Hubert Schäfer

A highly sensitive cytotoxicity assay based on the release of reporter enzymes, from stably transfected cell lines

The well-established methods of generating stably transfected cell lines, and the detection of nanomolar amounts of an enzyme in a fast and reproducible assay, were utilised to establish non-radiometric cytotoxicity assays. In these assay systems, the detection of released enzymes was used to quantitate the leakage of intracellular proteins after membrane disintegration. Target cell lines were transfected with a luciferase reporter gene under the control of a strong eucaryotic promoter. Release of the intracellular expressed enzyme into the culture supernatant occurred after membrane perforation and was measured as an indicator of cellular death. The quantitation of released enzyme was a reliable indicator of cell death initiated either by complement-mediated killing, or by cell-mediated cytotoxicity. This system was initially established with P815 mastocytoma cells as an example of a target cell line. Transfection with the firefly luciferase gene provided an intracellular enzyme absent in mammalian cells. In a parallel approach, P815 and BW5147 target cells were transfected with bacterial beta-galactosidase to provide a similar cytotoxicity system. This enzyme, however, has a considerably longer half life in tissue culture medium than luciferase. In a direct comparison between the standard 51Cr release and beta-galactosidase release, the enzyme release showed a much higher signal-to-noise ratio, i.e., low background and high induced release if spontaneous release and detergent induced maximal lysis were measured. Since a wide range of human and murine cell lines can be stably transfected and several reporter genes are available, the system should provide an alternative for conventional cytotoxicity assays. The detection of released enzymes by colorimetric or luminometric methods makes this cytotoxicity assay independent of radionuclides. The sensitivity of luminometric enzyme detection systems should also permit the measurement of apoptotic processes and might make in vivo studies of cellular death using transgenic animals feasible.

Cytopathogenicity of Balamuthia mandrillaris, an Opportunistic Causative Agent of Granulomatous Amebic Encephalitis

ABSTRACT. Balamuthia mandrillaris is a free‐living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis in humans and other mammals. Balamuthia mandrillaris is highly cytopathic but, in contrast to the related Acanthamoeba, does not feed on bacteria and seems to feed only on eukaryotic cells instead. Most likely, the cytopathogenicity of B. mandrillaris is inseparable from its infectivity and pathogenicity. To better understand the mechanisms of B. mandrillaris cytopathogenicity, an assay for measuring amebic cytolytic activity was adapted that is based on the release of a reporter enzyme by damaged target cells. The ameba is shown to lyse murine mastocytoma cells very efficiently in a time‐ and dose‐related manner. Furthermore, experiments involving semipermeable membranes and phagocytosis inhibitors indicate that the cytolytic activity of B. mandrillaris is essentially cell contact‐dependent. Standard and fluorescence light microscopy, as well as scanning and transmission electron microscopy support and extend these findings at the ultrastructural level.

Immune reactions of CD4- and CD8-positive T cell subpopulations in spleen and lymph nodes of guinea pigs after vaccination with Bacillus Calmette Guerin.

Vaccination of guinea pigs with Mycobacterium bovis BCG confers partial resistance against infection with Mycobacterium tuberculosis. Induction of immunity is associated with a strong T cell response. The reactions of the cytotoxic and helper T lymphocyte subsets after BCG vaccination were analyzed by cytofluorometry and in functional tests. The relative number of CD8(+) T cells in the spleen increased substantially after injection of BCG. In vitro restimulation after immunization induced a strong proliferative response but no cytotoxic reactions of CD8(+) T cells against BCG-infected macrophages. A specific induction of IFN-gamma and RANTES mRNA was observed after vaccination particularly in CD8(+) but not in CD4(+) T cells of the lymph nodes.

Tools for cellular immunology and vaccine research the in the guinea pig: Monoclonal antibodies to cell surface antigens and cell lines

The use of monoclonal antibodies directed against membrane proteins of leukocytes has greatly contributed to our understanding of the function and development of the immune system. Meanwhile these reagents provide valuable tools in many fields of research, stretching far beyond immunology and hematology. For guinea pigs only a limited number of such reagents have been described, and the information about availability and specificity is scattered over many years and journals. We provide an overview on the monoclonal antibodies produced since the technique was applied first in this species, with a focus on those reagents which have been characterized in more detail, and which should still be available either commercially or directly from the labs that created them.

T-lymphocyte responses in guinea pigs vaccinated with foot-and-mouth disease virus

The guinea pig provides an alternative experimental model for analysis of the immune response against foot-and-mouth disease virus (FMDV). The cellular immune response against FMDV in this experimental animal is unknown and was analyzed by in vivo and in vitro studies. In guinea pigs immunized with an FMDV A5 vaccine, a marked change in T-lymphocyte count appeared. For analyzing which functional T-cell compartment was affected, immunofluorescence studies, using monoclonal antibodies directed against differentiation antigens on guinea pig lymphoid cells, were performed. The proliferating T-cells were predominantly CD4-positive and, therefore, helper cells. T-cells from these animals were re-stimulated in vitro with homologous inactivated virus. The antigen-specific proliferative response of the T-cells in vitro was measured using the thymidine incorporation assay. A proliferative response to FMDV was observed that depended on the dose of the antigen. High concentration of virus had an inhibitory effect on T-cell proliferation. These data indicate that the guinea pig is a useful model for analysis of T-cell mediated mechanisms in the pathogenesis and immunity of foot-and-mouth disease.

Analysis of guinea pig leukocyte antigens using interspecies T cell hybrids

In the guinea pig system, there are no T lymphocyte tumors available. In addition, in this species the production of T cell lines in vitro for analysis of T cell markers or function proved to be difficult. As an alternative approach, guinea pig X mouse and guinea pig X rat T cell hybridomas were obtained by fusion of guinea pig T cells with HAT-sensitive mouse or rat thymomas. In ten fusions a total of 578 hybrids were produced and kept in culture for periods ranging from weeks up to several months. The highest yield was obtained when the Mb C 12 line was used as tumor parent. BW 5147 and the rat thymoma W/Fu(C58NT)D gave slightly lower fusion efficiencies. The yield depended also on the pretreatment of the parental cells. Activation by MLR proved to be most effective. A number of interspecies hybrids expressed guinea pig T cell differentiation antigens as detected by a cell ELISA. The positive hybrids were recloned several times and exhibited a stable expression of these markers, even after continuous culture for more than 2 months. Western blot analysis was used to confirm antigen expression at the protein level by comparing the hybrids with both parental cells. The hybrids expressed the proteins of the guinea pig lineage. No reaction was observed with the murine or rat thymomas. Therefore, the application of interspecies T cell hybrids provides an alternative for studies of guinea pig T cells. The cells are easy to grow and to clone and may be stored frozen in liquid nitrogen. The hybrids should permit analysis at the clonal level also for functional studies.

Temporal changes in the gene signatures of BCG-vaccinated guinea pigs in response to different mycobacterial antigens

Mycobacterium bovis BCG-vaccination in the guinea pig model of tuberculosis (TB) is sufficiently protective that candidate TB vaccines are judged against this. Little is understood about how the BCG vaccine works and, in the absence of a definitive correlate of protection, it is difficult to interpret the significance of novel vaccine induced host responses. Here an extended custom-made microarray (86 guinea pig genes) was used to dissect temporal changes in BCG-vaccine induced gene signatures to different mycobacterial antigens. Initially at 4h, pro-inflammatory genes such as IL-1α, IL-1β, IL-8 and GRO were up-regulated (P<0.001) and these were then superseded by IFN-γ and GM-CSF (at 12 and 20h) post-stimulation, ex vivo with PPD. Similar genes were seen following stimulation with viable BCG but with the addition of IL-23 (P<0.01) after 8h. Our results suggest that temporal changes in the up- and down-regulation of a variety of genes are required to trigger a successful protective response to TB in guinea pigs. This provides base-line information against which new TB vaccines can be compared.

Effector functions of CD8-positive guinea pig T lymphocytes

The response of guinea pig T lymphocytes to different stimuli was analysed with focus on the functions of CD8-positive T cells, which so far had been poorly defined in this animal model. For identification and purification of guinea pig cytotoxic T lymphocytes, three monoclonal antibodies, directed against the CD8 differentiation antigen were characterized and compared with respect to expression pattern and biochemical characteristics of the corresponding cell surface antigen. The antibodies were used for the identification of the cytotoxic T lymphocyte subpopulation within alloreactive T cell lines, and for the depletion of CD8-positive cells in in vitro assays. Purified CD4- and CD8-positive cells were tested for their ability to proliferate in response to antigen, mitogen or anti-guinea pig Thy-1 monoclonal antibodies. Both, CD4- and CD8-positive cells showed IL-2 release and subsequent proliferation after polyclonal stimulation. Cytotoxic activity in CD8-positive alloreactive T cells was expressed in vitro only after repeated stimulation.

Identification and comparative analysis of a genomic island in Mycobacterium avium subsp. hominissuis

Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacterium causing opportunistic infections. The objective of this study was to identify flexible genome regions in MAH isolated from different sources. By comparing five complete and draft MAH genomes we identified a genomic island conferring additional flexibility to the MAH genomes. The island was absent in one of the five strains and had sizes between 16.37 and 84.85 kb in the four other strains. The genes present in the islands differed among strains and included phage‐ and plasmid‐derived genes, integrase genes, hypothetical genes, and virulence‐associated genes like mmpL or mce genes.

Production of antibodies to the constant region of the mouse T‐cell‐receptor β‐chain

To generate antibodies to the constant region of the mouse T‐cell‐receptor β chain, the corresponding cDNA clone 86T5 was subcloned into the bacterial expression vector pEX2. The induced hybrid protein consisting of a 30 kDa T‐cell‐receptor segment and a β‐galactosidase carrier moiety was used for immunization. In Western blots using lysates of a T‐cell‐receptor‐positive T‐cell hybrid, the antisera obtained reacted with a 42 kDa protein under reducing and a 85 kDa protein under nonreducing conditions. The antisera did not react in binding assays with intact T lymphocytes, i.e. membrane‐associated T‐cell‐receptor protein.