Crf2 - A novel antigen of Aspergillus fumigatus

Aspergillus fumigatus ist ein ubiquitärer Schimmelpilz, der durch Inhalation seiner Sporen bei immunsupprimierten und immungeschwächten Patienten eine letale Invasive Aspergillose (IA) auslöst. Aus der mRNA eines klinischen Patientenisolats wurde die cDNA einer neuen, sequenzhomologen Variante der Glykosylhydrolase Crf1 isoliert, die als Crf2 bezeichnet wurde. Rekombinant hergestelltes Crf2 erwies sich in einer aktiven Immunisierung gegen die IA im Mausmodell als protektiv und die Mausseren wurden zur Kartierung der immunogenen Epitope von Crf2 und der sequenzhomologen Glykosylhydrolasen Crf1 und Asp f16 eingesetzt. In der Immunfluoreszenzmikroskopie wurden mit den Immunseren ausschließlich Bereiche aktiven Wachstums auf den Hyphen von A. fumigatus gefärbt und nicht seine ruhenden Sporen. Für die Generierung rekombinanter Crf2-spezifischer Antikörper wurde die Phagen-Display-Technologie eingesetzt. Die Antikörperselektion wurde mit der Antikörpergenbibliothek HAL4/7 und der neu generierten Makaken-Immunantikörpergenbibliothek MAYKI, mit jeweils zwei unterschiedlichen Selektionsstrategien, durchgeführt. Die Wahl der Selektionsmethode hatte für die isolierten Antikörper einen erheblichen Einfluss auf die Art der von ihnen erkannten Epitope. Es wurden gegen direkt immobilisiertes Crf2-Protein nur scFv-Fragmente gegen lineare Epitope und gegen das biotinylierte Antigen in Lösung nur Antikörperfragmente gegen konformationelle Epitope isoliert. Für Antikörperfragmente gegen lineare Epitope des Crf2-Proteins, wurde im capture-ELISA eine geringere Nachweisgrenze festgestellt und zudem eine intensivere Färbung der Hyphen in der Immunfluoreszenz. Die rekombinanten anti-Crf2 Antikörper zeigten ein ähnliches Färbemuster wie die anti-Crf2 Mausseren und wiesen keine Kreuzreaktionen mit anderen humanpathogenen Pilzen auf. Abschließend wurde die Affinität der isolierten Antikörper mittels Oberflächenplasmonresonanztechnologie bestimmt.Aspergillus fumigatus is an ubiquitous mould that causes a lethal invasive aspergillosis (IA) in immunosuppressed and immunocompromised persons via the inhalation of its spores. The cDNA of a new sequence homologue variant of the glycosylhydrolase crf1, called crf2, was isolated from the mRNA of a clinical isolate. The recombinant Crf2-protein was found to be protective in an active immunisation with mice against IA. The immunogenic epitopes of the Crf2-protein and its sequence homologues glycosylhydrolases Crf1 and Asp f16 were determined using these mice sera. Contrary to dormant spores, with these sera were in the immunofluorescence microscopy exclusevely areas of active growing, on hyphaes of A. fumigatus, stained. The phage-display-technology was used to generate recombinant anti-Crf2 antibodies. The antibody selection was performed with the antibody gene library HAL 4/7 and the new generated immune antibody gene library MAYKI, derived from a macaque, in two different selection stratagies. The choice of selection strategy had an enormous influence on the character of the bound epitopes. ScFvs against linear epitopes were only isolated on direct coated Crf2-protein and scFvs against conformational epitopes were isolated when the selection was performed with in vivo biotinylated protein in solution. The detection limit of soluble antigen in the Crf2-capture-ELISA was lower when using scFvs binding linear epitopes and additional showed these scFvs a more intensive staining of hyphae in the immunofluorescence. The recombinant anti-Crf2 antibodies had a similar staining pattern as the anti-Crf2 immune sera of Crf2-immunised mice and did not show any cross reaction with other humanpathogenic fungi. Finally, the affinity for some antibody fragments were determined via the surface plasmon resonance technolog

Crf2 - A novel antigen of Aspergillus fumigatus

Aspergillus fumigatus ist ein ubiquitärer Schimmelpilz, der durch Inhalation seiner Sporen bei immunsupprimierten und immungeschwächten Patienten eine letale Invasive Aspergillose (IA) auslöst. Aus der mRNA eines klinischen Patientenisolats wurde die cDNA einer neuen, sequenzhomologen Variante der Glykosylhydrolase Crf1 isoliert, die als Crf2 bezeichnet wurde. Rekombinant hergestelltes Crf2 erwies sich in einer aktiven Immunisierung gegen die IA im Mausmodell als protektiv und die Mausseren wurden zur Kartierung der immunogenen Epitope von Crf2 und der sequenzhomologen Glykosylhydrolasen Crf1 und Asp f16 eingesetzt. In der Immunfluoreszenzmikroskopie wurden mit den Immunseren ausschließlich Bereiche aktiven Wachstums auf den Hyphen von A. fumigatus gefärbt und nicht seine ruhenden Sporen. Für die Generierung rekombinanter Crf2-spezifischer Antikörper wurde die Phagen-Display-Technologie eingesetzt. Die Antikörperselektion wurde mit der Antikörpergenbibliothek HAL4/7 und der neu generierten Makaken-Immunantikörpergenbibliothek MAYKI, mit jeweils zwei unterschiedlichen Selektionsstrategien, durchgeführt. Die Wahl der Selektionsmethode hatte für die isolierten Antikörper einen erheblichen Einfluss auf die Art der von ihnen erkannten Epitope. Es wurden gegen direkt immobilisiertes Crf2-Protein nur scFv-Fragmente gegen lineare Epitope und gegen das biotinylierte Antigen in Lösung nur Antikörperfragmente gegen konformationelle Epitope isoliert. Für Antikörperfragmente gegen lineare Epitope des Crf2-Proteins, wurde im capture-ELISA eine geringere Nachweisgrenze festgestellt und zudem eine intensivere Färbung der Hyphen in der Immunfluoreszenz. Die rekombinanten anti-Crf2 Antikörper zeigten ein ähnliches Färbemuster wie die anti-Crf2 Mausseren und wiesen keine Kreuzreaktionen mit anderen humanpathogenen Pilzen auf. Abschließend wurde die Affinität der isolierten Antikörper mittels Oberflächenplasmonresonanztechnologie bestimmt.Aspergillus fumigatus is an ubiquitous mould that causes a lethal invasive aspergillosis (IA) in immunosuppressed and immunocompromised persons via the inhalation of its spores. The cDNA of a new sequence homologue variant of the glycosylhydrolase crf1, called crf2, was isolated from the mRNA of a clinical isolate. The recombinant Crf2-protein was found to be protective in an active immunisation with mice against IA. The immunogenic epitopes of the Crf2-protein and its sequence homologues glycosylhydrolases Crf1 and Asp f16 were determined using these mice sera. Contrary to dormant spores, with these sera were in the immunofluorescence microscopy exclusevely areas of active growing, on hyphaes of A. fumigatus, stained. The phage-display-technology was used to generate recombinant anti-Crf2 antibodies. The antibody selection was performed with the antibody gene library HAL 4/7 and the new generated immune antibody gene library MAYKI, derived from a macaque, in two different selection stratagies. The choice of selection strategy had an enormous influence on the character of the bound epitopes. ScFvs against linear epitopes were only isolated on direct coated Crf2-protein and scFvs against conformational epitopes were isolated when the selection was performed with in vivo biotinylated protein in solution. The detection limit of soluble antigen in the Crf2-capture-ELISA was lower when using scFvs binding linear epitopes and additional showed these scFvs a more intensive staining of hyphae in the immunofluorescence. The recombinant anti-Crf2 antibodies had a similar staining pattern as the anti-Crf2 immune sera of Crf2-immunised mice and did not show any cross reaction with other humanpathogenic fungi. Finally, the affinity for some antibody fragments were determined via the surface plasmon resonance technolog

Thin-disk laser-pumped OPCPA system delivering 4.4 TW few-cycle pulses

We present an optical parametric chirped pulse amplification (OPCPA) system delivering 4.4 TW pulses centered at 810 nm with a sub-9 fs duration and a carrier-envelope phase stability of 350 mrad. The OPCPA setup pumped by sub-10 ps pulses from two Yb:YAG thin-disk lasers at 100 Hz repetition rate is optimized for a high conversion-efficiency. The terawatt pulses of the OPCPA are utilized for generating intense extreme ultraviolet (XUV) pulses by high-order harmonic generation, achieving XUV pulse energies approaching the microjoule level. © 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreemen

Microbiological, histological, immunological, and toxin response to antibiotic treatment in the mouse model of Mycobacterium ulcerans disease.

Mycobacterium ulcerans infection causes a neglected tropical disease known as Buruli ulcer that is now found in poor rural areas of West Africa in numbers that sometimes exceed those reported for another significant mycobacterial disease, leprosy, caused by M. leprae. Unique among mycobacterial diseases, M. ulcerans produces a plasmid-encoded toxin called mycolactone (ML), which is the principal virulence factor and destroys fat cells in subcutaneous tissue. Disease is typically first manifested by the appearance of a nodule that eventually ulcerates and the lesions may continue to spread over limbs or occasionally the trunk. The current standard treatment is 8 weeks of daily rifampin and injections of streptomycin (RS). The treatment kills bacilli and wounds gradually heal. Whether RS treatment actually stops mycolactone production before killing bacilli has been suggested by histopathological analyses of patient lesions. Using a mouse footpad model of M. ulcerans infection where the time of infection and development of lesions can be followed in a controlled manner before and after antibiotic treatment, we have evaluated the progress of infection by assessing bacterial numbers, mycolactone production, the immune response, and lesion histopathology at regular intervals after infection and after antibiotic therapy. We found that RS treatment rapidly reduced gross lesions, bacterial numbers, and ML production as assessed by cytotoxicity assays and mass spectrometric analysis. Histopathological analysis revealed that RS treatment maintained the association of the bacilli with (or within) host cells where they were destroyed whereas lack of treatment resulted in extracellular infection, destruction of host cells, and ultimately lesion ulceration. We propose that RS treatment promotes healing in the host by blocking mycolactone production, which favors the survival of host cells, and by killing M. ulcerans bacilli

Opportunities for organoids as new models of aging.

The biology of aging is challenging to study, particularly in humans. As a result, model organisms are used to approximate the physiological context of aging in humans. However, the best model organisms remain expensive and time-consuming to use. More importantly, they may not reflect directly on the process of aging in people. Human cell culture provides an alternative, but many functional signs of aging occur at the level of tissues rather than cells and are therefore not readily apparent in traditional cell culture models. Organoids have the potential to effectively balance between the strengths and weaknesses of traditional models of aging. They have sufficient complexity to capture relevant signs of aging at the molecular, cellular, and tissue levels, while presenting an experimentally tractable alternative to animal studies. Organoid systems have been developed to model many human tissues and diseases. Here we provide a perspective on the potential for organoids to serve as models for aging and describe how current organoid techniques could be applied to aging research

Targeting Aspergillus fumigatus Crf Transglycosylases With Neutralizing Antibody Is Relevant but Not Sufficient to Erase Fungal Burden in a Neutropenic Rat Model

Aspergillus fumigatus is an airborne opportunistic fungal pathogen responsible for severe infections. Among them, invasive pulmonary aspergillosis has become a major concern as mortality rates exceed 50% in immunocompromised hosts. In parallel, allergic bronchopulmonary aspergillosis frequently encountered in cystic fibrosis patients, is also a comorbidity factor. Current treatments suffer from high toxicity which prevents their use in weakened subjects, resulting in impaired prognostic. Because of their low toxicity and high specificity, anti-infectious therapeutic antibodies could be a new alternative to conventional therapeutics. In this study, we investigated the potential of Chitin Ring Formation cell wall transglycosylases of A. fumigatus to be therapeutic targets for therapeutic antibodies. We demonstrated that the Crf target was highly conserved, regardless of the pathophysiological context; whereas the CRF1 gene was found to be 100% conserved in 92% of the isolates studied, Crf proteins were expressed in 98% of the strains. In addition, we highlighted the role of Crf proteins in fungal growth, using a deletion mutant for CRF1 gene, for which a growth decrease of 23.6% was observed after 48 h. It was demonstrated that anti-Crf antibodies neutralized the enzymatic activity of recombinant Crf protein, and delayed fungal growth by 12.3% in vitro when added to spores. In a neutropenic rat model of invasive pulmonary aspergillosis, anti-Crf antibodies elicited a significant recruitment of neutrophils, macrophages and T CD4 lymphocytes but it was not correlated with a decrease of fungal burden in lungs and improvement in survival. Overall, our study highlighted the potential relevance of targeting Crf cell wall protein (CWP) with therapeutic antibodies

Identification of Functional Networks of Estrogen- and c-Myc-Responsive Genes and Their Relationship to Response to Tamoxifen Therapy in Breast Cancer

BACKGROUND: Estrogen is a pivotal regulator of cell proliferation in the normal breast and breast cancer. Endocrine therapies targeting the estrogen receptor are effective in breast cancer, but their success is limited by intrinsic and acquired resistance. METHODOLOGY/PRINCIPAL FINDINGS: With the goal of gaining mechanistic insights into estrogen action and endocrine resistance, we classified estrogen-regulated genes by function, and determined the relationship between functionally-related genesets and the response to tamoxifen in breast cancer patients. Estrogen-responsive genes were identified by transcript profiling of MCF-7 breast cancer cells. Pathway analysis based on functional annotation of these estrogen-regulated genes identified gene signatures with known or predicted roles in cell cycle control, cell growth (i.e. ribosome biogenesis and protein synthesis), cell death/survival signaling and transcriptional regulation. Since inducible expression of c-Myc in antiestrogen-arrested cells can recapitulate many of the effects of estrogen on molecular endpoints related to cell cycle progression, the estrogen-regulated genes that were also targets of c-Myc were identified using cells inducibly expressing c-Myc. Selected genes classified as estrogen and c-Myc targets displayed similar levels of regulation by estrogen and c-Myc and were not estrogen-regulated in the presence of siMyc. Genes regulated by c-Myc accounted for 50% of all acutely estrogen-regulated genes but comprised 85% (110/129 genes) in the cell growth signature. siRNA-mediated inhibition of c-Myc induction impaired estrogen regulation of ribosome biogenesis and protein synthesis, consistent with the prediction that estrogen regulates cell growth principally via c-Myc. The 'cell cycle', 'cell growth' and 'cell death' gene signatures each identified patients with an attenuated response in a cohort of 246 tamoxifen-treated patients. In multivariate analysis the cell death signature was predictive independent of the cell cycle and cell growth signatures. CONCLUSIONS/SIGNIFICANCE: These functionally-based gene signatures can stratify patients treated with tamoxifen into groups with differing outcome, and potentially identify distinct mechanisms of tamoxifen resistance

Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus fumigatus

BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus

The Lancet Countdown: tracking progress on health and climate change

The Lancet Countdown: tracking progress on health and climate change is an international, multidisciplinary research collaboration between academic institutions and practitioners across the world. It follows on from the work of the 2015 Lancet Commission, which concluded that the response to climate change could be “the greatest global health opportunity of the 21st century”. The Lancet Countdown aims to track the health impacts of climate hazards; health resilience and adaptation; health co-benefits of climate change mitigation; economics and finance; and political and broader engagement. These focus areas form the five thematic working groups of the Lancet Countdown and represent different aspects of the complex association between health and climate change. These thematic groups will provide indicators for a global overview of health and climate change; national case studies highlighting countries leading the way or going against the trend; and engagement with a range of stakeholders. The Lancet Countdown ultimately aims to report annually on a series of indicators across these five working groups. This paper outlines the potential indicators and indicator domains to be tracked by the collaboration, with suggestions on the methodologies and datasets available to achieve this end. The proposed indicator domains require further refinement, and mark the beginning of an ongoing consultation process—from November, 2016 to early 2017—to develop these domains, identify key areas not currently covered, and change indicators where necessary. This collaboration will actively seek to engage with existing monitoring processes, such as the UN Sustainable Development Goals and WHO's climate and health country profiles. The indicators will also evolve over time through ongoing collaboration with experts and a range of stakeholders, and be dependent on the emergence of new evidence and knowledge. During the course of its work, the Lancet Countdown will adopt a collaborative and iterative process, which aims to complement existing initiatives, welcome engagement with new partners, and be open to developing new research projects on health and climate change