Florentine Marx

Lack of Antibody Production Following Immunization in Old Age: Association with CD8+CD28− T Cell Clonal Expansions and an Imbalance in the Production of Th1 and Th2 Cytokines

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.

Antifungal Protein PAF Severely Affects the Integrity of the Plasma Membrane of Aspergillus nidulans and Induces an Apoptosis-Like Phenotype

ABSTRACT The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the β-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.

Characterization of the Penicillium chrysogenum antifungal protein PAF.

The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF (Penicillium antifungal protein). In this study, the antifungal activity of PAF is described. PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected. PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching. Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi. In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations.

Flatworm stem cells and the germ line: Developmental and evolutionary implications of macvasa expression in Macrostomum lignano

We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA interference were developed for M. lignano to study gene expression and function. Macvasa expression was followed during postembryonic development, regeneration and in starvation experiments. We were able to follow gonad formation in juveniles and the reformation of gonads from stem cells after amputation by in situ hybridization and a specific Macvasa antibody. Expression of macvasa in the germ cells was highly affected by feeding conditions and correlated with the decrease and regrowth of the gonads. RNA interference showed specific down-regulation of macvasa mRNA and protein. The absence of Macvasa did not influence gonad formation and stem cell proliferation. Our results corroborate the exclusive nature of the flatworm stem cell system but challenge the concept of a solely postembryonic specification of the germ line in Platyhelminthes. We address the transition of somatic stem cells to germ cells and speculate on Macrostomum as a system to unravel the mechanisms of preformation or epigenesis in the evolution of germ line specification from somatic stem cells.

The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies

Abstract.In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed.

Small, basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression, structure, function and potential application

Peptides and proteins with antimicrobial activity are produced throughout all kingdoms in nature, from prokaryotes to lower and higher eukaryotes, including fungi, plants, invertebrates and vertebrates. These proteins contribute to an important constitutive or induced defense mechanism of the producer against microorganisms. According to their variety in structure and function, these proteins are classified arbitrarily into groups that are based on their mechanism of action, their structure and their similarity to other known proteins. The present review focuses on a new group of antimicrobial proteins, namely small, basic and cysteine-rich antifungal proteins, which are secreted from filamentous fungi of the group Ascomycetes. These proteins are encoded by orthologous genes and exhibit both similarities and differences concerning their species-specificity, primary structure, protein activity and target sites. The properties of these proteins, their possible mode of action and their potential application for human benefits are discussed in comparison with other already well known antimicrobial proteins.

Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity

An abundantly secreted, highly basic 12-kDa protein (PAF) was purified from the culture medium of Penicillium chrysogenum (Pc). Based on the N-terminal amino acid (aa) sequence of the protein, an oligodeoxyribonucleotide probe was derived and used for amplification of the encoding cDNA by PCR. This cDNA fragment encodes a Cys-rich preproprotein of 92 aa which appears to be processed to a mature product of 55 aa. The deduced aa sequence of the preproprotein reveals 42.6% identity to an antifungal protein (AFP) of Aspergillus giganteus. Agar diffusion tests confirmed that the Pc protein exhibits antifungal activity. In order to investigate the promoter region and the structural organization of the paf gene, a genomic 6-kb fragment was isolated and partially sequenced. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of a coding region of 279 bp which is interrupted by two introns of 76 and 68 bp in length. In the promoter region, a typical TATA box, a motif resembling the fungal carbon catabolite repression element, as well as several putative GATA factor binding motifs, were found. Northern blot analysis indicated that the regulation of paf expression occurs at the level of mRNA transcription and is under control of carbon catabolite and nitrogen metabolite repression regulatory circuits.

Immunization with β-amyloid: could T-cell activation have a harmful effect?

We read with interest the comments of Karen Duff regarding the article by Schenk et al. on the prevention of Alzheimer’s disease (AD)-like pathology in the PDAPP-mouse by immunization with β-amyloid (Aβ)1xCuring amyloidosis: will it work in humans?. Duff, K. Trends Neurosci. 1999; 22: 485–486Abstract | Full Text | Full Text PDF | PubMed | Scopus (14)See all References, 2xImmunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Schenk, D. et al. Nature. 1999; 400: 173–177Crossref | PubMed | Scopus (2477)See all References. We agree with most of the issues raised, but would like to make one additional point. According to recent research, human autoreactive lymphocytes can indeed recognize Aβ and might therefore be important for its elimination3xAPP peptides stimulate lymphocyte proliferation in normals, but not in patients with Alzheimer’s disease. Trieb, K. et al. Neurobiol. Aging. 1996; 17: 541–547Abstract | Full Text PDF | PubMedSee all References, 4xThe possible role of the immune system in Alzheimer’s disease. Marx, F. et al. Exp. Gerontol. 1998; 33: 871–881Crossref | PubMed | Scopus (39)See all References. This recognition mechanism seems impaired in individuals with AD, and, on the basis of these findings, the possibility has been raised that immunization against Aβ might prevent and treat AD in humans4xThe possible role of the immune system in Alzheimer’s disease. Marx, F. et al. Exp. Gerontol. 1998; 33: 871–881Crossref | PubMed | Scopus (39)See all References4. However, the likelihood that such immunization might lead to harmful side effects is of great concern. In particular, the consequences of T-lymphocyte activation after immune-system stimulation with Aβ.In the article by Schenk et al.2xImmunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Schenk, D. et al. Nature. 1999; 400: 173–177Crossref | PubMed | Scopus (2477)See all References2, no information was provided on the exact nature of the immune response that occurs in mice. As expected for a peptide antigen, and confirmed by the presence of Aβ-reactive T cells, Aβ is certain to trigger T-cell activation and antibody production in humans. Aβ-reactive T cells might be part of a natural line of defence against the accumulation of Aβ, which is a toxic metabolite. Aβ-directed T-cell reactivity is relatively weak in healthy individuals3xAPP peptides stimulate lymphocyte proliferation in normals, but not in patients with Alzheimer’s disease. Trieb, K. et al. Neurobiol. Aging. 1996; 17: 541–547Abstract | Full Text PDF | PubMedSee all References3 and, therefore, unlikely to cause tissue damage. In order to achieve substantial antibody production after immunization, a much stronger T-cell response would have to be induced. This could be detrimental for several reasons. Aβ-specific T-cell lines contain a high percentage of CD8-positive cytotoxic T cells, which are capable of lysing cells that overproduce Aβ (Ref.5xTransfected human B cells: a new model to study the functional and immunostimulatory consequences of APP production. Marx, F. et al. Exp. Gerontol. 1999; 34: 783–795Crossref | PubMed | Scopus (13)See all ReferencesRef.5). When activated in vivo, these cells are likely to cause tissue damage in peripheral organs and in the brain, which they can enter as activated cells6xImmune reactivity in the nervous system: modulation of T-lymphocyte activation by glial cells. Wekerle, H. et al. J. Exp. Biol. 1987; 132: 43–57PubMedSee all References6. Cytokine production by T cells will presumably represent another problem, as Aβ-specific T cells produce a large amount of interferon γ (IFNγ; Ref. 5xTransfected human B cells: a new model to study the functional and immunostimulatory consequences of APP production. Marx, F. et al. Exp. Gerontol. 1999; 34: 783–795Crossref | PubMed | Scopus (13)See all ReferencesRef. 5). This cytokine triggers the production of Aβ40 and Aβ42 , in combination with tumour necrosis factor α (TNFα), and inhibits the secretion of soluble amyloid precursor proteins (APPs) by human neuronal and extraneuronal cells7xTNFalpha plus IFNgamma induce the production of Alzheimer beta-amyloid peptides and decrease the secretion of APPs. Blasko, I. et al. FASEB J. 1999; 13: 63–68PubMedSee all References7, thus completing a vicious circle. IFNγ can also be harmful in other respects, as it increases the production of TNFα and oxygen radicals by microglial cells after they have been stimulated with Aβ (Ref.8xActivation of microglial cells by beta-amyloid protein and interferon-gamma. Meda, L. et al. Nature. 1995; 374: 647–650Crossref | PubMedSee all ReferencesRef.8).For this and other reasons, great caution will be needed when immunization strategies against amyloidosis are designed for humans. It will have to be the goal of immunological interventions to achieve maximal clearance of the pathogen while minimizing side-effects, such as those caused by inappropriate T-cell activation. This might be achieved by the diversion of the immune response from a type 1 (IFNγ and TNFα) to a type 2 (interleukin 4) cytokine production pattern9xThe mechanism of in vitro T helper cell type 1 to T helper cell type 2 switching in highly polarized Leishmania major-specific T cell populations. Mocci, S. et al. J. Immunol. 1997; 158: 1559–1564PubMedSee all References9, the administration of certain Aβ epitopes that can stimulate those T cells that provide help to B cells (but no cytotoxic cells), or by similar responses. The success of a potentially revolutionary approach to prevent degenerative diseases might otherwise be endangered.

The Antifungal Protein AFP from Aspergillus giganteus Inhibits Chitin Synthesis in Sensitive Fungi

ABSTRACT The antifungal protein AFP from Aspergillus giganteus is highly effective in restricting the growth of major human- and plant-pathogenic filamentous fungi. However, a fundamental prerequisite for the use of AFP as an antifungal drug is a complete understanding of its mode of action. In this study, we performed several analyses focusing on the assumption that the chitin biosynthesis of sensitive fungi is targeted by AFP. Here we show that the N-terminal domain of AFP (amino acids 1 to 33) is sufficient for efficient binding of AFP to chitin but is not adequate for inhibition of the growth of sensitive fungi. AFP susceptibility tests and SYTOX Green uptake experiments with class III and class V chitin synthase mutants of Fusarium oxysporum and Aspergillus oryzae showed that deletions made the fungi less sensitive to AFP and its membrane permeabilization effect. In situ chitin synthase activity assays revealed that chitin synthesis is specifically inhibited by AFP in sensitive fungi, indicating that AFP causes cell wall stress and disturbs cell integrity. Further evidence that there was AFP-induced cell wall stress was obtained by using an Aspergillus niger reporter strain in which the cell wall integrity pathway was strongly induced by AFP.

Functional aspects of the solution structure and dynamics of PAF – a highly‐stable antifungal protein from Penicillium chrysogenum

Penicillium antifungal protein (PAF) is a promising antimycotic without toxic effects on mammalian cells and therefore may represent a drug candidate against the often lethal Aspergillus infections that occur in humans. The pathogenesis of PAF on sensitive fungi involves G‐protein coupled signalling followed by apoptosis. In the present study, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by NMR. We demonstrate that PAF belongs to the structural classification of proteins fold class of its closest homologue antifungal protein from Aspergillus giganteus. PAF comprises five β‐strands forming two orthogonally packed β‐sheets that share a common interface. The ambiguity in the assignment of two disulfide bonds out of three was investigated by NMR dynamics, together with restrained molecular dynamics calculations. The clue could not be resolved: the two ensembles with different disulfide patterns and the one with no S–S bond exhibit essentially the same fold. 15N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time are appropriate for a compact monomeric protein of this size. Using site‐directed mutagenesis, we demonstrate that the highly‐conserved and positively‐charged lysine‐rich surface region enhances the toxicity of PAF. However, the binding capability of the oligosaccharide/oligonucleotide binding fold is reduced in PAF compared to antifungal protein as a result of less solvent‐exposed aromatic regions, thus explaining the absence of chitobiose binding. The present study lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins.