Quality control for biomarker determination in oncology: the experience of the Italian Network for Quality Assessment of Tumor Biomarkers (INQAT)

Biomarker analysis and evaluation in oncology is the product of a number of processes (including managerial, technical and interpretation steps) which need to be monitored and controlled to prevent and correct errors and guarantee a satisfactory level of quality. Several biomarkers have recently moved to clinical validation studies and successively to clinical practice without any definition of standard procedures and/or quality control (QC) schemes necessary to guarantee the reproducibility of the laboratory information. In Italy several national scientific societies and single researchers have activated -- often on a pilot level -- specific external quality assessment protocols, thereby potentially jeopardizing the clinical reality even further. In view of the seriousness of the problem, in 1998 the Italian Ministry of Health sponsored a National Survey Project to coordinate and standardize the procedures and to develop QC programs for the analysis of cancer biomarkers of potential clinical relevance. Twelve QC programs focused on biomarkers and concerning morphological, immunohistochemical, biochemical, molecular, and immunoenzymatic assays were coordinated and implemented. Specifically, external QC programs for the analytical phase of immunohistochemical p53, Bcl-2, c-erb-2/neu/HER2, and microvessel density determination, of morphological evaluation of tumor differentiation grade, and of molecular p53 analysis were activated for the first time within the project. Several hundreds of Italian laboratories took part in these QC programs, the results of which are available on the web site of the Network (www.cqlaboncologico.it). Financial support from the Italian Government and the National Research Council (CNR) will guarantee the pursuit of activities that will be extended to new biomarkers, to preanalytical phases of the assays, and to revision of the criteria of clinical usefulness for evaluating the cost/benefit ratio.     

Differential roles of N- and C-terminal immunoreceptor tyrosine-based inhibition motifs during inhibition of cell activation by killer cell inhibitory receptors

Killer cell inhibitory receptors (KIRs) inhibit NK and T cell cytotoxicity when recognizing MHC class I molecules on target cells. They possess two tandem intracytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) that, when phosphorylated, each bind to the two Src homology 2 domain-bearing protein tyrosine phosphatases SHP-1 and SHP-2 in vitro. Using chimeric receptors having an intact intracytoplasmic KIR domain bearing both ITIMs (N + C-KIR), a deleted domain containing the N-terminal ITIM only (N-KIR), or a deleted domain containing the C-terminal ITIM only (C-KIR), we examined the respective contributions of the two ITIMs in the inhibition of cell activation in two experimental models (a rat mast cell and a mouse B cell line) that have been widely used to analyze KIR functions. We found that the two KIR ITIMs play distinct roles. When coaggregated with immunoreceptor tyrosine-based activation motif-bearing receptors such as high-affinity IgE receptors or B cell receptors, the N + C-KIR and the N-KIR chimeras, but not the C-KIR chimera, inhibited mast cell and B cell activation, became tyrosyl-phosphorylated, and recruited phosphatases in vivo. The N + C-KIR chimera recruited SHP-1 as expected, but also SHP-2. Surprisingly, the N-KIR chimera failed to recruit SHP-1; however, it did recruit SHP-2. Consequently, the N-terminal ITIM is sufficient to recruit SHP-2 and to inhibit cell activation, whereas the N-terminal and the C-terminal ITIMs are both necessary to recruit SHP-1. The two KIR ITIMs, therefore, are neither mandatory for inhibition nor redundant. Rather than simply amplifying inhibitory signals, they differentially contribute to the recruitment of distinct phosphatases that may cooperate to inhibit cell activation.     

Iron uptake by the yeast Pichia guilliermondii. Flavinogenesis and reductive iron assimilation are co-regulated processes

Pichia guilliermondii cells overproduce riboflavin (vitamin B2) in responce to iron deprivation. The increase in ferrireductase activity in iron-starved P. guilliermondii cells correlated with the increase in flavin excretion. As in Saccharomyces cerevisiae, a typical b-type cytochrome spectrum was associated with the plasma membrane fraction of P. guillermondii and the cell ferrireductase activity was strongly inhibited by diphenylene-iodonium, an inhibitor of flavoproteins, in both yeasts. Mutants of P. guilliermondii with increased ferrireductase activity were selected for further investigation of the relationship between iron reduction/uptake and flavin production. The obtained mutation has been called hit (high iron transport). A hit mutant with a single recessive mutation showed the following phenotype: high ferrireductase activity, increased rate of iron uptake and elevated flavinogenic activity. Cu(II) (50 m) strongly inhibited the growth of the hit mutant compared to the wild-type. The mutant cells grown in copper-supplemented medium (5–25 m) showed an increase of the ferrireductase activity (up to 2–3 fold). The copper content of the mutant cells grown under these conditions was also higher (1.5–2 fold) than that of the wild-type. The role of the HIT gene of P. guillermondii in the regulation of iron, copper and flavin metabolisms is discussed.     

GNA33 from Neisseria meningitidis serogroup B encodes a membrane-bound lytic transglycosylase (MltA).

In a previous study, we used the genome of serogroup B Meningococcus to identify novel vaccine candidates. One of these molecules, GNA33, is well conserved among Meningococcus B strains, other Meningococcus serogroups and Gonococcus and induces bactericidal antibodies as a result of being a mimetic antigen of the PorA epitope P1.2. GNA33 encodes a 48-kDa lipoprotein that is 34.5% identical with membrane-bound lytic transglycosylase A (MltA) from Escherichia coli. In this study, we expressed GNA33, i.e. Meningococcus MltA, as a lipoprotein in E. coli. The lipoprotein nature of recombinant MltA was demonstrated by incorporation of [3H]palmitate. MltA lipoprotein was purified to homogeneity from E. coli membranes by cation-exchange chromatography. Muramidase activity was confirmed when MltA was shown to degrade insoluble murein sacculi and unsubstituted glycan strands. HPLC analysis demonstrated the formation of 1,6-anhydrodisaccharide tripeptide and tetrapeptide reaction products, confirming that the protein is a lytic transglycosylase. Optimal muramidase activity was observed at pH 5.5 and 37 degrees C and enhanced by Mg2+, Mn2+ and Ca2+. The addition of Ni2+ and EDTA had no significant effect on activity, whereas Zn2+ inhibited activity. Triton X-100 stimulated activity 5.1-fold. Affinity chromatography indicated that MltA interacts with penicillin-binding protein 2 from Meningococcus B, and, like MltA from E. coli, may form part of a multienzyme complex.     

Human leukemic K562 cells: suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor

The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression.     

A frequent factor XII gene mutation in Hageman trait

Summary An additionalTaqI restriction site was mapped in intron 2 of the factor XII gene. The site was found only in subjects with total or partial factor XII deficiency and thus represents the true gene lesion or a very tightly linked restriction fragment length polymorphism. The altered gene identified by this marker is present in four (three heterozygotes and one homozygote) of five unrelated Hageman trait subjects from different Italian regions. In the homozygous state the altered gene gives rise to a very marked reduction of factor XII activity. No deletion was found in the deficient factor XII genes.     

Analysis of Yeast Flora Associated with Grape Sour Rot and of the Chemical Disease Markers

The frequency and the density of the species associated with grape sour rot in different cultivars were determined. The most frequent species in the rotten grapes, Candida krusei, Kloeckera apiculata, and Metschnikowia pulcherrima, and a less frequent species, Issatchenkia occidentalis, when inoculated with Saccharomycopsis crataegensis were able to induce in vitro the symptoms of the disease. The gas chromatographic determination of the volatile compounds in the headspace was used to evaluate the metabolic role of the different species associated with the disease. These analyses made it possible to presume that, whereas some species, such as Candida krusei and Hanseniaspora uvarum, can be considered responsible for these modifications and in particular for the ethyl acetate production, others, such as Saccharomycopsis crataegensis, can promote the development of the former species.     

Binding constants of soluble NGF-receptors in rat oligodendrocytes and astrocytes in culture

The neuronotrophic factor NGF binds to peripheral neurons of the dorsal root ganglion and the sympathetic nervous system. NGF binds to a cell surface receptor, NGFR, on these cells and displays Kd's of 10(-9) and 10(-11)M. NGF receptors have also been reported for basal forebrain magnocellular neurons. In addition, NGF specifically binds to NGFR on Schwann cells although the biological significance of this binding is not known. Here we report that NGF binds in a saturable and specific fashion to receptors on cultured isolated populations of rat astrocytes but not to oligodendrocytes. The binding to astrocytes in culture displayed a Kd of 2.7 +/- 1.0 nM with 36,000 receptors per cell.     

Preliminary assessment on the use of immobilized yeast cells in sodium alginate for sparkling wine processes

Summary Use of alginate-immobilized yeasts in the production of sparkling wine using the champenois method was investigated. The results indicate that there are no variations in the principal chemical-physical characteristics between sparkling wines obtained through immobilized yeast and traditional sparkling wines.