H-imidazole  reacts with AgBF4 to give [Ag(MET)2]BF4, in which the Ag atom is coordinated by two MET ligands with a trans arrangement.1-(2-Hydroxyethyl)-2-methyl-5-nitro-1 H-imidazole  is a medication that is used to treat infections by a variety of anaerobic organisms, but there are relatively few reports of the structures of metal compounds that exhibit coordination of metronidazole. We have demonstrated that MET reacts with AgBF4 to give [Ag(MET)2]BF4·CH3OH, in which the AgI cation is coordinated by two MET ligands with a trans arrangement. The structure of [Ag(MET)2]BF4 exhibits some interesting differences from its nitrate counterpart, [Ag(MET)2]NO3 . For instance, although the two MET ligands of both [Ag(MET)2]BF4 and [Ag(MET)2]NO3 are almost coplanar, the former compound has an anti-like geometry with a molecular inversion center, but the latter has a syn-like arrangement. In the crystal, the BF4− anion is linked by an O—H⋯F hydrogen bond to the methanol solvent molecule, which is, in turn, linked to the cation by an O—H⋯O hydrogen bond; the components of the structure are linked by O—H⋯O hydrogen bonds, forming chains along [001]. One of the MET ligands and the BF4− anion are disordered over two sets of sites with ratios of refined occupancies 0.501 (17):0.499 (17) and 0.539 (19):0.461 (19), respectively.1-(2-Hydroxyethyl)-2-methyl-5-nitro-1 H-imidazole, also known as metronidazole (MET) or Flagyl, is a medication used particularly for treatment of parasitic infections, such as trichomoniasis, amoebiasis and giardiasis, but is also effective against anaerobic bacteria .1-(2-Hydroxyethyl)-2-methyl-5-nitro-12]BF4·MeOH were obtained from a solution in methanol. The asymmetric unit consists of a silver cation, [Ag(MET)2]+, a tetrafluoridoborate anion, BF4−, and a solvent methanol molecule. The silver atom of [Ag(MET)2]+ is coordinated by two MET ligands in a trans manner by their N3 nitrogen atoms, as illustrated in Fig. 1Crystals of composition [Ag(MET)A = 2.163 (16) and Ag—N21 = 2.1193 (15) Å] are comparable to those values in the nitrate derivative, . The Ag—N bond lengths BF4 and [Ag(MET)2]NO3 are almost coplanar, the former compound has an anti-like geometry, and the latter has a syn-like arrangement. Thus, the C13—N11⋯N21—C23 torsion angle for [Ag(MET)2]BF4 is 160.8 (9)° [148.6 (11)° for the minor component of disorder], while the value for [Ag(MET)2]NO3 is 24.10° 2]BF4 has an approximate inversion center at the AgI ion, whereas [Ag(MET)2]NO3 does not.First, while the two MET ligands of both [Ag(MET)2]BF4 is much closer to 180° than is the corresponding value for [Ag(MET)2]NO3 NO3 exhibits Ag⋯O contacts of 2.63 and 2.67 Å, which are comparable to distances in other silver nitrate compounds ° for [Ag(MET)The hydroxyethyl group of one of the MET ligands [O21—H] serves as a donor in an intermolecular hydrogen-bonding interaction with the other hydroxyethyl group [O11—H] of an adjacent molecule. In turn, the latter hydroxyethyl group serves as a hydrogen-bond donor to a methanol molecule, which also hydrogen bonds to a tetrafluoridoborate anion. In the crystal, the components of the structure are linked into chains along [001] by the O—H⋯O hydrogen bonds Table 1.et al., 2002et al., 2002et al., 1991et al., 2005et al., 1998et al., 2002et al., 2002et al., 2003et al., 2006et al., 1990et al., 1994et al., 2002et al., 1994et al., 1993et al., 2002et al., 1983In addition to coordination to silver, metronidazole has also been shown to coordinate to other metals, and structurally characterized compounds have been reported for Co Uiso(H) = 1.2Ueq. One of the MET ligands was refined as rotationally disordered with occupancies of 0.501 (17) and 0.499 (17) and the configurations were modeled using the SAME command in SHELXL2013  I. DOI: 10.1107/S2056989015002819/lh5749Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015002819/lh5749Isup3.molSupporting information file. DOI: 10.1107/S2056989015002819/lh5749Isup4.txtSupporting information file. DOI: 1048516CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Immunoglobulin G4 related disease (IgG4-RD) is a poorly understood chronic inflammatory disorder affecting the middle-aged and elderly that can present to the otolaryngologist. We aim to summarize the current literature regarding the manifestations and management of IgG4-RD in the head and neck.Pubmed and EMBASE were searched using the term relevant search algorithm utilizing keywords such as: IgG4 related disease, head and neck, orbit, salivary glands, sialadenitis, Kuttner, angiocentric eosinophilic fibrosis, submandibular, lacrimal, thyroid, dacryoadenitis, nasal, sinus, and Mikulicz’s. Reference lists were searched for identification of relevant studies.Case reports, original research and review articles published in English from 1964 to 2014 whose major topic was IgG4-RD affecting the head and neck were included. Data regarding patient demographics, presentation, histopathology, management and treatment outcomes of IgG4-RD were extracted. Level of evidence was also assessed and data were pooled where possible. Three independent reviewers screened eligible studies; extracted relevant data and discrepancies were resolved by consensus, where applicable. Descriptive and comparative statistics were performed.Fourty-three articles met our inclusion criteria. IgG4-RD most often presents as a mass lesion in the head and neck region. Common diagnostic features include: 1) elevated serum IgG4 level, 2) marked infiltration of exocrine glands by IgG4-positive plasma cells with fibrosis, and 3) marked improvement with corticosteroid therapy and additional immunosuppressive therapy in corticosteroid refractory cases. Early diagnosis and involvement of rheumatology is important in management.IgG4-RD is a challenging non-surgical disease that has multiple manifestations in the head and neck. It must be distinguished from various mimics including malignancy, systemic diseases, and infectious. Otolaryngology-Head and Neck surgeons should be aware of this condition and its management. IgG4-RD was first recognized after a connection between elevated serum IgG4 levels and inflammatory mass lesions in the pancreas causing autoimmune pancreatitis was made by Hamano  of 2011 .The exact etiology of IgG4-RD is unknown and no known role of the IgG4 molecule itself has been identified. It is postulated that the inflammatory and fibrotic processes that drives IgG4-RD are propagated by a combination of Th2 cells and regulatory T cells (Treg cells) . HistoloCurrently, the literature proposes that IgG4-RD could be both over and under recognised . This stThis systematic review was performed using the following search strategy and study selection criteria.The databases PubMed (1966-December 2014) and Embase (1988-December 2014) were searched using an algorithm designed from an extensive list of relevant search terms . Relevant articles were obtained and reviewed in full. Discrepancies were resolved by consensus amongst the reviewers. The inclusion criteria comprised of all original clinical studies, case series and case reports of histologically confirmed IgG4-RD in the head and neck. Histologic diagnosis of IgG4-RD required identification of >10 IgG4 positive plasma cells per high powered field, IgG4 + plasma cell to IgG containing cell ratio 40 % or greater, and characteristic finding of fibrosis, sclerosis and phlebitis. Articles were excluded on the basis of biopsy information not being from a head and neck site, insufficient histologic information, inability to extract head and neck specific information and narrative review and expert opinions.The information gathered for each study included study design, country of publication and number of patients. Where possible, patient-specific data was extracted, including age at presentation, duration and nature of symptoms. Specifically, we collected data regarding head and neck manifestations, laboratory and histologic findings as well as specific treatments and outcome information.Basic statistical analysis, including descriptive statistics was performed using Excel .We initially identified 1592 articles through a combination of literature search and citation review. After reviewing abstracts, 247 articles of interest were identified. Three authors independently reviewed the articles and 43 articles met inclusion criteria  received and their outcomes are summarized in Table This is the first systematic review of IgG4-RD presentation in the head and neck. Our study demonstrates a strong propensity for IgG4-RD to present in the head and neck region. We included 43 articles containing 484 patients. The most common site of presentation was the orbit, followed by the submandibular gland – with many patients having presentations in multiple head and neck and distant sites. Treatment information was also collected, showing that majority of patients receiving corticosteroids responded very well to treatment.The vast majority of the included cases were from Asia (429 of 484 or 88.6 %) and more specifically Japan (419 of 484 or 86.6 %). Indeed, much of the literature on IgG4-RD originates from Japan . While cWhile systemic presentations of IgG4-RD favor males over females with a reported ratio of 2.8-3.5:1, our study demonstrates an almost 1 to 1 ratio of head and neck manifestations , 9, 10. Few patients in our series received surgical excision alone. The majority cases received some form of medical management comprising of high-dose corticosteroids. Patients had excellent response to medical therapy alone with full remission rate of 90 %. A consensus statement from 17 referral centres in Japan developed a treatment regime of 0.6 mg/Kg prednisolone for 2 to 4 weeks with a taper over 3 to 6 months and a low daily dose for 3 years. Importantly, this regime was developed for treatment of autoimmune pancreatitis, where consequences of not treating are associated with significant morbidity and mortality . HoweverStrict selection criteria was used in article selection. Histologic diagnosis was based on IgG4 + ve cells/ HPF and the IgG4 + ve/IgG ratio. This is considered the most rigorous definition of IgG4-RD . HoweverDue to the numerous potential manifestations of IgG4-RD in the head and neck, it is crucial for otolaryngologists to be aware of this condition. A high index of suspicious is required particularly in the setting of patients who present with recurrent salivary and lacrimal gland swelling, lymphadenopathy, along with fibroinflammatory systemic involvement. This disease process remains under recognized and poorly understood. Future studies are necessary to better understand the pathophysiology and natural history of this disease.


IACIgG4‐associated cholangitisIgG4‐RDIgG4‐related diseasePSCprimary sclerosing cholangitisTo the Editor:IgG4‐associated cholangitis (IAC) and autoimmune pancreatitis are the predominant manifestations of IgG4‐related diseases (IgG4‐RD); their pathogenesis is enigmatic.Using a questionnaire, we carefully investigated the job history of our mostly retired IgG4‐RD patients. Of 25 patients with IAC and/or autoimmune pancreatitis, 88% had a history of blue‐collar work of at least 1 year, but often of a whole career  and no histological evidence of IAC, the percentage of blue‐collar workers was 22%. Among the PSC patients, 7% reported any  exposure to these compounds.Our earlier finding of clonal expansions of IgG4‐switched B cells in patients with IgG4‐RD1*Emma L. Culver, M.D.2*Ulrich Beuers, M.D.1Lucas J. Maillette de Buy Wenniger, M.D.1Department of Gastroenterology and HepatologyTytgat Institute for Liver and Intestinal ResearchAcademic Medical Center, University of AmsterdamAmsterdam, The Netherlands2Department of Gastroenterology and HepatologyOxford UniversityOxford, UK


Bjerkandera adusta CCBAS 930 decolorizing, in stationary cultures, 0.01 % solutions of carminic acid and Poly R-478, were characterised by a strong increase in the activity of the horseradish peroxidase (HRP-like) and manganese-dependent peroxidase (MnP) at a low activity of lignin peroxidase. Genotypically modified mutants of B. adusta CCBAS 930: 930-5 and 930-14, with total or partial loss of decolorization capabilities relative to anthraquinonic dyes, showed inhibition of the activity of HRP-like peroxidase and MnP. Whereas, compared to the parental strain, in the mutant cultures there was an increase in the activity of lignin peroxidase and laccase. The paper presents a discussion of the role of the studied enzymatic activities in the process of decolorization of anthraquinonic dyes by the strain B. adusta CCBAS 930.Cultures of the anamorphic fungus  Dyes are substances used in various branches of industry . Ineffectiveness of the process of dyeing causes that, depending on the type of dye applied, from 2 to 50 % of the dye used can find its way to wastewater , while MnP does not display such properties . That dye (RBBR) was decolorized faster than azo-dyes, from five- to ten-fold, by laccase of T.versicolor ATCC 20869  traits, that strain was classified as Geotrichum-like (strain R59) as it produced conidia (arthrospores) of the Geotrichum type. However, it differed from Geotrichum by the presence of abundant, fluffy and hyalinic aerial mycelium. Analysis of internal transcribed sequences (ITS): ITS1 and ITS2 and of the 5,8S rDNA region revealed the species identity of the isolated conidial fungus among the B. adusta (Willd ex Fr)P. Karst (Basidiomycota)  and Poly R-478, with the use of mutants of the fungus.The aim of this study was to characterise activities of phenoloxidases of B. adusta CCBAS 930 and its two phenotypic mutants: 930-5 and 930-14. The isolation from soil, identification, morphological characterisation and identification sequences of the gene fragment rRNA: ITS1, 5,8S rRNA and ITS2 of parental strain B. adusta CCBAS 930 are presented in the papers by Korniłłowicz-Kowalska et al. (B. adusta CCBAS 930. Induction and selection of mutants of B. adusta CCBAS 930 with modified ligninolytic activity after the application of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and UV radiation are described by Korniłłowicz-Kowalska and Iglik  in a maltose extract medium . The fungus mycelium was separated from liquid phase using sterile filter paper and washed by sterile distilled water. The fresh mycelium was ground in a mortar with quartz sand and DNA was extracted with the use of DNA Plant mini Kit .The isolation of genomic DNA of the parental strain 2, 1 × PCR buffer). The stages of the reaction of amplification, comprising initial denaturation at 95 °C (15 min) for HotStarTaq polymerase activation, 35 cycles: denaturation at 94 °C (1 min), primer annealing at 38 °C (1 min), elongation at 72 °C (2 min) and final elongation at 72 °C (10 min) were conducted in the Mastercycler personal (Eppendorf). The products of the PCR-RAPD reaction were separated electrophoretically in 1.5 % agarose gel with an addition of 10 μg/ml ethidium bromide in buffer 1 × TAE  and run at 8 V/cm of gel, then visualised in UV light and photographed. Characterization of amplified products was designed for each RAPD marker based on the molecular size and primer used GelScan 2.0 software  with used DNA GeneRuler Ladder 100 bp . Similarities among fungal genotypes were deduced on the basis of the number of shared amplification products ; pH 6. Dyes were added in such amounts as to achieve their final concentration of 0.01 %. The inoculum was constituted three circles of mycelium with ∅ = 1 cm, obtained from 7-day culture on glucose-potato agar medium . The control for decolorization and enzymatic activity was respectively non-inoculated medium with dyes and cultures of B. adusta CCBAS 930 and its mutants without dyes. The cultures and the control were maintained for 2 weeks at 28 °C, under stationary conditions.The fungi  were cultured in 50 cmg for 5 min HRP-like activity was estimated according to Maehly and Chance (460nm = 11.3 M/cm) as substrate in 0.1 M acetate buffer with pH 5.5, in the presence of 0.1 mM H2O2. Manganese-dependent peroxidase (MnP) activity was measured through oxidation with 1 mM MnSO4 in 50 mM sodium malonate, pH 4.5, and in the presence of 0.2 mM H2O2 followed by determination of the Mn+3—malonic acid complex (ε270nm = 11.59 M/cm) according to Wariishi et al. (310nm = 9.3 M/cm) in 40 mM tartrate buffer, pH 3, in the presence of 0.4 mM H2O2. Laccase activity was estimated according to Leonowicz and Grzywnowicz (525nm = 6.5 M/cm) in 0.1 M citrate–phosphate buffer, pH 5.The enzyme activities were determined spectrophotometrically. Clear extracellular culture fluids were obtained through centrifuging of the cultures at 7000×d Chance  as modifd Chance , in the i et al. . Lignin i et al.  in the pywnowicz , in the In all assays, one unit of enzymatic activity (U) was defined as the amount of enzyme that oxidized 1 μmol substrate per minute under defined condition and activities was reported as U/l.495nm (maximum of absorbance), for Poly R-478—A519nm (maximum of absorbance). The concentration of dyes in the solution was determined from a model curve plotted for Ac and for Poly R-478.The decolorization activity was determined by measuring the lowering of absorbance: for Ac at AB. adusta CCBAS 930 strain with use the use of NTG and UV radiation induced changes in the genetic material of the wild strain which affected the quality and quantity of the products of amplification and indicated differentiation of the genetic profile of parental strain and the mutants. The RAPD analysis applied, with the use of molecular typing of OPN starters, confirmed the existence of genotypic differences between the parental strain of B. adusta CCBAS930 and the phenotypic mutants studied: 930-5 and 930-14  in profile of 930-14 mutants. With the use of OPN14 primer for all three strain were amplified six product with the values of range 300–1000 bp. In the genetic profile of 930-14 mutants characteristic was present additional binding loci of OPN14 primer and band with size of 190 bp , up to complete inhibition in 14-day cultures  in control (cultures without dyes) and in cultures with Ac. Whereas, the enzyme was detected in cultures of those strains growing in the presence of Poly R-478  of strain CCBAS 930 increased the biosynthesis of laccase in cultures with Poly R-478. It was showed that the parental strain synthesised laccase only on the 12th and 14th days of culturing, while the mutants did it throughout (930-5) or during most of (930-14) the period of incubation with that dyes.In the cultures of all strains the highest laccase activity was observed after 12 days of growth Table B. adusta CCBAS 930 and modified its decolorization properties.Mutagenesis with the use of nitrosoguanidine (NTG) and UV radiation induced changes in DNA of strain A. tenuissima FCBP-252 and T. viride FCBP obtained after UV irradiation as compared to mutants produced through the effect of chemical factors alone.The RAPD-PCR method is used for the purposes of identification of genetic differentiation between a wild strain and its mutants obtained through the effect of chemical and physical factors : 930-5 (R59-5) and 930-14 (R59-14) decolorized post-industrial lignin more effectively than the parental strain  was, however, from two- (in the presence of Ac) to four-fold (in the presence of Poly R-478) lower as compared with HRP-like peroxidase. The process of decolorization of the studied dyes by CCBAS 930 strain was not accompanied by changes in the activity of lignin peroxidase which was very low throughout the period of culturing. Laccase activity was detected only towards the end of the culturing, and only in the presence of Poly R-478. However, induction of that enzyme did not contribute to the decolorization of the remaining content (ca. 30 %) of that dyes. The relation between decolorization of anthraquinonic dyes and the HRP-like peroxidase activity of B. adusta CCBAS 930 was demonstrated also in our previous publication  and MnP peroxidases and the decolorization of anthraquinonic dyes by the strainB. adusta CCBAS 930 is also indicated by the study of mutants of the fungus. It was demonstrated that the loss of decolorization abilities of the mutants towards Ac was coupled with a lack of HRP-like peroxidase and MnP activity. Mutant 930-5, that less effective decolorized Poly R-478, was also characterised by inability to produce HRP-like peroxidase, but it did produce MnP, though in small amounts. Whereas, the study excluded the participation of laccase and lignin peroxidase in decolorization of both dyes by B. adusta CCBAS 930, as the stimulation of the activity of those enzymes in the cultures of 930-5 and 930-14 did not contribute to the decolorization of the culture fluids.B. adusta CCBAS 930 shows similarity to the strain Irpex lacteus KR35 W studied by Shin , is also characterised by three different peroxidase activities: the activity of substrate-nonspecific peroxidase (non-dependent with manganese), MnP and LiP. In view of our own research, however, the similarity of the strain B. adusta CCBAS 930 and the strain B. adusta Dec 1, being the subject of long-term research (since 1995) by Japanese researchers: Kim et al. B. adusta. Initially, the strainch since  by JapanB. adusta Dec 1 . Subsequently, based on molecular criteria , it was included among the Basidiomycota as Thanatephorus cucumeris Dec 1  and B. adusta . Due to the fact that T. cucumeris (which is a teleomorph of Rhizoctonia solani) does not produce any conidial spores, while B. adusta produces conidia of the type of arthrospores, the studied strain CCBAS 930 was finally classified as an anamorphic stadium of Bjerkandera adusta.1 Sugano . In turna et al. , yieldedB. adusta CCBAS 930 and B. adusta Dec 1, is related with their specialisation in enzymatic decolorization of derivatives of anthraquinone—a process in which the key role is played by peroxidases of those fungi. Sugano , their activity, estimated on the basis of the rate of decolorization, was also the highest with relation to derivatives of anthraquinone: daunomycin and anthraquinonic dyestuffs  , further studies are required, comprising the isolation, characterisation of properties and estimation of application possibilities of purified preparations of peroxidase (peroxidases) of the investigated strain.The physiological similarity of the two strains: . Sugano  reports s Sugano . AlthougB. adusta CCBAS 930 including empirical modeling technique. In is regard of key importance are Response Surface Methodology. There are statistically designed experimental models, which allow for optimization of the culture studies using biological decolorization (Sharma et al. It is also necessary to optimize decolorization anthraquinone dyes by strain of B. adusta CCBAS 930 decolorization process occurs by co-metabolism. With respect to the anthraquinone dyes decolorization by white rot fungi, the essential parameters for controlling the course of decolorization should include the concentration of the dye, a source of C and N and their concentrations, pH medium and temperature of incubation.


Clostridium difficile infections and other intestinal or extra-intestinal disorders. Nonetheless, adverse events (AEs) remain a major challenge in the application of FMT.Fecal microbiota transplantation (FMT) is a microbiota-based therapy that shows therapeutic potential in recurrent or refractory To review the AEs of FMT and to address the concerns of safety during the procedure.Publications were retrieved in the databases of Medline, Embase and Cochrane Library. AEs were classified according to their causality with FMT or their severity.Clostridium difficile infection .A total of 7562 original articles about FMT were identified in this study, 50 of them fulfilled the inclusion criteria. Totally 78 kinds of AEs were revealed enrolled in these 50 selected publications. The total incidence rate of AEs was 28.5%. Among the 42 publications, 5 kinds were definitely and 38 kinds were probably related to FMT. The commonest FMT-attributable AE was abdominal discomfort, which was reported in 19 publications. For upper gastrointestinal routes of FMT, 43.6% (89/204) patients were compromised by FMT-attributable AE, while the incidence dropped to 17.7% (76/430) for lower gastrointestinal routes. In contrast, the incidences of serious adverse events (SAEs) were 2.0% (4/196) and 6.1% (40/659) for upper and lower gastrointestinal routes, respectively. A total of 44 kinds of SAEs occurred in 9.2% patients, including death , infection , relapse of inflammatory bowel diseases  and Consequently, both AEs and SAEs are not rare and should be carefully monitored throughout FMT. However, high quality randomized controlled trials are still needed for the more definite incidence of AEs of FMT. The maj 10-fold . Alterat 10-fold –12. Noveet al applied FMT to treat antibiotic-associated diarrhea [et al reported the first FMT therapy for CDI in 1983 [Fecal microbiota transplantation (FMT), also known as fecal bacteriotherapy or intestinal microbiota transplantation, is defined as the perfusion of treated feces from a healthy donor via the upper or lower gastrointestinal route . About 1diarrhea . Since S in 1983 , the app in 1983 –19. The  in 1983 , 21. Alt in 1983 . Moreove in 1983 , 23 incl in 1983 , 25, irr in 1983 –28, meta in 1983 , 29, 30, in 1983 , autoimm in 1983 , 33, all in 1983 , 35, and in 1983 .Although patients benefit from FMT, concerns about this emerging strategy remain to be addressed, including long-term outcomes of FMT and the AEs. So far, the exact roles of the gut microbiota in FMT are not yet fully understood. And the AEs that happen during or after FMT still perplex clinicians and fundamental researchers. Hence, we systematically reviewed the AEs of FMT in all related publications aiming to elucidate the causality between FMT and the AEs. Furthermore, the AEs of FMT were divided into different degrees according to the severity and SAEs were emphatically introduced to arouse attention in FMT.Electronic databases for literature search included the Medline, Embase, and Cochrane Library. The last search was run on July 2015. The complete string used for the electronic search is shown in Titles, abstracts, and keywords were independently assessed by two investigators (WSN and XMQ) to determine the appropriateness of the publications. Both investigators checked all the articles in accordance with the inclusion criteria and exclusion criteria. Disagreement was resolved by a third investigator (CHL). Original full-text articles, letters to the editor, abstracts of scientific conferences, case reports and case series which were published between 1913 and 2015 were reviewed. Studies involving the AEs of FMT for human sujects of any age were included into this study. Studies evaluating treatments with cultured bacteria other than human feces, animal studies and non-original reports  were excluded.Data extraction was conducted according to the above mentioned inclusion and exclusion criteria and cross-checked by the two independent investigators (WSN and XMQ). When publications included patients from a previous study and newly enrolled ones, only the latter were brought into the study. Items of this study were listed as follows: (i) the study characteristics ; (ii) the patients ; (iii) the relationship between donors and recipients; (iv) the FMT procedure ; (v) the detailed descriptions of AEs .The relationship between the AEs and FMT were categorized into four types as previously dsecribed with minor modifications: definitely related, probably related, possibly related, and unrelated to FMT .AEs caused by endoscopic procedure during FMT; an event that follows a reasonable temporal sequence from FMT exposure; that follows a known or expected response pattern to the FMT; that is confirmed by stopping the FMT procedure; and that is not explained by any other reasonable hypothesis.An event that follows a reasonable temporal sequence from FMT procedure; that follows a known or expected response pattern to the FMT; that is confirmed by stopping the FMT procedure; and that is unlikely to be explained by the known characteristics of the subject’s clinical state or by other interventions.An event that follows a reasonable temporal sequence from FMT procedure; that follows a known or expected response pattern to FMT; but that could readily have been caused by a number of other factors.An event that can be determined with certainty to have no relationship to FMT.AE is defined as any untoward medical occurrence in a patient after administration of FMT that does not necessarily have a causal relationship with this treatment. Therefore, an AE can be any unfavorable and unintended sign , symptom, or disease temporally associated with FMT, whether or not related to the FMT .A SAE is any adverse experience occurring during or after FMT that results in any of the following outcomes: death, life-threatening experience, inpatient hospitalization or prolongation of existing hospitalization, persistent or significant disability or incapacity, congenital anomaly or birth defect, or an important medical event .A total of 7562 original articles about FMT were identified. Among them, 534 were selected for further assessment according to the titles. After reviewing the full text articles, 50 of them fulfilled the inclusion criteria among which 16 were case series, 9 were case reports and 4 were randomized controlled trials . The excIn the selected 50 publications, a total of 1089 patients were treated with FMT (age range: 1–95 years). Among them, 831 patients were affected by refractory or recurrent CDI, of whom 106 had concomitant IBD; 235 were affected by independent IBD; 1 affected by early onset colitis; 9 affected by antibiotic-associated diarrhea (AAD), of whom 6 had concomitant CDI; and 13 affected by IBS. Overall, 78 kinds of AEs were reported to happen on 310 patients during or after FMT. The majority of AEs were presented as mild symptoms such as abdominal disomfort, diarrhea, transient fever, nausea, vomiting and constipation. Each AE was cited once for one patient since the AE always recorded only once during a multiple FMT in one course of treatment.Since AEs usually overlapped, we could not obtain the exact total number of patients with AEs in a publication. Hence, we took the number of patients with the most frequent kind of AEs from each publication for further calculating the overall incidence of AEs. Based on the above mentioned statistical principles, the overall incidence of AEs was 28.5% (310/1089). The incidences of AEs in CDI and non-CDI  were 28.0% (233/831) and 29.8% (77/258) respectively.Many factors could be involved in the development of AEs, including the individual difference of recipients, donors, methods of administration and regimen of FMT. The causality between AEs (including SAEs) and FMT was analyzed according to the description as above . As a reClostridium difficile toxin, and routine bacterial culture for enteric pathogens , parasites and ova. However, the donors who were in the latent period of infection could not be excluded by the above screening tests and thus these donors might contribute to the development of infectious AEs.So far, there is no unified standard to screen the stool samples from donors. The following donor screening tests were applied to the donors in the selected 50 publications: viral screenings , stool tests for In addition, individual differences of donors may also lead to AEs. For example, one patient (a 1-year-old girl) developed fever, vomiting and tachycardia after receiving the fecal transplant from her brother, while she well tolerated the transplant from her niece . For theRelated donors (family members) for FMT were reported in 11 publications. Unrelated donors were reported in 9 publications.For FMT via upper gastrointestinal routes, the recipients were prepared in fasting condition. For FMT via lower gastrointestinal routes, bowel lavage and/or antibiotics were given to the recipients before FMT. However, no association of AEs with the preparation of FMT was found in the 50 publications. Of note, in Bota Cui’s report, the recipients who took metoclopramide before FMT manifested fewer AEs, suggesting metoclopramide might potentially help avoid the AEs .The routes of administration are listed as follows according to the frequency that they were used: colonoscopy (26 publications), retention enema (8 publications), nasogastric tube (6 publications), nasojejunal tube (5 publications), gastroscopy (2 publications), sigmoidoscopy (1 publication), nasoduodenal tube (4 publications), enteroscopy (1 publication), esophagogastroduodenoscopy (1 publication) and capsule (1 publication). Among the above routes of administration, lower gastrointestinal routes include colonoscopy, sigmoidoscopy and retention enema. Upper gastrointestinal routes include the remaining means. Compared with upper gastrointestinal routes, lower gastrointestinal routes were more widely used. After exclusion of the publications in which the routes of administration were not clearly stated, the proportion of patients affected by FMT-attributable AE is 43.6% (89/204) for upper gastrointestinal routes of FMT administration, while the incidence dropped to 17.7% (76/430) for lower gastrointestinal routes. The FMT-attributable AEs were grouped by routes of administration  patients (in 9 publications) were reported to suffer abdominal discomfort after FMT. For lower gastrointestinal routes in 22 publications, 13.0% (56/430) patients (in 10 publications) developed abdominal discomfort after FMT. The upper gastrointestinal routes were therefore more likely to develop abdominal discomfort compared with lower gastrointestinal. The second commonest attributable AE was transient fever which was happened on 3.4% (7/204) and 2.8% (12/430) patients for upper and lower gastrointestinal routes of FMT administration, respectively .Mild to moderate AEs such as abdominal pain, abdominal cramping, flatulence, increased stool frequency, constipation, vomiting, belching, fever and transient increase of C-reactive protein (CRP) were reported in most of the selected 50 publications and ususally did not cause critical clinical outcome. Hence, we paid emphatic attention to SAEs and listed 44 kinds of SAEs that were reported in 27 publications , of whicThe FMT-attributable  SAEs included death, pathogen infections, IBD flare, auto-immune diseases, and FMT procedure related injury, etc, while the FMT unrelated SAEs covered death or hospitalization caused by underlying conditions. The commonest SAEs were death, severe infections and relapse of CDI and IBD.As the most devastating SAEs, death happened on 38 patients in 10 publications  and the Escherichia coli, Proteus mirabilis, Citrobacter koseri, and Enterococcus faecium [Twenty-seven patients were reported to be hospitalized or die for infection in 12 publications (CDI was not included) . The inc faecium , 45. TheAlthough unrelated to FMT, CDI relapse was still another unignorable SAE, the incidence of which reached 0.9% (10/1089) in the selected 50 publications .The human gastrointestinal tract harbors the largest number of microbes in the human body, which is referred to as the gut microbiota. Perturbations in the gut microbiota have been associated with conditions as diverse as gastrointestinal diseases and even systemic disorders . As a miHere, we selected 50 publications in which AEs of FMT were reported. Totally 78 kinds of AEs happened on 310 patients following FMT treatment. The AEs of FMT were divided into two major categories, namely related and unrelated to FMT. Our analytical results showed that the AEs related to FMT  accounted for a larger proportion than the AEs unrelated to FMT. Moreover, we found that SAEs related to FMT such as death, viral and bacterial infections, transient relapse of IBD, were not rare and therefore deserved attention and consideration in the procedure of FMT.Human Microbiome Project (HMP) has sampled the microbiome of many people to get a better idea of variability, and how microbes work together in complex communities. HMP implied that because the microbiome is more varied than the genome, and easier to modify, it gives a more logical starting point for individual treatments . As obseRoute of fecal infusion is another concern in FMT that may lead to AEs. Lower gastrointestinal routes, including colonoscopy, sigmoidoscopy, and retention enema, were more widely used than upper gastrointestinal routes. We found that the patients who received FMT treatment via upper gastrointestinal routes were more likely to develop AEs than those who received FMT treatment via lower gastrointestinal routes (43.9% vs. 20.6%). To avoid injury associated AEs during endoscopic process, noninvasive and patient-acceptable routes can be chosen for FMT treatment. Actually, a recent pilot study in which frozen capsules FMT was administered orally for patients with recurrent CDI demonstrated a high incidence of diarrhea resolution  but few mild AEs such as abdominal cramping and bloating . TherefoThough this systematic review provides a handful of valuable messages for clinical application of FMT, some limitations need to be addressed. First, the incidences of AEs might be underestimated. On one hand, since AEs usually overlapped, we took the number of patients with the most frequent kind of AE from a publication for further calculating the overall incidence of AEs. On the other hand, transient or mild AEs were sometimes ignored by researchers, resulting in the missing data of AE occurrence. Secondly, some potential confounding factors such as the health conditions of the donors, the time span from FMT exposure to the onset of the AEs and the outcomes of AEs could have substantial impacts on the classification of AEs. Thirdly, there was subjective nature in the classification of AEs. In most instances, it is impossible for us to obtain the original data of the publications we selected. Therefore we categorized AEs according to the authors’ subjective description or FDA general definitions of AEs for drugs or therapy .Though FMT was validated to be a beneficial therapeutic strategy, we should pay enough attention to AEs of FMT. In order to prevent or treat AEs during or after FMT, more clinical trials and fundamental research are urged to elucidate the exact mechanism of how FMT causes AEs and set up a guideline on how to handle FMT-related AEs in different situations.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOC)Click here for additional data file.S3 Appendix(DOC)Click here for additional data file.


Management of acute respiratory tract infection varies substantially despite this being a condition frequently encountered in pediatric emergency departments. Previous studies have suggested that the use of antibiotics was higher when chest radiography was performed. However none of these analyses had considered the inherent indication bias of observational studies.The aim of this work was to assess the relationship between performing chest radiography and prescribing antibiotics using a propensity score analysis to address the indication bias due to non-random radiography assignment.We conducted a prospective study of 697 children younger than 2 years of age who presented during the winter months of 2006–2007 for suspicion of respiratory tract infection at the Pediatric Emergency Department of an urban general hospital in France (Paris suburb). We first determined the individual propensity score (probability of having a chest radiography according to baseline characteristics). Then we assessed the relation between radiography and antibiotic prescription using two methods: adjustment and matching on the propensity score.We found that performing a chest radiography lead to more frequent antibiotic prescription that may be expressed as OR = 2.3, CI [1.3–4.1], or as an increased use of antibiotics of 18.6% [0.08–0.29] in the group undergoing chest radiography.Chest radiography has a significant impact on the management of infants admitted for suspicion of respiratory tract infection in a pediatric emergency department and may lead to unnecessary administration of antibiotics. Lower respiratory tract infection (LRTI) is one of the most common conditions encountered in pediatric emergency departments (PED) during the winter months, and its management consumes substantial health-care resources.propensity score (PS), defined as the probability of receiving a particular treatment or procedure conditional on the observed covariates: two subjects from the two groups compared (CR or no CR) with the same propensity score can be considered as if they had been randomly assigned to the two groups.Although radiographic findings are often nonspecific, observational studies have reported a higher prevalence of prescription of antibiotics among children in whom CR has been performed.The aim of this study was to assess the effects of CR on the likelihood of antibiotic therapy, using PS-based methods to minimize bias due to non-random radiography assignment.The ethics committee, Comité de Protection des Personnes Ile de France 3, reviewed this project and concluded that this research conformed to generally accepted scientific principles, and to French laws and regulations Informed oral consent was obtained from parents and refusal to participate did not alter the clinical care provided to the patients.We conducted a prospective study in the PED of an urban general hospital in France (Paris suburb). All infants (less than 2 years of age) who presented for suspicion of LRTI between October 2006 and February 2007 were included. Suspicion of LRTI was defined as one or more of the following clinical findings: hypoxia (<95%), age-related tachypnea, signs of respiratory distress  or abnormal breath sounds  obtained by auscultation.Physicians filled in a short questionnaire with a fixed-choice format, collecting information about demographics, case history, physical examination and management. Demographic data included age, gender, history of prematurity, known chronic illness, and previous wheezing episodes. Case history information included duration of symptoms, history of fever, feeding difficulties, and antibiotic use before PED admission. Physicians were also asked to report temperature at first evaluation, respiratory rate, oxygen saturation, retraction signs, auscultation findings, presence of acute otitis media, conjunctivitis or apparent toxicity. They also recorded the following details of the management: CR, antibiotic prescription, and decision about hospitalization. Patients who received antibiotics before admission were excluded from the study because prior antibiotic therapy undoubtedly affects final treatment decision. CR were interpreted by the emergency pediatrician.Descriptive and multivariate data analyses were performed with Stata 11.2 software .As recommended, the propensity score (PS), i.e. the probability to undergo CR, was calculated with a non-parsimonious multivariate logistic regression model  with baseline characteristics at admission as independent covariates (14 variables included) and CR as the dependent dichotomized variable. These baseline characteristics were chosen because they were related to the prescription of both CR and antibiotics.To study the association between CR and antibiotic prescription, and controlling for indication bias, we used two methods: adjustment for and matching on PS.double-robust model as described by D’Agostino and al.The adjustment method consisted of including PS as a continuous adjustment variable in a logistic model with antibiotic prescription as the dependent variable and CR as an independent variable. To ensure that confounders are taken into account as best as possible, we also adjusted on a subset of nine covariates linked to antibiotic prescription, leading to a The matching method consisted of matching two subjects on PS  so as to obtain pairs with the same set of baseline covariates.Missing data (less than 5% per variable) were imputed using a method based on multiple chained equations.n = 697) are shown in Demographic and clinical characteristics of the sample (Distributions of PS are displayed by group (CR or no CR) in The crude OR between CR and antibiotic prescription was 4.9 .After adjustment for PS and for the subset of nine covariates linked to antibiotic therapy, the association between CR and antibiotics prescription remained significant: OR 2.3 .With the matching method, we found a significantly higher rate (18.6% higher) of antibiotic prescription in the group of patients with than in the group without CR .Our study showed that children presenting with LRTI to an emergency department were more likely to receive antibiotics if they underwent CR as part if their initial evaluation. Using PS, this association remained statistically significant after taking into account a possible indication bias. Indeed, antibiotics were prescribed in 18.6% more o infants with CR than in those without CR. This finding suggested that the management of two infants with identical LRTI prognostic baseline characteristics differed, depending on whether or not they had CR: therefore CR had a significant effect on care and might have lead to unnecessary antibiotic therapy. For the purpose of this study, CR images were independently reviewed by two radiologists who were blinded for clinical history. They were simply aware that the imaging was obtained as part of LRTI evaluation but were blinded to other clinical history and outcomes. The Emergency Department practitioners made clinical decisions based on their own interpretation of the CR. This was very important because one of the aim of our study was to determine if the prescription of CR influenced antibiotic prescription, especially without interpretation by a pediatric radiologist.Our findings are consistent with previous studies. A randomized control trial published in 1998 reported that performing CR had consequences for both management and clinical outcome in 522 children who met the WHO case definition of pneumonia.Our study had two main strengths: the choice of the study population and the control of indication bias using PS. We analyzed a large sample of infants under two years with various clinical forms and severity scores of respiratory distress. Our study population was not selected, so it was likely to represent the population encountered in clinical practice. We focused on infants because clinical forms and causes of respiratory tract infections in children under 2 years of age are generally homogeneous and comparable. We had a CR rate of 51% that was consistent with rates reported in previous observational studies Using PS (i.e. the probability that a patient would have a CR) to adjust the association between CR and antibiotic use, we mimicked a “quasi-randomized” experiment in which two subjects with the same baseline characteristics are randomly assigned to two groups (CR or no CR). It can be shown that PS provides an unbiased estimate of the association between CR and antibiotic use, if all confounders are considered.There were several potential limitations to our study. Some relevant factors may have been unobserved or unmeasured. Consequently, our results were not as reliable as those of a randomized trial. Data were collected in only one PED such that extrapolation of the results to other centers or to infants consulting in private practice may be questionable. We also observed a higher rate of antibiotic use in the group that underwent CR (30.9%) than in another previous study reporting antibiotic therapy in 15% of the CR group.Finally, we stressed that CR prescriptions must be limited for several reasons, other than reducing the rate of unnecessary antibiotic therapy. Although the radiation dose associated with CR is small , a recent report on exposure of the French population to ionizing radiation related to acts of medical diagnosis reported a mean of 0.2 CR per year per child under one year old .Our study confirmed the previously observed increase of antibiotic use among children undergoing CR, even when indication bias was taken into account. We can assume that part of this antibiotherapy was unnecessary, in the context of an emergency room. Guidelines should be updated to reduce CR prescriptions in PED for children with suspected LRTI.


Many antibiotic stewardship interventions are not well-enough grounded in theory to understand why they succeed or fail.To understand impressions and antibiotic decision processes during a hospital’s transition to an informatics-supported antibiotic stewardship program that was based on Dual Process theories.A multi-pronged informatics intervention for antibiotic stewardship was implemented in a large US Veterans Affairs Medical Center. The objective of the program was to encourage providers to discontinue unnecessary antibiotics on or after antibiotic day three. It required the use of a template note for self-approval of antibiotics to nudge teams away from continuing vancomycin and piperacillin/tazobactam. It also included a paper handout on antibiotic day three to facilitate the cognitive process of determining whether antibiotics were still needed. During early intervention, six focus groups were conducted with users or potential users. Interviews were recorded and transcribed. Iterative thematic analysis was used to identify recurrent themes from feedback.Themes emerged around alerting and orienting (“it reminds us to think about it”), deliberative reasoning , post-hoc justification and rationalization , impact on role responsibilities and relationships , and change management and impact on workflow processes .Requiring a simple template nudged providers to think about whether they needed antibiotics. Thinking about antibiotics was further aided by clinical information. Nudges can be used to make it harder to continue undesired behaviors and easier to move to desired behaviors without seriously curtailing autonomy. When designing informatics interventions, a knowledge of clinician time, workflow, and thought processes could result in the design of effective nudges.None declared.


Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan fibroinflammatory condition with lymphoplasmacytic infiltrates containing abundant IgG4-positive plasma cells. The immunopathogenesis of the disease and the potential role of triggering autoantigens or infectious factors have not been clearly defined. Immunoglobulin G4-related lung disease is a new and emerging condition in pediatric patients and to date, there have been only two reports regarding pulmonary manifestation of IgG4-RD in children recently published. This is the first report of IgG4-related lung disease in an immunodeficient child with Epstein-Barr virus infection.We report on the case of a 7-year old atopic boy who was hospitalized with an initial clinical and radiological diagnosis of pneumonia, positive Epstein-Barr virus (EBV)-DNA in the blood and defective adaptive immunity. The lung CT showed a consolidated mass lesion adjacent to the posterior wall of the chest and the diaphragm. The child underwent surgical resection of the tumor, and the histologic examination of the lung specimens revealed lymphoplasmacytic infiltrates with fibrosis and vasculitis correlating with IgG4-related lung disease. Subsequent monitoring of the patient with lung CT, pulmonary function tests and IgG4 levels did not show signs of active disease.The diagnosis of IgG4-related lung disease in children is challenging because of its rarity, nonspecific symptomatology and heterogeneous morphological manifestations. Further studies are required in children with pulmonary presentation of IgG4-RD to better understand pathogenesis of this condition, possible immunological or infectious triggering factors, and finally, to determine pediatric patient-targeted therapeutic interventions.The online version of this article (doi:10.1186/s13052-016-0269-0) contains supplementary material, which is available to authorized users. Immunoglobulin G4-related disease (IgG4-RD) is a systemic fibroinflammatory condition with elevated serum IgG4 concentration in most cases and multiorgan involvement. It is characterized by lymphoplasmacytic infiltrates with fibrosis, phlebitis and an accumulation of IgG4-expressing plasma cells at the affected sites. IgG4-related disease is an emerging and increasingly recognized condition with a broad spectrum of clinicopathologic features and various clinical phenotypes, including manifestations in the digestive system, the exocrine and endocrine glands, kidneys, soft tissues, the skin, the cardiovascular system and the respiratory tract , 2. The We report on the case of a 7-year-old boy referred to our pediatric pneumonology, allergology and clinical immunology department because of chest pain and fever. He had the history of a blunt injury to the right side of his chest while he was training in martial arts a few days before admission. He did not manifest either cough, dyspnea, problems with breathing or hemoptysis nor had night sweats, weight loss or chronic loss of appetite.His past medical history was significant for allergic rhinitis and a few episodes of bronchitis which have been treated with inhaled bronchodilators  showed a round lesion in the right lower lobe consistent with parenchymal infiltrations Fig.  and hypoImmunoglobulin G4-related lung disease manifests with a spectrum of nonspecific symptoms such as cough, dyspnea, fever, and hemoptysis which along with pulmonary consolidations may mimic pneumonia or pleural effusion , 6, 9. IAFP, alpha-fetoprotein; C, complement; CD, cluster of differentiation; CEA, carcinoembryonic antigen; CRP, C-reactive protein; CT, computed tomography; CXR, chest X-ray; DNA, deoxyribonucleic acid; EBV, Epstein-Barr virus; EMA, epithelial membrane antigen; EvG, Elastica van Gieson; HPF, high-power field; Ig, immunoglobulin; IgG4-RD, immunoglobulin G4-related disease; IHC, immunohistochemistry; SMA, smooth muscle actin; VATS, video-assissted thoracoscopy; VMA, vanillylmandelic acid; VRT, volume-rendering technique; WT, Wilm’s tumor; β-HCG, beta-human chorionic gonadotropin


IgG4-related disease is a novel disease entity characterized by diffuse lymphoplasmacytic infiltration rich in IgG4-positive plasma cells and fibrosis into multiple organs. There is still controversy over whether some thyroid diseases are actually IgG4-related disease. The objective of this study was to elucidate the clinicopathological features of Graves’ disease with diffuse lymphoplasmacytic infiltration in the thyroid.Among 1,484 Graves’ disease patients who underwent thyroidectomy, we examined their histopathological findings including the degree of lymphoplasmacytic and fibrotic infiltration and levels of IgG4-positive plasma cells in the thyroid. Their clinical pictures were defined by laboratory and ultrasonographic evaluation.A total of 11 patients (0.74%) showed diffuse lymphoplasmacytic infiltration in the stroma of the thyroid gland. Meanwhile, other patients showed variable lymphoid infiltration ranging from absent to focally dense but no aggregation of plasma cells in the thyroid gland. Based on the diagnostic criteria of IgG4-related disease, 5 of the 11 subjects had specifically increased levels of IgG4-positive plasma cells in the thyroid. Fibrotic infiltration was present in only 1 patient developing hypothyroidism after anti-thyroid drug treatment for 4 years, but not in the other 10 patients with persistent hyperthyroidism. Obliterative phlebitis was not identified in any of the 11 subjects. Thyroid ultrasound examination showed 1 patient developing hypothyroidism who had diffuse hypoechogenicity, but the other hyperthyroid patients had a coarse echo texture.In our study, Graves’ disease patients with persistent hyperthyroidism who had diffuse lymphoplasmacytic infiltration rich in IgG4-positive plasma cells in the thyroid showed no concomitant fibrosis or obliterative phlebitis. IgG4-related disease is a novel disease entity characterized by diffuse lymphoplasmacytic infiltration rich in IgG4-positive plasma cells into multiple organs. Concomitant fibrosis and obliterative phlebitis are usually detected around IgG4-positive plasma cells. An elevated concentration of serum IgG4 beyond 135 mg/dL) is helpful to identify IgG4-related disease before histopathological examination from biopsy or surgical specimens [35 mg/dL Among autoimmune thyroid diseases, diffuse lymphoplasmacytic infiltration is the most characteristic feature of Hashimoto’s thyroiditis, in which the follicular epithelium can be quite scant in areas of intense lymphoplasmacytic infiltration . Plasma Here, we screened for the degree of lymphoplasmacytic infiltration using thyroid specimens of Graves’ disease patients. Levels of IgG4-positive plasma cells and further clinicopathological features were evaluated among subjects with diffuse lymphoplasmacytic infiltration.From 2004 through 2012, a total of 1,647 patients with Graves’ disease ; 11–87 years (range)) underwent total or near-total thyroidectomy at Kuma Hospital. The diagnosis of Graves’ disease was based on the presence of hyperthyroidism, positive thyroid stimulating antibody (TRAb), and increased radioiodine uptake by the thyroid. Among them, 163 patients were excluded due to the main reason for surgical resection of accompanied thyroid tumors. Consequently, we examined the degree of lymphoplasmacytic infiltration in the stroma and other histopathological findings using the thyroid specimens of 1,484 patients. The present study was approved by the ethics committee of Kuma Hospital, and written informed consent was obtained from all the adult subjects as well as the next of kin on behalf of minors for the use of samples for research purposes and for publication of accompanying images. A copy of the written consent is available for review upon requests.Concentrations of serum TSH, free triiodothyronine (FT3), and free thyroxine (FT4) were measured with a chemiluminescent immunoassay . The reference ranges used for serum TSH, FT3, and FT4 were 0.30–5.00 μIU/mL, 1.70–3.70 pg/mL, and 0.70–1.60 ng/dL, respectively. Serum TRAb were measured with a second-generation enzyme-linked immunoassay  through July 2008 and with a third-generation electro-chemiluminescent immunoassay  from August 2008 through December 2012. The upper cut-off limits of values of the second- and third-generation TRAb are 15% and 1.9 IU/L, respectively. Serum anti-thyroglobulin (TgAb) and anti-thyroid peroxidase (TPOAb) antibodies were measured with commercially available hemagglutination methods  or with an electro-chemiluminescent immunoassay . Serum IgG4 levels were measured by nephelometry using IgG subclass (BS-NIA) kits . Ultrasonographic examinations were performed as previously described [After surgical resection, thyroid tissues were routinely fixed in 10% neutral buffered formalin and specimens were embedded in paraffin. Serial sections (4 μm thick) were cut from each paraffin block. For light-microscopic examination, the sections were stained with hematoxylin-eosin (HE). All HE sections of the 1,484 surgical specimens were reviewed by a pathologist (M.H.) to evaluate the histopathological findings, including the levels of lymphoid and plasmacytic cells. The severities of stromal fibrotic changes and obliterative phlebitis were examined and expressed as 3+, severe; 2+, moderate; 1+, mild;-, negative.Immunostaining for IgG4  and IgG  was performed following a previously reported method . In 3 hiP<0.05 was accepted as indicating significance.The age at surgery, duration of Graves’ disease, levels of TRAb, TgAb, and TPOAb, and weight of the resected thyroid tissue were compared between the subjects and controls employing the Student’s t-test. Among the 1,484 patients with Graves’ disease, 11 subjects (0.74%) showed diffuse lymphoplasmacytic infiltration in the stroma of the thyroid gland . FurtherSerum IgG4 measurement results before and after thyroidectomy were available for 4 of the 11 patients. Two patients who presented with more than 135 mg/dL serum IgG4 before thyroidectomy had higher levels of IgG4-positive plasma cells in the thyroid . Levels To compare several clinical findings between the 11 subjects with diffuse plasmacytic infiltration and controls lacking plasmacytic aggregation, 80 patients were enrolled as a control group by the consecutive extraction of 40 patients from January 2007 and by that of 40 patients from January 2011 . Titers of third-generation TRAb, TgAb, and TPOAb were significantly elevated in the 11 subjects, but the age at surgery, duration of Graves’ disease, weight of the resected thyroid tissue, and levels of second-generation TRAb showed no significant difference between the subjects and controls . ThyroidIn the spectrum of autoimmune thyroid diseases, several histopathological and immunological findings overlap between Graves’ disease and Hashimoto’s thyroiditis ,13. In tA dense lymphoplasmacytic infiltration is the key morphologic feature of IgG4-related disease . In HashIn our study, no persistent hyperthyroid patients with Graves’ disease who had high levels of IgG4-positive plasma cells in diffuse lymphoplasmacytic infiltration showed concomitant fibrosis or obliterative phlebitis . The diaWhile persistently hyperthyroid patients with Graves’ disease presented with no association with IgG4 thyroiditis in our study, 1 Graves’ patient (case 1) who rapidly developed hypothyroidism with a large goiter showed marked stromal fibrosis along with IgG4-positive plasma cells in the thyroid, which is consistent with IgG4 thyroiditis . It is wIn conclusion, persistent hyperthyroid patients with Graves’ disease who show the intense infiltration of IgG4-positive plasma cells in the thyroid manifest no histopathological association with IgG4 thyroiditis or IgG4-related disease. However, some Graves’ patients who rapidly develop hypothyroidism with high levels of IgG4 in serum or tissue and a diffuse hypoechoic structure by ultrasound are likely to present with histopathological compatibility.S1 TableGraves’ ophthalmopathy (GO) was diagnosed based on more than 3 by clinical active score and/or CT images such as extraocular muscles enlargement or increased orbital fat in clinical records.(DOCX)Click here for additional data file.


Variables derived from electroencephalogram like cerebral state index (CSI) have been used to monitor the anesthesia depth during general anesthesia. Observed evidences show such variables have also been used as a detector of brain death or outcome predictor in traumatic brain-injured (TBI) patients.The current study was designed to determine the correlation between Glasgow coma score (GCS) and CSI among TBI patients.In 60 brain-injured patients who did not need and receive sedatives, GCS and CSI were daily measured during the first ten days of their hospital stay. Correlation between GCS and CSI was studied using the Pearson's correlation test. The Gamma agreement coefficient was also calculated between the two variables for the first day of hospitalization.A significant correlation coefficient of 0.611-0.796 was observed between CSI and GCS in a ten-day period of the study (P < 0.001). Gamma agreement coefficient was 0.79 (P < 0.001) for CSI and GCS for the first day of hospitalization. An increased daily correlation was observed in both CSI and GCS values. However, this increase was less significant in CSI compared with the GCS.A statistically significant correlation and agreement was found between GCS and CSI in the brain-injured patients and GCS was also found to be more consistent and reliable compared with CSI. Traumatic brain injury (TBI) is one of the major causes of post-injuries mortality and disability, worldwide . TBI is The purpose of this study was to assess the degree of agreement and correlation between GCS as a subjective and CSI as an objective method to evaluate the consciousness in TBI patients.™Danmeter, Odese, Denmark). To measure CSI, the patient's skin was prepared by swapping with alcohol and wiping. Standard electrocardiogram electrodes  were then positioned as recommended by the manufacturer. After controlling of electrode impedance, the numerical value of CSI, ranging 0-100, was recorded for each patient by a nurse who was blinded to the study. Patients' neurological status was assessed using the GCS and recorded, at the moment of CSI measuring.This prospective observational study was performed after approval and informed consent in patients with mild (GCS 13-15), moderate (GCS 9-12) and severe (GCS 3-8) head injury. All patients had been admitted to the neurosurgical ICU of Shahid-Beheshti Hospital, Kashan University of Medical Sciences, Iran, between December 2011 and March 2012. Demographic characteristics, mechanism of head injury, GCS, computed tomography scan, and neurological examination at the time of admission were recorded. Patients demanding sedatives were excluded from the study. Since some of the patients underwent intracranial surgical operation, to eliminate the effect of anesthesia on consciousness indices, GCS and CSI were measured three hours after recovery from general anesthesia in these cases. Respiratory and metabolic disorders were considered as exclusion criteria, due to their potential effects on GCS, and patients with such problems did not enter the study. CSI was measured after the ICU admission and afterwards on a daily basis for the next nine days. CSI was measured using CSI monitor  aged (mean ± SD) 33.4 ± 17.1 years were included in the study. The injury mechanism was road traffic accident in 49 cases, falling in nine and assault in two patients. Fourteen patients had mild injury, 13 had moderate and 33 had severe injuries. The majority of cases were admitted to the ICU due to subdural hematoma and during the course of the study 26 cases underwent surgical operation due to intracranial mass lesion and 17 patients (28.3%) expired due to their condition deterioration . Ten setThe present study showed that there is a statistically significant correlation and agreement between GCS and CSI in patients with traumatic brain injury. Although we have found a correlation between GCS and CSI in patients during all days of hospitalization, interestingly, we found GCS was more consistent than the CSI.Basically, cerebral state index has been developed for monitoring the depth of anesthesia during operations. Its predecessor, BIS, has been also used for monitoring of consciousness during the management of both traumatic and non-traumatic coma , 17. The


Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains . These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged. Ligand-stimulated Met acts, furthermore, as an angiogenic and neurotrophic factor.3 HGF/SF and Met are essential to several steps of embryogenesis, experiments on transgenic mice having shown that they are necessary for formation of the placenta, liver, limb muscle, neurons, and lung airspace.8 In adults, HGF/SF and Met promote regeneration of several organs, including the liver, kidneys, and thymus.13Met is a receptor tyrosine kinase expressed predominantly by epithelial cells and activated by its stromal ligand, hepatocyte growth factor/scatter factor (HGF/SF). Met activation stimulates a biological program called invasive growth,et al.).14 A direct link between Met and cancer has been evidenced by observation of Met germinal mutations linked to hereditary papillary renal carcinoma.15 Met and/or HGF/SF are/is also overexpressed in several human cancers.16 Given its important oncogenic activity, Met is the target of many therapeutic agents currently under clinical investigation.17Aberrant Met and HGF/SF signaling contributes to promoting tumorigenesis and metastasis -10,20 generating a soluble N-terminal fragment (Met-NTF), which is released into the extracellular space, and a membrane-anchored C-terminal Met fragment (Met-CTF). The latter is in turn efficiently degraded by the lysosome and by further γ-secretase cleavages. Constitutive degradation of the Met receptor by PS-RIP contributes to regulating its half-life.Downregulation of Met following its activation by HGF/SF is an important negative regulatory mechanism preventing receptor overactivation. We have previously shown that Met expression and activity are also controlled by proteolytic cleavages. Under steady-state conditions, Met is processed by PS-RIP (presenilin-regulated intramembrane proteolysis).21 within its C-terminal tail and its intracellular juxtamembrane domain. These cleavages remove the C-terminal tail of Met and separate the extracellular ligand-binding domain from the intracellular kinase domain. The generated 40-kDa intracellular fragment, previously called ‘p40Met' and here called p40Metcaspase, can increase cell death by promoting mitochondrial permeabilization.23 Removal of the C-terminal tail of Met is required for the efficient pro-apoptotic action of the fragment. This pro-apoptotic function of Met makes it a member of the dependence receptor family.24 Met cleavages are illustrated in Figure 6a.Under apoptotic conditions, Met is cleaved by caspases25 Necrosis is an adenosine triphosphate (ATP)-independent cell death mechanism featuring early plasma membrane and organelle disruption. Many pathways can lead to cell necrosis, including calcium overload. This type of cell stress has been amply described in the nervous system, where an increase in intracellular calcium results in neuronal injury and neurodegenerative diseases. In many other cell types, calcium ionophores such as ionomycin can induce rapid necrosis. An increase in intracellular calcium triggers activation of several proteases, including calpains and cathepsins.28 Calpains are calcium-dependent proteases capable of cleaving multiple substrates and involved in regulating various cellular processes, including migration, autophagy, apoptosis, and necrosis. Interestingly, the effector role of calpains during necrosis is reminiscent of the function of caspases during apoptosis. Caspases are directly involved in morphological changes observed during apoptosis, while calpains can cleave cytoskeletal proteins such as spectrin and tubulin, thus favoring dismantling of cell structure during necrosis.31Although the mechanisms underlying apoptosis have been studied extensively, necrosis has only recently been described as a regulated cell death mechanism.32 some such cells remain sensitive to other cell death processes, including necrosis.33 Thus, a better understanding of the mechanisms underlying necrosis is important, as it could help to elaborate novel therapeutic strategies. Here we show that calcium stress induced by calcium ionophores triggers Met degradation during necrotic cell death. This loss of Met receptor occurs early during the process and is mediated by Met cleavages: by calpains in its intracellular part and by metalloproteases in its extracellular part. These cleavages generate an extracellular fragment and an intracellular fragment with a molecular weight close to that of p40Metcaspase.Although apoptosis resistance is a hallmark of many cancer cells,caspase  polymerase (PARP), Annexin V staining, along with degradation of full-length Met, and production of the pro-apoptotic fragment p40Metcaspase . In contcaspase . Ionomyc and AKT . It is wTo see whether the 40-kDa fragment observed during calcium stress is generated by proteolytic cleavage, we incubated epithelial cells with various protease inhibitors before ionomycin treatment. In contrast to caspase and cathepsin inhibitors , the calcaspase, this novel fragment was called p40Metcalpain.To confirm the involvement of calpains, we incubated purified calpain 1 with cell extracts or with the recombinant intracellular domain of Met. In the presence of calcium, cleavage of full-length Met in MCF-10A lysate by purified calpain 1 generated a Met-CTF fragment of the same size as the calcium stress fragment . Similar35 To determine the calpain cleavage sequence, we analyzed the in vitro Met cleavage product by mass spectrometry. AspN digestion followed by mass spectrometry revealed that the first N-terminal peptide begins at amino acid D1041, suggesting that cleavage occurs before this sequence , but not by the calpain inhibitor calpeptin , which is efficiently degraded by the lysosome or further cleaved by necrosis . To assenecrosis .18, 19 A chimera . In cont21 Although many immunohistochemistry (IHC) studies have shown Met to be overexpressed in a variety of cancers,14 the Met cleavage state was never characterized in these studies. About half of all non-small-cell lung cancers (NSCLCs) are known to overexpress Met. We thus analyzed the amount and state of Met by both IHC and western blotting in a library of 13 surgically resected NSCLCs , and p40Metcaspase and p40Metcalpain were generated by inducing apoptosis or necrosis, respectively,  and Bax (Bcl-2-associated X-protein), which are resistant to many apoptosis inducers, have been found to die from necrosis following calcic stress.33 Yet the mechanisms underlying calcium-stress-induced necrosis are still poorly understood. As Met is actively involved in both survival and apoptosis, we have sought to determine its fate and potential role during calcium-stress-induced necrosis.Necrosis is a caspase- and ATP-independent cell death process characterized by a loss of plasma membrane and organelle integrity. It lacks features of apoptosis, such as phosphatidylserine externalization and apoptotic body release. Although first defined as accidental cell death, necrosis is currently regarded as a programmed cell death mechanism, as pharmacological inhibitors or gene deletions can protect cells from this demise. Some necrotic cell death can be induced by calcium overload, for example, in neural cells in the case of neurodegenerative disorders or following ischemia.calpain generation can be inhibited efficiently by treatment with a pharmacological calpain inhibitor and partially by means of interfering RNAs targeting calpains 1 and 2. This demonstrates that these two proteases, and likely other calpains, are involved in Met cleavage. We demonstrate as well that calcium stress induces autocatalytic cleavage of calpain 1, consistent with activation of calpain by the intracellular calcium level.38In vitro cleavage of Met by calpain 1 generates a main fragment of ~40 kDa, confirming the involvement of calpain 1.We demonstrate here that calcium-stress-induced necrosis causes a substantial decrease in Met within minutes following treatment with a calcium ionophore. Consistently with this decrease, HGF stimulation can no longer induce Met phosphorylation or activation of downstream signaling pathways under these conditions. This Met degradation might prevent cell protection triggered by ligand-dependent activation of Met. Rapid Met degradation, associated with detection of fragments, suggests that Met undergoes proteolytic cleavages during this process. Accordingly, we have found that Met is cleaved by both calpains and membrane metalloproteases. We show that p40Met1036-S1037 sequence, and the presence of the Met C-terminal tail in p40Metcalpain. Thus, the p40Metcaspase and p40Metcalpain fragments differ at both their N- and C-terminal ends. Consistently with the fact that p40Metcalpain retains the C-terminal tail of Met, known to prevent its pro-apoptotic action,22 we show here that it does not induce increased caspase 3 activity and is thus not a pro-apoptotic fragment.Our mass spectrometry and immunoblot analyses reveal juxtamembrane cleavage, possibly at the LTcalpain, calcium overload induces an increase in two other Met fragments: Met-NTF (in conditioned medium) and the labile Met-CTF, both known to be generated by PS-RIP.18 A metalloprotease inhibitor was found to impair production of these fragments and to rescue the full-length Met receptor, demonstrating that calcium stress triggers Met shedding. It is worth noting that inhibition of calpain activity did not rescue full-length Met, suggesting that PS-RIP is the main contributor to the latter's disappearance. The contribution of calpain is hard to assess, as calpain inhibitors only reduced, but did not totally prevent, p40Metcalpain generation.In addition to intracellular p40Met41 During calcium-stress-induced necrosis, the amount of released Met-NTF strongly increased, suggesting an increased decoy effect. We thus propose that during calcium-stress-induced necrosis, cleavages decrease the amount of Met receptor and generate a potential extracellular decoy fragment. Interestingly, the N-terminal counterpart of p40Metcaspase is a membrane-anchored fragment that can act as a decoy for HGF/SF.42 Hence, the stable N-terminal Met fragment generated during apoptosis could be a membrane-anchored decoy, while the fragment generated during calcium stress could be a released decoy, both decoys being able to cause inhibition of an HGF/SF-induced survival response. In the future, it will thus be important to evaluate the decoy role of Met-NTF and its importance in necrosis expansion to the surrounding tissues.Although Met PS-RIP is mainly involved in Met degradation through generation of labile intracellular fragments, the released Met-NTF is stable and can act as a decoy for HGF/SF.43 Although the anti-apoptotic properties of HGF/SF have been well described,44 further work is needed to elucidate the necrosis-countering pro-survival mechanisms of this ligand.HGF/SF stimulation partially protects cells against calcium-ionophore-induced necrosis and reduces Met cleavage by calpain. Other growth factors have also been shown to protect cells against necrosis, such as the insulin-like growth factor-1, which inhibits calcium-ionophore-induced photoreceptor cell death through expression of the calpain inhibitor calpastatin.caspase, and p40Metcalpain. These fragments were mainly detected in tumors showing high levels of full-length Met, except for p40Metcalpain, detectable in low-scoring tumors. Thus, full-length Met is responsible for only part of the detected signal, as its presence is accompanied by that of multiple C-terminal fragments. This suggests that the level of full-length Met could be overestimated in tumor cells evaluated by IHC. In addition, although the subcellular localization of Met is difficult to establish by IHC, these tumor cells display both plasma membrane and cytoplasmic staining. The latter might be associated with detection of Met C-terminal fragments such as the p40Metcaspase and p40Metcalpain. Consistently, a cytoplasmic and even a nuclear localization of Met have been described in breast cancer samples and mesothelioma tumors.46In NSCLC tumors overexpressing Met, we have identified several C-terminal fragments, including the previously identified Met-CTF (p55 Met), p45 Met generated in highly confluent cells (manuscript under revision), p40Metcaspase and p40 Metcalpain are generated by different molecular mechanisms, and that they do not coexist in the same cell. Because Met fragments produced in NSCLC samples were detected by western blot, which involve the lysis of a cell population, we cannot assert that p40 Metcaspase and p40 Metcalpain are not produced in the same cells of the tumor. However, it is well established that tumor tissues undergo various stresses influencing differently the cell fates. Thus, the diversity of detected Met fragments might correspond to a diversity of cellular conditions within lung tumors. For instance, the presence of p40Metcaspase might be associated with apoptotic cells, whereas the presence of p40Metcalpain might be associated with a necrotic area. In these tumor samples, necrotic areas are indeed frequently observed and correlate with a strong decrease of total Met (data not shown). Detection of the labile Met-CTF (p55 Met) in tumor samples suggests that lysosomal degradation is altered in these tumors or that the amount of generated fragment exceeds the capacity of the cells to degrade it. We have demonstrated in a cell model that most of the Met fragments are associated with degradation of the full-length Met receptor. Thus, in tumor samples overexpressing Met, several proteolytic degradation mechanisms might compensate for receptor overexpression, thus limiting the amount of receptor.We demonstrated in cell lines that p40 Metcaspase somewhat resembles p40Metcalpain, these two fragments appear to be functionally different: the former can favor cell death while the latter is unable to promote such a response.At first glance, the mechanism of Met degradation appears similar during apoptosis and calcium-stress-induced necrosis: in both cases, it involves generation of an intracellular fragment of ~40 kDa. In both processes, the extracellular ligand-binding domain of Met is separated from the intracellular kinase domain, preventing survival. Calcium-stress-induced necrosis, however, triggers a more complex proteolytic process, also involving cleavage by membrane metalloproteases. Consequently, the cleavages occurring during necrosis are quicker than caspase cleavages, leading to Met degradation within minutes of stress induction. In addition, while p40MetN-(2-quinolyl)-l-valyl-l-aspartyl- methylketone), the calpain inhibitors ALLN  and calpeptin, the metalloprotease inhibitor TAPI-1, and the H+-pump inhibitor bafilomycin A1 were purchased from Calbiochem. The cathepsin inhibitor Z-FA-FMK (Z-Phe-Ala fluoromethyl keton) was obtained from Bachem . The proteasome inhibitor lactacystin was from Sigma-Aldrich. The γ-secretase inhibitor compound E was from Alexis/Coger .Ionomycin and A23187 were purchased from Santa Cruz Biotechnology . Staurosporine was obtained from Sigma-Aldrich . Purified calpain 1 was obtained from Calbiochem . Recombinant human HGF was obtained from Peprotech . The caspase inhibitor QVD-OPh (μg/ml); Life Technologies). HEK-293T cells were cultured in DMEM supplemented with 10% FBS, 1% non-essential amino acids (Life Technologies), and antibiotics. MCF-10 A cells were cultured in DMEM and HAM's F12  supplemented with 5% horse serum (Life Technologies), 500 ng/ml hydrocortisone , 20 ng/ml epidermal growth factor (Peprotech), 10 μg/ml insulin (Sigma-Aldrich), 100 ng/ml cholera toxin , and 1% antibiotics. Calcium-free DMEM was purchased from Life Technologies.MDA-MB-231 and GTL-16 cells were cultured in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum  and 1% antibiotics (penicillin (10 000 U/ml)–streptomycin  Met (no. 3126), phosphorylated (S473) Akt (no. 9271), and active caspase 3 (no. 9661) and mouse monoclonal antibody directed against phosphorylated (T202/Y204) Erk were purchased from Cell Signaling Technology . Mouse monoclonal antibody against the kinase domain of Met (3D4) was purchased from Life Technologies. Mouse monoclonal antibody against glyceralehyde-3-phosphate dehydrogenase , rabbit polyclonal antibodies against the C-terminal domain of human PARP-1 (H250), against Akt (H136), and against ERK2 (C14), and goat polyclonal antibodies against the calpain 1 large subunit (C-20) and the calpain 2 large subunit (C-19) were purchased from Santa Cruz Biotechnology. Rabbit monoclonal antibody directed against the C-terminal tail of Met (SP44) was purchased from Roche . Mouse monoclonal antibody directed against the extracellular domain of Met (MAB3582) and goat polyclonal antibody directed against the extracellular domain of Met (AF276) were purchased from R&D Systems . Mouse monoclonal antibody directed against the C-terminal domain of Met (DL-21) was kindly provided by Dr. Sylvia Giordano . Regions recognized by the anti-Met antibodies are shown in the caspase was constructed as described previously.22 The vector expressing p40Metcalpain (Met S1037–S1390) was constructed as follows. The portion of Met between D1030 and S1390 was amplified by PCR from pRS2 hu Met  used as template, with the following primers 5′-AGGGATCCATGGACATGTCCCCCATCCTAACTAG-3′ containing a BamH1 restriction site and 5′-AGCTCGAGCTATGATGTCTCCCAGAAGGAG-3′ containing a Xho1 restriction site. The PCR product was subcloned into pcDNA3 between the BamH1 and Xho1 restriction sites. The amino acids D1030–S1037 were removed with the QuikChange site-directed mutagenesis system of Stratagene , with the following primers: 5′-GTACCGAGCTCGGATCCATGAGTGGGGACTCTGATATATC-3′ and 5′- GATATATCAGAGTCCCCACTCATGGATCCGAGCTCGGTAC-3′. The vector expressing Flag-p40Metcalpain was constructed as follows. pcDNA3 p40Metcalpain was subcloned into pcDNA3 FLAG between the BamH1 and Xho1 restriction sites.The vector expressing Flag-p40Met23 For gene silencing, a suspension of 400 000 cells was incubated for 20 min with a mix of 3 μl/ml Lipofectamine 2000 (Invitrogen) and 60 nM small interfering RNA (siRNA), and then plated in a six-well plate in complete medium. The Met-targeting siRNAs were a pool of three stealth siRNAs (Invitrogen) . The corresponding pool (3 mM) was mixed with control siRNA so as to achieve a total siRNA concentration of 60 nM. The siRNAs targeting calpain 1 and calpain 2 (Sigma-Aldrich) were pools of two siRNAs: calpain 1: (5′-CUAUUGGCUUCGCGGUCUAdTdT-3′ and 5′-GGAACAACGUGGACCCAUAdTdT-3′) and calpain 2: (5′-CGCUAUUCAAGAUAUUUAAdTdT-3′ and 5′-GAAACUGAUCCGCAUCCGAdTdT-3′). Mixtures totaling 60 nM siRNA, containing each pool at 30 nM or one pool at 30 nM and a control siRNA at 30 nM, were prepared.Transfections of HEK-293T and MCF-10 A cells with the reagents polyethyleneamine Exgen 500 , and Jet Prime  were performed as previously described.47Western blotting was performed as previously described.μg/ml) or goat anti-extracellular Met antibody (4 μg/ml)) or in combination (anti-Flag antibody (1 μg/ml) and anti-active caspase 3 antibody (1 : 500)). The cells were washed with PBS and incubated for 60 min at room temperature with secondary antibodies diluted to 2 μg/ml. Secondary antibodies were used alone (green-fluorescent Alexa Fluor 488-conjugated anti-goat IgG (H+L), far red-fluorescent Alexa Fluor 647-conjugated anti-rabbit IgG (H+L)) or in combination (green-fluorescent Alexa Fluor 488-conjugated anti-mouse IgG (H+L) and red-fluorescent Alexa Fluor 594-conjugated anti-rabbit IgG (H+L)). The cells were washed with PBS and the nuclei were counterstained with Hoechst 33258. Coverslips were mounted with glycergel mounting medium . Slides were observed in oil immersion with an AxioImager Z1 (Carl Zeiss), numerical aperture, ECL-PLAN NEOFLUAR × 40 NA 1.3, with a monochrome Zeiss AxioCam MRm camera and Zen Blue acquisition software.Cells were grown on glass coverslips for 24 h and then washed, fixed in 4% paraformaldehyde at room temperature for 10 min, and permeabilized in PBS 1 × and 0.5% Triton X-100 at room temperature for 10 min. The cells were then washed and blocked in PBS and 0.2% casein for 30 min. Primary antibodies were incubated for 1 h at room temperature, either alone . Briefly, cells were harvested by centrifugation and incubated with Alexa Fluor 488-conjugated Annexin V and PI. Then staining was measured with a Tali image-based cytometer (Life Technologies) to determine the amounts of unstained, singly stained, and doubly stained cells.2. The reaction was stopped by adding 3 × Laemmli buffer and heating at 95 °C for 4 min. Samples were analyzed by western blotting.Cells were lysed in lysis and reaction (LR) buffer . Equal amounts of cell lysate or 5 ng recombinant human Met were incubated at 30 °C with the indicated concentrations of purified calpain 1 and CaClμM CaCl2 and 2.5 nM purified calpain 1. The reaction was stopped by adding 3 × Laemmli buffer and heating the sample at 95 °C for 4 min. After electrophoresis, the polyacrylamide gel was fixed in 40% methanol, 7% acetic acid solution and stained with Coomassie blue. Stained band corresponding to Met fragment was excised from the gel, reduced, alkylated with iodoacetamide (10 mg/ml in 20 mM NH4HCO3), and digested overnight with 100 ng AspN endoproteinase . The resulting peptide mixture was eluted from the gel, desalted, and spotted onto a MALDI plate with freshly dissolved α-cyano-4-hydroxycinnaminic acid . Mass spectrometry was performed by MALDI-TOF-TOF Autoflex Speed . MS and MS/MS data were analyzed with BioTools software . Peptides were identified with Mascot, http://www.matrixscience.com/.For mass spectrometry, 900 ng recombinant human Met was incubated at 30 °C for 30 min in LR buffer with 250 g for 30 min and proteins in the supernatant were quantified by the BCA Protein Assay (Pierce). Equal amounts of protein were analyzed by western blotting. For IHC, FFPE tissue sections were stained with hematein/eosin/safran and IHC was performed with an antibody against the intracellular domain of Met . Met expression was scored according to the study of Spigel et al.48 .After surgery, tumor samples were divided into two parts. One was frozen in a Snapfrost fast freezing system  and stored at −80 °C, and the other was formaldehyde fixed and paraffin embedded (FFPE). For western blot analysis, frozen tumor samples were sliced into pieces ~1.2 mm in diameter and transferred into Lysing Matrix type D tubes containing ceramic beads  in the presence of RIPA buffer . The samples were lysed with a FastPrep homogenizer . The samples were then centrifuged at 20 000 ×