Jörg Steinmann

Anti-infective properties of epigallocatechin-3-gallate (EGCG), a component of green tea.

The consumption of green tea (Camellia sinensis) has been shown to have many physiological and pharmacological health benefits. In the past two decades several studies have reported that epigallocatechin‐3‐gallate (EGCG), the main constituent of green tea, has anti‐infective properties. Antiviral activities of EGCG with different modes of action have been demonstrated on diverse families of viruses, such as Retroviridae, Orthomyxoviridae and Flaviviridae and include important human pathogens like human immunodeficiency virus, influenza A virus and the hepatitis C virus. Furthermore, the molecule interferes with the replication cycle of DNA viruses like hepatitis B virus, herpes simplex virus and adenovirus. Most of these studies demonstrated antiviral properties within physiological concentrations of EGCG in vitro. In contrast, the minimum inhibitory concentrations against bacteria were 10–100‐fold higher. Nevertheless, the antibacterial effects of EGCG alone and in combination with different antibiotics have been intensively analysed against a number of bacteria including multidrug‐resistant strains such as methicillin‐resistant Staphylococcus aureus or Stenotrophomonas maltophilia. Furthermore, the catechin EGCG has antifungal activity against human‐pathogenic yeasts like Candida albicans. Although the mechanistic effects of EGCG are not fully understood, there are results indicating that EGCG binds to lipid membranes and affects the folic acid metabolism of bacteria and fungi by inhibiting the cytoplasmic enzyme dihydrofolate reductase. This review summarizes the current knowledge and future perspectives on the antibacterial, antifungal and antiviral effects of the green tea constituent EGCG.

The green tea polyphenol, epigallocatechin‐3‐gallate, inhibits hepatitis C virus entry

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin‐3‐gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell‐culture–derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell‐to‐cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co‐)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation. (HEPATOLOGY 2011)

Impaired neutral sphingomyelinase activation and cutaneous barrier repair in FAN‐deficient mice

The WD‐40 repeat protein FAN binds to a distinct domain of the p55 receptor for tumor necrosis factor (TNF) and signals the activation of neutral sphingomyelinase (N‐SMase). To analyze the physiological role of FAN in vivo, we generated FAN‐deficient mice by targeted gene disruption. Mice lacking a functional FAN protein do not show any overt phenotypic abnormalities; in particular, the architecture and cellular composition of lymphoid organs appeared to be unaltered. An essential role of FAN in the TNF‐induced activation of N‐SMase was demonstrated using thymocytes from FAN knockout mice. Activation of extracellular signal‐regulated kinases in response to TNF treatment, however, was not impaired by the absence of the FAN protein. FAN‐deficient mice show delayed kinetics of recovery after cutaneous barrier disruption suggesting a physiological role of FAN in epidermal barrier repair. Although FAN exhibits striking structural homologies with the CHS/Beige proteins, FAN‐deficient mice did not reproduce the phenotype of beige mice.

Emergence of azole-resistant invasive aspergillosis in HSCT recipients in Germany

OBJECTIVES Aspergillus fumigatus is the most common agent of invasive aspergillosis (IA). In recent years, resistance to triazoles, the mainstay of IA therapy, has emerged in different countries worldwide. IA caused by azole-resistant A. fumigatus (ARAF) shows an exceedingly high mortality. In this study, IA due to ARAF isolates in HSCT recipients in Germany was investigated. METHODS The epidemiology of azole resistance in IA was analysed in two German haematology departments. Between 2012 and 2013, 762 patients received HSCT in Essen (n = 388) and Cologne (n = 374). Susceptibility testing of A. fumigatus isolates was performed by Etest, followed by EUCAST broth microdilution testing if elevated MICs were recorded. In all ARAF isolates the cyp51A gene was sequenced and the genotype was determined by microsatellite typing using nine short tandem repeats. RESULTS In total, A. fumigatus was recovered from 27 HSCT recipients. Eight patients had azole-resistant IA after HSCT, and seven of the cases were fatal (88%). All except one patient received antifungal prophylaxis (in five cases triazoles). TR34/L98H was the most common mutation (n = 5), followed by TR46/Y121F/T289A (n = 2). In one resistant isolate no cyp51A mutation was detected. Genotyping revealed genetic diversity within the German ARAF isolates and no clustering with resistant isolates from the Netherlands, India and France. CONCLUSIONS This report highlights the emergence of azole-resistant IA with TR34/L98H and TR46/Y121F/T289A mutations in HSCT patients in Germany and underscores the need for systematic antifungal susceptibility testing of A. fumigatus.

How Stable Is the Hepatitis C Virus (HCV)? Environmental Stability of HCV and Its Susceptibility to Chemical Biocides

BACKGROUND In the absence of a cell culture system for propagation of the hepatitis C virus (HCV), the antiviral activity of disinfectants against HCV was extrapolated from studies with the bovine viral diarrhea virus. The recent development of an HCV infection system allowed the direct assessment of environmental stability and susceptibility to chemical disinfectants. METHODS Studies were performed using cell-culture grown HCV. Infectivity was determined by limiting dilutions. HCV RNA levels were analyzed by quantitative real-time polymerase chain reaction. Genome stability was determined by transfection of recovered RNA into Huh7.5 cells and immunostaining. RESULTS HCV infectivity in a liquid environment was detectable for up to 5 month at lower temperatures. The risk of HCV infections may not accurately be reflected by determination of HCV RNA levels, because viral infectivity and HCV RNA copy numbers did not directly correlate. Different alcohols and commercially available antiseptics reduced the infectivity of HCV to undetectable levels. However, diluting the hand disinfectants abrogated the virucidal activity. CONCLUSIONS This study assessed the environmental stability and susceptibility to chemical biocides of HCV. The results should be useful in defining rigorous disinfection protocols to prevent nosocomial transmission of HCV.

Inactivation and Survival of Hepatitis C Virus on Inanimate Surfaces

BACKGROUND Hepatitis C virus (HCV) cross-contamination from inanimate surfaces or objects has been implicated in transmission of HCV in health-care settings and among injection drug users. We established HCV-based carrier and drug transmission assays that simulate practical conditions to study inactivation and survival of HCV on inanimate surfaces. METHODS Studies were performed with authentic cell culture derived viruses. HCV was dried on steel discs and biocides were tested for their virucidal efficacy against HCV. Infectivity was determined by a limiting dilution assay. HCV stability was analyzed in a carrier assay for several days or in a drug transmission assay using a spoon as cooker. RESULTS HCV can be dried and recovered efficiently in the carrier assay. The most effective alcohol to inactivate the virus was 1-propanol, and commercially available disinfectants reduced infectivity of HCV to undetectable levels. Viral infectivity on inanimate surfaces was detectable in the presence of serum for up to 5 days, and temperatures of about 65-70°C were required to eliminate infectivity in the drug transmission assay. CONCLUSIONS These findings are important for assessment of HCV transmission risks and should facilitate the definition of stringent public health interventions to prevent HCV infections.

Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukaemia effect in mice.

Allogeneic lymphocytes administered with an unmanipulated bone marrow transplant provide a strong antileukaemic effect, the so‐called graft‐versus‐leukaemia (GVL) effect. On the other hand, T‐cell‐mediated graft‐versus‐host‐disease (GVHD) observed after transplantation of unmanipulated BM graft causes substantial morbidity and mortality. The aim of the present study was to determine the antileukaemic potential of enriched IL‐2 activated NK cells administered 2 h after BMT. Balb/c (H‐2d) mice were given a dose of A20 (H‐2d, B‐cell leukaemia) cells 2 d prior to lethal total body irradiation (TBI) and transplantation of either syngeneic or allogeneic anti‐Thy1.2 (CD90) depleted bone marrow cells. Either syngeneic (Balb/c, H‐2d) or allogeneic (C57BL/6, H‐2b) enriched and IL‐2 (200 U/ml for 24 h) activated NK cells were given 2 h after BMT. Injection of A20 leukaemia into normal Balb/c recipients led to death after a median of 14 d. A lethal dose of TBI followed by either syngeneic or allogeneic Thy1.2‐depleted BMT resulted in a modest antileukaemic effect. The adoptive transfer of syngeneic enriched and IL‐2 preincubated NK cells given at time of BMT exerted a significantly better GVL effect. However, the infusion of allogeneic enriched NK cells resulted in a stronger GVL effect. These results clearly demonstrate that allogeneic NK cells are superior to syngeneic NK cells in their potential to eradicate residual leukaemia cells after BMT without mediating clinical overt GVHD. This experimental setting may offer a strategy for treatment of haematological malignancies in a phase of minimal residual disease.

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24–33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance.

Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota

Molecular phylogenies based on the small subunit ribosomal RNA gene (SSU or 18S ribosomal DNA (rDNA)) revealed recently the existence of a relatively large and widespread group of eukaryotes, branching at the base of the fungal tree. This group, comprising almost exclusively environmental clones, includes the endoparasitic chytrid Rozella as the unique known representative. Rozella emerged as the first fungal lineage in molecular phylogenies and as the sister group of the Microsporidia. Here we report rDNA molecular phylogenetic analyses of two endonuclear parasites of free-living naked amoebae having microsporidia-like ultrastructural features but belonging to the rozellids. Similar to microsporidia, these endoparasites form unflagellated walled spores and grow inside the host cells as unwalled nonphagotrophic meronts. Our endonuclear parasites are microsporidia-like rozellids, for which we propose the name Paramicrosporidium, appearing to be the until now lacking morphological missing link between Fungi and Microsporidia. These features contrast with the recent description of the rozellids as an intermediate wall-less lineage of organisms between protists and true Fungi. We thus reconsider the rozellid clade as the most basal fungal lineage, naming it Rozellomycota.

Sphingoid long chain bases prevent lung infection by Pseudomonas aeruginosa

Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P. aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P. aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.