Validité de la somme angulaire comme technique d’extraction d’images planaires à partir de données tomoscintigraphiques pulmonaires de ventilation-perfusion

The aim of our study was to assess the concordance between the results of angular summed images obtained from lung SPECT data versus true planar images in the diagnosis of pulmonary embolism. One hundred and ten patients suspected of pulmonary embolism included in a prospective study underwent a planar lung scan and a lung SPECT. Planar images were extracted from SPECT data using an angular summing technique. Both modalities were reviewed according to revised Pioped criteria by two nuclear medicine physicians. Concordance between modalities and observers was compared with a weighted kappa test. A good degree of agreement between the two modalities (κ = 0.696) were found. Interobserver degree of agreement was similar in planar images (κ = 0.919) and in angular summed images (κ = 0.903). These results suggest that angular sum images from SPECT data are usable and concordant with ventilation perfusion planar images.     

Effect of IL-1 on the hydrolysis of the tumor antigen epitope gp100(280-288) by fibroblast-expressed enzymes

The role of proinflammatory cytokines in increasing the activity of specific proteases suggests the hypothesis that, by altering the expression of these mediators, adjuvants may modulate the effectiveness of peptides used as vaccines. The possible effect of IL-1 on fibroblast-expressed, peptidases was, thus, investigated by analyzing the degradation of a tumor antigen epitope (gp100(280-288), YLEPGPVTA) in the presence of cultured human fibroblasts. The data obtained indicate an increase of substrate hydrolysis after IL-1 treatment as compared with non-treated controls. Hydrolysis increase was accompanied by defined changes in the population of the by-products formed: specifically, the amount of peptidic by-products increased more than the amount of single amino acids, and the amount of the C-terminal by-products increased more than the amount of their N-terminal counterpart. These data appear to indicate that the positive effect of IL-1 on the activity of substrate-active enzymes is function of modified expression of a number of these enzymes by fibroblasts. From these data it can be inferred that the use of IL-1-inducing adjuvants, increasing the activity of peptidases expressed by bystander cells, may reduce the bio-availability of peptides used for immunization.     

Apoptosis in human atherosclerotic plaques

Intimal cell death has been a recognized feature of advanced atherosclerotic disease. With the advent of DNA in situ end labelling and/or ultrastructural techniques, recent findings suggest that cells of an atheroma undergo programmed cell death or apoptosis. The pathophysiologic relevance of apoptosis in atherosclerotic disease is debatable. Apoptotic cell death may influence lesion progression and thus reduce overall plaque burden. Alternatively, apoptosis may prove a means of quenching the inflammation, converting cellular-rich lesions to so-called stable fibrous hypocellular plaques or conversely weaken the fibrous cap causing plaque rupture, a major cause of acute coronary syndromes. Apoptotic cells within plaques are typically macrophages, smooth muscle cells and T-cells and the frequency of death varies in the different regions of the lesion. The precise signalling pathways of apoptosis in plaques are unknown. There is however, some evidence that production of immune cytokines may promote apoptosis through activation of the Fas ligand-mediated death pathway. Genetic signals that regulate apoptosis in the atheroma, at least in smooth muscle cells, may involve the tumour suppressor genes p105 ^RB and p53. Further studies as to the relevance of apoptosis in acute coronary syndromes and potential mechanisms are emerging.     

Total biosynthesis of hydrocortisone from a simple carbon source in yeast

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.     

Synchronized onset of nuclear and cell surface modifications in U937 cells during apoptosis

In this study we investigated the relationship between nuclear and cell surface modifications (i.e. blebbing, phosphatidylserine [PS] and sugar residues exposure) in a monocytic cell line, U937, during apoptosis induced by oxidative stress (1 mM H2O2) or inhibition of protein synthesis (10 microg/ml puromycin). Dying cells were simultaneously observed for nuclear modifications, presence of superficial blebs and plasma membrane alterations. Morphological analysis performed by conventional fluorescence microscopy, or by transmission and scanning electron microscopy showed that the courses of nuclear and membrane alterations occured concomitantly, but the phenotype was dependent on the stage of the apoptotic process and the type of apoptogenic inducer used. The progression of apoptosis in U937 cells beyond early stages resulted in the extensive formation of blebs which concomitantly lost some typical markers of apoptosis, such as PS and sugar residues. Therefore, the modality by which the nucleus condenses, or the amount and the pattern of distribution of PS on the cell surface were, for each cell line, strictly related to the apoptogenic inducer. The morphological data reported in the present paper should lead to a more precise quantification of apoptosis by improving the detection of apoptotic cells in vivo (i.e. in tissue, organs), which is a crucial point in the evaluation of efficiency of antiproliferative drugs, such as antiblastic or immunosuppressive compounds.     

Metabolism of 125I-labeled lipoproteins by the isolated rat lung

The capacity of the isolated perfused rat lung to metabolize the protein moieties of serum lipoproteins was assessed using homologous (rat) and heterologous (human) plasma lipoproteins. The protein and lipid moieties of the plasma lipoproteins were labeled in vivo with Na[125I]. In selected cases the lipoprotein peptides were labeled in vivo with 14C- or 3H-labeled amino acids. Uptake of lipoprotein label during perfusion was monitored by measure of losses in perfusate label and by rises in pulmonary tissue labeling as shown by radioassay and by light and electron microscope radioautography. Lipoprotein degradation was assessed by fractionation of perfusate and lung tissue radioactive material into trichloroacetic acid (TCA)-isoluble, TCA-soluble, and ether-ethanol-soluble fractions. When heparin was included in the perfusion medium, there was selective degradation of the protein portion of very low density lipoprotein (VLDL) in the perfusate and concomitant uptake of radioactive label by the lungs. Low density lipoprotein (LDL)) was neither taken up nor catabolized by the isolated rat lung in the absence or presence of heparin. By light and electron microscopy, the label was localized over the interalveolar septa, predominantly the capillary endothelium. Disappearance of TCA-insoluble radioactivity from the perfusate was associated with the generation of both TCA-soluble iodide and noniodide radioactivity. Greater than 50% of the radioactive label taken up by the lungs was found in the delipidated TCA-insoluble fraction. This study provides in vitro evidence for pulmonary catabolism of VLDL apolipoproteins and uptake of peptide catabolic products of VLDL by the lung.     

Induction of potent antitumor CTL responses by recombinant vaccinia encoding a melan-A peptide analogue

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.     

Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma

Recent studies have shown that CTL epitopes derived from tumor-associated Ags can be encoded by both primary and nonprimary open reading frames (ORF). In this study we have analyzed the HLA-A2-restricted CD8(+) T cell response to a recently identified CTL epitope derived from an alternative ORF product of gene LAGE-1 (named CAMEL), and the highly homologous gene NY-ESO-1 in melanoma patients. Using MHC/peptide tetramers we detected CAMEL(1-11)-specific CD8(+) T cells in peptide-stimulated PBMC as well as among tumor-infiltrated lymph node cells from several patients. Sorting and expansion of tetramer(+) CD8(+) T cells allowed the isolation of tetramer(bright) and tetramer(dull) populations that specifically recognized the peptide Ag with high and low avidity, respectively. Remarkably, only high avidity CAMEL-specific CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression of LAGE-1 and NY-ESO-1 mRNA and protein and tested for recognition by CAMEL-specific CTL as well as CTL that recognize a peptide (NY-ESO-1(157-165)) encoded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes. This analysis revealed that tumor-associated CD8(+) T cell epitopes are simultaneously and efficiently generated from both primary and nonprimary ORF products of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the majority of melanoma tumors. These findings underscore the in vivo immunological relevance of CTL epitopes derived from nonprimary ORF products and support their use as candidate vaccines for inducing tumor specific cell-mediated immunity against cancer.