1,8-cineol potentiates IRF3-mediated antiviral response in human stem cells and an ex vivo model of rhinosinusitis

Common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial super-infections resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential of 1,8-cineole for treating primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates Poly(I:C)-induced activity of the anti-viral transcription factor Interferon Regulatory Factor 3, while simultaneously reducing pro-inflammatory NF-κB-activity in human cell lines, inferior turbinate stem cells (ITSCs) and ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with Poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared to Poly(I:C) alone, whereas NF-κB-activity was reduced. Accordingly, 1,8-cineole- and Poly(I:C)-treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared to the Poly(I:C)-treated approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with LPS and 1,8-cineole compared to the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on pro-inflammatory NF-κB-signalling and may thus broaden its field of application

Autologous lipoinjection of the patulous Eustachian tube: Harvesting, cellular analysis, clinical application and preliminary outcome

Sudhoff H, Schürmann M, Brotzmann V. Autologous lipoinjection of the patulous Eustachian tube: Harvesting, cellular analysis, clinical application and preliminary outcome. Otorhinolaryngology-Head and Neck Surgery. 2016;1(5)

Identification of a Novel High Yielding Source of Multipotent Adult Human Neural Crest-Derived Stem Cells

Schürmann M, Brotzmann V, Bütow M, et al. Identification of a Novel High Yielding Source of Multipotent Adult Human Neural Crest-Derived Stem Cells. Stem Cell Reviews and Reports. 2018;14(2):277–285.Due to their extraordinarily broad differentiation potential and persistence during adulthood, adult neural crest-derived stem cells (NCSCs) are highly promising candidates for clinical applications, particularly when facing the challenging treatment of neurodegenerative diseases or complex craniofacial injuries. Successful application of human NCSCs in regenerative medicine and pharmaceutical research mainly relies on the availability of sufficient amounts of tissue for cell isolation procedures. Facing this challenge, we here describe for the first time a novel population of NCSCs within the middle turbinate of the human nasal cavity. From a surgical point of view, high amounts of tissue are routinely and easily removed during nasal biopsies. Investigating the presence of putative stem cells in obtained middle turbinate tissue by immunohistochemistry, we observed Nestin+/p75NTR+/S100+/α smooth muscle actin (αSMA)− cells, which we successfully isolated and cultivated in vitro. Cultivated middle turbinate stem cells (MTSCs) kept their expression of neural crest and stemness markers Nestin, p75 and S100 and showed the capability of sphere formation and clonal growth, indicating their stem cell character. Application of directed in vitro differentiation assays resulted in successful differentiation of MTSCs into osteogenic and neuronal cell types. Regarding the high amount of tissue obtained during surgery as well as their broad differentiation capability, MTSCs seem to be a highly promising novel neural crest stem cell population for applications in cell replacement therapy and pharmacological research

Stem cells in middle ear cholesteatoma contribute to its pathogenesis.

Nagel J, Wöllner S, Schürmann M, et al. Stem cells in middle ear cholesteatoma contribute to its pathogenesis. Nature Scientific Reports. 2018;8(1): 6204.Cholesteatoma is a potentially life-threatening middle ear lesion due to the formation of an inflamed ectopic mass of keratinizing squamous epithelium. Surgical removal remains the only treatment option, emphasizing the need to gain a better understanding of this severe disease. We show for the first time that stem cells residing in cholesteatoma tissue contribute to disease progression. Cells expressing the "stemness" markers Nestin and S100B were detected in middle ear cholesteatoma and auditory canal skin. Isolated Nestin + /S100B + -cells showed the capability for self-renewal, neurosphere formation and differentiation into mesodermal and ectodermal cell types. Compared to auditory canal skin stem cells middle ear cholesteatoma-derived stem cells displayed an enhanced susceptibility to inflammatory stimuli, and this suggested a possible contribution to the inflammatory environment in cholesteatoma tissue. Cholesteatoma derived stem cells were able to differentiate into keratinocyte-like cells using factors mimicking the microenvironment of cholesteatoma. Our findings demonstrate a new perspective on the pathogenesis of cholesteatoma and may lead to new treatment strategies for this severe middle ear lesion

1,8-Cineol Reduces Mucus-Production in a Novel Human <i>Ex Vivo</i> Model of Late Rhinosinusitis

<div><p>Inflammatory diseases of the respiratory system such as rhinosinusitis, chronic obstructive pulmonary disease, or bronchial asthma are strongly associated with overproduction and hypersecretion of mucus lining the epithelial airway surface. 1,8-cineol, the active ingredient of the pharmaceutical drug Soledum, is commonly applied for treating such inflammatory airway diseases. However, its potential effects on mucus overproduction still remain unclear.In the present study, we successfully established <i>ex vivo</i> cultures of human nasal turbinate slices to investigate the effects of 1,8-cineol on mucus hypersecretion in experimentally induced rhinosinusitis. The presence of acetyl-α-tubulin-positive cilia confirmed the integrity of the <i>ex vivo</i> cultured epithelium. Mucin-filled goblet cells were also detectable in nasal slice cultures, as revealed by Alcian Blue and Periodic acid-Schiff stainings. Treatment of nasal slice cultures with lipopolysaccharides mimicking bacterial infection as observed during late rhinosinusitis led to a significantly increased number of mucin-filled goblet cells. Notably, the number of mucin-filled goblet cells was found to be significantly decreased after co-treatment with 1,8-cineol. On a molecular level, real time PCR-analysis further showed 1,8-cineol to significantly reduce the expression levels of the mucin genes MUC2 and MUC19 in close association with significantly attenuated NF-κB-activity. In conclusion, we demonstrate for the first time a 1,8-cineol-dependent reduction of mucin-filled goblet cells and MUC2-gene expression associated with an attenuated NF-κB-activity in human nasal slice cultures. Our findings suggest that these effects partially account for the clinical benefits of 1,8-cineol-based therapy during rhinosinusitis. Therefore, topical application of 1,8-cineol may offer a novel therapeutic approach to reduce bacteria-induced mucus hypersecretion.</p></div

Stem cells in middle ear cholesteatoma contribute to its pathogenesis

Cholesteatoma is a potentially life-threatening middle ear lesion due to the formation of an inflamed ectopic mass of keratinizing squamous epithelium. Surgical removal remains the only treatment option, emphasizing the need to gain a better understanding of this severe disease. We show for the first time that stem cells residing in cholesteatoma tissue contribute to disease progression. Cells expressing the “stemness” markers Nestin and S100B were detected in middle ear cholesteatoma and auditory canal skin. Isolated Nestin +/S100B +-cells showed the capability for self-renewal, neurosphere formation and differentiation into mesodermal and ectodermal cell types. Compared to auditory canal skin stem cells middle ear cholesteatoma-derived stem cells displayed an enhanced susceptibility to inflammatory stimuli, and this suggested a possible contribution to the inflammatory environment in cholesteatoma tissue. Cholesteatoma derived stem cells were able to differentiate into keratinocyte-like cells using factors mimicking the microenvironment of cholesteatoma. Our findings demonstrate a new perspective on the pathogenesis of cholesteatoma and may lead to new treatment strategies for this severe middle ear lesion

Primer Sequences.

<p>Primer Sequences.</p

1,8-cineol-treamtent leads to significantly decreased levels of MUC gene expression after their LPS-dependent stimulation.

<p><b>A</b>: Real time PCR analyses of nasal slice culture depicted increased levels of MUC2 after LPS-treatment, which were significantly reduced in LPS- and 1,8-cineol-treated approaches. <b>B</b>: No significant changes in gene expression level of MUC5AC in nasal slice cultures after LPS- as well as LPS- and 1,8-cineol-treatment shown by real time PCR. <b>C</b>: Real time PCR analyses revealed decreased levels of MUC19 in nasal slice cultures co-treated with LPS- and 1,8-cineol in comparison to LPS-treated approaches. <b>D</b>: Real time PCR analyses showed decreased expression levels of TNFα in nasal slice cultures co-treated with LPS- and 1,8-cineol compared to LPS-treated approaches. *p < 0.5, **p < 0.01 were considered significant (t-test); ns: not significant (t-test). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.</p

Cultured human nasal slices show unimpaired epithelium containing mucus-filled goblet cells.

<p><b>A,B</b>: Overview images of the established nasal slice culture system showing sliced nasal tissue cultured in culture plate inserts within a 12-well-plate. <b>C,D</b>: Immunohistochemical staining revealed the presence of acetyl-α-tubulin-positive cilia in nasal slice cultures. <b>E</b>: Hematoxylin and eosin-staining displayed the integrity of the <i>ex vivo</i> cultured epithelium containing ciliated epithelial cells (arrowheads), goblet cells (arrows) and a basal membrane (BM). Scale Bar: 20 μm. <b>F, G</b>: Mucin-filled goblet cells (arrows) were detected in cultivated nasal slices by Alcian Blue-staining and Periodic acid-Schiff stain. Scale Bar: 20 μm.</p