Study of the capacity of Toll-like receptors to modulate pro-inflammatory responses mediated by receptors for the complement anaphylatoxin C5a

Toll-like receptors (TLRs) and the complement system play a crucial role in the innate immune response by mediating the initial recognition of, and prompt response to a variety of microorganisms. The concerted activation of TLRs and complement ensures efficient clearance of infection. Previous studies have documented synergism between TLRs and the receptor for the pro-inflammatory peptide C5a (C5aR/CD88), and regulation of TLR-induced pro-inflammatory responses by C5aR, suggesting crosstalk between TLRs and C5aR. However, it is unclear whether and how TLRs modulate C5a-induced pro-inflammatory responses. This study tested the hypothesis that a genuine, bi-directional signalling crosstalk between TLRs and C5a receptors exists, involving not only modulation of TLR-mediated responses by C5a receptor activation, but also modulation by TLR activation of the extent and/or quality of cellular responses to C5a. The experiments described in this thesis confirmed this hypothesis by demonstrating a marked positive modulatory effect of TLR activation on cell sensitivity to C5a in vitro and ex vivo and identifying underlying mechanistic targets. Pre-exposure of peripheral blood mononuclear cells and whole blood to diverse TLR ligands or bacteria enhanced C5a-induced pro-inflammatory responses. This effect was not observed in TLR4-signalling-deficient mice. TLR-induced hypersensitivity to C5a did not result from C5aR up-regulation or modulation of C5a-induced calcium mobilization. Rather, TLRs targeted the second C5a receptor, C5L2 (acting as a negative modulator of C5aR) by reducing C5L2 expression and activity. TLR-induced hypersensitivity to C5a was mimicked by blocking C5L2 and was not observed in C5L2KO mice. Furthermore, TLR activation inhibited C5L2 expression upon C5a stimulation. Expression of the key adaptor molecule β-arrestin 1, which mediates the inhibitory effects of C5L2 on C5aR, was also found to be negatively regulated by TLR activation. These findings identify a novel pathway of crosstalk within the innate immune system that amplifies innate host defence at the TLR-complement interface. Unravelling the mutually regulated activities of TLRs and complement may reveal new therapeutic avenues to control inflammation

Do glucagonomas always produce glucagon?

Pancreatic islet α-cell tumours that overexpress proglucagon are typically associated with the glucagonoma syndrome, a rare disease entity characterised by necrolytic migratory erythema, impaired glucose tolerance, thromboembolic complications and psychiatric disturbances. Paraneoplastic phenomena associated with enteric overexpression of proglucagon-derived peptides are less well recognized and include gastrointestinal dysfunction and hyperinsulinaemic hypoglycaemia. The diverse clinical manifestations associated with glucagon-expressing tumours can be explained, in part, by the repertoire of tumorally secreted peptides liberated through differential post-translational processing of tumour-derived proglucagon. Proglucagon-expressing tumours may be divided into two broad biochemical subtypes defined by either secretion of glucagon or GLP-1, GLP-2 and the glucagon-containing peptides, glicentin and oxyntomodulin, due to an islet α-cell or enteroendocrine L-cell pattern of proglucagon processing, respectively. In the current review we provide an updated overview of the clinical presentation of proglucagon-expressing tumours in relation to known physiological actions of proglucagon-derived peptides and suggest that detailed biochemical characterisation of the peptide repertoire secreted from these tumours may provide new opportunities for diagnosis and clinical management.   

Generation of two isogenic iPSC lines with either a heterozygous or a homozygous E280A mutation in the PSEN1 gene.

Alzheimer's disease (AD) is the most common form of dementia. Mutations in the gene PSEN1 encoding Presenilin1 are known to cause familial forms of AD with early age of onset. The most common mutation in the PSEN1 gene is the E280A mutation. iPSCs are an optimal choice for modeling AD, as they can be differentiated in vitro into neural cells. Here, we report the generation of two isogenic iPSC lines with either a homozygous or a heterozygous E280A mutation in the PSEN1 gene. The mutation was introduced into an iPSC line from a healthy individual using the CRISPR-Cas9 technology.Resource tableUnlabelled TableUnique stem cell lines identifier 1. BIONi010-C + homozygous E280A = BIONi010-C-29 2. BIONi010-C + heterozygous E280A = BIONi010-C-30Alternative names of stem cell lines 1. BIONi010-C E280 +/+ (homozygous line) 2. BIONi010-C E280 +/− (heterozygous line)InstitutionBioneer A/S Hørsholm Denmark and University of Copenhagen (UCPH) Copenhagen DenmarkContact information of distributorContact at Bioneer: Benjamin Schmid,[email protected]– Contact at UCPH: Kristine Freude,[email protected] of cell linesiPSCsOriginHumanCell SourceFibroblastsClonalityClonalMethod of reprogrammingEpisomal plasmids (Okita et al., 2011)Multiline rationaleMutated isogenic clonesGene modificationYESType of modificationInduced point mutationAssociated diseaseAlzheimer's disease (AD)Gene/locusPSEN1/Chr14:73664808Method of modificationCRISPR-Cas9Name of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateSeptember 2017Cell line repository/bank 1. BIONi010-C-29:https://hpscreg.eu/cell-line/BIONi010-C-29 2. BIONi010-C-30:https://hpscreg.eu/cell-line/BIONi010-C-30Ethical approvalThe study was approved by the Ethics Committee of the Capital Region of Denmark (H-4-2011-157), and written informed consent was obtained from the participant before enrolmen

Targeting of non-dominant antigens as a vaccine strategy to broaden T-cell responses during chronic viral infection

In this study, we compared adenoviral vaccine vectors with the capacity to induce equally potent immune responses against non-dominant and immunodominant epitopes of murine lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that vaccination targeting non-dominant epitopes facilitates potent virus-induced T-cell responses against immunodominant epitopes during subsequent challenge with highly invasive virus. In contrast, when an immunodominant epitope was included in the vaccine, the T-cell response associated with viral challenge remained focussed on that epitope. Early after challenge with live virus, the CD8+ T cells specific for vaccine-encoded epitopes, displayed a phenotype typically associated with prolonged/persistent antigenic stimulation marked by high levels of KLRG-1, as compared to T cells reacting to epitopes not included in the vaccine. Notably, this association was lost over time in T cells specific for the dominant T cell epitopes, and these cells were fully capable of expanding in response to a new viral challenge. Overall, our data suggests a potential for broadening of the antiviral CD8+ T-cell response by selecting non-dominant antigens to be targeted by vaccination. In addition, our findings suggest that prior adenoviral vaccination is not likely to negatively impact the long-term and protective immune response induced and maintained by a vaccine-attenuated chronic viral infection

Physical and mental recovery after aortic valve surgery in non-elderly patients: native valve-preserving surgery vs. prosthetic valve replacement

Background: Exercise capacity and patient-reported outcomes are increasingly considered crucial following aortic valve (AV) surgery in non-elderly adults. We aimed to prospectively evaluate the effect of native valve preservation compared with prosthetic valve replacement. Methods: From October 2017 to August 2020, 100 consecutive non-elderly patients undergoing surgery for severe AV disease were included. Exercise capacity and patient-reported outcomes were evaluated upon admission, and 3 months and 1 year postoperatively. Results: In total, 72 patients underwent native valve-preserving procedures (AV repair or Ross procedure, NV group), and 28 patients, prosthetic valve replacement (PV group). Native valve preservation was associated with an increased risk of reoperation (weighted hazard ratio: 10.57 (95% CI: 1.24–90.01), p = 0.031). The estimated average treatment effect on six-minute walking distance in NV patients at 1 year was positive, but not significant (35.64 m; 95% CI: −17.03–88.30, adj. p = 0.554). The postoperative physical and mental quality of life was comparable in both groups. Peak oxygen consumption and work rate were better at all assessment time points in NV patients. Marked longitudinal improvements in walking distance (NV, +47 m (adj. p < 0.001); PV, +25 m (adj. p = 0.004)) and physical (NV, +7 points (adj. p = 0.023); PV, +10 points (adj. p = 0.005)) and mental quality of life (NV, +7 points (adj. p < 0.001); PV, +5 points (adj. p = 0.058)) from the preoperative period to the 1-year follow-up were observed. At 1 year, there was a tendency of more NV patients reaching reference values of walking distance. Conclusions: Despite the increased risk of reoperation, physical and mental performance markedly improved after native valve-preserving surgery and was comparable to that after prosthetic aortic valve replacement

Soluble toll-like receptor 2 is a biomarker for sepsis in critically ill patients with multi-organ failure within 12 h of ICU admission

Soluble TLR2 levels are elevated in infective and inflammatory conditions, but its diagnostic value with sepsis-induced multi-organ failure has not been evaluated. 37 patients with a diagnosis of severe sepsis/septic shock (sepsis) and 27 patients with organ failure without infection (SIRS) were studied. Median (IQR) plasma sTLR2 levels were 2.7 ng/ml (1.4–6.1) in sepsis and 0.6 ng/ml (0.4–0.9) in SIRS p < 0.001. sTLR2 showed good diagnostic value for sepsis at cut-off of 1.0 ng/ml, AUC:0.959. We report the ability of sTLR2 levels to discriminate between sepsis and SIRS within 12 h of ICU admission in patients with multi-organ failure

Pre-Existing Vector Immunity Does Not Prevent Replication Deficient Adenovirus from Inducing Efficient CD8 T-Cell Memory and Recall Responses

Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity

Generation of spinocerebellar ataxia type 2 patient-derived iPSC line H271

AbstractSpinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease primarily affecting the cerebellum. Very little is known about the molecular mechanisms underlying the disease and, to date, no cure or treatment is available. Here, we demonstrate the generation of an induced pluripotent stem cell (iPSC) line of a SCA2 patient. The selected clone has been proven to be a bona fide iPSC line, which retains a normal karyotype. Due to its differentiation potential into neurons, this iPSC line will be a valuable tool in studying a disease-specific phenotype of SCA2