@article{PrustyGulveGovindetal.2018, author = {Prusty, Bhupesh K. and Gulve, Nitish and Govind, Sheila and Krueger, Gerhard R. F. and Feichtinger, Julia and Larcombe, Lee and Aspinall, Richard and Ablashi, Dharam V. and Toro, Carla T.}, title = {Active HHV-6 Infection of Cerebellar Purkinje Cells in Mood Disorders}, series = {Frontiers in Microbiology}, volume = {9}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2018.01955}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-369222}, year = {2018}, abstract = {Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD.}, language = {en} } @article{JohnAbrantesPrustyetal.2019, author = {John, Cathy N. and Abrantes, Pedro M. D. S. and Prusty, Bhupesh K. and Ablashi, Dharam V. and Africa, Charlene W. J.}, title = {K21 Compound, a Potent Antifungal Agent: Implications for the Treatment of Fluconazole-Resistant HIV-Associated Candida Species}, series = {Frontiers in Microbiology}, volume = {10}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2019.01021}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323505}, year = {2019}, abstract = {Background/Objectives: With mucocutaneous candidiasis being highly prevalent in HIV patients, the emergence of fluconazole-resistant Candida species forms a major challenge in treating and eradicating these infections. The objective of this study was to establish the antifungal activity of K21, a membrane-rupturing antimicrobial compound derived from a silica quaternary ammonium compound (SiQAC) with tetraethoxysilane (TEOS). Methods: The study sample included 81 Candida species of which 9 were type strains and 72 were clinical isolates. Minimum inhibitory concentrations, synergy, fractional inhibitory concentration index (FICI), and time kill assays were determined by broth microdilution. Electron microscopy (EM) was used to determine the qualitative changes brought about after treatment with K21. Results: K21 inhibited the growth of all fluconazole-resistant and susceptible Candida strains with only 2 h of exposure required to effectively kill 99.9\% of the inoculum, and a definite synergistic effect was observed with a combination of K21 and fluconazole. EM demonstrated the presence of two forms of extracellular vesicles indicative of biofilm formation and cell lysis. Conclusion: The study established the efficacy of K21 as an antifungal agent and with fluconazole-resistant candidiasis on the increase, the development of K21 can provide a promising alternative to combat acquired drug resistance.}, language = {en} } @article{MedlerNelkeWeisenbergeretal.2019, author = {Medler, Juliane and Nelke, Johannes and Weisenberger, Daniela and Steinfatt, Tim and Rothaug, Moritz and Berr, Susanne and H{\"u}nig, Thomas and Beilhack, Andreas and Wajant, Harald}, title = {TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity}, series = {Cell Death \& Disease}, volume = {10}, journal = {Cell Death \& Disease}, doi = {10.1038/s41419-019-1456-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223948}, year = {2019}, abstract = {Antibodies specific for TNFRSF receptors that bind soluble ligands without getting properly activated generally act as strong agonists upon FcγR binding. Systematic analyses revealed that the FcγR dependency of such antibodies to act as potent agonists is largely independent from isotype, FcγR type, and of the epitope recognized. This suggests that the sole cellular attachment, achieved by Fc domain-FcγR interaction, dominantly determines the agonistic activity of antibodies recognizing TNFRSF receptors poorly responsive to soluble ligands. In accordance with this hypothesis, we demonstrated that antibody fusion proteins harboring domains allowing FcγR-independent cell surface anchoring also act as strong agonist provided they have access to their target. This finding defines a general possibility to generate anti-TNFRSF receptor antibodies with FcγR-independent agonism. Moreover, anti-TNFRSF receptor antibody fusion proteins with an anchoring domain promise superior applicability to conventional systemically active agonists when an anchoring target with localized disease associated expression can be addressed.}, language = {en} } @article{BaurOttoStegeretal.2018, author = {Baur, Johannes and Otto, Christoph and Steger, Ulrich and Klein-Hessling, Stefan and Muhammad, Khalid and Pusch, Tobias and Murti, Krisna and Wismer, Rhoda and Germer, Christoph-Thomas and Klein, Ingo and M{\"u}ller, Nora and Serfling, Edgar and Avots, Andris}, title = {The transcription factor NFaTc1 supports the rejection of heterotopic heart allografts}, series = {Frontiers in Immunology}, volume = {9}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2018.01338}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221530}, year = {2018}, abstract = {The immune suppressants cyclosporin A (CsA) and tacrolimus (FK506) are used worldwide in transplantation medicine to suppress graft rejection. Both CsA and FK506 inhibit the phosphatase calcineurin (CN) whose activity controls the immune receptor-mediated activation of lymphocytes. Downstream targets of CN in lymphocytes are the nuclear factors of activated T cells (NFATs). We show here that the activity of NFATc1, the most prominent NFAT factor in activated lymphocytes supports the acute rejection of heterotopic heart allografts. While ablation of NFATc1 in T cells prevented graft rejection, ectopic expression of inducible NFATc1/αA isoform led to rejection of heart allografts in recipient mice. Acceptance of transplanted hearts in mice bearing NFATc1-deficient T cells was accompanied by a reduction in number and cytotoxicity of graft infiltrating cells. In CD8\(^+\) T cells, NFATc1 controls numerous intracellular signaling pathways that lead to the metabolic switch to aerobic glycolysis and the expression of numerous lymphokines, chemokines, and their receptors, including Cxcr3 that supports the rejection of allogeneic heart transplants. These findings favors NFATc1 as a molecular target for the development of new strategies to control the cytotoxicity of T cells upon organ transplantation.}, language = {en} } @article{KrausBrinkSiegel2019, author = {Kraus, Amelie J. and Brink, Benedikt G. and Siegel, T. Nicolai}, title = {Efficient and specific oligo-based depletion of rRNA}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-48692-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224829}, year = {2019}, abstract = {In most organisms, ribosomal RNA (rRNA) contributes to >85\% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5\% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.}, language = {en} } @article{LauruschkatMuchsinReinetal.2023, author = {Lauruschkat, Chris David and Muchsin, Ihsan and Rein, Alice and Erhard, Florian and Grathwohl, Denise and D{\"o}lken, Lars and K{\"o}chel, Carolin and Falk, Christine Susanne and Einsele, Hermann and Wurster, Sebastian and Grigoleit, G{\"o}tz Ulrich and Kraus, Sabrina}, title = {CD4+ T cells are the major predictor of HCMV control in allogeneic stem cell transplant recipients on letermovir prophylaxis}, series = {Frontiers in Immunology}, volume = {14}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2023.1148841}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-316982}, year = {2023}, abstract = {Introduction Human cytomegalovirus (HCMV) causes significant morbidity and mortality in allogeneic stem cell transplant (alloSCT) recipients. Recently, antiviral letermovir prophylaxis during the first 100 days after alloSCT replaced PCR-guided preemptive therapy as the primary standard of care for HCMV reactivations. Here, we compared NK-cell and T-cell reconstitution in alloSCT recipients receiving preemptive therapy or letermovir prophylaxis in order to identify potential biomarkers predicting prolonged and symptomatic HCMV reactivation. Methods To that end, the NK-cell and T-cell repertoire of alloSCT recipients managed with preemptive therapy (n=32) or letermovir prophylaxis (n=24) was characterized by flow cytometry on days +30, +60, +90 and +120 after alloSCT. Additionally, background-corrected HCMV-specific T-helper (CD4+IFNγ+) and cytotoxic (CD8+IFNγ+CD107a+) T cells were quantified after pp65 stimulation. Results Compared to preemptive therapy, letermovir prophylaxis prevented HCMV reactivation and decreased HCMV peak viral loads until days +120 and +365. Letermovir prophylaxis resulted in decreased T-cell numbers but increased NK-cell numbers. Interestingly, despite the inhibition of HCMV, we found high numbers of "memory-like" (CD56dimFcεRIγ- and/or CD159c+) NK cells and an expansion of HCMV-specific CD4+ and CD8+ T cells in letermovir recipients. We further compared immunological readouts in patients on letermovir prophylaxis with non/short-term HCMV reactivation (NSTR) and prolonged/symptomatic HCMV reactivation (long-term HCMV reactivation, LTR). Median HCMV-specific CD4+ T-cell frequencies were significantly higher in NSTR patients (day +60, 0.35 \% vs. 0.00 \% CD4+IFNγ+/CD4+ cells, p=0.018) than in patients with LTR, whereas patients with LTR had significantly higher median regulatory T-cell (Treg) frequencies (day +90, 2.2 \% vs. 6.2 \% CD4+CD25+CD127dim/CD4+ cells, p=0.019). ROC analysis confirmed low HCMV specific CD4+ (AUC on day +60: 0.813, p=0.019) and high Treg frequencies (AUC on day +90: 0.847, p=0.021) as significant predictors of prolonged and symptomatic HCMV reactivation. Discussion Taken together, letermovir prophylaxis delays HCMV reactivation and alters NK- and T-cell reconstitution. High numbers of HCMV-specific CD4+ T cells and low numbers of Tregs seem to be pivotal to suppress post-alloSCT HCMV reactivation during letermovir prophylaxis. Administration of more advanced immunoassays that include Treg signature cytokines might contribute to the identification of patients at high-risk for long-term and symptomatic HCMV reactivation who might benefit from prolonged administration of letermovir.}, language = {en} } @article{AintablianStrozniakHeueretal.2023, author = {Aintablian, Arpa and Strozniak, Sandra and Heuer, Marion and Lutz, Manfred B.}, title = {M-MDSC in vitro generation from mouse bone marrow with IL-3 reveals high expression and functional activity of arginase 1}, series = {Frontiers in Immunology}, volume = {14}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2023.1130600}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-317769}, year = {2023}, abstract = {Myeloid-derived suppressor cells (MDSC) represent major regulators of immune responses, which can control T cells via their inducible nitric oxide synthase (iNOS)- and arginase 1 (Arg1)-mediated effector functions. While GM-CSF is well documented to promote MDSC development, little is known about this potential of IL-3, an established growth factor for mast cells. Here, we show that IL-3, similar to GM-CSF, generates monocytic MDSC (M-MDSC) from murine bone marrow (BM) cells after 3 days of in vitro culture. At this time point, predominantly CD11b+ CD49a+ monocytic and CD11b+ CD49a- FcεR I- neutrophilic cells were detectable, while CD11blow/neg FcεR I+ mast cells accumulated only after extended culture periods. Both growth factors were equivalent in generating M-MDSC with respect to phenotype, cell yield and typical surface markers. However, IL-3 generated M-MDSC produced less TNF, IL-1β and IL-10 after activation with LPS + IFN-γ but showed higher Arg1 expression compared to GM-CSF generated M-MDSC. Arg1 was further induced together with iNOS after MDSC activation. Accordingly, an increased Arg1-dependent suppressor activity by the IL-3 generated M-MDSC was observed using respective iNOS and Arg1 inhibitors. Together, these data indicate that M-MDSC can be generated in vitro by IL-3, similar to GM-CSF, but with increased Arg1 expression and Arg1-mediated suppression capacity. This protocol now allows further in vitro studies on the role of IL-3 for MDSC biology.}, language = {en} } @article{DiesendorfRollGeigeretal.2023, author = {Diesendorf, Viktoria and Roll, Valeria and Geiger, Nina and F{\"a}hr, Sofie and Obernolte, Helena and Sewald, Katherina and Bodem, Jochen}, title = {Drug-induced phospholipidosis is not correlated with the inhibition of SARS-CoV-2 - inhibition of SARS-CoV-2 is cell line-specific}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {13}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2023.1100028}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-326202}, year = {2023}, abstract = {Recently, Tummino et al. reported that 34 compounds, including Chloroquine and Fluoxetine, inhibit SARS-CoV-2 replication by inducing phospholipidosis, although Chloroquine failed to suppress viral replication in Calu-3 cells and patients. In contrast, Fluoxetine represses viral replication in human precision-cut lung slices (PCLS) and Calu-3 cells. Thus, it is unlikely that these compounds have similar mechanisms of action. Here, we analysed a subset of these compounds in the viral replication and phospholipidosis assays using the Calu-3 cells and PCLS as the patient-near system. Trimipramine and Chloroquine induced phospholipidosis but failed to inhibit SARS-CoV-2 replication in Calu-3 cells, which contradicts the reported findings and the proposed mechanism. Fluoxetine, only slightly induced phospholipidosis in Calu-3 cells but reduced viral replication by 2.7 orders of magnitude. Tilorone suppressed viral replication by 1.9 orders of magnitude in Calu-3 cells without causing phospholipidosis. Thus, induction of phospholipidosis is not correlated with the inhibition of SARS-CoV-2, and the compounds act via other mechanisms. However, we show that compounds, such as Amiodarone, Tamoxifen and Tilorone, with antiviral activity on Calu-3 cells, also inhibited viral replication in human PCLS. Our results indicate that antiviral assays against SARS-CoV-2 are cell-line specific. Data from Vero E6 can lead to non-transferable results, underlining the importance of an appropriate cell system for analysing antiviral compounds against SARS-CoV-2. We observed a correlation between the active compounds in Calu-3 cells and PCLS.}, language = {en} } @article{KarunakaranSubramanianJinetal.2023, author = {Karunakaran, Mohindar M. and Subramanian, Hariharan and Jin, Yiming and Mohammed, Fiyaz and Kimmel, Brigitte and Juraske, Claudia and Starick, Lisa and N{\"o}hren, Anna and L{\"a}nder, Nora and Willcox, Carrie R. and Singh, Rohit and Schamel, Wolfgang W. and Nikolaev, Viacheslav O. and Kunzmann, Volker and Wiemer, Andrew J. and Willcox, Benjamin E. and Herrmann, Thomas}, title = {A distinct topology of BTN3A IgV and B30.2 domains controlled by juxtamembrane regions favors optimal human γδ T cell phosphoantigen sensing}, series = {Nature Communications}, volume = {14}, journal = {Nature Communications}, doi = {10.1038/s41467-023-41938-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358179}, year = {2023}, abstract = {Butyrophilin (BTN)-3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members.}, language = {en} } @article{DjakovicHennigReinischetal.2023, author = {Djakovic, Lara and Hennig, Thomas and Reinisch, Katharina and Milić, Andrea and Whisnant, Adam W. and Wolf, Katharina and Weiß, Elena and Haas, Tobias and Grothey, Arnhild and J{\"u}rges, Christopher S. and Kluge, Michael and Wolf, Elmar and Erhard, Florian and Friedel, Caroline C. and D{\"o}lken, Lars}, title = {The HSV-1 ICP22 protein selectively impairs histone repositioning upon Pol II transcription downstream of genes}, series = {Nature Communications}, volume = {14}, journal = {Nature Communications}, doi = {10.1038/s41467-023-40217-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358161}, year = {2023}, abstract = {Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) when transcription termination is disrupted by the viral ICP27 protein. This is accompanied by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperone FACT is not sufficient to induce dOCRs in ΔICP22 infection but increases dOCR induction in wild-type HSV-1 infection. Interestingly, this is accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone repositioning downstream of genes in the wake of Pol II in HSV-1 infection.}, language = {en} }