Werner Haas

Poly- and monoclonal antibodies against recombinant rat brain calbindin D-28K were produced to map its selective distribution in the central nervous system.

Abstract: Many processes in the CNS depend on calcium. The calcium signal is transduced into an intracellular response via Ca2+‐binding proteins, including calbindin D‐28K. In many laboratories, polyclonal antibodies against chicken in testinal calbindin D‐28K have been used to study its localization in the brain (normal and degenerated) of various species, including humans, but some of these antisera cross‐reacted with other proteins, including calretinin. We purified recombinant rat brain calbindin D‐28K to raise antisera in rabbits and purified a recombinant rat–chicken calbindin D28K hybrid protein to immunize mice for the generation of monoclonal antibodies. These antisera were highly specific for calbindin D‐28K, as demonstrated by two‐dimensional Western blotting analysis. Immunohistochemical analyses combined with in situ hybridization studies demonstrated that calbindin D‐28K in the Purkinje cells of the cerebellum is independent of vitamin D. The antibodies described here will be important tools for studying the regulation of expression of calbindin D‐28K and its biological function in the brain and in the PNS.

The development and function of γδ T cells

In June of this year, a small group of researchers came together at the inspiring location of Segovia, Spain to exchange information and opinions on gamma delta T cells. In this report the new, unpublished findings presented, and the ideas that germinated during the meeting are discussed in the context of recent gamma delta literature.

Haptenation of Viable Biological Carriers

Publisher Summary This chapter discusses the problem of attaching defined molecules to complex carriers such as proteins, bacteriophages, or cells without interfering with the functional integrity of the carriers. If the hapten-carrier complex is to be used as a tool for the analysis of immunological specificity, it must be made certain that the observed biological effects are because of the attached hapten and not because of the side-effects of the procedure. Although many haptens may be coupled via the activated N-hydroxysuccinimide carboxyl ester, there are some restrictions. Many haptens bear a carboxyl group that may be converted immediately to the activated ester. Those with no carboxyl but some other reactive group may be attached to an appropriate bridging molecule bearing both an acceptor group for the hapten and a carboxyl group that is subsequently esterified. Haptens presented on intermediate molecules may have antigenic properties different from those coupled directly.

MURINE CYTOLYTIC T-CELL LINES : STABILITY OF FUNCTIONAL PHENOTYPE AND EXPRESSION OF CELL SURFACE MARKERS

ABSTRACT The stability of the lytic activity of two cloned murine cytolytic T-cell lines was investigated by analysis of subclones. In one line a large degree of interclonal variation persisted even after repeated sub-cloning. Variation among subclones of the other line was lower. The frequency of totally inactive variants does not exceed a few percent in subclones of either line several months after derivation. These lines express Thy-1 and Ly-2 antigens but not Ly-1. Initial expression of Ly-3 was lost in one line.

Knock out Mice Models for Immunodeficiency Diseases

The impressive progress in transgenic techniques in mice within the past ten years has opened up valuable new experimental approaches to immunologists. For many research purposes it is of great interest to eliminate a specific gene or at least to prevent the normal function of its product. A well known example are transgenic mice with rearranged antigen receptor genes preventing the diversification of whole lymphocyte subsets via allelic exclusion. Such antigen receptor transgenic mice have been successfully used to study T- and B-cell development (Storb 1987, Goodnow et al 1988, von Boehmer 1990). The direct modification of genes in the mouse germline could be achieved in the past only by insertional mutagenesis using transgenes or retroviruses, and chemical- or radiation-induced mutagenesis (for review see Jaenisch 1988). The limitation of these strategies is that the gene to be mutated and the exact nature of the mutation cannot be predetermined. The accuracy by which mutations can be introduced into the mouse genome has been greatly improved with the development of gene targeting techniques within the last four years allowing the modification of a particular gene in a predetermined manner (for review see Capecchi 1989, Gridley et al 1991, Bradley et al 1992).

17 – Cytolytic T Cell Clones and Hybridomas

Publisher Summary This chapter provides an overview of cytolytic T-cell clones and hybridomas. Cloned T-cell populations are required for the biochemical and genetic analysis of antigen receptors, lymphokine receptors, and of secreted lymphokines. T-cell clones can also be used to study the antigen-dependent regulation of lymphokine secretion and expression of receptors for lymphokines. The two types of CTL clones are distinguished according to their growth requirements: (1) CTL-A clones that are maintained in culture by the regular addition of stimulator cells and T-cell growth factor (TCGF) and (2) CTL-B clones that are grown in medium with TCGF in the absence of any stimulator or feeder cells. More types of CTL clones are currently distinguished according to their growth requirements and antigen-regulated expression of lymphokine receptors, in addition to their secretion of lymphokines. The chapter describes the methodology used to obtain the various types of CTL clones. It also describes the methods used to generate cytolytic T-cell hybridomas. It has been recently found that antigenic stimulation regulates the level of expression of receptors for T-cell growth factor.