Δ7−8. METΔ7−8 is located predominantly in the cytosol and is constitutively active. The auto-activating nature of METΔ7−8, in combination with a lack of transmembrane localization, renders METΔ7−8 not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6 % of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named MET MET proto-oncogene (chromosome 7q31.2) encodes the tyrosine kinase membrane receptor MET , which is essential during development. Signaling from the receptor controls epithelial-to-mesenchymal transition (EMT) of myogenic precursor cells during differentiation into skeletal muscle cells . Blots were scanned on the Odyssey imager (LI-COR Biosciences).A homology model for the new hybrid IPT-domain using the WHAT IF & YASARA Twinset was generated , 60. We E98 and U87 cells, grown on Nunc Lab-Tek chamber slides (Sigma-Aldrich) to 40 % confluence, were fixed with 2 % PFA in 0.1 M phosphate buffer (pH 7.4) for 15 min at RT, followed by three washes with PBS, 30 min glycine incubation (100 mM in PBS) to quench PFA-induced autofluorescence, three PBS washes and permeabilization with digitonin for 10 min (100 μM in PBS). Aspecific binding was blocked by incubation with 20 % normal goat serum in PBS/1 %BSA for 20 min, and cells were incubated overnight at 4 °C in a humidified chamber with PBS/1 %BSA containing (combinations of): mouse monoclonal anti-CD44 antibody ; rabbit anti-MET ; rabbit anti-P-MET ; mouse anti-EEA-1 ; anti-CLIMP-63 . Primary antibodies were detected using Alexa Fluor 488- or 594-labeled secondary goat-anti-mouse and Alexa Fluor 488- or 568-labeled goat-anti-rabbit IgGs , 1:200 diluted in PBS/1 %BSA. Q-nuclear deep red  was used to stain nuclei, and cells were mounted in Fluoromount G with DAPI . Cells were analyzed using a confocal laser scanning microscope (Leica SP2 CLSM) and Leica confocal software.E98 and A549 cells were allowed to adhere and grown to 80 % confluence in a 6-well plate . Cells were washed 3 times with ice-cold PBS and incubated for 30 min at 4 °C with 0.5 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (ThermoFisher Scientific). This compound does not penetrate cells and biotinylation is therefore confined to exposed membrane proteins. In parallel, Tris-free RIPA extracts, prepared from equivalent numbers of E98 or A549 cells by repeated washing over Amicon Ultra K10 centrifugal filters , were treated similarly to biotinylate all cellular proteins. The reaction was quenched by washing cells or cytosolic proteins three times with 100 mM glycine/PBS and two times with PBS (using K10 columns in the case of protein lysates). Intact biotinylated cells were then subjected to lysis with RIPA buffer. Protein concentrations in all lysates were determined using the BCA protein concentration assay (ThermoFisher Scientific). MET was immunoprecipitated from 200 µg total protein in a volume of 200 μl using anti-MET  for 1 h at 4 °C. Immune complexes were captured by incubation for 30 min at 4 °C with 10 μl prot A agarose slurry , followed by centrifugation  and three PBS washes. Immune complexes were solubilized by heating (5 min 95 °C) in 30 μl 2× SDS-PAGE sample buffer . Samples were subjected to 10 % SDS-PAGE and western blotting as described above.n = 80) were obtained from the Radboud UMC and from the University of Cologne. Sarcoma (n = 25) and castration-resistant prostate cancer (CRPC) tissues (n = 43) were obtained from Radboud UMC. RNA was isolated from tissues using the mirVANA RNA isolation kit (Life Technologies) according to standard procedures. For cDNA synthesis, 1 μg RNA was reverse transcribed using the Quantitect Reverse Transcription Kit  using hexanucleotides. To distinguish wtMET from METΔ7−8-transcripts, a PCR was performed in AmpliTaq Gold 360 mastermix  using primer MET1997Fw , and primer MET2342Rv . This PCR results in a 105-bp product for METΔ7–8 and a 345-bp product for wtMET. Conditions were: denaturation 95 °C, followed by 35 cycles of 95 °C denaturation; annealing at 58 °C, 30 s; elongation at 72 °C, 1 min. A final elongation step of 7 min was done at 72 °C.Glioma tissues  was about 14-fold. FISH analysis using probes specific for MET and chromosome 7 centromere confirmed the amplification . The MET PCR product from E98 was 240 bp smaller than that from U87 and CaCo2 cDNA  marker CLIMP-63 suggested that METΔ7–8 in E98 cells is predominantly retained in the RER , containing a C-terminal biotin tag. Whereas the extracellular domain of wtMET was properly processed and secreted into the culture medium . Intragene deletions spanning multiple exons are, however, in general more difficult to recognize in whole-exome sequencing (WES). Furthermore, the coverage of the exact location of the deletion is poor in the mostly used WES protocols, and as can be seen in Fig. wt and METΔ7–8. FISH analysis confirmed the presence of the MET amplification in the original tumor  and a number of other tumor types in which MET has been suggested to play an important role  [Δ7–8 was found in 6 out of 102 gliomas, both grade III and IV , and was not detected in the other tumor types tested  , 28. METomas n = 02 and a Δ7–8 resulted from a mutation or from exon skipping or alternative splicing, such as suggested for MET variants lacking exon 10 or 14 [Sanger sequencing of the smaller PCR products of one patient with glioma confirmed the deletion of exons 7 and 8 (not shown). Of note, due to the lack of high-quality genomic DNA from these clinical samples we could not discriminate in these samples whether MET10 or 14 .MET oncogene is causally involved in this phenotype in subsets of tumors [A major problem in glioma treatment is diffuse growth in the neuropil, and it has been suggested that the f tumors . Severalf tumors . Also, tf tumors , but thif tumors .Δ7–8 and we therefore hypothesize that the novel IPT1-2 fusion domain in METΔ7–8 is involved in defective furin cleavage, intracellular retention of the uncleaved perform and auto-activation of the uncleaved preform.We previously reported that MET is crucially implicated in proliferation, survival and migration of (EGFR-negative) E98 glioma cells in vitro. To further investigate the involvement of MET in the biological behavior of E98 cells, we attempted to knock down the protein by a lentiviral shRNA approach. Attempts to generate stable MET knockdowns using 2 different MET shRNAs failed since both shRNA encoding lentiviruses were lethal to E98 cells, in contrast to control viruses (data not shown). In agreement with a crucial role of MET for cell survival, we showed that treatment with the MET tyrosine kinase inhibitor cabozantinib prolongs survival of mice carrying orthotopic E98 xenografts . Based oΔ7–8 did not accumulate in early endosomes, suggesting that the underlying mechanism of intracellular retention is different [Δ7–8 that is cleaved reaches the cell membrane, but this fraction is insignificant with respect to the auto-active component in the cytosol.Recently, effects of various activating mutations in the MET TK domain have been analyzed in detail. These mutants are expressed on the cell surface but are subject to increased rates of turnover, resulting in accumulation in early endosomes where they can still signal . METΔ7–8ifferent . Our conMETΔ7–8 mutant resembles the auto-active EGFRvIII variant which occurs concomitant with EGFR amplification in 25–64 % of all GBMs [EGFRvIII mutation [Δ7–8 alteration appears to be restricted to only few cancer types, as we detected it in glioma but not in a number of other tumor types. The mutation was detected in grade III gliomas of both oligodendroglial and astrocytic origin and GBMs. Investigations of METΔ7–8 expression in other tumor types is however warranted and is ongoing in our lab.From a genetic perspective, the all GBMs , 59. Thimutation , the METPDGFRA. Forty percent of the GBMs with amplified PDGFRA contain an in-frame deletion in the ectodomain, leading to constitutive activation of the TK domain [PDGFRA deletion variants have been reported [Δ7–8 mutation in that it also involves the first IPT domain [Another example of a tyrosine kinase receptor which often carries deletions in GBM is K domain . Recentlreported , 58. DatT domain . Of notewt transcript in E98 cells, in contrast to the clinical gliomas which were analyzed in this study and which all showed abundant wtMET, also in METΔ7–8 tumors. We formally cannot exclude that the wild-type MET amplicons in our PCR derive from ‘contaminating’ non-neoplastic stromal cells in the tumor biopsies that were tested. There may, however, be another explanation for the relatively low levels of METΔ7–8 in clinical tumors: in the patient tumor from which the E98 model was generated, a low percentage of MET-expressing tumor cells was detected, although it is impossible to determine whether these cells carry the METΔ7–8 mutation because the antibodies used do not discriminate between MET and METΔ7–8. It is tempting to speculate that during the generation of the E98 model, a small subset of METΔ7–8 tumor cells in the primary tumor experienced a growth advantage, ultimately resulting in clonal outgrowth during xenograft formation. This scenario fits with the notion that clinical tumors not only show inter- but also intratumoral heterogeneity [Importantly, we did not detect a METogeneity , and derΔ7–8 may have important consequences for choice of therapy. MET is increasingly recognized as an important target in multiple tumor types, including glioma, and therapeutic antibodies against HGF or the HGF binding site on MET have been developed. Since HGF is not involved in METΔ7–8 activation and METΔ7–8 is retained intracellular, MET-mutated cells will not be responsive to these therapies. Indeed, we were able to show that E98 cells do not respond to the anti-MET VHH G2. Selection of MET-mutated cells in tumors that initially respond to antibody-based MET-directed treatment is expected to result in recurrence of treatment-resistant clones. In this respect, it will be important in future studies to assess the occurrence of the Δ7–8 mutation in paired samples of primary and recurrent tumors after MET antibody-based therapies, but also anti-EGFR therapies since cells may use METΔ7–8 to bypass EGFR signaling [Δ7–8 is sensitive to these inhibitors [Expression of METignaling , 53. Witignaling , 57, 61 hibitors . Such inhibitors , 45. A rhibitors . A studyhibitors .In conclusion, we describe a highly active, non-ligand-dependent mutant of MET in 6 % of gliomas, which is not exposed on the cell surface and is predicted to be non-targetable with therapeutic antibodies against MET and/or HGF.


P < 0.01). Serum levels of antinuclear IgG4 in SLE patients were positively correlated with serum levels of total IgG4, albumin, and C3  and negatively correlated with 24-hour urinary protein . Serum levels of IgG4-specific IgM-RF were higher in RA patients than in SLE patients (P < 0.001). Also, the ratio of the deposition score for IgG4/(IgG1 + IgG2 + IgG3 + IgG4) was negatively correlated with the score for C1q and C3 deposition in LN . In summary, the IgG4 autoantibody may dampen the inflammatory response in SLE, thus maybe providing a novel therapeutic target for SLE.In order to investigate the association between IgG4 autoantibody and complement abnormalities in systemic lupus erythematosus (SLE), 72 newly diagnosed SLE patients, 67 rheumatoid arthritis (RA) patients, and 41 healthy normals were employed. Serum levels of antinuclear IgG4 and IgG4-specific IgM-rheumatoid factor (RF) were measured, and the correlations between serum levels of antinuclear IgG4 and several clinical parameters were analyzed. Also, the levels of IgG subclasses, C1q, and C3 deposition in lupus nephritis (LN) were detected. The results showed that serum levels of antinuclear IgG4 were higher in SLE patients relative to healthy normals ( Systemic lupus erythematosus (SLE) is a systemic disease that can affect multiple organs, such as lupus nephritis (LN) resulting from autoantibody and complement-fixing immune complexes (ICs) deposition . Also, c2 and CH3), determine the ability of IgG4 to bind to C1q and Fcγ receptors, and this region in IgG4 is significantly different from the hinge regions in the other IgG subclasses, resulting in a lower binding ability. Thus, IgG4 cannot stimulate the classical pathway of complement activation, even though the binding ability of IgG4 toward targeted antigen is the same as those of the other subclasses of IgG [Human IgG is the main component of serum immunoglobulin, which can be divided into four subclasses: IgG1 60–70%), IgG2 15–20%), IgG3 (5–10%), and IgG4 (4–6%) [–20%, IgG0–70%, Igs of IgG . It can In this study, serum levels of anti-nuclear IgG4 and IgG4-specific IgM-rheumatoid factor (IgM-RF) were detected, and the correlations between the serum levels of anti-nuclear IgG4 and several clinical parameters of SLE patients were analyzed. Additionally, the levels of IgG subclass, C1q, and C3 deposition in the kidney were detected by immunofluorescence staining to investigate the association between IgG4 autoantibody (anti-nuclear IgG4) and complement abnormalities in SLE.Seventy-two newly diagnosed and untreated SLE patients  and 67 RA patients  were selected from the Kidney Disease Department of Internal Medicine, Affiliated Hospital of Guangdong Medical University, from March 2013 to December 2014. Forty-one healthy normals  were included as normal control subjects. All SLE patients were diagnosed according to the 1997 revised criteria of the American College of Rheumatology (ACR). Lupus nephritis (LN) was diagnosed in accordance with the ISN/RPS 2003 classification of LN, and rheumatoid arthritis (RA) was diagnosed in accordance with the 2009 criteria of the ACR/European League Against Rheumatism (EULAR). To eliminate the influence of IgG4-RD on IgG4 in SLE, patients who had IgG4-RD were excluded from the study. The clinical data and laboratory results of the SLE patients were collected. Written informed consent was obtained, and the protocol of this study was approved by the Medical Ethics Committee of the Affiliated Hospital of Guangdong Medical University.™ Kit, ZEUS Scientific, Inc., USA), Pierce™ BCA Protein Assay Kit , mouse anti-human IgG1, IgG2, IgG3, and IgG4-FITC , rabbit anti-human C1q and goat anti-human C3 , and Multiskan MK3 microplate reader .Antibodies and reagents were obtained as follows: mouse anti-human IgG4 antibody , HRP-labeled rabbit anti-human IgG4 antibody , ANA Screen Test System ; 24-hour urinary protein was detected using an automatic biochemistry analyzer (Olympus AU2700); and serum C3 was detected by rate nephelometry using a Beckman Coulter IMMAGE 800.After obtaining plasma from SLE patients, the anti-human IgG4 affinity column was manufactured using an established method  by conjuμm sections, which were then stained with diluted mouse anti-human IgG1, IgG2, IgG3, IgG4, C3, or C1q-FITC using an established direct IF staining method [One hundred forty-one cases, including 46 newly diagnosed and 65 treated SLE patients, were diagnosed with LN by renal biopsy. Kidney tissue specimens were divided and processed for immunofluorescence. Fragments from each biopsy were snap-frozen and cut using a Leica-CM1510-freezing microtome into 5 g method –15. Immut-test. Multiple-group comparisons were performed using ANOVA, followed by the Bonferroni or Dunnett post hoc tests. The correlations between different parameters were analyzed using Spearman's rank correlation coefficient. The P value was considered statistically significant if it was less than 0.05.All statistical tests were performed using SPSS version 16  and Prism 5 software . All data are expressed as the mean ± standard deviation (SD). Two-group comparisons were carried out using our independent sample P < 0.01). However, there was no significant difference between the SLE patients and RA patients (P > 0.05)  .r = 0.55, P < 0.05; r = 0.42, P < 0.05)   (P > 0.05).Serum levels of anti-nuclear IgG4 in SLE patients were positively correlated with serum levels of C3 and albumin  , and pos < 0.05) . HoweverP < 0.001, P < 0.001), but there was no significant difference between the SLE patients and the healthy normals (P > 0.05)  .r = 0.34, P < 0.05; r = 0.51, P < 0.01, resp.)  .In SLE, autoreactive B cells produce various autoantibodies against autoantigens. Anti-nuclear antibody is a disease activity marker in SLE  and in lThere is still controversy regarding serum levels of IgG4 in SLE, and the serum levels of anti-nuclear IgG4 have not been reported. Morland et al., Lin et al., and Sun et al. reported that serum levels of IgG4 in SLE patients had a declining trend compared to those of normal subjects –21. Addi2, the Fc fragment of IgG is recognized by rheumatoid factor (RF), and the resulting formation of RF-IgG immune complexes (ICs) continues to activate complement, leading to tissue and organ damage [Because denatured IgG (including IgG4) exposes CHn damage . One stun damage . Howeverγ receptors and the C1 complement molecules [Complement abnormalities  and the olecules –35. van olecules . Anotherolecules . Therefoγ receptor and the C1 complement molecule, thus maybe providing a novel therapeutic target for SLE.This study firstly investigated the association between IgG4 autoantibody (anti-nuclear IgG4) and complement abnormalities in SLE and found that the IgG4 autoantibody (anti-nuclear IgG4) may dampen the inflammatory response in SLE by competitively binding to autoantigens to form nonpathogenic ICs that result from the low affinity of IgG4 for both the Fc


Streptomyces. These include: (i) elucidation of the signals that trigger the antibiotic biosynthetic pathways to improve culture media, (ii) bacterial hormone studies aiming to reproduce intra and interspecific communications resulting in antibiotic burst, (iii) co-cultures to mimic competition-collaboration scenarios in nature, and (iv) the very recent in situ search for antibiotics that might be applied in Streptomyces natural habitats. These new research strategies combined with new analytical and molecular techniques should accelerate the discovery process when the urgency for new compounds is higher than ever.Emergence of antibiotic resistant pathogens is changing the way scientists look for new antibiotic compounds. This race against the increased prevalence of multi-resistant strains makes it necessary to expedite the search for new compounds with antibiotic activity and to increase the production of the known. Here, we review a variety of new scientific approaches aiming to enhance antibiotic production in  Streptomyces genus. Following this initial discovery, thousands of compounds produced by these microorganisms have been described and utilized in order to fight infections, and they comprise over two-thirds of all known antibiotic compounds  one of the most feasible methods for antibiotic(s) discovery in this counter clock race against multi-resistant strains. Moreover, new approaches designed with a view to awake silent or cryptic pathways will also surely result in the discovery of highly valuable secondary metabolites with antifungal, herbicidal, anti-cancer, immunosuppressive, anti-inflammatory, antihelmintic, or antiviral activities, amongst others, widening the interest for the research in this area.Genome sequencing combined with Streptomyces  remain elusive. Phosphate, nitrogen and iron are compounds present in laboratory culture media. However, other compounds found in nature but absent in lab culture might also act as the signals triggering other TCSs responses (or different regulatory mechanisms) and therefore controlling antibiotic production. So, the addition of low concentration of rare earth elements to the culture medium may activate the secondary metabolism in S. coelicolor and in other Streptomyces species. Scandium has been the one studied more in depth but yttrium, lanthanum, cerium, and europium can also provoke antibiotic production boost  are the main signaling pathways in e Figure . As an eStreptomyces research, also linked with nutrients, since their depletion is coupled with sporulation, are exploring new solutions in order to wake silent pathways through morphological differentiation, namely sporulation recovery. As has been recently described, physiological differentiation is tightly linked to secondary metabolism and therefore recovering sporulation capacities of some Streptomyces might also lead to the discovery of new compounds  or even to coordinate efforts with other antibiotic producing neighbors (combining strategies in order to repel a common menace). Therefore, the addition of certain antibiotics or bacterial hormones in low concentrations to the culture medium might also be an alternative for antibiotic production stimulation Figure . Over thNodwell, . For exa Takano,  a recentStreptomyces, as with other microorganisms, has been basically done in axenic cultures. However, antibiotic functions can only be understood in the context of Streptomyces' habitat. Traditionally, antibiotics have been considered biological weapons that allow the bacteria to compete with others microorganisms, either by killing them or inhibiting their growth. Nevertheless, antibiotics are also signals that trigger adaptive responses  although not mediated via any chemical substance  and matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) imaging mass spectrometry that allow researchers to gain an Streptomyces.A new universe of possibilities for antibiotic discovery Figure  is openiThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There was no significant difference between genders, different age groups, or different parenthood status. The determined level of self–medication with antibiotics was 31.0%. The men , the respondents from rural areas , and those without children  were more likely to use antibiotics in self-medication. Lithuanian residents’ knowledge of antibiotics is insufficient. More information about antibiotic use should be provided by physicians and pharmacists. Self-medication with antibiotics is a serious problem in Lithuania and requires considerable attention.Irrational antibiotic use has led society to antibiotic resistance—a serious health problem worldwide. This study aimed to assess public knowledge, beliefs, and behavior concerning antibiotic use and self-medication in Lithuania. The cross-sectional survey method was processed using a validated questionnaire in different regions of Lithuania. In total, 1005 adults completed the questionnaire and were included in the study. More than half of the respondents (61.1%) had poor knowledge of antibiotics. Almost half of the respondents incorrectly identified antibiotics as being effective either against viral (26.0%) or mixed  infections (21.7%). The respondents with lower educational qualifications (OR = 2.515; 95% CI 1.464–4.319;  Irrational antibiotic use has led society to antibiotic resistance—a serious health problem worldwide, which is now trying to be solved by many various approaches. In 2011, the World Health Day theme was “Combat drug resistance: no action today means no cure tomorrow” and for this occasion the World Health Organization introduced a six-point policy package to fight against the spread of antimicrobial resistance [There are various factors which may influence an increase in irrational antibiotic use. Many studies have reported that antibiotic regime non-adherence and inappropriate antibiotic use are strongly associated with public awareness and knowledge of antibiotics ,3,4,5. FAnother important issue related to the increase in antibiotic resistance is self-medication, which is defined as the acquisition of antibiotics and self-administering them (or administering them to children) with the aim of treating perceived infection . There aIrrational antibiotic use reflects not only patients’ failure to comply with physician’s instructions on how to use antibiotics adequately, but is also associated with inappropriate antibiotic prescribing. Rational antibiotic therapy should be based on the correct indication, the right drug and dosage, the drug of the first choice, the appropriate period of use, and the lowest treatment costs . All antDue to wide cross-national differences in antibiotic use and public knowledge of antibiotics, determination of key populations requiring educational interventions is needed in each country prior to the establishment of effective educational programs. Studies regarding public knowledge of antibiotics and self-medication with antibiotics are rare ,25. Our A prospective cross-sectional study was designed based on a validated anonymous self-administered questionnaire. Ethical approval was obtained from the Lithuanian University of Health Sciences Bioethics Centre. To be eligible for this study, participants had to provide signed informed consent. The anonymous survey was processed in 16 community pharmacies located in 4 different regions of Lithuania, covering the whole country. All the selected pharmacies were similar in terms of size, location , and patient load per month. Respondents were recruited by our research team. All the patients who came to the selected community pharmacies during the study period were asked to fill out the questionnaire at the pharmacy, regardless of antibiotic acquisition at the time of visit or antibiotic use at any time in life. Respondents under 18 and those with occupation related to health care were not included in this study. A total of 1,005 respondents were eligible for the final analysis. The anonymous survey method was performed using the original validated 37-item questionnaire (in Lithuanian) containing both open- and close-ended (multiple-choice) questions. The questionnaire was developed by our research group based on a previously conducted literature review ,23,24,28There were six questions in Part 1 regarding the demographic characteristics of the study population, including gender, age, level of education, occupation, place of residence, and parenthood status. Urban place of residence was defined as living in a city or town with the population size of more than 3000 inhabitants . Parenthood status was defined as having children under 18. The data regarding occupation were only used to exclude respondents with the occupation related to health care. Part 2 consisted of seven questions. The respondents were asked to indicate prior antibiotic use at any time in life and the approximate frequency of antibiotic use (if applicable). The antibiotic users were also asked about their most recent antibiotic use . They were also asked to indicate the main sources of information regarding antibiotic use. Before filling out Part 3, the respondents had to evaluate themselves and to assess their knowledge of antibiotics using a 6-point numerical rating scale (0–5), where the end points were the extremes of no knowledge and excellent knowledge. According to their self-evaluation, we divided the respondents into three groups as follows: 4–5 points—good knowledge, 2–3 points—average knowledge, and 0–1 point—poor knowledge of antibiotics. The results of self-evaluation were later compared with the actual knowledge of antibiotics.There were nine questions in Part 3 regarding knowledge and beliefs related to antibiotic use. We selected five questions from this part to assess respondents’ actual knowledge of antibiotics. The questions inquired information about rational antibiotic use, antibiotic effectiveness against bacterial and/or viral infections, indications for antibiotic use, and the appropriate period of antibiotic use. The number of correct answers to these questions (0–5) indicated the actual knowledge score as follows: 4–5 points—good knowledge, 2–3 points—average knowledge, and 0–1 point—poor knowledge of antibiotics. Part 3 also included the questions regarding respondents’ beliefs on safety of antibiotic use and possible adverse drug reactions (ADRs). Part 4 consisted of eight questions related to antibiotic use behavior, antibiotic acquisition, and self-medication with antibiotics. Self-medication prevalence was measured among the antibiotic users. Self-medication was determined based on the answers to questions in Part 4 and considered as the selection and use of antibiotics by the study participants (or their family members) to treat self-recognized or self-diagnosed condition at any time in life. The last part of the questionnaire was designed for parents and included seven questions. The parents were asked about their children’s most recent antibiotic use if applicable . Other questions in Part 5 assessed how the parents supervised their children’s health problems related to antibiotic use.A sample size calculation was performed using the Raosoft sample size calculator  with a 52 tests were performed to identify variables associated with dependent variables—knowledge of antibiotics and self-medication. Risk factors of poor antibiotic knowledge  and self-medication (among the antibiotic users) was first evaluated using bivariate logistic regression. Variables with p < 0.25 in the bivariate analysis were included in the multivariate logistic regression analysis to determine the factors that are independently associated with each dependent variable. Only the results of multivariate logistic regression analysis are reported. Odds ratios (OR), 95% confidence intervals (CI), and p-values were calculated for each independent variable. A p-value less than 0.05 was considered to be statistically significant.A descriptive and comparative statistical data analysis was processed with the SPSS 17.0 . Frequencies and cross tables of preselected variables were calculated and χA total of 1005 out of 1300 administered questionnaires were returned fully completed and met the inclusion criteria, giving a response rate of 77.3%. Less than half of the respondents (42.1%) were men. The mean age of the respondents was 38.6 ± 13.9 . Most respondents were from urban places of residence (74.3%) and had university or college education (70.3%). Study population characteristics compared with the Lithuanian general population are detailed in Nearly all the respondents (93.7%) claimed that they had used antibiotics at some point in their life. One-tenth (9.8%) indicated that they had used antibiotics at least once in 6 months, and one-quarter (24.9%) at least once a year. The main sources of antibiotic information were physicians (64.5%), pharmacists (45.0%), family members or friends (41.8%), and the Internet (40.5%) (study participants could choose more than one possible source of information). Even though physicians were reported to be the main source of information related to antibiotic use by most respondents, one-third of them (34.0%) stated that they got too little or no information about antibiotics from their physician.The antibiotic users had to identify health problems and antibiotics used for the most recent antibiotic therapy. Almost one-third of the respondents (29.7%) did not indicate the disease or the antibiotic (most of the answers in these cases were “I don’t know” or “I don’t remember”); 18.6% of the respondents indicated that they had used antibiotics for various noninfectious diseases; 5.3% of the respondents confused antibiotics with other medications such as nonsteroidal anti-inflammatory drugs or antipyretics, which are usually used for the symptomatic treatment of common colds. p < 0.001). Though almost half of the respondents (42.0%) evaluated their knowledge of antibiotics accurately, a similar proportion (44.6%) overestimated their knowledge. Moreover, better self-evaluation was associated with higher prevalence of self-medication (p < 0.001).Before answering the questions related to antibiotic use, the respondents evaluated their knowledge of antibiotics; 22.8% of the respondents rated their antibiotic knowledge as good, 43.8% as average, and 33.4% as poor . Poor knAccording to our scoring of respondents’ knowledge of antibiotics, only one-third of them had good (10.3%) or average (28.6%) knowledge. Almost half of the respondents incorrectly identified antibiotics as being effective either in treating viral (26.0%) or mixed  infections (21.7%); 41.7% noted common cold as an appropriate indication for antibiotic use. A considerably low percentage of the respondents (15.1%) indicated 3 days or less to be an appropriate duration of antibiotic use. The majority of the respondents (92.9%) knew that antibiotics could cause ADRs, and most of them were aware of such ADRs as allergic reactions (62.3%), dysbacteriosis (45.7%), nausea and/or vomiting (41.4%), diarrhea (24.4%), and headache (25.0%). The respondents’ knowledge and beliefs of antibiotics are shown in p = 0.001) and the respondents from rural places of residence  were significantly less knowledgeable of antibiotics. There was no significant difference between men and women, different age groups, or respondents with a different parenthood status.p = 0.011), the respondents from rural place of residence , and the respondents without children  were more likely to use antibiotics in self-medication. Higher education level or better knowledge of antibiotics were not associated with the increased risk of self-medication. Interestingly, the younger respondents  were less likely to use antibiotics in self-medication. More than one-quarter of the antibiotic users (27.8%) stated that they had bought or used antibiotics without prescription at some point in life. The results showed that the main sources of antibiotic supply without prescription were community pharmacies (72.7%) or leftover of antibiotics stored in the household (14.8%). Supplies by family members and friends accounted for 9.9%. The determined level of self-medication with antibiotics among the study population was 31.0%. The independent risk factors of self-medication were found to be gender, place of residence, and parenthood status  stated that their children had used antibiotics at some point in life. More than one-tenth of the respondents (12.3%) indicated that their children had used antibiotics at least once in 6 months, and almost half of them (43.2%)—at least once a year. Almost half of the parents (41.3%) did not indicate the disease or the antibiotic that was administrated to their children during the most recent antibiotic therapy; 14.4% of the respondents indicated that their children had used antibiotics for noninfectious diseases and only 1.4% of respondents confused antibiotics with other medications. Most parents noted supervising the use of antibiotics according to the instructions given by a physician (58.0%) or a pharmacist (23.1%). More than one-tenth of the parents reported administering antibiotics to children according to their own knowledge (8.5%) or advice given by family members and friends (5.6%); 4.8% parents stated that they would never give antibiotics to their children.This study revealed important findings, related to inadequate public knowledge of antibiotics. Our results showed that antibiotic knowledge among Lithuanian population was insufficient as almost two-thirds of the respondents (61.1%) had poor knowledge of antibiotics. A large proportion of the respondents (41.7%) thought that antibiotics worked on common cold, 26.0% that antibiotics were effective in treating viral infections, and 21.7% that antibiotics were effective in treating mixed  infections. Moreover, people tended to overestimate their knowledge of antibiotics, which might lead to increased non-adherence and self-medication. The respondents with lower educational qualifications and those from rural places of residence were less knowledgeable about antibiotics. Self-medication behavior was identified in almost one-third of the antibiotic users (31.0%) and was found to be significantly associated with male gender, rural place of residence, and absence of children. Insufficient public knowledge of antibiotics has been previously reported in various countries and regions ,17,19,30Our study showed pleasing results that health care professionals were the main source of antibiotic information. As physicians and pharmacists play an important role in contributing to public knowledge of antibiotics, they must be considered as a powerful instrument for the increase in rational antibiotic use in society and the improvement of patients’ behavior regarding antibiotic use. Both, physicians and pharmacists must be encouraged by national health programs to provide more information about appropriate antibiotic use and its’ importance while prescribing or dispensing these medications. Moreover, all academic institutions should pay huge attention to education of future health care professionals, providing them good rational antibiotic therapy knowledge and effective patient consultation skills ,33,34,35One of the most important and novel findings in our study is the relationship between self-evaluation of antibiotic knowledge and the actual knowledge of antibiotics. Interestingly, the respondents with poor knowledge of antibiotics tended to overestimate their knowledge. Such overestimation and confidence in self-knowledge was found to negatively affect the prevalence of self-medication behavior. We were not able to find any previous surveys investigating respondents’ opinion on their knowledge of antibiotics and requiring study participants to perform self-assessment. This finding seems to be very substantial, demonstrating the need for broad educational interventions. Various studies have presented local, national, and regional educational programs and public campaigns, processed to increase antibiotic awareness with various success rates ,36,37. TOur study results show that there is still an easy access to antibiotics without supervision of a physician, as 27.8% of the respondents stated that they had bought or used antibiotics without prescription, despite the national regulation that strictly defines antibiotics as prescription-only medicines. The main source of such antibiotic supply was a community pharmacy. This may indicate that the Lithuanian health care system and especially community pharmacies are still struggling to cope with their task in enhancing rational antibiotic use. It should be noted that a similar situation has been reported by many previous studies ,23,28,38According to our results, the estimated self-medication with antibiotics in Lithuania remains high 31.0%). In comparison with previously reported data, this level has increased by 10% since 2006 . However1.0%. In The results of our study revealed that self-medication with antibiotics in Lithuania was significantly higher in the men, the respondents from rural places of residence, and the respondents without children. Higher prevalence of self-medication with antibiotics in men has been reported by several previous studies ,38,39. HStudy participants demonstrated sufficiently appropriate behavior regarding supervision of antibiotic use of their children. Most parents followed the instructions given by a health care professional (physician or pharmacist), while only a small percentage of parents reported self-administering antibiotics to their children or trusting family members’ and friends’ advice. These results demonstrate that parents take their children’s health problems more seriously and responsibly than their own. In our study, we obtained a high response rate (77.3%), which minimizes potential response bias because a response rate greater than 50%–60% is considered to be sufficient . To dateIn spite of the large sample size and high response rate, several limitations of this study should be noted. As the study was based on a self-administered questionnaire, the data were presented from recollection of respondents’ memory, and, thus, in some cases it was subjective. The distribution of the respondents with high and low education level was not even. We obtained a small percentage of the respondents over 60 years old. Other limitations include education level and previous use of antibiotics being self-reported. Family income, employment status, number of children, marital status, and health status were not analyzed as covariates possibly associated with knowledge of antibiotics and self-medication, although several previous studies have suggested this association ,24,28,38In conclusion, this study revealed a high percentage of inappropriate antibiotic knowledge and a high rate of self-medication with antibiotics among Lithuanian population. Education programs should be developed, targeting specific public groups identified in this study, with lower antibiotic knowledge and higher self-medication risk. Finally, the attention of health care policy makers should be focused on physicians and pharmacists, as the main information providers of rational antibiotic use, as well as on community pharmacies, identified as the main source of nonprescription antibiotics.


IgG4-related disease (IgG4-RD) is a recently recognized group of conditions, characterized by tumor-like swelling of involved organs, lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, variable degrees of fibrosis, and elevated serum IgG4 concentrations. Currently IgG4-RD is recognized as a systemic condition that can affect several organs and tissues. Herein we report the case of a 34-year-old male patient who was admitted to our hospital with diffuse abdominal pain, weight loss, and painful stiffness in his neck. He had a history of tumoral mass of the left maxillary region, right palpebral ptosis with protrusion of the eyeball, and chronic dry cough for about 6 years. Laboratory tests revealed polyclonal hypergammaglobulinemia and increased serum IgG4 levels. Immunohistochemical staining of the maxillary biopsy was compatible with IgG4-RD. He had an excellent response to corticosteroid therapy. This case highlights that IgG4-RD should be included in the differential diagnosis with multisystem diseases. IgG4-related disease (IgG4-RD) is a recently recognized fibroinflammatory group of disorders of unknown etiology, characterized by tumor-like swelling of the involved organs, tissue lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, variable degrees of fibrosis, and, often, elevated serum IgG4 concentrations. It was recognized as a systemic condition in 2003, in patients with autoimmune pancreatitis with extrapancreatic manifestations . In thisSeveral clinical conditions encompass the IgG4-RD: Type 1 autoimmune pancreatitis; sclerosing cholangitis; dacryoadenitis and sclerosing sialadenitis; inflammatory orbital pseudotumor; idiopathic retroperitoneal fibrosis; chronic sclerosing aortitis; Riedel's thyroiditis; IgG4-related interstitial pneumonitis, pulmonary inflammatory pseudotumors, tubulointerstitial nephritis, hypophysitis, and pachymeningitis and many other associations are likely to be revealed .The aim of the present report was to illustrate a case, in which diagnosis was delayed for years before IgG4-RD was recognized, drawing attention to the multidisciplinary nature of this condition and the necessity for suspecting its presence in patients with unspecific signs and symptoms, particularly when associated with histological findings of “chronic inflammatory process.”A 34-year-old man was admitted to our hospital with a 4-month history of diffuse abdominal pain and body weight loss, progressing in 3 months to muscle weakness, abdominal distension, nausea, vomiting, and stiff painful swelling of his neck. His previous history included a fluctuating tumoral mass in the left maxillary region for the last six years, causing mild dysphagia and limiting movement of the temporomandibular joint. The pathological examination was consistent with a chronic inflammatory process. Later on he presented right palpebral ptosis with protrusion of the eyeball and chronic dry cough, with transbronchial biopsy examination revealing chronic pneumonitis and irregular septal fibrosis.On examination he was afebrile, with blood pressure 120/80 mmHg, with pale mucous membranes, anterior neck swelling and stiffness with local hyperemia, without palpable lymph nodes, reduced pulmonary expansion, diffuse abdominal pain, and rigidity on palpation. The remainder of the physical examination was unremarkable.3, platelet count 808,000/mm3, total bilirubin 2.89 mg/dL (direct bilirubin 2.26 mg/dL), calcium 8.2 mg/dL, creatinine 0.4 mg/dL, aspartate transaminase 25.7 mg/dL, alanine transaminase 16.6 mg/dL, and alkaline phosphatase 417 mg/dL. Search for antinuclear antibodies (ANA), anti-SSA/Ro, and ANCA showed negative results. Serum protein electrophoresis and immunofixation showed polyclonal hypergammaglobulinemia. Serum IgG4 was 264 mg/dL . Ultrasonography of the neck showed soft tissues swelling. Abdominal ultrasonography was normal.On admission laboratory tests revealed the following: hemoglobin 9.0 g/dL, white blood cell count 21,390/mmThe patient developed daily fever, asthenia, diarrhea, profuse coughing, and intense dyspnea. Chest radiography revealed bilateral parenchymal infiltration, multiple pulmonary nodules, and consolidation in the lower right hemithorax. As the patient's clinical condition was not improving, despite the use of broad spectrum antibiotics  and mechanical ventilation, pulse therapy with methylprednisolone 1 g was started and used for three days, with considerable clinical and radiological improvement. After this, prednisone 50 mg/day was initiated and he was discharged in good general condition. After two-year follow-up, he has been well on lower dose (5 mg/day) of prednisone. In the last few months, after abrupt voluntary discontinuation of his medication, there was a mild relapse of the disease which remitted with the reintroduction of 20 mg/day of prednisone. The patient remains asymptomatic with low doses of steroids, maintaining complete resolution of respiratory, eye, and neck symptoms which were assumed to be manifestations of the same disease.A reanalysis  of the histological sample from previous maxillary biopsy revealed chronic inflammatory infiltrate rich in plasma cells with areas of fibrosis and erosion. Immunohistochemistry showed mixed population of B and T lymphocytes and an increased amount of positive IgG4 cells  . Helicobacter pylori; immune complex deposition in some affected organs; increased levels of Th2 cytokines, T regulatory cells, interleukin-10, and transforming growth factor; evidences of allergic response with peripheral eosinophilia.The pathogenesis of IgG4-RD is poorly understood; however some findings are consistent with multiple immune-mediated mechanisms : class IThe overall epidemiology of the disease remains largely undefined. IgG4-RD is typically described in middle-aged and older men, mostly in autoimmune pancreatitis; the disease prototype and patients usually have a history of atopic disease –11.Multiorgan disease can be evident at diagnosis, but it usually evolves metachronously over a variable time. Estimates of the relative frequency of different manifestations depend upon the perspective of the index illness being analyzed.Sometimes IgG4-RD is a diagnostic challenge, because it has multiple clinical manifestations and a wide spectrum of differential diagnosis. The confirmation of diagnosis is based on histopathological findings of the affected organ with immunohistochemical staining. The serum IgG4 level should be measured as it may strengthen the diagnostic hypothesis, although about 30% of patients may have normal serum IgG4 concentrations, despite classic histopathological findings of IgG4-RD .This report well illustrates a common clinical situation of delayed diagnosis of IgG4-RD. Many reports of inflammatory pseudotumor in different sites may be examples of undiagnosed IgG4-RD , 14. In This case highlights the need to recognize IgG4-RD as an important differential diagnosis with multisystem diseases as the treatment may lead to remission and prevent significant morbidity and mortality.


IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition, characterised by an elevated serum IgG4 concentration and abundant IgG4-positive plasma cells in the involved organs. An important question is whether the elevated IgG4 response is causal or a reflection of immune-regulatory mechanisms of the disease.To investigate if the IgG4 response in IgG4-RD represents a generalised polyclonal amplification by examining the response to common environmental antigens.Serum from 24 patients with IgG4-RD , 9 patients with primary sclerosing cholangitis and an elevated serum IgG4 (PSC-high IgG4), and 18 healthy controls were tested against egg white and yolk, milk, banana, cat, peanut, rice and wheat antigens by radioimmunoassay.We demonstrated an elevated polyclonal IgG4 response to multiple antigens in patients with IgG4-RD and in PSC-high IgG4, compared with healthy controls. There was a strong correlation between serum IgG4 and antigen-specific responses. Responses to antigens were higher in treatment-naive compared with treatment-experienced patients with IgG4-RD. Serum electrophoresis and immunofixation demonstrated polyclonality.This is the first study to show enhanced levels of polyclonal IgG4 to multiple antigens in IgG4-RD. This supports that elevated IgG4 levels reflect an aberrant immunological regulation of the overall IgG4 response, but does not exclude that causality of disease could be antigen-driven. IgG4-related disease (IgG4-RD) is a multisystem fibroinflammatory condition, characterised by the development of mass lesions with similar histopathological findings in the involved organs.4Helicobacter pylori plasminogen-binding peptide, through a process of antibody cross-reactivity with ubiquitin-protein ligase E3 component n-recognin 2 (molecular mimicry) in genetically predisposed individuals, has been suggested in AIP.10An immune-mediated pathogenesis has been suggested in IgG4-RD, supported by a human leucocyte antigen type II association, presence of autoantibodies and elevated levels of IgG4 and a dramatic response to corticosteroid therapy.Our alternative hypothesis is that the elevated IgG4 may not be (primarily) triggered by specific (auto)-antigens, but be an indirect consequence of the expansion of pre-existing IgG4-switched B cells as being responsible for IgG4-RD. In this case, one would expect to find a more generalised and  a more transient increase in IgG4 antibodies against different antigens that are known to elicit an IgG4 antibody response in the general population. We tested this hypothesis by investigating the level and persistence of the IgG4 response to a variety of known IgG4-inducing non-infectious environmental antigens. We analysed patients with IgG4-RD, patients with primary sclerosing cholangitis (PSC) and elevated IgG4 , and healthy controls in a UK cohort.Detailed description of patient inclusion criteria and methodology can be found in the online supplementary repository. Antigen-specific IgG4 responses to egg, milk, peanut, banana, rice, wheat and cat were quantified using a previously developed radioimmunoassay.11Demographics, clinical characteristics and serum immunoglobulin values of patients and controls are shown in the online supplementary table S1. Most patients with IgG4-RD (83%) had pancreatic (AIP) and/or biliary (IRC) involvement, with 71% having other systemic organ involvement. The concentration of serum total IgG, IgG4, IgE and, to a lesser extent IgG1, was higher in patients with IgG4-RD versus healthy controls, as well as in the patients with PSC-high IgG4 versus healthy controls (see online supplementary figure S1).We analysed the IgG4 response to proteins from egg, milk, peanut, banana, rice, wheat and cat. In line with our hypothesis, the response to egg (p=0.004), milk (p=0.04), peanut (p=0.0003), cat dander and serum (p=0.012), rice and wheat (0.006) antigens was found to be higher in patients with IgG4-RD than in healthy controls, and the response to egg (p=0.03), cat dander and serum (p=0.04), and rice and wheat (p=0.01) antigens was higher in patients with PSC-high IgG4 than in healthy controls .We further examined the correlation of serum immunoglobulin levels with antigen-specific responses. In patients with IgG4-RD, there was a positive correlation between serum IgG4 levels and IgG4 responses to banana , peanut , cat , rice and wheat  antigens (see online supplementary figure S2). By contrast, total serum IgE levels did not correlate with antigen-specific responses (data not shown).We also tested the differences in immunoglobulin levels and IgG4-specific antigen responses in 14 treatment-naive and 10 treatment-experienced patients with IgG4-RD receiving corticosteroid therapy. Levels of antibodies to banana (p=0.001), egg (p=0.039), peanut (p=0.003) and cat (p=0.006) antigens were lower in treatment-experienced compared with treatment-naive patients , as wereWe analysed serum electrophoresis data for evidence of monoclonality in patients and controls, but no monoclonal bands in the polyclonal gamma region were observed (see online supplementary figure S4). All individuals showed polyclonality; 11/14 (78.6%) patients with IgG4-RD, 3/9 (33.3%) PSC-high patients with IgG4, and 0/18 healthy controls showed hypergammaglobulinaemia. Further immunofixation analysis of patient samples with a diffuse increase in immunoglobulins, using IgG, IgA, IgM, kappa and lambda light-chain antisera, confirmed no monoclonal bands in the gamma region (see online supplementary figure S4).Our data show that IgG4-RD is associated with an elevated IgG4 response to diverse antigens. Therefore, elevated levels of IgG4 in patients with IgG4-RD may be the result primarily of a polyclonal expansion of many IgG4 B cells irrespective of their specificity. This would imply that factors other than antigen induce an expansion and differentiation of IgG4 B cells, but does not exclude the possibility that a single antigen (self or exogenous) serves as the initial trigger of disease. Antigen-independent involvement of many IgG4-switched B cells implies differential selection via signals that uniquely act on IgG4 B cells. We have recently demonstrated differences in phenotype between IgG1 and IgG4 B cells,The fall in IgG4-specific antigen responses after corticosteroids may be explained by suppressed proliferation of expanded IgG4-switched B cells or suppressed differentiation into IgG4 plasmablasts. Corticosteroids interfere with the production of cytokines critical for T cell proliferation and interaction, and the binding of interleukins to B cells suppressing proliferation and antibody production. The steroid effect highlights the rapid turnover of the IgG4-producing plasma cells, as opposed to production by long-lived plasma cells in the bone marrow.Evidence for polyclonality in IgG4-RD is supported by the diversity of antigen specificity, and by serum electrophoresis and immunofixation analysis no monoclonal bands in the polyclonal gamma region was observed (see online supplementary figure S4). However, this is complicated by the ability of the bispecific IgG4 antibody to undergo Fab-arm exchange  may also increase IgE B cell expansion in certain individuals. In this instance, one may expect an increased predisposition to allergies in later years of life. However, the frequency of allergy/atopy was similar in patient and control groups (see online supplementary table S1). Furthermore, there was no significant difference between total and antigen-specific IgG4 responses in patient and control groups (not shown). However, serum IgE levels in patients with IgG4-RD with allergy/atopy was higher than in those without (p=0.0255) (see online supplementary figure S3); consistent with Della Torre To summarise, this is the first study to show an enhanced polyclonal IgG4 response to multiple non-infectious environmental antigens in IgG4-RD. Multiple different antigen responses are higher in treatment-naive compared with treatment-experienced patients. The elevated IgG4 levels may reflect the aberrant immunological regulation of the overall IgG4 response in the disease.


A 44-year-old man consulted an internist because of abnormalities in an upper gastrointestinal series. It showed an elevated lesion with central depression in the greater curvature of the middle part of the stomach. Upper gastrointestinal endoscopy showed an elevated lesion with central depression, bridging hold, and no abnormalities of the gastric mucosa in the greater curvature of the middle part of the stomach. Endoscopic ultrasonography showed a submucosal tumor derived from the muscle layer of the stomach. Computed tomography showed a 22-mm tumor in the upper part of the stomach. Integrated position emission tomography/computed tomography (PET/CT) showed an intense tracer uptake by the tumor. Based on these findings, a gastrointestinal stromal tumor was suspected and laparoscopic endoscopic cooperative surgery was performed. A histopathological examination showed lymphoplasmacytic infiltration and fibrosis, and an immunohistochemical analysis showed the infiltration of IgG4-positive lymphoplasmacytic cells. The probable diagnosis was IgG4-related sclerosing disease of the stomach. We herein describe a rare case of probable IgG4-related sclerosing disease which presented as a gastric submucosal tumor. PET/CT is a useful imaging technique for the diagnosis and follow-up of this disease. IgG4-related sclerosing disease is a systemic disease characterized by the extensive infiltration of IgG4-positive plasma cells and lymphocytes in various organs . AlthougWe present a case of probable IgG4-related sclerosing disease presenting as a gastric submucosal tumor with an intense tracer uptake on PET/CT.max = 6.8)  were almost within the normal limits. Upper gastrointestinal endoscopy showed an elevated lesion with central depression, bridging hold, and no abnormalities of the gastric mucosa in the greater curvature of the middle part of the stomach Fig. . EndoscoThe resected specimen showed a tumor measuring 20 × 18 mm in diameter, and its divided face was solid and poorly marginated. A histopathological examination showed lymphoplasmacytic infiltration and fibrosis Fig. , and an The patient made an uneventful recovery and was discharged on postoperative day 7. He was examined in a clinic 7 weeks after surgery and was found to have made a full recovery. Subsequent blood tests revealed that his serum IgG4 level was 98.1 mg/dL (reference range 4.8–105 mg/dL); however, the levels had not been examined prior to resection as an IgG4-related sclerosing disease had not been suspected.The gastric manifestations of IgG4-related sclerosing disease are rare, and PET/CT is a useful imaging technique for their diagnosis and follow-up. The organ that is most commonly involved in IgG4-related sclerosing disease is the pancreas; such involvement is termed autoimmune pancreatitis (AIP) . AIP is A diagnosis of IgG4-related disease is definitive in patients with the following findings: (1) organ enlargement, mass or nodular lesions, or organ dysfunction; (2) a serum IgG4 concentration of >135 mg/dL; and (3) histopathological findings of >10 IgG4 cells/HPF and an IgG4+/IgG+ cell ratio of >40 %. A diagnosis of IgG4-related disease is possible in patients who fulfill criteria (1) and (2), but with negative results on histopathology or without a histopathologic examination, whereas a diagnosis of IgG4-related disease is probable in patients with organ involvement (1) and in whom the histopathologic criteria are fulfilled, without an increase in serum IgG4 concentration (2) [There are few reports of IgG4-related sclerosing disease presenting as a gastric lesion. Rollins et al. reported a gastric midbody mass which was histologically characterized as an IgG4-related autoimmune fibrosclerosing pseudotumor . Fujita Some reports have shown the usefulness of PET/CT imaging in IgG4-related sclerosing disease , 9. PET/As corticosteroid therapy is effective in IgG4-related sclerosing disease, it may be considered the first treatment option. However, a therapeutic diagnosis with corticosteroid dosage is not recommended when differentiation with a malignant tumor is difficult [IgG4-related sclerosing disease is a relatively new disease concept, and its prognosis has not been well described. Additional studies of its long-term prognosis are needed.In summary, we herein described a rare case of probable IgG4-related sclerosing disease presenting as a gastric submucosal tumor. PET/CT is a useful imaging technique for the diagnosis and follow-up of this disease.Written 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.


As cholangiographic features of IgG4-related sclerosing cholangitis (IgG4-SC) resemble those of cholangiocarcinoma, it is highly confusing between the 2 conditions on the basis of cholangiographic findings. This study presents a case of extensive metastatic cholangiocarcinoma with IgG4-SC misdiagnosed as isolated IgG4-SC, and reviews recent studies of the 2 diseases.A 56-year-old man with no family history of malignant tumors or liver diseases presented with recurrent mild abdominal pain and distention for 3 months. Magnetic resonance cholangiopancreatography showed a 3.7 cm nodular lesion with unclear boundary in segment VI of the liver. Serum IgG4 and CA19-9 were slightly elevated. Histopathological examination was consistent with the consensus statement on the pathology of IgG4-SC. IgG4-SC was thus considered. Due to his mild symptoms, glucocorticoid was not given at first. However, 3 months after his first admission, he had more severe abdominal pain and further elevated serum CA19-9. Actually he was found suffering from extensive metastatic cholangiocarcinoma with IgG4-SC by exploratory laparotomy.The present case serves as a reminder that extensive metastatic cholangiocarcinoma with or without IgG4-SC may be misdiagnosed as an isolated IgG4-SC case if one relies solely on elevated serum and tissue IgG4 levels. We emphasize on the importance of repeated core needle biopsy or exploratory laparoscopy/laparotomy before immunosuppressive drugs are given, and on follow-up of imaging findings and serum CA19-9 once immunosuppressive therapy is started. Characteristic features of the entity include abdominal pain, biliary strictures, and jaundice.3 Although unspecific, serological studies, such as serum bilirubin, liver enzymes , tumor markers (carbohydrate antigen 19-9 [CA19-9] and carcinoembryonic antigen [CEA]), are useful as a diagnostic guide.Cholangiocarcinoma, first reported by Durand-Fardel in 1840,4 Given that fact that clinical cholangiographic features of IgG4-SC resemble those of cholangiocarcinoma,6 it is highly confusing between the 2 conditions in cholangiographic findings. Herein, we describe a case of extensive metastatic cholangiocarcinoma with IgG4-SC that was wrongly diagnosed as isolated IgG4-SC solely on the basis of serum and tissue IgG4 levels, and conduct an overview of the published literature on this issue.IgG4-related sclerosing cholangitis (IgG4-SC) is a new emerging disease which was identified by Hamano et al at 2001.A 56-year-old man with no family history of malignant tumors or liver diseases presented with recurrent mild abdominal pain and distention for 3 months. Duration of pain may last from hours to days, and the pain caused a loss of appetite. He lost 3 kg in body weight in half a month before admission. Physical examination showed mild tenderness in the upper abdomen, without rebound tenderness, guarding, mass, or hepatomegaly. Serum tests revealed that slightly elevated IgG4  and CA19-9 . Total bilirubin, conjugated bilirubin, and liver function parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALP, and albumin, remained normal  investigation showed a solid nodular lesion at 3.7 cm in size with unclear boundary in segment VI of the liver Fig. A, left. 7 and therefore, IgG4-SC was highly considered as a diagnosis.Based on the imaging results, a liver core needle biopsy under ultrasound guide was performed with the aim to exclude malignancy. Histopathological examination revealed conspicuous inflammatory cell infiltration consisting largely of plasmacytes, macrophages, and lymphocytes on a background of fibrous interstices with fibrosis and fibroblast proliferation. Plasmatic infiltrates were particularly apparent, with a portion showing atypia such as polynuclear cells, and represented reactive growth. Proliferative storiform fibrosis was found, which is characterized by a cartwheel sign with spindle cells radiating from a center Fig. A. Immuno10 However, 3 months after his first MRCP, he had more severe abdominal pain and further elevated serum CA19-9, and was admitted to our hospital again. At this time, the results of MRCP were similar to that of the first one. But enlargement of supraclavicular lymph nodes was suspected by physical examination, which was confirmed by ultrasonography examination (largest 0.7 cm). Due to suspected spread of malignancy accompanied with intraabdominal inflammation and adhesion, laparoscopy was not considered to avoid unnecessary pain and expense. Thus, an exploratory laparotomy in the liver was performed in the surgery department of this hospital.In the view of his slight symptoms, glucocorticoid was not given at first. Considering that liver-occupying lesion and chronic cholecystitis may further lead to abnormal liver function, treatment was initially started with liver protective drug .During the surgery, a tumor at 5 cm in size in the segment VI of the liver was found to invade into the whole layer of the duodenum and transverse colon wall, as well as the serous membrane of the gallbladder wall. Diffuse miliary nodules were observed on abdominal wall, parietal peritoneum, omentum, and diaphragm Table . These nInstitutional approval of what was given by Institutional Ethics Committee of Zhongshan Hospital of Fudan University , and informed consent was given by the patient.11 Clinically, symptoms , are all nonspecific, and could also be found in both IgG4-SC and cholangiocarcinoma.12 It is not easy to distinguish IgG4-SC from primary sclerosing cholangitis (PSC), pancreatic cancer, and cholangiocarcinoma on the basis of cholangiographic findings alone.15 Radiologic features of IgG4-SC, such as thickening bile duct wall, mimic that of cholangiocarcinoma, and other inflammatory or immune-mediated disorders, such as PSC.18 For laboratory tests of IgG4-SC, abnormal liver functions, such as elevated bilirubin (65%), ALP (84%), ALT (62%), and AST (32%), could be seen.12 However, these abnormal values varied widely among IgG4-SC patients, thus it is hard to find a proper cut-off value to differentiate IgG4-SC from cholangiocarcinoma.IgG4-related disease (IgG4-RD) is a fibroinflammatory, multiorgan condition of unknown etiology with commonly shared features that include elevated serum IgG4 concentrations, tumefactive lesions, storiform fibrosis, as well as dense lymphocyte and IgG4-positive plasmacyte infiltrates.12 were presented in this case. The pathophysiology is unclear but seems to reflect a direct gastrointestinal effect in this part of patients and in our case. However, jaundice was absent in our case, which was presented in most IgG4-SC patients. It is possible that IgG4-SC accompanied with autoimmune pancreatitis increases the chance of obstructive jaundice. Further, tumor was found in segment VI of the liver, while the intrahepatic bile ducts were partly dilated in our case. This may partly explain normal total bilirubin and conjugated bilirubin.In spite of large similarities in clinical, imaging, and biochemical characteristics, notably, there are still significant differences between IgG4-SC and cholangiocarcinoma Table . Two gas19 In our study, serum IgG4 level of this patient was slightly rising (216 mg/dL); while Straub et al20 reported that serum IgG4 level (84 mg/dL) is within the normal range in the co-occurrence of cholangiocarcinoma with IgG4-SC. By comparison, a cross-sectional study of 53 IgG4-SC patients found that mean serum IgG4 level is 516 ± 98 mg/dL (mean ± SE), and the level over 280 mg/dL accounts for up to 50%.12 It seems that serum IgG4 level in cholangiocarcinoma is not as high as that in IgG4-SC. Ohara et al21 conducted a cohort study to establish a cut-off value for IgG4 level with the expectation of distinguishing IgG4-SC from other non-IgG4-related hepatopancreatobiliary diseases. The cut-off level of serum IgG4 was set at 135 mg/dL to differentiate all the 344 IgG4-SC patients from pancreatic cancer, PSC, and cholangiocarcinoma controls. However, it is noteworthy that the specificity of the value is low for distinguishing IgG4-SC from cholangiocarcinoma, especially those with cholangiocarcinoma and PSC. Moreover, Oseini et al22 suggested that serological IgG4 concentrations above 4 times the upper limit of the normal range is more specific for IgG4-SC.Although molecular mechanisms for elevated serum IgG4 and IgG4-positive plasma cells remains largely unknown, serum IgG4 level could be considered as a noninvasive and proven test for the diagnosis of IgG4-SC.25 It was also elevated both in IgG4-SC and in cholangiocarcinoma  during follow-up evaluations after surgery, radiotherapy, and/or chemotherapy.18F-fluorodeoxyglucose (18F-FDG) has been widely used in the diagnosis, staging, and relapse monitoring of malignancies. Recently, a few case reports have suggested the value of 18F-FDG PET/CT in IgG4-RD diagnosis.30 Zhang et al31 conducted a prospective research in 35 patients underwent 18F-FDG PET/CT who were diagnosed with IgG4-RD. The results indicated that 18F-FDG uptake lesion in bile duct and liver, without signs of infection, is a moderate indicator for IgG4-SC, and that 18F-FDG PET/CT may serve as a powerful tool by assessing disease distribution, thus helping to guide the biopsy site. Further, Ozaki et al32 indicated that 18F-FDG uptake by the hilar lymph node is significantly more frequent in autoimmune pancreatitis (a major constituent of IgG4-RD) than in pancreatic cancer. Based on the experience in the management of this case, we feel that 18F-FDG PET/CT is likely to be one potential key modality in the assessment of the overall condition of patients with IgG-SC. However, 18F-FDG PET/CT does not appear to have a well-established role in the diagnosis of patients with IgG4-SC in this case. Future prospective studies are required to define the cost-effectiveness and clinical impact in the management of patients with IgG4-SC.Integrated positron emission tomography and computed tomography (PET/CT) with 33 A variety of cytokines and growth factors involved in proliferation, apoptosis, senescence, and cell-cycle regulation are required for the development of cholangiocarcinoma genesis. It is well-known that PSC, another autoimmune biliary disease, has been regarded as one of the most common predisposing condition for cholangiocarcinoma with 0.5% to 1.5% of PSC patients developing into cholangiocarcinoma per year.34 As a result, the chronic inflammation microenvironment of IgG4-SC may also be involved in the pathogenesis of cholangiocarcinoma. In this case, immunohistochemical staining of interleukin-4 , IL-17, interferon-γ , and Foxp3 (the marker of Tregs) was performed. IL-4+ cells scattered in the germinal centers and the Disse space without preference  has not been reported in IgG4-SC previously, but was detected in the germinal centers and the Disse space without preference in this patient  and human epidermal growth factor receptor type 2 (HER2) kinase inhibitor, was significantly effective in inhibiting of the growth of human cholangiocarcinoma cell lines.43 A phase I study is still underway, investigating the role of Dovitinib, small molecule fibroblast growth factor receptor (FGFR) inhibitor, in combination with gemcitabine and capecitabine in patients with biliary cancers.44 Additionally, a phase I trial of everolimus, a mammalian target of rapamycin (mTOR) inhibitor, in combination with gemcitabine and cisplatin, points to a promising future for cholangiocarcinoma patients who are refractory to conventional therapies.45Curative surgery is the best way to obtain good survival for resectable cholangiocarcinoma. However, chemotherapy was the optimal therapy for this extensive metastatic patient. Combination chemotherapy with gemcitabine and cisplatin has become the practical therapies for cholangiocarcinoma.13 Thus, although low incidence of the cooccurrence of IgG4-SC and cholangiocarcinoma exits, we highly recommend repeated core needle biopsy or even laparoscopy/laparotomy before immunosuppressive drugs are given in addition to the laboratory test of serum and tissue IgG4 levels. Follow-up of the serum CA19-9 and imaging evidences should continue once immunosuppressive therapy starts. 18F-FDG PET/CT should also take into consideration when imaging examinations (such as CT or MRI scanning) fail to help.In conclusion, we surmised that IgG4-SC and cholangiocarcinoma are not 2 separate entities; investigating whether they share common molecular abnormalities may lead to a better understanding of their relationship. At present, it is hard to make it clear whether these 2 diseases have a causal or just a coincidental association.