Harmonisation of product notification to Poisons Centres in EU Member States.

INTRODUCTION: In the European Union (EU), notification of product information by industry to poisons centres and/or competent authorities is a legal obligation for mixtures classified as hazardous. However, EU legislation does not specify the precise information needed for this product notification. As a consequence, varying requirements have been developed in different EU Member States. The European Commission (EC) carried out an assessment of whether harmonisation of product notification can be achieved. This manuscript provides an overview of the most important (discussion) points to reach harmonisation. COMPOSITION AND CONCENTRATION OF INGREDIENTS: Discussions have focused mainly on whether non-classified ingredients should be notified only above a concentration threshold and on the use of defined, narrow concentration ranges instead of exact concentrations for hazardous ingredients. ELECTRONIC DATA EXCHANGE FORMAT: All stakeholders agree to the development of an electronic data exchange format for product notification and identify the eXtensible Markup Language (XML) as the most appropriate format. EUROPEAN PRODUCT DATABASE: Instead of multiple notifications to national databases, the EC will analyse the benefits, feasibility and costs of a European product database to provide a centralised portal for companies to upload their product information. Poisons centres and competent authorities need to have access to this information. UNIQUE PRODUCT IDENTIFIER: A Unique Product Identifier (UPI) on the product label can unambiguously identify the product and its formula and links it to the corresponding notified product information. A procedure for the creation of a UPI by companies has already been proposed. PRODUCT CATEGORY SYSTEM: There is broad support for the development of a hierarchical product category system to facilitate statistical analyses and comparability of poisoning incidents in EU Member States. OUTLOOK: Following a 3-year assessment period, the EC concluded that harmonisation of product notification is an achievable goal. In order to draft an Annex to the CLP Regulation concerning this topic, a new working group with representatives of EU Member States, European Association of Poisons Centres and Clinical Toxicologists (EAPCCT) and other stakeholders will attempt to find consensus on harmonisation of product notification.

Transferrin receptor expression as a marker of immature cycling thymocytes in the mouse.

Dividing cells require iron and, therefore, express the transferrin receptor (CD71) on the cell surface to enable internalization of transferrin-bound iron. Since early T cell development is marked by intense proliferation, we questioned whether CD71 might serve as a marker of immature T cells. Therefore, we analyzed the expression of CD71 on fetal, neonatal, and adult thymocytes in correlation with cell size, cell cycle status, and expression of CD3, CD4, CD8, alpha beta TcR, and gamma delta TcR. Phenotypic analysis showed that only the large, immature CD4-8-3-, CD4-8+3-, and CD4+8+3- cells in fetal, neonatal, and adult thymus expressed CD71. In addition, DNA analysis showed that all CD71+ large adult thymocytes were cycling. Downregulation of CD71 occurs when proliferation ceases, i.e., within the CD4+8+3- thymocyte subpopulation. The gradual changes in size and CD71 expression suggest a sequential development within this CD4+8+3- subpopulation from large CD71+ via small CD71+/- to small CD71- cells. As a consequence, CD71 expression is downregulated, in adult T cell development as well as in ontogeny, before the alpha beta TcR appears on the cell surface of the thymocyte. Together, our findings show that CD71 is a marker of immature, proliferating T cells.

Inhibition of proliferation and differentiation during early T cell development by anti-transferrin receptor antibody.

Proliferating cells require iron and, therefore, express the transferrin receptor (CD71) that mediates cellular iron uptake. Cycling thymocytes, which have the CD4-8-3-, CD4-8+3-, or CD4+8+3- phenotypes, also express CD71. The importance of CD71-mediated iron uptake for proliferation and maturation of thymocytes was studied using fetal thymus organ cultures at day 14 of gestation and treating them for 7 days with a CD71 monoclonal antibody (mAb). The intracellular iron deficiency caused by this treatment, inhibits both proliferation and maturation of the thymocytes. Cell recovery was reduced by 60%, but cells still expanded tenfold during the culture. Remarkably, the final maturation of alpha beta T cells was completely blocked as no thymocytes with low or high CD3/alpha beta TcR expression developed. Moreover, only few cells reached the CD4+8+3- stage of T cell development. CD4-8-3- thymocytes, however, as well as its CD44-25+ subset developed in normal numbers, suggesting that CD44-25+ CD4-8-3- cells, or their immediate progeny, were most vulnerable to CD71 mAb treatment. The development of gamma delta T cells, which also express CD71, was not affected in these cultures. This suggests that gamma delta T cells are either less iron-dependent or possess alternative iron-uptake mechanisms. Thus, our observations indicate that CD71 treatment, causing decreased intracellular iron levels, severely inhibits the major proliferation phase from the CD44-25+ CD4-8-3- to the CD4+8+3- cells, and completely abrogates the final maturation of CD4+8+3- cells into alpha beta TcR-expressing cells. In contrast, proliferation and differentiation of the earliest thymic precursors into CD44-25+ CD4-8-3- cells is not affected by CD71 treatment.

Characterization of the neutral aminopeptidase activity associated to the mouse thymocyte-activating molecule.

We previously characterized a dimeric, Mr = 115,000, developmentally regulated mouse T cell-activating molecule (THAM). We show in this report that the THAM-specific mAb H194-112 exhibits strong reactivity with several nonlymphoid tissues including polarized enterocytes from small intestine, kidney cortical tubuli, and liver bile ductuli, as well as kidney glomeruli and lung alveoloar pneumocytes. Both the tissue distribution and the structural features of THAM made it likely that this molecule belongs to the ectoenzyme family. This was confirmed by the following experimental evidence: 1) the H194-112+ molecules from enterocyte brush borders (BB) or M14.T thymoma cells were recognized by several antisera specific for intestinal aminopeptidase N (AP-N); 2) mAb H194-112 was found to immunodeplete the AP-N activity from both thymic or enterocyte BB detergent extracts; 3) the hydrophilic, Mr = 115,000 form obtained by papain treatment of thymoma or enterocyte BB could be immunopurified on H194-112 column and exhibited, after hypotonic elution, strong enzymatic activity on the AP-N substrates (i.e., leucyl or alanyl beta-derivatives); 4) mAb H194-112 was found to inhibit the AP-N activity when assayed on alanyl but not leucyl beta-naphthylamide substrate; and 5) preincubation of AP-N with mAb H194-112 prevented the inhibiting effects of bestatin and D,L-methionyl hydroxamate on AP-N activity. These data add a new member to the list of functional ectoenzymatic markers of lymphoid cells (i.e., CD10, CD13, CD26, CD55, and CD73). In view of the known immunomodulating properties of bestatin, one may speculate that the T cell-activating effects of mAb H194-112 is related to an impairment of a surface enzymatic function regulating lymphoid cell activation.

Phenotypic characterization of murine thymic microenvironments.

The thymus provides the necessary microenvironments for the differentiation of T lymphocytes. Thymic non-lymphoid cells, such as epithelial cells, macrophages and interdigitating cells are thought to promote sequential stages in T cell differentiation. However, their specific role in each step of T cell differentiation remains to be established. With the development of new monoclonal antibodies it has now become possible to characterize the different thymic stromal cell types. In this review, various aspects of thymic stromal cells and their functions in T cell differentiation are discussed, such as: (1) phenotypic analysis of stromal cells in situ; (2) the application of new "chimeric' monoclonal antibodies which "link' developing thymocytes and stromal cells; (3) perturbation of thymic microenvironments after cyclosporin-A treatment; (4) perturbation of thymic microenvironments in new transgenic mouse lines; (5) phenotypic analysis of in vitro growing stromal cell lines.

Thymic epithelial antibodies: immunohistological analysis and introduction of nomenclature.

During the Workshop "The Thymus. Histophysiology and Dynamics in the Immune System', Rolduc, april 1989, a special workshop was hold to characterize monoclonal antibodies (mAb) to thymic epithelial cells (TEC) in thymus of man, mice, and rat. Twenty-five TEC-specific mAb's were evaluated for their immunohistological staining patterns on reference thymus, thymuses during ontogeny, various other organs (all tissues obtained from the species against the mAb was raised) and thymuses of other species. In this report only immunohistological results of reference thymuses and thymuses of other species are described. Based on the staining patterns on reference thymuses, mAb could be subdivided in 5 main groups. It is proposed to use these clusters of thymic epithelial staining patterns (CTES) to designate individual mAb, awaiting the possible incorporation in existing CD nomenclature for leucocyte differentiation antigens. The present immunohistological approach will be extended by additional analysis for which a protocol was designed. The ultimate goal of this TEC-mAb workshop is to get well-characterized reagents in the analysis of TEC-associated molecules with putative function in intrathymic T-cell processing.

Establishment and characterization of mouse thymic epithelial cell lines.

Primary stromal cell cultures from fetal day-16 thymuses of Swiss mice were developed using a combination of D-valine-containing DMEM and Ham's F-12 medium supplemented with epidermal growth factor, insulin and cortisone. Using cloning cylinders and subsequent limiting dilution techniques, we obtained two clones, MTE-1 and MTE-2. The presence of cytokeratin filaments established their epithelial origin. These cells expressed class I and class II MHC antigens after induction by gamma-interferon, and lacked conventional lymphoid cell-surface markers. Their ability to form rosettes with thymocytes should allow us to identify cell-surface antigens involved in thymocyte-epithelial cell interaction. Moreover, these lines will be used to set up in vitro thymocyte maturation assays.

Lymphoid microenvironments in the thymus and lymph node.

The three-dimensional architecture of the thymus and mesenteric lymph node reveals several different stromal cell types important in the development and function of T cells. In the thymic cortex, T cells proliferate and differentiate in a meshwork of epithelial-reticular cells. They then migrate towards the medulla where they may interact with interdigitating cells. T cells migrate from the thymus through perivascular spaces, surrounding large vessels at the cortico-medullary boundary. In this area also large thymic cystic cavities are found, their function remains at present unclear. Mature "selected" T cells leave the thymus most probably by the venous bloodstream, to enter peripheral lymph nodes. Upon entering the lymph node they cross the wall of high endothelial venules. On the other hand, lymph enters the node by afferent lymphatics draining into various types of sinuses. Here, macrophages are strategically located to phagocytose and process antigen. These cells then expose antigen to T cells and B cells within the lymph node parenchyma, thus creating a microenvironment for the onset of an immune response. The various microenvironments important in T cell development and T cell function are shown in this paper using scanning electron microscopy as a dissecting tool. We discuss our morphological findings in the light of recent data on the physiology of T cell differentiation and function.

A novel T cell-activating molecule (THAM) highly expressed on CD4-CD8- murine thymocytes.

Recent studies have focused on the potential role of accessory molecules such as CD2, CD28, Thy-1, or TAP in the delivery of activating signals to thymocytes through antigen-independent pathways. To better understand the molecular interactions involved in the expansion of early thymic immigrants, rat mAb were raised against murine thymocyte-surface molecules and screened for their capacity to trigger thymocyte proliferation. One of these mAb (H194-112, IgG2a) was found to recognize a novel heterodimeric thymocyte-activating molecule (THAM) of Mr = 110,000 to 128,000. Flow cytometric analyses and staining patterns on frozen thymus sections subdivided adult thymocytes in three subsets expressing THAM at either low (10%), moderate (80%), or high (5 to 8%) cell-surface density; these cell groups were found to correspond, respectively, to the medullary, the cortical, and the immature CD4-CD8-, J11d+ thymocytes, in which the T cell precursor pool is included. Moreover, most (90%) day 16 fetal thymocytes were also found to upregulate THAM cell-surface expression. The THAMhigh cells were localized in the subcapsular area of the neonatal thymus and scattered throughout the adult organ. Cross-linked mAb H194-112 induced the proliferation of both immature and mature thymocytes in the presence of either PMA or IL-1 and IL-2. The observation that early thymocytes up-regulate THAM along with the IL-2R suggests that this molecule might be involved in an important activation pathway during thymocyte differentiation.

Liver parenchymal cells differ from the fat-storing cells in their lipid composition.

The neutral lipid and phospholipid compositions of purified sinusoidal (fat-storing, endothelial and Kupffer) cells, parenchymal cells and liver homogenates were determined by thin layer chromatography. In addition, the retinoid content of the same purified cell populations was determined by high performance liquid chromatography. From each cell type, both a lipid droplet fraction and a pellet fraction (containing the majority of the remaining cell organelles) were prepared by differential centrifugation. Electron microscopic analysis showed that lipid droplets isolated from fat-storing cells were larger (up to 8 microns) than those isolated from parenchymal cells (up to 2.5 microns). Moreover, the parenchymal lipid droplets seemed to be surrounded by a membranous structure, while the fat-storing lipid droplets seemed not to be. Both fat-storing and parenchymal cells contained high concentrations of neutral lipids, 57.9 micrograms and 71.0 micrograms/10(6) cells, respectively, while endothelial and Kupffer cells contained only 8.6 micrograms and 13.8 micrograms/10(6) cells of neutral lipids, respectively. Sixty-five percent of fat-storing cell lipid droplet fractions comprised esters of retinol and cholesterol. This combined ester fraction contained mainly retinyl esters. In addition, considerable quantities (20%) of triglycerides were present. Parenchymal cell lipid droplet fractions comprised triglycerides (62%) and cholesteryl esters (up to 30%). The pellet fractions prepared from all four cell types consisted mainly of cholesterol (41-67%) and free fatty acids (20-28%). The phospholipid content was much higher in parenchymal cells than in the sinusoidal liver cell types. The relative proportions of the four major phospholipid classes were comparable in all liver cell types analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte maturation in the human thymus. Relevance of purine nucleotide metabolism for intrathymic T cell function.

The combination of centrifugal elutriation as an efficient and reproducible method to separate thymocytes by size, micromethods to assess purine interconversion enzymes, and assessment of purine (deoxy)nucleoside inhibition of mitogen responses enabled us to study purine metabolism at the intrathymic level. Out of six fractions, four (nos. 3-6), containing medium- and large-sized lymphocytes, showed a proliferative response after stimulation with phytohaemagglutinin (PHA). In fractions 1-6 the number of cells with an immature immunological phenotype gradually decreased, and cells with the phenotype of mature cells gradually increased. The enzyme activity ratio of adenosine deaminase to purine nucleoside phosphorylase gradually decreased from 21 in fraction 1 to 7 in the last fraction (blood T-cell value, 0.7). We conclude that this enzyme activity ratio is a useful marker for intrathymic T-cell maturation stages. In PHA-responsive cell fractions (3-6), the sensitivity to inhibition of the PHA response by (deoxy)adenosine and deoxyguanosine was inversely related to the enzyme activity ratio of ecto-5'-nucleotidase to deoxycytidine kinase. These findings are compatible with the hypothesis that intracellular concentrations of phosphorylated (deoxy)nucleosides are related to this inhibition. We conclude that the differences in purine metabolism among the various (mitogen-responsive) human thymocyte fractions are related to lymphoid cell function. Since the number of cells contributing to the enzyme activities and the number of cells contributing to the proliferative response (about 15% of unseparated cells) differ considerably, it is not possible to evaluate enzyme activities in unseparated thymocytes in terms of relationships between purine metabolism and lymphocyte function.

T-cell maturation in the human thymus and tonsil: peanut agglutinin binding T lymphocytes in thymus and tonsil differ in maturation stage.

The finding of peanut agglutinin (PNA) binding capacity, supposed to be a marker of immature lymphocytes, within the T-cell population of the human thymus (58%) and tonsil (10%) prompted the comparison of maturation stages of PNA binding (PNA+) and nonbinding (PNA-) T cells in both organs. The proliferative response after mitogenic stimulation of purified PNA+ fractions was significantly less than that of purified PNA- fractions. The results of mitogen dose-response experiments, of variation in time of culture harvest, and of addition of irradiated allogeneic peripheral blood non-T cells indicated the intrinsic mitogen unresponsiveness of cells in the PNA+ fractions. The mitogen response of tonsil fractions was higher than that of thymocyte fractions. Cells with an immature immunologic phenotype were enriched in the thymocyte PNA+ fraction, and almost absent in the tonsil fractions. Both tonsil fractions contained cells with the immunologic phenotype of mature T cells, and showed a purine interconversion enzyme makeup comparable to mature T lymphocytes. It is concluded that the tonsil PNA+ T cell is a functionally immature lymphocyte which is in a further maturation stage than PNA+ or PNA- thymocytes. The presence of PNA+ T cells outside the thymus is of relevance for the clinical evaluation of PNA binding assays and suggests the occurrence of T-cell maturation within the tonsil environment.