Catalase causes detoxification of H2O2 in cells, and it is an indispensable antioxidant enzyme. The study was designed to measure erythrocyte catalase activity (ECAT) in patients with IDA (10) or BTT (21), to relate it with thalassemia mutation type (β0 or β+) and to compare it with normal subjects (67). Ninety-eight individuals were analyzed since September 2013 to June 2014 in Tucumán, Argentina. Total blood count, hemoglobin electrophoresis at alkaline pH, HbA2, catalase, and iron status were performed. β-thalassemic mutations were determined by real-time PCR. Normal range for ECAT was 70,0–130,0 MU/L. ECAT was increased in 14% (3/21) of BTT subjects and decreased in 40% (4/10) of those with IDA. No significant difference  was shown between normal and BTT groups, while between IDA and normal groups the difference was proved to be significant . In β0 and β+ groups, no significant difference  was observed. An altered ECAT was detected in IDA and BTT. These results will help to clarify how the catalase activity works in these anemia types.Most common microcytic hypochromic anemias are iron deficiency anemia (IDA) and  Normal erythrocytes are protected against potentially dangerous combination of oxygen and iron (hemichromes and heme associated iron) for extremely efficient endogenous mechanisms, such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), reduced glutathione, and vitamin E. Microcytosis is the physiological consequence of reduced hemoglobin content in the red blood cell (RBC) due to a synthesis defect of globin chains or heme . ExperimMicrocytosis is defined by MCV lower than 80 fL and hypochromia through mean corpuscular hemoglobin lower than 27 pg. Hypochromic microcytic anemia may result from an iron deficiency (iron deficiency anemia), a defect in the globin genes , a defect in heme synthesis (sideroblastic anemia), or a defect in iron availability and acquisition by erythroblast (anemia of chronic disease).β-) thalassemia is the most common hemoglobinopathy in the Mediterranean basin, the Middle East, and Asia [β-thalassemia is the most common inherited anemia [β-thalassemia.According to World Health Organization (WHO), the primary cause of anemia is iron deficiency, especially in pregnant women and children . Regardiand Asia . Accordid anemia –8. Thereβ-thalassemia, β+ thalassemia in which a small amount of β-globin chains are detectable and β0 thalassemia, in which they are undetectable. Till this day there are over 200 gene mutations capable of producing thalassemia phenotypes [β-thalassemia is classified as minor (asymptomatic) or major (severe anemia), depending on the mutation responsible for this alteration is present in heterozygous or homozygous state. There is also a moderate clinical features syndrome known as thalassemia intermedia (homozygous or double heterozygous). In β-thalassemia, as a result of β-chains decrease, a relative alpha- (α-) chains excess occurs [α-chains are unable to form viable tetramers and precipitate in RBC precursors in bone marrow forming inclusion bodies called hemichromes. They are responsible for the large intramedullary destruction of erythroblasts and therefore for ineffective erythropoiesis in β-thalassemia [2O2), and hydroxyl radicals [There are two molecular forms of enotypes . Clinicas occurs . Free α-lassemia . Hemichrlassemia . In bothradicals .β-thalassemia it is difficult to evaluate the role played by the antioxidant enzymes because of the relevant proportion of normal red cells due to multiple transfusions. Furthermore little is known about the oxidative status in β-thalassemia trait (BTT) subjects and its relation with different β-thalassemia mutations. Such assessment is important due to the large genotypic and phenotypic heterogeneity of different populations.Several studies have evaluated the oxidant and antioxidant status in thalassemia major and intermedia , 15, and2O2 in cells [2O2 concentration and damage of oxidation sensitive tissues that may contribute to the manifestation of various diseases such as diabetes mellitus and anemia [The antioxidant system has been proposed as a biomarker of OxS through measuring detoxifying enzymes such as catalase . The enzin cells . Decreasd anemia . in vitro exposure to H2O2 [Iron deficiency affects activity of many iron-dependent enzymes , and in iron deficiency anemia (IDA) RBCs are more susceptible to oxidation. RBCs from IDA subjects lyse more readily than normal cells on to H2O2 , suggest to H2O2 . The lit to H2O2  and decr to H2O2  have beeβ-thalassemia mutation with the catalase activity.The present study was designed to measure catalase activity in individuals suffering from some of the most common microcytic hypochromic anemia, that is, IDA or BTT, and to compare it with normal subjects. It was also proposed to relate the type of Design. A descriptive cross-sectional study was conducted.Subjects. The sample consisted of 31 patients who attended Instituto de Bioquímica Aplicada , for the diagnosis of hereditary anemia during the period of September 2013 up to June 2014. Sixty-seven normal individuals, whose participation was voluntary, were also included. Blood was drawn and placed in two different tubes, one containing K2-EDTA anticoagulant and the other without anticoagulant with the purpose of obtaining serum.Inclusion Criteria. Patients diagnosed with β-thalassemia minor or iron deficiency anemia and normal subjects that needed to be older than 1 year old were included.Exclusion Criteria. People under vitamin intake, people who smoke, with diabetes, coronary heart disease, rheumatoid arthritis, dyslipidemia, hypertension, malignancy, chronic liver disease, and renal dysfunction, and patients under iron therapy during 21 days prior to analysis or who have received transfusions in the last three months were excluded.Hematological Studies. Total blood count was performed in hematology analyzer Sysmex KX-21N . The diagnosis of the β-thalassemia trait was performed by cellulose acetate hemoglobin (Hb) electrophoresis at alkaline pH and HbA2 quantification by microcolumn chromatography . IDA diagnosis was made by determining serum iron, total iron binding capacity (TIBC), and transferrin saturation (SAT) through colorimetric method . SAT lower than 16% was considered diagnostic of IDA.Erythrocyte Catalase Activity (ECAT). The enzyme was analyzed in K2-EDTA anticoagulated whole blood using Góth technique [A) of the yellow complex of molybdate and hydrogen peroxide was measured at 405 nm against blank 3. Sample contained 1,0 mL substrate  and 30 μL hemolysate, and after 60 seconds the enzymatic reaction was stopped by adding 1,0 mL of 32,4 mmol/L ammonium molybdate. Blank 1 contained 1,0 mL substrate, 1,0 mL molybdate, and 30 μL hemolysate; blank 2 contained 1,0 mL substrate, 1,0 mL molybdate, and 30 μL buffer; blank 3 contained 1,0 mL buffer, 1.0 mL molybdate, and 30 μL buffer. One unit of catalase decomposes 1 μmol of H2O2 in 1 minute under these conditions and it is related to 1 L of whole blood. Catalase activity was expressed in Mega Units/L (MU/L) and was calculated with the following formula:echnique . AbsorbaMolecular Analysis. Characterization of β-thalassemic mutations was realized by real-time PCR. Genomic DNA isolation was performed with High Pure PCR Template Preparation Kit (Roche Diagnostics). PCR, dissociation curves, and subsequent analysis were executed on the LightCycler 2.0 (Roche) equipment, simultaneously measuring signals from two different fluorophores. Primers were designed to amplify a 587 bp region of β-globin gene: Forward Primer, 5′-gctgtcatc act acctca tag-3′; Reverse Primer, 5′-gct gcaagtcaccactca g-3′. Two combinations of hybridization probes labeled with different fluorophores were used [ere used .Statistical Analysis. Results were analyzed using SPSS 21.0 statistical program. The results were reported as Media ± Standard Deviation. For comparison Student t-Test and ANOVA were used. A significance level of p < 0.05 was adopted. The influence of iron serum on catalase values, independent of the group , was evaluated by simple regression analysis.β-thalassemia trait (BTT group), and 10 with iron deficiency anemia (IDA group). p = 0,187) in ECAT between the stated groups.Ninety-eight individuals were studied of which 67 were normal (N group), 21 with β-thalassemia carriers , and 2 IDA . Only one of these subjects, which belonged to the normal group, showed increased catalase activity (139 MU/L). Apparently, age has no influence in the ECAT of this population.Five individuals, who were more than 60 years old, were normal , 2 In order to establish the normal range of catalase activity, 5th and 95th percentiles of N group results were determined. A range from 70,0 to 130,0 MU/L was obtained.p = 0,245) were observed when N and BTT groups were compared.Increased catalase activity in 14% (3/21) of BTT subjects was observed. No significant differences  was observed, since the IDA subjects had lower values than N group  showed decreased catalase activity. No significant differences between men and women were detected in all groups. When N and IDA groups were compared, significant difference  with normal group in all parameters, except TIBC. Catalase in male IDA patients did not demonstrate significant differences  when compared with normal and BTT groups.β-thalassemia mutations detected in order of frequency were codon 39 (C→T) (5 subjects), IVS-I-110 (G→A) (5 subjects), IVS-I-1 (G→A) (4 subjects), and IVS-I-6 (T→C) (2 subjects), and in 5 cases the mutation could not be assigned. The small number of patients for each group prevented the comparative study between them. Therefore differences between β0  and β+  groups were studied, which were not significant .The r2 = 0,153) in these samples.There was no influence of iron levels on catalase values  of catalase activity in β-thalassemia major and intermedia. The present results, in concordance with Gerli et al. [Kósa et al.  revealedpatients . They cor et al.  observedi et al. , showed Several authors reported increased antioxidant capacity in BTT individuals, but they do not measured catalase activity , 29. Insβ0 and β+ thalassemia traits, the results were not significant. Also, Kósa et al. [β-thalassemia mutations. Furthermore, Labib et al. [β0 and β+ thalassemia traits.In the catalase activity comparison between a et al.  did not b et al.  reportedThe normal range of erythrocyte catalase activity was slightly lower than the one reported by another authors , which wβ-thalassemia major and intermedia, iron excess can lead to organ damage, especially in liver and heart, and to endocrine dysfunction [In this study, like Ondei et al. , there wfunction . Recent function .Decreasing serum iron concentration causes insufficient Hb synthesis with subsequent reduction of erythrocytes proliferation. Iron deficiency also affects the production of other iron-containing proteins, such as cytochrome, myoglobin, catalase, and peroxidase. Therefore, a decreased catalase activity could be anticipated in iron deficiency and has been corroborated in this study, in agreement with other works , 34, 35.Increased OxS have been reported in patients with IDA , 21, 35,


Hepatic inflammatory pseudotumor (IPT) is a rare disease which often mimics a malignant tumor and is therefore often misdiagnosed and surgically resected. Recently, a concept of IgG4-related diseases (IgG4-RD) has been proposed that is becoming widely recognized and includes IgG4-related hepatic IPT. Corticosteroids are widely accepted as the standard treatment.A 72-year-old Japanese man, who had been followed for ten years after surgery and chemotherapy for treatment of hilar and lower bile duct cancers, developed intermittent fever and abdominal pain and visited this hospital. Blood examinations revealed an inflammatory reaction, worsened glucose intolerance, and an increased level of serum IgG4 (137 mg/dL). Computed tomography (CT) revealed a 5 cm-sized mass in hepatic segment 7. Because of his cancer history, not only was a benign mass suspected, but there was also the possibility of a recurrent biliary malignancy. Liver biopsy was performed and the histology met the criteria for IgG4-related IPT. Corticosteroid therapy was initiated and his symptoms quickly resolved. However, two months later, a repeat CT demonstrated that the hepatic mass had been replaced by an abscess. The abscess was initially refractory, despite tapering corticosteroid treatment, controlling diabetes by intensive insulin therapy, administration of antibiotics, and percutaneous abscess drainage. Finally, after six months, the condition resolved.The diagnosis of hepatic IPT is sometimes difficult. To differentiate it from a malignant tumor, histological examination is necessary. Although corticosteroids are recognized as the standard therapy, unexpected and critical complications can develop in cases of IgG4-related hepatic IPT. Hepatic inflammatory pseudotumor (IPT) is a rare entity that has been thought to be a composition which develops through a variety of pathogeneses, such as infections with a virus  or bacte1c (HbA1c): 6.4–7.4 %, normal range: 4.6–6.2 %].A Japanese man was being followed after he underwent left hepatectomy and pancreatoduodenectomy for a mucin-producing hilar cholangiocarcinoma  and lower bile duct carcinoma  10 years ago when he was 62 years of age. He had also undergone chemoradiation therapy during the year following surgery . Follow up was continued after this chemoradiation management, and the patient’s course was uneventful without recurrence, except for new onset of diabetes after surgery [serum hemoglobin A1c (8.8 %), and IgG4 . Serum levels of hepatobiliary enzymes, carcinoembryonic antigen, carbohydrate antigen 19-9, and α-fetoprotein were within the normal range. Computed tomography (CT) revealed a 5-cm mass lesion in hepatic segment 7  , alkaline phosphatase , C-reactive protein (CRP) , HbAt 7 Fig. , but no t 7 Fig. . No canct 7 Fig.  and the 1c (9.5 %), but normalized IgG4 (84.5 mg/dL). CT revealed an extensive low-density area in the hepatic segment 7–8 . Concerning about exacerbation of his hyperglycemia, we accelerated the steroid tapering. After treatment with oral steroids, he had no symptoms and his laboratory data reached the nearly normal levels  and the shrinkage of the IPT was recognized by US. However, after three months from starting the steroid tapering, his fever and abdominal pain recurred afterward. Again, blood tests were abnormal as follows: elevated levels of WBCs (15000 /μL with 94.1 % neutrophils), CRP (12.7 mg/dL), HbA7–8 Fig. . The lesIn general, hepatic IPTs are difficult to differentiate from malignant tumors, as their incidence is rare, their pathogenesis is diverse, and imaging findings are varied , 7, 11. From the viewpoints of differentiating their pathogenesis or subtypes, biopsy is recommended in cases of a suspected hepatic IPT. As described above, this tumor is caused by a variety of pathogeneses –7, 11, aAccording to a keyword search in PubMed, a total of 11 cases of IgG4-related hepatic IPT were identified –23  and steroid withdrawal syndrome. In autoimmune pancreatitis or one of the most popular subtypes of IgG4-RD, many cases actually show improvement in diabetes by steroid treatment , as theyThe diagnosis of hepatic IPT is difficult, especially in terms of differentiation from malignant tumors. Histological diagnosis is essential and percutaneous needle biopsy is effective for the diagnosis of IgG4-related IPT. Clinicians must bear in mind that corticosteroid treatment is not always ideal and may cause critical adverse events in cases with IgG4-RD.CT, computed tomography; HbA1c, hemoglobin A1c; HPF, high power field; IgG4, immunoglobulin G4; IgG4-RD, IgG4-related disease; IPT, inflammatory pseudotumor; US, ultrasonography


Background. The study was designed to investigate if H2S could inhibit high-salt diet-induced renal excessive oxidative stress and kidney injury in Dahl rats. Methods. Male salt-sensitive Dahl and SD rats were used. Blood pressure (BP), serum creatinine, urea, creatinine clearance rate, and 24-hour urine protein were measured. Renal ultra- and microstructures were observed. Collagen-I and -III contents the oxidants and antioxidants levels in renal tissue were detected. Keap1/Nrf2 association and Keap1 s-sulfhydration were detected. Results. After 8 weeks of high-salt diet, BP was significantly increased, renal function and structure were impaired, and collagen deposition was abundant in renal tissues with increased renal MPO activity, H2O2, MDA, GSSG, and •OH contents, reduced renal T-AOC and GSH contents, CAT, GSH-PX and SOD activity, and SOD expressions in Dahl rats. Furthermore, endogenous H2S in renal tissues was decreased in Dahl rats. H2S donor, however, decreased BP, improved renal function and structure, and inhibited collagen excessive deposition in kidney, in association with increased antioxidative activity and reduced oxidative stress in renal tissues. H2S activated Nrf2 by inducing Keap1 s-sulfhydration and subsequent Keap1/Nrf2 disassociation. Conclusions. H2S protected against high-salt diet-induced renal injury associated with enhanced antioxidant capacity and inhibited renal oxidative stress. Numerous studies have demonstrated that high salt not only caused hypertension, but also resulted in kidney injury –8. The k2S) is produced by the metabolism of sulfur-containing amino acid. The key enzymes of synthesis, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and mercaptopyruvate transsulphurase (MPST), are abundantly expressed in the kidney [2S has various pathophysiological roles including relaxing blood vessels, lowering blood pressure [2S might protect neurons against oxidative injury by promoting the generation of antioxidants—glutamine [2S could inhibit oxidative stress of lung tissue [2S had a protective effect on gentamicin-induced kidney injury [2S could alleviate the oxidative stress by upregulating catalase (CAT), Superoxide dismutase (SOD), and glutathione (GSH), thereby improving renal fibrosis [2S could protect against high-salt-induced kidney injury and what are the possible mechanisms remain unclear. Therefore, the present study was undertaken to explore the protective effects of H2S on high-salt-induced kidney injury in Dahl rats and its possible mechanisms.As the third gaseous signal molecule after nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide  rats and 40 male SD rats  were fed in Animal Center of Peking University First Hospital. They were randomly divided into three groups after adapting to the environment for one week. Dahl rats were divided into control group (Dahl + NS), high-salt group (Dahl + HS), and high-salt + NaHS group (Dahl + HS + NaHS), while SD rats were divided into control group (SD + NS), high-salt group (SD + HS), high-salt + NaHS group (SD + HS + NaHS), and high-salt + hydroxylamine group (SD + HS + HA), with 10 rats in each group. The rats in Dahl + NS and SD + NS groups were fed with normal diet and 0.9% normal saline was given via intraperitoneal injection daily; the rats in Dahl + HS and SD + HS groups were fed with the diet containing 8% salt and intraperitoneally injected with 0.9% normal saline every day; the rats in Dahl + HS + NaHS and SD + HS + NaHS group were fed with high-salt diet while 90 very day ; and thevery day . The NaHAfter eight weeks of experiment, 25% urethane (0.5 mL/100 g) was given to rats through intraperitoneal injection for anesthesia. Physiological multilead recorder  was used to record arterial blood pressure. Automatic biochemical analyzer  was used to detect serum creatinine, creatinine clearance rate, serum urea, and 24-hour urine protein.2 mm × 2 mm × 2 mm of renal cortical tissue was put in 3% glutaraldehyde to fix, making electron microscope specimens and observing the changes in kidney ultrastructure.μm of section for Periodic acid-Schiff staining  and Masson staining  in strict accordance with the kit experimental steps. Leica Q550CW Image Processing and Analysis System was used for image acquisition and semiquantitative analysis of the results of kidney PAS and Masson staining. The semiquantitative analysis of kidney PAS staining was used to evaluate glomerulosclerosis. Depending on the severity of glomerulosclerosis, it is divided into four grades: (0) normal; (1) glomerulosclerosis area <25%; (2) 25%–50%; (3) 50%–75%; and (4) >75%. Ten visual fields were randomly selected for each rat to calculate the proportion of sclerotic glomeruli. Five visual fields of renal cortex were randomly selected for each rat to calculate the average index number of renal fibrosis [0.2 cm of tissue specimens from kidney transverse section was taken and fixed in 4% paraformaldehyde, paraffin embedding, taking 0.5 fibrosis .μL), fully ground to renal tissue homogenate, and centrifuged at 12000 g/min for 10 min, taking the supernatant. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect the content of collagen-I and collagen-III  and hydroxyproline content was measured by colorimetry . The experimental procedures were in strict accordance with the kit instructions.The appropriate renal tissue was taken, adding PBS buffer  according to the mass volume ratio of 1 : 10 , ground on the ice at 4°C, and centrifuged at 12000 g/min for 10 min, taking the supernatant. Free Radical Detection Analyzer TBR4100  was used to measure H2S content in the supernatant of renal tissue homogenates [2S-100 sensor  was placed in PBS buffer solution  for polarization. When the stable reference current appeared , the sensor was calibrated. Approximate 10–15 mm of the top of the sensor was immersed in 20 mL of PBS buffer solution , until the stable current appeared on the display. Then, the detecting sensors were successively inserted in six kinds of different concentrations of Na2S solution formulated by PBS buffer . The current output would rise rapidly to a plateau after each sample was added. As long as the current reached a plateau, the next sample was detected. Then, the calibration curve was made based on the output signal (pA) and the corresponding H2S concentration (μmol/L). There was about 10–15 mm of the sensor that was immersed in each sample. H2S content of each sample was calculated according to pA- H2S concentration calibration curve.ogenates . FirstlyμL), fully ground to renal tissue homogenate, and centrifuged at 12000 g/min for 10 min, taking the supernatant. Biochemical colorimetry was used to detect the following indices: SOD, myeloperoxidase (MPO), CAT, glutathione peroxidase (GSH-PX) activity and oxidized glutathione (GSSG), GSH, hydrogen peroxide (H2O2), malondialdehyde (MDA), hydroxyl radical (OH) content, and total antioxidant capacity (T-AOC). The experimental procedures were in strict accordance with the kit instructions .The right amount of renal tissue was taken; PBS buffer  was added according to the mass volume ratio of 1 : 10 (mg/μL) and then centrifuged at 12000 g/min for 10 min, taking the supernatant. The equal amounts of protein mixture samples were added into each channel for polyacrylamide gel electrophoresis and then transferred onto a membrane. After closing by milk at room temperature for 1 h, primary antibodies SOD1 , SOD2 , and β-actin (Santa Cruz Biotechnology) were added, incubated at 4°C overnight, and washed by TTBS, 10 min/times, for a total of four times. Then, HRP-labeled secondary antibody  was added, incubated at room temperature for 1 h, washed by TTBS, 10 min/times, for a total of four times, and then incubated with chemiluminescent reagents for 1 min before being exposed, developed, and fixed. AlphaImager gel imaging system was used to scan protein bands and measure optical density of protein bands, and β-actin was used as interior reference to correct [The renal tissues were homogenized at 4°C by adding 1x tissue lysates according to the mass volume ratio of 1 : 10 (mg/ correct .The coimmunoprecipitation was performed as described previously . BrieflyμL of EZ-Link iodoacetyl-PEG2 biotin (10 mg/mL) for 12 h at 4°C. Biotinylated proteins were precipitated by 30 μL of ultralink immobilized NeutrAvidin for 4 h at 4°C. After supernatant removal, the protein beads were washed with PBS buffer  for 3 times and boiled for 10 min at 100°C mixed with loading buffer without β-mercaptoethanol. Then, the biotinylated proteins were detected with Keap1 antibody by Western blotting [Keap1 s-sulfhydration was performed by the modified biotin switch assay as described previously . The renblotting .t-test, and the means among many groups were compared by one-way ANOVA. P < 0.05 was considered significant.SPSS13.0 software was used for statistical analysis. The results are expressed as mean ± standard error, the means between the two groups were compared by independent sample P < 0.01, P < 0.01, P < 0.01, Figures P > 0.05, Figures Compared with the rats of Dahl + NS, blood pressure of rats in Dahl + HS group was significantly increased , •OH−, and H2O2, and RNS includes NO, nitrogen dioxide, and peroxynitrite. There are two types of enzymatic and nonenzymatic antioxidant systems in the body, and the former includes SOD, CAT, and GSH-PX, while the latter includes vitamin C, vitamin E, and glutathione peptide [2S has antioxidative stress effect [2O2, •OH, and GSSG were increased, and MPO activity was also increased. The results suggested that high salt could lead to oxidative stress of Dahl rat renal tissue. After giving NaHS, an H2S donor, into Dahl rats, the renal T-AOC and GSH content were enhanced, the renal activity of CAT, GSH-PX, and SOD increased, and expression of SOD1 and SOD2 protein was upregulated, while the content of MDA, H2O2, •OH, GSSG, and MPO activity decreased. The results suggested that H2S could increase the renal antioxidative ability and reduce the oxidative injury.Up to now, the mechanism by which H peptide , 42. Stus effect , 43, 44.2S donor. Furthermore, the increase in combination of Keap1 with Nrf2 in the renal tissues of Dahl + HS rats was mimicked by giving HA, an inhibitor of endogenous H2S generation, in the renal tissues of SD rats with high-salt diet. The above results suggested that H2S might decrease the combination of Keap1 with Nrf2, thereby releasing the free Nrf2 and subsequently activating Nrf2. Regarding the mechanism by which H2S reduced the association of Keap1 with Nrf2, previous studies have reported that Keap1 s-sulfhydration could destroy the combination of Keap1 with Nrf2 and then enhance the Nrf2 nuclear translocation [2S donor enhanced Keap1 s-sulfhydration in the renal tissues of Dahl + HS rats, CBS inhibitor HA increased Keap1 s-sulfhydration in the renal tissues of SD + HS rats. However, further studies are needed to deepen the understanding of the mechanism by which H2S plays an important role in the antioxidation.As well known, Keap1/Nrf2 is one of the main pathways for antioxidative system. It regulates expression of many antioxidant enzymes and plays a pivot role in the homeostasis of oxidative and antioxidative responses , 45–47. location , 46. Whi2S upregulated the expression of SOD protein, improved antioxidant capacity, removed ROS, and thereby improved renal function and renal structural injury. s-Sulfhydration of Keap1 and thereby dissociation of Keap1 with Nrf2 might be involved, the mechanisms by which H2S enhanced the expression of antioxidant enzyme. In conclusion, H2S could reduce the high-salt-induced renal injury in association with inhibiting renal oxidative stress in Dahl rats. Keap1/Nrf2 pathway might mediate the antioxidant role of H2S.Renal oxidative injury and collagen deposition were observed in Dahl rats with hypertension induced by high salt. H


Erwinia amylovora. E. amylovora initiates infection by colonizing the stigmata of apple and pear trees, and entering the plants through natural openings. Epiphytic populations of the related enteric bacterium, Pantoea, reduce the incidence of disease through competition and antibiotic production. In this study, we identify an antibiotic from Pantoea ananatis BRT175, which is effective against E. amylovora and select species of Pantoea. We used transposon mutagenesis to create a mutant library, screened approximately 5,000 mutants for loss of antibiotic production, and recovered 29 mutants. Sequencing of the transposon insertion sites of these mutants revealed multiple independent disruptions of an 8.2 kb cluster consisting of seven genes, which appear to be coregulated. An analysis of the distribution of this cluster revealed that it was not present in any other of our 115 Pantoea isolates, or in any of the fully sequenced Pantoea genomes, and is most closely related to antibiotic biosynthetic clusters found in three different species of Pseudomonas. This identification of this biosynthetic cluster highlights the diversity of natural products produced by Pantoea.Fire Blight is a destructive disease of apple and pear caused by the enteric bacterial pathogen,  Erwinia amylovora, is an epiphytic bacterium that relies on nutrients from the plants for survival E. amylovora enters the plants through natural openings and spreads to various parts of the plants through the vascular system, allowing for widespread colonization E. amylovora is established, disease symptoms such as flower necrosis and fruit rot develop, ultimately resulting in major losses Fire blight is a destructive disease known to plague many rosaceous plants, in particular apple and pear plants E. amylovora have been the subject of many studies. Initially, antibiotics like streptomycin were shown to be an effective means of controlling bacterial colonization when applied to plants during early bloom E. amylovoraPantoea agglomerans (formerly Erwinia herbicola), to apple and pear trees in early bloom may help to reduce the disease caused by E. amylovoraPantoea vagans C9-1 and P. agglomerans E325 have been commercialized as the active agents in BlightBan C9-1 and Bloomtime Biological, respectively E. amylovoraMethods of control for Pantoea isolates have been shown to produce a variety of antibiotics, such as pantocins E. amylovora. These antibiotics have been classified into five distinct groups based on whether they are effective in the presence of specific amino acids E. amylovora by inhibiting L-histidinol phosphate aminotransferase, a critical step in histidine biosynthesis, resulting in a histidine deficiency Pantoea ananatis strain BRT175 (PanBRT175) that is effective against E. amylovora, which we have called Pantoea Natural Product 1 (PNP-1). We evaluate the impact of nutritional changes on production, including whether PNP-1 activity is affected by amino acids. We demonstrate that the activity appears to be relatively narrow spectrum affecting Erwinia and Pantoea, and that resistance in Pantoea appears to have evolved in the lineage leading to the Pantoea agglomerans and Pantoea eucalyptii groups. We also identify the biosynthetic cluster responsible for PNP-1 production, and evaluate its distribution in Pantoea and other species.In this study, we identify an antibiotic from Pantoea and Erwinia strains were incubated at 30°C. E. coli strains VPE42 (pBSL118) and HB101 (RK600), Staphylococcus aureus K1-7, Pseudomonas aeruginosa ATCC 27853, Streptococcus mutans UAIS9:wt, and Lactococcus lactis were incubated at 37°C. Where appropriate, antibiotics were supplemented in the following final concentrations: kanamycin, 50 µg/ml, chloramphenicol, 50 µg/ml, and rifampicin, 50 µg/ml.All strains used were cultured and maintained on lysogeny broth (LB) media . PantoeaPanBRT175 was evaluated using the agar overlay method on E. coli minimal media with peptone concentrations of 2.5 mg/ml, 5.0 mg/ml, 10.0 mg/ml, and 15.0 mg/ml, and tryptone concentrations of 5.0 mg/ml, 10.0 mg/ml, 15.0 mg/ml, and 20.0 mg/ml, both with and without glucose. Antibiotic activity was also evaluated in the presence of the following amino acids: alanine, β-alanine, asparagine, aspartic acid, arginine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, orinithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. All amino acids were obtained from Sigma-Aldrich®, except for β-alanine, asparagine, and arginine, which were obtained from Alfa Aesar®. The amino acids were added into separate 5X Glycerol Salt Solutions at a concentration of 1.5 mg/ml, which was then added into the top layer.The antibiotic activity of PanBRT175 was carried out with a triparental mating involving the helper E. coli HB101 (RK600) and the mini-Tn5 donor E. coli VPE42 (pBSL118). Overnight LB broth cultures of each strain were aliquoted in 1 mL volumes, centrifuged at 13,500 g for 1 minute, and resuspended in 100 µL of 10 mM Mg2SO4. All three were combined, vortexed briefly, and three 100 µL volumes were spotted on to a single LB agar plate, which was incubated overnight at 30°C. Following 16-24 hours of incubation, small samples of each triparental spot were spread on to individual LB agar plates containing kanamycin and incubated at 30°C for 48 hours.Transposon mutagenesis of PanBRT175 mutants was performed using an agar overlay method. The bottom agar was E. coli Minimal Medium 2SO4, 0.002 g MgSO4*7H2O, 20 mL glycerol) with a top layer of 0.9% agar containing 5X Glycerol-Arginine Salt Solution  seeded with E. amylovora EA321, resuspended in 300 µL of 5 mM K2HPO4. PanBRT175 mutants were picked from the triparental spread plates based on pigmentation and spotted on to the E. coli minimal media plate. The plates were incubated at 30°C overnight. Mutants that did not inhibit the EA321 indicator in the agar overlay were selected, retested, and cultured on LB agar containing kanamycin.Screening for PanBRT175 mutants were inoculated into 3 mL of LB broth and incubated overnight at 30°C. Genomic DNA was then extracted using E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek®). The genomic DNA was digested in a reaction containing 5 units HincII (New England Biolabs), 0.2 µL 10x Buffer 3, 2.0 µL BSA (10 mg/ml), 13.8 µL dH2O, 2.0 µL gDNA (280-440 ng/µL) and a unimolecular ligation performed in a reaction containing 3 units T4 DNA Ligase (NEB), 162.0 µL dH2O, 20.0 µL T4 DNA Ligase Buffer, and 15.0 µL digested gDNA. Samples at both steps were cleaned using E.Z.N.A. Purification Kit (Omega Bio-Tek®). An inverse PCR was then performed  (5 U/µL), 0.5 µL forward primer (50 µM), 0.5 µL reverse primer (50 µM), 2.0 µL 10X EconoTaq Buffer, 1.2 µL 25 mM MgCl2, 2.0 µL dNTPs (200 µM each), 5.0 µL template DNA) and resulting samples were purified directly, or extracted from the gel using Omega E.Z.N.A. Gel Extraction Kit. Sequencing was performed by Eurofins MGW Operon .All selected PanBRT175 genome (Accession: ASJH00000000) Pantoea septica X44686, Pantoea agglomerans Eh318, Pantoea ananatis BRT98, Pantoea agglomerans TX10, Pantoea eucalyptii F9026, Pantoea ananatis 15320, Pantoea agglomerans DC432, Pantoea dispersa 625, Pantoea eucalyptii 299R, Pantoea eucalyptii SP03391, Pantoea agglomerans SP04022, Pantoea eucalyptii SP03372, Pantoea eucalyptii SP04013, Pantoea eucalyptii SP02021, Pantoea agglomerans SP00101, Pantoea agglomerans VB39851-A, Pantoea calida B021323, Pantoea eucalyptii B011489, Pantoea agglomerans B025670, Pantoea brenneri B024858, Pantoea brenneri B016381, Pantoea dispersa M1657A, and Pantoea calida BB957621-B2 using standalone BLAST with an e-value cutoff of 0.0001 to identify even the most divergent potential homologs. The NCBI Conserved Domain Database (CDD) was used to assign each protein to a family.Mutant sequences were queried against GenBank at NCBI using the Basic Local Alignment Search Tool (BLAST). Mutant sequences were also queried against a draft sequence of the 50 of 376,883 bp and an estimated genome size of 5,036,004 bp at 319X coverage. Contigs that were 200 bp or larger  were submitted to the NCBI Prokaryotic Genome Automatic Annotation Pipeline version 2.0. The genome has been deposited under accession number AXOF00000000.Total DNA was sequenced using Illumina HiSeq 2000, 100-bp paired-end sequencing, resulting in 16,055,112 reads, with an average Phred quality score of 32. ABySS version 1.3.5 was used for de novo paired-end assembly using the default parameters and an optimized k-mer value of 87. This resulted in 51 contigs with an Npnp1A, pnp1C, pnp1D, and pnp1F were used to examine the distribution of the biosynthetic cluster in Pantoea. Colony PCR was performed for each Pantoea isolate by toothpick inoculation of a single colony into a PCR reaction containing 13.6 µL dH2O, 0.2 µL EconoTaq DNA Polymerase (5 U/µL), 0.5 µL forward primer (50 µM), 0.5 µL reverse primer (50 µM), 2.0 µL 10X EconoTaq Buffer, 1.2 µL 25 mM MgCl2, and 2.0 µL dNTPs (200 µM each), on each Pantoea strain  in a lawn of E. amylovora. To determine how antibiotic production and/or activity was affected by nutritional changes, the assay was attempted on the rich medium, LB. The ZOI was reduced to approximately half the size of that formed on minimal medium with glucose  and phosphoribosylformylglycinamidine synthase (purL) genes, which are necessary components in purine and pyrimidine metabolism PanBRT175 genome Transposon mutagenesis was used to identify the antibiotic biosynthetic cluster in pnp1A, which belongs to COG1167 (ARO8), a family whose members contain both a winged helix-turn-helix domain of the GntR family of transcriptional regulators (Conserved Domain Database Accession: cl17414), as well as an aspartate aminotransferase domain (CDD Accession:cl00321). The GntR transcriptional regulators have been implicated in bacterial growth and development, as well as antibiotic production pnp1A mutants could be restored with the exogenous supplementation of histidine. Transposon insertion in this mutant occurred in the aminotransferase region, possibly indicating that histidine is a PNP-1 precursor, and that the product of this gene plays a role in regulating histidine availability. The supplementation of histidine would have bypassed the requirement for this locus, thereby restoring antibiotic biosynthesis. The pdxR regulator of Streptomyces venezuelae, which also contains both an aminotransferase and helix-turn-helix domain was shown to complement pyridoxal (vitamin B6) auxotrophy by regulating the availability of pyridoxal phosphate and related co-enzymes The first gene identified is the divergently transcribed pnp1B, pnp1C, and pnp1D are the next three genes of the predicted biosynthetic cluster (pnp1B belongs to the SGNH/GDSL family of hydrolases (CDD Accession: cl01053), which include esterases and lipases that have known activity towards esters, acyl-CoAs, and amino acid derivatives. Applications for these hydrolytic enzymes include synthesis of pharmaceuticals, including phospholipase A and B pnp1C is a 2111 bp-long predicted coding region; however, it has no identifiable protein domain that could indicate biochemical function. Finally, pnp1D codes for a carbamoyltransferase of the NodU/CmcH family , which is responsible for post-transcriptional modifications of late gene products Rhizobium, Bradyrhizobium, and StreptomycesPseudomonas syringae pv. maculicola ES4326, Pseudomonas fluorescens WH6, and Pseudomonas chlororaphis PCL1391, as well as P. agglomerans Eh318 (discussed later) ( cluster . pnp1B bd later) , Table 4pnp1E falls under the aspartate aminotransferase (AAT) superfamily (fold type I) of pyridoxal phosphate (PLP)-dependent enzymes (Accession: cl00321), members of which are known to play a role in transamination, racemization, decarboxylation, and various other side chain reactions pnp1F belongs to the formyltransferase, catalytic core domain superfamily (CDD Accession: cl00395), which is known to be involved in purine biosynthesis, formate biosynthesis, translation initiation, and modification of lipid A, leading to resistance to cationic antimicrobial peptides and clinical antimicrobials The product of PanBRT175 cluster, pnp1G, is a member of the MFS transporter family that includes the LysE type translocator family (pfam01810) and the RhtB homoserine/threonine transport family . LysE exporters have been noted to transport L-lysine, cadmium, and various quaternary amines in bacterial species such as E. coli, Mycobacterium tuberculosis, and Bacillus subtilisThe final annotated gene in the Pantoea strains, a PCR-based survey was conducted on four of the seven genes . A total of 117 strains were evaluated, all of which were negative for all four genes in the antibiotic cluster. Standalone BLASTs were also performed for each gene in the PNP-1 cluster against a draft genome sequence of P. agglomerans Eh318, an isolate known to produce at least two antibiotics, pantocins A and B, which are effective against E. amylovorapnp1B, pnp1C, and pnp1D were identified as the first genes in a seven-gene 10 kb cluster in Eh318, while the remaining genes of the cluster include an MFS transporter, ATP-grasp/glutathione synthase, sulfotransferase, and aminotransferase . These results suggest that if the cluster in ES4326 also produces an antimicrobial compound, it may be differentially regulated, such that it is not induced under the same conditions as PNP-1. Alternatively, the chemical structure and/or spectrum may be different than PNP-1, due to the differing composition of their gene clusters.Because the PNP-1 cluster was unique and not found complete in any other isolate in our collection, we examined its distribution in other species. We queried GenBank and identified similar gene clusters in  PCL1391 . The clu PCL1391 . Four ge cluster . Since tP. chlororaphis PCL1391 directs the production of a phenazine antibiotic, phenazine-1-carboxamide  The chemical nature of the PNP-1 antibiotic is still not known. The homologous cluster in boxamide . PhenaziP. ananatis BRT175 whose biosynthesis is determined by an 8.2 kb biosynthetic gene cluster consisting of seven genes. The loss of antibiotic production and/or export in 24 independent transposon mutants is strong evidence that the seven-gene cluster identified is likely responsible for the production of the antibiotic. Although this cluster has yet to be reported within Pantoea, the identification of homologous antibiotic biosynthetic gene clusters in Pseudomonas that have been shown to produce an antimicrobial phenazine adds further support to our conclusion. Although there are several conserved genes between this cluster and those of the pseudomonads, differences in the genetic composition of the clusters, along with the spectrum of activity, suggest that PNP-1 is likely structurally and functionally different from phenazines. Still, further characterization of the gene cluster, including its role in antibiotic biosynthesis, and the determination of the biosynthetic steps and chemical structure of PNP-1 are presently being pursued. PNP-1 has the potential to be used to control E. amylovora, as well as clinically and agriculturally relevant Pantoea isolates.This work has identified an antibiotic in Figure S1Effects of nutrition on antibiotic production. A) LB medium with and without glucose, B) E. coli minimal medium, with and without glucose; C) E. coli minimal medium with varying peptone concentrations, with and without glucose; D) E. coli minimal medium with varying tryptone concentrations, with and without glucose.(DOC)Click here for additional data file.


Archaea were Deep-sea Hydrothermal Vent Euryarchaeotic Group 6 (DHVEG 6), Marine Group I (MGI) and Deep Sea Archaeal Group (DSAG), and those in Bacteria were Alpha-, Gamma-, Delta- and Epsilonproteobacteria, Chloroflexi, Bacteroidetes, Planctomycetes and Acidobacteria. Diversity and richness were examined by 8,709 and 7,690 tag-sequences from sediments at 5 and 25 cm below the seafloor (cmbsf), respectively. The estimated diversity and richness in the methane seep sediment are as high as those in soil and deep-sea hydrothermal environments, although the tag-sequences obtained in this study were not sufficient to show whole microbial diversity in this analysis. We also compared the diversity and richness of each taxon/division between the sediments from the two depths, and found that the diversity and richness of some taxa/divisions varied significantly along with the depth.Microbial community structures in methane seep sediments in the Nankai Trough were analyzed by tag-sequencing analysis for the small subunit (SSU) rRNA gene using a newly developed primer set. The dominant members of  Euryarchaeota and syntrophic sulfate-reducing Deltaproteobacteria oxidize methane anaerobically with the following equation:In deep-sea anoxic methane seep sediments, methanotrophic Gammaproteobacteria and Epsilonproteobacteria that utilize the high sulfide fluxes derived from anaerobic oxidation of methane (AOM) , Marine Benthic Group D (MBGD) and Miscellaneous Crenarchaeotic Group  and diverse bacterial groups such as the Chloroflexi, Planctomycetes, WS1, Bacteroidetes, Firmicutes, JS1, OP11 and other taxa/divisions have been observed in methane seep sediments (and this syntrophic reaction is one of the most abundant processes of methane seep ecosystems . In contne (AOM) , 20, 31,bacteria , 40, andmilales’ , 11, 14 milales’ , 34. In ediments , 29, 31.e.g., In this study, we found atypical microbial communities in a sediment core from a microbial mat site on a deep-sea methane seep in the Nankai Trough by quantitative PCR techniques and geochemical analyses, and then applied SSU rRNA gene tag-sequencing analysis. Pyrosequencing technique applied to SSU rRNA gene tag-sequencing analysis of marine and soil biospheres, including hydrocarbon seep sediments, retrieved more than 5,000 sequences and revealed a ‘rare biosphere’ e.g., , 32, 35.Shinkai 6500’ (dive #6K949) on the YK06-03 cruise (April 2006) of R/V Yokosuka . The sediments consisted of blackish-gray sandy-silt and contained hydrogen sulfide. The core was subsampled by sterilized top-cut syringes and spatulas at 5 cm intervals on board, stored at −80°C for microbiology and used for interstitial water geochemistry. Sulfate concentration in core 949C3 decreased with increasing depth and more than 5.4×102 μmol kg−1 methane was detected through the sediment core column  and will be published elsewhere.Sediment core 949C3 (25 cm in length) was taken by a push corer from a bacterial mat in an active methane seep site at the Omine Ridge  , Nankai Trough off Kumano area with a manned submersible ‘e column . Geochemg sediments using an Ultra Clean Power Max Soil DNA Purification Kit . The archaeal and total prokaryotic SSU rRNA genes from environmental microbial DNA were quantified using a quantitative fluorescent PCR method with a 7500 Real Time PCR System  described previously  in the presence of MgCl2  as described previously  based on the population of sequences covered by each primer sequence, predominant primer sequences were determined; 3) based on the number of mismatch positions between the predominant primer sequence and minor sequences, the coverage of each primer sequence ratio between the predominant primer and minor primers was assessed. The coverage of the primer set was reconfirmed at the end of 2011 targeting sequences longer than 1.2 and 0.9 kb in length of bacterial and archaeal sequences, respectively, in SILVA SSU Ref NR database (http://www.arb-silva.de/download/arb-files/) , we surveyed 530F and 907R primer sites  correspo−1 LA Taq, 0.4 pmol μL−1 of each primer mixture and 1.6 nmol μL−1 dNTP. The DNA amplification condition was 5 min of denaturation at 96°C, and 25 cycles at 96°C for 20 s, 48°C for 70 s and 72°C for 30 s. Each of the ‘530F’ and ‘907R’ oligonucleotide primers harbored 24 bases of non-palindromic sequencing “key” sequences ‘CCATCTGTTCCCTCCCTGTCTCAG’ and ‘CCTATCCCCTGTTGCGTGTCTCAG’ for pyrosequencing at their 5′ terminal end, respectively. These primers were mixed as shown in −1 of Ex Taq (Takara), 0.4 pmol μL−1 of each primer, 0.8 nmol μL−1 dNTP and 0.01 pmol SSU rRNA gene fragments. The amplification condition was 4 min of denaturation at 96°C, and 4 cycles at 96°C for 30 s, 64°C for 30 s and 72°C for 1 min. The biotin-labeled SSU rRNA gene fragments were purified by gel electrophoresis. The SSU rRNA gene fragments were analyzed by 454 pyrosequencer GS20  in Takara. A gasket separated into 16 regions was used for pyrosequencing and we applied one sample per region in this study.DNA assemblages for SSU rRNA gene tag-sequencing analysis were extracted from sediments at 5 and 25 cm below the seafloor (cmbsf). PCR amplification of SSU rRNA gene fragments using the primer set of ‘530F’ and ‘907R’ described above was conducted under the following conditions. The PCR mixture contained 0.5 volumes of 2×GC buffer I for LA Taq polymerase (Takara), 0.1 U μLhttp://www.arb-silva.de/aligner/) with a reference multiple alignment SILVA SSU Ref NR. Then, tags were clustered into operational taxonomic units (OTU) by 98% sequence identity using MOTHUR 3.6 with default parameters   ranged from 92 to 97%. Most of the zetaproteobacterial SSU rRNA gene sequences have been detected from hydrothermal environments with a few exceptions, such as the brine-seawater interface in the Red Sea , Actinobacteria, Chlamidiae, Chlorobi, Deferribacteres, Elusimicrobia (TM1), Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Spirochaetes, Tenericutes and Verrucomicrobia were detected as minor populations  was observed even at 25 cmbsf. At 5 cmbsf, only one each of the clones of ANME-1 and Lost City Methanosarcinales sequences and 3 ANME-3/Methanococcoides sequences was found, and 2 ANME-1, 13 ANME-2a, 14 ANME-2c and 21 ANME-3/Methanococcoides sequences were detected at 25 cmbsf. The ANME-3 and Methanococcoides methanogens could not be distinguished by short tag-sequences. In addition, the DSEG, MEG, MCG, Ancient Archaeal Group (AAG), Marine Hydrothermal Vent Group (MHVG) and Marine Benthic Group D (MBGD) were detected as minor populations in the archaeal tag population  was apparently smaller than those in previously analyzed methane seep sediments (core 744C1) taken from the other microbial mat formation site on the same ridge in April 2003; archaeal SSU rRNA gene abundance at 5 cmbsf was 9.0×106 and 4.6×108 copies g−1 sediment and at 25 cmbsf was 2.9×109 and 3.4×107 copies g−1 sediment, in cores 949C3 and 744C1, respectively, and mcrA gene abundance at 5 cmbsf was 2.8×105 and 4.6×107 copies g−1 sediment, and at 25 cmbsf was 3.4×107 and 2.6×109 copies g−1 sediment, in cores 949C3 and 744C1, respectively (2-1.2×103 μmol kg−1) were higher than those of 744C1 (4.2–6.0×101 μmol kg−1)  , 36. There 949C3  suggest bacteria  below thEpsilonproteobacteria (From another aspect, the microbial communities analyzed are likely analogues of microbial communities just beneath the seafloor under the microbial mat formation site that harbors typical mature AOM-dependent microbial communities. In typical methane seep sediments presenting geochemical signatures of sulfate reduction and methane oxidation, the transition of the sulfur-oxidizing community to sulfate-reducing ANME community occurs within a few cm, and the ANME community predominates in the core column , 17, 29.bacteria .


Microglia and astrocytes contribute to Alzheimer's disease (AD) etiology and may mediate early neuroinflammatory responses. Despite their possible role in disease progression and despite the fact that they can respond to amyloid deposition in model systems, little is known about whether astro- or microglia can undergo proliferation in AD and whether this is related to the clinical symptoms or to local neuropathological changes. Previously, proliferation was found to be increased in glia-rich regions of the presenile hippocampus. Since their phenotype was unknown, we here used two novel triple-immunohistochemical protocols to study proliferation in astro- or microglia in relation to amyloid pathology. We selected different age-matched cohorts to study whether proliferative changes relate to clinical severity or to neuropathological changes. Proliferating cells were found across the hippocampus but never in mature neurons or astrocytes. Almost all proliferating cells were colabeled with Iba1+, indicating that particularly microglia contribute to proliferation in AD. Proliferating Iba1+ cells was specifically seen within the borders of amyloid plaques, indicative of an active involvement in, or response to, plaque accumulation. Thus, consistent with animal studies, proliferation in the AD hippocampus is due to microglia, occurs in close proximity of plaque pathology, and may contribute to the neuroinflammation common in AD. Its two main pathological lesions are amyloid- disease –3. In aduring AD , 5, and uring AD , 7.β lesions [β peptide in vitro [β from the brain [Neuroinflammation is prominent during AD –10 and m lesions –23, degrin vitro , 25, andhe brain .β; their proliferation index increases and they release tumor necrosis factor α (TNFα) in response to Aβ [β plaques [Earlier work had established that microglia can also respond to damage by undergoing proliferation. Indeed, cultured microglia become activated by Ase to Aβ . Microglse to Aβ , 29 and  plaques , 31.β plaque pathology [β lesions in human brain.Another common feature of AD is extensive astrogliosis. Astrocytes have been classically known to change in AD and show, for example, hypertrophy or changes in the expression of specific GFAP isoforms, parallel to the development of Aathology , 32. Amyathology –37. Alsoathology , 39. Whiathology , their rβ plaques were unknown, we therefore developed two triple-immunohistochemical stainings to detect astrocyte or microglial proliferation in combination with Aβ pathology. To further assess whether the clinical or neuropathological aspects of dementia influence proliferation, we here studied older age-matched individuals that differed in cognitive and neuropathological staging: (1) a control (Con) cohort without any clinical or neuropathological signs of dementia, (2) a cohort that had low levels of AD pathology at autopsy (Braak stages 1-2) but was clinically demented during their lives (Dem), and (3) a confirmed Alzheimer cohort (AD) that was clinically demented and exhibited severe neuropathology at autopsy (Braak stages 4-5). As followup to our study on presenile cases, we here asked (a) whether also elderly cohorts (>80 years) of AD or demented subjects exhibit increases in proliferation, (b) if astrocytes (GFAP+) or microglia (Iba1+) contribute to these proliferative changes, and (c) if proliferation is anatomically related to Aβ pathology or to clinical severity.We previously documented an increase in proliferation in the hippocampus of presenile AD patients (≤70 years of age) . Interesμm sections were mounted on Superfrost Plus slides. Hippocampal tissue was selected from 8 age-matched, nondemented controls (Con), 8 clinically demented (Dem) cases with low Braak scores, and 8 confirmed Alzheimer disease (AD) cases. Braak staging of the three groups was, respectively, Con: 1.4 ± 0.2, Dem: 1.5 ± 0.2, and AD: 4.8 ± 0.3. Mean age (y) was, respectively, Con: 80 ± 3 y, Dem: 84 ± 2, and AD: 81 ± 2. Brain weight (g) was Con: 1208 ± 65, Dem: 1238 ± 87, and AD: 1125 ± 50. No significant differences in postmortem delay (between 5–7 hours) or fixation duration  were present and the groups were further balanced male : female in a 1 : 1 ratio  supplemented with 10% fetal calf serum and incubated for 1 hr at room temperature (RT) and then overnight at 4°C.Sections were deparaffinized in xylene and rehydrated through graded ethanol solutions and then washed in 0.05 M PBS prior to antigen retrieval by heating in a food steamer  for 60 minutes at 100°C in citrate buffer (pH = 6.0). Sections were cooled to room temperature prior to incubation with 0.3% Hβ antibody (Ab)  was reacted with biotinylated sheep anti-mouse , followed by incubation with avidin-peroxidase  and development with diaminobenzidine  substrate. After washing, sections were incubated with antiproliferating cell nuclear antibody (PCNA) Ab  overnight, followed by donkey anti-mouse alkaline phosphatase-conjugated Ab and NBT/BCIP substrate to produce a blue stain. In order to inactivate the binding properties of the first round of antibodies and to retrieve additional GFAP and Iba1 epitopes, sections were then treated with EDTA  in TRIS buffer for 30 minutes followed by washing and final incubation with rabbit polyclonal anti-GFAP  or anti-Iba1 Ab . Subsequently, sections were incubated with alkaline phosphatase-conjugated donkey anti-rabbit  and developed with Fast Red substrate to stain, in separate series, astrocytes and microglia dark red.Triple immunostaining was performed through sequential development steps. First, anti-Aβ 4G8 antibody was found previously to provide the most consistent staining of diffuse plaques in formalin-fixed, paraffin embedded human tissue [β peptide) recognizes extracellular APP domains containing the Aβ epitope and thus labels full length APP as well as Aβ plaques. PCNA has been validated and used before extensively and the anti-PCNA antibody identifies proliferating cell profiles in human brain processed in a similar manner [All secondary antibodies were incubated for 1 hr at room temperature. The anti-An tissue . 4G8 Ab r manner –54.Glial fibrillary acidic protein (GFAP) is a classic member of the intermediate filament protein family involved in astrocyte cytoarchitecture and generally considered a sensitive marker of reactive astrogliosis in the CNS , 56. TheFor the detection of microglia, several markers, like Iba1 and CD68 antibodies , are ava2. Quantification of PCNA+ and Iba1+ cells was obtained from the PCNA/Iba1/Aβ stained slides, while quantification of GFAP+ cells was obtained from the PCNA/GFAP/Aβ triple stained sections. Values for cell numbers are expressed as averages ± SE and values for plaques are expressed as plaques per mm2.Cross-sectional areas for the dentate gyrus and CA subregions were determined using StereoInvestigator software  linked to a Zeiss Axiophot microscope  to outline the appropriate hippocampal subregions. All phenotypic quantification was performed in midlevel sections of the hippocampus. Cell count values were normalized to the surface area of each anatomical subregion and expressed per mmβ plaques were not colabeled with PCNA in any hippocampal subregion  and subgranular zone (SGZ) of the dentate gyrus (DG) Figures , areas p Figures . Regardlβ pathology (P = 0.07).Within the CA regions, PCNA+ cells were also found in astrocyte-rich regions that lacked Aathology , and, coathology . The popathology . Quantifβ plaques  showed this hypertrophic morphology. GFAP expression was enriched in the stratum moleculare and stratum lacunosum of the AD hippocampus, where astrocytes were found preferentially with Aβ plaques (P = 0.02).GFAP+ astrocytes showed a characteristic, spider-like cellular morphology. They were seen in close proximity to neurons and blood vessels with endfeet clearly visible that contacted blood vessel walls. In the CA regions and subiculum, hypertrophic astrocytes were observed with processes infiltrating diffuse, primitive, and dense-core A Figures . These c plaques . A signi plaques  (one-wayP < 0.01) and GFAP-associated, that is, degraded plaque subtypes (one-way ANOVA P < 0.001). For the dense-core plaques, averaged numbers were as follows: control: 0.25+/−0.2, dementia: 0.25+/−0.2, and AD: 2.37+/−0.8. For the degraded plaques, these numbers were control: 0.37+/−0.2, dementia: 1.37+/−1.1, and AD: 7.25+/−1.89. The population of Aβ plaques with invasive hypertrophic GFAP+ astrocytes was increased in the AD population (one-way ANOVA P < 0.001). However, we could not observe any indication that mature astrocytes underwent proliferation in the presence of Aβ plaques.Plaque values were low across the Braak 1 stage cases, the control cohort, and dementia cohort. Morphologically, the plaque pathology in the AD cohort was distinct from the control and dementia sections; AD cases had a significant increase in dense-core (one-way ANOVA P = 0.43)  (one-way ANOVA P < 0.001) (P < 0.01) and GFAP-associated, that is, degraded plaque subtypes (one-way ANOVA P < 0.001).Similar to earlier studies, the 4G8 antibody labeled diffuse, primitive, dense-core, and remnant Aprofiles . Remnant< 0.001) . Morpholβ dense-cored plaques and were generally found in close association to plaques .Despite this evidence that microglia proliferate directly at sites of AP = 0.14, After the surface areas of the hippocampal subregions were measured, no significant differences were observed across subregions; however, surface areas of the CA1/2 subregion were somewhat smaller, but not significantly, in the AD cohort  , we here Ex vivo microglia cultures isolated from aged mice, for example, show elevated production of the proinflammatory molecules Il-6 and TNF-α, while microglia from old animals have a decreased ability to internalize Aβ compared to cells isolated from young animals [β plaques [α, Il-1β, Il-6, and IP-10 by microglia [β plaques, microglial proliferation and astrocytic activation may be complementary processes that could enhance local inflammation at Aβ plaque sites. Activation of astrocytes is generally reflected by increased expression of GFAP per cell or proteasome activation [Whereas, in general, aged microglia do not show major morphological changes, there is evidence that these cells, in functional terms, are highly proinflammatory. animals . Microgl animals . For ins plaques . Interesicroglia . Hence, tivation , 31 and  in vitro studies have shown that the recruitment of monocytes across the BBB is increased after an interaction of the Aβ peptide with RAGE receptor [While proliferating microglia are seen in the hippocampus, the source of these cells remains to be determined. CNS microglia are unique because two populations exist, that is, resident cells present since early development and infiltrating microglia that pass the blood-brain barrier (BBB). Iba1 identifies both types of microglia in the brain regardless of their source. Determining the kinetics of microglial infiltration during a chronic disease like AD is challenging, but so far resident microglia were shown to incorporate the thymidine analog BrdU+ in AD mice  and durireceptor . In AD mreceptor . Hence, Another relevant issue in this respect is the turnover of the microglia population. Although limited information is available on their precise kinetics, glial cells exhibit remarkable homeostasis and strive to maintain a constant density in response to internal or external perturbations. In a recent paper by Elmore et al. , selectiOur results on proliferation in brain microvessels are in agreement with earlier studies in which proliferating cells were found associated with the vasculature , 77. TheAfter careful study, PCNA+ profiles were never present in mature hippocampal neurons. Earlier studies using various cell cycle markers on human AD or Lewy body dementia tissues suggested that a subset of pyramidal neurons in the affected human hippocampus may reenter the cell cycle during early stages , 80–82. β plaques, without astrocyte proliferation in these animals; indeed, age-related increases in proliferation in this mouse model were due to increases in newborn BrdU+ cells coexpressing markers for activated microglia [β plaques in transgenic mice that lacked microglia [β plaques seen in human AD, suggesting that inbred mouse models are not always optimal for studying the complex in situ microglial responses in human brain [Our study further agrees with recent findings from the APPswePS1dE9 AD mouse model. It has been established that CD11b microglia cell clusters were observed as early as 4 months of age . Other sicroglia . Despiteicroglia  whereas icroglia  with theicroglia , 90–92. an brain .β plaque pathology.In conclusion, we demonstrate that proliferation in the human hippocampus is due to microglia and not to that of astroglia or mature neurons. These results are consistent with recent observations in AD mouse models. Although our data do not exclude that stimulation of microglia can also occur through additional mechanisms, they further suggest that microglial proliferation is not influenced by cognitive decline but occurs in close association with, and possibly in response to, local A


Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury. However, exactly how DLK fulfills these functions remains to be determined. Using RNA-seq profiling, we evaluated the global changes in gene expression that are caused by shRNA-mediated knockdown of endogenous DLK in differentiated Neuro-2a neuroblastoma cells.Recent genetic studies in model organisms, such as Our analysis led to the identification of numerous up- and down-regulated genes, among which several were found to be associated with system development and axon guidance according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, respectively. Because of their importance in axonal growth, pruning and regeneration during development and adult life, we then examined by quantitative RT-PCR the mRNA expression levels of the identified axon guidance genes in DLK-depleted cells. Consistent with the RNA-seq data, our results confirmed that loss of DLK altered expression of the genes encoding neuropilin 1 (Nrp1), plexin A4 (Plxna4), Eph receptor A7 (Epha7), Rho family GTPase 1 (Rnd1) and semaphorin 6B (Sema6b). Interestingly, this regulation of Nrp1 and Plxna4 mRNA expression by DLK in Neuro-2a cells was also reflected at the protein level, implicating DLK in the modulation of the function of these axon guidance molecules.Collectively, these results provide the first evidence that axon guidance genes are downstream targets of the DLK signaling pathway, which through their regulation probably modulates neuronal cell development, structure and function.The online version of this article (doi:10.1186/s13064-016-0068-8) contains supplementary material, which is available to authorized users. The mitogen-activated protein kinases (MAPKs) are important regulators of fundamental biological processes, such as cell proliferation, differentiation, cell survival, migration and apoptosis. These enzymes are activated in response to extracellular stimuli by upstream kinases termed MAPKKs, which are themselves activated by the third component of the MAPK system, the MAPKKKs . Based oin situ hybridization and immunolocalization, DLK has a tissue-specific expression pattern in both mouse and human, being mainly expressed in brain, kidney and skin [DLK gene in mice results in perinatal death [DLK display abnormal brain development, characterized by defects in axon growth, neuron migration, apoptosis and axon degeneration [One MAPKKK that emerges as a pivotal component of the MAPK pathways is dual leucine zipper kinase , which was originally identified in a screen for proteins differentially expressed during the retinoic acid-induced differentiation of human NT2 teratocarcinoma cells –6. Basedand skin –10. Preval death . Embryosneration –15. Aparneration , 15–17. neration –19. Receneration , 20, 21.Because one way to unravel the mode of action of DLK is to identify genes critical for its function in neurons, we characterized by next-generation sequencing (RNA-seq) the transcriptome of differentiated Neuro-2a neuroblastoma cells in which DLK has been depleted by RNA interference. Our results led to the identification of many genes whose expression was significantly altered upon DLK knockdown. Notably, among the identified genes, we focused on those encoding axon guidance molecules due to their crucial roles in many aspects of neuronal development, including axon pathfinding, axon growth, neuronal polarization, neuronal migration and dendrite formation, as well as in axon regeneration in the adult nervous system –25.The polyclonal antiserum used for detection of DLK was described previously . The polMouse Neuro-2a neuroblastoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. When indicated, cells were differentiated by incubation in DMEM containing 0.1 % bovine serum albumin for 24 h.6 cells per well in six-well dishes, 0.5 × 106 cells in 60-mm dishes or 2.0 × 106 cells in 100-mm dishes 24 h before, were infected with viral supernatants supplemented with 8 μg/ml polybrene. Two days later, infected cells were treated with puromycin (2.5 μg/ml) and selected for 2 days, after which they were induced to differentiate as mentioned above.HEK 293T cells grown in DMEM supplemented with 10 % (v/v) FBS and antibiotics were cotransfected with the envelope protein expressing vector pMD2.G and the packaging protein expressing vector psPAX2,  and with either the transfer pLKO.1 empty lentiviral vector   in combination with TRIzol , following the manufacturer’s protocol. A 30 min on-column DNase treatment was performed before elution according to manufacturer’s instructions. RNA was quantified on a NanoDrop (Thermo Scientific) spectrophotometer. Total RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs (Roche Diagnostics), and 10 units of RNAseOUT (Invitrogen) following the manufacturer’s protocol in a total volume of 20 μl. All forward and reverse primers were individually resuspended to 20–100 μM stock solution in Tris-EDTA buffer (IDT) and diluted as a primer pair to 1 μM in RNase DNase-free water (IDT). Quantitative PCR (qPCR) reactions were performed in 10 μl in 96 well plates on a CFX-96 thermocycler (BioRad) with 5 μL of 2X iTaq Universal SYBR Green Supermix (BioRad), 10 ng (3 μl) cDNA, and 200 nM final (2 μl) primer pair solutions. The following cycling conditions were used: 3 min at 95 °C; 50 cycles: 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C. Relative expression levels were calculated using the qBASE framework  and the lsewhere . In evert test.Images of control and DLK-depleted Neuro-2a cells were acquired in bright-field using a Zeiss AxioObserver.Z1 inverted microscope equipped with a 20x/0.30 NA objective and a Zeiss Axiocam 506 camera. The length of approximately 200 neurites was measured from five random microscope fields per sample using the open source software Fiji . GraphPahttps://bitbucket.org/mugqic/mugqic_pipelines). Briefly, the reads were trimmed using Trimmomatic version 0.32 [Total RNA was isolated as described above from control, sh73/DLK- and sh69/DLK-depleted cells. The quality of total RNA was evaluated using the Agilent 2100 BioAnalyzer and the Agilent RNA 6000 Nano Kit (#5067-1511) according to the manufacturer’s instructions. For all samples, RNA integrity numbers were sufficiently high (>9.5) to perform mRNA sequencing. mRNA was purified from 5 μg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module  following manufacturer’s instructions. cDNA libraries were then prepared with 25 ng mRNA of each sample using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina  and barcoded by PCR for subsequent multiplexed sequencing. The quality and quantity of the librairies were assessed using the Agilent High Sensitivity DNA Kit (#5067-4626) on Agilent 2100 BioAnalyzer. Sequencing of the multiplexed cDNA libraries was performed on an Illumina HiSeq 2500 system  in two lanes at the Institut de Recherches Cliniques de Montréal (IRCM). The obtained raw reads were processed using the McGill University and Génome Québec Innovation Centre (MUGQIC) RNA-seq pipeline version 1.4  using GraphPad Prism software. p-values of < 0.05 were considered to be statistically signifiant.qRT-PCR and immunoblot data represent the mean ± SEM of at least three independently performed experiments. The statistical significance between mean values was determined by unpaired Neuro-2a is an established mouse neural crest-derived cell line that has been used extensively for studying neuronal differentiation, axon growth and signaling pathways . Upon trSince DLK is an upstream activator of the JNK signaling pathway , we testPrevious studies have shown that DLK is required for axon outgrowth both in vivo and in vitro , 14, 44.To identify downstream effector genes of the DLK signaling pathway, we carried out RNA-seq experiments on both control and DLK-depleted Neuro-2a cells after differentiation Fig. . In ordeThe RNA-seq data were subsequently analyzed using the HTSeq , DESeq  and edgeEpha7, Nfasc and Nrp1, which undergo up-regulation by two-fold or more in DLK-depleted cells   to identP-value, we decided to focus our experimental efforts on this group of genes for further characterization. These putative DLK-regulated genes fell into three distinct functional categories of axon guidance molecules, namely membrane-bound ligand (Sema6b), cell surface receptors  and GTP-binding protein (Rnd1) . One ofromoters , 49.Interestingly, decrease of DLK expression in Neuro-2a cells resulted in transcriptional dysregulation of a subset of genes involved in neuronal function, such as migration, differentiation, axonogenesis, and axon guidance, an observation that supports its critical role in nervous system development (Additional file Epha7, Nrp1, Plxna4 and Unc5a, showed an increase in expression level after DLK depletion, whereas two, Rnd1 and Sema6b, were down-regulated (Additional file Epha7, Nrp1, Plxna4, Rnd1 and Sema6b (Fig. Unc5a, we currently cannot draw any conclusion for this gene. A part of our findings was further validated by Western blot analysis, which shows that Nrp1 and Plxna4 protein levels were significantly induced in DLK-depleted cells (Fig. Epha7 and Rnd1 as up- and down-regulated genes, respectively, in DLK-depleted cells is also of great interest in regard to the contribution of DLK to neuron projection development. Indeed, a recent study revealed that Epha7, which generally transduces repulsive signals [Sema6b, whose expression decreased upon DLK knockdown in Neuro-2a cells, results obtained from loss-of-function experiments demonstrated its requirement for proper projection of hippocampal granule cell axons [As stated above, the current results highlight for the first time the involvement of DLK in expression of axon guidance genes. This family of genes encodes proteins that act as attractants or repellents for axons, thereby guiding them towards or away from a specific region , 50. The signals , impairs signals , while k signals . Finallyll axons . Taken tll axons , 62.Although further work will be required to investigate the biological consequences of dysregulated axon guidance gene expression in DLK-depleted Neuro-2a cells, we anticipate as a potential outcome an alteration of the cytoskeleton. This prediction is supported by the fact that axon guidance cues and their receptors modulate actin and microtubule dynamics in the growth cone via activation of downstream signaling molecules, such as protein kinases, small GTPases and cytoskeleton-associated proteins , 63, 64.Nrp1, Plxna4, Epha7, Rnd1 and Sema6b as well as the consequences of their up- or down-regulation on axon growth. Further experiments using the DLK knockout mice will also be required to assess the in vivo relevance of our in vitro findings.We found that DLK plays an important role in regulating expression of genes recognized for their contribution in nervous system development and function. Future studies will be dedicated to understand how DLK influences the expression of DLK, dual leucine zipper kinase; Epha7, eph receptor A7; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; Nrp1, neuropilin 1; Plxna4, plexin A4; Rnd1, Rho family GTPase 1; Sema6b, semaphorin 6B; Unc5a, Unc-5 homolog A


Thrombomodulin alfa  significantly improved disseminated intravascular coagulation (DIC) when compared with heparin therapy in a phase III study. Post-marketing surveillance of TM-α was performed to evaluate the effects and safety in patients with sepsis-induced DIC.From May 2008 to April 2010, a total of 1,787 patients with sepsis-induced DIC treated with TM-α were registered. DIC was diagnosed based on the Japanese Association for Acute Medicine (JAAM) criteria. The DIC resolution and survival rates on day 28 after the last TM-α administration, and changes in DIC, systemic inflammatory response syndrome (SIRS), and sequential organ failure assessment (SOFA) scores and coagulation and inflammation markers were evaluated.p < 0.001). DIC resolution and 28-day survival rates were 44.4% and 66.0%, respectively. The 28-day survival rate decreased significantly according to the duration of DIC before TM-α administration (p < 0.001). Total adverse drug reactions (ADRs), bleeding ADRs, and serious bleeding adverse events occurred in 126 (7.1%), 98 (5.5%), and 121 (6.8%) subjects, respectively. On day 28, after the last TM-α administration available for an antibody test, only one patient was positive for anti-TM-α antibodies (0.11%).The most frequent underlying disease was infectious focus-unknown sepsis (29.8%). The mean ± SD values of age, dose, and the duration of TM-α administration were 64.7 ± 20.3 years, 297.3 ± 111.4 U/kg/day, and 5.6 ± 3.4 days, respectively. A total of 1,320 subjects (73.9%) received combined administration with other anticoagulants. Both coagulation and inflammation markers, such as fibrin/fibrinogen degradation products, prothrombin time ratio, thrombin-antithrombin complex, and C-reactive protein, as well as JAAM DIC, SIRS, and SOFA scores, significantly and simultaneously decreased after TM-α administration (Our results suggest that TM-α is most effective for treating patients with sepsis-induced DIC when administered within the first 3 days after diagnosis. In severe sepsis, disseminated intravascular coagulation (DIC) occurs in about 35% of patients . The morThrombomodulin (TM) is a thrombin receptor on the endothelial cell surface that plays an important role in the regulation of intravascular coagulation . ThrombiThrombomodulin alfa  is a recombinant, human, soluble TM possessing an extracellular domain that includes a functional domain. The effects of TM-α on DIC have been examined in a multicenter, randomized, clinical trial in Japan , 8. In pThe present PMS sub-population analysis evaluated the effects and safety of TM-α in 1,787 sepsis-induced DIC patients who were prescribed TM-α according to the Japanese Association for Acute Medicine (JAAM) DIC criteria for early diagnosis of DIC.The subjects in the present observational study were a subset of those in the PMS reported by Mimuro et al. . In respThe data of patients re-treated with TM-α were excluded from this analysis. TM-α was administered by intravenous infusion for 30 min once a day at a dose of 380-U/kg body weight . Since TSerum samples collected at baseline and on day 28 after the last TM-α administration were tested by enzyme-linked immunosorbent assay (ELISA) for anti-TM-α antibodies. This PMS study was conducted in accordance with the guidelines for Good Post-Marketing Surveillance Practices as required by the Japanese Ministry of Health, Labor, and Welfare. All subjects were treated according to the attending physicians' decisions, and no limitations were placed on the concomitant use of other anticoagulants or medicine for the treatment of underlying diseases and complications. In addition, personal data anonymization was carried out upon data collection. Therefore, approval of this surveillance by ethical committees and institutional review boards or informed consent acquisition was not necessary.n = 1,771).Organ dysfunction was assessed using the SOFA score. SIRS, sepsis, and septic shock were defined according to the American College of Chest Physicians/Society of Critical Care Medicine consensus conference  and SurvSubjects were followed until day 28 after the last administration. At the start of TM-α administration, information about the following 14 baseline characteristics was collected: age, sex, severity of underlying disease, history of bleeding, duration of DIC, kidney injury, liver injury, bleeding or symptoms of organ dysfunction, platelet count, fibrin/fibrinogen degradation products (FDP) or fibrinogen or antithrombin levels, and PT ratio. Furthermore, the JAAM DIC, SIRS, and SOFA scores were added as a factor relevant to sepsis . MultiplBlood collection for clinical laboratory tests and coagulation studies was performed at the following three time points: before, during, and after TM-α administration. However, the TM-α administration period was excluded from the data analysis because the time varied by case. Safety data were coded using the preferred terms from version 13.1 of the Japanese version of the Medical Dictionary for Regulatory Activities (MedDRA/J) . The safp < 0.05 was considered significant. Statistical testing was performed with JMP version 9.0 software .In the descriptive analysis of baseline characteristics, numerical data are expressed as means ± standard deviation (SD) or medians . Statistical analysis was performed to compare values using the chi-square test, the Wilcoxon signed rank test, and the Cochran-Armitage test. To identify risk factors, DIC resolution and survival at 28 days after starting TM-α administration were stratified and tabulated by the 15 baseline characteristics. Differences in frequency by strata were tested for significance by the chi-square test. Relative risk factors for the DIC resolution rate and the survival rate after TM-α administration for DIC treatment were analyzed by multiple logistic regression analysis. Relative risk factors for serious bleeding AEs were identified using multiple logistic regression analysis. Odds ratios (ORs) with 95% confidence intervals (CIs) for selected variables were calculated. A value of n = 533, 29.8%). The DIC score  was 6.0 (5.0–7.0). There were 816 (58.3%) subjects with a DIC score over 6 points and thus considered to be critically ill , but serious bleeding AEs did . No subject was found to have antibodies that neutralized TM-α activity.To investigate the risk factors before TM-α administration for serious bleeding AEs, a multiple logistic regression analysis was performed Table . HistoryIn this PMS sub-population analysis, in which DIC was diagnosed according to the JAAM DIC criteria and TM-α was administered at 4 days or later, the 28-day survival rate was significantly decreased as compared with TM-α administration within 3 days of diagnosis. These findings suggest that early use of TM-α could be useful for treating sepsis-induced DIC.The survival rate in this PMS study was somewhat lower than that of the phase III clinical trial . In the p = 0.086) [The interaction between inflammation and coagulation involves significant cross-talk between systems , and the= 0.086) . Taken t= 0.086) .On the other hand, as for the present result, the WBC count in association with inflammation was not affected by TM-α administration. WBC counts in patients with severe infection sometimes show levels below the normal range, which then increase according to the attenuation of inflammation. These phenomena may, in part, explain the unchanged WBC levels before and after TM-α administration.The rates of serious bleeding events and intracranial hemorrhage were somewhat high in the present PMS sub-population analysis. Khan et al. reported that both serious bleeding events and intracranial hemorrhage in patients receiving activated protein C were significantly higher in the retrospective observation studies than in the randomized controlled trial (RCT) . A recenin vitro study. Thus, it appears that treatment with TM-α in this subject did not decrease the effect of TM-α.In order to estimate the frequency of anti-TM-α antibody production, especially neutralizing antibody that neutralizes the activity of TM-α and/or endothelial cell thrombomodulin, anti-TM-α antibody was measured before, and on day 28 after the last TM-α administration by ELISA for anti-TM-α antibodies. In the 1,787 subjects, there were 945 serum samples taken on day 28 after the last TM-α administration available for an antibody test, and only one patient was positive for anti-TM-α antibodies (0.11%). However, the subject was not found to possess antibodies that neutralized TM-α activity in an There were several limitations in the present PMS sub-population analysis. First, this PMS study was an examination of a single arm with no comparison arms. Second, this PMS study was performed under daily clinical practice conditions, with restrictions on neither the treatment of underlying diseases nor the usage of other anticoagulants. However, this investigation offers new information about the effect and safety profile of TM-α for sepsis-induced DIC in daily clinical practice, and it collected prospective data and analyzed sepsis-induced DIC diagnosed according to the JAAM DIC criteria, which was not fully elucidated by the phase III clinical study. The findings of a recent retrospective cohort study revealed that TM-α administration improved sepsis-induced DIC diagnosed according to the JAAM DIC criteria . DespiteThe present findings suggest that TM-α is most effective for treating patients with sepsis-induced DIC when administered within the first 3 days after diagnosis. To verify the curative effect of TM-α on sepsis-induced DIC, a large-scale RCT is required.The JAAM DIC criteria are presented in Table 8.