• C-Type Lectin Receptor (CLR)-Fc Fusion Proteins As Tools to Screen for Novel CLR/Bacteria Interactions: An Exemplary Study on Preselected Isolates.

      Mayer, Sabine; Moeller, Rebecca; Monteiro, João T; Ellrott, Kerstin; Josenhans, Christine; Lepenies, Bernd; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (2018-01-01)
      C-type lectin receptors (CLRs) are carbohydrate-binding receptors that recognize their ligands often in a Ca2+-dependent manner. Upon ligand binding, myeloid CLRs in innate immunity trigger or inhibit a variety of signaling pathways, thus initiating or modulating effector functions such as cytokine production, phagocytosis, and antigen presentation. CLRs bind to various pathogens, including viruses, fungi, parasites, and bacteria. The bacterium Campylobacter jejuni (C. jejuni) is a very frequent Gram-negative zoonotic pathogen of humans, causing severe intestinal symptoms. Interestingly, C. jejuni expresses several glycosylated surface structures, for example, the capsular polysaccharide (CPS), lipooligosaccharide (LOS), and envelope proteins. This “Methods” paper describes applications of CLR–Fc fusion proteins to screen for yet unknown CLR/bacteria interactions using C. jejuni as an example. ELISA-based detection of CLR/bacteria interactions allows a frst prescreening that is further confrmed by flow cytometry-based binding analysis and visualized using confocal microscopy. By applying these methods, we identifed Dectin-1 as a novel CLR recognizing two selected C. jejuni isolates with different LOS and CPS genotypes. In conclusion, the heredescribed applications of CLR–Fc fusion proteins represent useful methods to screen for and identify novel CLR/bacteria interactions.
    • C-X-C Motif Chemokine Receptor 4 Blockade Promotes Tissue Repair After Myocardial Infarction by Enhancing Regulatory T Cell Mobilization and Immune-Regulatory Function.

      Wang, Yong; Dembowsky, Klaus; Chevalier, Eric; Stüve, Philipp; Korf-Klingebiel, Mortimer; Lochner, Matthias; Napp, L Christian; Frank, Heike; Brinkmann, Eva; Kanwischer, Anna; et al. (Lippinscott, Williams & Wilkins; American Heart Association, 2019-01-30)
      Acute myocardial infarction (MI) elicits an inflammatory response that drives tissue repair and adverse cardiac remodeling. Inflammatory cell trafficking after MI is controlled by C X-C motif chemokine ligand 12 (CXCL12) and its receptor, C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 antagonists mobilize inflammatory cells and promote infarct repair, but the cellular mechanisms are unclear. We investigated the therapeutic potential and mode of action of the peptidic macrocycle CXCR4 antagonist POL5551 in mice with reperfused MI. We applied cell depletion and adoptive transfer strategies using lymphocyte-deficient Rag1 knockout mice; DEREG mice, which express a diphtheria toxin receptor-enhanced green fluorescent protein fusion protein under the control of the promoter/enhancer region of the regulatory T (T Intraperitoneal POL5551 injections in wild-type mice (8 mg/kg at 2, 4, 6, and 8 d) enhanced angiogenesis in the infarct border-zone, reduced scar size, and attenuated left ventricular remodeling and contractile dysfunction at 28 d. Treatment effects were absent in splenectomized wild-type mice, Rag1 knockout mice, and T Our data confirm CXCR4 blockade as a promising treatment strategy after MI. We identify dendritic cell-primed splenic T
    • Cell therapy products: focus on issues with manufacturing and quality control of chimeric antigen receptor T-cell therapies

      Eyles, Jim E; Vessillier, Sandrine; Jones, Anika; Stacey, Glyn; Schneider, Christian K; Price, Jack; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany.
      Recent accelerated approvals of Chimeric Antigen Receptor T‐cell (CAR‐T) therapies targeting refractory haematological malignancies underscore the potential for this novel technology platform to provide new therapeutic options for oncology areas with high unmet medical needs. However, these powerful ‘living drugs’ are markedly different to conventional small molecule and biologic therapies on several levels. The highly complex nature and varied composition of CAR‐T based products still requires considerable investigation to resolve the best approaches to ensure reproducible and cost‐effective manufacture, clinical development, and application. This review will focus on key issues for manufacturing and quality control of these exciting new therapeutic modalities, preceded by a brief description of CAR principals and clinical development considerations. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
    • Cell therapy products: focus on issues with manufacturing and quality control of chimeric antigen receptor T-cell therapies

      Eyles, Jim E; Vessillier, Sandrine; Jones, Anika; Stacey, Glyn; Schneider, Christian K; Price, Jack; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (2018-12-17)
    • A combined in silico and in vitro study on mouse Serpina1a antitrypsin-deficiency mutants.

      Eggenschwiler, Reto; Patronov, Atanas; Hegermann, Jan; Fráguas-Eggenschwiler, Mariane; Wu, Guangming; Cortnumme, Leon; Ochs, Matthias; Antes, Iris; Cantz, Tobias; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Springer-Nature, 2019-05-16)
      Certain point-mutations in the human SERPINA1-gene can cause severe α1-antitrypsin-deficiency (A1AT-D). Affected individuals can suffer from loss-of-function lung-disease and from gain-of-function liver-disease phenotypes. However, age of onset and severity of clinical appearance is heterogeneous amongst carriers, suggesting involvement of additional genetic and environmental factors. The generation of authentic A1AT-D mouse-models has been hampered by the complexity of the mouse Serpina1-gene locus and a model with concurrent lung and liver-disease is still missing. Here, we investigate point-mutations in the mouse Serpina1a antitrypsin-orthologue, which are homolog-equivalent to ones known to cause severe A1AT-D in human. We combine in silico and in vitro methods and we find that analyzed mutations do introduce potential disease-causing properties into Serpina1a. Finally, we show that introduction of the King’s-mutation causes inactivation of neutrophil elastase inhibitory-function in both, mouse and human antitrypsin, while the mouse Z-mutant retains activity. This work paves the path to generation of better A1AT-D mouse-models.
    • Commonly setting biological standards in rare diseases

      O’Connor, Daniel J.; Buckland, Jenny; Almond, Neil; Boyle, Jennifer; Coxon, Carmen; Gaki, Eleni; Martin, Javier; Mattiuzzo, Giada; Metcalfe, Clive; Page, Mark; et al. (Taylor& Francis, 2019-01-01)
      Introduction: Standardization is important across the life cycle of medicinal products, supporting the diagnosis, treatment, and prevention of a wide range of diseases. For rare diseases, standardization is even more important, as patient groups are small, presenting significant challenges in the design, conduct, analysis, and interpretation of clinical studies. It is here that standardization institutions, including the UK’s National Institute for Biological Standards and Control (NIBSC), can have a key role. Areas covered: A considerable proportion of NIBSC’s work supports the better understanding, diagnosis, treatment, and prevention of rare diseases. NIBSC is also part of the UK’s Medicines and Healthcare products Regulatory Agency (MHRA), creating an agency that is uniquely placed to combine scientific and regulatory expertize for the benefit of public health. This review provides an overview of NIBSC’s work in rare diseases and highlights the positive impact of the work of standardization institutions in this field. Expert opinion: Standardization in product development is key for patients with rare diseases. The work of standardization institutions is increasingly being recognized as crucial for supporting scientific and clinical advancements, and early and collaborative interactions can provide drug developers with the necessary expertize, when standards matter most.
    • 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.
    • 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.
    • Efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins.

      Serradell, Marianela C; Rupil, Lucía L; Martino, Román A; Prucca, César G; Carranza, Pedro G; Saura, Alicia; Fernández, Elmer A; Gargantini, Pablo R; Tenaglia, Albano H; Petiti, Juan P; et al. (Springer-Nature, 2019-01-21)
      Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.
    • 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.
    • 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.
    • Homologous recombination mediates stable Fah gene integration and phenotypic correction in tyrosinaemia mouse-model.

      Junge, Norman; Yuan, Qinggong; Vu, Thu Huong; Krooss, Simon; Bednarski, Christien; Balakrishnan, Asha; Cathomen, Toni; Manns, Michael P; Baumann, Ulrich; Sharma, Amar Deep; et al. (2018-02-27)
      To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout (Fah-/-)mice by homologous-recombination-mediated targeted addition of theFahgene.
    • 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.
    • Parasites in brains of wild rodents (Arvicolinae and Murinae) in the city of Leipzig, Germany

      Waindok, Patrick; Özbakış-Beceriklisoy, Gökben; Janecek-Erfurth, Elisabeth; Springer, Andrea; Pfeffer, Martin; Leschnik, Michael; Strube, Christina; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Elsevier, 2019-12-01)
      Small rodents serve as intermediate or paratenic hosts for a variety of parasites and may participate in thetransmission of these parasites into synanthropic cycles. Parasites with neuroinvasive stages, such asToxoplasmagondiiorToxocara canis, can cause detrimental damage in the brain of intermediate or paratenic hosts.Therefore, the occurrence of neuroinvasive parasite stages was evaluated in brains of wild rodents captured inthe city of Leipzig, Germany. In addition, a few specimens from the cities of Hanover, Germany, and Vienna,Austria were included, resulting in a total of 716 rodents collected between 2011 and 2016. Brains were in-vestigated for parasitic stages by microscopic examination of native tissue, artificially digested tissue as well asGiemsa-stained digestion solution to verify positive results. Infective stages of zoonotic ascarids or other hel-minths were not detected in any sample, while coccidian cysts were found in 10.1% (95% CI: 7.9–12.5%; 72/716) of examined brains. The most abundant rodent species in the study was the bank vole (Myodes glareolus;Arvicolinae), showing an infection rate with cerebral cysts of 13.9% (95% CI: 11.0–17.8%; 62/445), while 2.7%(95% CI: 1.0–5.8%; 6/222) of yellow-necked mice (Apodemusflavicollis; Murinae) were infected. Generalizedlinear modelling revealed a statistically significant difference in prevalence betweenM. glareolusandA.flavi-collis, significant local differences as well as an effect of increasing body mass on cyst prevalence. Coccidian cystswere differentiated by amplification of the18S rRNAgene and subsequent sequencing. The majority of iden-tifiable cysts (97.9%) were determined asFrenkelia glareoli, a coccidian species mainly circulating betweenM.glareolusas intermediate and buzzards (Buteospp.) as definitive hosts. The zoonotic pathogenToxoplasma gondiiwas confirmed in oneM. glareolusoriginating from the city of Leipzig. Overall, it can be concluded that neu-roinvasion of zoonotic parasites seems to be rare inM. glareolusandA.flavicollis.
    • 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.
    • The role of epigenetics in the development of childhood asthma.

      Qi, Cancan; Xu, Cheng-Jian; Koppelman, Gerard H; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (2019-11-10)
      Introduction: The development of childhood asthma is caused by a combination of genetic factors and environmental exposures. Epigenetics describes mechanisms of (heritable) regulation of gene expression that occur without changes in DNA sequence. Epigenetics is strongly related to aging, is cell-type specific, and includes DNA methylation, noncoding RNAs, and histone modifications.Areas covered: This review summarizes recent epigenetic studies of childhood asthma in humans, which mostly involve studies of DNA methylation published in the recent five years. Environmental exposures, in particular cigarette smoking, have significant impact on epigenetic changes, but few of these epigenetic signals are also associated with asthma. Several asthma-associated genetic variants relate to DNA methylation. Epigenetic signals can be better understood by studying their correlation with gene expression, which revealed higher presence and activation of blood eosinophils in asthma. Strong associations of nasal methylation signatures and atopic asthma were identified, which were replicable across different populations.Expert commentary: Epigenetic markers have been strongly associated with asthma, and might serve as biomarker of asthma. The causal and longitudinal relationships between epigenetics and disease, and between environmental exposures and epigenetic changes need to be further investigated. Efforts should be made to understand cell-type-specific epigenetic mechanisms in asthma.
    • Sialylation Is Dispensable for Early Murine Embryonic Development in Vitro.

      Abeln, Markus; Borst, Kristina M; Cajic, Samanta; Thiesler, Hauke; Kats, Elina; Albers, Iris; Kuhn, Maike; Kaever, Volkhard; Rapp, Erdmann; Münster-Kühnel, Anja; et al. (2017-07-04)
      The negatively charged nonulose sialic acid (Sia) is essential for murine development in vivo. In order to elucidate the impact of sialylation on differentiation processes in the absence of maternal influences, we generated mouse embryonic stem cell (mESC) lines that lack CMP-Sia synthetase (CMAS) and thereby the ability to activate Sia to CMP-Sia. Loss of CMAS activity resulted in an asialo cell surface accompanied by an increase in glycoconjugates with terminal galactosyl and oligo-LacNAc residues, as well as intracellular accumulation of free Sia. Remarkably, these changes did not impact intracellular metabolites or the morphology and transcriptome of pluripotent mESC lines. Moreover, the capacity of Cmas
    • Targeting Antigens to Dendritic Cells the DC-Specific-ICAM3-Grabbing-Nonintegrin Receptor Induces Strong T-Helper 1 Immune Responses.

      Velasquez, Lis Noelia; Stüve, Philipp; Gentilini, Maria Virginia; Swallow, Maxine; Bartel, Judith; Lycke, Nils Yngve; Barkan, Daniel; Martina, Mariana; Lujan, Hugo D; Kalay, Hakan; et al. (2018-01-01)
      Tuberculosis remains a major global health problem and efforts to develop a more effective vaccine have been unsuccessful so far. Targeting antigens (Ags) to dendritic cells (DCs) in vivo has emerged as a new promising vaccine strategy. In this approach, Ags are delivered directly to DCs via antibodies that bind to endocytic cell-surface receptors. Here, we explored DC-specifc-ICAM3-grabbing-nonintegrin (DC-SIGN) targeting as a potential vaccine against tuberculosis. For this, we made use of the hSIGN mouse model that expresses human DC-SIGN under the control of the murine CD11c promoter. We show that in vitro and in vivo delivery of anti-DC-SIGN antibodies conjugated to Ag85B and peptide 25 of Ag85B in combination with anti-CD40, the fungal cell wall component zymosan, and the cholera toxin-derived fusion protein CTA1-DD induces strong Ag-specifc CD4+ T-cell responses. Improved anti-mycobacterial immunity was accompanied by increased frequencies of Ag-specifc IFN-γ+ IL-2+ TNF-α+ polyfunctional CD4+ T cells in vaccinated mice compared with controls. Taken together, in this study we provide the proof of concept that the human DC-SIGN receptor can be effciently exploited for vaccine purposes to promote immunity against mycobacterial infections.
    • TGFβ-activation by dendritic cells drives Th17 induction and intestinal contractility and augments the expulsion of the parasite Trichinella spiralis in mice.

      Steel, Nicola; Faniyi, Aduragbemi A; Rahman, Sayema; Swietlik, Stefanie; Czajkowska, Beata I; Chan, Bethany T; Hardgrave, Alexander; Steel, Anthony; Sparwasser, Tim D; Assas, Mushref B; et al. (PLOS, 2019-01-01)
      Helminths are highly prevalent metazoan parasites that infect over a billion of the world’s population. Hosts have evolved numerous mechanisms to drive the expulsion of these parasites via Th2-driven immunity, but these responses must be tightly controlled to prevent equally devastating immunopathology. However, mechanisms that regulate this balance are still unclear. Here we show that the vigorous Th2 immune response driven by the small intestinal helminth Trichinella spiralis, is associated with increased TGFβ signalling responses in CD4+ T-cells. Mechanistically, enhanced TGFβ signalling in CD4+ T-cells is dependent on dendritic cell-mediated TGFβ activation which requires expression of the integrin αvβ8. Importantly, mice lacking integrin αvβ8 on DCs had a delayed ability to expel a T. spiralis infection, indicating an important functional role for integrin αvβ8-mediated TGFβ activation in promoting parasite expulsion. In addition to maintaining regulatory T-cell responses, the CD4+ T-cell signalling of this pleiotropic cytokine induces a Th17 response which is crucial in promoting the intestinal muscle hypercontractility that drives worm expulsion. Collectively, these results provide novel insights into intestinal helminth expulsion beyond that of classical Th2 driven immunity, and highlight the importance of IL-17 in intestinal contraction which may aid therapeutics to numerous diseases of the intestine.