(20). Disc diffusion, as per CLSI guidelines (14), and MIC, by the macrodilution method, were assessed. The isolates were also subjected to MIC testing for meropenem using the broth macrodilution technique. The organism was considered sensitive if the MIC was < 4 μg/mL and resistant if it was selleck inhibitor > 16 μg/mL according to

CLSI guidelines. The choice of meropenem was based on information from the clinicians in Mangalore that meropenem is the drug of choice in multidrug resistant Acinetobacter infections, rather than imipenem and ertapenem. Four CRA primers (21, Table 1) were initially tested for their specificity in RAPD-PCR. Of these, the primer CRA 22 was found to be most suitable as it generated polymorphic bands and the results obtained were reproducible. The banding patterns were compared using Gelcompar II software version 2.5 (Applied Maths, Sint-Martens-Latem, Belgium). The levels of similarities between different profiles were calculated using the Pearson coefficient correlation and clustered by the UPGMA algorithm. In this study, identification of A. baumannii was based both on

the basis of phenotypic tests and the on the presence of blaOXA-51 gene, which has been reported to be intrinsic to this species. Out of 62 Acinetobacter isolates included in this study, 48 were identified as A. baumannii and 14 as other Acinetobacter spp. (Table click here 2). Of the 48 A. baumannii, 15 were from the respiratory tract, 15 from skin and soft tissues, 11 from blood, 5 from urine and Phloretin 2 from other sources. Among the other Acinetobacter spp., the majority of the isolates (9/14) were from blood (Table 2). Multiplex PCR-based analysis of the isolates for the four major classes of carbapenemase genes (Fig. 1) revealed the presence of blaOXA-23-like genes in 27 isolates, of which 23 were A. baumannii and 4 comprised other Acinetobacter spp. (Table 2). Of the 20 isolates that were positive for blaOXA-24-like genes, 11 were A. baumannii

and 9 were other Acinetobacter spp. Only seven isolates had blaOXA-58-like genes, among these two were A. baumannii and five were other Acinetobacter spp. The prevalence of blaOXA-23-like genes in A. baumannii was 47.9% while in other Acinetobacter spp. it was 28.5%. On the other hand the prevalence of blaOXA-24-like genes in A. baumannii was only 22.9% and in other Acinetobacter spp. it was as high as 64.3%. A low prevalence of blaOXA-58-like genes (4.2%) was seen in A. baumannii, whereas for other Acinetobacter spp. it was 35.7%. Polymerase chain reaction for the presence of the insertional sequence ISAba1 using specific primers (Table 1) showed 33.3% (16/48) of A. baumannii isolates harbored this gene. None of the other Acinetobacter spp. were positive for this gene. The presence of ISAba1 in A. baumannii was detected only in the upstream region of blaOXA-23-like gene (Fig.

It will not become a grave menace to the poultry industry and human health. Several studies have tested using live E. coli as vaccines against colibacillosis (22, 23, 25). In almost all selleck screening library cases, live bacteria were delivered by spray, allowing stimulation of eye-, conjunctiva-, and bronchus-associated

lymphoid tissue. Use of fine sprays, which penetrate deep into the lower respiratory system, lungs, and air sacs, may result in a stronger immune response than coarse sprays, which do not penetrate as deeply into the respiratory system (43). In the current study, we observed that AESN1331 administered via fine spray colonized the avian respiratory tract, but disappeared within a few days. Administration of AESN1331 by fine spray, coarse spray, Selleck Navitoclax or eye drop induced equivalent protection against challenge with an APEC wild strain. These results show that the AESN1331 strain is attenuated and safe, yet immunogenic and extremely effective against avian colibacillosis. We also demonstrated that AESN1331 partially protected chickens that had been immunized as 19-day-old embryonated eggs. Our mutant provided protection without impairing hatching or chick survival, although a small number of the challenge strain was recovered from some in ovo-immunized chickens that had survived exposure to challenge. Given that the poultry industry is moving towards greater use of in ovo vaccination, administration

of the mutant via this route may be of value. We did not detect the

major virulence-associated genes in AESN1331, as is true for J29. AESN1331 remained susceptible to all tested antibiotics except for nalidixic acid. These properties are appropriate for a live vaccine candidate, since field usage of such a mutant would not spread virulence-associated or drug resistance-encoding genes to wild APEC. Emergence of drug resistance (4, 5, 8–12) and costs associated with administration of drugs have led to increased medical costs worldwide. Ozawa et al. (11) reported APEC isolates in Japan have moderate- or high-level check details resistance to many antimicrobials, including fluoroquinolones. Antimicrobial susceptibility is critical for the adequate treatment of colibacillosis. AESN1331 is resistant only to nalidixic acid. This resistance resulted from the construction of AESN1331; it was introduced by the amino acid change at position 87 (Asp to Gly) on the gyrA gene of chromosomal DNA. Unlike quinolone resistance caused by qnr plasmid, this resistance will not disseminate to APEC wild strains and other Enterobacteriaceae. Resistance to nalidixic acid, a drug that is not commonly used to treat colibacillosis in the poultry industry, is not a serious obstacle to the treatment, elimination and prevention of colibacillosis. Administration of the AESN1331 strain via various routes evoked an effective immune response that protected against a virulent, wild-type E. coli O78 APEC isolate.

Parasite burdens were determined at wk 3 (Fig. 4B), and wk 6 and 13 (data not shown). As expected from the lesion data, parasite loads were higher in Lm/CpG-vaccinated IL-17R−/− mice if compared with WT at the early time point. No differences in parasite burdens between the two groups were observed at later time points (data not shown). The analysis of the find more dermal site during the “silent” phase (2 wk) revealed, as expected, that the frequency of CD4+Th17 cells is elevated in the ears of Lm/CpG-vaccinated

WT animals. In contrast, the frequency of these cells was decreased fourfold in IL-17R−/− mice vaccinated with the same vaccine (Fig. 5A). The same trend was observed for IFN-γ expression, with an even more dramatic decrease in frequency of Th1 cells in the IL-17R−/− mice (tenfold). The absolute number of IL-17+ and IFN-γ+ T cells is shown in Fig. 5B, and confirms that Th1 cells are the most decreased population in the deficient mice. The frequency of Treg was increased in Seliciclib purchase IL-17R−/− mice (Supporting Information Fig. 3). A decrease in effector numbers concomitant to an increase in Treg could explain the elevated parasite burdens in the ears of IL-17R−/− mice 2 wk post vaccination. Because IL-17 has been reported to contribute to inflammatory immune response by recruiting neutrophils, which are implicated in the control of leishmaniasis, we wanted to determine

whether decreased frequencies of Th17 cells would result in differences in neutrophil accumulation in the vaccination site. To determine this, we quantified the relative percentage of different cell populations in cytospin preparations generated from the vaccinated ears at wk 2. Figure 5C confirmed that neutrophils frequencies were significantly increased in WT mice vaccinated with Lm/CpG and

decreased in the IL-17R−/− animals. The relative frequencies of mast cells and melanocytes were significantly decreased in the Lm/CpG-vaccinated WT mice, probably a reflexion of the relative higher numbers of neutrophils in the skin of these animals. We also did not detect infected neutrophils, and very few infected macrophages (5%), in the cytospin preparations from Lm/CpG-vaccinated mice. Infected cells were more prominent (5% neutrophils, 21% macrophages) in L. major-infected mice (data not shown). This suggests that the phagocytic ability of these cell types was enhanced Tangeritin by Lm/CpG vaccination. Cytokine levels were also determined in the draining lymph nodes of WT and IL-17R−/− mice at wk 2 post inoculation. IL-6 and TGF-β production, which in conjunction causes Th17 development, was significantly increased in WT mice vaccinated with Lm/CpG (p=0.0001 and p=0.001, Fig. 6A and B), but not in IL-17R−/− animals. IL-17 was only detected in WT mice vaccinated with Lm/CpG (Fig. 6C). IL-12 was secreted by lymph node cells of all mice vaccinated with Lm/CpG (Fig. 6D). Interestingly, IFN-γ could not be detected in IL-17R−/− immunized with this vaccine (Fig. 6F).

To clarify further the background of the differential activation of PKCα in macrophages of susceptible and resistant mouse strains, it will be crucial to analyse the precise binding site of LPG to PKCα. It is noteworthy that Pritelivir in vivo the inhibition of PKCα by Gö6976 is achieved through binding of the

inhibitor to the C3 domain of PKCα, thereby achieving the same degree of inhibition in both mouse strains (28). Even though we found that the modulation of PKCα by LPG affects parasite survival through the modulation of oxidative burst, it is possible that this is not the only mechanism affecting parasite survival, as this enzyme also affects other macrophage effector functions. To the best of our knowledge, this is the first comparative report that shows a differential modulation of PKCα by L. mexicana and by the parasite LPG in macrophages of susceptible BALB/c and the more resistant C57BL/6 mice, which correlates with the oxidative burst and with parasite survival. To date, it is not clear if the different activation of PKCα by LPG in both mouse strains is possibly related to different binding domains or possibly to other mechanisms such as polymorphisms PD98059 or SNPs in the genes

associated with the signalling pathway of this enzyme. It will be interesting to analyse the response of PKCα to L. mexicana LPG in macrophages of patients with DCL. In addition, it remains to be determined whether the inhibition that LPG exerts on the enzymatic activity of PKCα in macrophages is solely responsible for the susceptibility of BALB/c mice towards infection with L. mexicana. José Delgado-Domínguez was a recipient of a CONACyT scholarship for the Posgrado en Ciencias Biológicas. We thank Marco Gudiño Zayas, Omar Agni García, Augusto González and Daniel Sánchez Almaraz for technical assistance, and Lucía Álvarez Trejo for excellent secretarial support. This work was supported by CONACyT: 45052-M, CONACyT: 102155 and DGAPA: IN221806-3 and IN220109. “
“Airway infections are known to cause exacerbations of allergy and asthma. Tonsils constitute a primary site for microbial recognition and triggering of the immune system in

the airways. Human β-defensins (HBDs) are Orotidine 5′-phosphate decarboxylase antimicrobial peptides with an important role in this defense. Our aim was to investigate HBD1-3 in tonsillar tissue and their potential role in allergic rhinitis (AR). Tonsils, obtained from patients with AR and non-allergic controls, and isolated tonsillar CD4+, CD8+ and CD19+ lymphocytes were analyzed for HBD1-3 expression using real-time RT-PCR and/or immunohistochemistry. Tonsillar tissue, mixed tonsillar lymphocytes and airway epithelial cells (AECs) were cultured with or without IL-4, IL-5, IL-13 or histamine followed by measurements of HBD1-3 release using ELISA. HBD1-3 were present in tonsillar tissue, including epithelial, CD4+, CD8+ and CD19+ cells. The expression was reduced in allergic compared to healthy tonsils.

Univariate analysis, which consisted of chi-squared or Fisher’s exact test for categorical independent variables and

logistic regression for continuous independent variables, was used to identify factors present at the time of initial clinical Y-27632 chemical structure presentation associated with 28-day crude mortality. A multivariable Cox Proportional Hazards Regression model was built in a forward stepwise fashion using biologically plausible variables identified by univariate analysis (P < 0.1) accounting for potential confounders. Continuous variables were analysed continuously or categorically using cut-off threshold values identified by classification and regression tree (CART) partitioning. Variables retained in the multivariate model were then assigned a weighted score based on the adjusted hazard ratios rounded to the nearest whole number from the regression model, which was then added to the baseline APACHE II score calculated at the time of PM diagnosis. Receiver–operator curves (ROC) were used to analyse the ability of the risk score https://www.selleckchem.com/products/PF-2341066.html to differentiate non-survivors from surviving patients

at 28 days, and assign a breakpoint score associated with high risk of early death. Antifungal and other treatment variables occurring after diagnosis were not included in the development of the model. Time to death following the initial clinical signs of PM were then compared in patients with low- vs. high-risk scores using Kaplan–Meier curves, and mortality rates were compared among groups using the log-rank test. All analysis was performed with spss version 20 (IBM, Armonck, NY) and Medcalc Software Packages (Ostend, Belgium). We identified 75 patients with PM over the 12-year study period (13 proven/62 probable) (Table 1). The male : female ratio was 2 : 1 (50 males and 25 female patients). The median Amino acid age at diagnosis was 57 years (range, 16–76 years). The vast majority of the

patients were Caucasians (81%). Thirty patients (40%) had a diagnosis of AML or myelodysplastic syndrome. Forty-three patients (57%) had active haematological malignancy at the time of diagnosis. Moreover, 36 patients (48%) were HSCT recipients. Of these, 29 (81%) received allogeneic stem cell transplants and 19 (66%) patients had developed severe GvHD. A history of diabetes mellitus and a serum glucose level higher than 200 mg dl−1 were present in 23 (31%) and 25 (34%) patients at the time of diagnosis respectively. Neutropenia and lymphopenia were present at diagnosis in 43 (57%) and 48 (64%) patients, respectively, whereas monocytopenia was present in 39 (52%) of the study cohort. Among patients with neutropenia, 28 (65%) had an ANC count less than 100 mm−3. The median duration of neutropenia before diagnosis was 10 days (range, 1–100 days). Only 18 patients (24%) recovered from neutropenia during the infection course. In addition, among patients with lymphopenia, 34 (71%) had severe lymphopenia.

A and A/J mice infected with Pb, mainly at the later phase of infection with Pb18. Tissue necrosis was also observed in association with fibrotic lesions, only in resistant mice infected with Pb18 and in both mouse strains infected with Pb265, see more leading to resolution of the infection. In this study, lymphomononuclear cells showed intense IFN-γ staining, distributed at the periphery of necrotic lesions

of resistant mice. Some authors have described the role of osteopontin (OPN) in the preferential activation of cellular immunity, increasing the cytokine expression of IL-12 and inhibiting IL-10, thus, leading to the immune response toward Th1 immune pattern, which is important to resistance for infection (Ashkar et al., 2000). Li

et al. (2003) demonstrated that IFN-γ stimulates the OPN expression, which in turn increases the IFN-γ production, suggesting a mechanism of positive regulation to development of Th1 immune response. Increased OPN expression, particularly observed in macrophages, was detected in the granulomatous lesions of mice, as previously described (Nishikaku et al., 2008). Although B10.A mice have shown increased cellular positivity of OPN and IFN-γ at the later stage of infection with Pb18, no association with the control of the infection was found. Decreased OPN immunostaining was detected in Pb265-infected mice in comparison with Pb18-infected mice. Furthermore, OPN reactivity decreased Temozolomide ic50 throughout the infection with Pb265, as observed for IFN-γ, probably due to resolution of the infection. Hence, presence of OPN and IFN-γ at paracoccidioidal granulomas suggests that both these components might be part of mechanisms to control the P. brasiliensis infection, particularly in resistant mice. Altogether, our data suggest the active participation Hydroxychloroquine price of IFN-γ, TNF-α, TGF-β, OPN, and of immune cell populations (macrophage, giant cells, and lymphocytes) in the tissue alterations observed during the development of granulomatous response and also in the immune effector

mechanisms against P. brasiliensis infection. This study characterized the presence of IFN-γ in lymphomononuclear cells indicating its participation in the tissue response developed during the experimental infection with P. brasiliensis. The findings suggest that intense expression of IFN-γ in lymphomononuclear cells of susceptible and resistant mice infected with the highly virulent Pb18, points toward a similar activation of cellular immune response in the early phase of the infection in these animals and to an absence of correlation between resistance and genetic background of the host at this time point of infection; however, the expressive increase of IFN-γ positive cells in resistant mice at the later time point, suggests that at this stage, this mouse strain has a more efficient capacity to activate phagocytes, mainly macrophages, to control the fungal dissemination.

However, in contrast to the increasing prevalence of diabetes and early stages of DKD, recent trends in the incidence selleck chemical of DM-ESKD suggest that better management in the earlier

stages of DKD has been successful in slowing rates of disease progression. Simultaneous improvements in use of renin–angiotensin inhibitors and improved glycaemic and blood pressure control are likely to be largely responsible for this trend. Primary prevention, maximizing early detection of DKD and optimal management of diabetes and kidney disease hold great potential to attenuate the future health burden attributable to DKD in Australia. Diabetes-related kidney disease (DKD) may be defined as the presence of persistent albuminuria, proteinuria and/or estimated glomerular filtration rates (eGFR) <60 mL/min per 1.73 m2 in a person with diabetes. As is the case in the non-diabetic population, both albuminuria and reduced eGFR are independently associated Vadimezan with increased risk of premature cardiovascular and all-cause mortality, and risk of progression to end-stage kidney disease (ESKD). The magnitude of this risk is proportional to the magnitude of the abnormality for both parameters, and is significantly greater in those with diabetes compared with those without.[1] Based on data from the United Kingdom Prospective Diabetes Study (UKPDS), conducted between 1977 and 1997, one quarter of the population with type 2

diabetes (T2DM) will develop albuminuria within 10 years of diabetes diagnosis.[2] This is consistent with earlier studies of the development of DKD in T1DM patients, showing onset at approximately 5–10 years post-diagnosis and peaking at 10–19 years diabetes duration.[3, 4] Younger age at diagnosis increases the probability of developing DKD over the life course, whereas the risk of reaching ESKD for those diagnosed with diabetes later in life may be relatively low.[2] Over the past two decades, increasing diabetes prevalence in Australia has produced a commensurate increase in the number of adults

with DKD and diabetes-related Urease ESKD (DM-ESKD). Here we review the current and the potential future burden of DKD and DM-ESKD in Australia, taking into account evolving practices in diabetes management and incidence trends in other high-income countries. The baseline AusDiab Study conducted in 1999/2000 found that among Australian adults (25 years and older) with diabetes, 27% had evidence of DKD (Table 1). These data suggest that approximately a quarter of a million Australians have DKD, and because of this are at high risk of progression to DM-ESKD, cardiovascular events and premature death. By comparison, the prevalence of DKD in the United States diabetic population was 40%, according to the results of the 2005–2010 NHANES survey.[5] Based on AusDiab data, the vast majority (94%) of the adult DKD population exhibited albuminuria, either alone or in combination with a low estimated eGFR.

Furthermore, BbGL-IIc induced iNKT cell activation occurs independently of MyD88 and TRIF signaling (49). These results show that BbGL-IIc is a bacterial antigen for the mouse iNKT cell TCR. BbGL-II compounds also stimulate human iNKT cells to release cytokines. Interestingly, BbGL-IIf, which contains linoleic acid (C18:2) in the sn-1 position and oleic acid in the sn-2 position, has been found to selleck products be the most potent

antigen for human iNKT cells (49). Data from another study suggest that the different iNKT cell responses to Borrelia glycolipids are due to a difference between human and mouse CD1d molecules (51). These studies show that iNKT cell TCR detects DAGs, another category of glycolipid, in addition to glycosphingolipids.

Moreover, DAG antigen induced iNKT cell activation is dependent on acyl chain length and saturation (49). The TCR of iNKT cells recognizes Sphingomonas GSL and B. burgdorferi DAG as well as αGalCer. Although the structures of these bacterial antigens are similar to that of αGalCer (Fig. Pembrolizumab supplier 5), there are several small structural differences. DAG belongs to a different category of glycolipid than do αGalCer and Sphingomonas GSL. Also, the bacterial antigens are less potent than αGalCer. What determines the antigenic potency of these glycolipids? To address this point, crystal structures of mouse CD1d in complex with Sphingomonas GalAGSL or B. burgdorferi DAG were determined (51, 52). GalAGSL binds to mouse CD1d similarly to αGalCer. Between the α1 and α2 helices, the CD1d molecule has two pockets (A′ and F′) which accommodate Org 27569 the lipid tails of antigens (Fig. 6a, b) (6, 7). The fatty acid and sphinganine tails of GalAGSL extend into the A′ and F′ pockets, respectively (52). However, because of an alternative hydrogen-bonding interaction, the sphinganine tail of GalAGSL, which lacks 4-OH, is more deeply inserted into the F′ pocket (52). The sugar head group of GalAGSL is present in the center of the binding groove at

the CD1d surface where an incoming TCR recognizes antigens (Fig. 6b, c), but it shows a slight lateral shift compared to αGalCer (52). These differences are thought to cause the difference in antigenic potency between Sphingomonas GalAGSL and αGalCer. The binding of B. burgdorferi galactosyl DAG is more flexible than that of Sphingomonas GalAGSL or αGalCer. The sn-1 linked oleic acid and the sn-2 linked palmitic acid of BbGL-IIc are inserted into the A′ and F′ pockets, respectively (51). The glycerol moiety of BbGL-IIc is tilted toward the α1 helix of the CD1d molecule, and the galactose of BbGL-IIc is pointed upward and away from the α2 helix of CD1d. These differences result in the loss of important hydrogen bonding interactions with the amino acids in the α2 helix that are present in the case of αGalCer (51).

Efforts of several research groups have been combined to identify the clinical[18-20] and molecular[21-24] selleck chemicals llc parameters that are associated with an insufficient

clinical response to RTX treatment. Our group has recently found a positive association between the presence of Epstein–Barr virus (EBV) genome in the BM of patients with RA and clinical response to RTX treatment.[25] Interestingly, RTX treatment was followed by complete clearance of EBV from the BM. The ability to respond to interferon stimulation, an essential mechanism of human anti-viral defence, may potentially predict clinical effect of RTX in patients with RA.[26, 27] Infection with EBV is one of the environmental risk factors for the development of RA.[28] The EBV glycoprotein gp110 contains a sequence identical to the motif of the HLA-DRB1 alleles within the MHC II complex; called ‘shared epitope’, it is the strongest known genetic factor for the development of RA.[29-31] Also, EBV infection in carriers of shared epitope greatly enhanced the development of RA.[30] Consequently, a compromised innate immune response towards Decitabine in vitro EBV and poor viral clearance are attributed

to RA patients and lead to a high load of EBV-infected cells in the circulating blood and in the synovial cells, impaired cytolytic activity of T cells to EBV proteins and high titres of anti-EBV antibodies compared with healthy subjects.[32-37] B cells are currently considered critical for the primary EBV infection and for its persistence. Epstein–Barr virus activates B cells and induces their proliferation and transformation into antibody-secreting cells.[38] It has the ability to infect almost all types of B cells in vivo but naive IgM+ IgD+ B cells are the major

target in tonsils, while the latent infection is found in the memory B-cell pool.[39-41] The naive B-cell subset seems to be the cell population that shares susceptibility to RTX and EBV, so we attempted to outline phenotypic and functional changes in the peripheral blood and bone marrow B cells of patients with RA following RTX buy Palbociclib treatment and during EBV infection. Samples of BM and PB were collected from 35 patients with established RA, diagnosed according to the ACR 1987 criteria[42] before B-cell depletion therapy with anti-CD20 antibodies.[13] All patients were recruited from the Rheumatology Clinic at Sahlgrenska University Hospital, Göteborg, Sweden, during the period from January 2007 to September 2008, and all patients gave written informed consent to participate. Additionally, 18 patients with RA donated PB samples for functional analysis. Another 10 patients with RA also donated PB and synovial fluids for phenotypic B-cell analysis. All patients with RA were receiving methotrexate treatment and had not been treated with RTX previously. Clinical and demographic characteristics of the patients and their immunosuppressive treatment are presented in Table 1.

0) for 40 min, followed by a blocking step for 1 h (Roti-Immuno Block, dilution 1:10; Roth, Karlsruhe, Germany). Primary anti-human FUBP1 antibody was incubated overnight at 4°C and subsequently labelled with a secondary antibody for 1 h at room temperature (RT) (dilution 1:500; Alexa Fluor568, donkey anti-goat IgG, Invitrogen, Darmstadt, Germany). Next, the primary antibodies for the double staining (as listed above) were added and incubated for 1 h at RT and specimens

were then labelled with an additional secondary antibody for 1 h at RT (dilution 1:500; Alexa Fluor488, goat anti-mouse IgG, Alexa Fluor488, goat anti-rabbit IgG, Invitrogen). Nuclear staining was performed with DAPI (dilution 1:1000, Invitrogen). The images were analysed and recorded on a Nikon Eclipse 80i fluorescence microscope (Nikon,

Düsseldorf, Germany). Digital images were then adjusted Daporinad research buy selleckchem with NIS elements imaging software (Nikon). Thirty samples were analysed by fluorescence in situ hybridization (FISH) to assess for 1p and 19q deletions. The two-colour FISH assay was performed on 3-μm-thick sections using a mixed 1p36/1q25 dual colour probe and 19p13/19q13 dual colour probe set (ZytoLight SPEC, Cat. No. Z-2075 and Z-2076, Zyto-Vision, Bremerhaven, Germany). The Histology Accessory FISH Kit (Dako) was used for slide pretreatment, probe hybridization and post-hybridization processing. Nuclei were counterstained with DAPI/Antifade-Solution (Zyto-Vision). Fluorescent signals were LY294002 analysed using an Olympus BX50 fluorescent microscope with the appropriate filters (Olympus, Hamburg, Germany). Samples displaying sufficient FISH efficiency (80% fluorescent nuclei) were evaluated. Signals were scored in at least 100 non-overlapping, intact nuclei. Deletions of 1p or 19q were defined by samples with over 50% of the tumour nuclei containing only one signal. The FUBP1 gene (ENSG00000162613; ENST00000370767) was analysed for mutations in 15 tumour samples using the primers shown in Table 1. All exons listed were amplified using GoTaq polymerase (Promega, Mannheim, Germany). PCR products were treated with

ExoSAP (ExoSAP-IT, GE Healthcare, Pittsburgh, PA, USA) and sequenced in both directions using an ABI PRISM 3100 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Additionally, the number of FUBP1 mutated gliomas was increased by samples deriving from a previously studied cohort [4]. A semiquantitative score was used for the analysis of FUBP1 protein expression. The immunohistochemical staining intensity value (no staining = 0, low = 1, moderate = 2, strong = 3) was multiplied with the assigned value for the proportion of positive tumour or vascular cells (0–1% = 0, 1–10% = 1, 10–25% = 2, 25–50% = 3, > 50% = 4). MIB-1-positive cells were analysed as a percentage of all cells within the tumour and then plotted against FUBP1 expression scores followed by the Wilcoxon rank sum test.