Mediastinal reinforcement after induction therapy and pneumonectomy: comparison of intercostal muscle versus diaphragm flaps.

OBJECTIVE: Prospective non-randomised comparison of full-thickness pedicled diaphragm flap with intercostal muscle flap in terms of morbidity and efficiency for bronchial stump coverage after induction therapy followed by pneumonectomy for non-small cell lung cancer (NSCLC). METHODS: Between 1996 and 1998, a consecutive series of 26 patients underwent pneumonectomy following induction therapy. Half of the patients underwent mediastinal reinforcement by use of a pedicled intercostal muscle flap (IF) and half of the patients by use of a pedicled full-thickness diaphragm muscle flap (DF). Patients in both groups were matched according to age, gender, side of pneumonectomy and stage of NSCLC. Postoperative morbidity and mortality were recorded. Six months follow-up including physical examination and pulmonary function testing was performed to examine the incidence of bronchial stump fistulae, gastro-esophageal disorders or chest wall complaints. RESULTS: There was no 30-day mortality in both groups. Complications were observed in one of 13 patients after IF and five of 13 after DF including pneumonia in two (one IF and one DF), visceral herniations in three (DF) and bronchopleural fistula in one patient (DF). There were no symptoms of gastro-esophageal reflux disease (GERD). Postoperative pulmonary function testing revealed no significant differences between the two groups. CONCLUSIONS: Pedicled intercostal and diaphragmatic muscle flaps are both valuable and effective tools for prophylactic mediastinal reinforcement following induction therapy and pneumonectomy. In our series of patients, IF seemed to be associated with a smaller operation-related morbidity than DF, although the difference was not significant. Pedicled full-thickness diaphragmatic flaps may be indicated after induction therapy and extended pneumonectomy with pericardial resection in order to cover the stump and close the pericardial defect since they do not adversely influence pulmonary function.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

Randomized Trial of prophylactic granulocyte colony-stimulating factor during rapid COJEC induction in pediatric patients with high-risk neuroblastoma: the European HR-NBL1/SIOPEN study.

Purpose To reduce the incidence of febrile neutropenia during rapid COJEC (cisplatin, vincristine, carboplatin, etoposide, and cyclophosphamide given in a rapid delivery schedule) induction. In the High-Risk Neuroblastoma-1 (HR-NBL1) trial, the International Society of Paediatric Oncology European Neuroblastoma Group (SIOPEN) randomly assigned patients to primary prophylactic (PP) versus symptom-triggered granulocyte colony-stimulating factor (GCSF; filgrastim). Patients and Methods From May 2002 to November 2005, 239 patients in 16 countries were randomly assigned to receive or not receive PPGCSF. There were 144 boys with a median age of 3.1 years (range, 1 to 17 years) of whom 217 had International Neuroblastoma Staging System (INSS) stage 4 and 22 had stage 2 or 3 MYCN-amplified disease. The prophylactic arm received a single daily dose of 5 μg/kg GCSF, starting after each of the eight COJEC chemotherapy cycles and stopping 24 hours before the next cycle. Chemotherapy was administered every 10 days regardless of hematologic recovery, provided that infection was controlled. Results The PPGCSF arm had significantly fewer febrile neutropenic episodes (P = .002), days with fever (P = .004), hospital days (P = .017), and antibiotic days (P = .001). Reported Common Toxicity Criteria (CTC) graded toxicity was also significantly reduced: infections per cycle (P = .002), fever (P < .001), severe leucopenia (P < .001), neutropenia (P < .001), mucositis (P = .002), nausea/vomiting (P = .045), and constipation (P = .008). Severe weight loss was reduced significantly by 50% (P = .013). Protocol compliance with the rapid induction schedule was also significantly better in the PPGCSF arm shown by shorter time to completion (P = .005). PPGCSF did not adversely affect response rates or success of peripheral-blood stem-cell harvest. Following these results, PPG-GSF was advised for all patients on rapid COJEC induction.

Salicylic Acid Biosynthetic Genes Expressed in Pseudomonas fluorescens Strain P3 Improve the Induction of Systemic Resistance in Tobacco Against Tobacco Necrosis Virus.

ABSTRACT Application of salicylic acid induces systemic acquired resistance in tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in tobacco against tobacco necrosis virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of tobacco, but did not improve the ability of CHA0 to induce systemic resistance in tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobacco in a gnotobiotic system.

Degradation of pheromone and plant volatile components by a same Odorant-Degrading Enzyme in the cotton leafworm, Spodoptera littoralis

Background: Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant. Methodology: Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis , a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants. Conclusion: SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

Carbonic anhydrase activity in primary sensory neurons. II. Influence of environmental factors on the phenotypic expression of the enzyme in dissociated cultures of chicken dorsal root ganglion cells.

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.

Induction of apoptosis by mercury compounds depends on maturation and is not associated with microglial activation

The earliest sign of neurotoxicity observed after exposure of three-dimensional brain cell cultures to low concentrations of mercury compounds is a microglial reaction. We hypothesized that an induction of apoptosis by mercury compounds could be an activating signal of the microglial reaction. Aggregating brain cell cultures of fetal rat telencephalon were treated for 10 days with either mercury chloride or monomethylmercury chloride at noncytotoxic concentrations during two developmental periods: from day 5 to 15, corresponding to an immature stage, and from day 25 to 35 corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. It was found that both mercury compounds caused a significant increase in the number of apoptotic cells, but exclusively in immature cultures exhibiting also spontaneous apoptosis. Double staining by the TUNEL technique combined with either neuronal or astroglial markers revealed that the proportion of cells undergoing apoptosis was highest for astrocytes. Furthermore neither an association nor a colocalization was found between apoptotic cells and microglial cells. In conclusion, it appears that the induction of apoptosis by mercury compounds in immature cells is only an acceleration of a spontaneously occurring process, and that it is not a directly related to the early microglial reaction.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

Induction of human papillomavirus oncogene-specific CD8 T-cell effector responses in the genital mucosa of vaccinated mice.

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV-16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70-80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7(1-98) polypeptide showed induction of 100% tumor protection against s.c. TC-1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7-specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7-specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high-avidity E7-specific INF-gamma secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV-tumors reside.

New wheat genotypes tolerant to aluminum toxicity obtained by mutation induction

Seed from the sensitive wheat (Triticum aestivumL.) cultivar Anahuac was treated to gamma-ray irradiation and eleven Al3+ tolerant mutants selected. The objective was to compare these mutants to the original Anahuac and to the tolerant wheat cultivars IAC-24 and IAC-60 from 1994 to 1996 in acid (Capão Bonito) and limed (Monte Alegre do Sul) soil field trials, in the State of São Paulo, Brazil. Grain yield and agronomic characteristics were analyzed. All the mutant lines yielded higher than the sensitive Anahuac cultivar in the acid soils of Capão Bonito. Under limed soil conditions, 10 mutants had a similar yield to the original sensitive cultivar and one a lower yield. The majority of the mutants were similar in yield to the tolerant cultivars IAC-24 and IAC-60 under both conditions. Some of the mutants showed altered agronomic characteristics, but these alterations did not generally influence the grain yield. The results indicated that tolerant lines with good characteristics may be obtained from a susceptible cultivar by mutation induction, thus allowing cropping under conditions where Al3 + is a limiting factor.

Comparison of a multiplexed bead-based assay with an immunofluorescence and an enzyme-immuno assay for the assessment of Epstein-Barr virus serologic status.

We have compared a multiplexed bead-based assay (BBA) with an enzyme immunoassay (EIA) and immunofluorescence assay (IFA) for the assessment of the Epstein-Barr virus (EBV) serostatus. Three hundred and ninety-three sera, classified according to IFA results as seronegative (n=100), acute infection (n=100), past infection (n=100) and indeterminate (n=93), were tested by BBA and EIA. Overall, the three methods gave similar results with a relatively high (75.2%) concordance with the consensus interpretation of the serostatus. The most significant discordances were: (i) 58 samples had uninterpretable results for BBA, in majority due to the detection of non-antigen specific antibody binding by control beads. (ii) almost half the samples positive for anti-Epstein-Barr nuclear antigen (EBNA) IgG by BBA or EIA were negative by IFA. Among the latter, only a minority had a history of immunocompromise or treatment, or detectable anti-early antigen antibody. This discrepancy probably reflects a poor sensitivity of IFA for anti-EBNA IgG detection. EIA and BBA had a similar performance and had substantial practical advantages over IFA with respect to testing for EBV serostatus.

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis.

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.

Relationship between estrogen receptor alpha location and gene induction reveals the importance of downstream sites and cofactors.

BACKGROUND: To understand cancer-related modifications to transcriptional programs requires detailed knowledge about the activation of signal-transduction pathways and gene expression programs. To investigate the mechanisms of target gene regulation by human estrogen receptor alpha (hERalpha), we combine extensive location and expression datasets with genomic sequence analysis. In particular, we study the influence of patterns of DNA occupancy by hERalpha on expression phenotypes. RESULTS: We find that strong ChIP-chip sites co-localize with strong hERalpha consensus sites and detect nucleotide bias near hERalpha sites. The localization of ChIP-chip sites relative to annotated genes shows that weak sites are enriched near transcription start sites, while stronger sites show no positional bias. Assessing the relationship between binding configurations and expression phenotypes, we find binding sites downstream of the transcription start site (TSS) to be equally good or better predictors of hERalpha-mediated expression as upstream sites. The study of FOX and SP1 cofactor sites near hERalpha ChIP sites shows that induced genes frequently have FOX or SP1 sites. Finally we integrate these multiple datasets to define a high confidence set of primary hERalpha target genes. CONCLUSION: Our results support the model of long-range interactions of hERalpha with the promoter-bound cofactor SP1 residing at the promoter of hERalpha target genes. FOX motifs co-occur with hERalpha motifs along responsive genes. Importantly we show that the spatial arrangement of sites near the start sites and within the full transcript is important in determining response to estrogen signaling.

Protective role of early aquaporin 4 induction against postischemic edema formation.

Aquaporin 4 (AQP4) is a water channel involved in water movements across the cell membrane and is spatially organized on the cell surface in orthogonal array particles (OAPs). Its role in edema formation or resolution after stroke onset has been studied mainly at late time points. We have shown recently that its expression is rapidly induced after ischemia coinciding in time with an early swelling of the ischemic hemisphere. There are two isoforms of AQP4: AQP4-M1 and AQP4-M23. The ratio of these isoforms influences the size of the OAPs but the functional impact is not known. The role of the early induction of AQP4 is not yet known. Thrombin preconditioning in mice provides a useful model to study endogenous protective mechanisms. Using this model, we provide evidence for the first time that the early induction of AQP4 may contribute to limit the formation of edema and that the AQP4-M1 isoform is predominantly induced in the ischemic tissue at this time point. Although it prevents edema formation, the early induction of the AQP4 expression does not prevent the blood-brain barrier disruption, suggesting an effect limited to the prevention of edema formation possibly by removing of water from the tissue.

An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major.

The possible immunomodulatory role of polymorphonuclear leukocytes (PMN) in CD4+ T lymphocyte differentiation in mice was examined by studying the effect of transient depletion of PMN during the early phase after Leishmania major delivery. A single injection of the PMN-depleting NIMP-R14 mAb 6 h before infection with L. major prevented the early burst of IL-4 mRNA transcription otherwise occurring in the draining lymph node of susceptible BALB/c mice. Since this early burst of IL-4 mRNA transcripts had previously been shown to instruct Th2 differentiation in mice from this strain, we examined the effect of PMN depletion on Th subset differentiation at later time points after infection. The transient depletion of PMN in BALB/c mice was sufficient to inhibit Th2 cell development otherwise occurring after L. major infection. Decreased Th2 responses were paralleled with partial resolution of the footpad lesions induced by L. major. Furthermore, draining lymph node-derived CD4+ T cells from PMN-depleted mice remained responsive to IL-12 after L. major infection, unlike those of infected BALB/c mice receiving control Ab. PMN depletion had no effect when the NIMP-R14 mAb was injected 24 h postinfection. The protective effect of PMN depletion was shown to be IL-12 dependent, as concomitant neutralization of IL-12 reversed the protective effect of PMN depletion. These results suggest a role for an early wave of PMN in the development of the Th2 response characteristic of mice susceptible to infection with L. major.