Porcine-derived collagen peptides promote re-epithelialisation through activation of integrin signalling.

Chronic non-healing cutaneous wounds represent a major burden to patients and healthcare providers worldwide, emphasising the continued unmet need for credible and efficacious therapeutic approaches for wound healing. We have recently shown the potential for collagen peptides to promote proliferation and migration during cutaneous wound healing. In the present study, we demonstrate that the application of porcine-derived collagen peptides significantly increases keratinocyte and dermal fibroblast expression of integrin α2ß1 and activation of an extracellular signal-related kinase (ERK)-focal adhesion kinase (FAK) signalling cascade during wound closure in vitro. SiRNA-mediated knockdown of integrin ß1 impaired porcine-derived collagen peptide-induced wound closure and activation of ERK-FAK signalling in keratinocytes but did not impair ERK or FAK signalling in dermal fibroblasts, implying the activation of differing downstream signalling pathways. Studies in ex vivo human 3D skin equivalents subjected to punch biopsy-induced wounding confirmed the ability of porcine-derived collagen peptides to promote wound closure by enhancing re-epithelialisation. Collectively, these data highlight the translational and clinical potential for porcine-derived collagen peptides as a viable therapeutic approach to promote re-epithelialisation of superficial cutaneous wounds.

A substantial neutrophilic inflammation as regular part of severe type 2 chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is generally associated with severe type 2 immune reactions in the white population. However, recent findings suggest an additional role for neutrophils in severe type 2 inflammation. OBJECTIVE: This study aimed to characterize the neutrophilic inflammation in CRSwNP and its relation to eosinophilic inflammation in severe type 2 immune reactions. METHODS: The presence and activation of neutrophils and eosinophils was analyzed in CRS without NP and CRSwNP by measuring cell and activation markers via immunohistochemistry, immunofluorescence, Luminex assay, ELISA, UniCAP, fluorescence-activated cell sorting, and PCR. Differential neutrophil migration was assessed via Boyden-chamber assay and neutrophil survival was analyzed via flow cytometry. RESULTS: Both CRS without NP and CRSwNP displayed variable degrees of eosinophilic and neutrophilic inflammation, with a profound neutrophilic infiltration and activation in type 2 CRSwNP, associated with eosinophil extracellular traps cell death and Charcot-Leyden crystals, but independent of IL-17. Neutrophil extracellular traps cell death in CRSwNP was associated with bacterial colonization, however, neutrophils were less prone to undergo neutrophil extracellular traps cell death in the tissue of patients with severe type 2 CRSwNP. Neutrophils did not show increased migration nor survival in the CRSwNP environment in vitro. CONCLUSIONS: This study demonstrated a severe neutrophilic inflammation associated with severe eosinophilic type 2 inflammatory CRSwNP, the role of which needs further study.

Biologics for chronic rhinosinusitis with nasal polyps.

With the increasing recognition of the role of type 2 immune responses in chronic rhinosinusitis, its severity, recurrence, and comorbidities, several biologics targeting IL-4, IL-5, and IL-13 as well as IgE have been administered in small proof-of-concept studies. Recently, the first phase 3 trials have been reported with dupilumab, an IL-4 receptor antagonist, demonstrating a significant and clinically relevant reduction of the disease burden from polyp size and sinus involvement to symptoms and smell; these changes consecutively led to an important increase in quality of life. Finally, the biologic versus placebo treatment reduced the need for systemic glucocorticosteroids and sinus surgery significantly and clinically meaningfully. Dupilumab today is registered for the treatment of chronic rhinosinusitis with nasal polyps in Europe and the United States. Within a year, 2 further phase 3 trials with omalizumab and mepolizumab will be reported. With this development, without any doubt, a new era for the treatment of severe uncontrolled chronic rhinosinusitis with nasal polyps has begun. Questions on the indication of the biologics, the selection of patients, and finally criteria for monitoring the efficacy in individual patients need to be urgently answered, and care pathways need to be established integrating the current standard of care including surgery.

Type 2 inflammation in chronic rhinosinusitis without nasal polyps: Another relevant endotype.

BACKGROUND: Chronic rhinosinusitis without nasal polyps (CRSsNP) is mainly considered a type 1 mediated disease. The role and clinical significance of type 2 immune responses in CRSsNP have not been addressed sufficiently; a recent cluster analysis for CRS described the existence of a subgroup of patients with CRSsNP with a type 2 inflammation. OBJECTIVE: We aimed to characterize the underlying type 2 immune response and its clinical significance in patients with CRSsNP. METHODS: A total of 240 patients with CRSsNP were endotyped and subdivided on the basis of expression of marker cytokines. Clinical data such as recurrence, comorbid asthma and allergy, and numbers of blood eosinophils and neutrophils were collected from all patients. A selection of 15 patients was further characterized for the presence of eosinophils, neutrophils, Charcot-Leyden crystals, and eosinophil extracellular traps in the mucosae. RESULTS: A type 2 immune response with increased levels of IL-4, IL-5, eosinophil cationic protein, IgE, and Staphylococcus aureus enterotoxin-specific IgE was observed in 49% of patients with CRSsNP. Those patients showed increased numbers of blood and tissue eosinophils, and they displayed a considerable eosinophilic inflammation associated with eosinophil extracellular trap cell death and Charcot-Leyden crystals. A significantly increased prevalence of recurrence and asthma was observed in patients with type 2 CRSsNP compared with in patients with non-type 2 CRSsNP. However, only 4 of 117 patients with type 2 CRSsNP developed nasal polyps within 12 years. CONCLUSION: This study shows that type 2 immune responses in CRSsNP follow similar patterns but are less pronounced than in chronic rhinosinusitis with nasal polyps. Also CRSsNP with a moderate type 2 immune response showed a considerable eosinophilic inflammation with clinical impact.

Chronic rhinosinusitis in Asia.

Chronic rhinosinusitis (CRS), although possibly overdiagnosed, is associated with a high burden of disease and is often difficult to treat in those truly affected. Recent research has demonstrated that inflammatory signatures of CRS vary around the world, with less eosinophilic and more neutrophilic inflammation found in Asia compared with Europe and North America. Although in the Western world about 80% of nasal polyps carry a type 2 signature, this might be between 20% and 60% in China and Korea or Thailand, respectively. These differences are associated with a lower asthma comorbidity and risk of disease recurrence after surgery in the Asian population. As a hallmark of severe type 2 inflammation, eosinophils attacking Staphylococcus aureus at the epithelial barrier have been described recently; they also can be found in a subgroup of Asian patients with nasal polyps. Furthermore, the percentage of type 2 signature disease in patients with CRS is dramatically increasing ("eosinophilic shift") in several Asian countries over the last 20 years. Establishing an accurate diagnosis along with considering the current and shifting patterns of inflammation seen in Asia will enable more effective selection of appropriate pharmacotherapy, surgical therapy, and eventually biotherapy. Determining the causes and pathophysiology for this eosinophilic shift will require additional research.

Extracellular eosinophilic traps in association with Staphylococcus aureus at the site of epithelial barrier defects in patients with severe airway inflammation.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is characterized by TH2-biased eosinophilic inflammation. Eosinophils have been shown to generate so-called extracellular eosinophilic traps (EETs) under similar pathologic conditions. OBJECTIVE: Our aim was to investigate a possible link between EET formation and the presence of Staphylococcus aureus, an organism frequently colonizing the upper airways, at the human mucosal site of the disease. METHODS: Tissue slides were investigated for the presence of EETs and S aureus by using immunofluorescent staining and the PNA-Fish assay, respectively. An ex vivo human mucosal disease tissue model was used for artificial infection with S aureus. Cell markers were analyzed by using immunohistochemistry, the Luminex Multiplex assay, ELISA, PCR, and immunoblotting and linked to the presence of EETs. RESULTS: About 8.8% ± 4.8% of the infiltrating eosinophils exhibited EETs in patients' nasal polyp tissues. Formation of EETs was associated with increased IL-5 (P < .05) and periostin (P < .05) tissue levels and colonization with S aureus (P < .05). By using an ex vivo human mucosal disease tissue model, EET formation was induced (4.2 ± 0.9-fold) on exposure to S aureus but not Staphylococcus epidermidis. Eosinophils were shown to migrate (P < .01) toward S aureus and entrap the bacteria both inside and outside the mucosal tissue. Blocking NAPDH oxidase activity led to a complete inhibition (P < .05) of EET formation by S aureus. CONCLUSION: Eosinophils are likely to be specifically recruited to S aureus and possibly other microorganisms and form EETs at sites of airway epithelial damage to protect the host from infections in patients with chronic rhinosinusitis with nasal polyps.

Advances in rhinitis and rhinosinusitis in 2015.

The last year has seen great progress in the understanding of upper airway disease and in its management. For allergic rhinitis, authors focused on the prediction of and effect on the natural course of disease. New evidence was published for the disease-modifying effect of allergen immunotherapy in terms of avoidance of new sensitizations and prevention of asthma in either randomized or real-life studies. Specifically, for patients with house dust mite allergies, which are often underestimated and difficult to diagnose, the efficacy of SQ house dust mite sublingual immunotherapy tablets has been demonstrated in patients with allergic rhinitis and asthma. For the first time, allergen immunotherapy significantly reduced asthma exacerbations. In patients with chronic rhinosinusitis, a novel endotyping approach purely based on T helper cell biomarkers has been developed and has shown clinical relevance through associations with asthma comorbidity and recurrence after surgery. Severe nasal polyposis with high risk for asthma comorbidity and disease recurrence is characterized by type 2 inflammatory patterns, including IgE antibodies to staphylococcal superantigens; several studies using biologic agents have targeted exactly this spectrum of mediators. This goes in parallel with new knowledge on even more type 2 mediators derived from epithelial cells, which will expand the number of possible candidates for innovative intervention.

An anti-inflammatory selective glucocorticoid receptor modulator preserves osteoblast differentiation.

Glucocorticoids (GCs) are in widespread use to treat inflammatory bone diseases, such as rheumatoid arthritis (RA). Their anti-inflammatory efficacy, however, is accompanied by deleterious effects on bone, leading to GC-induced osteoporosis (GIO). These effects include up-regulation of the receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratio to promote bone-resorbing osteoclasts and include inhibition of bone-forming osteoblasts. We previously identified suppression of osteoblast differentiation by the monomer glucocorticoid receptor (GR) via the inhibition of Il11 expression as a crucial mechanism for GIO. Here we show that the GR-modulating substance compound A (CpdA), which does not induce GR dimerization, still suppresses proinflammatory cytokines in fibroblast-like synovial cells from patients with RA and in osteoblasts. In contrast to the full GR agonist dexamethasone, it does not unfavorably alter the RANKL/OPG ratio and does not affect Il11 expression and subsequent STAT3 phosphorylation in these cells. Notably, while dexamethasone inhibits osteoblast differentiation, CpdA does not affect osteoblast differentiation in vitro and in vivo. We describe here for the first time that selective GR modulators can act against inflammation, while not impairing osteoblast differentiation.

Unravelling the expression of interleukin-9 in chronic rhinosinusitis: A possible role for Staphylococcus aureus.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a Th2 biased inflammation, associated with nasal colonization of Staphylococcus (S.) aureus. Interleukin (IL)-9 is a pro-inflammatory Th2 cytokine with a pivotal role in asthma, allergy and chronic obstructive pulmonary disease (COPD), but is less studied in CRSwNP. We aimed to characterize the expression and cellular source of IL-9 and examined S. aureus as potential local trigger in CRSwNP. We showed increased numbers of interleukin-9 producing neutrophils and mononuclear cells in the tissue of CRSwNP patients. This interleukin-9 production was stimulated by S. aureus and its enterotoxin B in vitro. These findings underline the contribution of S. aureus and define IL-9 as another relevant cytokine in type 2 CRSwNP.

Protein crystallization promotes type 2 immunity and is reversible by antibody treatment.

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability.

In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10-20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed.

Galactose-functionalized gelatin hydrogels improve the functionality of encapsulated HepG2 cells.

The present study investigates the effect of galactosylated gelatin on encapsulated HepG2 cells. Methacrylamide modified gelatin is evaluated and compared with its galactosylated counterpart with respect to effects on viability, morphological characteristics, proliferation, and the expression of hepatocyte specific markers. The research reveals that further modifications of methacrylamide modified gelatin are possible without affecting the survival of the encapsulated cells (viability of 90%). Moreover, the study demonstrates a clear and long-term (up to 21 d) improvement in hepatocyte specific gene expression when the cells are encapsulated in the galactosylated gelatin. It is concluded that the use of galactosylated gelatin derivates supports the hepatocyte phenotype.

High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

Quantitative contrasts in the photopolymerization of acrylamide and methacrylamide-functionalized gelatin hydrogel building blocks.

The synthesis and evaluation of gelatin acrylamide as an alternative to the well-established gelatin methacrylamide are reported. High-resolution magic angle spinning NMR is used for the quantitative determination of the chemical cross-linking density. The gelatin acrylamide precursors reveal enhanced cross-linking in terms of reactivity and double bond conversion, resulting in stronger networks. Remarkably, even at very low double bond conversions, ≈5%, gel fractions of ≈40% are obtained. The cross-linked networks are also reviewed in the framework of the rubber elasticity and thermodynamic swelling theories to estimate important nanostructural properties. Preliminary cell tests revealed that highly viable (>90%) cell-laden constructs are obtained.