Friedrich Bootz

Tissue-resident mesenchymal stem cells attract peripheral blood neutrophils and enhance their inflammatory activity in response to microbial challenge.

Human MSCs may respond to TLR ligation, and recent research has suggested that many tissues contain tissue‐specific MSCs, possibly located in periendothelial and perivascular regions. At present, the functional consequences of these findings are unclear. We hypothesized that tissue‐specific MSCs could play an instructional role during early phases of bacterial challenge. To investigate this hypothesis further, we set up a coculture system of glandular MSCs and peripheral blood neutrophils so that we could analyze the cellular interactions of these cells in response to LPS challenge. We found that stimulation with bacterial endotoxin induced chemokine receptor expression and mobility of MSCs. Activated MSCs secreted large amounts of inflammatory cytokines and recruited neutrophils in an IL‐8‐ and MIF‐dependent manner. Recruited and activated neutrophils showed a prolonged lifespan, an increased expression of inflammatory chemokines, and an enhanced responsiveness toward subsequent challenge with LPS. Our findings demonstrate a complex, functional interaction between tissue‐resident MSCs and peripheral blood neutrophils upon bacterial challenge and suggest a role for MSCs in the early phases of pathogen challenge, when classical immune cells have not been recruited yet.

Mesenchymal stem cells augment the anti-bacterial activity of neutrophil granulocytes.

Background Mesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs. Methodology/Principal Findings We isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN. Conclusions/Significance This study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue.

Free-Circulating Methylated DNA in Blood for Diagnosis, Staging, Prognosis, and Monitoring of Head and Neck Squamous Cell Carcinoma Patients: An Observational Prospective Cohort Study

BACKGROUNDnCirculating cell-free DNA methylation testing in blood has recently received regulatory approval for screening of colorectal cancer. Its application in other clinical settings, including staging, prognosis, prediction, and recurrence monitoring is highly promising, and of particular interest in head and neck squamous cell carcinomas (HNSCCs) that represent a heterogeneous group of cancers with unsatisfactory treatment guidelines.nnnMETHODSnShort stature homeobox 2 (SHOX2) and septin 9 (SEPT9) DNA methylation in plasma from 649 prospectively enrolled patients (training study: 284 HNSCC/122 control patients; testing study: 141 HNSCC/102 control patients) was quantified before treatment and longitudinally during surveillance.nnnRESULTSnIn the training study, 59% of HNSCC patients were methylation-positive at 96% specificity. Methylation levels correlated with tumor and nodal category (P < 0.001). Initially increased methylation levels were associated with a higher risk of death [SEPT9: hazard ratio (HR) = 5.27, P = 0.001; SHOX2: HR = 2.32, P = 0.024]. Disease recurrence/metastases were detected in 47% of patients up to 377 days earlier compared to current clinical practice. The onset of second cancers was detected up to 343 days earlier. In the testing study, sensitivity (52%), specificity (95%), prediction of overall survival (SEPT9: HR = 2.78, P = 0.022; SHOX2: HR = 2.50, P = 0.026), and correlation with tumor and nodal category (P <0.001) were successfully validated.nnnCONCLUSIONSnMethylation testing in plasma is a powerful diagnostic tool for molecular disease staging, risk stratification, and disease monitoring. Patients with initially high biomarker levels might benefit from intensified treatment and posttherapeutic surveillance. The early detection of a recurrent/metastatic disease or a second malignancy could lead to an earlier consecutive treatment, thereby improving patients outcomes.

RIG-I Activation Protects and Rescues from Lethal Influenza Virus Infection and Bacterial Superinfection

Influenza A virus infection causes substantial morbidity and mortality in seasonal epidemic outbreaks, and more efficient treatments are urgently needed. Innate immune sensing of viral nucleic acids stimulates antiviral immunity, including cell-autonomous antiviral defense mechanisms that restrict viral replication. RNA oligonucleotide ligands that potently activate the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are promising candidates for the development of new antiviral therapies. Here, we demonstrate in an Mx1-expressing mouse model of influenza A virus infection that a single intravenous injection of low-dose RIG-I ligand 5-triphosphate RNA (3pRNA) completely protected mice from a lethal challenge with influenza A virus for at least 7xa0days. Furthermore, systemic administration of 3pRNA rescued mice with pre-established fulminant influenza infection and prevented the fatal effects of a streptococcal superinfection. Type I interferon, but not interferon-λ, was required for the therapeutic effect. Our results suggest that the use of RIG-I activating oligonucleotide ligands has the clinical potential to confine influenza epidemics when a strain-specific vaccine is not yet available and to reduce lethality of influenza in severely infected patients.

PD-1 ( PDCD1 ) Promoter Methylation Is a Prognostic Factor in Patients With Diffuse Lower-Grade Gliomas Harboring Isocitrate Dehydrogenase ( IDH ) Mutations

Immune checkpoints are important targets for immunotherapies. However, knowledge on the epigenetic modification of immune checkpoint genes is sparse. In the present study, we investigated promoter methylation of CTLA4, PD-L1, PD-L2, and PD-1 in diffuse lower-grade gliomas (LGG) harboring isocitrate dehydrogenase (IDH) mutations with regard to mRNA expression levels, clinicopathological parameters, previously established methylation subtypes, immune cell infiltrates, and survival in a cohort of 419 patients with IDH-mutated LGG provided by The Cancer Genome Atlas. PD-L1, PD-L2, and CTLA-4 mRNA expression levels showed a significant inverse correlation with promoter methylation (PD-L1: p = 0.005; PD-L2: p < 0.001; CTLA-4: p < 0.001). Furthermore, immune checkpoint methylation was significantly associated with age (PD-L2: p = 0.003; PD-1: p = 0.015), molecular alterations, i.e. MGMT methylation (PD-L1: p < 0.001; PD-L2: p < 0.001), ATRX mutations (PD-L2: p < 0.001, PD-1: p = 0.001), and TERT mutations (PD-L1: p = 0.035, PD-L2: p < 0.001, PD-1: p < 0.001, CTLA4: p < 0.001) as well as methylation subgroups and immune cell infiltrates. In multivariate Cox proportional hazard analysis, PD-1 methylation qualified as strong prognostic factor (HR = 0.51 [0.34–0.76], p = 0.001). Our findings suggest an epigenetic regulation of immune checkpoint genes via DNA methylation in LGG. PD-1 methylation may assist the identification of patients that might benefit from an alternative treatment, particularly in the context of emerging immunotherapies.

Robot-assisted endoscope guidance versus manual endoscope guidance in functional endonasal sinus surgery (FESS)

Abstract Background: Having one hand occupied with the endoscope is the major disadvantage for the surgeon when it comes to functional endoscopic sinus surgery (FESS). Only the other hand is free to use the surgical instruments. Tiredness or frequent instrument changes can thus lead to shaky endoscopic images. Methods: We collected the pose data (position and orientation) of the rigid 0° endoscope and all the instruments used in 16 FESS procedures with manual endoscope guidance as well as robot-assisted endoscope guidance. In combination with the DICOM CT data, we tracked the endoscope poses and workspaces using self-developed tracking markers. Results: All surgeries were performed once with the robot and once with the surgeon holding the endoscope. Looking at the durations required, we observed a decrease in the operating time because one surgeon doing all the procedures and so a learning curve occurred what we expected. The visual inspection of the specimens showed no damages to any of the structures outside the paranasal sinuses. Conclusion: Robot-assisted endoscope guidance in sinus surgery is possible. Further CT data, however, are desirable for the surgical analysis of a tracker-based navigation within the anatomic borders. Our marker-based tracking of the endoscope as well as the instruments makes an automated endoscope guidance feasible. On the subjective side, we see that RASS brings a relief for the surgeon.

Posttherapeutische Lebensqualität beim Nasopharynxkarzinom

BACKGROUNDnThis retrospective study analysed patient characteristics and quality of live (QoL) for patients with nasopharyngeal carcinoma (NPC) after treatment.nnnMATERIAL AND METHODSnA cross-sectional investigation was conducted to assess the QoL of 20 NPC patients with cancer-free survival of more than one year, which were treated with radiotherapy (RT) or chemoradiotherapy (RCT) during the period 2001-2009 at the University Hospital Bonn, Germany. The QoL was assessed by the FACT-NP (functional assessment of cancer therapy-nasopharyngeal) questionnaire.nnnRESULTSnThe median age of the patients was 57 ± 13 years and the male/female ratio was 2.33/1.3 (15%) patients were treated with RT and 17 (85%) with RCT. The global QoL was good in our patients. Xerostomia, chewing, decrease of gustatory sense, discontent with sexual life and ear problems were of major concern with the majority of patients and affected the QoL negatively. Pain, lost of working ability, emotional distress, or family problems were no significant factors.nnnCONCLUSIONnThe expected reduction of QoL after treatment must be explained in detail to the NPC patient. The integration of the family and partner, an antidepressant therapy or psycho-oncological support can be useful and necessary.

Comparative functional cell biological analysis of mesenchymal stem cells of the head and neck region: potential impact on wound healing, trauma, and infection.

Mesenchymal stem cells (MSCs) are multipotent mesenchymal progenitor cells, originally identified in bone‐marrow. Little is known about MSCs of the head and neck region. We investigated cell biological properties with a potential impact on wound healing of 2 different tissue‐resident MSC populations.

Potential of quantitative SEPT9 and SHOX2 methylation in plasmatic circulating cell-free DNA as auxiliary staging parameter in colorectal cancer: a prospective observational cohort study

BackgroundSeptin 9 (SEPT9) and short stature homeobox 2 (SHOX2) methylation in circulating cell-free DNA (ccfDNA) are powerful biomarkers for colorectal cancer (CRC) screening, as well as head and neck squamous cell carcinoma staging and monitoring. In the present study, we investigated SEPT9 and SHOX2 ccfDNA methylation as auxiliary pre and post-therapeutic staging parameters in CRC patients.MethodsccfDNA methylation was quantified in 184 prospectively enrolled patients prior to and 3–10 days after surgery, and biomarker levels were associated with clinico-pathological parameters.ResultsPre-therapeutic levels of SHOX2 and SEPT9 ccfDNA methylation were strongly associated with Union for International Cancer Control (UICC) stages, tumour (T), nodal (N), and metastasis (M) categories, and histological grade (all Pu2009≤u20090.001), as well as lymphatic invasion and extracapsular lymph node extension (all P<u20090.05). Post-therapeutic SHOX2 and SEPT9 ccfDNA methylation levels correlated with UICC stage (all Pu2009u2009<0.01). SEPT9 ccfDNA methylation further allowed for an accurate pre- and post-therapeutic detection of distant metastases (AUCpre-therapeuticu2009=u20090.79 (95%CI 0.69–0.89), AUCpost-therapeuticu2009=u20090.93 (95% CI 0.79–1.0)).ConclusionsDNA methylation analysis in plasma is a powerful pre and post-therapeutic diagnostic tool for CRC and may add valuable information to current TNM staging, thereby holding the potential to assist in the development of individually tailored treatment protocols.

Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients

BackgroundSHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance.MethodsMethylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [meanSEPT9/SHOX2] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated.ResultsSporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61–63% sensitivity/90–92% specificity (SEPT9/training), 53–57% sensitivity/87–90% specificity (SHOX2/training), and 64–65% sensitivity/90–91% specificity (meanSEPT9/SHOX2/training). Results were confirmed in a testing cohort with 54–56% sensitivity/88–90% specificity (SEPT9/testing), 43–48% sensitivity/93–95% specificity (SHOX2/testing), and 49–58% sensitivity/88–94% specificity (meanSEPT9/SHOX2/testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals (SEPT9: AUCtrainingu2009=u20090.79–0.80; AUCtestingu2009=u20090.74–0.75; SHOX2: AUCtrainingu2009=u20090.78–0.81, AUCtestingu2009=u20090.77–0.79; meanSEPT9/SHOX2: AUCtrainingu2009=u20090.81–0.84, AUCtestingu2009=u20090.80). The accurate prediction of overall survival was possible with all three algorithms (training cohort: HRSEPT9u2009=u20091.23-1.90, HRSHOX2u2009=u20091.14-1.85, HRmeanSEPT9/SHOX2u2009=1.19-1.89u2009; testing cohort: HRSEPT9u2009=1.22-1.67, HRSHOX2u2009=u20091.15-1.71, HRmeanSEPT9/SHOX2u2009=u20091.12-1.77).ConclusionThe concordant clinical performance based on different quantification algorithms allows for the application of various diagnostic platforms for the analysis of ccfDNA methylation biomarkers.