Isolation of chromosome clusters from metaphase-arrested HeLa cells

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosomes from a cell remain together in a bundle which we call a “metaphase chromosome cluster”. Cells are arrested to 90–95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a Percoll TM density gradient. — This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca ++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47359/1/412_2004_Article_BF00327351.pd

Target cell-induced T cell activation with bi- and trispecific antibody fragments.

Previously we proposed a concept for tumor immunotherapy in which two different bispecific antibody conjugates, an anti-target x anti-CD3 and an anti-target x anti-CD28 conjugate, induce the activation of resting humanTcells upon binding to the respective tumor target cells. After in vivo application of these reagents, this model of a "target cell-induced T cell activation" envisages the destruction of target cells by in situ activated T cells. Obviously however, for in vivo application, the use of Fc-free antibody fragments is mandatory to prevent binding of the conjugates to Fc receptor-positive cells which would lead to Fc-mediated T cell activation. Here we report a simplification of published procedures for the generation of bispecific Fab-hybrid fragments, univalent for each antigen. We demonstrate that an anti-target x anti-CDS/anti-target X antiCD28 combination of such hybrids, as well as an identical combination of covalently coupled F(ab′)2 fragments, mediate "target cell-induced Tcell activation" in an in vitro test system.Thus, these reagents may be capable of inducing an in situ activation of human T cells upon systemic in vivo application according to the concept outlined above. A trispecific conjugate with anti-target, anti-CD3-and anti-CD28 specificity appears to be unsuitable for this purpose because it activates resting T cells in soluble form without requiring immobilization through binding via its anti-target portion

Intralesional Targeted Alpha Therapy for Metastatic Melanoma

The paper reports the development and application of intralesional targeted alpha therapy (TAT) for melanoma, being the first part of a program to establish a new systemic therapy.JRC.E.5-Nuclear chemistr