PREPARATION AND CHARACTERIZATION OF AN IMMUNOELECTRON MICROSCOPE TRACER CONSISTING OF A HEME-OCTAPEPTIDE COUPLED TO Fab

A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent Km of 0.2 M, an apparent vmax of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of ∼2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 Å, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent Km of 0.4 M, an apparent vmax of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits

A Heterogeneous In Vitro Three Dimensional Model of Tumour-Stroma Interactions Regulating Sprouting Angiogenesis

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model – a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis

Localization of secretory IgA, secretory component, and alpha chain in the mammary gland of lactating rabbits by immunoelectron microscopy

The different steps in the processing of secretory IgA (sIgA) of the mammary gland of lactating rabbits have been investigated with immunoelectron microscopy. The method employed consists of an indirect localization sequence, in which the immunologic reagents are allowed to diffuse into the fixed tissue prior to embedding and thin sectioning. In the first step, goat F(ab')2 fragments directed against sIgA, secretory component (SC), or alpha chain were used. To visualize the location of these first-step antibody fragments, a second Fab fragment directed against goat F(ab')2 antibody was prepared and coupled to a small heme octapeptide that possessed peroxidatic activity, the reaction product of which is visible by electron microscopy. Both the epithelial cell and the local plasmacytes of the mammary gland contain alpha chain, whereas SC is exclusively located in the epithelial cells. In the plasmacytes, both the cisternae of the rough endoplasmic reticulum and the saccules and vesicles of the Golgi complex contain alpha chain. In the epithelial cells, the elements of the Golgi complex and large apical vacuoles situated in the apical region of the cell. Based on these results, a model for processing of sIgA is proposed and discussed