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Hoffmann, C.

Publications and source records attributed to Hoffmann, C..

48 records · Page 3Linked to original sources

Selectively expressed RNA molecules: a new dimension infunctionalized cell targeting

Exact targeting of specific mammalian cell types or diseased cells is one of the most urgently needed prerequisites for a new generation of potent pharmaceuticals. Different approaches have been pursued, failing mainly due to a lack of specific surface markers in most cases. Developing a completely novel RNA-based methodology, we can now ensure exact cell targeting and simultaneously combine this with selective expression of effector proteins, thereby functionalization of the target cell for therapy, diagnostics or cell steering. The specific combination of the molecular properties of antisense technology and mRNA therapy with functional RNA secondary structures allowed us to develop selectively expressed RNA molecules for medical applications. These so-called seRNAs remain inactive in non-target cells and are only activated by partial degradation to induce translation in preselected cell types of interest. Cell type specificity and type of functionalization are easily adaptable based on a simple modular system. In proof of concept in vitro and in vivo studies we used seRNAs as a highly selective platform technology for powerful glioblastoma cancer cell targeting and significantly reduce brain tumors of mice without detectable side effects with just a single treatment within days. Our data open up new potential avenues for the efficient treatment of various cancers and other human diseases.

cell biology↗

GM-CSF-activated human dendritic cells promote type1 T follicular helper cells (Tfh1) polarization in a CD40-dependent manner

T follicular helper (Tfh) cells are specialized CD4+ T cells that regulate humoral immunity by providing B cell help. Tfh1 sub-population was recently identified and associated with severity in infection and autoimmune diseases. The cellular and molecular requirements to induce human Tfh1 differentiation are unknown. Our work investigated the role of human dendritic cells (DC) in promoting Tfh1 differentiation and their physiopathological implication in mycobacterium tuberculosis and mild COVID-19 infection. Activated human blood CD1c+ DC were cocultured with allogeneic naive CD4+ T cells. Single-cell RNA sequencing was then used alongside protein validation to define the induced Tfh lineage. DC signature and correlation with Tfh1 cells in infected patients was established through bioinformatic analysis. Our results show that GM-CSF-activated DC drove the differentiation of Tfh1 cells, displaying typical Tfh molecular features, including 1) high levels of PD-1, CXCR5, and ICOS expression; 2) BCL6 and TBET co-expression; 3) IL-21 and IFN-{gamma} secretion. Mechanistically, GM-CSF triggered the emergence of two distinct DC sub-populations defined by their differential expression of CD40 and ICOS-ligand (ICOS-L), and distinct phenotype, morphology, transcriptomic signature, and function. We showed that Tfh1 differentiation was efficiently and specifically induced by CD40highICOS-Llow DC in a CD40-dependent manner. Tfh1 cells were positively associated with a CD40highICOS-LLow DC signature in patients with latent mycobacterium tuberculosis and mild COVID-19 infection. Our study uncovers a novel CD40-dependent human Tfh1 axis. Immunotherapy modulation of Tfh1 activity might contribute to control diseases where Tfh1 are known to play a key role, such as infections. Significance StatementDendritic cells (DC) play a central role in triggering the adaptive immune response due to their T cell priming functions. Among different T cell subsets, it is still not clear how human type1 T follicular helper cells (Tfh1) differentiate. Tfh1 cells are implicated in several physiopathological conditions, including infections. Here we show that GM-CSF induces diversification of human DC. Only CD40highICOS-LLow DC were able to drive Tfh1 cell differentiation. We found that CD40highICOS-LLow DC signature was associated to Tfh1 cells in mycobacterium tuberculosis and COVID-19 patients. Our data reveal a previously undescribed pathway leading to human Tfh1 cell differentiation and highlight the importance of GM-CSF and CD40 as potential targets for the design of anti-infective therapies.

immunology↗

iTRVZ: liquid nano-platform for signal integration on the plasma membrane

Cell survival in immune-competent organisms relies on both growth and immune evasion, yet these processes have been studied as independent cellular programs. Here, using super-resolution single-molecule imaging, we identify GEM (Growth and Evasion Metastable hub), a {approx}34-nm, metastable, nano-liquid, plasma membrane hub that physically integrates receptor-type tyrosine kinase (RTK) inputs with CD59-mediated immune-evasion signaling within the same assembly. GEM is formed by LLPS-like protein clustering on PI(4,5)P2-containing raft-nanodomains. Upon concurrent RTK and CD59 stimulations, GEM recruits both receptors and downstream kinases, enabling reciprocal activation via nano-liquid confinement that supralinearly amplifies PLC{gamma}-IP3-Ca2+ and PI3K-Akt survival signaling outputs. In mice, disrupting GEM suppresses tumor growth in vivo, establishing nano-liquid GEM as an integrator that physically couples growth and CD59-mediated immune-evasion signaling for cell survival.

cell biology↗

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model

Deposition of amyloid beta (A{beta}) along with glia cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimers disease (AD). In recent years, impairment of autophagy has been found to be another important feature, contributing to AD progression and aging. Therefore, we assessed the effect of the autophagy activator Spermidine, a small body-endogenous polyamine often used as dietary supplement and known to promote longevity, on glia cell-mediated neuroinflammation. Spermidine reduced TLR3- and TLR4- mediated inflammatory processes in microglia and astrocytes by decreasing cytotoxicity, inflammasome activity and NF-{kappa}B signaling. In line with these anti-inflammatory effects, oral treatment of the amyloid prone AD-like APPPS1 mice with Spermidine reduced neuroinflammation and neurotoxic soluble A{beta}. Mechanistically, single nuclei sequencing revealed microglia as one of the main targets of Spermidine treatment, with increased expression of genes implicated in cell motility and phagocytosis. Thus, Spermidine provides a promising therapeutic potential to target glia cells in AD progression.

neuroscience↗

Suitability of GRK antibodies for individual detection and quantification of GRK isoforms in western blots

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This leads to the recruitment of arrestins resulting in desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffold. This multifunctionality is also reflected by their diverse roles in pathological conditions like cancer, influenza infection, malaria, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRK2, GRK3, GRK5, and GRK6 in western blot analysis. We thereby identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross reactivity. Thus, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained western blot results. Utilizing the most suitable antibodies we established the western blot-based, cost-effective, simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in five commonly used cell lines revealing differential isoform expression.

molecular biology↗

Bacteriocinogenic lactic acid bacteria in the traditional cereal-based beverage Boza: a genomic and functional approach

Boza is a traditional low-alcohol fermented beverage from the Balkan Peninsula, frequently explored as a functional food product. The product is rich in Lactic Acid Bacteria (LAB) and some of them can produce bacteriocins. In this study, a sample of Boza from Belogratchik, Bulgaria, was analyzed for the presence of bacteriocinogenic LAB, and after analyses by RAPD-PCR, three representative isolates were characterized by genomic analyses, using whole genome sequencing. Isolates identified as Pediococcus pentosaceus ST75BZ and Pediococcus pentosaceus ST87BZ contained operons encoding for bacteriocins pediocin PA-1 and penocin A, while isolate identified as P. acidilactici ST31BZ contained only the operon for pediocin PA-1 and a CRISPR/Cas system for protection against bacteriophage infection. The antimicrobial activity of bacteriocins produced by the three isolates was inhibited by treatment of the cell-free supernatants with proteolytic enzymes. The produced bacteriocins inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp., among other tested species. The levels of bacteriocin production varied from 3200 AU/ml to 12800 AU/ml recorded against L. monocytogenes 104, 637 and 711, measured at 24 h of incubation at 37{degrees}C. All bacteriocins remained active after incubation at pH 2.0 to 10.0. The activity mode of the studied bacteriocins was bactericidal, as determined against L. monocytogenes 104, 637 and 711. In addition, bactericidal activity was demonstrated using a cell leakage {beta}-galactosidase assay, indicating a pore formation mechanism as a mode of action. The present study highlights the importance of combining metagenomic analyses and traditional microbiological approaches as way of characterizing microbial interactions in fermented foods.

microbiology↗

Lung epithelial CYP1 activity regulates aryl hydrocarbon receptor dependent allergic airway inflammation

The lung epithelial barrier serves as a guardian towards environmental insults and responds to allergen encounter with a cascade of immune reactions that can possibly lead to inflammation. Whether the environmental sensor aryl hydrocarbon receptor (AhR) together with its downstream targets cytochrome P450 (CYP1) family members contribute to the regulation of allergic airway inflammation remains unexplored. By employing knockout mice for AhR and for single CYP1 family members, we found that AhR-/- and CYP1B1-/- but not CYP1A1-/- or CYP1A2-/- animals display enhanced allergic airway inflammation compared to WT. Expression analysis, immunofluorescence staining of murine and human lung sections and bone marrow chimeras suggest an important role of CYP1B1 in non-hematopoietic lung epithelial cells to prevent exacerbation of allergic airway inflammation. Transcriptional analysis of murine and human lung epithelial cells indicates a functional link of AhR to barrier protection/inflammatory mediator signaling upon allergen challenge. In contrast, CYP1B1 deficiency leads to enhanced expression and activity of CYP1A1 in lung epithelial cells and to an increased availability of the AhR ligand kynurenic acid following allergen challenge. Thus, differential CYP1 family member expression and signaling via the AhR in epithelial cells represents an immunoregulatory layer protecting the lung from exacerbation of allergic airway inflammation.

immunology↗

High Level of Interaction between Phages and Bacteria in an Artisanal Raw Milk Cheese Microbial Community

Endogenous starter cultures are used in the production of several cheeses around the world, such as Parmigiano-Reggiano, in Italy, Epoisses, in France, and Canastra, in Brazil. These microbial communities are responsible for many of the intrinsic characteristics of each of these cheeses. Bacteriophages are ubiquitous around the world, well known to be involved in the modulation of complex microbiological processes. However, little is known about phage-bacteria growth dynamics in cheese production systems, where phages are normally treated as problems, as the viral infections can negatively affect or even eliminate the starter culture during production. Furthermore, a recent metagenomic based meta-analysis has reported that cheeses contain a high abundance of phage-associated sequences. Here, we analyse the viral and bacterial metagenomes of Canastra cheese, a tradition artisanal cheese produced using an endogenous starter culture. We observe a very high phage diversity level, mostly composed of novel sequences. We detect several metagenomic assembled bacterial genomes at strain level resolution, and several putative phage-bacteria interactions, evidenced by the recovered viral and bacterial genomic signatures. We postulate that at least one bacterial strain detected could be endogenous to the Canastra region, in Brazil, and that its growth seems to be modulated by native phages present in this artisanal production system. This relationship is likely to influence the fermentation dynamics and ultimately the sensorial profile of these cheeses, with implication for all cheeses that employ similar production processes around the world.

microbiology↗

GRK2/3/5/6 knockout: The impact of individual GRKs on arrestin-binding and GPCR regulation

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and represent major drug targets. Upon ligand stimulation, GPCRs activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and formation of receptor-arrestin complexes. For many GPCRs, this mechanism triggers receptor desensitisation, internalisation, and possibly a second intracellular signalling wave. Here we created eleven different HEK293 knockout cell clones for GRK2, 3, 5, and 6 individually and in combination. These include four single, two double, four triple, and the quadruple GRK knockout. The statistical evaluation of {beta}-arrestin1/2 interactions for twelve different receptors grouped the tested GPCRs into two main subsets: those for which {beta}-arrestin interaction was mediated by either GRK2, 3, 5, or 6 and those that are mediated by GRK2 or 3 only. Interestingly, the overexpression of specific GRKs was found to induce a robust, ligand-independent {beta}-arrestin interaction with the V2R and AT1R. Finally, using GRK knockout cells, PKC inhibitors, and {beta}-arrestin mutants, we present evidence for differential AT1R-{beta}-arrestin2 complex configurations mediated by selective engagement of PKC, GRK2, or GRK6. We anticipate our novel GRK-knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and {beta}-arrestin complex formation.

cell biology↗

MMP2 As An Independent Prognostic Stratifier In Oral Cavity Cancers

BackgroundAround 25% of oral cavity squamous cell carcinoma (OCSCC) are not controlled by standard of care. Identifying those patients could offer them possibilities for intensified and personalized regimen. However, there is currently no validated biomarker for OCSCC patient selection in a pre-treatment setting.\n\nPatients and methodsOur objectives were to determine a robust and independent predictive biomarker for disease related death in OCSCC treated with standard of care. Tumor and juxtatumor secretome were analyzed in a prospective discovery cohort of 37 OCSCC treated by primary surgery. Independent biomarker validation was performed by RTqPCR in a retrospective cohort of 145 patients with similar clinical features. An 18-gene signature (18G) predictive of the response to PD-1 blockade was evaluated in the same cohort..\n\nResultsAmong 29 deregulated molecules in a secretome analysis, we identified soluble MMP2 as a prognostic biomarker. In our validation cohort (n=145), high levels of MMP2 and CD276, and low levels of CXCL10 and STAT1 mRNA were associated with poor prognosis in univariate analysis (Kaplan-Meier). MMP2 (p = 0.001) and extra-nodal extension (ENE) (p = 0.006) were independent biomarkers of disease-specific survival (DSS) in multivariate analysis, and defined prognostic groups with 5-year DSS ranging from 36% (MMP2highENE+) to 88% (MMP2lowENE-). The expression of 18G was similar in the different prognostic groups, suggesting comparable responsiveness to anti-PD-1.\n\nConclusionHigh levels of MMP2 was an independent and validated prognostic biomarker, which may be used to select poor prognosis patients for intensified neoadjuvant or adjuvant regimens.

cancer biology↗

PDL1 AND ICOSL DISCRIMINATE HUMAN SECRETORY AND HELPER DENDRITIC CELLS

Dendritic cells (DC) are described as immature at the steady state, with a high antigen capture capacity, turning into a mature state with a strong T cell stimulatory capacity upon activation. Using 16 different stimuli in vitro (130 observations), we describe two states of human activated dendritic cells. PDL1highICOSLlow \"secretory DC\" produced large amounts of inflammatory cytokines and chemokines but induced very low levels of T helper (Th) cytokines following DC-T co-culture; conversely PDL1lowICOSLhigh \"helper DC\" produced low levels of secreted factors but induced high levels of Th cytokines characteristic of a broad range of Th subsets. Secretory DC were phenotypically identified in T cell inflamed primary head and neck squamous cell carcinoma. RNAseq analysis showed that they expressed a typical secretory DC signature, including CD40, PVR, IL1B, TNF, and CCL19. This novel and universal functional dichotomy of human DC opens broad perspectives for the characterization of inflammatory diseases, and for immunotherapy.

immunology↗

Transcriptional profiling reveals extraordinary diversity among skeletal muscle tissues

Skeletal muscle comprises a family of diverse tissues with highly specialized morphology, function, and metabolism. Many acquired diseases - including HIV, COPD, cancer cachexia, critical illness myopathy, and sepsis - affect specific muscles while sparing others. Even monogenic muscular dystrophies tend to selectively affect certain muscle groups, despite their causative genetic mutations being present in all tissues. These observations suggest that factors intrinsic to muscle tissues influence their susceptibility to various disease mechanisms. Nevertheless, most studies have not addressed transcriptional diversity among skeletal muscles. Here we use RNA sequencing (RNA-seq) to profile global mRNA expression in a wide array of skeletal, smooth, and cardiac muscle tissues from mice and rats. Our data set, MuscleDB, reveals extensive transcriptional diversity, with greater than 50% of transcripts differentially expressed among skeletal muscle tissues. This diversity is only partly explained by fiber type composition and developmental history, suggesting that specialized transcriptional profiles establish the functional identity of muscle tissues. We find conservation in the transcriptional profiles across species as well as between males and females, indicating that these data may be useful in predicting gene expression in related species. Notably, thousands of differentially expressed genes in skeletal muscle are associated with human disease, and hundreds of these genes encode targets of drugs on the market today. We detect mRNA expression of hundreds of putative myokines that may underlie the endocrine functions of skeletal muscle. In addition to demonstrating the intrinsic diversity of skeletal muscles, these data provide a resource for generating testable hypotheses regarding the mechanisms that establish differential disease susceptibility in muscle.\n\nSignificance StatementSkeletal muscles are a diverse family of tissues with a common contractile function but divergent morphology, development, and metabolism. One need only reflect on the different functions of limb muscles and the diaphragm to realize the highly specialized nature of these tissues. Nevertheless, every study of global gene expression has analyzed at most one representative skeletal muscle. Here we measure gene expression from 11 different skeletal muscles in mice and rats. We show that there is no such thing as a representative skeletal muscle, as gene expression profiles vary widely among the tissues analyzed. These data are an important resource for pharmacologists, tissue engineers, and investigators studying the mechanisms of cellular specialization.

systems biology↗