Recently, incretin hormones, including glucagon-like peptide-1 (GLP-1) analogue and dipeptidyl peptidase-4 (DPP-4) inhibitor, have been found to regulate glucose metabolism. The aim of this study was to elucidate the efficacy and safety of the clinical usage of DPP-4 inhibitors in Japan.This study was designed as a prospective, open-label, multi-center trial. Patients with diabetes mellitus type 2 (T2DM) with poor glycemic profiles (HbA1c ≥ 6.2%) in spite of receiving a medical diet, therapeutic exercise, and/or medications were eligible for this study. The participants received 50 to 100 mg of the DPP-4 inhibitor sitagliptin once daily for 12 months.One hundred and eighty-eight subjects were enrolled. After 12 months of sitagliptin treatment, HbA1c levels decreased  as well as fasting plasma glucose (FPG) . The rate of glycemic control achieved (in accordance with the guidelines of the Japanese Diabetes Society) significantly increased. Blood pressure and serum levels of triglycerides and total cholesterol decreased significantly. Furthermore, the Pittsburgh Sleep Quality Index (PSQI) and Diabetes Symptomatic Scores improved significantly. Adverse events such as hypoglycemia and loss of consciousness occurred in twenty three subjects (11%).These results suggest that the actions of DPP-4 inhibitors improve not only glycemic control, but also blood pressure, lipid profiles, and quality of life (QOL). Sitagliptin is a sound agent for use in the comprehensive treatment of patients with T2DM. In Japan, the Ministry of Health, Labour and Welfare published a report on health and nourishment in 2007 that estSitagliptin binds to DPP-4 and prevents the breakdown of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Both GLThis study is a single-arm, prospective, multi-center trial conducted to evaluate the efficacy and safety of the DPP-4 inhibitor sitagliptin in clinical use. In this trial, we particularly focused on the effects of sitagliptin on quality of life (QOL).The Institutional Review Board of Human Research at Saga University approved this study and informed consent was obtained from all participants. Patients with T2DM (age ≥ 20 years) with poor glycemic control profiles  in spite of receiving a medical diet, therapeutic exercise, and/or conventional anti-diabetic medications were recruited. The exclusion criteria were treatment with insulin, a history of severe diabetic ketoacidosis or coma, severe infection, perioperative state, severe trauma, pregnancy, breast-feeding, renal dysfunction , a history of experiencing side effects to sitagliptin or other unsuitableness. For the participants, sitagliptin was given as either a new prescription, as an additional prescription to other conventional anti-diabetic agents, or replaced other anti-diabetic drugs.The subjects received 50 mg sitagliptin, once a day for the first 3 months. After 3 months, the dose of sitagliptin was changed to between (and including) 25 mg/day and 100 mg/day, and other oral hypoglycemic drugs were added according to the discretion of each physician. The observation period was 12 months.After 12 months of treatment with sitagliptin, changes in HbA1c, fasting plasma glucose (FPG), blood pressure, body weight (BW), body mass index (BMI), total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), triglycerides (TG), 1.5-anhydro-D-glucitol (1.5-AG), microalbuminuria, and homeostasis model assessment analyses of beta cell function (HOMA-β) and insulin resistance (HOMA-IR) were assessed. We also assessed changes in the subjects’ quality of life (QOL) using the Euro QOL (EQ)-5 Dimensions (EQ-5D), the EQ Visual Analogue Scale (EQ-VAS), the Pittsburgh Sleep Quality Index (PSQI), and the Diabetes Symptomatic Score.The EQ-5D is a generic instrument for measuring health-related QOL that has been developed and validated in a number of European countries,7. The Et-test. To compare changes in the values of the EQ-5D, EQ-VAS, PSQI, and Diabetes Symptomatic Score, we used the Wilcoxon signed-rank test. Values of p < 0.05 were considered to be statistically significant.Values are expressed as the mean ± SD. To compare changes in the values of HbA1c, FPG, BW, BMI, BP, lipids, 1.5AG, and HOMA from baseline to after 12 weeks of treatment, we used the paired A total of 221 patients agreed to participate in this study. Of the 221 patients, 14 were excluded due to protocol violation. Among the 207 enrolled subjects, seven were excluded due to discontinuing sitagliptin within the first 3 months, and 12 were excluded because data acquisition to evaluate the efficacy of the drug failed. Therefore, sitagliptin efficacy over 3 months was evaluated in 188 subjects as efficacy population. The safety of sitagliptin over 12 months was also evaluated in the 207 enrolled subjects as safety population  and 12 months  of sitagliptin treatment  and those with a BMI ≥ 25 kg/m2 (n = 80), a decrease in HbA1c levels was observed after 12 months of sitagliptin treatment: 7.59% ± 1.16% to 7.06% ± 1.12%, p < 0.001 and 7.68% ± 1.47% to 7.05% ± 1.03%, p < 0.001, respectively , 6.9% ≤ baseline HbA1c < 8.4% (n = 89), and 8.4% ≤ baseline HbA1c (n = 43)], the HbA1c levels were decreased at 3 months  Figure d. FPG wa2, p = 0.006). At 12 months, these values had returned to baseline levels   in all subjects  of the 207 enrolled subjects suffered adverse events (AEs) Table . Two subThree subjects (1.15%) developed AEs that were sus-pected to have a causal relationship with sitagliptin. Hypoglycemia and loss of consciousness occurred in two subjects (0.96%). Pneumonia/intestinal tuberculosis and stool abnormality were recognized in one subject.In our study, the HbA1c and FPG levels were reduced at 3 months  and at 12 months  after treatment with sitagliptin at a dose of 25 to 100 mg/day. Our results are similar to those of previous studies reported in the US and JapaIn our study, AEs after sitagliptin treatment were seen in 23 (11%) of the 207 enrolled subjects. In particular, direct sitagliptin-related AEs such as hypoglycemia and loss of consciousness were observed in only two subjects (0.96%). A previous pooled analysis reportedSystolic and diastolic blood pressure decreased after 3 months of treatment with sitagliptin. The active isoforms of GLP-1 include GLP-1(7–36) amide and glycine-extended GLP-1(7–37). GLP-1: Shigeki Gondoh, Haruda Yoshio, Minekazu Hashimoto, Hideo Ikeda, Takahiko Imamura, Taketo Iwamoto, Ryota Kaihara, Hideyuki Kamachi, Yoshiyuki Koga, Shigetaka Kuroki, Kazuo Matsunaga, Tadahiro Mizukami, Taizo Minami, Hiroshi Nakanishi, Hirofumi Naito, Masanori Nakao, Masayuki Nakayama, Shinichi Nakayama, Akira Takahashi, Norio Takeda, Otohisa Tajiri, Satoshi Tamesue, Toshifumi Uchida, Yasufumi Uchida, Tetsushi Wakiyama, Tsuneko Yamaguchi, Kenichi Yamamoto and principal investigator Koichi Node.


Cacospongia mycofijiensis, is a potent inhibitor of metabolic activity and mitochondrial electron transport chain complex I in sensitive cells, but other cells are relatively insensitive to the drug. Sensitive cell lines (IC50 0.36–13.8 nM) include HeLa, P815, RAW 264.7, MDCK, HeLa S3, 143B, 4T1, B16, and CD4/CD8 T cells. Insensitive cell lines (IC50 12.2–26.5 μM) include HL-60, LN18, and Jurkat. Thus, there is a 34,000-fold difference in sensitivity between HeLa and HL-60 cells. Some sensitive cell lines show a biphasic response, suggesting more than one mechanism of action. Mitochondrial genome-knockout ρ0 cell lines are insensitive to mycothiazole, supporting a conditional mitochondrial site of action. Mycothiazole is cytostatic rather than cytotoxic in sensitive cells, has a long lag period of about 12 h, and unlike the complex I inhibitor, rotenone, does not cause G2/M cell cycle arrest. Mycothiazole decreases, rather than increases the levels of reactive oxygen species after 24 h. It is concluded that the cytostatic inhibitory effects of mycothiazole on mitochondrial electron transport function in sensitive cell lines may depend on a pre-activation step that is absent in insensitive cell lines with intact mitochondria, and that a second lower-affinity cytotoxic target may also be involved in the metabolic and growth inhibition of cells.Mycothiazole, a polyketide metabolite isolated from the marine sponge  Spongia mycofijiensis in 1988 D20 −19.3° ; HRESIMS [M + H]+, observed m/z 405.2215, calculated m/z 405.2212 for C22H33N2O3S, Δ = +0.7 ppm.Methanolic extracts of the marine sponge 0 , P815 , HL-60 , Jurkat , LN18, 4T1, B16, MDCK, and RAW264.7 . The parent lines are all phenotypically stable and exhibit properties characteristic of their cell or tissue of origin. HL-60 is regularly tested in our laboratory for respiratory burst activity and differentiation into neutrophils (retinoic acid) or monocytes (PMA or vitamin D3). Four of the five ρ0 cell lines lacking mitochondrial DNA  used in this study, with the exception of 143Bρ0, were generated in our laboratory by prolonged culture of the parental cell lines in the presence of ethidium bromide, followed by culture in its absence [0 cells were tested at regular intervals for loss of MET function by FCCP resistance of WST-1 reduction, and by PCR for absence of mitochondrially-encoded cytochrome b. Cell lines used in this study included murine splenic T-cells, adherent cell lines of human , mouse , and dog origin , and non-adherent cell lines of human  and mouse origin . Cell lines were obtained from the following sources: HeLa , HeLa S3 , 143B and 143Bρ®, 10% fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin with 50 μg/mL uridine and 1 mM pyruvate. RAW264.7 cells and CD4/CD8 T cells isolated from the mouse spleen were cultured in a DMEM-based complete T cell medium containing 10% FCS and other supplements as previously described [2 in air and high humidity. Adherent cell lines were passaged using TrypLE™ Express (Invitrogen) to detach the cells, washed with medium, and then passaged into fresh medium.The standard culture medium consisted of RPMI-1640 medium supplemented with 5–10% fetal calf serum, 2 mM glutamate, 25 μg/mL penicillin, 25 μg/mL streptomycin, 50 μg/mL uridine, and 1 mM pyruvate. Murine B16 cells, however, were cultured in complete medium (cIMDM) containing Iscove’s modified Dulbecco’s medium (IMDM), 2 mM Glutamaxescribed . All cel4 cells/well in a 96-well plate and treated with varying concentrations of MYZ for different lengths of time. MTT solution (20 μL of 5 mg/mL in PBS) was added to each well, and after 2 h incubation, 100 μL of solubilizer  was added to lyse the cells and dissolve the blue crystals. The absorbance was measured at 570 nm using a multi-well plate reader. Absorbances were then used to generate concentration-response curves and to calculate IC50 values (the concentration at which MTT reduction is 50% of a non-treated control) using SigmaPlot software.An MTT cell proliferation assay ,21 was u5 cells/mL and treated with MYZ. For the last 8 h of culture, 1 μCi 3H-thymidine was added to each well, and the plates were frozen to stop cell proliferation. After thawing, the cells in each well were transferred to filters using a cell harvester  and the radioactivity determined in a liquid scintillation counter . Cells were seeded in 96-well plates at a density of 1 × 1050 for growth inhibition, as previously described [In primary cultures of splenic T cells, a carboxyfluorescein succinimidyl ester (CFSE) proliferation assay was used to determine the ICescribed . Briefly5/mL) were cultured with varying concentrations of MYZ and then stained with trypan blue and counted in a hemocytometer.A trypan blue dye exclusion assay was used to determine cell viability. Cells . Samples were scanned and analyzed on a FACScan flow cytometer with BD Cell Quest Pro software.Flow cytometric analysis of cell DNA content following staining with propidium iodide (PI) was used to assess progression through the cell cycle. Cells were seeded at 1 × 10Mitochondrial ROS was measured by flow cytometry using changes in fluorescence of dichlorofluorescein (DCF). Briefly, cells were prepared as for cell cycle analysis, then 230 μL of 10 μM DCF in PBS was added and left for 30 min. PI (20 μL of 100 μg/mL) was also added to allow gating on only live, PI-negative cells. The samples were then scanned on a FACScan flow cytometer with BD Cell Quest Pro software.The main finding of this study was that MYZ requires mitochondrial function to exert its nanomolar effects, providing support for its action as a MET complex I inhibitor, but that some cell lines display a biphase response or are resistant to the action of the compound. Studies on isolated mitochondria and individual MET complex activities may better define the cellular mechanism of action of MYZ. It would be interesting to compare the effect of MYZ on mitochondrial oxygen consumption in sensitive and insensitive cell lines. This would clarify whether differences in sensitivity to the drug are due to drug uptake/efflux mechanisms or different targets. It would also be interesting to explore the secondary cytotoxic mechanism of action of MYZ that occurs in cells at high, micromolar concentrations.


Bacillus subtilis strain S1-4, with the capacity to efficiently degrade feathers, was isolated from chicken feathers. Sequencing showed that the genome of strain S1-4 differs from that of other B. subtilis strains, with limited insertions and deletions. The genome encodes multiple extracellular proteases and keratinases. Bacillus subtilis strain S1-4, which was isolated from chicken feathers from a local poultry farm in China, degrades chicken feathers efficiently. It can secrete several extracellular proteases which hydrolyze various substrates  .Feathers from the poultry industry are a valuable protein source and can be transformed into soluble peptides and free amino acids , 2. Manygelatin) . In ordeN50 of 43,179 bp, and the largest contig was 280,067 bp. The draft genome sequence contains a total of 4,447,441 bp with a G+C content of 43.1%, about 0.23 Mb more than that of the model strain B. subtilis 168. In addition, a circular plasmid with a length of 6,609 bp was assembled. The genome phylogenetic trees inferred from the whole orthologs and constructed using PhyML , B. amyloliquefaciens strain FZB52, and B. licheniformis strain ATCC 14580.The sequencing generated about 1.0 Gb paired-end reads with lengths of 90 bp, which covered the whole genome about 200 times. The clean reads were assembled using ABySS software . We obtang PhyML  showed tB. subtilis 168. In contrast, the CDS in the S1-4 genome sequence belonging to the subsystems of membrane transport, sulfur metabolism, mobility and chemotaxis, regulation, and cell signaling were much more numerous than those in strain 168. However, the putative genes encoding extracellular proteases and peptidases did not show any significant differences between strains S1-4 and 168, except that some single-nucleotide polymorphisms (SNPs) occurred between these genes. In addition, some CDS encoding putative reductases were present in S1-4. This genome sequence information indicated that the keratinolytic activities of strain S1-4 may be derived from proteases and putative reductases. Using Mauve software (Bacillus. Overall, the sequences of S1-4 and 168 were collinear, with only limited insertions and deletions. Several regions containing a putative prophage, a type II restriction-modification system, and mobile elements, which may be obtained from horizontal gene transfer in natural niches, were present in the S1-4 genome sequence.The draft genome sequence was annotated with the RAST system . A totalsoftware , we carrBacillus subtilis S1-4 under this whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under accession number ANIP00000000.The sequence of


The mechanisms of the latter effects are unclear. The aim of this study was to determine the mechanisms by which pioglitazone (PGZ), a member of the thiazolidinedione class of antidiabetic agents, decreases the activity of the MRC. In isolated mitochondria from mouse liver, we measured the effects of PGZ treatment on MRC complex activities, fully-assembled complex I and its subunits, gene expression of complex I and III subunits, and PGZ binding to subunits NDUFA9, NDUFB6, and NDUFA6. Treatment with PGZ increased mitochondrial gene transcription in mice liver and HepG2 cells. In these cells, PGZ decreased intracellular ATP content and enhanced gene expression of specific protein 1 and peroxisome-proliferator activated receptor (PPAR)γ coactivator 1α (PGC-1α).PGZ binds complex I subunits, which induces disassembly of this complex, reduces its activity, depletes cellular ATP, and, in mice and HepG2 cells, upregulates nuclear DNA-encoded gene expression of complex I and III subunits. As shown in Figure 3H]PGZ binds to mitochondrial proteins of about 980, 600, and 400 kDa molecular weight. To identify subunits that PGZ was bound to, we treated isolated mitochondria with 10 μM [3H]PGZ and complex I subunits were separated in parallel on two SDS/BN-PAGE gels as indicated in Methods. Autoradiography showed that [3H]PGZ was bound particularly to a subunit of 39 kDa, but also to subunits of 17 and 15 kDa that overlapped the signals originated by NDUFA9, NDUFB6, and NDUFA6, respectively  mRNA and mitochondrial DNA (mtDNA)-encoded  mRNA in the liver from ten control mice and in ten mice treated with 10 mg PGZ/kg/day for 12 weeks. This experiment revealed that gene expression of complex I subunits encoded by the nDNA was increased by 30% to 50% over control levels in PGZ-treated mice -encoded . To examine whether PGZ modifies gene expression of transcription factors involved in the regulation of gene encoding MRC proteins, we exposed HepG2 cells to 10 μM PGZ for 5 days and measured gene expression of Sp1, estrogen related receptor α (ERRα), and PGC-1α of the MRC by real-time polymerase chain reaction (RT-PCR). We found that PGZ treatment increased significantly gene expression of Sp1 and PGC-1α, but not of ERRα  PGZ (PGZ 3H(G) hydrochloride; Hartmann Analytic, Grupo Taper SA, Alcobendas, Spain) at 27°C for 30 minutes. Afterwards, proteins were separated in parallel, simultaneously, on three one-dimensional BN-PAGE as described above. Following electrophoresis, proteins from one of these three gels were transferred to a polyvinyl difluoride membrane (0.45-μm pore size)  as previously described [A total of 100 μg of liver mitochondria isolated from a C57BL/6J mouse were incubated with 10 μM [escribed . Western3H]PGZ for 30 minutes and lysed in radioimmunoprecipitation assay (RIPA) buffer . A total of 800 μg of mitochondrial lysates were incubated with 2 μg monoclonal antibody  at 4°C overnight. Following incubation, the immune complexes were precipitated with 50 μl protein G  or protein A  MicroBeads  for 4 h on ice. Columns were placed in the magnetic field of the μMACS separator and rinsed with 200 μl PBS buffer (pH 7.5). Precipitates were placed onto the columns and let to run through. The columns were rinsed four times with 200 μl PBS buffer and once with 100 μl low salt buffer (20 mM Tris/HCl (pH 7.5)). Afterwards, 20 μl of preheated 95°C hot 1 × SDS gel loading buffer (50 mM Tris/HCl (pH 6.8); 50 mM dithiothreitol; 1% SDS; 0.005% bromophenol blue; 10% glycerol) was applied to the column and incubated for 5 minutes at room temperature. Following incubation, 50 μl of preheated 95°C hot SDS gel loading buffer was added and elute was collected as immunoprecipitate. This was separated into two aliquots. In one of these, the radioactivity of the sample was quantitated by scanning laser densitometry . The second aliquot was electrophoresed in a 10% acrylamide gel and transferred to a nitrocellulose membrane. Subunits were analyzed by western blot. In addition, the individual bands in the blot containing subunits were excised and their radioactivity was measured by liquid scintillation spectrophotometry.Liver mitochondria were isolated from liver homogenates by differential centrifugation as described elsewhere . Mitochoin vitro experimental condition was repeated three to four times. Quantitative data are expressed as the mean ± SD unless otherwise mentioned. P values <0.05 were considered significant.Statistical analysis was performed using SPSS Statistical Software for Windows, version 9 . The unpaired t test was used to assess the significance of differences between means. Each ATP5A1: Subunit α complex V; BN-PAGE: Blue native polyacrylamide gel electrophoresis; CS: Citrate synthase; ERRα: Estrogen related receptor α; FADH2: Reduced flavin adenine dinucleotide; MRC: Mitochondrial respiratory chain; mtDNA: Mitochondrial DNA; nDNA: Genomic DNA; PGC-1α: PPARγ coactivator 1α; PGZ: Pioglitazone; PPARγ: Peroxisome proliferator-activated receptor γ; SDHA: Subunit 70 kDa complex II; Sp1: Specific protein-1; TZD: Thiazolidinediones; UQCRC2: Subunit core 2 complex III.The authors declare that they have no competing interests.IG-R performed many of the experiments and contributed to design, analysis and interpretation of data. PS-M was involved in the acquisition, analysis and interpretation of data, critical revision of the manuscript for important intellectual content. DF-M performed many of the experiments and analyzed and interpreted data. He also reviewed and approved the final version of the manuscript. TM-Y contributed by designing the study, in acquisition of data, and critical revision of the manuscript. JAS-H was involved in the conception, design and supervision of the study, in the analysis and interpretation of data, and writing the manuscript. All authors read and approved the final manuscript.


Male breast cancer accounts for 0.5–1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer.Tissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers.Growth factor receptors were variably expressed in 4.5% (MET) up to 38.5% (IGF1-R) of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII) were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers.Expression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of molecular tracers for male breast cancer. Breast cancer in males is a rare disease, accounting for 0.5–1% of all breast cancer cases With regard to potential druggable targets, knowledge on the expression of individual tumor markers is limited and variable. Estrogen Receptor α (ERα) and progesterone receptor (PR) expression in male breast cancer is present in around 90% of patients Nowadays, antibody-based molecular therapies have been developed for e.g. HER2 Since male breast cancer patients have more frequently lymph node metastases, molecular imaging may be of benefit for males to assess stage of disease and monitor disease progression or response to therapy. In the present study, we therefore investigated by immunohistochemistry the expression of growth factor receptors and membrane markers in male breast cancer and compared the results with those we observed in female breast cancer and gynecomastia, in order to find a panel of potential markers for therapy and molecular imaging of male breast cancer.The origin and composition of the male breast cancer study population was described before In addition, 32 gynecomastia cases from 2000–2010 were retrieved from the archives of the Department of Pathology of the University Medical Center Utrecht. Original HE slides were reviewed by two observers  to confirm the diagnosis, and to subtype gynecomastia  as described before Immunohistochemistry was carried out on 4 µm thick sections for a panel of growth factor receptors. After deparaffination and rehydration, endogenous peroxidase activity was blocked for 15 min in a buffer solution pH5.8 containing 0.3% hydrogen peroxide. After antigen retrieval, i.e. boiling for 20 min in 10 mM citrate pH6.0 (for progesterone receptor (PR), Hepatocyte growth factor receptor (MET), Fibroblast growth factor receptor 2 (FGFR2), CD44v6, CAXII, carbonic anhydrase 9 (CAIX), and GLUT1), or Tris/EDTA pH9.0 (estrogen receptor α (ERα), HER2, and Insulin-like Growth Factor-1 receptor (IGF1-R)) or Prot K (0.15 mg/ml) for 5 min at room temperature (EGFR), a cooling off period of 30 min preceded the primary antibody incubation. ERα  1∶200; PR  1∶100; HER2  1∶100; IGF1-R  1∶400; FGFR2  1∶800; CD44v6  1∶500; GLUT1  1∶200; CAXII  1∶200; CAIX  1∶1,000 were incubated for 1 h at room temperature. Primary antibodies against EGFR  1∶30; MET  1∶100 were incubated overnight at 4°C. All primary antibodies were diluted in PBS containing 1% BSA.The signal was amplified using Brightvision poly-HRP anti-mouse, rabbit, rat  or the Novolink kit  (in the case of EGFR) and developed with diaminobenzidine, followed by counterstaining with haematoxylin, dehydration in alcohol and mounting.All stainings were compared to normal breast tissue  and scored as positive when a clear membrane staining (2+ or 3+) was observed, except for HER2 where only a score of 3+ was considered positive. All scoring was done by JFV, RK and PJvD who were blinded to patient characteristics and results of other stainings. Expression data for the female breast cancers and for the hypoxia proteins in male breast cancer were derived from our previous studies Statistical analysis was performed using IBM SPSS Statistics version 18.0 . Associations between categorical variables were examined using the Pearson’s Chi-square test and associations between continuous variables using Student’s T-test. Logistic regression was used to correct for differences in ERα expression, tumor size (≤2 cm vs. >2 cm), age (<60 years vs. ≥60 years), and histological type  between male and female breast cancers. P-values <0.05 were considered to be statistically significant.In our study population of 133 cases of male breast cancer, we found 4 cases (3.0%) expressing HER2, 15 cases (11.4%) EGFR, 6 cases (4.5%) MET, 16 cases (12.1%) FGFR2, and 50 cases (38.5%) IGF1-R . RepreseCompared to female breast cancer, IGF1-R was more frequently expressed in male breast cancer (p<0.001), while MET and FGFR2 were less frequently expressed  and female (20.7%) breast cancer (p = 0.178). As shown in Like in female breast cancer, expression of HER2 in male breast cancer correlated with high histological grade (p = 0.023) and tumor-size (p<0.001). In contrast to female breast cancer, expression of EGFR, GLUT1, and CAIX in male breast cancer did not correlate with any clinicopathological feature. Expression of IGF1-R, MET, FGFR2, and CD44v6 was not correlated to clinicopathological features in both male and female breast cancer. Nevertheless, expression of HER2, MET, EGFR, GLUT1, and CAIX was, as expected, significantly associated with loss of ERα and PR expression in female breast cancer , whereas FGFR2 and CAXII were associated with ERα and PR expression , however the specificity (as compared to gynecomastia) would be high. Due to low sensitivity, expression patterns of hypoxia markers and CD44v6 were studied in male breast cancer. We found that the ERα and hypoxia associated marker CAXII was more frequently expressed in male compared to female breast cancer. This could not be explained by ERα expression or a different regulation of the hypoxia response, because expression of CAIX and GLUT1 was not significantly different in male and female breast cancer In conclusion, we found that expression of individual growth factor receptors  and CD44v6, CAXII in male breast cancer is different compared to female breast cancer. However, when used as a panel of markers for molecular imaging strategies, the potential detection rate is similar for male and female breast cancer. This implies that membrane targets for molecular imaging of female breast cancer can also be used for detecting male breast cancer. The feasibility of molecular imaging (and therapy) of male breast cancer requires further study, but the present study thereby serves as a starting point for development of a set of antibody-based therapeutics and molecular tracers for male breast cancer.


Diaphragmatic dysfunction found in the patients with acute lung injury required prolonged mechanical ventilation. Mechanical ventilation can induce production of inflammatory cytokines and excess deposition of extracellular matrix proteins via up-regulation of transforming growth factor (TGF)-β1. Lumican is known to participate in TGF-β1 signaling during wound healing. The mechanisms regulating interactions between mechanical ventilation and diaphragmatic injury are unclear. We hypothesized that diaphragmatic damage by short duration of mechanical stretch caused up-regulation of lumican that modulated TGF-β1 signaling.lumican-null, aged 3 months, weighing between 25 and 30 g, were exposed to normal tidal volume (10 ml/kg) or high tidal volume (30 ml/kg) mechanical ventilation with room air for 2 to 8 hours. Nonventilated mice served as control groups.Male C57BL/6 mice, either wild-type or High tidal volume mechanical ventilation induced interfibrillar disassembly of diaphragmatic collagen fiber, lumican activation, type I and III procollagen, fibronectin, and α-smooth muscle actin (α-SMA) mRNA, production of free radical and TGF-β1 protein, and positive staining of lumican in diaphragmatic fiber. Mechanical ventilation of lumican deficient mice attenuated diaphragmatic injury, type I and III procollagen, fibronectin, and α-SMA mRNA, and production of free radical and TGF-β1 protein. No significant diaphragmatic injury was found in mice subjected to normal tidal volume mechanical ventilation.Our data showed that high tidal volume mechanical ventilation induced TGF-β1 production, TGF-β1-inducible genes, e.g., collagen, and diaphragmatic dysfunction through activation of the lumican. T) in normal animals mimics this overdistention of the normal lung. Previous studies of human dermal fibroblasts has shown that TGF-β1 caused a marked increase of the production of type I and III collagens, fibronectin, and α-smooth muscle actin (α-SMA) Acute lung injury (ALI) and its most severe manifestation, acute respiratory distress syndrome (ARDS), are inhomogeneous lung diseases characterized by the initial diffuse inflammatory reactions, neutrophil influx into the lungs, loss of epithelial and endothelial integrity, the development of noncardiogenic pulmonary edema and is followed by fibroblast proliferation and extracellular matrix accumulation Lumican belongs to the family of small leucine-rich repeat proteoglycans (SLRPs) binding collagen fibrils and are important in regulating collagen fibrillogenesis, i.e., fibril diameter and interfibrillar spacings No statistical difference was found in pH, PaO2, PaCO2, mean arterial pressure, and peak inspiratory pressure at the beginning versus the end of 8 hours of mechanical ventilation . The norlumican-null mice, there was a reduction of damage of diaphragmatic fiber. This suggested that lumican may be involved in the pathogenesis of ventilator-induced diaphragmatic injury. No significant disruptions of diaphragmatic collagen fiber and increase of interfibrillar spacing were observed in mice subjected to normal tidal volume mechanical ventilation (10 ml/kg) for 8 hours as compared to that of the control, non-ventilated mice.Electromicroscpy was used to determine the effects of mechanical ventilation on the ultrastructures of diaphragmatic collagen fibers. Disruptions of diaphragmatic collagen fiber and increase of interfibrillar spacing were observed in mice subjected to high tidal volume mechanical ventilation for 8 hours as compared to that of control, nonventilated mice . In the T 10 ml/kg, 2 h = 1.3±0.3*, VT 30 ml/kg, 4 h = 1.1±0.4; VT 10 ml/kg, 8 h = 1.0±0.2, *P<0.05 versus Control). With immunohistochemistry, we then further examined the role of lumican up-regulation and determined the cells types involved in the ventilation-induced diaphragmatic injury (T) 30 ml/kg increased as compared to control, nonventilated mice, and that of lumican null mice ventilated at VT 30 ml/kg. However, no significant increase of staining of lumican was found in mice ventilated at VT 10 ml/kg. Nonimmune rabbit immunoglobulin G control showed no detectable staining . To determine if the increase of mechanical ventilation-induced diaphragmatic damage was accompanied by up-regulation of procollagens, we measured type I and type III procollagen, fibronectin, and α-SMA mRNA  and angiostatic (IP-10 and CXCL11) chemokines may promote aberrant angiogenesis/fibrosis in vivo mouse model, we have demonstrated that high tidal volume mechanical ventilation induces diaphragmatic dysfunction, which are associated with activation of type I and III procollagen, fibronectin, and α-SMA mRNA, TGF-β1 and free radical production. This process was dependent, at least in part, on the lumican pathway. Our data add to the understanding of the effects of mechanical forces in the diaphragmatic injury in the lumican-null mice. In patients with ARDS in the early fibroproliferative phase, the inhibition of lumican may offer new treatment options for these patients.Though the ARDS network trial demonstrated that low is safer than high tidal volume ventilation, these findings have been questioned and the mechanisms of injury and protection need to be further examined All procedures for handling mice were conformed to ARVO . Statements for the use of animals in research are approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital (Permit Number: 2008090102). All surgery was performed under ketamine and xylazine anesthesia, and all efforts were made to minimize suffering.lumican-null (Lum−/−) mice 3 months old, weighing between 25 and 30 g, were used. Lumican−/− mice line, were generated by targeted gene disruption as previously described −/− and their wild-type littermates) were of a C57BL/6 genetic background and were identified by polymerase chain reaction. Mice that are homozygous for the targeted mutation are viable and fertile. The lumican deficient mice have serious functional defects including corneal opacity and fragile skin and tendon associated with disorganized and loosely packed collagen fibers. This proteoglycan has also been shown to participate in the regulation of many cellular functions including cell proliferation, migration, adhesion, and gene expression −/− mice were confirmed using Western blot analysis.Male C57BL/6, either wild-type or −/− mice ; group 3, VT 30 ml/kg wild-type mice ; group 4, VT 30 ml/kg lumican−/− mice ; group 5, VT 10 ml/kg wild-type mice  ; group 6, VT 10 ml/kg lumican−/− mice .Animals were randomly distributed into six groups in each experiment: group 1, control, nonventilated wild-type mice ; group 2, control, nonventilated lumicanWe used an established mouse model of ventilator-induced lung injury (VILI), as previously described The diaphragms were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer, (pH 7.4) for 1 hour at 4°C. The diaphragms were then postfixed in 1% osmium tetroxide (pH 7.4), dehydrated in a graded series of ethanol, and embedded in EPON-812. Thin sections (70 nm) were cut, stained with uranyl acetate and lead citrate, and examined on a Hitachi H-7500 EM transmission electron microscope.The diaphragms were homogenized in 0.5 ml of lysis buffer -N, N, N′, N′-tetraacetic acid, 50 µM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiotreitol, 400 µM aprotinin, and 400 µM phenylmethylsulfonyl fluoride), transferred to eppendorff tubes and placed on ice for 15 min. Tubes were centrifuged at 14,000 rpm for 10 min at 4°C and supernatant was flash frozen. Same amount of proteins from crude cell lysates determined by the Bradford method were resolved by 10% bis-acrylamide gel electrophoresis and electrotransferred to Immobilon-P membranes . For assay of lumican and glyceraldehydes-phosphate dehydrogenase (GAPDH) protein expression, immunoanalyses were performed with antibodies against lumican and GAPDH . Blots were developed by enhanced chemiluminescence .5′-TGTGCCACTCTGACTGGAAGA-3′ and reverse primer 5′-CAGACGGCTGAGTAGGGAACA-3′; type III procollagen, forward primer 5′-GGAAAGGATGGAGAGTCAGGAA-3′ and reverse primer 5′-CATTGCGTCCATCAAAGCCT-3′; fibronectin: forward primer 5′-CGCTGTGACAACTGCCGTA-3′and reverse primer 5′-TTGTAGTTGTGGCCGGTGG -3′; α-SMA: forward primer 5′-GAACCCTGAGACGCTGCTCCAGCTATGTG-3′ and reverse primer 5′-CAGTAGTCACGAAGGAATAGCCACGC-3′, and GAPDH as internal control by using the following primers: forward primer 5′-AATGCATCCTGCA CCACCAA-3′ and reverse primer 5′-GTAGCCATATTCATTGTCATA-3′  For isolating total RNA, the diaphragms were homogenized in TRIzol reagents  according to the manufacturer's instructions. Total RNA (1 µg) was reverse transcribed by using a GeneAmp PCR system 9600 , as previously described The diaphragms were homogenized in phosphate buffered saline containing butylated hydroxytoluene. The malondialdehyde in the protein extracts was measured using the Oxiselect TBARS assay kit  containing thiobarbituric acid reactive substances. Each sample was run in duplicate and expressed as µmole/g protein according to the manufacturer's instructions.At the end of the study period, the lungs were lavaged via tracheostomy with a 20-gauge angiocatheter (sham instillation) 3 times with 0.6 ml of 0.9% normal saline. The effluents were pooled and centrifuged at 2,000 rpm for 10 minutes. Supernatants were frozen at −80°C for further analysis of the cytokine. TGF-β1 with a lower detection limit of 4.61 pg/ml was activated from the latent form and measured in BAL fluid by using a commercially available immunoassay kit containing primary polyclonal anti-mouse TGF-β1 antibodies . Each sample was run in duplicate according to the manufacturer's instructions.The diaphragms from control, nonventilated mice, and ventilated mice exposed to 10 ml/kg or 30 ml/kg ventilation for 8 hours while breathing room air were paraffin embedded, sliced at 4 µm, deparaffinized, antigen unmasked in 10 mM sodium citrate (pH 6.0), incubated with normal rabbit IgG or anti-lumican primary antibody (1∶100), and biotinylated goat anti-rabbit secondary antibody (1∶100) according to the manufacturer's instruction for an immunohistochemical kit . The specimens were further incubated with horseradish peroxidase-streptoavidin complex, the immune reaction was visualized with diaminobenzidine (DAB), and counterstained by hematoxylin. A dark-brown DAB signal, identified by arrows, indicated positive staining of lumican of skeletal muscle cells, whereas shades of light blue signified nonreactive cells.The type I procollagen, type III procollagen, fibronectin, and α-SMA mRNA and Western blots were quantitated by using a National Institutes of Health (NIH) image analyzer, ImageJ 1.27z  and presented as arbitrary units. Values were expressed as the mean ± SD for at least 5 separate experiments. The MDA and TGF-β1 were conducted by using Statview 5.0 . All results of type I procollagen, type III procollagen, fibronectin, and α-SMA mRNA and Western blots were normalized to control, nonventilated mice breathing room air. ANOVA was used to assess the statistical significance of the differences, followed by multiple comparisons with a Scheffe′s test, and a P value <0.05 was considered statistically significant.


Staphylococcus pseudintermedius strain K7, isolated from the nares of a veterinarian in Seoul, South Korea.We report the genome sequence of the methicillin-resistant  Staphylococcus pseudintermedius is a commensal in dogs and an important opportunistic pathogen commonly associated with skin and ear infections. S. pseudintermedius is not part of the normal human microflora, and occurrence in humans is limited mainly to individuals who have regular contact with pets, such as pet owners and veterinarians. Methicillin-resistant S. pseudintermedius (MRSP) is a serious veterinary problem due to its characteristic multidrug-resistance phenotype , was isolated from the nares of a healthy female veterinarian at a small animal veterinary teaching hospital in Seoul, South Korea, in April 2008 (ant(6′)-Ia-aph(3′)-III (aminoglycoside resistance), mecA and blaZ (β-lactam resistance), erm(B) , lnu(A) (lincosamide resistance), tet(M) (tetracycline resistance), and dfrG (trimethoprim resistance), and the exfoliative toxin gene set.MRSP strain K7, belonging to ril 2008 . This stDe novo genome assembly using Geneious v6.1.3 produced 77 contigs of >1,000 bp, with a mean length of 37,767 bp. Automated annotation of genes was performed on the Rapid Annotations using Subsystems Technology (RAST) server , generating 101,942 reads with an average length of 437 bp. S. pseudintermedius strains, MRSP strain E140 (accession no. ANOI01000001) and the two methicillin-susceptible S. pseudintermedius strains HKU 10-03 (accession no. CP002439) and ED99 (accession no. CP002478). The K7 genome is 6 kb smaller than that of MRSP E140 but is 146 and 191 kb larger than those of HKU 10-03 and ED99, respectively. Methicillin resistance is associated with the presence of staphylococcal cassette chromosome mec element (SCCmec) type V (28 kb). K7 is predicted to have 2,937 protein-coding sequences and 85 tRNAs.The draft genome of K7 is estimated to be 2,763,498 bp and has a G+C content of 37.4%. K7 has a slightly lower G+C content (0.2 to 0.8%) than the other sequenced S. pseudintermedius strains from different countries, as well as aid in the identification of factors related to host range.This K7 MRSP genome of human origin will facilitate future comparative genomic analyses of BARM01000001 to BARM01000077.The K7 genome has been deposited at DDBJ/EMBL/GenBank under the accession no.


Immunoglobulins, antigens and complement can assemble to form immune complexes (IC). ICs can be detrimental as they propagate inflammation in autoimmune diseases. Like ICs, submicron extracellular vesicles termed microparticles (MP) are present in the synovial fluid from patients affected with autoimmune arthritis. We examined MPs in rheumatoid arthritis (RA) using high sensitivity flow cytometry and electron microscopy. We find that the MPs in RA synovial fluid are highly heterogeneous in size. The observed larger MPs were in fact MP-containing ICs (mpICs) and account for the majority of the detectable ICs. These mpICs frequently express the integrin CD41, consistent with platelet origin. Despite expression of the Fc receptor FcγRIIa by platelet-derived MPs, we find that the mpICs form independently of this receptor. Rather, mpICs display autoantigens vimentin and fibrinogen, and recognition of these targets by anti-citrullinated peptide antibodies contributes to the production of mpICs. Functionally, platelet mpICs are highly pro-inflammatory, eliciting leukotriene production by neutrophils. Taken together, our data suggest a unique role for platelet MPs as autoantigen-expressing elements capable of perpetuating formation of inflammatory ICs. Upon activation, cells of many different lineages bud small extracellular vesicles called microparticles (MP)  from the cytoplasmic membrane. Given the universality of this process, MPs likely play essential functions that include cell–cell communication and transportation of mediators  represent immunoglobulins and complement clustered about target antigen. They are extremely potent at eliciting inflammatory responses and implicated in response to infections as well as multiple autoimmune diseases such as Sjögren's syndrome , transmission electron microscopy and proteomic approaches, we found that ICs in RA synovial fluid frequently include MPs, thereby forming true mpICs. Although we observed that MPs originating from platelets express FcγRIIa, mpICs formed not through binding to this receptor but rather by recognition of citrullinated autoantigens such as vimentin and fibrinogen. Together, these results identify a unique site of autoantigen expression in RA and suggest that MPs, including those derived from platelets, may participate in the generation of a novel type of potent ICs in this disease.MPs are widely recognized as ∼100–1000 nm extracellular vesicles that can exhibit phosphatidylserine. These uncommon features permit the detection of MPs cytofluorometrically by employing Annexin-V (which recognizes phosphatidylserine) conjugated to fluorochromes and size discrimination  parameters to distinguish small particles , we asse+ particles are promptly solubilized by Triton X-100 treatment, a finding consistent with the membrane composition present in MPs (+ MPs/µl in SF of RA patients (n = 23). For comparison, we find 193,000 ± 20,000 Annexin-V+ MPs/µl in SF of PA patients (n = 18), significantly lower than the concentrations detected in RA , ruling out the involvement of freezing–thawing in an artifactual generation of mpICs.Intrigued by the presence of large dimension MPs in RA SF, we hypothesized that MPs could bind immunoglobulins and thereby form large MP-containing ICs (mpICs). To assess the presence of mpICs in RA SF, we used cryo-TEM, which allows direct visualization of small particles and their membrane bilayers. We further examined the exposure of phosphatidylserine in RA SF mpICs using Annexin-V conjugated to 4 nm gold nanoparticles and identified immunoglobulin containing ICs using larger gold nanoparticles (10 nm) conjugated to protein A. Using this method, we identify macromolecular structures up to 2 µm in diameter that contain both ICs and MPs (mpICs). Interestingly, these mpICs often harbour multiple MPs at the periphery , ∼65% of+ MPs  of MPs in SF. Considering their role in arthritis  or PA SF (n = 18). In this assay, the fluorescent label present in in vitro MPs affords discrimination of newly formed mpICs from those present endogenously. As anticipated, the fluorescent platelet MPs are easily detected by hs-FCM, are smaller than intact platelets, the vast majority of them having dimensions of ∼100–300 nm in diameter .To assess a contribution of platelet MP CD32a in mpIC formation, we incubated diameter . Strikindiameter , includidiameter . Interesdiameter . To explin vitro and those in PA SF, the platelet MPs in RA SF contain citrullinated epitopes , were applied to protein G affinity columns and the IgG within mpICs were isolated. Using an autoantigen microarray  following the citrullination of its surface and (ii) upon binding to citrullinated proteins. Autoantigens other than vimentin and fibrinogen may also be expressed on surface of platelet MPs during RA. Indeed, multiple citrullinated proteins derived from PAD4-treated MPs are recognized by RA autoantibodies. Further, we also identify autoantibodies against canonical citrullinated RA autoantigens such as filaggrin (including cfc), enolase and histone H2A/a in mpICs. Among these other proteins, only histones were also identified in our mass spectrometry analyses. Histones may be associated with DNA, itself bound to MPs. Alternatively, histones are recognized potent platelet stimuli and can associate with platelets directly . Fresh SF harvested without anticoagulant were cleared of leukocytes by centrifugation at 19006/ml in Tyrode's buffer and stimulated using 0.5 µg/ml cross-linked collagen related peptide  for 15 min prior to stimulation.Platelets were obtained from citrated blood of healthy human volunteers under an approved Institutional Review Board protocol . Platelets were isolated   and 0.5 µg of PE-conjugated mouse anti-human CD41 (M148) (anti-CD41-PE)  in combination to 5 µl of FITC or V450-conjugated Annexin-V (BD). The samples were labelled during 30 min in a volume of 100 µl of Annexin-V buffer 1× and diluted to 600 µl before hs-FCM analysis. To process the data quantitatively, 100,000 polystyrene microsphere  were added to each tube and 1000 microspheres were acquired. The included Annexin-V+ or CD41+ events were portrayed as FSC-PMT versus side scatter (SSC) graph and the relative dimensions were displayed according to the acquisition of Sky Blue microspheres of mean diameter of 90, 220, 450, 840 and 3200 nm . The sensitivity of the MPs to detergent was assessed by addition of Triton X-100 to the SF samples (1:5 in PBS) prior to addition of the antibodies  were incubated with 0.5 µg of Cy5-conjugated F(ab′)6) were incubated in presence of 50 µl RA or PA SF in final volume 100 µl PBS for 48 h at 4°C. Then, 50 µl of the cocktail were incubated with anti-IgG-Cy5 as described above. When the contribution of CD32a was evaluated, 20 µg/ml of CD32a blocking Fab fragment antibody  was added 15 min prior to MP addition to SF.Fluorescent MPs . One microgram of sample was lysed in Laemmli buffer, electrophoresed, transferred onto membranes and incubated in 0.2% milk/TBS-Tween solution containing a serum raised against a peptide in the cytoplasmic domain of CD32a  or RA SF (5 µl pre-incubated with Annexin-V-V450) were incubated with 2 µl of PE-conjugated anti-human CD32 (clone IV.3) . To determine the capability of MPs to associate with exogenous fibrinogen, 15 µg/ml of Alexa Fluor-647-conjugated fibrinogen (Invitrogen) was incubated with 10 × 106 platelet MPs or added to 10 µl of RA SF in a final volume of 100 µl for 48 h at 4°C. Their associations were evaluated using Cell-Tracker fluorescence (for platelet MPs) or anti-CD41-PE (for SF). To detect vimentin and fibrinogen on surface of MPs, platelet MPs (20 × 106) and RA SF (20 µl) were pre-incubated with 20 µg/ml of CD32a blocking Fab fragment antibody for 15 min to abrogate any risk of potential non-specific binding of primary/secondary antibodies to platelet Fc receptor. MPs were then incubated with 25 µg/ml goat anti-fibrinogen (Abcam) or 1:100 goat anti-human vimentin  for 48 h at 4°C. Samples were washed, centrifuged and pellets retrieved in 100 µl of PBS. For flow cytometry detection, 6.25 µg of DyLight 649-conjugated F(ab)′2 anti-goat IgG (Jackson ImmunoResearch) and anti-CD41-PE were added.Fluorescent platelet MPs . 350 × 106 MPs were incubated with/without 3 U of PAD for 18 h as described  were incubated in presence of 1.2 µg of anti-CD41-PE for 30 min, then with 50 µl anti-PE magnetic beads (Miltenyi Biotec (MACS), CA, USA) for 20 min at RT and diluted to 1 ml in PBS. The preparations were applied to LS magnetic MACS columns, washed with 3 ml PBS 3 times and eluted with 4.5 ml PBS. The eluted CD41+ MPs were centrifuged at 18,000g for 90 min at 18°C and pellets retrieved in 100 µl of PBS containing 0.5% Triton X-100 and 0.5% NP40. The CD41+ MP lysates were applied to a G-protein column, washed with 7 ml PBS, then with 2 ml PBS pH 5.0 and the IgG eluted with 100 mM glycine pH 2.5. The pH of the fraction was adjusted to 7.4 and conserved at −80°C. The reactivity of the antibodies retrieved from the mpICs against forty candidate RA autoantigens  are formed after antibody deposition against antigens and propagate inflammation in autoantibody-driven autoimmune diseases. Extracellular vesicles of submicron dimensions, called microparticles, are also often present in the same pathologies where ICs are observed. Understanding how ICs continuously form is of high relevance to human diseases.RESULTS:We used a new generation of flow cytometer and sophisticated cryo-transmission electron microscopy approaches to characterize and quantify ICs and microparticles in the synovial fluid from patients affected with rheumatoid arthritis (RA). We identify a new class of ICs that predominates in RA; the microparticle-associated immune complexes (mpICs). We further reveal that platelet microparticles participate to the formation of mpICs via exposure of the canonical RA autoantigens. We demonstrate that these platelet mpICs are highly active, eliciting pro-inflammatory leukotriene release by neutrophils.IMPACT:Considering the abundance of platelet microparticles in autoimmune disease such as RA, this suggests that microparticles may serve to the constant autoantibody deposition and perpetuation of inflammation in chronic autoimmune inflammatory disorders.2 and 10 mM HEPES, pH 7.4. A 10 µl aliquot of SF was mixed with 1 µl of 10 nm Protein A-conjugated gold nanoparticles , incubated for 30 min, mixed with 1 µl of 4 nm Annexin-V-conjugated gold nanoparticles suspension  and further incubated for 15 min. Aliquots (4 µl) were deposited on TEM grids coated with a perforated carbon film and quickly frozen in liquid ethane. TEM grids were mounted on a Gatan 626 cryoholder and transferred into a Tecnai F20 (FEI) microscope operated at 200 kV. Images were recorded with an USC1000-SSCCD camera (Gatan). The synthesis and characterization of Annexin-V-conjugated gold nanoparticles will be presented elsewhere (patent application Mornet and Brisson WO2007122259).For cryo-Transmission Electron Microscopy (TEM) experiments, samples were diluted 10× in a buffer containing 150 mM NaCl, 2 mM CaCl+ MPs (isolated using MACS as described above) was measured using Dc Protein Assay . Twenty-five micrograms proteins from individual SF CD41+ MP sample were reduced and alkylated by using 21 mM triethyl phosphine (TEP) and 58 mM iodoethanol (IE). The samples were speed dried in a high speed vacuum centrifuge and redissolved in 100 µl of 0.1 M ABC buffer containing 0.1% perflurooctanoic acid (PFOA). Trypsin was added in the ratio of 1 µg per 50 µg of proteins. The samples were placed in a 37°C water bath for 16 h. The reaction was stopped by drying the samples and adding 70% IPA in ammonium hydroxide (50 mM) solution, pH 10. The enzyme was inactivated by reducing (TEP/acetonitrile) at 45°C for 1 h and then alkylating (iodoethanol/CH3CN) in the dark at 45°C for 2 h. A 1:1:1 solution of ethanol, ethyl acetate and HPLC grade water with 0.1% TFA was added to the dried peptide digest and subjected to high speed vacuum centrifuge to remove PFOA. The samples were then analysed by LC–MS/MS. A total of four SF biological replicates were analysed by mass spectrometry. PFOA was purchased from TCI America . All other reagents were of the highest quality commercially available.We used mass spectrometry to survey the presence of known autoantigens in platelet MPs in RA SF  equilibrated with 0.1% formic acid/2% acetonitrile v/v and washed for 5 min with the equilibration solvent at a flow rate of 25 µl/min, using an isocratic loading pump operated through an auto-sampler. After the washing step, the trapping column was switched in-line with a reversed-phase C18 Acclaim PepMap 100 column  and the peptides were chromatographed using a linear gradient of acetonitrile from 4.8 to 40% in aqueous 0.1% formic acid over a period of 96 min at the above-mentioned flow rate such that the eluate was directly introduced to the mass spectrometer. An 80% acetonitrile elution step was subsequently performed for 5 min prior to resetting the analytical column to the initial equilibration conditions for 11 additional minutes at the end of the chromatographic run. The mass spectrometer was operated in a data-dependent MS to MS/MS switching mode, with the five most intense ions in each MS scan subjected to MS/MS analysis. The full scan was performed at 60,000 resolution in the Orbitrap detector and the MS/MS fragmentation scans were performed in the ion trap detector (IT) CID mode such that the total scan cycle frequency was approximately 1 s. The threshold intensity for the MS/MS trigger was always set at 1000 and the fragmentation was carried out using the CID mode using normalized collision energy (NCE) of 35. The data was collected in the profile mode for the full scan and centroid mode for the MS/MS scans. The dynamic exclusion function for previously selected precursor ions was enabled during the analysis such that the following parameters were applied: repeat count of 2, repeat duration of 45 s, exclusion duration of 60 s and exclusion size list of 150. Xcalibur software (version 2.0.7), Thermo-Finnigan, Inc., San Jose, CA) was used for instrument control, data acquisition, and data processing. MS raw data files were searched against Swiss-Prot (version 57) database using Mascot database search software  to identify peptides/proteins with the following parameters: S-hydroxyethylation of cysteine as a fixed modification, and oxidation of methionine to methionine sulfoxide as variable modifications. The mass tolerance for the precursor ion was set to 10 ppm, and for the product ion it was set to 0.8 Da. Strict trypsin specificity was applied, allowing for one missed cleavage. Peptides with a minimum score of 20 were considered as significant.The protein digests were analysed by LC–MS/MS using an Ultimate 3000 LC system  interfaced to an LTQ-Orbitrap XL mass spectrometer . The platform operated in the nano-LC mode using the standard nano-ESI API stack fitted with a picotip emitter . The solvent flow rate through the column was maintained at 300 nl/min using a 1:1000 splitter system. The protein digests (5 µl) were injected into a reverse-phase C18 PepMap trapping column (0.3 × 5 mm9 MPs/ml), anti-fibrinogen (50 µg/ml), or in vitro mpICs. RA SF mpICs were enriched by centrifugations of 100 µl of RA SF  and resuspended in 10 µl of PBS. In vitro mpICs were generated by pre-incubation of MPs (1 × 109/ml) and goat anti-fibrinogen from Abcam (50 µg/ml) at RT for 30 min. Reactions were stopped by addition of 500 µl cold methanol/acetonitrile (1:1) containing 12.5 ng of internal standards PGB2 and 19-OH-PGB2. Samples were stored at −20°C O/N then analysed by reverse-phase HPLC as previously described .Statistical significance was determined using non-parametric Mann-Whitney test. When indicated, unpaired Student's NC: study conception, study design, acquisition of data, analyses and interpretation of data, manuscript preparation, statistical analyses; ST: acquisition of data, analyses and interpretation of data, manuscript preparation; LHB: study design, acquisition of data, analyses and interpretation of data, manuscript preparation, statistical analyses; CC: acquisition of data, manuscript preparation, statistical analyses; RSu: acquisition of data, manuscript preparation, statistical analyses; LL: acquisition of data, manuscript preparation, statistical analyses; AA: acquisition of data, analyses and interpretation of data; RSh: acquisition of data, analyses and interpretation of data; RG: interpretation of data, manuscript preparation; PAN: contributed critical reagent, analyses and interpretation of data, manuscript preparation; RWF: generated critical reagent, manuscript preparation; WHR: analyses and interpretation of data, manuscript preparation; AB: acquisition of data, analyses and interpretation of data, manuscript preparation; DML: study conception, analyses and interpretation of data, manuscript preparation; EB: study conception, study design, acquisition of data, analyses and interpretation of data, manuscript preparation.


As previously reported, high temperature- and high pressure-treated red ginseng (HRG) contain higher contents of phenolic compounds and protect C2C12 muscle cells and 3T3-L1 adipocytes against oxidative stress. This study investigated the effect of HRG on oxidative stress using a mouse model. Our results show that the levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, hepatic malondialdehyde in the HRG group were significantly lower than those of the exercise groups supplemented with commercial red ginseng (CRG) or not supplemented. The muscular glycogen level, glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities of the HGR group were higher than that of the CGR group. Furthermore, the HRG treatment group displayed upregulated mRNA expression of Cu/Zn-SOD and muscle regulatory factor 4. These results indicate that HRG may protect oxidative stress induced by exercise as well as improve exercise performance capacity. In general, appropriate exercise is helpful to prevent cancer, diabetes, stroke, and hypertension, but the habit of excessive training promotes the generation of reactive oxygen species (ROS), and then leads to oxidative stress . ROS, byPanax ginseng C.A. Meyer, Araliaceae Family) is a popular traditional herbal medicine that has been used for centuries in Korea, China, and Japan. Over 30 different ginsenosides from saponin in ginseng have been isolated and identified. Major ginsenosides can be separated into protopanaxadiol-type  and protopanaxatriol-type . Korean red ginseng (KRG) is processed through steaming and drying processes, and has some distinct ginsenosides, such as Rg3, Rg5, Rh1 and Rk1 [2) [p-hydroxybenzoic acid (823.3 μg/100 g) and syringic acid (6938.8 μg/100 g) than that found in white ginseng or commercial red ginseng (CRG) [Ginseng  . The HRGng (CRG) . Furtherng (CRG) .Therefore, to elucidate the inhibitory effect of HRG on oxidative stress, we investigated the oxidative stress indices in mice treated with HRG.Anthrone, potassium chloride (KCl), 1,1,3,3-tetraethoxypropane (TEP), potassium hydroxide, potassium phosphate, sodium chloride (NaCl), β-nicotinamide adenine dinucleotide phosphate hydrate (β-NADP), β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH), glucose-6-phosphate, bradford reagent, maleimide, glucose, and glacial acetic acid were purchased from Sigma . Ethylenediaminetetraacetic acid (EDTA), bovine serum albumin, and trizma base were obtained from Bio-Rad . Pyruvate was purchased from Fluka AG .2) for 20 min. The steamed HRG was dried at 50 °C, and the dried HRG was ground to 20~30 mesh, and refluxed with 10 volumes (v/w) of hot water at 80 °C for 3 h. After reflux, the extract was filtered two times and concentrated at 55 °C. Then, the concentrate was freeze-dried and stored at −20 °C until used.The red ginseng treated with high temperature and high pressure was produced according to the method developed by Korea Food Research Institute. Four-year-old ginsengs were first dried at 50 °C, and then again steamed at high temperature (140 °C) and high pressure (3 kg/cmp-hydroxybenzoic acid (823.3 μg/100 g), caffeic acid + vanillic acid (307.8 μg/100 g), syringic acid (6938.8 μg/100 g), p-coumaric acid (901.2 μg/100 g), ferulic acid (3796.7 μg/100 g), and cinnamic acid (3737.1 μg/100 g). Phenolic compounds were substantially increased compared with CRG and white ginseng as described previously [HPLC was used to determine phenolic acids in HRG, which contain maltol (6213.2 μg/100 g), ad libitum and weighed periodically during the experimental period . Male ICR mice were purchased at three weeks-old from DooYeol Biotech . The animals were housed in individual cages and maintained at 23–25 °C with 50%–60% relative humidity. Feed and water were provided l period .At the age of four weeks, the mice were randomly divided into four groups; no exercise group (CON), high intensity exercise group (E), high intensity exercise group supplemented with commercial red ginseng extract (E + CRG), high intensity exercise group supplemented with high temperature and high pressure-treated red ginseng extract (E + HRG); each group contained eight mice.g for 10 min to separate the serum, and then stored at −70 °C for analysis. The tissues were washed with 0.9% NaCl, and then stored at −70 °C until used.High intensity exercise was induced by a treadmill for three weeks. As shown in g for 5 min using a tissue grinder . The ground tissues were centrifuged at 12,000× g for 15 min at 4 °C. The each supernatant was placed in new vials and stored at −20 °C until used.The gastrocnemius tissues were placed in vials, and 2 mL of 50 mM potassium phosphate buffer (pH 7.5) containing 0.15 M NaCl and 1 mM EDTA were added to the vials. The tissues were homogenized at 2000× y = 0.3633x + 0.004) was prepared using BSA.The protein content was based on the method of Bradford . Three mThe activities of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in blood serum were analyzed by AST and ALT kit . One milliliter of each substrate solution of GOT and GPT was placed in a tube, incubated at 37 °C for 5 min, and then 200 μL of serum was added. The GOT mixture was incubated for 60 min and the GPT mixture was incubated for 30 min, respectively. Then, color reagent was added to the mixture, vortexed and incubated at room temperature for 20 min. After incubation, 10 mL of 0.4 N NaOH was added, vortexed, and incubated for 10 min, and then the absorbance of mixture against distilled water was monitored at 505 nm. GOT and GPT activities in serum were expressed as U/L, respectively.et al. [y = 3.8167x + 0.0014) was prepared using 1,1,3,3-tetraethoxypropane (TEP) and expressed as nmol MDA/g.Hepatic MDA content was measured by the method of Ohkawa et al.  with somet al. [2SO4 was added. The tube was heated at 100 °C for 10 min, and then the absorbance of the mixture was monitored at 620 nm. The calibration curve (y = 1.6136x − 0.0211) was prepared using glucose and expressed as mg/g.Muscular glycogen content was determined by the method of Seifer et al. . 100 mg 2, 53.7 μM glucose-6-phosphate and 0.77 mM maleimide. The 50 μL of tissue extracts was added to 1 mL of the assay medium to initiate the reaction. The assay medium was used to obtain baseline of the spectrophotometer at 340 nm.G6PDH activity of the tissue was determined as described by Deutsch  with somLDH activity of the tissue was measured by the method of Narang . The assTotal RNA was isolated from gastrocnemius tissues as described previously . First, p < 0.05 level.Data were expressed as means ± standard error (SEM), and the results were taken from at least three independent experiments performed in triplicate. The data were analyzed by one-way analysis of variance (ANOVA) procedure of Statistical Analysis System  with DunBody weight and feed intake of mice were determined every Monday morning during the experiment periods containing adaptation period for treadmill. Body weight and feed intake of mice were not measured during the acclimation period of seven days. As shown in The indices of oxidative stress in blood and tissues were analyzed . Figure To determine the effect of HRG extracts on oxidative damage of liver, hepatic MDA levels were measured. It was observed that the MDA content of the CON group (4.9 ± 1.5 nmol/g) was the lowest, but the MDA content of the E group (6.6 ± 1.0 nmol/g) was the highest. The MDA content (5.1 ± 1.0 nmol/g) of the HRG group was similar to the CON group but the CRG group (5.4 ± 1.2 nmol/g) was higher than the HRG group c.The effect of HRG extracts on skeletal muscle glycogen content was described in To investigate whether HRG extract has an effect on enzyme activities induced by exercise, the enzyme activities of G6PDH and LDH were determined in gastrocnemius tissue . The HRGIn KGR is produced through drying and steaming processes, and is used by many Asians to improve health and physical fitness ,22. ReceIt was reported that the enzymes’ activity of GOT and GPT were increased by acute physical exercise . The levWe have investigated the muscular glycogen content, which plays an important role as a fuel source in muscle contraction . During LDH induced by high intensity exercise is associated with both the generation of lactate and muscle damage . MoreoveIn conclusion, HRG protected tissue damage resulting from oxidative stress induced by high intensity exercise. Consequently, our results indicate that HRG, which contains phenolic compounds increased by high heat and high-pressure processes, may be responsible for the enhanced antioxidative activity.


Anthroposophic treatment includes special artistic and physical therapies and special medications. We here report an update to a previously published study of anthroposophic treatment for chronic diseases, including more patients and a longer follow up. The Anthroposophic Medicine Outcomes Study (AMOS) was a prospective observational cohort study of anthroposophic treatment for chronic indications in routine outpatient settings in Germany. Anthroposophic treatment was associated with improvements of symptoms and quality of life. Previous follow-up-analyses have been performed after 24 months or, in subgroups of patients enrolled in the period 1999-2001, after 48 months. We conducted a 48-month follow-up analysis of all patients enrolled in AMOS in the period 1999-2005.1,510 outpatients aged 1-75 years, starting anthroposophic treatment for chronic conditions in routine German outpatient settings, participated in a prospective cohort study. Main outcomes were Symptom Score , SF-36 Physical and Mental Component scores in adults, and disease-specific outcomes in the six most common diagnosis groups: asthma, anxiety disorders and migraine , depression , attention deficit hyperactivity symptoms , and low back pain .Median disease duration at baseline was 3.5 years. From baseline to 48-month follow-up all ten outcomes improved significantly . Standardised Response Mean effect sizes were large (range 0.84-1.24 standard deviations) for seven comparisons, medium for two comparisons , and small for one comparison . Symptom Score improved significantly with large effect sizes in adults and children, and in the four main anthroposophic therapy modality groups .This 48-month follow-up analysis confirmed previous analyses from the AMOS study. Outpatients receiving anthroposophic treatment for chronic indications had sustained, clinically relevant improvements of symptoms and quality of life. We here report an update to a previously published study of anthroposophic treatment for chronic diseases , includiChronic diseases are the most common cause of disease burden worldwide and are rarely completely cured . Many paAnthroposophic medicine (AM), founded by Rudolf Steiner and Ita Wegman in the 1920s , is a coAM is provided by physicians  in collaboration with non-medical practitioners  in hospitals and outpatient settings. AM treatment is used alone or in combination with conventional treatment as needed. In Europe 24 hospitals (including 14 with accident & emergency services) offer AM therapy, and an estimated 17.000 physicians in outpatient settings prescribe AM medications .Since AM therapy aims for sustained improvement ,7, long-This is the final follow-up analysis of a prospective observational cohort study of AM therapy for chronic diseases in routine outpatient settings (AMOS) . Compare•recruitment period: 1999-2005 vs. 1999-2001 in previous analyses •last follow-up: 48 months vs. 24 months in previous analyses ,25-30The research questions concerned the following items:A. Disease symptoms and quality of life at 48- month follow-up (main research question)B. Patient satisfaction after 6 and 12 monthsC. Safety in months 0-24D. Continuity of the physician-patient relationship at 48-month follow-up.For items A-C, the research question was: Can findings from previous analyses that were restricted to patient subgroups (items A-C) or earlier follow-ups (item A) be confirmed in the present full sample and at 48 months follow-up, respectively?For item D, previously not analysed, the research question was: Which proportion of the patients are, after 48 months, still being treated by the same physician who had enrolled them into the study?All physicians certified by the Physicians’ Association for Anthroposophical Medicine in Germany and working in an office-based practice or outpatient clinic in Germany were invited to participate in the AMOS study. The participating physicians were asked to recruit consecutive outpatients starting AM therapy under routine clinical conditions. Patients enrolled from 1 January 1999 to 31 December 2005 were included in the present analysis if they fulfilled the eligibility criteria (patients enrolled before 1 January 1999 had no follow-up documentation beyond 12 months). Inclusion criteria were:1. Outpatients aged 1-75 years.2. Starting AM therapy for any indication (main diagnosis)2a: AM-related consultation of at least 30 minutes followed by new prescription of AM medication, or2b: referral to AM treatment by non-medical therapist: art therapy, eurythmy therapy or rhythmical massage therapy.3. Duration of main diagnosis of at least 30 days at study enrolment.Patients were excluded if they had previously received the AM therapy in question (see inclusion criteria no. 2) for their main diagnosis. AM therapy was evaluated as a whole system  with subClinical outcomes were documented after 0, 3, 6, 12, 18, 24, and 48 months. In this analysis clinical outcomes were assessed at 48-month follow-up.•Primary outcome was Symptom Score, a compound measure of the symptoms for which the patients had sought medical attention. At baseline, the patients (caregivers in children) documented one to six symptoms, ranked in order of decreasing importance, and assessed the intensity of each symptom on a numerical rating scale  from 0 (•First ranked symptom at baseline (see above) on a numerical rating scale 0-10 •Generic quality of life: SF-36 Physical and Mental Component summary measures and the eight SF-36 scales (0-100)  for adul•Depressive symptoms: Center for Epidemiological Studies Depression Scale, German version  ,35 for a•Disease-specific outcomes for the most frequent diagnosis groups: Anxiety Disorders, Asthma, Attention Deficit Hyperactivity Symptoms, Depression, Low Back Pain, and Migraine•Use of AM art, eurythmy and rhythmical massage therapy in months 0-24 was documented by the therapists in a therapy diary.•Therapy outcome rating and satisfaction with therapy were documented by the patients on numerical rating scales (0-10) after 6 and 12 months.•The continuity of the physician-patient relationship was documented after 6, 12, 18, 24, and 48 months.•Suspected adverse reactions to medications or therapies were documented by the patients after 6, 12, 18, and 24 months, and by the physicians after 6 months . The documentation included suspected cause, intensity , and therapy discontinuation because of adverse reactions.•Serious adverse events  were documented by the physicians throughout the study months 0-24.All data were documented with questionnaires returned in sealed envelopes to the study office. The physicians documented eligibility criteria; all other items were documented by the patients or caregivers, unless otherwise stated. The patient responses were not made available to the physicians. The physicians were compensated 40 Euro (after March 2001: 60 Euro) per included and fully documented patient, while the patients received no compensation.The data were entered twice by two different persons into Microsoft® Access 97. The two datasets were compared and discrepancies resolved by checking with the original data.The study was approved by the Ethics Committee of the Faculty of Medicine Charité, Humboldt University, Berlin, Germany, and was conducted according to the Declaration of Helsinki and largely following the ICH Guideline for Good Clinical Practice E6. Written informed consent was obtained from all patients before enrolment.The data analysis was performed on all patients fulfilling the eligibility criteria, using PASW® Statistics 18.0  and StatXact® 9.0.0 . Diagnoses were coded according to the International Classification of Diseases, Tenth Revision (ICD-10).t-test was used; for independent samples with skewed data the Mann–Whitney U-test was used. For binominal data the two-tailed McNemar test and Fisher’s exact test were used. Significance criteria were p < 0.05. Since this was a descriptive study, no adjustment for multiple comparisons was performed [For bivariate analyses of continuous data with approximately normal distribution the two-tailed erformed .Pre-post effect sizes were calculated as Standardised Response Mean (= mean change score divided by the standard deviation of the change score) and classified as minimal (< 0.20), small (0.20-0.49), medium (0.50-0.79), and large (≥ 0.80),38. ClinFor analysis of clinical outcomes, missing values were replaced with the last value carried forward. Accordingly, each clinical outcome and subgroup analysis comprised all patients in the respective group with available baseline scores . For other analyses, missing data were not replaced.All suspected adverse reactions were classified as reported adverse reactions and subject to descriptive analysis. The frequency of reported reactions from AM medications in this analysis (patients enrolled January 1999 to December 2005) were compared to the frequency of confirmed reactions in a previously published detailed safety analysis from AMOS (patients enrolled January 1999 to March 2001). SeriousA total of 151 physicians enrolled patients. These physicians did not differ significantly from eligible physicians without study patients (n = 167) regarding gender  and setting . Significant differences were found regarding the number of years in practice  and age .The patients who had been referred to AM art, eurythmy or rhythmical massage therapy were treated by 275 different therapists. Comparing these therapists to eligible therapists without study patients (n = 911), no significant differences were found regarding age , gender  or the number of years since therapy school graduation .From 1 January 1999 to 31 December 2005, a total of 1,678 patients aged 1-75 years were assessed for eligibility. Of these patients, 1,510 fulfilled all eligibility criteria and were included in the present analysis. Of the 168 patients who were not included, 32 did not fulfil the eligibility criteria for the analysis (disease duration < 30 days: n = 32) and 136 patients were potentially eligible but were not included in the AMOS study for the following reasons: patients’ baseline questionnaire missing (n = 57), patients’ and physicians’ baseline questionnaire dated > 30 days apart (n = 49), previous or ongoing use of AM therapy (n = 19), no informed consent (n = 8), other reasons (n = 3). The included patients  and the patients not included but potentially eligible (n = 136) did not differ significantly regarding age, gender, diagnosis, disease duration or baseline Symptom Score.A total of 71.7%  of patients were enrolled by general practitioners, 12.2% by paediatricians, 6.4% by internists, 6.1% by gynaecologists, and 3.6% by other specialists. The physicians’ settings were primary care practices , referral practices (11.2%), and outpatient clinics (6.6%). Each physician enrolled 1-4 patients , 5-9 patients , 10-19 patients  or ≥ 20 patients , with a median of 4.0 patients enrolled per physician .The last patient follow-up ensued on 16 May 2010. A total of 96.0%  of patients returned at least one follow-up questionnaire. The patients were administered a total of 9,060 follow-up questionnaires, out of which 7,114 (78.5%) were returned. Follow-up rates were 94.5% , 91.2%, 86.7%, 82.9%, 76.6%, 73.2%, and 60.5% after 3, 6, 12, 18, 24, and 48 months, respectively. Respondents  and non-respondents (n = 596) of the 48-month follow-up did not differ significantly regarding age, gender, diagnosis or disease duration. Baseline Symptom Score was 6.0 ±1.8 points in respondents and 6.3 ±1.7 points in non-respondents . For non-respondents of the 48-month follow-up, the last available questionnaire was returned after average 15.7 months. In respondents of the 48-month follow-up Symptom Score was (interpolated average) 3.40 points after 15.7 months and 2.48 points after 48 months.The patients were recruited from 15 of 16 German federal states. Age groups were 1-19 years: 29.8% , 20-39 years: 25.4%, 40-59 years: 35.2%, and 60-75 years: 9.6% with a median age of 37.0 years . A total of 69.8%  of all patients and 81.5%  of adults aged 17-75 were women.Compared with the German population, adult patients had higher educational and occupational levels and were less frequently regular smokers, daily alcohol consumers, overweight, and unemployed, whereas more patients engaged in sports than in the population. Socio-demographic status was similar to the population regarding income, severe disability status, and the proportion living alone, and less favourable for work disability pension and sick-leave Table . Socio-dMost frequent main diagnoses, classified by the ICD-10 diagnosis chapters were F00-F99 Mental and behavioural disorders , M00-M99 Diseases of the musculoskeletal system and connective tissue (15.4%), J00-J99 Diseases of the respiratory system (9.9%), and G00-G99 Diseases of the nervous system (7.2%). The most frequent diagnosis groups are listed in Table At enrolment 19.4%  of patients fulfilled inclusion criterion 2a (AM-related consultation of ≥ 30 minutes followed by new prescription of AM medication), 44.3% fulfilled inclusion criterion 2b , and 36.3% fulfilled inclusion criteria 2a and 2b. The duration of the consultation with the AM physician at enrolment was < 30 min in 51.4%  of patients, 30-44 min in 23.6%, 45-59 min in 11.5%, and ≥ 60 min in 13.5% of patients.Of the 1,217 patients who were referred to AM art, eurythmy or massage therapy, 86.8%  had the planned AM therapy within the first 24 months, 0.5% did not have AM therapy, and for 12.6% the AM therapy documentation is incomplete. AM therapies used were eurythmy therapy , rhythmical massage therapy (10.9%), and art therapy  with the therapy modalities painting/drawing/clay , speech exercises (33.5%), and music (12.4%). The AM therapy started median 13 (IQR 2-41) days after enrolment. Median therapy duration was 119 days (IQR 84-190 days), median number of therapy sessions was 12 (IQR 10-20). AM medications were used by 61.2%  of patients in months 0-6 and by 71.7% in months 0-24.At 48-month follow-up 62.8% of the evaluable patients (n = 575/915) were still being treated by the AM physician who had enrolled them into the study. Reasons for no longer being treated by the AM physician were “positive”: full recovery or improvement , “negative”: choice of other treatment or dissatisfaction with the physician , and “neutral”: practical reasons, e. g. patient or physician had moved, physician had stopped practicing or financial reasons , and other reasons . The 0-48 month Symptom Score improvement did not differ significantly between patients with or without ongoing treatment by the AM physician (p = 0.234), while in the latter group, patients with “positive” reasons for not being treated had more improvement than patients with “negative” reasons   were mean ± standard deviation 7.23 ± 2.39 points; patient satisfaction with therapy  was 7.94 ± 2.20 points. Between 6- and 12-month follow-ups therapy outcome ratings did not change significantly, while therapy satisfaction decreased by average 0.31 points .Adverse reactions from AM therapies and medications were infrequent and mostly of mild-to-moderate intensity Table . AdverseSerious adverse events occurred in 2.3%  of patients. The events were acute hospital admissions (n = 16), deaths (n = 15), new malignancy (n = 1), life-threatening ileus (n = 1), and permanent disability from whiplash injury (n = 1). Death causes in the 15 patients who died were malignant neoplasms (n = 12), AIDS (n = 1), pneumonia (n = 1), and accident or suicide in a patient hospitalised for severe depression (n = 1). None of the serious adverse events were causally related to any medications or therapies.This is a four-year follow-up analysis of the largest clinical outcome study of AM treatment for chronic disease so far. We studied 1,510 outpatients starting comprehensive AM treatment  for mental, musculoskeletal, respiratory or neurological disorders or other chronic conditions. At four-year follow-up, 76% of the evaluable patients were either still being treated by the AM physician who had enrolled them into the study (63% of the patients) or were no longer receiving treatment because they had improved or recovered (13%). Previous analyses of different age, diagnosis and therapy modality groups had shown clinically relevant improvements of symptoms and quality-of-life following AM treatment -30. The Strengths of the AMOS study and this analysis include a large sample size, a long follow-up period, the combination of generic and disease-specific outcome measures, the assessment of a broad range of AM therapy modalities, and a high representativeness due to the participation of 47% of eligible AM physicians and 23% of eligible AM therapists in Germany. The participating physicians and therapists resembled eligible but not participating AM physicians and AM therapists with respect to demographic characteristics, and the included patients resembled not included patients regarding baseline characteristics. These features suggest that the study to a high degree mirrors contemporary AM use in German outpatient settingsThe main research question of the present analysis concerned the magnitude of long-term improvements. A limitation in this respect is the increasing long-term nonrespondent rate . However, nonrespondents and respondents at 48-month follow-up did not differ regarding baseline characteristics (except nonrespondents had 5% higher symptom intensity at baseline), and in a telephone survey of AMOS patients, the proportion with clinical deterioration at 24-month follow-up was comparable in nonrespondents and respondents . MoreoveThis was an exploratory analysis and a total of 19 clinical outcomes were analyzed with a total of 26 pre-post comparisons Table , therefoIn this analysis, improvements following AM treatment persisted for 48 months. This is in keeping with the aims of AM therapy to bring about sustained improvement,7,56. LoIn this study, adverse reactions to AM treatment were infrequent and mostly of mild-to-moderate intensity. This confirms findings from other prospective studies,65-68 asTo sum up: This study confirms the findings of similar research conducted elsewhere,57-73 anThis 48-month follow-up analysis confirmed previous analyses from the AMOS study. Outpatients receiving comprehensive AM treatment for chronic indications had sustained, clinically relevant improvements of symptoms and quality of life. Adverse reactions to AM treatment were infrequent and mostly of mild-to-moderate intensity; patient satisfaction was high.± and SD: Standard deviation; ADHD: Attention deficit hyperactivity disorder; AM: Anthroposophic medicine; AMOS: Anthroposophic medicine outcomes study; CES-D: Center for epidemiological studies depression scale, German version; CI: Confidence interval; ICD-10: International classification of diseases, Tenth Edition; IQR: Interquartile range; NRS: Numerical rating scale.All authors declare that they have no competing interests.HJH, HK and GSK contributed to study design. HJH, AG, and HK contributed to data collection. HJH, RZ, and HK wrote the analysis plan, HJH and AG analysed data. HJH was principal author of the paper, had full access to all data, and is guarantor. All authors contributed to manuscript drafting and revision and approved the final manuscript.


In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs) are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor- Inflammatory response is a basic protective immune process of the organism and is accompanied by symptoms such as redness, heat, swelling, and pain associated with damage to tissues or organs . This isα (TNF-α)), other chemical mediators, and extracellular matrix proteins. A variety of membrane receptors are expressed on the surfaces of macrophages, including pattern recognition receptors (PRRs) such as dectin-1 and Toll-like receptors (TLRs) ; h; h111]; p38 signaling and specific p38 inhibitors were identified simultaneously. A series of pyridinyl imidazole anti-inflammatory agents, such as bicyclic pyridinyl imidazoles SKF-86002, SB203580, and SB202190 , 112–116 Scutellaria baicalensis extract attenuates MAPK phosphorylation, especially p38 activity, resulting in inhibition of inflammatory mediators such as COX-2, iNOS, L-1β, IL-12, IL-6, IL-2, PGE2, and TNF-α in RAW 264.7 cells treated with LPS [ Phaseolus angularis ethanol extract suppressed the release of PGE2 and NO in macrophages activated by LPS-, Poly(I:C)-, or pam3CSK through regulation of TAK1/p38 pathways and, moreover, it ameliorated gastritis induced by EtOH/HCl in mice, which implies a close relationship between p38 and gastritis [ Archidendron clypearia extract suppressed the production of PGE2 in activated RAW264.7 and peritoneal macrophages, as well as gastritis lesions in mouse stomachs exposed to EtOH/HCl [Natural plant extracts that target p38 are promising therapeutics for the treatment of inflammatory diseases . For exawith LPS . Phaseolastritis . ArchideEtOH/HCl . UnfortuEtOH/HCl –123. MeaEtOH/HCl , during EtOH/HCl . In addiEtOH/HCl , 29. Pla Calocedrus formosana, effectively decreased the production of intracellular reactive oxygen species (ROS), IL-1β, and TNF-α in LPS-stimulated macrophages through regulation of MAPKs [α by targeting both MAPK (ERK and p38) and IκBα signaling pathways [2, and proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in macrophages by suppressing p38 transduction pathways [Several compounds from natural products inhibit p38 activity and inflammatory responses . Sugiol,of MAPKs . Quercetpathways , 127. Supathways . A summaα in inflammation, people have focused on inhibitors for this isoform rather than the other isoforms. ML3403, a SB203580 analogue, represses the expression of TNF-α, IL-6, and IL-8. It can bind to both active and inactive forms of p38α kinase, which may reduce asthma-induced airway inflammation and remodeling [α, IL-1β, IL-6, PEG2, and MMP3 at very low concentrations. Moreover, it can reduce the enzyme activity of both p38 α and β but has no effect on 100 other kinases, including p38γ and δ. It has been shown in rats experiment that low doses of this compound can also reduce the expression of LPS- and Con A-stimulated proinflammatory cytokines, including TNF-α and IL-6 [α and β activity and therefore the expression of TNF-α, IL-1β, and IL-6. It also shows high specific activity; when tested for binding to 350 kinases, it only bound to four other kinases in addition to p38. Furthermore, it can reduce clinical signs of inflammatory diseases, such as arthritis, bone loss, and renal diseases. Consistent with this, it inhibited TNF-α production in RA synovial explants and reduced bone loss in murine collagen-induced arthritis. Meanwhile, it increased pain tolerance in a rat model of hyperalgesia [Pharmaceutical companies and researchers have worked hard to develop novel, safe, and specific p38 inhibitors. Based on the importance of p38modeling . AS19404and IL-6 . Pamapimralgesia . Exampleralgesia . In summα level were clearly seen by p38 MAPK inhibitor adalimumab in lesional psoriatic skin [Based on the importance of p38 MAPK in disease development, inhibition of p38 was regarded as a promising therapeutic strategy to control inflammatory diseases. Right now, effectiveness of some p38 inhibitors is currently under evaluation in clinical trials to treat human diseases. For example, it has been reported that PH797804 and losmapimod were able to improve lung function parameters and to attenuate dyspnoea in patients with chronic obstructive pulmonary disease symptoms , 133. Altic skin . Moreovetic skin . Besidestic skin –140.Inflammation has attracted great interest because of its significant role in several major diseases and the need to develop better ways to treat these diseases. Importantly, because inflammatory responses are largely mediated by macrophages, functional studies of macrophages in inflammation are crucial. Investigation of the roles of p38 MAPKs is particularly relevant as these are essential protein kinases in macrophage-mediated inflammatory responses. A number of studies have indicated that p38 plays a significant role in inflammatory diseases mediated by macrophages, and, as a consequence, several p38 inhibitors have been developed to treat inflammatory diseases. However, most of these inhibitors have shortcomings, such as low specificity, low efficacy, and high toxicity. As a result, new p38 inhibitors are urgently required. We are optimistic that novel and safe p38 inhibitors that possess strong anti-inflammatory properties will be developed in the near future to treat inflammatory diseases.