Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.epidermal growth factor receptor | transactivation T he ectodomains of many cell surface proteins are shed from the surface (i.e., "ectodomain shedding") by metalloproteases. Ectodomain shedding generates many diverse bioactive cytokines and growth factors, and governs important cellular processes in the developing and adult organism, including the control of growth, adhesion, and motility of cells (reviewed in refs. 1-3). EGF receptor activation generates signals for cell proliferation, migration, differentiation, or survival. The 12 EGF family members are synthesized as cell surface transmembrane precursors. The active growth factors are released by A disintegrin and metalloproteinases (ADAMs) and activate specific heterodimeric EGF receptors on the cell surface connected to diverse intracellular signaling pathways (4, 5). Increased shedding of EGF ligands has been linked to different clinical pathologic processes (6-10); hence, therapeutic control of ligand release would be beneficial. Of the 12 functional ADAMs encoded in the human genome (3) only two-ADAM10 and ADAM17-handle most of the numerous ADAM substrates, in particular, the EGF ligands. However, broad-spectrum metalloprotease inhibitors tested for clinical use have failed as a result of indiscriminate blockade of substrate cleavage, leading to clinical side effects (11). Even recently developed selective ADAM inhibitors still affect the cleavage of many substrates (12). Modulation of the release of specific ADAM substrates has been impossible to date because it is unknown how cleavage specificity is regulated on the molecular level. It is therefore necessary to identify key signals that determine substrate specificity of cleavage.Ectodomain cleavage is made specific by a number of intracellular signals; e.g., by calcium influx, by activation of G protein-coup...

: There is a global need for effective and affordable rabies vaccines, which is unmet by current vaccines due to limitations in their production capacities, required administration schedules, storage requirements, and cost. Many different experimental approaches previously used for bacterial and viral vaccines have been applied to rabies, but with variable success. One of the most promising new concepts is the use of messenger RNA (mRNA) in encoding the main rabies virus antigen, the envelope glycoprotein (RABV-G). CureVac has applied their proprietary technology platform for the production of mRNA to this problem, resulting in the rabies vaccine candidate CV7201. Following preclinical studies in mice and pigs showing that CV7201 could induce neutralizing immune responses that protected against rabies virus, different dosages and routes of administration of CV7201 were tested in a phase 1 human study. This clinical study proved that mRNA vaccination was safe and had an acceptable reactogenicity profile, but immune responses depended on the mode of administration, and they did not unequivocally support CV7201 for further development as a prophylactic vaccine with this particular formulation. Further, preclinical studies using RABV-G mRNA encapsulated in lipid nanoparticles (LNPs) showed an improved response in both mice and nonhuman primates, and these encouraging results are currently being followed up in clinical studies in humans. This review summarizes the recent advances in mRNA vaccines against rabies.

Defensins represents an integral part of the innate immune system serving to ward off potential pathogens and to protect the intestinal barrier from microbial encroachment. In addition to their antimicrobial activities, defensins in general, and human β-defensin 2 (hBD2) in particular, also exhibit immunomodulatory capabilities. In this report, we assessed the therapeutic efficacy of systemically administered recombinant hBD2 to ameliorate intestinal inflammation in three distinct animal models of inflammatory bowel disease; i.e., chemically induced mucosal injury (DSS), loss of mucosal tolerance (TNBS), and T-cell transfer into immunodeficient recipient mice. Treatment efficacy was confirmed in all tested models, where systemically administered hBD2 mitigated inflammation, improved disease activity index, and hindered colitis-induced body weight loss on par with anti-TNF-α and steroids. Treatment of lipopolysaccharide (LPS)-activated human peripheral blood mononuclear cells with rhBD2 confirmed the immunomodulatory capacity in the circulatory compartment. Subsequent analyzes revealed dendritic cells (DCs) as the main target population. Suppression of LPS-induced inflammation was dependent on chemokine receptor 2 (CCR2) expression. Mechanistically, hBD2 engaged with CCR2 on its DC target cell to decrease NF-κB, and increase CREB phosphorylation, hence curbing inflammation. To our knowledge, this is the first study showing in vivo efficacy of a systemically administered defensin in experimental disease.

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID50s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P< 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.

Myocardial inflammation is critical for ventricular remodeling after ischemia. Phospholipid mediators play an important role in inflammatory processes. In the plasma membrane they are degraded by phospholipase D1 (PLD1). PLD1 was shown to be critically involved in ischemic cardiovascular events. Moreover, PLD1 is coupled to tumor necrosis factor-α signaling and inflammatory processes. However, the impact of PLD1 in inflammatory cardiovascular disease remains elusive. Here, we analyzed the impact of PLD1 in tumor necrosis factor-α-mediated activation of monocytes after myocardial ischemia and reperfusion using a mouse model of myocardial infarction. PLD1 expression was highly up-regulated in the myocardium after ischemia/reperfusion. Genetic ablation of PLD1 led to defective cell adhesion and migration of inflammatory cells into the infarct border zone 24 hours after ischemia/reperfusion injury, likely owing to reduced tumor necrosis factor-α expression and release, followed by impaired nuclear factor-κB activation and interleukin-1 release. Moreover, PLD1 was found to be important for transforming growth factor-β secretion and smooth muscle α-actin expression of cardiac fibroblasts because myofibroblast differentiation and interstitial collagen deposition were altered in Pld1(-/-) mice. Consequently, infarct size was increased and left ventricular function was impaired 28 days after myocardial infarction in Pld1(-/-) mice. Our results indicate that PLD1 is crucial for tumor necrosis factor-α-mediated inflammation and transforming growth factor-β-mediated collagen scar formation, thereby augmenting cardiac left ventricular function after ischemia/reperfusion.

Spinal muscular atrophy (SMA) is an autosomal recessive disease of variable severity caused by mutations in the SMN1 gene. Deficiency of the ubiquitous SMN function results in spinal cord α-motor neuron degeneration and proximal muscle weakness. Gene replacement therapy with recombinant adeno-associated viral (AAV) vectors showed therapeutic efficacy in several animal models of SMA. Here, we report a study aimed at analyzing the efficacy and biodistribution of a serotype-9, self-complementary AAV vector expressing a codon-optimized human SMN1 coding sequence (coSMN1) under the control of the constitutive phosphoglycerate kinase (PGK) promoter in neonatal SMNΔ7 mice, a severe animal model of the disease. We administered the scAAV9-coSMN1 vector in the intracerebroventricular (ICV) space in a dose-escalating mode, and analyzed survival, vector biodistribution and SMN protein expression in the spinal cord and peripheral tissues. All treated mice showed a significant, dose-dependent rescue of lifespan and growth with a median survival of 346 days. Additional administration of vector by an intravenous route (ICV+IV) did not improve survival, and vector biodistribution analysis 90 days postinjection indicated that diffusion from the cerebrospinal fluid to the periphery was sufficient to rescue the SMA phenotype. These results support the preclinical development of SMN1 gene therapy by CSF vector delivery.

In contrast to other retroviruses, foamy viruses (FVs) generate their Pol protein precursor independently of the Gag protein from a spliced mRNA. The exact mechanism of Pol protein incorporation into the viral capsid is poorly understood. Previously, we showed that Pol encapsidation critically depends on the packaging of (pre-) genomic RNA and identified two distinct signals within the cis-acting sequences (CASI and CASII), Pol encapsidation sequences (PESI and PESII), which are required for Pol capsid incorporation. Here, we investigated whether the presence of PESI and PESII in an FV vector is sufficient for Pol encapsidation and whether the rather extended CASII element can be shortened without loss of functionality. Our results indicate that (i) the presence of PESI and II are not sufficient for Pol encapsidation, (ii) prototype FV vectors with a shortened CASII element retain Pol incorporation and full functionality, in particular upon transducing fibroblasts and primary human mesenchymal stem cells, (iii) the presence of the central poly purine tract significantly increased the transduction rates of FV vectors and (iv) Pol encapsidation and RNA packaging can be clearly separated. In essence, we designed a new FV vector that bears approximately 850 bp less of CAS than previously established vectors and is fully functional when analysed to transduce cell lines and primary human cells. INTRODUCTIONSpumaretroviruses or foamy viruses (FVs) constitute one of the two subfamilies of retroviruses (Linial et al., 2005). The replication pathway of FV differs from what has been established for orthoretroviruses (Linial, 2007;Rethwilm, 2005). Despite the conservation of the general physical order of the provirus with long-terminal repeat (LTR)-gag-pol-env-accessory genes-LTR virtually all aspects of the replication pathway have their own facets that separate FVs from orthoretroviruses (Rethwilm, 2003). Among these, the ways that FVs express and encapsidate their reverse transcriptase proteins are central and pose some unresolved questions. The FV pol gene is expressed independently of the gag gene essentially from a spliced mRNA and gives rise to a large precursor protein comprised of protease (PR), reverse transcriptase/RNaseH (RT) and integrase (IN) (Bodem et al., 1996;Enssle et al., 1996;Yu et al., 1996). While IN is cleaved from the rest of the protein, no cleavage of PR from RT occurs (Flügel & Pfrepper, 2003). The capsid protein-independent translation of the pol gene raises the question of how the Pol protein is incorporated into the newly formed capsid.FV genomes bear two cis-acting sequences (CASI and II), which are required for transfer of a vector (Erlwein et al., 1998;Heinkelein et al., 1998;Wu et al., 1998). These elements are located in the 59 region of the (pre-) genomic RNA extending some hundred nucleotides into the gag gene and in the 39 pol genomic region (Heinkelein et al., , 2002aRussell et al., 2001;Trobridge et al., 2002). Both regions (together more than 2.5 kb) have complex functions in virus ...

The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow–derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38–CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.