Behring Institute Mitteilungen最新文献

Outer membrane protein OprF is the major outer membrane of Pseudomonas aeruginosa, and has been expressed to a similar high level in Escherichia coli from the cloned gene. It contains conserved surface epitopes, and antibodies against these epitopes can protect mice from P. aeruginosa infections. To develop the oprF gene as a carrier for foreign epitopes, linker insertion mutagenesis has been performed to introduce 12 nucleotide inserts marked by a unique PstI site. Nine such sites can accept and express a foreign epitope within the surface loop regions of OprF on the surface of E. coli. The antigenicity at a given insertion site, and the influence of the length of a model repeating malarial epitope on antigenicity, have been shown to be site-specific and apparently dependent on the nature of the surrounding amino acids at the insertion site. Immunization of mice with OprF containing a highly antigenic inserted epitope led to an epitope-specific antibody response. These data suggest that OprF has potential for use as a carrier for foreign epitopes.

The epithelial glycoprotein called secretory component (SC) is quantitatively the most important receptor of the immune system because it is responsible for external transport of locally produced polymeric IgA (pIgA) to generate remarkably large amounts of secretory IgA. Antibodies of this type constitute the major mediators of specific humoral immunity. Transmembrane SC belongs to the Ig supergene family and functions as a common pIg receptor, also translocating pentameric IgM externally to form secretory IgM. The B cells responsible for mucosal pIg production are initially stimulated in organized mucosa-associated lymphoepithelial structures, particularly the Peyer's patches in the distal small intestine; from these inductive site they migrate as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory effector site. There is currently great interest in exploiting this integrated or "common" mucosal immune system for oral vaccination against pathogenic infectious agents and also to induce tolerance in T cell-mediated autoimmune diseases. However, much remains to be learned about mechanisms for antigen uptake and processing necessary to elicit stimulatory or suppressive mucosal immune responses. Moreover, evidence is emerging for the existence of considerable regionalization with regard to functional links between inductive sites and effecter sites of mucosal immunity.

The Bcl-2 protein blocks a distal step in an evolutionarily conserved pathway for programmed cell death and apoptosis. The gene encoding this protein was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in B-cell lymphomas, where it contributes to neoplastic cell expansion by preventing cell turnover due to programmed cell death. Overexpression of BCL-2 also occurs in many other types of human tumors, including cancers of the prostate, colon, and lung, and has been associated with chemoresistance and radioresistance in some types of malignancy. Conversely, expression of BCL-2 is frequently reduced in the circulating lymphocytes of persons infected with Human Immunodeficiency Virus (HIV), which are prone to apoptotic cell death. Since the discovery of Bcl-2 a decade ago, several other cellular and viral genes encoding homologous proteins have been identified, some of which suppress cell death akin to Bcl-2 (Bcl-XL, Mcl-1, A1/Bfl-1, Nr13, Ced-9, BHRF-1) and others which promote apoptosis (Bax, Bcl-Xs, Bak, Bik, Bad). Several of these Bcl-2 family proteins are capable of physically interacting with each other through a complex network of homo- and heterodimers. The expression of some of these other BCL-2 family genes becomes altered in human cancers, as well as in the setting of ischemia and some other pathological conditions, suggesting a potentially important role for these Bcl-2 homologs in human diseases characterized by either insufficient or excessive cell death. Despite intensive investigation, the mechanisms by which Bcl-2 and its homologs control cell life and death largely remain enigmatic. Knowledge about the specific domains in Bcl-2 family proteins that are required for interactions with other proteins and for function however is beginning to provide insights into the molecular mechanisms through which these proteins regulate the programmed cell death pathway in normalcy and disease.

The ability of ligands of the tumor necrosis factor (TNF) family to induce death of cells independently of new protein synthesis provides a unique approach to molecular analysis of programmed cell death mechanisms. Sequential analysis of the protein-protein interactions by which these receptors signal, allows identification of specific molecules that participate in the cell death process and unequivocal definition of cause-effect relationships between them. Several receptors of this family, with structurally unrelated intracellular domains, have the ability to trigger cell death. some intracellular proteins that bind to the receptors and participate in the induction of their effects have been identified. Association of the Fas/APO1-interacting protein MORT1/FADD with the p55 TNF receptor-interacting protein TRADD, and the association of both MORT1/FADD and TRADD with a third protein, RIP, provide potential cross-talk mechanisms between Fas/APO1 and the p55 TNF receptor. TRAF2, a cytoplasmic protein that binds to the p75 TNF receptor, as well as to several other receptors of the TNF/NGF family, also binds to TRADD, thus further extending the range of receptors of this family that can share common signaling mechanisms. The N-terminal part of MORT1/FADD binds to a protease of the CED3/ICE family, MACH alpha. Activation of MACH alpha by the TNF/NGF receptors appears to be the most upstream enzymatic activity in the cascade of signaling for cell death.

Apoptotic cell death is used as a defence against infection by viruses. To counter this protective mechanism, some viruses carry genes whose products can inhibit progression of the apoptotic process in the host cell. As it is clear that the core cell death mechanisms have been conserved through evolution, viral genes from various sources can be used to unravel these mechanisms in mammalian cells. We have produced transgenic mice that express the cowpox gene crmA in their T cell compartment, and analysed their susceptibility to apoptosis. We have studied the effects of the baculovirus genes p35 from Autographa californica nuclear polyhedrosis virus and IAP from Orgyia pseudotsugata nuclear polyhedrosis virus on cell death induced in HeLa cells by over-expression of interleukin-1 beta converting enzyme (ICE), overexpression of the CD95-associated protein FADD, or cell death induced by treatment with TNF plus cycloheximide. These experiments indicate that viral anti-apoptosis proteins target both the activation and effector phases of the physiological cell death process.

Peripheral deletion of activated T cells has an important function in the regulation of the extent of an immune response. Upon restimulation through the T cell receptor previously stimulated cells have been shown to die by activation-induced cell death. Recent data indicate that this process is mediated by a CD95 (Fas/APO-1)/CD95 ligand interaction which induces apoptosis of the T cell. CD95 ligand (CD95-L) is absent on unactivated T cells but is readily expressed upon stimulation. Here we discuss evidence that CD95-L expression is induced by T cell receptor-mediated signals and is regulated at different levels. Different inhibitors of activation-induced cell death have been found to directly or indirectly act on the signal transduction pathway leading to CD95-L expression. CD95-L seems not only to be induced in T cells after activation but is also found constitutively expressed in many non-lymphoid tissues. This indicates that CD95-L is not only critically involved in activation-induced T cell death, but may have other functions as well. One such function is in the maintenance of immunological privilege, the protection of some tissues from potentially destructive immune responses. Thus, the regulation of CD95 expression in lymphoid and non-lymphoid cells appears to represent a key element in immune regulation.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis which have been shown to be implicated in peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. Analysis of FasL expression during mouse embryogenesis and in adult tissues reveals that FasL, although initially thought to be restricted to lymphoid cells, is constitutively expressed in a wide array of non lymphoid tissues. FasL mRNA is detectable in mouse embryos from 16.5-d onwards in epithelial cells of the submaxillary gland, and neurons of the developing nervous system. In general, FasL mRNA was not detectable in characteristic sites of embryonic programmed cell death. In the adult mouse, by RNase protection analysis, FasL mRNA is detectable in all 20 tissues tested except for the heart and pancreas. Similar analysis performed simultaneously for Fas indicates that several tissues, including the thymus, lung, spleen, small intestine, liver, seminal vesicle, prostate and uterus co-express the two genes. Most tissues constitutively co-expressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune-privileged. The pattern of FasL expression in mice suggests that FasL may be implicated in the regulation of physiological cell turnover, and the protection of tissues against potential lymphocyte mediated damage.

The p53 tumor suppressor gene is a key target for inactivation in human cancer. One of the main biological functions of the p53 protein is the positive regulation of apoptosis in response to signals such as genomic damage and the aberrant activation of certain oncogenes. A transient transfection assay was utilized in order to study the mechanism and regulation of p53-mediated apoptosis in human cancer cells. It was found that the sequence specific transcriptional activation (SST) function of p53 is essential for apoptosis in certain cell types, but not in others. This implies the existence of at least two distinct mechanisms for p53-mediated apoptosis, one requiring the activation of specific target genes, and the other being SST-independent. Typically, both mechanisms may be triggered simultaneously, and their cooperation may be required for maximal apoptotic effects. In addition, in cells lacking the function of the Rb tumor suppressor, the apoptotic activity of p53 could be inhibited by reconstitution of active Rb. p53-mediated apoptosis could also be inhibited by the protein encoded by the mdm2 oncogene. The latter inhibition required the formation of complexes between the Mdm2 protein and p53, and operated only on SST-dependent apoptosis but not SST-independent apoptosis. Together, the data imply that p53 induces apoptosis through the activation of multiple biochemical pathways, and that the efficiency of the process is dictated by the cellular context.