Recent Submissions

  • Clarithromycin impairs tissue-resident memory and Th17 responses to macrolide-resistant Streptococcus pneumoniae infections.

    Lindenberg, Marc; Almeida, Luis; Dhillon-LaBrooy, Ayesha; Siegel, Ekkehard; Henriques-Normark, Birgitta; Sparwasser, Tim; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Springer, 2021-02-17)
    The increasing prevalence of antimicrobial resistance in pathogens is a growing public health concern, with the potential to compromise the success of infectious disease treatments in the future. Particularly, the number of infections by macrolide antibiotics-resistant Streptococcus pneumoniae is increasing. We show here that Clarithromycin impairs both the frequencies and number of interleukin (IL)-17 producing T helper (Th) 17 cells within the lungs of mice infected with a macrolide-resistant S. pneumoniae serotype 15A strain. Subsequently, the tissue-resident memory CD4+ T cell (Trm) response to a consecutive S. pneumoniae infection was impaired. The number of lung resident IL-17+ CD69+ Trm was diminished upon Clarithromycin treatment during reinfection. Mechanistically, Clarithromycin attenuated phosphorylation of the p90-S6-kinase as part of the ERK pathway in Th17 cells. Moreover, a strong increase in the mitochondrial-mediated maximal respiratory capacity was observed, while mitochondrial protein translation and mTOR sisgnaling were unimpaired. Therefore, treatment with macrolide antibiotics may favor the spread of antimicrobial-resistant pathogens not only by applying a selection pressure but also by decreasing the natural T cell immune response. Clinical administration of macrolide antibiotics as standard therapy procedure during initial hospitalization should be reconsidered accordingly and possibly be withheld until microbial resistance is determined. KEY MESSAGES: • Macrolide-resistant S. pneumoniae infection undergoes immunomodulation by Clarithromycin • Clarithromycin treatment hinders Th17 and tissue-resident memory responses • Macrolide antibiotics impair Th17 differentiation in vitro by ERK-pathway inhibition.
  • Selective Host Cell Death by Staphylococcus aureus : A Strategy for Bacterial Persistence.

    Missiakas, Dominique; Winstel, Volker; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Frontiers, 2021-01-21)
    Host cell death programs are fundamental processes that shape cellular homeostasis, embryonic development, and tissue regeneration. Death signaling and downstream host cell responses are not only critical to guide mammalian development, they often act as terminal responses to invading pathogens. Here, we briefly review and contrast how invading pathogens and specifically Staphylococcus aureus manipulate apoptotic, necroptotic, and pyroptotic cell death modes to establish infection. Rather than invading host cells, S. aureus subverts these cells to produce diffusible molecules that cause death of neighboring hematopoietic cells and thus shapes an immune environment conducive to persistence. The exploitation of cell death pathways by S. aureus is yet another virulence strategy that must be juxtaposed to mechanisms of immune evasion, autophagy escape, and tolerance to intracellular killing, and brings us closer to the true portrait of this pathogen for the design of effective therapeutics and intervention strategies.
  • Regulatory T Cells in an Endogenous Mouse Lymphoma Recognize Specific Antigen Peptides and Contribute to Immune Escape.

    Ahmetlić, Fatima; Riedel, Tanja; Hömberg, Nadine; Bauer, Vera; Trautwein, Nico; Geishauser, Albert; Sparwasser, Tim; Stevanović, Stefan; Röcken, Martin; Mocikat, Ralph; et al. (American Association for Cancer Research (AACR), 2019-03-20)
    Foxp3+ regulatory T cells (Tregs) sustain immune homeostasis and may contribute to immune escape in malignant disease. As a prerequisite for developing immunologic approaches in cancer therapy, it is necessary to understand the ontogeny and the antigenic specificities of tumor-infiltrating Tregs. We addressed this question by using a λ-MYC transgenic mouse model of endogenously arising B-cell lymphoma, which mirrors key features of human Burkitt lymphoma. We show that Foxp3+ Tregs suppress antitumor responses in endogenous lymphoma. Ablation of Foxp3+ Tregs significantly delayed tumor development. The ratio of Treg to effector T cells was elevated in growing tumors, which could be ascribed to differential proliferation. The Tregs detected were mainly natural Tregs that apparently recognized self-antigens. We identified MHC class II-restricted nonmutated self-epitopes, which were more prevalent in lymphoma than in normal B cells and could be recognized by Tregs. These epitopes were derived from proteins that are associated with cellular processes related to malignancy and may be overexpressed in the tumor.
  • HBV evolution and genetic variability: Impact on prevention, treatment and development of antivirals.

    Glebe, Dieter; Goldmann, Nora; Lauber, Chris; Seitz, Stefan; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Elsevier, 2020-11-06)
    Hepatitis B virus (HBV) poses a major global health burden with 260 million people being chronically infected and 890,000 dying annually from complications in the course of the infection. HBV is a small enveloped virus with a reverse-transcribed DNA genome that infects hepatocytes and can cause acute and chronic infections of the liver. HBV is endemic in humans and apes representing the prototype member of the viral family Hepadnaviridae and can be divided into 10 genotypes. Hepadnaviruses have been found in all vertebrate classes and constitute an ancient viral family that descended from non-enveloped progenitors more than 360 million years ago. The de novo emergence of the envelope protein gene was accompanied with the liver-tropism and resulted in a tight virus-host association. The oldest HBV genomes so far have been isolated from human remains of the Bronze Age and the Neolithic (~7000 years before present). Despite the remarkable stability of the hepadnaviral genome over geological eras, HBV is able to rapidly evolve within an infected individual under pressure of the immune response or during antiviral treatment. Treatment with currently available antivirals blocking intracellular replication of HBV allows controlling of high viremia and improving liver health during long-term therapy of patients with chronic hepatitis B (CHB), but they are not sufficient to cure the disease. New therapy options that cover all HBV genotypes and emerging viral variants will have to be developed soon. In addition to the antiviral treatment of chronically infected patients, continued efforts to expand the global coverage of the currently available HBV vaccine will be one of the key factors for controlling the rising global spread of HBV. Certain improvements of the vaccine (e.g. inclusion of PreS domains) could counteract known problems such as low or no responsiveness of certain risk groups and waning anti-HBs titers leading to occult infections, especially with HBV genotypes E or F. But even with an optimal vaccine and a cure for hepatitis B, global eradication of HBV would be difficult to achieve because of an existing viral reservoir in primates and bats carrying closely related hepadnaviruses with zoonotic potential.
  • Cholesterol sensing by CD81 is important for hepatitis C virus entry.

    Palor, Machaela; Stejskal, Lenka; Mandal, Piya; Lenman, Annasara; Alberione, Maria Pia; Kirui, Jared; Moeller, Rebecca; Ebner, Stefan; Meissner, Felix; Gerold, Gisa; et al. (DeGruyter, 2020-09-08)
    CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease.
  • MicroRNA-342-3p is a potent tumour suppressor in hepatocellular carcinoma.

    Komoll, Ronja-Melinda; Hu, Qingluan; Olarewaju, Olaniyi; von Döhlen, Lena; Yuan, Qinggong; Xie, Yu; Tsay, Hsin-Chieh; Daon, Joel; Qin, Renyi; Manns, Michael P; et al. (Elsevier, 2020-07-30)
    Background & aims: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. Methods: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. Results: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. Conclusions: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. Lay summary: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.
  • MicroRNA-342-3p is a potent tumour suppressor in hepatocellular carcinoma

    Komoll, Ronja Melinda; Hu, Qingluan; Olarewaju, Olaniyi; von Döhlen, Lena; Yuan, Qinggong; Xie, Yu; Tsay, Hsin Chieh; Daon, Joel; Qin, Renyi; Manns, Michael P.; et al. (2021-01-01)
    Background & aims: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. Methods: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. Results: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. Conclusions: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. Lay summary: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.
  • MicroRNA-221: A Fine Tuner and Potential Biomarker of Chronic Liver Injury.

    Markovic, Jovana; Sharma, Amar Deep; Balakrishnan, Asha; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-07-23)
    The last decade has witnessed significant advancements in our understanding of how small noncoding RNAs, such as microRNAs (miRNAs), regulate disease progression. One such miRNA, miR-221, has been shown to play a key role in the progression of liver fibrosis, a common feature of most liver diseases. Many reports have demonstrated the upregulation of miR-221 in liver fibrosis caused by multiple etiologies such as viral infections and nonalcoholic steatohepatitis. Inhibition of miR-221 via different strategies has shown promising results in terms of the suppression of fibrogenic gene signatures in vitro, as well as in vivo, in independent mouse models of liver fibrosis. In addition, miR-221 has also been suggested as a noninvasive serum biomarker for liver fibrosis and cirrhosis. In this review, we discuss the biology of miR-221, its significance and use as a biomarker during progression of liver fibrosis, and finally, potential and robust approaches that can be utilized to suppress liver fibrosis via inhibition of miR-221.
  • Notch and TLR signaling coordinate monocyte cell fate and inflammation.

    Gamrekelashvili, Jaba; Kapanadze, Tamar; Sablotny, Stefan; Ratiu, Corina; Dastagir, Khaled; Lochner, Matthias; Karbach, Susanne; Wenzel, Philip; Sitnow, Andre; Fleig, Susanne; et al. (elife Sciences, 2020-07-29)
    Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.
  • Strategic Anti-SARS-CoV-2 Serology Testing in a Low Prevalence Setting: The COVID-19 Contact (CoCo) Study in Healthcare Professionals.

    Behrens, Georg M N; Cossmann, Anne; Stankov, Metodi V; Schulte, Bianca; Streeck, Hendrik; Förster, Reinhold; Bosnjak, Berislav; Willenzon, Stefanie; Boeck, Anna-Lena; Thu Tran, Anh; et al. (Springer Healthcare, 2020-09-04)
    Background: Serology testing is explored for epidemiological research and to inform individuals after suspected infection. During the coronavirus disease 2019 (COVID-19) pandemic, frontline healthcare professionals (HCP) may be at particular risk for infection. No longitudinal data on functional seroconversion in HCP in regions with low COVID-19 prevalence and low pre-test probability exist. Methods: In a large German university hospital, we performed weekly questionnaire assessments and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) measurements with various commercial tests, a novel surrogate virus neutralisation test, and a neutralisation assay using live SARS-CoV-2. Results: From baseline to week 6, 1080 screening measurements for anti-SARS CoV-2 (S1) IgG from 217 frontline HCP (65% female) were performed. Overall, 75.6% of HCP reported at least one symptom of respiratory infection. Self-perceived infection probability declined over time (from mean 20.1% at baseline to 12.4% in week 6, p < 0.001). In sera of convalescent patients with PCR-confirmed COVID-19, we measured high anti-SARS-CoV-2 IgG levels, obtained highly concordant results from enzyme-linked immunosorbent assays (ELISA) using e.g. the spike 1 (S1) protein domain and the nucleocapsid protein (NCP) as targets, and confirmed antiviral neutralisation. However, in HCP the cumulative incidence for anti-SARS-CoV-2 (S1) IgG was 1.86% for positive and 0.93% for equivocal positive results over the study period of 6 weeks. Except for one HCP, none of the eight initial positive results were confirmed by alternative serology tests or showed in vitro neutralisation against live SARS-CoV-2. The only true seroconversion occurred without symptoms and mounted strong functional humoral immunity. Thus, the confirmed cumulative incidence for neutralizing anti-SARS-CoV-2 IgG was 0.47%. Conclusion: When assessing anti-SARS-CoV-2 immune status in individuals with low pre-test probability, we suggest confirming positive results from single measurements by alternative serology tests or functional assays. Our data highlight the need for a methodical serology screening approach in regions with low SARS-CoV-2 infection rates.
  • Staphylococcus aureus Alpha-Toxin Limits Type 1 While Fostering Type 3 Immune Responses.

    Bonifacius, Agnes; Goldmann, Oliver; Floess, Stefan; Holtfreter, Silva; Robert, Philippe A; Nordengrün, Maria; Kruse, Friederike; Lochner, Matthias; Falk, Christine S; Schmitz, Ingo; et al. (Frontiers, 2020-08-07)
    Staphylococcus aureus can cause life-threatening diseases, and hospital- as well as community-associated antibiotic-resistant strains are an emerging global public health problem. Therefore, prophylactic vaccines or immune-based therapies are considered as alternative treatment opportunities. To develop such novel treatment approaches, a better understanding of the bacterial virulence and immune evasion mechanisms and their potential effects on immune-based therapies is essential. One important staphylococcal virulence factor is alpha-toxin, which is able to disrupt the epithelial barrier in order to establish infection. In addition, alpha-toxin has been reported to modulate other cell types including immune cells. Since CD4+ T cell-mediated immunity is required for protection against S. aureus infection, we were interested in the ability of alpha-toxin to directly modulate CD4+ T cells. To address this, murine naïve CD4+ T cells were differentiated in vitro into effector T cell subsets in the presence of alpha-toxin. Interestingly, alpha-toxin induced death of Th1-polarized cells, while cells polarized under Th17 conditions showed a high resistance toward increasing concentrations of this toxin. These effects could neither be explained by differential expression of the cellular alpha-toxin receptor ADAM10 nor by differential activation of caspases, but might result from an increased susceptibility of Th1 cells toward Ca2+-mediated activation-induced cell death. In accordance with the in vitro findings, an alpha-toxin-dependent decrease of Th1 and concomitant increase of Th17 cells was observed in vivo during S. aureus bacteremia. Interestingly, corresponding subsets of innate lymphoid cells and γδ T cells were similarly affected, suggesting a more general effect of alpha-toxin on the modulation of type 1 and type 3 immune responses. In conclusion, we have identified a novel alpha-toxin-dependent immunomodulatory strategy of S. aureus, which can directly act on CD4+ T cells and might be exploited for the development of novel immune-based therapeutic approaches to treat infections with antibiotic-resistant S. aureus strains.
  • Establishment of porcine and human expanded potential stem cells.

    Gao, Xuefei; Nowak-Imialek, Monika; Chen, Xi; Chen, Dongsheng; Herrmann, Doris; Ruan, Degong; Chen, Andy Chun Hang; Eckersley-Maslin, Melanie A; Ahmad, Shakil; Lee, Yin Lau; et al. (Nature publishing group(NPG), 2019-06-03)
    We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.
  • Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B.

    Krooss, Simon; Werwitzke, Sonja; Kopp, Johannes; Rovai, Alice; Varnholt, Dirk; Wachs, Amelie S; Goyenvalle, Aurelie; Aarstma-Rus, Annemieke; Ott, Michael; Tiede, Andreas; et al. (PLOS, 2020-04-08)
    Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3' untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins.
  • Let-7c inhibits cholangiocarcinoma growth but promotes tumor cell invasion and growth at extrahepatic sites.

    Xie, Yu; Zhang, Hang; Guo, Xing-Jun; Feng, Ye-Chen; He, Rui-Zhi; Li, Xu; Yu, Shuo; Zhao, Yan; Shen, Ming; Zhu, Feng; et al. (Springer Nature, 2018-02-14)
    Cholangiocarcinoma (CCA) is a cancer type with high postoperative relapse rates and poor long-term survival largely due to tumor invasion, distant metastasis, and multidrug resistance. Deregulated microRNAs (miRNAs) are implicated in several cancer types including CCA. The specific roles of the miRNA let-7c in cholangiocarcinoma are not known and need to be further elucidated. In our translational study we show that microRNA let-7c expression was significantly downregulated in human cholangiocarcinoma tissues when compared to adjacent tissues of the same patient. Let-7c inhibited the tumorigenic properties of cholangiocarcinoma cells including their self-renewal capacity and sphere formation in vitro and subcutaneous cancer cell growth in vivo. Ectopic let-7c overexpression suppressed migration and invasion capacities of cholangiocarcinoma cell lines in vitro, however, promoted distant invasiveness in vivo. Furthermore, we found that let-7c regulated the aforementioned malignant biological properties, at least in part, through regulation of EZH2 protein expression and through the DVL3/β-catenin axis. The miRNA let-7c thus plays an important dual role in regulating tumorigenic and metastatic abilities of human cholangiocarcinoma through mechanisms involving EZH2 protein and the DVL3/β-catenin axis.
  • The Dynamics of Interleukin-10-Afforded Protection during Dextran Sulfate Sodium-Induced Colitis.

    Cardoso, Ana; Gil Castro, Antonio; Martins, Ana Catarina; Carriche, Guilhermina M; Murigneux, Valentine; Castro, Isabel; Cumano, Ana; Vieira, Paulo; Saraiva, Margarida; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Frontiers, 2018-03-01)
    Inflammatory bowel disease encompasses a group of chronic-inflammatory conditions of the colon and small intestine. These conditions are characterized by exacerbated inflammation of the organ that greatly affects the quality of life of patients. Molecular mechanisms counteracting this hyperinflammatory status of the gut offer strategies for therapeutic intervention. Among these regulatory molecules is the anti-inflammatory cytokine interleukin (IL)-10, as shown in mice and humans. Indeed, IL-10 signaling, particularly in macrophages, is essential for intestinal homeostasis. We sought to investigate the temporal profile of IL-10-mediated protection during chemical colitis and which were the underlying mechanisms. Using a novel mouse model of inducible IL-10 overexpression (pMT-10), described here, we show that mice preconditioned with IL-10 for 8 days before dextran sulfate sodium (DSS) administration developed a milder colitic phenotype. In IL-10-induced colitic mice, Ly6C cells isolated from the lamina propria showed a decreased inflammatory profile. Because our mouse model leads to transcription of the IL-10 transgene in the bone marrow and elevated seric IL-10 concentration, we investigated whether IL-10 could imprint immune cells in a long-lasting way, thus conferring sustained protection to colitis. We show that this was not the case, as IL-10-afforded protection was only observed if IL-10 induction immediately preceded DSS-mediated colitis. Thus, despite the protection afforded by IL-10 in colitis, novel strategies are required, specifically to achieve long-lasting protection.
  • Expansion of functional personalized cells with specific transgene combinations.

    Lipps, Christoph; Klein, Franziska; Wahlicht, Tom; Seiffert, Virginia; Butueva, Milada; Zauers, Jeannette; Truschel, Theresa; Luckner, Martin; Köster, Mario; MacLeod, Roderick; et al. (Springer Nature, 2018-03-08)
    Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.
  • TLR4 abrogates the Th1 immune response through IRF1 and IFN-β to prevent immunopathology during L. infantum infection.

    Sacramento, Laís Amorim; Benevides, Luciana; Maruyama, Sandra Regina; Tavares, Lucas; Fukutani, Kiyoshi Ferreira; Francozo, Marcela; Sparwasser, Tim; Cunha, Fernando Queiroz; Almeida, Roque Pacheco; da Silva, João Santana; et al. (PLOS, 2020-03-25)
    A striking feature of human visceral leishmaniasis (VL) is chronic inflammation in the spleen and liver, and VL patients present increased production levels of multiple inflammatory mediators, which contribute to tissue damage and disease severity. Here, we combined an experimental model with the transcriptional profile of human VL to demonstrate that the TLR4-IFN-β pathway regulates the chronic inflammatory process and is associated with the asymptomatic form of the disease. Tlr4-deficient mice harbored fewer parasites in their spleen and liver than wild-type mice. TLR4 deficiency enhanced the Th1 immune response against the parasite, which was correlated with an increased activation of dendritic cells (DCs). Gene expression analyses demonstrated that IRF1 and IFN-β were expressed downstream of TLR4 after infection. Accordingly, IRF1- and IFNAR-deficient mice harbored fewer parasites in the target organs than wild-type mice due to having an increased Th1 immune response. However, the absence of TLR4 or IFNAR increased the serum transaminase levels in infected mice, indicating the presence of liver damage in these animals. In addition, IFN-β limits IFN-γ production by acting directly on Th1 cells. Using RNA sequencing analysis of human samples, we demonstrated that the transcriptional signature for the TLR4 and type I IFN (IFN-I) pathways was positively modulated in asymptomatic subjects compared with VL patients and thus provide direct evidence demonstrating that the TLR4-IFN-I pathway is related to the nondevelopment of the disease. In conclusion, our results demonstrate that the TLR4-IRF1 pathway culminates in IFN-β production as a mechanism for dampening the chronic inflammatory process and preventing immunopathology development.
  • Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction.

    Paul-Heng, Moumita; Leong, Mario; Cunningham, Eithne; Bunker, Daniel L J; Bremner, Katherine; Wang, Zane; Wang, Chuanmin; Tay, Szun Szun; McGuffog, Claire; Logan, Grant J; et al. (NLM (Medline), 2018-08-09)
    Adeno-associated viral vector–mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.
  • Ex Vivo/In vivo Gene Editing in Hepatocytes Using "All-in-One" CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template.

    Krooss, Simon Alexander; Dai, Zhen; Schmidt, Florian; Rovai, Alice; Fakhiri, Julia; Dhingra, Akshay; Yuan, Qinggong; Yang, Taihua; Balakrishnan, Asha; Steinbrück, Lars; et al. (Cell Press/Elsevier, 2020-01-24)
    Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.
  • Groundwater, soil and compost, as possible sources of virulent and antibiotic-resistant Pseudomonas aeruginosa.

    Kaszab, Edit; Radó, Júlia; Kriszt, Balázs; Pászti, Judit; Lesinszki, Virág; Szabó, Ádám; Tóth, Gergő; Khaledi, Ariane; Szoboszlay, Sándor; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Taylor & Francis, 2019-11-18)
    Pseudomonas aeruginosa is a major public health concern all around the world. In the frame of this work, a set of diverse environmental P. aeruginosa isolates with various antibiotic resistance profiles were examined in a Galleria mellonella virulence model. Motility, serotypes, virulence factors and biofilm-forming ability were also examined. Molecular types were determined by pulsed-field gel electrophoresis (PFGE). Based on our results, the majority of environmental isolates were virulent in the G. mellonella test and twitching showed a positive correlation with mortality. Resistance against several antibiotic agents such as Imipenem correlated with a lower virulence in the applied G. mellonella model. PFGE revealed that five examined environmental isolates were closely related to clinically detected pulsed-field types. Our study demonstrated that industrial wastewater effluents, composts, and hydrocarbon-contaminated sites should be considered as hot spots of high-risk clones of P. aeruginosa.

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