Radiotherapy and temozolomide for newly diagnosed glioblastoma: recursive partitioning analysis of the EORTC 26981/22981-NCIC CE3 phase III randomized trial

PURPOSE: The European Organisation for Research and Treatment of Cancer and National Cancer Institute of Canada trial on temozolomide (TMZ) and radiotherapy (RT) in glioblastoma (GBM) has demonstrated that the combination of TMZ and RT conferred a significant and meaningful survival advantage compared with RT alone. We evaluated in this trial whether the recursive partitioning analysis (RPA) retains its overall prognostic value and what the benefit of the combined modality is in each RPA class. PATIENTS AND METHODS: Five hundred seventy-three patients with newly diagnosed GBM were randomly assigned to standard postoperative RT or to the same RT with concomitant TMZ followed by adjuvant TMZ. The primary end point was overall survival. The European Organisation for Research and Treatment of Cancer RPA used accounts for age, WHO performance status, extent of surgery, and the Mini-Mental Status Examination. RESULTS: Overall survival was statistically different among RPA classes III, IV, and V, with median survival times of 17, 15, and 10 months, respectively, and 2-year survival rates of 32%, 19%, and 11%, respectively (P < .0001). Survival with combined TMZ/RT was higher in RPA class III, with 21 months median survival time and a 43% 2-year survival rate, versus 15 months and 20% for RT alone (P = .006). In RPA class IV, the survival advantage remained significant, with median survival times of 16 v 13 months, respectively, and 2-year survival rates of 28% v 11%, respectively (P = .0001). In RPA class V, however, the survival advantage of RT/TMZ was of borderline significance (P = .054). CONCLUSION: RPA retains its prognostic significance overall as well as in patients receiving RT with or without TMZ for newly diagnosed GBM, particularly in classes III and IV.

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

Analysis of plasma tocopherols alpha, gamma, and 5-nitro-gamma in rats with inflammation by HPLC coulometric detection

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that gamma-tocopherol (gammaT), the major form of vitamin E in the North American diet, may play an important role in these diseases. GammaT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-gammaT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, alphaT, and gammaT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (approximately 10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing approximately 4% of gammaT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to gammaT or corrected for total neutral lipids, while alpha- and gammaT levels remained unchanged. These results demonstrate that nitration of gammaT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of gammaT.

Prevention of brain injury by the nonbacteriolytic antibiotic daptomycin in experimental pneumococcal meningitis

Bacteriolytic antibiotics cause the release of bacterial components that augment the host inflammatory response, which in turn contributes to the pathophysiology of brain injury in bacterial meningitis. In the present study, antibiotic therapy with nonbacteriolytic daptomycin was compared with that of bacteriolytic ceftriaxone in experimental pneumococcal meningitis, and the treatments were evaluated for their effects on inflammation and brain injury. Eleven-day-old rats were injected intracisternally with 1.3 x 10(4) +/- 0.5 x 10(4) CFU of Streptococcus pneumoniae serotype 3 and randomized to therapy with ceftriaxone (100 mg/kg of body weight subcutaneously [s.c.]; n = 55) or daptomycin (50 mg/kg s.c.; n = 56) starting at 18 h after infection. The cerebrospinal fluid (CSF) was assessed for bacterial counts, matrix metalloproteinase-9 levels, and tumor necrosis factor alpha levels at different time intervals after infection. Cortical brain damage was evaluated at 40 h after infection. Daptomycin cleared the bacteria more efficiently from the CSF than ceftriaxone within 2 h after the initiation of therapy (log(10) 3.6 +/- 1.0 and log(10) 6.3 +/- 1.4 CFU/ml, respectively; P < 0.02); reduced the inflammatory host reaction, as assessed by the matrix metalloproteinase-9 concentration in CSF 40 h after infection (P < 0.005); and prevented the development of cortical injury (cortical injury present in 0/30 and 7/28 animals, respectively; P < 0.004). Compared to ceftriaxone, daptomycin cleared the bacteria from the CSF more rapidly and caused less CSF inflammation. This combined effect provides an explanation for the observation that daptomycin prevented the development of cortical brain injury in experimental pneumococcal meningitis. Further research is needed to investigate whether nonbacteriolytic antibiotic therapy with daptomycin represents an advantageous alternative over current bacteriolytic antibiotic therapies for the treatment of pneumococcal meningitis.

Standardization of infusion solutions to reduce the risk of incompatibility

PURPOSE: Although critically ill patients usually have various central intravenous (i.v.) lines, numerous drugs have to be infused simultaneously through the same lines. This can result in potentially harmful in-line incompatibility that can cause decreased drug effectiveness or increased microparticle load. To minimize the risk of these medication errors at an anesthesia intensive care unit (ICU), the preparation and administration of continuously infused drugs were standardized and the practicability in daily clinical routine was evaluated. SUMMARY: The concentration and diluent of continuously administered i.v. drugs were standardized. The drugs were grouped according to pH, medical indication, and chemical structure. The ICU staff decided to use multilumen central venous catheters, and each group of drugs was assigned to one lumen. Only drugs that belonged to the same group were infused simultaneously through the same lumen; therefore, intragroup incompatibilities had to be excluded before establishing the new drug administration plan at the ICU. The visual compatibility of 115 clinically reasonable intragroup drug mixtures was investigated. All drug combinations were compatible for six hours except mixtures containing thiopental, which was reassigned to a single-line use. In the following year, the practicability of this drug administration plan was evaluated. No deviations were found in the compliance of the staff prescribing and preparing only standardized concentrations and diluents. Further research to investigate the chemical compatibility of the drugs in these multiple mixtures will follow. CONCLUSION: A project intended to avoid incompatibility among i.v. drugs infused in the intensive care setting included steps to standardize solutions and determine which could be given together.

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.

Metabolomics reveals a novel vitamin E metabolite and attenuated vitamin E metabolism upon PXR activation

Pregnane X receptor (PXR) is an important nuclear receptor xenosensor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. In this study, ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS), revealed altered urinary metabolomes in both Pxr-null and wild-type mice treated with the mouse PXR activator pregnenolone 16alpha-carbonitrile (PCN). Multivariate data analysis revealed that PCN significantly attenuated the urinary vitamin E metabolite alpha-carboxyethyl hydroxychroman (CEHC) glucuronide together with a novel metabolite in wild-type but not Pxr-null mice. Deconjugation experiments with beta-glucuronidase and beta-glucosidase suggested that the novel urinary metabolite was gamma-CEHC beta-D-glucoside (Glc). The identity of gamma-CEHC Glc was confirmed by chemical synthesis and by comparing tandem mass fragmentation of the urinary metabolite with the authentic standard. The lower urinary CEHC was likely due to PXR-mediated repression of hepatic sterol carrier protein 2 involved in peroxisomal beta-oxidation of branched-chain fatty acids (BCFA). Using a combination of metabolomic analysis and a genetically modified mouse model, this study revealed that activation of PXR results in attenuated levels of the two vitamin E conjugates, and identification of a novel vitamin E metabolite, gamma-CEHC Glc. Activation of PXR results in attenuated levels of the two vitamin E conjugates that may be useful as biomarkers of PXR activation.

Infection of primary canine duodenal epithelial cell cultures with Neospora caninum

According to current knowledge, sexual development of the apicomplexan parasite Neospora caninum takes place in the canine intestine. However, to date there is no information on the interaction between the parasite and the canine intestinal epithelium, and, next to the clinical and in vivo research tools, an in vitro model comprised of canine intestinal cells infected with N. caninum would be very helpful for investigations at the cellular level. Following the isolation of cells of neonatal canine duodenum and growth of cell cultures to monolayers for 5-6 days, canine intestinal epithelial cells were exposed to cell culture-derived N. caninum tachyzoites and bradyzoites. The host cells remained viable during in vitro culture for an average of 2 wk. During this time span, N. caninum was found to readily adhere to any surface area of these cells, but infection took mostly place at sites where microvilli-like structures were missing, e.g., at the cell periphery, with tachyzoites exhibiting at least 3-4 times increased invasive capacities compared to bradyzoites. Once intracellular, parasites resided within a parasitophorous vacuole, moved toward the vicinity of the nucleus and the more distal portion of the epithelial cells, and proliferated to form vacuoles of not more than 2-4 parasites, which were surrounded by numerous mitochondria. Immunofluorescence staining and TEM of infected cells showed that the expression of cytokeratins and the structural integrity of desmosomes and tight junctions were not notably altered during infection. Furthermore, no changes could be detected in the alkaline phosphatase activities in cell culture supernatants of infected and noninfected cells. Canine duodenal epithelial cell cultures represent a useful tool for future studies on the characteristics of the intestinal phases of N. caninum infection.

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects.

1-deamino-8-d-arginine vasopressin (desmopressin [DDAVP]) is clinically efficacious in patients with mild platelet function disorders but it is not known which mechanisms mediate this effect. Our aim was to evaluate the impact of in vivo DDAVP administration in these patients. We assessed von Willebrand factor (VWF), factor VIII, platelet activation and aggregation, platelet-dependent thrombin generation, and platelet intracellular Na(+)/Ca(2+) fluxes, before and 2 and 4 hours after DDAVP (0.3 µg/kg). We found (1) no significant changes for P-selectin expression, PAC-1 binding, δ-granule content and secretion, and platelet-aggregation; (2) significant decreases of secretion of α-granules and GPIIb-IIIa activation induced by adenosine 5'-diphosphate, convulxin, and thrombin; (3) significant increases of procoagulant platelets induced by convulxin/thrombin and platelet-dependent thrombin generation; and (4) significant increases of intracellular Na(+)/Ca(2+) concentrations. We show that in vivo DDAVP selectively and markedly enhances the ability to form procoagulant platelets and increases platelet-dependent thrombin generation by enhancing Na(+)/Ca(2+) mobilization. This report indicates that the beneficial hemostatic effect of DDAVP is not limited to an increase in large VWF multimers. An enhancement of platelet procoagulant activity appears to be an additional and (at least in platelet disorders) -possibly clinically relevant mechanism of DDAVP's action.

Glomerular and renal vascular structural changes in alpha8 integrin-deficient mice

Integrins are matrix receptors that regulate cell-matrix interactions during development and in adult tissue. In the adult kidney, the alpha8 chain is specifically expressed in glomerular mesangial cells and vascular smooth muscle cells. alpha8-deficient (alpha8-/-) mice demonstrate reductions in renal mass, which can range from complete renal agenesis to the development of kidneys that are only slightly smaller than wild-type kidneys. No histologic abnormalities of these kidneys have been described. However, considering the prominent expression of alpha8 in glomeruli and renal vessels, it seemed unlikely that the kidneys of alpha8-/- mice would be completely normal. Therefore, the renal phenotype of adult alpha8-/- mice was investigated, for assessment of more subtle morphologic alterations in kidney tissue. alpha8-/- mice displayed a significant reduction in nephron number and an increase in glomerular volume, compared with wild-type control animals. Albuminuria was not different in wild-type and alpha8-/- mice. Quantitative morphologic analyses revealed that the glomeruli of alpha8-/- mice were hypercellular, with an increased number of mesangial cells, compared with wild-type mice. Mesangial matrix deposition (as demonstrated for collagen IV and the alpha8 ligand fibronectin) was expanded in alpha8-/- mice, compared with wild-type mice. Collagens I and III, which are not normally present in glomeruli, were detected in the glomeruli of alpha8-/- mice. Staining for other glomerular integrins demonstrated an increased abundance of the collagen receptor alpha2 integrin in alpha8-/- mice. The glomerular capillary length density was significantly greater in alpha8-/- mice than in wild-type mice. Cortical arterial vessel walls were not altered in alpha8-/- mice, but the capillaries of the peritubular network were widened. Despite the strong mesangial and vascular expression of alpha8, glomerular and renal vascular alterations in alpha8-/- mice were relatively mild. Only aged alpha8-/- mice demonstrated increased glomerular capillary widening, compared with control animals. The results suggest that the lack of alpha8 can be largely compensated for, at least in younger alpha8-/- mice. It is not yet clear whether the occurrence of collagens that are not normally present in glomeruli and the increased abundance of the collagen receptor alpha2 contribute to maintaining the glomerular structure in alpha8-/- mice. The compensatory mechanisms involved will be the subject of future research.

Prognostic factors for advanced-stage human immunodeficiency virus-associated classical Hodgkin lymphoma treated with doxorubicin, bleomycin, vinblastine, and dacarbazine plus combined antiretroviral therapy: A multi-institutional retrospective study.

BACKGROUND The treatment and outcomes of patients with human immunodeficiency virus (HIV)-associated Hodgkin lymphoma (HL) continue to evolve. The International Prognostic Score (IPS) is used to predict the survival of patients with advanced-stage HL, but it has not been validated in patients with HIV infection. METHODS This was a multi-institutional, retrospective study of 229 patients with HIV-associated, advanced-stage, classical HL who received doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) plus combination antiretroviral therapy. Their clinical characteristics were presented descriptively, and multivariate analyses were performed to identify the factors that were predictive of response and prognostic of progression-free survival (PFS) and overall survival (OS). RESULTS The overall and complete response rates to ABVD in patients with HIV-associated HL were 91% and 83%, respectively. After a median follow-up of 5 years, the 5-year PFS and OS rates were 69% and 78%, respectively. In multivariate analyses, there was a trend toward an IPS score >3 as an adverse factor for PFS (hazard ratio [HR], 1.49; P=.15) and OS (HR, 1.84; P=.06). A cluster of differentiation 4 (CD4)-positive (T-helper) cell count <200 cells/μL was associated independently with both PFS (HR, 2.60; P=.002) and OS (HR, 2.04; P=.04). The CD4-positive cell count was associated with an increased incidence of death from other causes (HR, 2.64; P=.04) but not with death from HL-related causes (HR, 1.55; P=.32). CONCLUSIONS The current results indicate excellent response and survival rates in patients with HIV-associated, advanced-stage, classical HL who receive ABVD and combination antiretroviral therapy as well as the prognostic value of the CD4-positive cell count at the time of lymphoma diagnosis for PFS and OS. Cancer 2014. © 2014 American Cancer Society.

Neurotropic astrovirus in cattle with nonsuppurative encephalitis in Europe

Encephalitis is a frequently diagnosed condition in cattle with neurological diseases. Many affected animals present with a nonsuppurative inflammatory reaction pattern in the brain. While this pattern supports a viral etiology, the causative pathogen remains unknown in a large proportion of cases. Using viral metagenomics, we identified an astrovirus (bovine astrovirus [BoAstV]-CH13) in the brain of a cow with nonsuppurative encephalitis. Additionally, BoAstV RNA was detected with reverse transcription-PCR and in situ hybridization in about one fourth (5/22 animals) of cattle with nonsuppurative encephalitis of unknown etiology. Viral RNA was found primarily in neurons and at the site of pathology. These findings support the notion that BoAstV infection is a common cause of encephalitis in cattle. Phylogenetically, BoAstV-CH13 was closely related to rare astrovirus isolates from encephalitis cases in animals and a human patient. Future research needs to be directed toward the pathogenic mechanisms, epidemiology, and potential cross-species transmission of these neurotropic astroviruses.

Rapid and accurate identification of Escherichia coli K-12 strains

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.