Ventricular stabilization with a customized decellularized cardiac ECM-based scaffold after myocardial infarction alters gene expression in a rodent LAD-ligation model.

Objectives: Decellularized extracellular matrix (dECM) is increasingly used in a wide range of regenerative medicine applications and may also offer the potential to support injured myocardium. Here, we evaluated the myocardial gene expression pattern after myocardial infarction (MI) in a standardized rodent LAD-ligation model with and without ventricular stabilization with a customized, cardiac dECM-based scaffold (cdECM). Methods: MI was induced in male Wistar rats by standard LAD-ligation and confirmed 14 days post-intervention by echocardiographic parameters (FAS<40%). Cardiac ECM from donor rats was used to generate individual cdECM-scaffolds (tissue engineered myocardial sleeve, TEMS), which were epicardially implanted after confirmed MI for ventricular stabilization. After 4 and 8 weeks heart function was assessed by echocardiography, rats were sacrificed and explanted hearts were analyzed. In addition to histological analysis, standardized anterior left ventricular wall myocardial tissue samples were assessed by quantitative real-time PCR evaluating the specific gene expression pattern for immunomodulatory (IL-10, TGFBR2, TNFα), pro-angiogenic (VEGFA, FGF2, PGF, PDGFB), pro-survival (HGF, SDF1, IGF1, AKT1), remodeling-associated (TIMP1, MMP2, MMP9) and infarction-specific (NPPA, NPPB) markers. Results: Ventricular stabilization led to integration of the TEMS-scaffold into the myocardial scar with varying degrees of cellular infiltration, as well as significantly improved echocardiographic parameters demonstrating attenuation of maladaptive cardiac remodeling. Further, TEMS implantation after MI altered the myocardial gene expression pattern. Differences in gene expression were most striking after 4 weeks with significantly reduced expression of NPPA (0.36 ± 0.26 vs 0.75 ± 0.40; p < 0.05), NPPB (0.47 ± 0.25 vs 0.91 ± 0.429; p < 0.01), TGFBR2 (0.68 ± 0.16 vs 0.90 ± 0.14; p < 0.01) and PDGFB (0.81 ± 0.13 vs 1.06 ± 0.14; p < 0.01) as well as increased expression of IL-10 (5.93 ± 5.67 vs 1.38 ± 0.60; p < 0.05), PGF (1.48 ± 0.38 vs 1.09 ± 0.25; p < 0.05) and IGF1 (1.67 ± 0.70 vs 1.03 ± 0.42; p < 0.05). However, after 8 weeks differences in the gene expression patterns of remodeling-associated, and pro-angiogenic markers could still be observed between groups. Conclusion: Ventricular stabilization via TEMS implantation after MI did not only led to biological integration of the cdECM-scaffolds into the host tissue and improved functional cardiac parameters, but also altered 4 and 8 week gene expression of infarcted myocardium, possibly contributing to reducing chronic deteriorating effects while increasing the potential for myocardial regeneration.

Generation of immune cell containing adipose organoids for in vitro analysis of immune metabolism.

Adipose tissue is an organized endocrine organ with important metabolic and immunological functions and immune cell-adipocyte crosstalk is known to drive various disease pathologies. Suitable 3D adipose tissue organoid models often lack resident immune cell populations and therefore require the addition of immune cells isolated from other organs. We have created the first 3D adipose tissue organoid model which could contain and maintain resident immune cell populations of the stromal vascular fraction (SVF) and proved to be effective in studying adipose tissue biology in a convenient manner. Macrophage and mast cell populations were successfully confirmed within our organoid model and were maintained in culture without the addition of growth factors. We demonstrated the suitability of our model for monitoring the lipidome during adipocyte differentiation in vitro and confirmed that this model reflects the physiological lipidome better than standard 2D cultures. In addition, we applied mass spectrometry-based lipidomics to track lipidomic changes in the lipidome upon dietary and immunomodulatory interventions. We conclude that this model represents a valuable tool for immune-metabolic research.

The envelope protein of tick-borne encephalitis virus influences neuron entry, pathogenicity, and vaccine protection.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection. METHOD: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated. RESULTS: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains. CONCLUSIONS: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.

Type I Interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1.

BACKGROUND: Although type I interferons (IFNs)-key effectors of antiviral innate immunity are known to be induced via different pattern recognition receptors (PRRs), the cellular source and the relative contribution of different PRRs in host protection against viral infection is often unclear. IPS-1 is a downstream adaptor for retinoid-inducible gene I (RIG-I)-like receptor signaling. In this study, we investigate the relative contribution of IPS-1 in the innate immune response in the different brain regions during infection with tick-borne encephalitis virus (TBEV), a flavivirus that causes a variety of severe symptoms like hemorrhagic fevers, encephalitis, and meningitis in the human host. METHODS: IPS-1 knockout mice were infected with TBEV/Langat virus (LGTV), and viral burden in the peripheral and the central nervous systems, type I IFN induction, brain infiltrating cells, and inflammatory response was analyzed. RESULTS: We show that IPS-1 is indispensable for controlling TBEV and LGTV infections in the peripheral and central nervous system. Our data indicate that IPS-1 regulates neuropathogenicity in mice. IFN response is differentially regulated in distinct regions of the central nervous system (CNS) influencing viral tropism, as LGTV replication was mainly restricted to olfactory bulb in wild-type (WT) mice. In contrast to the other brain regions, IFN upregulation in the olfactory bulb was dependent on IPS-1 signaling. IPS-1 regulates basal levels of antiviral interferon-stimulated genes (ISGs) like viperin and IRF-1 which contributes to the establishment of early viral replication which inhibits STAT1 activation. This diminishes the antiviral response even in the presence of high IFN-ß levels. Consequently, the absence of IPS-1 causes uncontrolled virus replication, in turn resulting in apoptosis, activation of microglia and astrocytes, elevated proinflammatory response, and recruitment of inflammatory cells into the CNS. CONCLUSIONS: We show that LGTV replication is restricted to the olfactory bulb and that IPS-1 is a very important player in the olfactory bulb in shaping the innate immune response by inhibiting early viral replication and viral spread throughout the central nervous system. In the absence of IPS-1, higher viral replication leads to the evasion of antiviral response by inhibiting interferon signaling. Our data suggest that the local microenvironment of distinct brain regions is critical to determine virus permissiveness.

Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses.

UNLABELLED: Vector-borne flaviviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus, and dengue virus, cause millions of infections in humans. TBEV causes a broad range of pathological symptoms, ranging from meningitis to severe encephalitis or even hemorrhagic fever, with high mortality. Despite the availability of an effective vaccine, the incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuroinflammation in LGTV-infected mice, characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis. IMPORTANCE: The type I interferon (IFN) system is important to control viral infections; however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system are poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system, is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection. The first phase is in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). In the second phase, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.

Ebselen improves ischemia-reperfusion injury after rat lung transplantation.

The heterocyclic organic compound ebselen (2-phenyl-1,2-benizsoselenazol-3(2H)-one) is a glutathione peroxidase mimick with protective properties against oxidative injury. Ebselen also has anti-inflammatory activity, including attenuation of tumor necrosis factor release and increase of interleukin-10, as shown in vivo, in inflammatory and ischemia-reperfusion injuries, including those of the lung. This study was designed to assess its effect on severe ischemia-reperfusion injury in a model of left-sided rat lung isotransplantation. Orthotopic single left-sided lung allotransplantation (Wistar to Wistar) was performed in female rats after a total ischemic time of 18 h. In nine recipients given 500 mg/kg oral ebselen 1 h before transplantation, graft PaO(2)/FiO(2) was improved 24 h after transplantation, as evidenced with a mean (standard deviation) PaO(2) of 139 (61) mmHg vs. eight controls with 65 (33) mmHg (p = 0.009). Bronchoalveolar PMN count was reduced to approximately 50% in the ebselen group compared with controls, whereas no difference in the tumor necrosis factor content was found. We conclude that the improvement of lung function in ebselen-treated transplanted rats is mainly the result of the anti-inflammatory activity of the drug during reperfusion.

A novel peptide motif binding to and blocking the intracellular activity of the human papillomavirus E6 oncoprotein.

Specific types of human papillomaviruses (HPVs) cause cervical cancer. The viral E6 oncogene is a critical factor for maintaining the malignant phenotype of HPV-positive tumour cells. By yeast two-hybrid screening of a randomised peptide expression library, we isolated linear short peptides, which specifically bind to the HPV16 E6 oncoprotein. Sequence alignments and mutational analyses of the peptides identified a hitherto undiscovered E6-binding motif. Intracellular expression of a peptide containing the novel E6-binding motif resulted in inhibition of colony formation capacity, specifically of HPV16-positive cancer cells. A solubility-optimised variant of the peptide was created, which binds to HPV16 E6 with high affinity. Its intracellular expression efficiently induced apoptosis in HPV16-positive cancer cells. This was linked to restoration of intracellular p53 activities. Thus, this newly identified E6-binding motif could form a novel basis for the development of rational strategies for the treatment of HPV16-positive preneoplastic and neoplastic lesions.

The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10.

Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.