Caveolin 1 (CAV1), a plasma membrane protein that modulates signal transduction molecules, is also overexpressed in HCC. The aim of this study was to investigate biological and clinical significance of co-expression and activation of c-Met and CAV1 in HCC. We showed that c-Met and CAV1 were co-localized in HCC cells and HGF treatment increased this association. HGF-triggered c-Met activation caused a concurrent rise in both phosphorylation and expression of CAV1. Ectopic expression of CAV1 accelerated c-Met signaling, resulted in enhanced migration, invasion, and branching-morphogenesis. Silencing of CAV1 downregulated c-Met signaling, and decreased migratory/invasive capability of cells and attenuated branching morphogenesis. In addition, activation and co-localization of c-Met and CAV1 were elevated during hepatocarcinogenesis. In conclusion reciprocal activating crosstalk between c-Met and CAV1 promoted oncogenic signaling of c-Met contributed to the initiation and progression of HCC. HCC is the sixth most common malignancy worldwide and the third most common cause of cancer related deaths c-Met was originally discovered as a proto-oncogene, expressed in epithelial cells and activated by its only known ligand, hepatocyte growth factor (HGF), which is secreted primarily by mesenchymal cells Experimental models of liver cancer and studies on human liver tissue samples have revealed that the outcome of HGF/c-Met activation would be both stimulation and inhibition of hepatocarcinogenesis Lipid rafts play an important role in signaling crosstalk via bringing different proteins into proximity and thus promoting interactions between receptors and intracellular signaling proteins The co-localization of CAV1 and c-Met was reported in osteosarcomas and in human embryonic kidney cells, however, there is no study evaluating CAV1 and c-Met interaction and its biological consequences in HCC. Furthermore, co-localization between phosphorylated c-Met and phosphorylated CAV1 has not been determined.In the present study, we report an association between CAV1 and c-Met modulated by HGF treatment, and the effects of this interaction on cellular motility, invasion and branching-morphogenesis. Additionally phosphorylated c-Met and phosphorylated CAV1 levels and their co-localization were determined in normal and cirrhotic liver and HCC tissues. These results add novel insights into the cooperativity of c-Met and CAV1 in HCC and the molecular mechanism behind the involvement of reciprocal activating crosstalk between c-Met and CAV1 in HCC progression.Human HCC cell lines HuH-7, and SNU-449, were cultivated as described HuH-7 cells were transfected with the plasmid pcDNA3.1/Myc-His (mock) and pcDNA3.1/-caveolin-1 (pCAV1). After transfection, cells were grown in selection medium containing 400 ug/mL geneticin (Life Technology 10131-027). Mock and CAV1 expressing cells both polyclonal and monoclonal were obtained and characterized by WB SNU-449 cells were transfected with 1 µM CAV1  (CAV1-siRNA) or non-targeting  (NT-siRNA) accell small interfering in Accell delivery media , according to the manufacturer's instructions. A NT-siRNA was used as a control. 72 hours post-transfection, the cells were used for protein studies, migration, invasion and branching-morphogenesis assays.In-vitro motility and invasion assays were performed as described previously Total cell lysates for IP and WB were prepared from SNU-449 cells with modified NP-40 buffer. Protein concentrations of samples were determined by the BCA assay following the manufacturer's instructions . Total cell lysates were used to analyze the interaction between c-Met and CAV1 in SNU-449 cells. Samples were incubated with agarose conjugated anti-c-Met (sc-161 AC) antibody and bound proteins were analyzed by WB.Total protein was prepared by using modified RIPA buffer as described previously 1 = pixelsCh1,coloc/pixelsCh1,total, where Ch1 and Ch2 are the first and second proteins to be detected. The given co-localization is expressed as the relative number of colocalizing pixels in channel 1 or channel 2, respectively, as compared to the total number of pixels above threshold. Threshold was set to 100 gray values. The entire TMA section was analyzed by acquisition of phospho-Met, phospho-CAV1 and DAPI signals by Immunofluorescence Microscopy using Olympus BX50 fluorescence microscope.Cells seeded on BD Falcon chamber slides  were grown for 24 hours. After overnight starvation, HuH-7-mock and HuH-7-pCAV1 were treated with HGF. Cells were washed three times with ice cold PBS, then fixed with a mixture of 50% acetone and 50% methanol. Fixed cells were then blocked with 1% Bovine Serum Albumine (BSA) in PBS with 0.1% Triton X-100 (PBS-T) at room temperature (RT) and were incubated with primary antibodies against phospho-Met (Y-1234/1235) (cs-3129), c-Met (sc-161), phospho-caveolin-1 (Y-14) BD611339, caveolin-1 (sc-894). Double immunofluorescence with phospho-Met and phospho-CAV1 was performed on a Tissue Microarray (TMA) (US Biomax (BC03117)). sections incorporating a series of 48 cases of HCC, 22 liver cirrhosis, and 5 normal hepatic tissues, together with pathology diagnosis, grade, stage, TNM, HBV, type and history of hepatitis (yr) data. After deparaffinization step antigen retrieval using proteinase K was performed. from After secondary fluorochrome-conjugated antibodies  treatment specimens were washed 6 times with PBS-T and slides were mounted with fluorescence mounting medium . Fluorophores from cell lines were visualized using Zeiss LSM 510 Meta confocal laser microscope  equipped with a Plan-Neofluar 40×/1.3NA oil objective at ambient temperature. Excitation and emission settings are as follows; for Alexa Flour 488: Ex: 488 nm/Em: 505–550 nm Band-pass filter; for Alexa Flour 555: Ex: 543 nm/Em: 560 nm Long-pass filter.DAPI was used to stain the cell nuclei. The detection parameters, such as laser intensity, amplifier offset and gain, and pinhole diameter, were fixed and kept at the same values for all specimens. Images were acquired using LSM510 ver.3.8 software and figures were generated using Adobe Photoshop. For co-localization analysis co-localization coefficients were calculated as cphospho-Met (Y1234/1235) and phospho-CAV1 (Y14) expression profiles were analyzed by using immunoperoxidase staining in the serial liver sections. Expression of phospho-CAV1 and phospho-Met were examined in normal liver (n = 11), cirrhotic liver (n = 42), and HCC (n = 60) tissue samples. The clinicopathological characteristics of HCC patients are shown in 2O2 for 4 minutes at 37°C. Endogenous avidin biotin activity was blocked by using the avidin-biotin block solution  and then primary antibodies anti-phospho-Met (Y-1234/1235) cs-3129, anti-phospho-Caveolin 1 (Y-14) BD-611339 were applied. The sections were stained with 3,3-diaminobenzidine tetrahydrocloride (DAB), a chromogen stain (brown in color), and counterstained with hematoxylin.Sections were deparaffinized and then rehydrated. Immunostaining was performed using an automated immunohistochemical stainer according to the manufacturer's guidelines . The antigen retriewal step was included in the automated programme. Endogenous peroxidase activity was blocked using 3% HAll staining were semi-quantitatively evaluated by certified pathologists  unaware of clinicopathological characteristics of the tumor. Expression of phospho-Met was defined as membranous and/or cytoplasmic when more than 10% of the hepatocytes stained positive for phospho-Met and phospho-CAV1. The extent of staining was scored as one positive (10 to 25% of cells were positively stained), two positive (26% to 50% of cells were positively stained) and three positive (more than 50% of the cells were positively stained). The intensity of phospho-Met and phospho-CAV1 immunostaining was semiquantitatively graded as follows: none (0), weak (+1), moderate (+2), and intense (+3).HuH-7-mock, HuH-7-pCAV1, NT-siRNA or CAV1- siRNA treated SNU-449 cells were embedded in three-dimensional collagen I gels (BD 354236) as previously described 2-test. Correlation between two groups was assessed by Pearson's correlation analysis. p<0.05 was considered statistically significant.Statistical analysis was performed using the GraphPad Prism and Statistical Package for Social Sciences 15.0 . Statistical methods included Analysis of variance (ANOVA), Student's t-test, χWe used SNU-449 cells that are known to express both c-Met and CAV1 endogenously, to test for an association between c-Met and CAV1. The effect of HGF stimulation on this putative interaction was also evaluated. As shown in HGF stimulation increased the phosphorylation of CAV1 on tyrosine-14 (pY-14) in a time dependent manner . WB analWe also evaluated the cellular distribution of phospho-Met and phospho-CAV1 in SNU-449 cells by CM . ConsistTo confirm CAV1 phosphorylation was mediated by c-Met activation, we used c-Met receptor kinase inhibitor, SU11274, to block c-Met activation and investigated the effect of c-Met inhibition on CAV1 phosphorylation. HGF-mediated CAV1 phosphorylation was decreased by SU11274 treatment, parallel to the decrease observed with c-Met phosphorylation . OvernigFindings obtained using SNU-449 cells suggested that c-Met positively regulated CAV1 activity. To examine the effect of CAV1 on the c-Met signaling pathway, we first introduced CAV1 gene into CAV1-negative and c-Met-positive cell lines, HuH-7 and Hep G2, by stable cloning to establish CAV1 overexpressing stable clones. Then, we treated HuH-7-pCAV1 and HuH-7-mock clones with HGF and subjected to WB analysis. We determined that HGF treatment increased CAV1 expression in CAV1 clones . Upon HGHGF-mediated c-Met activation generates a variety of cellular responses, including motility, invasion and branching morphogenesis. When we tested the role of CAV1 in HGF/c-Met induced motility and invasion we observed that HGF treatment caused approximately an 84-fold increase in the motility of HuH-7 mock cells compared to non-induced ones (p<0.0001) . RemarkaWe also tested the ability of these clones to undergo tubular morphogenesis in collagen gel. At basal conditions, mock-transfected cells formed mainly cysts, whereas cells expressing CAV1 formed branches and tubules. Representative clones are shown in Based on the observation that c-Met activation accelerated both the expression and phosphorylation of CAV1 protein, we hypothesized that the silencing of CAV1 had an effect on the c-Met transactivation pathway. To test this, we used siRNA to deplete CAV1 expression in SNU-449 cells and analyzed c-Met activation. We determined that siRNA treatment abolished both basal and HGF induced CAV1 phosphorylation . As showAs CAV1 knockdown suppressed the activation of c-Met signaling pathway, we next asked whether CAV1 silencing inhibits cell migration, invasion and branching morphogenesis in response to HGF. As shown in i) cirrhotic tissues staining with phospho-Met and phospho-CAV1; ii) HCC tissues staining with phospho-Met and phospho-CAV1 analysis revealed that phospho-CAV1 expression was correlated positively with phospho-Met in cirrhosis  and HCC . We next investigated the association between phospho-Met and phospho-CAV1 in normal, cirrhotic and HCC liver sections. Consistent with the data obtained from IHC, no phospho-Met and phospho-CAV1 expression were detected by IF microscopy (data not shown). Interestingly, while no co-localization was detected between phospho-CAV1 and phospho-Met . phospho-CAV1 positive HCC tissues were grouped as follows: 0/15 (0%) well-, 7/33 (21%) moderate-, and 5/16 (31%) poor-differentiated   . There wDespite extensive efforts by many researchers, treatment with several different agents for HCC has been ineffective. Current use of sorafenib, a multikinase inhibitor, in treatment of advanced HCC, have focused research efforts on several key molecular pathways implicated in the pathogenesis of HCC in vitro migration and invasion in vivo tumorigenicity and metastasis Others and we reported that the overexpression of c-Met in HCC is linked to an unfavorable clinicopathological status, including a low degree of differentiation, vascular invasion and metastasis Although there are many studies reporting the overexpression of either c-Met or CAV1 in HCC, the activation status of c-Met and CAV1 remains unclear. Our IHC analysis revealed a lack of c-Met and CAV1 activation in normal hepatocytes. However, activating phosphorylation levels of both CAV1 and c-Met as well as their co-localizations were increased in primary cirrhotic liver, and HCC tissues. To our knowledge, this is the first report that showed the activation levels and co-localization of c-Met and CAV1 in HCC tissues. Furthermore, increased activation of both phospho-Met and phospho-CAV1 in poorly-differentiated HCC tissues supported the role of this cooperation in the progression of HCC.Studies performed in different cancer models revealed that insulin treatment, c-Src kinase activation and integrin-mediated mechanotransduction cause tyrosine phosphorylation of CAV1 on tyrosine 14 (Y14), which appears to be essential for CAV1-driven cell migration It has been reported that, endocytic trafficking of c-Met induces sustained p44/42-MAPK activation and its translocation to focal adhesion site and increases cell migration We evaluated the impact of CAV1 on c-Met signaling and its functional impotance by examining c-Met driven cellular activities. In accordance with the higher degree of c-Met activation upon stimulation with HGF; higher migration and invasion rates were determined for CAV1-overexpressing cells under HGF stimulation. The overexpression of CAV1 drastically increased branching-morphogenesis. On the other hand CAV1 silencing impaired HGF-mediated branching-morphogenesis and reduced the migratory and invasive potential of SNU-449 cells. Although CAV1 has been reported to require for vascular morphogenesis Taken together in this study we described a reciprocal activating crosstalk between c-Met and CAV1 that modulates motility, invasion and branching-morphogenesis of HCC cells. These conclusions are supported by the following findings: i) c-Met co-localized with CAV1 and this association increased by HGF; ii) HGF induced the activation of c-Met and CAV1; iii) association of activated c-Met with CAV1 resulted in increased migration, invasion and branching morphogenesis; iv) co-existence and colocalization of activated c-Met and CAV1 in HCC tissues .Conclusively, our results suggest that the bidirectional interactions between c-Met and CAV1 reciprocally and positively regulate each other's activity and have a critical influence on the outcome of c-Met signaling pathway in HCC. We propose CAV1 as a potential factor that triggers c-Met signal transduction and contributes to the initiation and progression of HCC. Therefore, targeting the cross-talk between c-Met receptor tyrosine kinase and CAV1 might be an important approach for the treatment of HCC.Figure S1HGF induced association of CAV1 with c-Met.A. Serum starved Mahlavu cells were exposed to HGF for 15, 30 and 60 min. Whole cell lysates were immunoprecipitated with anti-c-Met antibody, resolved by SDS-PAGE and immunoblotted with antibody to CAV1 and c-Met. Anti-c-Met antibody was probed to the membrane as a loading control. There was no detectable c-Met and CAV1 band in immunoprecipitates prepared with IgG as an IP-control. B. Mahlavu cells were analyzed for c-Met phosphorylation and expression by WB analysis by using anti-phospho-Met and anti-c-Met antibodies with or without HGF stimulation. Immunoblot analysis showed a gradual increase in c-Met phosphorylation in response to 15, 30, 60 min HGF induction. The expression level of CAV1 was also determined by WB under the described conditions. Calnexin was used as a loading control.(TIF)Click here for additional data file.Figure S2HGF mediated c-Met and p44/42-MAPK activation. WB showing the activation of the indicated proteins in HuH-7 and HuH-7-pCAV1 cells, overnight starved and treated with or without HGF at different time points. Calnexin was used as a loading control.(TIF)Click here for additional data file.Figure S3Immunofluorescence analysis of co-localization between c-Met and CAV1. IF microscopy acquisition of three fluorescent signals: c-Met (green Alexa 488) , CAV1 (red Alexa 555)  and DAPI (blue nuclear staining). Co-localization of c-Met and CAV1 was shown in yellow  (Bar = 200 µm).(TIF)Click here for additional data file.Table S1Clinicopathological characteristics and phospho-Met and phospho-CAV1 immunohistochemical staining of tumors from primary HCC patients.(DOCX)Click here for additional data file.


Medical emergency team (MET) calls are quickly becoming an integral part of the response to a deteriorating patient in Australia. Conceptually the MET call response incorporates a structured approach, but in practice this can quickly disintegrate. This collapse of method can leave patients without clear treatment plans and staff disenfranchised. We sought to improve the process of the MET call response at our regional hospital by introducing targeted interventions focused on teamwork, communication, leadership and role allocation.We invited junior doctors and nurses to complete a survey designed by a multidisciplinary MET Call Working Group; 138 staff (40% of population) completed the survey. Based on analysis of responses, a focused three-pronged intervention was formulated and implemented hospital wide. The arms of the intervention were: identification of the name and role of each staff member using highly visible labels; role allocation according to policy written through a multidisciplinary working group; and a time out during the response allowing a structured synopsis of the patient's current status to be communicated to the team. The intervention was preceded by extensive staff education, and 175 staff (50%) completed the survey 6 months later to assess its success.The intervention significantly increased satisfaction amongst staff regarding: identification of the team leader and other key staff members at the response; and time out effectiveness in reducing repetition and improving staff understanding of the patient's status and medical issues. We found no significant change in staff perceptions regarding the clarity of the ongoing treatment plan at the end of the MET call response.Utilising a low-cost intervention in a regional setting, we were successful in improving staff perceptions of role allocation and communication within our MET call responses. The intervention also led to significantly increased overall satisfaction with the MET call system. Through our surveys we have identified other facets of the MET call response that also require attention. Given our encouragingwe are designing a follow-up intervention incorporating structured multidisciplinary training in MET call scenarios.


Physcomitrella patens, in which they act as signaling factors for controlling filament extension and possibly interaction with neighboring individuals. To gain a better understanding of SL action at the cellular level, we investigated the effect of exogenously added molecules  in moss growth media. We used the previously characterized Ppccd8 mutant that is deficient in SL synthesis and showed that SLs affect moss protonema extension by reducing caulonema cell elongation and mainly cell division rate, both in light and dark conditions. Based on this effect, we set up bioassays to examine chemical structure requirements for SL activity in moss. The results suggest that compounds GR24, GR5, and 5-deoxystrigol are active in moss (as in pea), while other analogs that are highly active in the control of pea branching show little activity in moss. Interestingly, the karrikinolide KAR1, which shares molecular features with SLs, did not have any effect on filament growth, even though the moss genome contains several genes homologous to KAI2 (encoding the KAR1 receptor) and no canonical homologue to D14 (encoding the SL receptor). Further studies should investigate whether SL signaling pathways have been conserved during land plant evolution.In vascular plants, strigolactones (SLs) are known for their hormonal role and for their role as signal molecules in the rhizosphere. SLs are also produced by the moss  Physcomitrella patens is a bryophyte from the moss lineage that is now a widespread plant model for studying evolution of plant development mechanisms and diversification of plant architecture P. patens has developmental mechanisms that are very similar to those of vascular plants, with hormones playing central roles as growth regulators Extant bryophytes are considered as descendants of the first plants that colonized land ca 460–470 million years ago. They were able to sustain growth and reproduction in an aerial environment due to their evolutionarily innovative features that could anchor the plant to the soil P. patens, haploid spores germinate and the first cells divide, producing chloronema filaments that very rapidly differentiate into a second type of filament, the caulonema. Chloronema filaments, rich in chloroplasts, only grow in the light. In contrast, caulonema filaments contain fewer chloroplasts, show faster apical cell division and ensure filament extension and colonization of the soil, in the light and in the dark In P. patens produces strigolactones (SLs) CCD7 and CCD8DWARF27 (D27) gene that encodes an isomerase responsible for the first step of trans-β-carotene isomerization Mosses possess most genes encoding the key enzymes of SL biosynthesis, namely both carotenoid cleavage dioxygenase (CCD) genes CCD8 gene, established that SLs regulate P. patens protonema (caulonema and chloronema) branching, and control plant size as quorum-sensing like molecules very likely by controlling caulonema radial extension PpCCD7 transcripts are upregulated in the SL-deficient Ppccd8 mutant and SL application decreased PpCCD7 transcript levels A study of a knock-out mutant for the Arabidopsis root hairs) or as a stimulant of parasitic plant seed germination or AM hyphal branching 23, 3′-methyl-GR24) are very active on pea buds but are poorly recognized by parasitic plant seeds, opening the possibility for the use of SLs in agronomy Exploring the links between the chemical structure of SL molecules and their activity on moss filament cells is useful for determining structural requirements for bioactivity. Comparison of those requirements with regard to hormonal bioactivity in vascular plants and non-vascular plants and with regard to other functions of SL in the rhizosphere may give indications on SL reception in the different systems. To date the SL-receptor has been identified only for the hormonal function in vascular plants Ppccd8 mutant P. patens compared to those required for hormonal activity in vascular plants, or for activity in the rhizosphere as part of parasitic and symbiotic relationships We investigated the cellular effects of SLs on moss in the light and in the dark. Dark-grown moss filaments show negative gravitropism P. patens protonema in the light, we compared the cell length of chloronema and caulonema filaments from WT and SL-deficient Ppccd8 mutants in light conditions. Given that the transition from chloronema to caulonema is progressive, chloronema cell length was first quantified on 7-day-old WT and Ppccd8 protonemata, before the initiation of caulonema cells. This seven-day growth period helped avoid confusion between chloronema cells and the first initiated caulonema cells. There were no statistical differences between WT and Ppccd8 in chloronema cell length , with a size similar to that of the WT  and a molecular assay on the expression of the Ppccd8 mutant at 0.1 µM, and in the WT and the Ppccd8 mutants at 1 µM . The same molecule was tested again at 1 µM along with SL analogs GR24, 23 (with two methyl groups on the D ring) and 31 (a thia-3′-methyl-debranone-like molecule) and two natural SLs. All but two tested molecules significantly decreased caulonema length  , but not strigol  . The GR5 strigol .KAI2 (encoding the KAR1 receptor) and no canonical homologue to D14 , it is tempting to presume an effect of the karrikins on moss development. The activity of KAR1 .As the moss genome contains several genes homologous to  of KAR1 , the firnot KAR1 . Because1 did not have any effect in the dark, a higher concentration of KAR1 was used for the light assay (5 µM instead of 1 µM). Eighteen-day-old individuals grown from spores of WT and Ppccd8 mutant were transferred to media containing KAR1 or GR24 and grown on in the light. Plant diameter was measured after 12 and 20 days .Given that KAR 20 days . After 1PpCCD7 transcripts in plants after the addition of SL. We have previously shown PpCCD7 gene expression in the Ppccd8 mutant, and thus restores the feedback control on SL synthesis genes observed in the WT.Because these bioassays on moss last several days, the stability of SLs may be an important factor of their activity. Furthermore, SLs are known for their instability in aqueous media PpCCD7 transcripts were measured 24 h after the addition of SLs (at 500 nM). The lack of feedback control in the Ppccd8 mutant led to a relative PpCCD7 transcript level almost twice as high as that in the WT , which was comparable to that of WT. Addition of 7-oxoorobanchyl acetate also showed a significant effect on PpCCD7 transcript levels . However, fabacyl acetate, orobanchyl acetate and strigol, all of which are natural SLs found in moss exudates 23, together with 5-deoxystrigol  were added at 1 µM to the medium, and the PpCCD7 transcript level was measured 6 h after application . In the third experiment, the activity of three natural molecules  was tested as early as 2 h following application of 100 nM SL , although the addition of 5-deoxystrigol also seemed to decrease PpCCD7 expression compared to the acetone-treated control.In the first experiment, the levels of n the WT . Additiolication . In thisn the WT . All fou00 nM SL . In thesPpCCD7 transcript level after transfer to SL-containing medium in the dark. Similar to results in light-grown caulonemata, the feedback control on PpCCD7 transcript levels was observed in the Ppccd8 mutant 6 h after the addition of 1 µM GR24 in the dark  had a significant effect on the length of caulonema cells, suggesting that GR24 has a stronger effect on cell division than on cell length (see below).When grown in the light, WT P. patens caulonemata, addition of GR24 promotes root-hair elongation in the vascular plant ArabidopsisP. patens than in Ppccd8 mutants, and the addition of GR24 (10 nM) restores the mutant phenotype, and also positively affects WT rhizoid length Contrary to what is observed on Arabidopsis, lateral root formation is enhanced in SL synthesis mutants, and to a greater extent in the SL perception mutant max2Arabidopsis SL synthesis mutants. However, SL effects on lateral root density depends on the nutrient  status of the plant, and contradictory observations have been reported under Pi-limited conditions 2PO4), and SLs inhibited caulonema growth and density, similar to the inhibitory effect observed on lateral roots in vascular plants. Hence, in moss as in vascular plants, SLs may enhance and inhibit organ size and number, depending upon the organ (rhizoid or caulonema).In moss, caulonemata are also involved in soil/medium exploration, a function that is fulfilled by roots in vascular plants. In Ppccd8 mutant compared to the WT Ppccd8 mutant. This effect of SLs on cell division rather than cell elongation was already suggested by the higher number of cells per chloronema filament at different times after germination, in Ppccd8 in comparison to the WTOur results on moss indicate that, at the cellular level, the caulonema cell division rate is inhibited by exogenously supplied SLs, in contrast to cell length that is only slightly affected. The higher branching rate of protonemata observed in the Arabidopsis, SL synthesis mutants show reduced cambium activity and local treatment of stems with GR24 induces cambium-like cell proliferation Arabidopsis and rice, both SL synthesis and perception mutants show a fewer root meristem cells compared to the WT Arabidopsis, the addition of GR24 in the growth medium restores the number of root meristem cells in SL-deficient plants In vascular plants, SLs have been reported to inhibit or enhance cell division, depending on the species and the tissue in question. In rice, on WT and SL synthesis mutant plants grown in the dark, addition of GR24 negatively regulates mesocotyl cell division but has no effect on cell elongation P. patens: one based on caulonema growth in dark conditions and one using the feedback control of SL on transcript levels of the biosynthesis gene PpCCD7. This feedback control is also observed in the dark, using synthetic GR24 or natural strigolactones (or their derivatives) exuded by the WT moss. Using these bioassays, the bioactivity of five natural SLs and four synthetic analogs were tested in moss to compare with SAR studies performed in pea with regard to branching inhibition We developed two relatively simple bioassays to test the activity of SL analogs in In pea, acetate-SLs are always more active than their corresponding hydroxyl-SLs. Fabacyl-acetate, orobanchyl acetate or solanacyl acetate are particularly active, occasionally even at a concentration of 10 nM P. patens. Therefore, ABC rings are not required for SL activity in moss, in contrast to SL structural requirements for AM fungi 23 and 3131 has been shown to be highly active MAX1 gene encoding a cytochrome P450 enzyme involved in the last steps of SL synthesis in vascular plants P. patensThe CD-rings analog GR5, which shows a similar level of activity as GR24 in pea, also showed activity comparable to GR24 in 1 and the synthetic SL GR24 both promote seed germination and inhibit hypocotyl elongation in Arabidopsis, and the MAX2 F-box protein is needed for these effects during Arabidopsis seedling development 1 needs MAX2 and AtD14-like/KARRIKIN INSENSITIVE 2 (KAI2) 1 was observed in our bioassay based on caulonema growth in the light and in the dark, despite the fact that there is one homolog of MAX2 as well as several homologs of AtD14-like/KAI2 genes in the genome of P. patensP. patens, the function of these genes is still unknown, in particular whether the PpMAX2 gene is indeed involved in the SL signaling pathway, together with one of the moss KAI2 homologs.The karrikinolide KARP. patens SL response mutant is needed. This kind of mutant is expected to show a phenotype similar to that of the Ppccd8 SL synthesis mutant, but the phenotype should not be affected by the addition of GR24. A response mutant would also allow the cloning of the gene(s) encoding the receptor(s) and thus represent an important step towards a better understanding of the SL signaling pathway in moss. SLs have been shown to stimulate rhizoid elongation in P. patensBecause SLs in moss inhibit caulonema growth, to distinguish between SL activity and toxicity, a Ppccd8 mutant 3 medium 3 medium supplemented with 2.7 mM NH4-tartrate for propagation. Plants were cultivated either in 9 cm round (for the light experiments) or 12 cm square (for the dark experiments) Petri dishes, on medium solidified with 0.7% agar  and overlaid with cellophane . For dark conditions, 0.5% glucose was added to the medium, 1% Phytoblend agar  was used, and Petri dishes were positioned vertically for better observation of caulonema growth. For the light experiments, cultures were placed in growth chambers set at 60% humidity, and with 16 h of light (quantum irradiance of 80 µE m−2 s−1) at 24°C and 8 hours of dark at 22°C.The Gransden WT strain 3 medium 1 was generously supplied by Gavin Flematti . SLs, SL analogs and KAR1 were dissolved in acetone and added to the liquid medium cooled to 45°C. Control treatments consisted in adding an equivalent amount of acetone. The Petri dishes were examined under a Leica MZ6 stereo microscope and images of caulonemata were captured by a Nikon CoolPix 4500 camera. Cell images were either captured directly under the microscope (Leitz), or following staining of the filaments with propidium iodide (10 µg/mL for 10 min) prior to transferring them to water on a slide for better visibility of the cross walls of the cells. Imaging of propidium iodide-stained tissue was performed with a Zeiss LSM 710 confocal microscope. The excitation wavelengths were 488 and 561 nm, and emission was collected at 565 to 720 nm. Chloroplasts were also visible, clearly distinguishing the chloronema from the caulonema cells. Measurements were performed with ImageJ software (http://rsbweb.nih.gov/ij/).Moss plants were either grown from spores, directly plated out on cellophane and grown for 2 weeks in light, or grown from fragmented protonema, using 7-day-old cultures. For dark-grown cultures, a horizontal band of fragmented moss was deposited on the cellophane and first grown in the light. After 8–10 days, each cellophane was transferred to square Petri dishes filled with PP-NOPpCCD7 gene expression was quantified using real-time PCR as described in PpAPT, which encodes ADENOSINE PHOSPHORIBOSYL TRANSFERASE was used as the constitutive gene.Tukey's multiple comparison ANOVAs were generally performed for statistical analyses using R Commander version 1.7–3 inf to LCAUsup for caulonema length and LCELLinf to LCELLsup for cell length, the minimum value for cell number was estimated by the ratio of LCAUinf to LCELLsup and the maximum value by the ratio of LCAUsup to LCELLinf.The mean number of caulonema cells produced in the dark experiments was estimated by the ratio of the mean caulonema length to the mean of cell length. To estimate the range of possible variation in the number of cells, the minimum and maximum values were estimated from the 95% confidence intervals of caulonema length (LCAU) and caulonema cell length (LCELL). Supposing that these confidence intervals range from LCAUFigure S1Effect of various strigolactones and analogues on relative PpCCD7 expression 2 h after application of 100 nM SL in WT and SL-deficient (Ppccd8) mutant plants in the light. Data are means ± SE . Asterisks denote significant differences from Ppccd8 treated with acetone .(TIF)Click here for additional data file.Figure S2Relative PpCCD7 expression of Ppccd8 mutant grown in the light (top) or in the dark (bottom), 6 h after addition of 1 µM GR24 (control: acetone) or following transfer to medium on which WT or Ppccd8 mutant plants (fragmented protonema using 7-day-old culture) had grown for 20 days. Plants from three Petri dishes were used for each condition. A biological replication of the experiment gave similar results.(TIF)Click here for additional data file.


The inappropriate use of antibiotics in the community is one of the major causes of antimicrobial resistance. This study aimed to explore the physician prescribing pattern of antibiotics for acute respiratory infections (ARIs) and to explore the knowledge, attitudes, and practices of patients regarding antibiotic use for ARIs. The study was conducted in Upper Egypt and used quantitative and qualitative research techniques. Eligible patients exiting outpatient clinics with ARIs were invited to participate in the study. A qualitative study was conducted through 20 focus group discussions. Out of 350 encounters for patients with various ARIs, 292 (83%) had been prescribed at least one antibiotic. Factors significantly associated with antibiotic prescribing for adults included patient preference that an antibiotic be prescribed. For children younger than 18, presentation with fever, cough, loss of appetite, and sore throat, along with the caregiver’s antibiotic preference, were associated with an antibiotic prescription. Several misconceptions regarding antibiotic use among community members were stated, such as the strong belief of the curing and prophylactic power of antibiotics for the common cold. Interventions to promote proper antibiotic use for ARIs need to be piloted, targeting both physicians and the public. Educational programs for physicians and campaigns to raise public awareness regarding proper antibiotic use for ARIs need to be developed. The inappropriate use of antibiotics is a worldwide public health problem contributing to the emergence of antimicrobial-resistant pathogens . The resThe objectives of the study were to examine physician antibiotic prescribing practices for acute respiratory tract infections (ARIs) in outpatient clinics of hospitals and primary care units. We also aimed to explore the knowledge, attitudes, and practices of patients regarding antibiotic use for ARIs, and to identify cultural and societal determinants contributing to the use of antibiotics in Egypt.The study was conducted from May–July 2011 in Minya district, Upper Egypt. Minya district  is the capital of Minya Governorate, located 280 km south of Cairo. The majority of the population lives in rural areas where agriculture is the predominant economic activity. Minya district has a network of government health care facilities composed of tertiary-care hospitals, district hospitals, rural hospitals, and primary health care units. Minya was chosen by the Ministry of Health and Population to conduct baseline studies exploring the extent of antibiotic use in Egypt and evaluate the impact of a pilot intervention to reduce unnecessary prescription and consumption of antibiotics.A survey was conducted at acute care hospitals and primary healthcare centers. Patients exiting outpatient clinics for internal medicine, pediatrics, and chest medicine; Ear, Nose, and Throat clinics; or general outpatient clinics in primary healthcare centers were screened through eligibility criteria for inclusion in the study. Screening was done by reviewing each patient’s prescription; patients with a physician diagnosis of common cold, otitis media, sinusitis, bronchitis, pharyngitis, influenza, or pneumonia were invited to be interviewed after consent. Adults were interviewed; children less than 18 years of age were represented by a parent or caretaker. Eight trained field investigators conducted a full-day visit to each of the selected healthcare facilities to interview all eligible patients during the day. A standardized data collection form was used for interviewing. Information collected included demographic characteristics , presenting symptoms, and characteristics of the current illness. Type and dosage of antibiotics prescribed were recorded on the questionnaire from the patient encounter. Additional information collected included patient knowledge, attitudes, and beliefs regarding antibiotic use.A sample size was estimated to allow for a subsequent study evaluating the impact of an educational intervention to reduce antibiotic use. The sample size was determined based on the predicted change in the prescribing behaviors of physicians as a result of the intervention. Assuming a change in prescribing behavior of 20%, a physician prescribing rate of antibiotics of 60% for acute respiratory infections, a significance level of 95%  and a power (Beta) of 90%, a total of 231 patients was required. A comprehensive list of governmental healthcare facilities that provide care for outpatients with respiratory diseases was provided by Minya Health Directorate. All four available acute care hospitals in Minya district were visited for one full working day. In addition, out of 42 primary healthcare units available in Minya, 50% were selected randomly and visited for patient exit interviews.p < 0.05.Data were analyzed using the Statistical Package for the Social Sciences (SPSS) . DescripThe qualitative study was undertaken in the village of Nazlet El Fellaheen . A total of 20 focus group discussions (FGDs) including 160 participants were conducted, equally divided among groups of women or men with higher or lower education levels. A less educated respondent was defined as a person with fewer than six years of schooling. A more educated respondent was defined as a person having either a diploma or university degree (12–16 years of education). Field workers experienced in qualitative research tools facilitated the focus group discussions after extensive training on the study objectives. The interviews were tape recorded, transcribed, and translated from Arabic to English. A qualitative research consultant did the data analysis of the transcribed text based on thematic analysis, which involved searching for meaningful segments of text and organizing them into categories based on emerging themes related to antibiotic use. The coded data was retrieved from the transcripts and placed in charts with quotes that allowed comparison across respondents within the target groups and across the groups. In the final stage, the data set was interpreted as a whole to answer the research questions .During the study period, a total of 387 patients were eligible for inclusion in the study; 350 (90%) consented to participate. A total of 64.8% (227/350) of the respondents were caregivers of children under 18. For adult patients, the median age was 37 years , and about 60% were female. Half of the adults (47%) did not receive any formal education. Thirty percent of adults had history of chronic conditions, mostly diabetes mellitus, hypertension, and liver and kidney diseases. The median age for children under 18 was 3.5 years , and 12.8% had chronic diseases . The description of the cohort of children under 18 and adults is presented in Out of 350 encounters for patients with ARIs, 292 (83.4%) received at least one antibiotic (82% of children and 85% of adults). The antibiotic prescription rates for the various diagnostic categories for children and adults are described in Several factors were significantly associated with high physician antibiotic prescribing rates on bivariate analysis for patients under 18 as well as adult patients . For chiA majority of respondents (54% of caregivers and 62% of adult patients) believed that overuse of antibiotics is a major problem. More than 50% of survey respondents also believed that too many people are treated with antibiotics when not necessary, and almost 90% of both groups (caregivers and adult patients) believed that physicians should not prescribe antibiotics when not indicated. About 50% of the cohort agreed that treatment with antibiotics is necessary when nasal discharge turns from yellow to green. Many caregivers (45%) and adult patients (35%) believed that antibiotics can be used to prevent the common cold. A total of 61% of caregivers and 44% of adult patients believed that antibiotics help cold symptoms clear up quickly.The practice of persons treating themselves with antibiotics for ARIs was very common and was mentioned by almost all participants of the focus groups. They had purchased antibiotics based either on the advice of pharmacists or that of neighbors or family. The results of the study identified four prevailing cultural beliefs influencing the use of antibiotics. These included beliefs regarding the effectiveness of antibiotics, perceived factors associated with effectiveness, the use of antibiotics as preventive medicine, and fear regarding overuse of antibiotics. Themes regarding beliefs and perceptions regarding antibiotic use did not appear to differ between higher and lower educated groups or between genders.powerful and magic medicines” that lead to rapid cure. They believed that antibiotics were the only tools to cure most infectious diseases and acute illnesses. Some respondents also believed that treatment with antibiotics leads to better health of the patient.“Nothing works like antibiotics. They have a great effect on any disease or illness.”Respondents expressed a strong belief in the curing power of antibiotics, describing antibiotics as “Cheap antibiotics have little effect on cure.”Respondents believed that the dose, route of administration and cost of the antibiotic were associated with the effectiveness and rapidity of cure. Injectable antibiotics were preferred over oral antibiotics, because they were considered to act more rapidly and effectively to cure infection as they flow directly in the bloodstream. Respondents favored more costly antibiotics, stating that a higher price indicated good quality medicines that should be used for severe illnesses or after surgery. “We don’t wait; we don’t risk getting sicker; we take antibiotics whenever we have any illness symptoms so microbes stay away.”“If I feel tired after returning from work in the field, I may just take an antibiotic to make sure that I won’t get sick and will be able to work the next day.”“Many respondents believed that antibiotics can prevent illnesses, and therefore they reported initiating antibiotic treatment early after the onset of minor symptoms such as cough, especially among children. Respondents mentioned that antibiotics are usually kept at home to be used immediately for any sudden onset of symptoms of respiratory infection.There is a possibility that antibiotics don’t work if we use them too much. I prefer taking one injection of antibiotics whenever I need it. If I feel better, I will not go for a second one. It is better to use them moderately.”“Respondents believed that the overuse of antibiotics could negatively impact their health by reducing their immune status or making them resistant to the antibiotic treatment for future infections. This argument indicated a preference for use of short courses of antibiotics, or stopping the antibiotic once symptoms started to resolve. Respondents also believed that using prophylactic single shots of antibiotics on a weekly or monthly basis was a better option compared to a long course of antibiotics, often prescribed by physicians for treatment of respiratory infections.This is one of the first studies from a developing country in the Eastern Mediterranean Region (EMR) describing the prescribing practices of physicians and the perception of community members regarding antibiotic use for ARIs. The study findings revealed that physician prescriptions of antibiotics for ARIs were extremely high in Minya, Egypt, ranging from 53% for the common cold to greater than 95% for pharyngitis, tonsillitis, and ear infections. The vast majority of these infections are caused by viruses and do not require antibiotic treatment . These fThe data in this report confirm the findings of other studies demonstrating that antibiotic prescribing for the common cold is high. The high prescription rates of antibiotics for the common cold (53% for children under 18 and 63% for adults) were comparable to those from a study from Jordan, where more than 51% of patients with the common cold received a physician’s prescription including antibiotics . As limiThe tendency to prescribe antibiotics for the common cold in Egypt may have similar drivers that lead to overprescribing in high-income countries, including patient demand, diagnostic uncertainty, and limited time . PhysiciIn a study conducted in the same district in Egypt, younger physicians were more likely to prescribe antibiotics for the common cold, in order to satisfy their patients and build a good reputation .Antibiotic treatment of pneumonia was lower than expected (89% for children and 70% for adult patients). This is likely explained by the study sites and population; government facility pharmacies provide inexpensive, older antibiotics which physicians perceive to be less effective than newer antibiotics. It is likely that patients were advised to purchase newer antibiotics from private pharmacies to treat pneumonia. This finding should be explored further in future studies to better understand if there is under-treatment of pneumonia.Almost all community members who participated in the FGDs harbored many misconceptions about antibiotic use that could contribute to inappropriate use. One of the most important findings was the belief that antibiotics are useful for prevention of the common cold, prompting early use of antibiotics for short durations to avoid developing a more severe infection. The perception that excessive antibiotic use leads to resistant organisms was not mentioned by the patients; rather, they thought that they themselves would develop resistance. Self-medication with antibiotics is facilitated by the availability of over-the-counter antibiotics in private pharmacies . SimilarThis study has several limitations. First, it was conducted in one district in Minya Governorate; thus the results may not be generalizable to areas outside Minya. Second, the diagnosis of ARIs was based on the physician’s judgment documented in the encounter, which may not be accurate; alternatively, the physician may have provided a diagnosis to justify antibiotic prescribing. Third, the presence of data collectors in the halls of the outpatient clinics might have influenced the prescribing practices of physicians, even though they did not know why the study was being conducted.This study provides evidence that antibiotics are commonly overprescribed and consumed for treatment and prevention of ARIs in the Minya district, driven by both inappropriate prescribing by physicians and excessive demand by the public. Educational interventions targeting providers and the public in Minya district should be piloted to measure the impact of interventions on promoting proper antibiotic use for ARIs. More widespread studies on antimicrobial use at the community level need to be conducted to determine the extent of the problem in Egypt. Reduction of antibiotic use for ARIs should be a priority for the MOHP in Egypt, as evidence exists that the incidence of antimicrobial-resistant infections is positively correlated with outpatient antibiotic use . ClinicaHigh inappropriate antibiotic prescription for ARIs existed in Minya. Several misconceptions regarding antibiotic use among community members were also stated. Data of this study could be used to design and pilot interventions to promote proper antibiotic use for ARIs targeting both physicians and the public.


The diagnostic criteria for IgG4-RD, proposed in 2011, were quite comprehensive and practical; however, it is important to remember that other diseases, such as hyper-interleukin (IL)-6 syndromes, may have common histopathological findings. Therefore, the histopathology of suspected IgG4-RD is occasionally not diagnostic. Here, we report a case of IgG4-related primary localized cervical lymphadenopathy without any other organ involvement. To our knowledge, there have been no previous reports of this. Additionally, the disease was associated with a 20-fold increase in IL-6 levels compared to that of the normal range.Immunoglobulin G4-related disease (IgG4-RD) is a recently recognized inflammatory condition with single- or multi-organ involvement. The disease is characterized by tumefactive lesions with dense IgG4 plasmacytic infiltration (an elevated IgG4We report the case of a 52-year-old Japanese man who presented with a painless, somewhat diffuse swelling in the left submandibular region. Although the case fulfilled diagnostic criteria for IgG4-RD, the diagnosis was not straightforward due to abnormally high levels of serum IL-6. After systematic evaluation of the patient, a final diagnosis of IgG4-RD was established. Since then, a specialist in connective tissue disorders has evaluated the patient on a regular basis. Two years after his initial visit, no disease progress or systemic involvement has been noted.We present a case of an IgG4-related primary localized cervical lymphadenopathy mimicking hyper-IL-6 syndrome. This case can serve as an excellent reminder that the definitive diagnosis of IgG4-RD should be established using a systematic approach, in particular when it appears as an atypical manifestation. Thus, the differential diagnosis of diseases involving cervical swelling is quite extensive. Immunoglobulin G4-related disease (IgG4-RD) is a recently recognized inflammatory condition that has single- or multi-organ involvement. The head and neck region is the second most common site for the development of IgG4-RD. The disease is characterized by tumefactive lesions with dense IgG4 plasmacytic infiltration -6 syndromes, such as Castleman’s disease [Increased numbers of IgG4 disease , all of  disease  proposedHere, we report a case of IgG4-related primary localized cervical lymphadenopathy without any other organ involvement. To our knowledge, there have been no previous reports of this. In addition, the disease was associated with 20-fold higher IL-6 levels than those of the normal range.A 52-year-old Japanese male with no significant past medical history visited our clinic in 2013 for evaluation of a swelling in the left submandibular region, which had increased in size over four years. The patient had not experienced any symptomatic manifestations.-weighted magnetic resonance imaging (MRI) demonstrated enlarged submandibular and submental lymph nodes and ipsilateral upper internal jugular vein , the patient was referred to the Department of Internal Medicine and Connective Tissue Disorders, Kurume University School of Medicine, two months after his initial visit. The patient has been under regular follow-up, and no disease progress or systemic involvement has been noted.IgG4-RD is a recently established immune-mediated, inflammatory condition that affects virtually every organ system. Lymphadenopathy, often associated with extranodal lesions, is frequently observed in patients with IgG4-RD. Lymph node involvement often occurs after or at the same time as the development of the extranodal lesion. The initial unifocal lymphadenopathy may progress to a multifocal lesion and/or develop to have extranodal involvement . The preIn the report by Sato et al. , IgG4-reThe head and neck region, including salivary, lacrimal, thyroid, and pituitary glands is the second most common site affected by IgG4-RD . IgG4-RDThe diagnostic dilemma in the present case was an extremely high level of serum IL-6 since hyper-IL-6 syndromes occasionally fulfill the histopathological diagnostic criteria for IgG4-RD and have elevated serum IgG4 levels , 4. A feBoth IgG4-RD and hyper-IL-6 syndrome share similar clinical and pathological manifestations and, therefore, it is quite difficult to differentiate them. As suggested by Sato et al. , diagnosWritten informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.We present a case of an IgG4-related primary localized cervical lymphadenopathy mimicking hyper-IL-6 syndrome. This case can serve as an excellent reminder that the definitive diagnosis of IgG4-RD should be established using a systematic approach, in particular when it appears as an atypical manifestation.


BALB/c nude mice were inoculated with MHCC97H cells or with tumor fragments of 10 patient-derived primary liver cancer explants selected according to c-Met/HGF expression levels. MSC2156119J  and sorafenib (50 mg/kg) were administered orally as single-agent treatment or in combination, with vehicle as control. Tumor response, metastases formation, and alpha fetoprotein (AFP) levels were measured. MSC2156119J inhibited tumor growth and induced complete regression in mice bearing subcutaneous and orthotopic MHCC97H tumors. AFP levels were undetectable after 5 weeks of MSC2156119J treatment, and the number of metastatic lung foci was reduced. Primary liver explant models with strong c-Met/HGF activation showed increased responsiveness to MSC2156119J, with MSC2156119J showing similar or superior activity to sorafenib. Tumors characterized by low c-Met expression were less sensitive to MSC2156119J. MSC2156119J was better tolerated than sorafenib, and combination therapy did not improve efficacy. These findings indicate that selective c-Met/HGF inhibition with MSC2156119J is associated with marked regression of c-Met high-expressing tumors, supporting its clinical development as an antitumor treatment for HCC patients with active c-Met signaling.The mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase with hepatocyte growth factor (HGF) as its only high-affinity ligand. Aberrant activation of c-Met is associated with many human malignancies, including hepatocellular carcinoma (HCC). We investigated the  The mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase with hepatocyte growth factor (HGF) as its only known high-affinity ligand. During embryonic development, c-Met controls morphogenesis, invasiveness, and migration of precursor cells. In adult life, the protein is typically expressed at low levels in a range of tissues, predominantly involved in tissue repair, and activated by pathologic stimulation. More specifically, c-Met is essential in liver development and regeneration. In conditional c-Met knockout mice, liver repair is delayed or absent after hepatectomy or chemically induced liver injury . In contin vivo, and to induce the regression of established tumors in xenograft models ), MSC2156119J , sorafenib , or rapamycin  as single-agent treatment. In addition, MSC2156119J (10 mg/kg) was given as a combination treatment with sorafenib  or rapamycin . Treatment was started seven days after orthotopic implantation of tumor fragments and terminated after five weeks. Six weeks after orthotopic inoculation, mice were sacrificed and whole liver weight, and primary tumor size and weight were measured. Tumor size was measured in two dimensions using a caliper and the volume was expressed in mm3 using the formula V = 0.5 a × b2, where a and b are the long and short diameters of the tumor, respectively.Mice were treated orally five days on/two days off with either vehicle combination  using a Synergy 2 Multi-Mode Microplate Reader  and analyzed by Gen5 Data Analysis software (BioTek).At necropsy, the lungs were inflated with 10 mL of 10% buffered formalin through the trachea and both the left and right lungs of each animal were collected, immediately fixed in 10% buffered formalin, and embedded in paraffin. Paraffin blocks were cut into serial sections and stained with hemotoxylin and eosin. For each animal, three sections cut at 6-μm intervals were examined for incidence of lung metastases and the number of metastatic foci.In addition to the patient-derived HCC xenograft model, 10 primary human liver cancer explant models  were selected on the basis of their c-Met expression levels: low expression models were LIM334, LIM1081, LIM348, LIM801, LIM941; intermediate expression models consisted of LIM752 and LIM574; and high expression models included, LIMsh050, LIM1098, and LIM612. The pathology diagnosis of these patient-derived HCC models is shown in 3 in volume, they were harvested for inoculation. Tumor fragments of 3 × 3 × 3 mm were implanted subcutaneously in the right rear flank of female BALB/c nude mice as described by the HuPrime tumor inoculation standard operating procedure .Tumor samples were derived from 10 HCC patients, and were thereafter maintained subcutaneously in nude mice. When tumors reached 500–700 mmn = 12), MSC2156119J , sorafenib , or the combination of MSC2156119J and sorafenib (n = 12) was initiated when the average tumor size reached approximately 100 mm3. Blood samples were taken before start of the treatment and end of the study for AFP determination.A five-days-on/two-days-off treatment schedule (up to five cycles) with vehicle , as described by Therasse et al.  and baseTreatment-naive patient-derived HCC explant models were immunostained for c-Met , phospho (Y1234/35)-Met , phospho (Y1349)-Met , and HGF  on formalin-fixed, paraffin-embedded sections. IHC was performed using the Discovery XT staining instrument , applying the OmniMap anti-Rb HRP Detection Kit , according to the manufacturer’s instructions. For the detection of HGF with the mouse IgG clone B-3, a rabbit monoclonal antimouse IgG antibody was used as secondary antibody followed by OmniMap anti-Rb HRP. Staining protocols were optimized using c-Met, phospho-Met and HGF-positive (high and medium expressing) and -negative cell lines. Sections were counterstained with hematoxylin.The IHC stains of c-Met, phospho-Met, and HGF were scored semiquantitatively by a surgical pathologist using the scoring system described for c-Met . Positivp value of ≤0.05 was considered to be statistically significant.The mean between-group differences for body weight, liver weight, and tumor size and weight were assessed using either the Student’s t-test (human MHCC97H HCC cell line xenograft model) or two-way RM- ANOVA (HuPrime models). Contingency table analysis and chi-square tests were used to determine the relationship between treatment and lung metastasis. The standard error of the mean is reported along with mean values. A In conclusion, these preclinical data show that selective c-Met/HGF inhibition with MSC2156119J is associated with marked regression of c-Met/HGF-expressing human tumors in mouse xenograft models. Superior efficacy of MSC2156119J compared to sorafenib was observed in c-Met high-expressing tumors, because selection of c-Met high-expressing HCC enriches for tumors that are driven by c-Met. Our data support the clinical development of MSC2156119J as an antitumor treatment for HCC patients with active c-Met signaling, and the enrichment strategy should enable selection for patients with the highest likelihood of response to MSC2156119J.


MET gene copy number is a common genetic alteration and inhibition o MET emerges as a promising targeted therapy in NSCLC. Here we aim to combine in a meta-analysis, data on the effect of high MET gene copy number on the overall survival of patients with resected NSCLC.MET is a receptor present in the membrane of NSCLC cells and is known to promote cell proliferation, survival and migration. MET gene copy number AND prognosis” in PubMed through January 2014. We selected the studies that investigated the association of MET gene copy number with survival, in patients who received surgery.Two independent investigators applied parallel search strategies with the terms “MET AND lung cancer”, “MET AND NSCLC”, “MET gene copy number on the survival of patients with NSCLC. Out of those, 6 used FISH and the remaining 3 used RT PCR to assess the MET gene copy number in the primary tumor. We calculated the I2 statistic to assess heterogeneity (I2 = 72%). MET gene copy number predicted worse overall survival when all studies were combined in a random effects model . When only the studies that had at least 50% of adenocarcinoma patients in their populations were included, the effect was significant . This was not true when we included only the studies with no more than 50% of the patients having adenocarcinoma histology .Among 1096 titles that were identified in the initial search, we retrieved 9 studies on retrospective cohorts with adequate retrievable data regarding the prognostic impact of MET gene copy number in the primary tumor at the time of diagnosis predicts worse outcome in patients with NSCLC. This prognostic impact may be adenocarcinoma histology specific.Higher  Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer related death worldwide The MET receptor has been characterized as a transmembrane receptor that is activated by the Hepatocyte Growth Factor (HGF). In NSCLC MET can be present in treatment naïve cases MET gene copy amplification in NSCLC. In this systematic review and meta-analysis, we aim to quantitatively review the effect of high vs. low MET gene copy number on the overall survival of patients with resected NSCLC.Gene copy amplification is a common mechanism of MET overexpression and it can be detected with reverse transcriptase polymerase chain reaction (RT PCR) or fluorescence in situ hybridization (FISH). A number of studies have explored the prognostic role of MET gene copy number with RT PCR, FISH or FISH equivalent in patients with NSCLC who underwent surgical resections. In addition, only the studies which reported the hazards ratio (HR) of increased MET gene copy number on survival or provided adequate data to calculate the HR were included. We excluded studies not published in full in the English language.We included studies which assessed MET gene copy number AND prognosis” in MEDLINE through January 2014. Two authors (A.D. and L.N.) reviewed the abstracts from the search and selected the eligible studies for full publication review. In the second phase of the review, the same authors selected the studies which meet all the study eligibility criteria. In addition, we included in the search all the bibliographies of the identified studies. In case of disagreement, the study was included or excluded on the basis of consensus by all the contributors. In the case of publications with overlapping populations, only the publication with more complete data was included.We entered the terms “met AND lung cancer”, “met AND NSCLC”, “Two authors (A.D. and L.N.) extracted the HR for overall survival with the accompanying 95% CI as the outcome measure from the identified studies. In addition relevant clinicopathologic data including the number of participants, the years of diagnosis, the year of publication, the histologic types, the TNM stage, the median age, the race and gender of the participants were collected. We noted patients who were initially included in individual studies but not in the final analysis. We reviewed the methods section in every included study and noted the exact assay of MET gene copy number assessment as well as the threshold of high MET gene copy number detection.Every included study was given a quality score on the basis of a quality assessment tool originally described in the study by Steels et al MET gene copy number on survival. The I2 test was used to assess heterogeneity between studies. In the case of I2>50%, the summary HR and the accompanying 95% CI were calculated with a random effects model whereas we used a fixed effects model in the case of low heterogeneity as defined by I2≤50%. We looked for publication bias with the aid of a funnel plot. Statistical significance was defined at the level of 0.05. All statistics were performed with RevMan 5.2.8 downloaded from the Cochrane website (http://tech.cochrane.org/revman/download).A forest plot was utilized to aggregate HRs from the univariate analysis of single studies in a summary HR of the effect of high MET gene copy number tumors but did not report the HR MET gene copy number in 136 surgically resected lung adenocarcinomas and used both PathVysion and the Cappuzo system to define high and low MET gene copy number. With the former methodology, the investigators identified a worse overall survival with higher MET gene copy number (p = 0.03), while with the latter methodology no association was found between MET gene copy number and overall survival (p = 0.22). Finally, Okuda et al MET gene copy number with RT PCR in an Asian population of 213 patients and by using a cut point of three MET gene copies reported a shorter overall survival in patients with higher gene copy number.Our initial search identified 1096 titles. After reviewing the corresponding abstracts we excluded 1020 publications for being reviews or case reports, for focusing on cell lines or non-human subjects, not being relevant to the MET receptor, not being relevant to NSCLC, or on the basis of language criteria . After tMET gene copy number with RT PCR, four with FISH and two with silver in situ hybridization (SISH). SISH utilizes light microscopy instead of fluorescence enabling the concomitant evaluation of gene copy number and cell morphology while at the same time allows for signal preservation for a longer time compared with FISH. Quality analysis revealed that all studies but one had comparable quality scores. In summary, most of the studies adequately described the eligibility criteria for the study population, included patient demographics, revealed the source of the tissue, described the method of MET gene copy number assessment, the study design, the objectives and the MET gene copy number positivity cut off  and the net HR and 95% CI is 1.78 (1.22–2.60) indicating a worse overall survival with higher MET gene copy number.We then quantified the summary effect of high MET gene copy number on survival in studies with Asian populations (HR = 2.31 95% CI 1.31–3.79), in studies where the reported histology was adenocarcinoma in more than half of the cases (HR = 1.55 95% CI 1.23–1.94), in studies which used FISH (HR = 1.48 95% CI 1.06–2.06) and in studies where the patients were diagnosed before 2004 (HR = 1.70 95% CI 1.34–2.16)  . In conty number .Finally, we attempted to estimate the risk for publication bias with a funnel plot . AlthougMET gene copy number and the potential association with survival in patients with surgically resected NSCLC. We further combined the available data in a meta-analysis and found a cumulative hazards ratio of 1.78 indicating worse overall survival with higher MET gene copy number in this patient population. In the subgroup analysis Asian ethnicity, FISH methodology, adenocarcinoma histology and year of diagnosis earlier than 2004 were indicators of a negative effect of MET gene copy number on survival. We assessed heterogeneity across different studies with the I2 statistic and found significant heterogeneity when all studies were included in the analysis. Interestingly, heterogeneity was low among studies including mostly adenocarcinoma cases and among studies including patients diagnosed before 2004.In the current study we reviewed the literature on MET gene copy number on the overall survival of patients with NSCLC can be explained by the role of MET in cancer biology. Among the different downstream pathways activated by MET, the JAK/STAT cascade is of particular importance for promoting cell motility, migration and metastasis MET gene copy number might identify a subgroup of patients with higher metastatic potential.The negative effect of higher MET gene copy number in this subset of studies and increased heterogeneity. Additionally, dual positivity for MET gene amplification and EGFR mutations might confound results after 2004 in case of treatment with an EGFR TKI inhibitor after disease progression. The effect of dual positivity for MET gene amplification and EGFR mutations might account for the different effects of increased MET gene copy number on prognosis between Asian and non-Asian populations. It is possible that different effect of high MET gene copy number on survival in patient populations with distinct clinicopathologic characteristics might reflect differences in other pathways which modify the MET pathway.There are a number of plausible explanations for the results of the sensitivity analysis in the present meta-analysis. First, studies that include patients diagnosed before 2004 were published earlier introducing a potential time lag bias in the interpretation of the data MET gene copy number and survival.It is not clear how the biology of MET receptor differs between adenocarcinoma and squamous cell carcinoma in NSCLC. A possibility is that additional histotype specific genetic alterations might interfere with the net effect of MET, or regulate MET expression. Krishnaswamy et al MET amplification in a phase I trial MET amplification status might be a better predictive biomarker for MET targeted therapies compared to protein expression by immunohistochemistry. We identified significant heterogeneity when all the identified studies are included in the meta-analysis. Possible reasons for heterogeneity might be populations with distinct demographics, possible interference of MET gene copy number prognostic impact and adjuvant chemotherapy as well as pre-analytical and analytical variance across different studies. For example, the cut point for high MET gene copy number ranges between 3 and 5, whereas some studies use in situ hybridization and others RT PCR. Additionally, the use of archived tissue poses a potential risk for loss of signal in FISH and RT PCR assays. Interestingly, we found that the studies where most of the patients were of adenocarcinoma histology were relatively homogeneous as were studies in which patients were diagnosed before 2004.Inhibition of MET is currently feasible small tyrosine kinase inhibitors like tivantinib or with monoclonal antibodies like onartuzumab. Tivantinib as shown in the MARQUEE trial MET gene copy number with overall survival, either a positive or negative. Therefore, we believe that heterogeneity across studies accounts partially for the observed asymmetry. Finally, there were three studies which were not involved in the meta-analysis because no hazards ratio was reported MET gene copy number on survival as they both report a positive significant effect on overall survival and they both include mostly or only patients with adenocarcinoma histology.There are a number of limitations in this meta-analysis. First, it is a meta-analysis of retrospective studies and therefore carries the biases of the retrospective design of the individual studies. Second, the number of studies is small, increasing the type I error. In addition, we observed an asymmetry pattern at the funnel plot. Asymmetry in funnel plots is illustrated by a correlation between the sample size and an estimate of the outcome effect and it indicates the presence of publication bias, heterogeneity or alternatively can be the result of random error MET gene copy number in NSCLC. We suggest that the worse overall survival observed in patients with higher gene copy number might be histotype specific and generates the hypothesis that patients with adenocarcinoma and high MET gene copy numbermight potentially benefit from adjuvant chemotherapy after resection.In conclusion, here we present a meta-analysis of nine retrospective studies which analyze the prognostic effect of Table S1(XLSX)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


IgG4-associated orbital and ocular inflammation is a relatively unknown entity characterized by sclerosing inflammation with infiltration of IgG4-positive plasma cells. Some so-called idiopathic inflammation syndromes are being re-classified as IgG4-associated inflammation with histopathologic evaluation.We report three cases with differing manifestations of IgG4-associated ocular and orbital inflammation: a case of recurrent, treatment-refractory sclero-uveitis that was diagnosed as granulomatosis with polyangiitis with an IgG4-related component, a case of pachymeningitis with optic neuritis that resulted in permanent visual loss, and a case of orbital inflammatory pseudotumor. All three would have been incompletely diagnosed without thorough histopathologic evaluation (including immunohistochemistry).IgG4-associated disease is an idiopathic, multi-organ inflammatory state that can manifest as chronic, relapsing, sclerosing inflammation in virtually any organ system. There is a wide range of presentations in ocular and orbital inflammation. Ophthalmologists should keep IgG4-associated inflammation in mind when examining chronic, sclerofibrosing inflammation with multi-system involvement. The histology of biopsy specimens is crucial in making the correct diagnosis. Timely assessment may lead to fewer diagnostic tests and more targeted therapy. IgG4-associated disease is a relatively newly discovered entity characterized by sclerosing inflammation, a lymphoplasmacytic infiltration full of IgG4-positive plasma cells and frequently associated with elevated serum IgG4 concentrations . It was A 79-year-old woman was referred to our institution with a 1-year history of tearing, blurry vision, pain, photophobia, and redness in her right eye. Past medical history was significant for breast cancer, which required right lumpectomy 14 years prior and left lumpectomy plus radiation that was completed about 1 year prior to referral. The patient’s symptoms in the right eye had begun about 1 month following the completion of the radiation treatment for breast cancer, at which point she was diagnosed with nodular scleritis inferotemporally in the right eye, and she was found to have elevated perinuclear accentuated anti-neutrophil cytoplasmic antibodies (p-ANCA), leading to suspicion for granulomatosis with polyangiitis. She was managed by multiple ophthalmologists and rheumatologists. Medical history also included insulin-dependent diabetes, hypertension, hyperlipidemia, anemia, and colon cancer about 16 years prior. There was a history of maxillary and sphenoid sinusitis, for which the patient had right mastoid surgery, as well as a history of bilateral ear tubes. The patient also had, at the time of presentation, bilateral renal oncocytomas treated with cryoablation, but renal function was normal. Past ocular history included open-angle glaucoma initially well-controlled with topical therapy alone but later exacerbated by an inflammatory/corticosteroid-induced component in the right eye. Prior to referral, the patient had been treated with oral prednisone, with good response, but experienced recurrences whenever the dose was tapered. She had also been treated with cyclophosphamide and indomethacin but had a history of poor response to azathioprine. Upon referral to our institution, best corrected visual acuity (BCVA) was 20/80 in the right eye. Slit-lamp examination demonstrated 360° of conjunctival and scleral injection, nodules in the temporal sclera, and 1+ nuclear sclerotic cataract. Dilated fundus examination and B-scan ultrasound showed panuveitis and serous retinal detachment associated with subretinal fluid . Her BCVA was 20/200. Slit-lamp examination showed mild scleral injection with a fibrotic-appearing lesion adherent to the medial sclera. Dilated fundus examination demonstrated chorioretinal scars with subretinal fibrosis and pale optic nerve. The infiltrative fibrotic lesion on the sclera was biopsied, and histologic examination revealed mixed chronic inflammatory infiltrate with fibrosis consistent with orbital inflammatory pseudotumor Fig. b–d. Althin situ hybridization demonstrating a relatively even mixture of kappa- and lambda-positive plasma cells (not shown). Many of the plasma cells stained positive with IgG4  of the brain revealed mild meningeal enhancement and a chronic subdural hematoma causing mild midline shift. Following surgical drainage, visual acuity remained unchanged, and his headaches worsened. Pertinent clinical findings were no light perception in both eyes, amaurotic pupils, and bilateral mild, temporal optic disc pallor. Ocular coherence tomography (OCT) of both optic nerves demonstrated normal nerve fiber layer thickness. Laboratory tests failed to suggest an etiology of his vision loss. Lumbar puncture showed lymphocytic pleocytosis but cytology and flow cytometry were negative for lymphoma. Repeat brain MRI was performed and demonstrated diffuse pachymeningeal enhancement Fig. a with poin situ hybridization revealing a kappa:lambda ratio of <4:1 among the plasma cells (not shown). In situ hybridization for Epstein-Barr virus was negative (not shown). Additionally, molecular genetic studies did not show any evidence of a clonal IgH gene rearrangement by polymerase chain reaction (PCR). However, IgG4 immunostain was markedly positive, with 40 IgG4-positive cells per high-power field (HPF), comprising over 40 % of the total plasma cell population . Accordingly, the diagnosis of IgG4-related meningeal disease was made. The patient was started on aggressive immunosuppression with high-dose corticosteroids to be followed by either cyclophosphamide or rituximab. However, due to insurance issues, he was able to receive only methotrexate. There was near-total resolution of meningeal enhancement on repeat MRI, but his visual acuity improved to only bare light perception in both eyes. He remains stable on methotrexate almost 1 year from his diagnosis.in situ hybridization, and flow cytometry were negative for lymphoma. The patient was lost to follow-up without any further work-up. He presented a year later due to recurrence of orbital inflammation, at which point the surgeon requested histologic work-up for IgG4-related disease. The slides from the prior orbital biopsy were requested and reviewed, showing lymphoid follicles with intervening bands of fibrosis and mixed chronic inflammatory cells. The tissue block was also received, and additional sections were cut for CD138 and IgG4 immunostaining. There were at least 150–180 IgG4-positive plasma cells per high-power field, comprising over 40 % of the total plasma cell population (as assessed by CD138). (Serum IgG4 was not obtained.) This was considered consistent with IgG4-associated orbital inflammatory pseudotumor with sclerosing features. The patient was initially treated with prednisone and then transitioned to intravenous rituximab as first-line therapy and had an excellent response with no recurrence at 2 years following therapy.A 76-year old man with a history of bilateral lacrimal gland masses and an otherwise unremarkable ocular examination underwent left orbital biopsy at another institution. Clinical suspicion was high for possible lymphoma due to the additional presence of cervical and axillary lymphadenopathy. The histologic diagnosis at that time was reactive lymphoid hyperplasia with fibrosis. Immunohistochemistry, kappa/lambda IgG4-associated disease is a relatively newly described, heterogeneous entity, initially described in autoimmune pancreatitis. Hamano et al. found elevated serum IgG4 level in patients with autoimmune pancreatitis, and since then, IgG4 has been used as an important marker to diagnose and re-classify select disease states previously regarded as idiopathic . The disImmune disregulation has been hypothesized as the most probable mechanism of IgG4-associated disease . IgG4 isThe treatment is mainly with oral corticosteroids, to which the inflammation is generally exquisitely responsive; nevertheless, patients remain on long-term steroid therapy, and relapse is common after cessation of corticosteroids . Other tOur first patient with necrotizing scleritis was refractory to oral corticosteroids and other immunomodulating agents such as methotrexate. Rituximab was not tried given that its possibility of therapeutic advantage was not known at the time of her treatment. The second patient underwent slow prednisone taper with a maintenance dose of methotrexate; however, the vision had minimal improvement despite the quiescent disease activity. Interestingly, only one of our patients (case 3) was treated with rituximab and had an excellent outcome. Even though rituximab was not an option due to insurance in case 2, this would have been our therapy of choice.Even though IgG4-related inflammation affects many different organs, the histopathology is remarkably similar in most cases, and this is critical in making the diagnosis. It should be noted, however, that besides the typical fibro-inflammatory histologic picture generally seen in IgG4-related disease, associations between granulomatous inflammatory diseases and IgG4 are also being discovered, thereby broadening our understanding of the potential scope of IgG4-related inflammation. For example, cases of sarcoidosis, adult orbital xanthogranuloma, and granulomatosis with polyangiitis involving relatively high amounts of IgG4-positive plasma cells on histology and/or elevated IgG4 concentrations have all been recently reported , 17–20. Without the assistance of immunohistochemistry on histopathology, our second patient would have been diagnosed as having idiopathic hypertrophic pachymeningitis, and our third case would have been diagnosed as idiopathic orbital inflammatory pseudotumor. There are several reported cases of IgG4-related sclerosing pachymeningitis, and many of them were previously diagnosed as idiopathic hypertrophic pachymeningitis –25. LindIgG4-related disease is an idiopathic, multi-organ inflammatory state that can manifest as chronic, relapsing sclerosing inflammation in virtually any organ system. Ocular/orbital manifestations include idiopathic orbital inflammation, pachymeningitis, or sclero-uveitis. IgG4-associated disease is an important diagnosis to make given that it can guide more specific management. Making the diagnosis prevents further extensive diagnostic testing and multiple biopsies while allowing more specific and aggressive immunosuppression. Treatment may be different depending on the involved organ systems. Systemic corticosteroids are generally the first-line therapy currently, but rituximab can be combined or used as an alternative if patients have contraindications to corticosteroids; furthermore, a shift toward rituximab as first-line therapy may be seen in the future if more data on its success accumulate . Physici