThere were some limitations to our study, which should be carefully considered in the interpretation of our findings. Firstly, due to the difficulty in collecting a large subject cohort with completed monthly follow-up data  the sample size used in this study was inevitably small and could have limited the significance of the study. More importantly, the small subject number may affect the efficacy of ICA due to the dimensional unbalance between component numbers and sample size. Thus, an obvious way of improving future studies would be to increase the sample size. Secondly, we only used a 6-month follow-up result to differentiate between heroin abstainers and relapsers. Although we tried to control a series of confounding factors by matching them between groups and adding as nuisance covariates, there are still some factors which could not be fully covered in this study and may preclude a definitive conclusion. For example, the abstainers were six years older than the relapse patients, a fact that could allude to some correlation between age and drug relapse in general. More importantly, the duration between last heroin use and MR scan was not involved, and this may affect the calculation of the abstinence duration. Therefore, the conclusion as currently drawn should be interpreted with caution. Finally, the HRL that was used as an indicator of the likelihood of drug relapse is an arbitrary measure that is based on interviews with heroin abusers (and should therefore be treated as such). Future research should agree on an objective measurement for the quantification of HRL.In conclusion, this study demonstrated that heroin users with a risk of relapse possibly show different DMN functional connectivity patterns compared with abstainers, which may suggested a potential biomarker of heroin relapse. These findings suggest that altered functional connectivity of DMN may contribute to the potential neurobiology mechanisms of heroin relapse and perhaps have a predictive value for heroin relapse probability under MMT. Moreover, these results may be helpful to predict the outcome of addiction treatment and to understand its related therapeutic mechanism(s).The present study was approved by the Institutional Board of the Fourth Military Medical University, China, and conducted in accordance with the Declaration of Helsinki.The participants were comprised of 13 heroin abstinent patients  and 13 heroin relapse patients . All the patients undergoing MMT were recruited from the Xi’an BaQiao Methadone Substitution Treatment Center. Primary inclusion criteria for the subjects were as follows: (1) met the DSM-IV (American Psychiatric Association) diagnostic criteria for heroin addiction; (2) came to the center to seek medical help of their own volition; (3) were under MMT and received stable treatment for at least three months. Participants of all study groups were excluded if they had any neuropsychiatric disorder including organic brain syndrome and schizophrenia. Other exclusion criteria for all subjects were: (1) had a current medical illness; (2) had a history of head trauma; (3) showed any contraindication during MRI examination. The comorbidity rate of anxiety and depression in drug addiction patients is high and the Beck depression Inventory (BDI) and Hamilton Anxiety Scale (HAMA) were performed respectively for the purpose of evaluating what, if any, influence these would have upon the resting state functional connectivity analysis.All participants gave written informed consent before entering the study in accordance with the requirements of the Institutional Review Board of the Tangdu Hospital.A six month’s follow-up occurred after fMRI scanning to determine the baseline (which was defined at the time of this MRI scan). For each of the subjects, a structured interview along with a urine test occurred monthly. Participants were considered as relapse patients if they had used heroin  at anytime during the six months. In this paper, in order to evaluate the severity of relapse in HR patients, heroin relapse level (HRL) was proposed based on the relapse frequency during the follow-up period.All MRI scans were performed at the same baseline levels in the Imaging Center of the Tangdu Hospital, the Fourth Military Medical University. Participants were instructed to lie still with their eyes opened, remain awake and not to think anything specific for the resting state session. A routine structure T2WI image was conducted to exclude any gross cerebral abnormality. All MR data were acquired with 3.0 T GE Signa Excite HD whole-body MRI system. The functional component of the image was acquired by using a spin-echo EPI (echo planar imaging) with following parameters: number of volumes 150, number of slices 32, repetition time 2000ms, echo time 30 ms, flip angle 90°, slice thickness 4mm with no gap, field-of-view 256*256 mm, matrix = 64*64. The whole scanning for each participant lasted for about 8 minutes. Foam pads were used for fixing the position of the head and reducing any artifacts due to movement.Data Processing Assistant for Resting-State fMRI (DPARSF) within Matlab©® software was used in the data preprocessing of the functional images. The functional scans were realigned to the first image with the volume of 150, and head motion correction was processed to get the corresponding mean functional image. The images were normalized with the voxel size of 3 × 3 × 3 mm by using an EPI template and spatially smoothed with a FWHM of 8 mm.The Group ICA of fMRI Toolbox (GIFT), as implemented in Matlab, was used to abstract independent components by recognizing the blood oxygen level-dependent (BOLD) signal for all participants. The number of components, being thirty in this study, was estimated using a minimum description length (MDL) criteria, modified to take into account spatial correlation. Corresponding ICA images were independently selected from the two groups according to the templates presented by GIFT. DMN was divided into two parts of posterior and anterior DMN, and between-group comparisons were performed respectively.The Resting State fMRI Data Analysis Toolkit (REST) was used for the statistical analysis. Corresponding individual subject components, representing the network of interest from the ICA back-reconstructed processes were entered into REST to facilitate group analysis. To test for differences in network homogeneity, individual-level network homogenous maps were entered using one-sample t-tests. For each selected component, a gray mask (threshold 0.2) was used to confine the one-sample test analysis with gray matter. A union mask of the significant voxels  of the one-sample t-test results from each group was made in order to limit between-group comparison. The two sample t-test analysis took age and pre-baseline methadone dosage as covariates and the significance level was set at p < 0.05 (FDR corrected), minimal cluster size was set to 20 voxels.To analyze the relationship between the brain network and HRL, the mean signal intensity of the regions of interest (ROI) was extracted for each HR. Partial correlation analysis were processed to analyze the correlation between ROI-signal and HRL taking age and pre-baseline methadone dosage as covariates. The demographic and correlation analysis was performed by the Statistical Package for the Social Sciences version 13.0  software.How to cite this article: Li, W. et al. Dysfunctional Default Mode Network in Methadone Treated Patients Who Have a Higher Heroin Relapse Risk. Sci. Rep.5, 15181; doi: 10.1038/srep15181 (2015).


Inappropriate antibiotic use in normally self-limiting acute respiratory tract infections (RTIs), such as sore throat and the common cold, is a global problem and an important factor for increasing levels of antibiotic resistance. A new group of international experts—the Global Respiratory Infection Partnership (GRIP)—is committed to addressing this issue, with the interface between primary care practitioners and their patients as their core focus. To combat the overuse of antibiotics in the community, and facilitate a change from prescribing empiric antibiotic treatment towards cautious deferment combined with symptomatic relief, there is a need to introduce and enhance evidence-based dialogue between primary care practitioners and their patients. Communication with patients should focus on the de-medicalisation of self-limiting viral infections, which can be achieved via a coherent globally endorsed framework outlining the rationale for appropriate antibiotic use in acute RTIs in the context of antibiotic stewardship and conservancy. The planned framework is intended to be adaptable at a country level to reflect local behaviours, cultures and healthcare systems, and has the potential to serve as a model for change in other therapeutic areas. Enterobacteriaceae spp. and extended-spectrum beta-lactamase-producing Enterobacteriaceae [Staphylococcus aureus, Escherichia coli, Enterobacter and Acinetobacter species and Pseudomonas [Antibiotic resistance exerts a significant and pressing global healthcare challenge, with 25,000 people in Europe dying each year as a direct result of resistant infection . Using 2eriaceae ,4,5,6. Ceriaceae ,8. In Soudomonas . The proNeisseriagonorrhoeae), typhoid fever and Group B streptococcus [While antibiotic resistance has predominantly been a clinical problem in hospital settings, recent data show resistant organisms have also been detected in patients outside of this setting , with hitococcus . Communitococcus . Infectitococcus ,14,15. Enterobacteriaceae, such as Escherichia coli and Klebsiellapneumoniae [K. pneumoniae infections diminishing [Some recent emerging trends that are of growing importance should also be considered. One is the rise in prescribing for urinary tract infections. Even in countries such as the Netherlands, which has a low and stable antibiotic prescribing rate, a 32% increase in prescribing for these infections was noted between 2007–2011 . In otheeumoniae . This poinishing . In the context of few innovative or new antibiotics in the drug development pipeline, the World Health Organization (WHO) describes a future of a post-antibiotic world and warns that not only will this eliminate the advances in healthcare made over the past 100 years, that have ensured longer life in most parts of the developed and developing worlds, but it may also result in simple infections becoming unmanageable and potentially fatal ,20. The GRIP is a new multi-disciplinary group of international experts, comprising of primary care and hospital physicians, microbiologists, researchers and pharmacists. GRIP members were selected due to their existing interest and work in the field of antibiotic use and resistance. In particular, all members are focused on implementing change in antibiotic use and the treatment of non-serious infections, such as respiratory tract infections (RTIs), in primary care. The need for a multidisciplinary group of experts with broad reach was also considered when identifying candidates, to maximise the impact GRIP is intending to have as a campaigning force for change. GRIP is committed to address antibiotic overuse-related problems using evidence-based studies to support suitable rationale for antibiotic use and promote antibiotic stewardship among healthcare professionals. A framework is being developed for non-antibiotic treatment options for symptoms of acute RTIs.In this first GRIP opinion paper, the group aims to stimulate debate between healthcare professionals and highlight the need for an integrated approach to facilitating behavioural change among antibiotic prescribers and users.A pilot for the development of such an integrated approach should be a single infection, the treatment of which is currently characterised by the overuse of antibiotics in the primary care sector—an ineffective and unnecessary treatment option for the majority of patients—and where alternative symptomatic relief options are available. GRIP identified that the majority of patients who develop acute RTIs, such as sore throat and common colds (with the exception of the more severe infections such as pneumonia), can adopt this approach. Once a coherent international approach has been developed to promote a change in the prescribing and use of antibiotics for the treatment of RTIs, this could serve as a model for change in other infections.S. pyogenes reducing in parallel with a decrease in antibiotic consumption. Antibiotic resistance dropped from 9.2% in 1997 to 7.4% in 2000, with a statistically significant association between regional macrolide resistance and consumption rates [Data show a direct correlation between the use of antibiotics and resistance. Countries where there is higher consumption of antibiotics show higher resistance rates ,21. The on rates .E. coli and/or K. pneumoniae varied markedly between countries. Resistance of both were among the highest in Greece and Slovakia and the lowest in Sweden and Norway in 2011 [E. coli varied by a factor of 18 between Sweden (1.0%) and Greece (18.2%); for K. pneumoniae the differences were even more pronounced, ranging from 0.7% in Sweden to 64.1% in Greece [Taking Europe as an example, antibiotic usage varies widely, as do resistance rates. The most recent data from the European Antibiotic Surveillance Reports found that antibiotic resistance rates of  in 2011 . Rates on Greece .E. coli and Klebsiella spp., are highest in India and China at ≥80% and ≥60%, respectively [The impact of high antibiotic use on antibiotic resistance is significant . Global ectively . Rates wectively . Europeaectively .This will exert a major adverse effect on treatment options. A recent review describing patients with urinary tract and respiratory tract bacterial infections treated with antibiotics reported that individual resistance may persist for up to 12-months post-treatment, thereby creating situations for the need of second-line antibiotics . When anS. pneumoniae among the main pathogenic species. This organism also demonstrates increasing resistance to a variety of antibiotic agents [In developing countries, the impact of rapidly increasing resistance rates is of particular importance and concern. Respiratory disease has overtaken diarrhoea as the most frequent cause of child death in these countries,  with comc agents . Misconceptions and uncertainties regarding the role of antibiotics exist among patients and primary care physicians . For exaRTIs are the most commonly treated acute problem in primary care , with thDespite this, antibiotic use for RTIs in primary care remains high. In Europe, upper RTIs accounted for 57% of antibiotics used, with a further 30% for lower RTIs; in contrast the next most common condition was urinary tract infections at 7% . In BangThere also appears to be a dissonance between physician and patient expectations during a RTI consultation. Primary care physicians say they often feel under pressure by the patient to prescribe antibiotics . Some stImproved communication in primary care can help bridge this gap between physician and patient expectations. This can be achieved using various approaches. In Germany, training physicians in patient-centered communication resulted in a 40% reduction in antibiotic prescribing, with results lasting for over one year . In compThere are various national and global initiatives to try to counteract the threat of antibiotic resistance. In 2010, the Infectious Diseases Society of America (IDSA) started a new campaign, the “10×'20” initiative, which is a global commitment to support the development of ten novel, effective antibiotics by 2020 . An updaIn many countries there are education campaigns that aim to change healthcare professional and patient behaviour on antibiotic consumption. These include initiatives from the US Center for Disease Control and Prevention, first launched in 1995, and the European Centre for Disease Prevention and Control that has been running a European-wide antibiotic awareness day since 2008. In addition, many countries have run their own national awareness measures, such as Australia (‘Resistance fighters’), France , Germany (the DART—Deutsche Antibiotika-Resistenzstrategie) and Thailand .The impact of these campaigns was diverse. An analysis of 22 national or regional level campaigns in high-income countries did find a reduction in antibiotic use. As all but one campaign targeted the patient and healthcare professional simultaneously , it cannYet, despite an 18-year awareness campaign in the US, antibiotic overprescribing for paediatric patients remains high at 60%, and research on a UK awareness campaign also found little impact. Polish data suggest that a third of the population changes behaviour due to the European Antibiotic Awareness day; prescribing rates however remain high .It is unclear from current study data whether these effects are sustainable. In France, the awareness initiative reduced antibiotic prescribing by 26.5% in the first five years . A more Despite the range of campaigns and initiatives, educating healthcare professionals, and also training them how to educate patients on antibiotic use is essential. More could be done to optimise the interaction between patient and healthcare professionals during the consultation for an acute RTI. Numerous regional, national and international guidelines are in place outlining appropriate prescribing and many educational initiatives exist that focus on raising awareness of antibiotic resistance globally. GRIP believes that this is where the focus in countering antibiotic resistance should lie and calls for the development of an international framework for the non-antibiotic management of RTIs that specifically addresses the physician-patient interaction.In addition to setting out the rationale for appropriate antibiotic use in RTIs and outlining non-antibiotic management strategies, the framework should cover several key topics . The development of this framework should be cognisant of effective behavioural change initiatives from various countries and involve multiple stakeholders. The framework has to be robust enough to set a benchmark for care, while allowing adaptation at country level to reflect local realities. For example, in some countries the enforcement of legal or fiscal measures would be of great value to restrict inappropriate antibiotic sales through pharmacies and to incentivise primary care physicians to reduce prescribing.GRIP is working to further develop this framework, which will be supplemented by practical materials to educate primary care healthcare professionals, pharmacists and patients in changing behaviour and perceptions around antibiotic use in RTI treatment, and to encourage symptomatic relief. A key component of this will be guidance for healthcare professionals on having a constructive dialogue with patients that allows patients’ needs for symptom relief to be met without the need for an antibiotic prescription. Materials will be available through a GRIP website.A summit meeting is to be held in the summer of 2013 to convene antibiotic resistance expertise from around the globe in order to finalise the framework into one that facilitates prudent antibiotic use via targeted, nationwide actions. Representatives will span both the developing world  and developed countries . The core GRIP group also aims to include members from developing countries where antibiotic resistance poses particular problems, such as India and Thailand. Inappropriate antibiotic use in normally self-limiting RTIs is common in many countries and is contributing to the increase in antibiotic resistance. To reverse this tendency, a multi-faceted international, collaborative framework needs to be developed that facilitates behavioural change towards a non-antibiotic, patient-centered symptomatic management strategy in primary care. This framework should not only set out the rationale for why appropriate antibiotic use in RTIs is essential, but it should particularly outline how to enforce its implementation to change practice through improved dialogue between the healthcare provider and patient, as articulated in the framework outline. The framework should be adaptable at country level to reflect cultural sensitivities, differing healthcare provision systems and national guidelines, and could serve as a model for change in other therapeutic sectors where overuse of antibiotics in the primary care is of concern. This framework will be supplemented with practical materials that facilitate conversations between healthcare professionals and patients to promote appropriate antibiotic use.