Identification of prognostic molecular features in the reactive stroma of human breast and prostate cancer.

Primary tumor growth induces host tissue responses that are believed to support and promote tumor progression. Identification of the molecular characteristics of the tumor microenvironment and elucidation of its crosstalk with tumor cells may therefore be crucial for improving our understanding of the processes implicated in cancer progression, identifying potential therapeutic targets, and uncovering stromal gene expression signatures that may predict clinical outcome. A key issue to resolve, therefore, is whether the stromal response to tumor growth is largely a generic phenomenon, irrespective of the tumor type or whether the response reflects tumor-specific properties. To address similarity or distinction of stromal gene expression changes during cancer progression, oligonucleotide-based Affymetrix microarray technology was used to compare the transcriptomes of laser-microdissected stromal cells derived from invasive human breast and prostate carcinoma. Invasive breast and prostate cancer-associated stroma was observed to display distinct transcriptomes, with a limited number of shared genes. Interestingly, both breast and prostate tumor-specific dysregulated stromal genes were observed to cluster breast and prostate cancer patients, respectively, into two distinct groups with statistically different clinical outcomes. By contrast, a gene signature that was common to the reactive stroma of both tumor types did not have survival predictive value. Univariate Cox analysis identified genes whose expression level was most strongly associated with patient survival. Taken together, these observations suggest that the tumor microenvironment displays distinct features according to the tumor type that provides survival-predictive value.

Clinical evolution after a non-reactive hypothermic EEG following cardiac arrest.

BACKGROUND: Lack of electroencephalography (EEG) background reactivity during therapeutic hypothermia (TH) has been associated with poor outcome in post-anoxic comatose patients. However, decision on intensive care withdrawal is based on normothermic (NT) evaluations. This study aims at exploring whether patients showing recovery of EEG reactivity in NT after a non-reactive EEG in TH differ from those remaining non-reactive. METHODS: Patients with non-reactive EEG during TH were identified from our prospective registry of consecutive comatose adults admitted after successful resuscitation from CA between April 2009 and June 2014. Variables including neurological examination, serum neuron-specific enolase (NSE), procalcitonin, and EEG features were compared regarding impact on functional outcome at 3 months. RESULTS: Seventy-two of 197 patients (37 %) had a non-reactive EEG background during TH with thirteen (18 %) evolving towards reactivity in NT. Compared to those remaining non-reactive (n = 59), they showed significantly better recovery of brainstem reflexes (p < 0.001), better motor responses (p < 0.001), transitory consciousness improvement (p = 0.008), and a tendency toward lower NSE (p = 0.067). One patient recovering EEG reactivity survived with good functional outcome at 3 months. CONCLUSIONS: Recovery of EEG reactivity from TH to NT seems to distinguish two patients' subgroups regarding early neurological assessment and transitory consciousness improvement, corroborating the role of EEG in providing information about cerebral functions. Understanding these dynamic changes encourages maintenance of intensive support in selected patients even after a non-reactive EEG background in TH, as a small subgroup may indeed recover with good functional outcome.

Application of a support system to the design of wastewater treatment plants

This paper presents a case study that explores the advantages that can be derived from the use of a design support system during the design of wastewater treatment plants (WWTP). With this objective in mind a simplified but plausible WWTP design case study has been generated with KBDS, a computer-based support system that maintains a historical record of the design process. The study shows how, by employing such a historical record, it is possible to: (1) rank different design proposals responding to a design problem; (2) study the influence of changing the weight of the arguments used in the selection of the most adequate proposal; (3) take advantage of keywords to assist the designer in the search of specific items within the historical records; (4) evaluate automatically the compliance of alternative design proposals with respect to the design objectives; (5) verify the validity of previous decisions after the modification of the current constraints or specifications; (6) re-use the design records when upgrading an existing WWTP or when designing similar facilities; (7) generate documentation of the decision making process; and (8) associate a variety of documents as annotations to any component in the design history. The paper also shows one possible future role of design support systems as they outgrow their current reactive role as repositories of historical information and start to proactively support the generation of new knowledge during the design process

A randomised clinical trial to assess the effect of total enteral and total parenteral nutritional support on metabolic, inflammatory and oxidative markers in patients with predicted severe acute pancreatitis (APACHE II >= 66)

Background: Total enteral nutrition (TEN) within 48 h of admission has recently been shown to be safe and efficacious as part of the management of severe acute pancreatitis. Our aim was to ascertain the safety of immediate TEN in these patients and the effect of TEN on systemic inflammation, psychological state, oxidative stress, plasma glutamine levels and endotoxaemia. Methods: Patients admitted with predicted severe acute pancreatitis (APACHE II score 15) were randomised to total enteral (TEN; n = 8) or total parenteral nutrition (TPN; n = 9). Measurements of systemic inflammation (C-reactive protein), fatigue ( visual analogue scale), oxidative stress ( plasma thiobarbituric acid- reactive substances), plasma glutamine and anti-endotoxin IgG and IgM antibody concentrations were made on admission and repeated on days 3 and 7 thereafter. Clinical progress was monitored using APACHE II score. Organ failure and complications were recorded. Results: All patients tolerated the feeding regime well with few nutrition-related complications. Fatigue improved in both groups but more rapidly in the TEN group. Oxidative stress was high on admission and rose by similar amounts in both groups. Plasma glutamine concentrations did not change significantly in either group. In the TPN group, 3 patients developed respiratory failure and 3 developed non-respiratory single organ failure. There were no such complications in the TEN group. Hospital stay was shorter in the TEN group [ 7 (4-14) vs. 10 (7-26) days; p = 0.05] as was time to passing flatus and time to opening bowels [1 (0-2) vs. 2 (1-5) days; p = 0.01]. The cost of TEN was considerably less than of TPN. Conclusion: Immediate institution of nutritional support in the form of TEN is safe in predicted severe acute pancreatitis. It is as safe and as efficacious as TPN and may be beneficial in the clinical course of this disease. Copyright (C) 2003 S. Karger AG, Basel and IAP.

Minimum recovery time between reactive hyperemia stimulus in the repeated measurement of brachial flow-mediated dilatation.

The ability to undertake repeat measurements of flow-mediated dilatation (FMD) within a short time of a previous measurement would be useful to improve accuracy or to repeat a failed initial procedure. Although standard methods report that a minimum of 10 min is required between measurements, there is no published data to support this. Thirty healthy volunteers had five FMD measurements performed within a 2-h period, separated by various time intervals (5, 15 and 30 min). In 19 volunteers, FMD was also performed as soon as the vessel had returned to its baseline diameter. There was no significant difference between any of the FMD measurements or parameters across the visits indicating that repeat measurements may be taken after a minimum of 5 min or as soon as the vessel has returned to its baseline diameter, which in some subjects may be less than 5 min.

Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells. (C) 2010 Elsevier Inc. All rights reserved.

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB`s cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.

Serum amyloid A induces reactive oxygen species (ROS) production and proliferation of fibroblast

Serum amyloid A (SAA) levels are elevated highly in acute phase response and elevated slightly and persistently in chronic diseases such as rheumatoid arthritis and diabetes. Given that fibroblasts exert profound effects on progression of inflammatory chronic diseases, the aim of this study was to investigate the response of fibroblasts to SAA. A dose-dependent increase in O(2)(-) levels was observed by treatment of fibroblasts with SAA (r = 0.99 and P <= 0.001). In addition, the expression of p47-phox was up-regulated by SAA (P < 0.001) and diphenyliodonium (DPI), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, reduced the release of O(2)(-) by 50%. Also, SAA raised fibroblast proliferation (P < 0.001) and this effect was completely abolished by the addition of anti-oxidants (P < 0.001). These findings support the notion that, in chronic inflammatory sites, SAA activated fibroblast proliferation and ROS production.

Cathodic stripping voltammetric detection and determination at a hanging mercury-drop electrode of dye contaminants in purified biomaterials: study of the human serum albumin and reactive dye 120 system

Procion red HE-3B (RR120) is an example of dye currently used in affinity purification. A method is described for determining trace amounts of RR120 dye contaminant in human serum albumin by cathodic stripping voltammetry. The method is based on a measure of a well-defined peak at -0.58 V, obtained when samples of HSA protein (0.01-2% w/v) containing dye concentrations are submitted to a heating time of 330 min at 80degreesC in NaOH, pH 12.0 and the samples are removed to a solution containing Britton-Robinson buffer, pH 4.0. Using an optimum accumulation potential and tune of 0 V and 240 s, respectively, linear calibration curves were obtained from 1.0 X 10(-9) to 1.0 X 10(-8) mol 1(-1) for RR120 dye. Leakage/hydrolysis of reactive red 120 from an agarose support (e.g. at pH 2 or 12) can also be conveniently determined at very low levels (sub-mug ml(-1)) by means of cathodic stripping voltammetry, which involves adsorptive accumulation of the dye onto the hanging mercury-drop electrode. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Use of commercial anion-exchange resins as solid support for peptide synthesis and affinity chromatography

This report demonstrates that due to the presence of residual reactive sites in their matrices, classical diethylaminoethyl-attaching commercial anion-exchanger resins such as DEAE-MacroPrep and DEAE-Sephadex A50 supports can be used for peptide synthesis. Moreover, due to the high stability of the peptide-resin bond in the final cleavage treatments, desired peptidyl-resins free of side-chain protecting groups, which enables them to be further used as solid support for affinity chromatography, can be obtained. To demonstrate this potentiality, a fragment corresponding to the antigenic and immunodominant epitope of sporozoites of the Plasmodium falciparum malaria parasite was synthesized in these traditional resins and antibody molecules generated against the peptide sequence were successfully retained in these peptidyl supports. Due to the maintenance of their original anion-exchange capacities, the present findings open the unique possibility of applying, simultaneously, dual anion-exchange and affinity procedures for purification of a variety of macromolecules. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Reactive halogen compounds in the marine boundary layer: method developments and field measurements

Reactive halogen compounds are known to play an important role in a wide variety of atmospheric processes such as atmospheric oxidation capacity and coastal new particle formation. In this work, novel analytical approaches combining diffusion denuder/impinger sampling techniques with gas chromatographic–mass spectrometric (GC–MS) determination are developed to measure activated chlorine compounds (HOCl and Cl2), activated bromine compounds (HOBr, Br2, BrCl, and BrI), activated iodine compounds (HOI and ICl), and molecular iodine (I2). The denuder/GC–MS methods have been used to field measurements in the marine boundary layer (MBL). High mixing ratios (of the order of 100 ppt) of activated halogen compounds and I2 are observed in the coastal MBL in Ireland, which explains the ozone destruction observed. The emission of I2 is found to correlate inversely with tidal height and correlate positively with the levels of O3 in the surrounding air. In addition the release is found to be dominated by algae species compositions and biomass density, which proves the “hot-spot” hypothesis of atmospheric iodine chemistry. The observations of elevated I2 concentrations substantially support the existence of higher concentrations of littoral iodine oxides and thus the connection to the strong ultra-fine particle formation events in the coastal MBL.

Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from Complex I

Evidence accumulated in the last ten years has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes or respirasomes. Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. Following this line of thought we have decided to directly investigate ROS production by Complex I under conditions in which the complex is arranged as a component of the supercomplex I1III2 or it is dissociated as an individual enzyme. The study has been addressed both in bovine heart mitochondrial membranes and in reconstituted proteoliposomes composed of complexes I and III in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. The results of this investigation provide experimental evidence that the production of ROS is strongly increased in either model; supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I . This is the first demonstration that dissociation of the supercomplex I1III2 in the mitochondrial membrane is a cause of oxidative stress from Complex I. Previous work in our laboratory demonstrated that lipid peroxidation can dissociate the supramolecular assemblies; thus, here we confirm that preliminary conclusion that primary causes of oxidative stress may perpetuate reactive oxygen species (ROS) generation by a vicious circle involving supercomplex dissociation as a major determinant.

Generation of FLT3-ITD-reactive T-cell populations from different sources

Approximately 25% of acute myeloid leukemias (AMLs) carry internal tandem duplications (ITD) of various lengths within the gene encoding the FMS-like tyrosine kinase receptor 3 (FLT3). Although varying duplication sites exist, most of these length mutations affect the protein´s juxtamembrane domain. FLT3-ITDs support leukemic transformation by constitutive phosphorylation resulting in uncontrolled activation, and their presence is associated with worse prognosis. As known form previous work, they represent leukemia- and patient-specific neoantigens that can be recognized by autologous AML-reactive CD8+ T cells (Graf et al., 2007; Graf et al., unpublished). Herein, in patient FL, diagnosed with FLT3-ITD+ AML and in first complete remission after induction chemotherapy, T cells against her leukemia´s individual FLT3-ITD were detected at a frequency up to 1.7x10-3 among peripheral blood CD8+ T lymphocytes. This rather high frequency suggested, that FLT3-ITD-reactive T cells had been expanded in vivo due to the induction of an anti-leukemia response.rnrnCell material from AML patients is limited, and the patients´ anti-leukemia T-cell repertoire might be skewed, e.g. due to complex previous leukemia-host interactions and chemotherapy. Therefore, allogeneic sources, i.e. buffy coats (BCs) from health donors and umbilical cord blood (UCB) donations, were exploited for the presence and the expansion of FLT3-ITD-reactive T-cell populations. BC- and UCB-derived CD8+ T cells, were distributed at 105 cells per well on microtiter plates and, were stimulated with antigen-presenting cells (APCs) transfected with in vitro-transcribed mRNA (IVT-mRNA) encoding selected FTL3-ITDs. APCs were autologous CD8- blood mononuclear cells, monocytes or FastDCs.rnrnBuffy coat lymphocytes from 19 healthy individuals were analyzed for CD8+ T-cell reactivity against three immunogenic FLT3-ITDs previously identified in patients VE, IN and QQ and designated as VE_, IN_ and QQ_FLT3-ITD, respectively. These healthy donors carried at least one of the HLA I alleles known to present an ITD-derived peptide from one of these FLT3-ITDs. Reactivities against single ITDs were observed in 8/19 donors. In 4 donors the frequencies of ITD-reactive T cells were determined and were estimated to be in the range of 1.25x10-6 to 2.83x10-7 CD8+ T cells. These frequencies were 1,000- to 10,000-fold lower than the frequency of autologous FLT3-ITD-reactive T cells observed in patient FL. Restricting HLA I molecules were identified in two donors. In one of them, the recognition of VE_FLT3-ITD was found to be restricted by HLA-C*07:02, which is different from the HLA allele restricting the anti-ITD T cells of patient VE. In another donor, the recognition of IN_FLT3-ITD was restricted by HLA-B*35:01, which also had been observed in patient IN (Graf et al., unpublished). By gradual 3´-fragmentation of the IN_FLT3-ITD cDNA, the 10-mer peptide CPSDNEYFYV was identified as the target of allogeneic T cells against IN_FLT3-ITD. rnLymphocytes in umbilical cord blood predominantly exhibit a naïve phenotype. Seven UCB donations were analyzed for T-cell responses against the FLT3-ITDs of patients VE, IN, QQ, JC and FL irrespective of their HLA phenotype. ITD-reactive responses against all stimulatory FLT3-ITDs were observed in 5/7 UCB donations. The frequencies of T cells against single FLT3-ITDs in CD8+ lymphocytes were estimated to be in the range of 1.8x10-5 to 3.6x10-6, which is nearly 15-fold higher than the frequencies observed in BCs. Restricting HLA I molecules were identified in 4 of these 5 positive UCB donations. They were mostly different from those observed in the respective patients. But in one UCB donation T cells against the JC_FLT3-ITD had exactly the same peptide specificity and HLA restriction as seen before in patient JC (Graf et al., 2007). Analyses of UCB responder lymphocytes led to the identification of the 10-mer peptide YESDNEYFYV, encoded by FL_FLT3-ITD, that was recognized in association with the frequent allele HLA-A*02:01. This peptide was able to stimulate and enrich ITD-reactive T cells from UCB lymphocytes in vitro. Peptide responders not only recognized the peptide, but also COS-7 cells co-transfected with FL_FLT3-ITD and HLA-A*02:01.rnrnIn conclusion, T cells against AML- and individual-specific FLT3-ITDs were successfully generated not only from patient-derived blood, but also from allogeneic sources. Thereby, ITD-reactive T cells were detected more readily and at higher frequencies in umbilical cord blood than in buffy coat lymphocytes. It occurred that peptide specificity and HLA restriction of allogeneic, ITD-reactive T cells were identical to autologous patient-derived T cells. As shown herein, allogeneic, FLT3-ITD-reactive T cells can be used for the identification of FLT3-ITD-encoded peptides, e.g. for future therapeutic vaccination studies. In addition, these T cells or their receptors can be applied to adoptive transfer.

Applying the reactive programming paradigm: Toward a more declarative application development approach

After almost 10 years from “The Free Lunch Is Over” article, where the need to parallelize programs started to be a real and mainstream issue, a lot of stuffs did happened: • Processor manufacturers are reaching the physical limits with most of their approaches to boosting CPU performance, and are instead turning to hyperthreading and multicore architectures; • Applications are increasingly need to support concurrency; • Programming languages and systems are increasingly forced to deal well with concurrency. This thesis is an attempt to propose an overview of a paradigm that aims to properly abstract the problem of propagating data changes: Reactive Programming (RP). This paradigm proposes an asynchronous non-blocking approach to concurrency and computations, abstracting from the low-level concurrency mechanisms.