Although it is generally recognized that the function of the immune system declines with age, the nature of the underlying defects is still poorly understood. We now demonstrate the predominance of CD8+CD28− T cell clonal expansions in elderly persons who fail to produce specific Abs following influenza vaccination. These clones express effector cell markers and are mostly CD45RA+. When isolated and put into culture, they are unable to proliferate, but produce IFN-γ (but no IL-5) upon stimulation with anti-CD3 or autoantigen. These autoreactive CD8+ type 1 effector cells seem to trigger a Th1 polarization, as CD4+ T cells from elderly persons without in vivo Ab production produce Th1, but only low amounts of Th2 cytokines upon in vitro stimulation with PHA. Therefore, the increased occurrence of CD8+CD28− clonal expansions may be decisive for the development of immune deficiency in the elderly.

The appearance of inflammatory markers associated with amyloid plaques indicates a state of chronic inflammation in Alzheimer's disease (AD). Multiple epidemiological studies also suggest that patients taking anti-inflammatory drugs have a decreased risk of developing AD. Here we present evidence that inflammatory cytokines can alter the metabolism of the beta-amyloid precursor protein (betaAPP). We show that the combination of tumor necrosis factor alpha and interferon gamma triggers the production of beta-amyloid peptides and inhibits the secretion of soluble APPs by human neuronal and extraneuronal cells. The results demonstrate a new mechanism by which inflammatory components can exacerbate the fundamental pathology in AD.

Small, cysteine-rich and cationic proteins with antimicrobial activity are produced by diverse organisms of all kingdoms and represent promising molecules for drug development. The ancestor of all industrial penicillin producing strains, the ascomycete Penicillium chryosgenum Q176, secretes the extensively studied antifungal protein PAF. However, the genome of this strain harbours at least two more genes that code for other small, cysteine-rich and cationic proteins with potential antifungal activity. In this study, we characterized the pafB gene product that shows high similarity to PgAFP from P. chrysogenum R42C. Although abundant and timely regulated pafB gene transcripts were detected, we could not identify PAFB in the culture broth of P. chrysogenum Q176. Therefore, we applied a P. chrysogenum-based expression system to produce sufficient amounts of recombinant PAFB to address unanswered questions concerning the structure and antimicrobial function. Nuclear magnetic resonance (NMR)-based analyses revealed a compact β-folded structure, comprising five β-strands connected by four solvent exposed and flexible loops and an "abcabc" disulphide bond pattern. We identified PAFB as an inhibitor of growth of human pathogenic moulds and yeasts. Furthermore, we document for the first time an anti-viral activity for two members of the small, cysteine-rich and cationic protein group from ascomycetes.The increasing incidence of fatal microbial infections due to the development of resistance against licensed antimicrobial drugs raises a strong demand for new antimicrobial treatment strategies. Filamentous ascomycetes are a rich source of antimicrobial bio-molecules that have the potential for wide application in medicine and agriculture to prevent and treat microbial infections 1 . As such, the industrially relevant fungus Penicillium chrysogenum is not only a well-known producer of the β-lactam antibiotic penicillin, but also secretes small, cysteine-rich and cationic proteins with antimicrobial activity. P. chrysogenum is an ideal producer of bio-products with beneficial potential to mankind as it is fermentable and bulk production is easy and cheap 2 . Most importantly, it is recognized as a "safe organism" by the US Food and Drug Administration.The P. chrysogenum strain Q176 is the ancestor of all industrial strains used for penicillin production today 2,3 and of the strains Wisconsin 54-1255 4 and P2niaD18 5 , whose genomes were sequenced and are publicly available. P. chrysogenum Q176 secretes the antifungal protein PAF whose structural and functional properties have been extensively studied [6][7][8] . PAF represents a promising bio-molecule for novel antifungal drug development as

The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the ␤-lactam producer Penicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treated Aspergillus nidulans hyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface of A. nidulans protoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism in A. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity of A. nidulans strain FGSC1053, which carries a dominant-interfering mutation in fadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.A large number of small, basic, cysteine-rich antimicrobial proteins are produced by organisms throughout all kingdoms. They display a great variety in their primary structure, in species specificity, and in the mechanism of action.Few ascomycetes secrete strongly related antifungal proteins, which do not show any sequence homology with other antimicrobial proteins, but most of these proteins exhibit structural similarities (46): a net positive charge and numerous cysteine residues that are involved in disulfide bond formation. These properties contribute to a compact tertiary structure and a high stability against environmental impact and finally support the model of a membrane-disturbing nature. The antifungal protein PAF from P. chrysogenum and AFP from A. giganteus are the two most intensively studied peptides in the group of antifungals from ascomycetes, but the information available on their exact mechanism of action is still rather limited (32,55,68,69). PAF inhibits the growth of various important plant pathogenic and zoopathogenic filamentous fungi, e.g., Aspergillus fumigatus, A. niger, A. nidulans, and Botrytis cinerea. Previous studies revealed the induction of multifactorial detrimental effects on target organisms that include growth inhibition, reduction of cellular metabolism, severe changes in hyphal morphology, increased K ϩ efflux, and the generation of intracellular reactive oxygen species (ROS) (32, 48). PAF was found to...