Comparative evaluation of the performance of two commercial kits for the detection of central nervous system tissue in meat.

The ScheBo Brainostic test, which detects neuron-specific enolase by Western blotting, and the r-Biopharm Ridascreen Risk Material ELISA test, which detects the presence of glial fibrillary acidic protein, were evaluated using meats containing spinal cord and brain central nervous system (CNS) tissue from ovine and bovine species. The meats were pork, cooked pork sausage, raw minced lamb and cooked minced lamb. Spiking of the CNS tissue ranged from 0.01 to 5%. No false-positives were observed with either test using the manufacturers' analytical protocols. The presence or absence of CNS tissue was correctly determined in 20 of 20 samples using the ScheBo Brainostic test and 18 of 20 samples using the Ridascreen tests. When results were placed in categories according to quantity of CNS tissue detected, 19 of 20 samples were classified correctly using the Brainostic test and 14 of 20 samples using the Ridascreen test. Both kits were considered appropriate for reporting the presence of 1% or more CNS tissue in meat products, but the ScheBo Brainostic test was more consistent at detecting the presence of CNS tissues below the 1% level. Overall, the format of the Ridascreen test was technically easier to use, and the data simpler to interpret.

Food allergy--towards predictive testing for novel foods.

The risks associated with IgE-mediated food allergy highlight the need for methods to screen for potential food allergens. Clinical and immunological tests are available for the diagnosis of food allergy to known food allergens, but this does not extend to the evaluation, or prediction of allergenicity in novel foods. This category, includes foods produced using novel processes genetically modified (GM) foods, and foods that might be used as alternatives to traditional foods. Through the collation and analysis of the protein sequences of known allergens and their epitopes, it is possible to identify related groups which correlate with observed clinical cross-reactivities. 3-D modelling extends the use of sequence data and can be used to display eptiopes on the surface of a molecule. Experimental models support sequence analysis and 3-D modelling. Observed cross-reactivities can be examined by Western blots prepared from native 2-D gels of a whole food preparation (e.g. hazelnut, peanut), and common proteins identified. IgEs to novel proteins can be raised in Brown Norway rat (a high IgE responder strain) and the proteins tested in simulated digest to determine epitope stability. Using the CSL serum bank, epitope binding can be examined through the ability of an allergen to cross-link the high affinity IgE receptor and thereby release mediators using in vitro cell-based models. This range of methods, in combination with data mining, provides a variety of screening options for testing the potential of a novel food to be allergenic, which does not involve prior exposure to the consumer.

Vectorial function of major histocompatibility complex class II in a human intestinal cell line.

This study explores the expression and the function of major histocompatibility complex class II in the intestinal epithelial cell line CaCo-2, which has been widely used as a model for the human gastrointestinal epithelium. Human leucocyte antigen (HLA)-DR expression on CaCo-2 cells is induceable by interferon-gamma (IFN-gamma), but responsiveness to IFN-gamma is dependent on cell differentiation and IFN-gamma availability at the basolateral cell surface. HLA-DR expression is concentrated in apical cytoplasmic vesicles and on the basolateral cell surface. Invariant chain is expressed in apical vesicles but is absent from the cell surface. Immunoprecipitation studies show a slow rate of dissociation of HLA-DR from Ii. Double labelling shows some overlap between HLA-DR expression and basolateral endosomal markers but no overlap with apical endosomal markers. Functional studies show processing and presentation of lysozyme endocytosed from the basolateral, but not apical surfaces. CaCo-2 cells may provide a useful model with which to dissect the antigen-processing pathways in polarized epithelial cells. The regulated access of antigens taken up from the gut lumen to the processing compartments may prevent overloading the immune system with antigens derived from normal gut contents.

Substratum of pleiotrophin (HB-GAM) stimulates rat CG-4 line oligodendrocytes to adopt a bipolar morphology and disperse: primary O-2A progenitor glial cells disperse similarly on pleiotrophin.

Pleiotrophin (HB-GAM), an extracellular matrix-associated protein with a high content of basic amino acid residues, is expressed in the central nervous system during late pre- and early post-natal development and promotes neurite outgrowth in vitro. Here, we show that, on a substratum of pleiotrophin formed from a 5 or 10 microg/ml solution, undifferentiated rat CG-4 line oligodendrocytes adopt a bipolar morphology and disperse over the substratum, as we have previously shown with poly-L-lysine (Rumsby et al. Neurosci. Res. Commun. 23:101-109, 1998). On pleiotrophin substrata formed from coating solutions of 1 microg/ml and below, CG-4 line cells form aggregates and do not disperse, as is also the case with poly-L-lysine. The same dispersing effect is observed with rat primary 0-2A progenitor glial cells on pleiotrophin substrata from solutions of 5 and 10 microg/ml: 0-2A cells aggregate together on pleiotrophin substrata formed from lower concentrations and do not disperse. A pleiotrophin substratum enhances proliferation of CG-4 line oligodendrocytes and primary 0-2A progenitor glial cells. The results show that pleiotrophin provides a substratum that can influence progenitor oligodendrocyte morphology, aid cell dispersion, and perhaps also enhance progenitor oligodendrocyte cell growth.

Rat oligodendrocyte O-2A precursor cells and the CG-4 oligodendrocyte precursor cell line express cadherins, beta-catenin and the neural cell adhesion molecule, NCAM.

Oligodendrocyte precursor cell migration throughout the developing central nervous system (CNS) and cessation of migration are poorly understood but are likely to involve cell adhesion molecules. The expression and distribution of neural cell adhesion molecule (NCAM), cadherins and beta-catenin were investigated in the CG-4 cell line and primary rat oligodendrocyte progenitor cells (O-2A) by immunofluorescence and Western blotting. NCAM was expressed by both cell types and was found all over the surface of both CG-4 cells and O-2A progenitor glia. The presence of a cadherin was detected in both CG-4 cells and O-2A progenitor glia, and this molecule was distributed all over the cell body and cell processes at different stages of differentiation. Beta-catenin showed a very similar distribution to that of the cadherin. We conclude that CG-4 cells are a valid model system to study cell adhesion molecule expression and function in oligodendrocyte progenitor cells.

Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells.

By raising monoclonal antibodies to the apical surface of Caco-2 cells we have identified a membrane protein (p100) that internalizes and recycles constitutively between the apical plasma membrane and endosomes in the apical cytoplasm. By applying tracers bound to the transferrin receptor, which internalizes and recycles back to the basolateral border, we demonstrate that the apical endosomes containing p100 include a subset of multivesticular bodies (MVB), which are also accessible to proteins arriving from the basolateral endosome. Tracers bound to EGF receptors and alpha-2-macroglobulin, which internalize from the basolateral border and are degraded, probably in lysosomes, also pass through the p100-containing MVB. These studies therefore suggest that the apical cytoplasm of Caco-2 cells contains a population of MVB capable of receiving membrane proteins trafficking in from both apical and basolateral borders and then routing them to a variety of cell surface and intracellular destinations. The differential distribution of apical and basolateral tracers within the 50-nm-diameter tubules connected to these p100-positive apical MVB suggests that the destination of proteins trafficking from the MVB back to apical and basolateral surfaces is determined by the tubules to which they gain access.

Transfection of vitamin D receptor cDNA into the monoblastoid cell line U937. The role of vitamin D3 in homotypic macrophage adhesion.

A 2-kB cDNA for the vitamin D receptor (VDR) was cloned in sense orientation into the plasmid pMEP4 (containing a cadmium-inducible metallothionein II promoter and a hygromycin-resistance selection gene) and transfected into monoblastoid U937 cells. The resultant cell line, DH39, expressed two species of VDR mRNA: 4.6-kb wild-type mRNA (present in native U937 cells or cells transfected with pMEP4 alone) and 2-kb transfected mRNA, which increased with cadmium treatment. Binding studies (using the active vitamin D metabolite, 1,25-dihydroxycholecalciferol (1,25-DHCC)) showed that DH39 cells contained five times more VDR per cell than controls, and ten times more after cadmium treatment. DH39 were sensitive to 1,25-DHCC: adding cadmium with 100 nM 1,25-DHCC for 72 h completely inhibited proliferation and induced concomitant differentiation. Unlike control cells, differentiation of DH39 by 1,25-DHCC led to homotypic cell-cell adhesion and formation of macrophage clusters. FACS analysis showed that 1,25-DHCC increased the number of cells expressing CD11b in both DH39 and controls, and the number of cells expressing CD11c in DH39. There was a quantitative increase in mean fluorescence intensity of expression of CD11a and CD18 in DH39. Northern blotting showed increased CD11a and CD18 mRNA in DH39. Ab inhibition of 1,25-DHCC-induced homotypic adhesion showed that CD11a/18 mediated the cell-cell clustering. CD50 expression was decreased on DH39, but the CD11a/18 ligand implicated was CD54. DH39 provides a model system not only for investigating the VDR role in 1,25-DHCC anti-proliferative effects, but also for regulation of homotypic macrophage adhesion mechanisms that are important in disease pathogenesis.

Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces.

The enterocyte-like cell line Caco-2 forms a polarized epithelium when grown on filters. We have investigated the interaction of endocytic pathways from the apical and basolateral surfaces. The transferrin receptor was an appropriate marker for the basolateral route; uptake of radiolabeled transferrin was highly polarized, and recycling of this ligand back to the basolateral surface occurred with an efficiency of 95%, even after prolonged incubations with transferrin. Using a transferrin-peroxidase conjugate to delineate the morphological pathway, we have identified an early endocytic compartment in the basolateral cytoplasm of the cells. Longer incubations revealed a deeper endocytic compartment in the apical cytoplasm. Concanavalin A complexed to gold was used to simultaneously label the apical endocytic route. After 60 min, extensive mixing of the two labels was seen in endocytic elements throughout the apical cytoplasm, including in the Golgi area, but never in the basal cytoplasm. Using a second double labeling procedure in which antitransferrin receptor antibody complexed to gold was applied to the basolateral surface for up to 2 h and free peroxidase applied to the apical surface for shorter periods, we demonstrated that this apical marker rapidly (within 5 min) reached endosomes containing antibody-gold. Our results indicate that, in Caco-2 cells, the endocytic pathways from the apical and basolateral surfaces meet in an endosomal compartment from which transferrin can still be recycled.

Basolateral secretion of kappa light chain in the polarised epithelial cell line, Caco-2.

The immunoglobulin kappa light chain is constitutively secreted in non-polarised cells. It is therefore unlikely to display any of the signals thought to be required for the selective delivery of proteins to the apical or basolateral borders of polarised epithelial cells. We have transfected the gene for the kappa light chain into a polarised epithelial cell line (Caco-2) and shown that it is secreted predominantly from the basolateral surface. Metabolically labelled endogenous secretory products show the same polarity and we conclude, therefore, that in Caco-2 cells there is a major intracellular trafficking route to the basolateral border that requires no sorting signal.