Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Review of potential medical treatments for middle ear cholesteatoma.

Middle ear cholesteatoma (MEC), is a destructive, and locally invasive lesion in the middle ear driven by inflammation with an annual incidence of 10 per 100,000. Surgical extraction/excision remains the only treatment strategy available and recurrence is high (up to 40%), therefore developing the first pharmaceutical treatments for MEC is desperately required. This review was targeted at connecting the dysregulated inflammatory network of MEC to pathogenesis and identification of pharmaceutical targets. We summarized the numerous basic research endeavors undertaken over the last 30+ years to identify the key targets in the dysregulated inflammatory pathways and judged the level of evidence for a given target if it was generated by in vitro, in vivo or clinical experiments. MEC pathogenesis was found to be connected to cytokines characteristic for Th1, Th17 and M1 cells. In addition, we found that the inflammation created damage associated molecular patterns (DAMPs), which further promoted inflammation. Similar positive feedback loops have already been described for other Th1/Th17 driven inflammatory diseases (arthritis, Crohn's disease or multiple sclerosis). A wide-ranging search for molecular targeted therapies (MTT) led to the discovery of over a hundred clinically approved drugs already applied in precision medicine. Based on exclusion criteria designed to enable fast translation as well as efficacy, we condensed the numerous MTTs down to 13 top drugs. The review should serve as groundwork for the primary goal, which is to provide potential pharmaceutical therapies to MEC patients for the first time in history. Video Abstract.

Middle ear cholesteatoma (MEC) is a destructive and locally invasive ulcerated lesion in the middle ear driven by inflammation which occurs in 10 out of 100,000 people annually. Surgical extraction/excision is the only treatment strategy available and recurrence is high (up to 40% after ten years), therefore developing the first pharmaceutical treatments for MEC is desperately required. This review is focused on the connections between inflammation and MEC pathogenesis. These connections can be used as attack points for pharmaceuticals. For this we summarized the results of research undertaken over the last 30 + years. MEC pathogenesis can be described by specific inflammatory dysregulation already known from arthritis, Crohn's disease or multiple sclerosis. A hallmark of this dysregulation are positive feedback loops of the inflammation further amplifying itself in a vicious circle-like manner. We have identified over one hundred drugs which are already used in clinic to treat other inflammatory diseases, and could potentially be repurposed to treat MEC. To improve and expedite clinical success rates, we applied certain criteria based on our literature searches and condensed these drugs down to the 13 top drugs. We hope the review will serve as groundwork for the primary goal, which is to provide potential pharmaceutical therapies to MEC patients for the first time in history.

Viral and Clinical Oncology of Head and Neck Cancers.

PURPOSE OF REVIEW: This study assesses the current state of knowledge of head and neck squamous cell carcinomas (HNSCC), which are malignancies arising from the orifices and adjacent mucosae of the aerodigestive tracts. These contiguous anatomical areas are unique in that 2 important human oncoviruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), are causally associated with nasopharyngeal and oropharyngeal cancers, respectively. Mortality rates have remained high over the last 4 decades, and insufficient attention paid to the unique viral and clinical oncology of the different subgroups of HNSCC. RECENT FINDINGS: We have compared and contrasted the 2 double-stranded DNA viruses and the relevant molecular oncogenesis of their respective cancers against other head and neck cancers. Tobacco and alcohol ingestion are also reviewed, as regard the genetic progression/mutation accumulation model of carcinogenesis. The importance of stringent stratification when searching for cancer mutations and biomarkers is discussed. Evidence is presented for a dysplastic/pre-invasive cancerous phase for HPV+ oropharyngeal cancers, and analogous with other HPV+ cancers. This raises the possibility of strategies for cancer screening as early diagnosis will undoubtedly save lives. Staging and prognostication have changed to take into account the distinct biological and prognostic pathways for viral+ and viral- cancers. Diagnosis of pre-cancers and early stage cancers will reduce mortality rates. Multi-modal treatment options for HNSCC are reviewed, especially recent developments with immunotherapies and precision medicine strategies. Knowledge integration of the viral and molecular oncogenic pathways with sound planning, hypothesis generation, and clinical trials will continue to provide therapeutic options in the future.

Chronic inflammation of middle ear cholesteatoma promotes its recurrence via a paracrine mechanism.

BACKGROUND: Cholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis alongside with other intracranial complications are found. No pharmaceutical treatment is available today and recurrence after surgical extraction occurs. We investigated possible TLR4-based mechanisms promoting recurrence and explore possible treatments strategies. METHODS: We isolated fibroblasts and epidermal stem cells from cholesteatoma tissue and healthy auditory canal skin. Subsequently, their expression under standard culture conditions and after stimulation with LPS was investigated by RT-qPCR. Cell metabolism and proliferation were analysed upon LPS treatment, with and without TLR4 antagonist. An indirect co-culture of fibroblasts and epidermal stem cells isolated from cholesteatoma tissue was utilized to monitor epidermal differentiation upon LPS treatment by RT-qPCR and immunocytochemistry. RESULTS: Under standard culture conditions, we detected a tissue-independent higher expression of IL-1ß and IL-8 in stem cells, an upregulation of KGF and IGF-2 in both cell types derived from cholesteatoma and higher expression of TLR4 in stem cells derived from cholesteatoma tissue. Upon LPS challenge, we could detect a significantly higher expression of IL-1α, IL-1ß, IL-6 and IL-8 in stem cells and of TNF-a, GM-CSF and CXCL-5 in stem cells and fibroblasts derived from cholesteatoma. The expression of the growth factors KGF, EGF, EREG, IGF-2 and HGF was significantly higher in fibroblasts, particularly when derived from cholesteatoma. Upon treatment with LPS the metabolism was elevated in stem cells and fibroblasts, proliferation was only enhanced in fibroblasts derived from cholesteatoma. This could be reversed by the treatment with a TLR4 antagonist. The cholesteatoma fibroblasts could be triggered by LPS to promote the epidermal differentiation of the stem cells, while no LPS treatment or LPS treatment without the presence of fibroblasts did not result in such a differentiation. CONCLUSION: We propose that cholesteatoma recurrence is based on TLR4 signalling imprinted in the cholesteatoma cells. It induces excessive inflammation of stem cells and fibroblasts, proliferation of perimatrix fibroblasts and the generation of epidermal cells from stem cells thru paracrine signalling by fibroblasts. Treatment of the operation site with a TLR4 antagonist might reduce the chance of cholesteatoma recurrence. Video Abstract.

Bone Allograft and Locking Plate for Severe Proximal Humeral Fractures: Early and Late Outcomes.

BACKGROUND Early failure of osteosyntheses is common even with use of locking plates. In patients with comminuted fractures and epiphyseal osseous defects, we performed a series of osteosyntheses by locking plate in combination with an allograft bone augmentation. Because of encouraging short-term results in the literature, we assumed that the method could be a potential alternative to a reverse shoulder prosthesis. MATERIAL AND METHODS Twenty-six patients with a dislocated proximal humeral fracture (Neer IV/V/VI) were studied. A lyophilized allogeneic bone graft was used to reinforce the humeral head fragments before locking plate osteosynthesis. The outcomes of fractures were assessed with Disabilities of the Arm, Shoulder and Hand (DASH) and Constant-Murley (Constant) scores, range of motion, a visual analog scale, and with radiological testing. The Constant-Murley scores were the endpoint of our study. RESULTS The Neer classification of the fractures was type IV in 4 patients, type V in 20 patients, and type VI in 2 patients. The mean DASH score was 52.85 (range, 4.17-79.3) and the mean Constant score was 39.26 (range, 17-88). We observed late necrosis of the humeral head in 15 of 24 patients (62.5%), although early radiological follow-up showed that the humeral head had been anatomically reconstructed. CONCLUSIONS Long-term follow-up demonstrated inferior functional results, as displayed by poor Constant scores. There was a high incidence of necrosis, in spite of initial anatomical reconstruction. Biointegration of the allogeneic bone graft and revascularization of the humeral head fragments could be impaired in geriatric patients who have gross dislocation. Therefore, augmentation of the humeral head with allogeneic bone grafts cannot be recommended in these patients.

Mouthrinses against SARS-CoV-2: anti-inflammatory effectivity and a clinical pilot study.

PURPOSE: The scope of this research endeavor was the determination of the applicability of over the counter mouthwash solutions in reducing the viral load in the saliva of COVID-19 patients and hence decreasing their infectivity. Beyond that, new experimental mouthwashes were investigated in terms of a possible positive immune modulation, which might offer an additional opportunity for a positive pharmaceutical effect. METHODS: The effectivity of the mouth washing solution was determined on 34 hospitalized COVID-19 patients by measuring the viral load by RT-qPCR in pharyngeal swabs, which were taken before and after rinsing. The inflammatory modulation thru the experimental solutions was assayed in an in vitro model of virus infected nasopharyngeal epithelium cells. RESULTS: The clinical pilot study demonstrated that the mouth rinsing solution was able to reduce the viral load by about 90% in the saliva of most patients. This reduction was determined to persist for about 6 h. In the experimental solutions, the ingredients dexpanthenol and zinc were able to reduce the expression of proinflammatory cytokines in the cell culture model, while the antiviral response was not altered significantly. CONCLUSION: We recommend the application of mouth wash solutions to COVID-19 patients, since our results indicate a reduction in infectivity and might govern the protection of health care professionals. Further improvement to the over the counter formulation can be made by utilizing zinc and dexpanthenol, as they which might be beneficial for the patients' health.

Helium Ion Microscopy Visualizes Lipid Nanodomains in Mammalian Cells.

Cell membranes are composed of 2D bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, direct visualization of cell membrane nanodomains by helium ion microscopy (HIM) is presented. It is shown that HIM is capable to image biological specimens without any conductive coating and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ≈ 15 nm.

First Clinical Experience with a New Device for the Removal of Cochlear Schwannomas.

Background: In most cases, intralabyrinthine schwannoma (ILS) occurs in patients with unilateral hearing deterioration or neurofibromatosis type II (NF II). The pattern of localization of these tumors varies but mostly affects the cochlea. Extirpation of the cochlear schwannoma, if hidden by the cochlea modiolus, is difficult under the aspect of complete removal. Therefore, a tissue removal device (TRD) was designed and tested in temporal bones. The principle of handling the new device is a pushing and pipe cleaner handling inside the cochlea. This present study aimed to describe the first in vivo experience with the newly developed TRD for removing cochlear intralabyrinthine schwannomas. Methods: In three patients, the TRD was used for the tumor removal of cochlear schwannomas. In two patients with a cochlear schwannoma in combination with a cochlea implantation and one patient suffering from NF II, a cochlear schwannoma was removed with the TRD. The access was performed with a posterior tympanotomy, an enlarged round window approach and an additional second turn access. The device was inserted and extracted gradually from the second turn access until the rings were visible in the second turn access. By pushing and pipe cleaner handling, the tumors were removed. An MRI control was performed on the day postoperatively with a T1 GAD sequence. Results: Tumor removal with the TRD was performed in a 15-min procedure without any complications. An MRI control confirmed complete removal on the postoperative day in all cases. Conclusions: In vivo handling of the device confirmed straightforward handling for the tumor removal. MRI scanning showed complete removal of the tumor by the TRD.

Characterization of non-invasive oropharyngeal samples and nucleic acid isolation for molecular diagnostics.

Molecular diagnostics is an increasingly important clinical tool, especially in routine sampling. We evaluated two non-invasive methods (oral swabs and mouthwashes) for sampling nucleic acids from the oral/pharyngeal area. We created a workflow from sample collection (n = 59) to RT-qPCR based analysis. The samples were further characterized in terms of their cellular composition as well as the purity, degradation and microbial content of the derived DNA/RNA. We determined the optimal housekeeping genes applicable for these types of samples. The cellular composition indicated that mouthwashes contained more immune cells and bacteria. Even though the protocol was not specifically optimized to extract bacterial RNA it was possible to derive microbial RNA, from both sampling methods. Optimizing the protocol allowed us to generate stable quantities of DNA/RNA. DNA/RNA purity parameters were not significantly different between the two sampling methods. Even though integrity analysis demonstrated a high level of degradation of RNA, corresponding parameters confirmed their sequencing potential. RT-qPCR analysis determined TATA-Box Binding Protein as the most favorable housekeeping gene. In summary, we have developed a robust method suitable for multiple downstream diagnostic techniques. This protocol can be used as a foundation for further research endeavors focusing on developing molecular diagnostics for the oropharyngeal cavity.

p53 Pathway Inactivation Drives SMARCB1-deficient p53-wildtype Epithelioid Sarcoma Onset Indicating Therapeutic Vulnerability Through MDM2 Inhibition.

Loss of the gene SMARCB1 drives the development of malignant rhabdoid tumors, epithelioid sarcomas, and other malignancies. The SMARCB1 protein is a core component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family of chromatin remodeling complexes, which are important regulators of gene expression and cell differentiation. Here, we use CRISPR-Cas9 to create germline smarcb1 loss of function in zebrafish. We demonstrate that the combination of smarcb1 deficiency with mutant p53 results in the development of epithelioid sarcomas, angiosarcomas, and carcinomas of the thyroid and colon. Although human epithelioid sarcomas do not frequently harbor p53 mutations, smarcb1-deficient tumors in zebrafish were only observed following disruption of p53, indicating that p53 signaling in human tumors might be attenuated through alternative mechanisms, such as MDM2-mediated proteasomal degradation of p53. To leverage this possibility for the treatment of human epithelioid sarcoma, we tested small molecule-mediated disruption of the p53-MDM2 interaction, which stabilized p53 protein leading to p53-pathway reactivation, cell-cycle arrest, and increased apoptosis. Moreover, we found that MDM2 inhibition and the topoisomerase II inhibitor doxorubicin synergize in targeting epithelioid sarcoma cell viability. This could be especially relevant for patients with epithelioid sarcoma because doxorubicin represents the current gold standard for their clinical treatment. Our results therefore warrant reactivating p53 protein in SMARCB1-deficient, p53-wildtype epithelioid sarcomas using combined doxorubicin and MDM2 inhibitor therapy.

Imaging of SARS-CoV-2 infected Vero E6 cells by helium ion microscopy.

Helium ion microscopy (HIM) offers the opportunity to obtain direct views of biological samples such as cellular structures, virus particles, and microbial interactions. Imaging with the HIM combines sub-nanometer resolution, large depth of field, and high surface sensitivity. Due to its charge compensation capability, the HIM can image insulating biological samples without additional conductive coatings. Here, we present an exploratory HIM study of SARS-CoV-2 infected Vero E6 cells, in which several areas of interaction between cells and virus particles, as well as among virus particles, were imaged. The HIM pictures show the three-dimensional appearance of SARS-CoV-2 and the surface of Vero E6 cells at a multiplicity of infection of approximately 1 with great morphological detail. The absence of a conductive coating allows for a distinction between virus particles bound to the cell membrane and virus particles lying on top of the membrane. After prolonged imaging, it was found that ion-induced deposition of hydrocarbons from the vacuum renders the sample sufficiently conductive to allow for imaging even without charge compensation. The presented images demonstrate the potential of the HIM in bioimaging, especially for the imaging of interactions between viruses and their host organisms.

Stem Cell-Induced Inflammation in Cholesteatoma is Inhibited by the TLR4 Antagonist LPS-RS.

Cholesteatoma is a severe non-cancerous lesion of the middle ear characterized by massive inflammation, tissue destruction, and an abnormal growth of keratinized squamous epithelium. We recently demonstrated the presence of pathogenic stem cells within cholesteatoma tissue, unfortunately their potential roles in regulating disease-specific chronic inflammation remain poorly understood. In the presented study, we utilized our established human in vitro cholesteatoma stem cell model for treatments with lipopolysaccharides (LPS), tumor necrosis factor α (TNFα), and the TLR4-antagonist LPS from R.sphaeroides(LPS-RS) followed by qPCR, western blot, and immunocytochemistry. Middle ear cholesteatoma stem cells (ME-CSCs) showed a significantly increased expression of TLR4 accompanied by a significantly enhanced LPS-dependent pro-inflammatory gene expression pattern of TNFα, IL-1α, IL-1ß, IL-6, and IL-8 compared to non-pathogenic control cells. LPS-dependent pro-inflammatory gene expression in ME-CSCs was driven by an enhanced activity of NF-B p65 leading to a TNFα-mediated feed-forward-loop of pro-inflammatory NF-B target gene expression. Functional inactivation of TLR4 via the TLR4-antagonist LPS-RS blocked chronic inflammation in ME-CSCs, resulting in a nearly complete loss of IL-1ß, IL-6, and TNFα expression. In summary, we determined that ME-CSCs mediate the inflammatory environment of cholesteatoma via TLR4-mediated NF-B-signaling, suggesting a distinct role of ME-CSCs as drivers of cholesteatoma progression and TLR4 on ME-CSCs as a therapeutic target.

An Effective Primary Head and Neck Squamous Cell Carcinoma In Vitro Model.

Head and neck squamous cell carcinoma is a highly malignant disease and research is needed to find new therapeutic approaches. Faithful experimental models are required for this purpose. Here, we describe the specific cell culture conditions enabling the efficient establishment of primary cell culture models. Whereas a classical 10% serum-containing medium resulted in the growth of fibroblast-like cells that outcompeted epithelial cells, we found that the use of specific culture conditions enabled the growth of epithelial tumor cells from HPV+ and HPV- head and neck cancer tissue applicable for research. EpCAM and high Thy-1 positivity on the cell surface were mutually exclusive and distinguished epithelial and fibroblast-like subpopulations in all primary cultures examined and thus can be used to monitor stromal contamination and epithelial cell content. Interestingly, cells of an individual patient developed tumor spheroids in suspension without the use of ultra-low attachment plates, whereas all other samples exclusively formed adherent cell layers. Spheroid cells were highly positive for ALDH1A1 and hence displayed a phenotype reminiscent of tumor stem cells. Altogether, we present a system to establish valuable primary cell culture models from head and neck cancer tissue at high efficiency that might be applicable in other tumor entities as well.

The Therapeutic Effect of 1,8-Cineol on Pathogenic Bacteria Species Present in Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is marked by an inflamed mucosa of sinuses and is accompanied by a significantly reduced quality of live. Since no guidelines for the treatment of CRS are available, long lasting clinical histories with health care costs adding up to dozens of billion $ annually are caused by CRS. The progression of CRS is often induced by bacterial infections and/or a shift in microbiome as well as biofilm formation. The exact microbiome alterations are still unclear and the impenetrable biofilm renders the treatment with common antibiotics ineffective. This study focuses on characterizing the microbiome changes in CRS and investigating the inhibition of biofilm growth by 1,8-Cineol, a small, non-polar and hence biofilm penetrating molecule with known antimicrobial potential. We performed MALDI-TOF MS based characterization of the microbiomes of healthy individuals and CRS patients (n = 50). The microbiome in our test group was shifted to pathogens (Staphylococcus aureus, Escherichia coli, and Moraxella catarrhalis). In contrast to published studies, solely based on cell culture techniques, we could not verify the abundance of Pseudomonas aeruginosa in CRS. The inhibition of bacterial proliferation and biofilm growth by 1,8-Cineol was measured for these three pathogens. Interestingly, S. aureus, the most prominent germ in CRS, showed a biofilm inhibition not simply correlated to its inhibition of proliferation. RT-qPCR confirmed that this was due to the downregulations of major key players in biofilm generation (agrA, SarA and σB) by 1,8-Cineol. Furthermore we verified this high biofilm inhibition potential in a model host system consisting out of S. aureus biofilm grown on mature respiratory epithelium. A second host model, comprising organotypic slices, was utilized to investigate the reaction of the innate immune system present in the nasal mucosa upon biofilm formation and treatment with 1,8-Cineol. Interestingly Staphylococcus epidermidis, the cause of very common catheter infections, possesses a biofilm generation pathway very similar to S. aureus and might be treatable in a similar fashion. The two presented in vitro model systems might be transferred to combinations of every biofilm forming bacterial with most kind of epithelium and mucosa.

Specific Targeting of Oncogenes Using CRISPR Technology.

In recent decades, tools of molecular biology have enabled researchers to genetically modify model organisms, including human cells. RNAi, zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats and CRISPR-associated protein 9), retro- or lentiviral gene transfer, and many other methods can be utilized to remove genes, add genes, or change their expression. Within the same timeframe, survival rates for many highly malignant tumor diseases have not improved substantially. If modern medicine could apply even a subset of research methods in clinical management, which are already well established and controllable in basic research laboratories, this could strongly impact patients' prognosis. CRISPR-Cas9 is a method to precisely target and manipulate genomic loci and recent studies have attempted to use this method as a genetic treatment for Duchenne muscular dystrophy, blood disorders, autosomal-dominant hearing loss, and cancer. Some of these approaches target mutant genomic sequences specifically and try to avoid affecting the respective normal loci. Considering obvious genetic risks opposing the objected benefits, data are needed to show whether CRISPR technology is suitable as a future cancer therapy approach or not. Here, we develop strategies for the specific targeting of viral cancer drivers and oncogenes activated by mutation, using the latest CRISPR technology. Cancer Res; 78(19); 5506-12. ©2018 AACR.

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

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 NTR 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.

Technical feasibility study for production of tailored multielectrode arrays and patterning of arranged neuronal networks.

In this manuscript, we first reveal a simple ultra violet laser lithographic method to design and produce plain tailored multielectrode arrays. Secondly, we use the same lithographic setup for surface patterning to enable controlled attachment of primary neuronal cells and help neurite guidance. For multielectrode array production, we used flat borosilicate glass directly structured with the laser lithography system. The multi layered electrode system consists of a layer of titanium coated with a layer of di-titanium nitride. Finally, these electrodes are covered with silicon nitride for insulation. The quality of the custom made multielectrode arrays was investigated by light microscopy, electron microscopy and X-ray diffraction. The performance was verified by the detection of action potentials of primary neurons. The electrical noise of the custom-made MEA was equal to commercially available multielectrode arrays. Additionally, we demonstrated that structured coating with poly lysine, obtained with the aid of the same lithographic system, could be used to attach and guide neurons to designed structures. The process of neuron attachment and neurite guidance was investigated by light microscopy and charged particle microscopy. Importantly, the utilization of the same lithographic system for MEA fabrication and poly lysine structuring will make it easy to align the architecture of the neuronal network to the arrangement of the MEA electrode.. In future studies, this will lead to multielectrode arrays, which are able to specifically attach neuronal cell bodies to their chemically defined electrodes and guide their neurites, gaining a controlled connectivity in the neuronal network. This type of multielectrode array would be able to precisely assign a signal to a certain neuron resulting in an efficient way for analyzing the maturation of the neuronal connectivity in small neuronal networks.

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.

Imaging in early posttraumatic complex regional pain syndrome: a comparison of diagnostic methods.

OBJECTIVES: The complex regional pain syndrome type I (CRPS I) still is difficult to diagnose in posttraumatic patients. As CRPS I is a clinical diagnosis the characteristic symptoms have to be differentiated from normal posttraumatic states. Several diagnostic procedures are applied to facilitate an early diagnosis, although their value for diagnosing posttraumatic CRPS I is unclear. METHODS: One hundred fifty-eight consecutive patients with distal radial fracture were followed up for 16 weeks after trauma. To assess the diagnostic value of the commonly applied methods a detailed clinical examination was carried out 2, 8, and 16 weeks after trauma in conjunction with bilateral thermography, plain radiographs of the hand skeleton, three phase bone scans (TPBSs), and contrast-enhanced magnetic resonance imaging (MRI). All imaging procedures were assessed blinded. RESULTS: At the end of the observation period 18 patients (11%) were clinically identified as having CRPS I and 13 patients (8%) revealed an incomplete clinical picture which were defined as CRPS borderline cases. The sensitivity of all diagnostic procedures used was poor and decreased between the first and the last examinations (thermography: 45% to 29%; TPBS: 19% to 14%; MRI: 43% to 13%; bilateral radiographs: 36%). In contrast a high specificity was observed in the TPBS and MRI at the eighth and sixteenth-week examinations (TPBS: 96%, 100%; MRI: 78%, 98%) and for bilateral radiographs 8 weeks after trauma (94%). The thermography presented a fair specificity that improved from the second to the sixteenth week (50% to 89%). DISCUSSION: The poor sensitivity of all tested procedures combined with a reasonable specificity produced a low positive predictive value (17% to 60%) and a moderate negative predictive value (79% to 86%). These results suggest, that those procedures cannot be used as screening tests. Imaging methods are not able to reliably differentiate between normal posttraumatic changes and changes due to CRPS I. Clinical findings remain the gold standard for the diagnosis of CRPS I and the procedures described above may serve as additional tools to establish the diagnosis in doubtful cases.

Analysis of efficacy and failure in proximal humerus fractures treated with locking plates.

OBJECTIVE: The purpose of this study was to determine the efficacy of proximal humerus locking plates (PHLP) and to clarify predictors of loss of fixation. DESIGN: Retrospective review of patients with proximal humerus fractures fixed with a PHLP. SETTING: Five Level 1 trauma centers. PATIENTS: One hundred fifty-three patients (111 female, 42 male) 18 years or older with a displaced fracture or fracture-dislocation of the proximal humerus treated with a PHLP between January 1, 2001 and July 31, 2005. INTERVENTION: Demographic data, trauma mechanism, surgical approach, and perioperative complications were collected from the medical records. Fracture classification according to the AO/OTA, radiographic head-shaft angle, and screw tip-articular surface distance in true anteroposterior (AP) and axillary lateral radiographs of the shoulder were measured postoperatively. Varus malreduction was defined as a head-shaft angle of <120 degrees. MAIN OUTCOME MEASUREMENTS: Statistical analysis was done to establish correlations between loss of fixation and postoperative head-shaft angle in the true AP radiograph, patient age, fracture type, trauma mechanism, number of locking head screws, and type of plate. RESULTS: The mean age was 62.3 +/- 15.4 years (22-92) and the mean injury severity score (ISS) was 9.5 +/- 10.16 (4-57; n = 73). The surgical approach was deltopectoral (90.2%) or transdeltoid (9.8%). No intraoperative complications were reported. The mean postoperative head-shaft angle was 130 degrees (95 degrees to 160 degrees; SD = 13). The overall incidence of loss of fixation was 13.7%. There was a statistically significant association between varus reduction (<120 degrees) and loss of fixation (30.4% when the head-shaft angle was <120 degrees versus 11% when the head-shaft angle was > or =120 degrees; P = 0.02). CONCLUSION: This series presents the experience using PHLP in 5 Level 1 trauma centers. There were no intraoperative complications related to the locking plate systems. Despite the use of fixed-angle devices, loss of fixation occurred, primarily in the presence of varus malreduction. Our findings suggest that avoiding varus should substantially decrease the risk of postoperative failures.