Specific nasopharyngeal Corynebacterium strains serve as gatekeepers against SARS-CoV-2 infection.

The SARS-CoV-2 virus is still causing a worldwide problem. The virus settles primarily on the nasal mucosa, and the infection and its course depend on individual susceptibility. Our aim was to investigate the nasopharynx composition's role in the individual susceptibility. During the first phase of SARS-CoV-2 pandemic, nasopharyngeal microbiome samples of close contact unvaccinated patients were investigated by 16S rRNA analysis and by culturing. The whole genome of cultured Corynebacteria was sequenced. The relative expression of ACE2, TMPRSS2, and cathepsin L on Caco-2 cells and the strength of S1-ACE2 binding were determined in the presence of Corynebacteria. From 55 close contacts exposed to identical SARS-CoV-2 exposure, 26 patients became infected and 29 remained uninfected. The nasopharyngeal microbiome analysis showed significantly higher abundance of Corynebacteria in uninfected group. Corynebacterium accolens could be cultivated only from uninfected individuals and Corynebacterium propinquum from both infected and uninfected. Corynebacteria from uninfected patient significantly reduced the ACE2 and cathepsin L expression. C. accolens significantly reduced the TMPRSS2 expression compared to other Corynebacteria. Furthermore, Corynebacterium spp. weakened the binding of the S1-ACE2. Most C. accolens isolates harbored the TAG lipase LipS1 gene. Based on these results, the presence of Corynebacterium spp. in the nasopharyngeal microbiota, especially C. accolens strains, could reduce the individual susceptibility to SARS-CoV-2 infection by several mechanisms: by downregulation the ACE2, the TMPRSS2 receptors, and cathepsin L in the host; through the inhibition of S1-ACE2 binding; and lipase production. These results suggest the use of C. accolens strains as probiotics in the nasopharynx in the future.

MEK Is a Potential Indirect Target in Subtypes of Head and Neck Cancers.

The poor prognosis of head-and-neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable prognostic and predictive markers. The Ras/Raf/MEK/ERK signaling pathway is often activated by overexpressed epidermal growth factor receptor (EGFR) and stimulates the progression of HNSCCs. Our research was performed on three human papillomavirus (HPV)-negative HNSCC-cell lines: Detroit 562, FaDu and SCC25. Changes in cell viability upon EGFR and/or MEK inhibitors were measured by the MTT method. The protein-expression and phosphorylation profiles of the EGFR-initiated signaling pathways were assessed using Western-blot analysis. The EGFR expression and pY1068-EGFR levels were also studied in the patient-derived HNSCC samples. We found significant differences between the sensitivity of the tumor-cell lines used. The SCC25 line was found to be the most sensitive to the MEK inhibitors, possibly due to the lack of feedback Akt activation through EGFR. By contrast, this feedback activation had an important role in the FaDu cells. The observed insensitivity of the Detroit 562 cells to the MEK inhibitors might have been caused by their PIK3CA mutation. Among HNSCC cell lines, EGFR-initiated signaling pathways are particularly versatile. An ERK/EGFR feedback loop can lead to Akt-pathway activation upon MEK inhibition, and it is related not only to increased amounts of EGFR but also to the elevation of pY1068-EGFR levels. The presence of this mechanism may justify the combined application of EGFR and MEK inhibitors.

The Potential Impact of Connexin 43 Expression on Bcl-2 Protein Level and Taxane Sensitivity in Head and Neck Cancers-In Vitro Studies.

The poor prognosis of head and neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable predictive markers. Connexin 43 (Cx43) protein and its cell-communication channels have been assigned tumor suppressor functions while the anti-apoptotic Bcl-2 (B-cell lymphoma-2) protein has been associated with negative prognostic significance in cancer. This study aimed to test the role of Cx43 protein on Bcl-2 expression, tumor progression and response to taxane-based treatment in HNSCC. Human papillomavirus (HPV) negative HNSCC cell lines were tested for paclitaxel sensitivity through measuring apoptosis induction, cell viability and changes in Cx43 and Bcl-2 levels using flow cytometry, cell viability assay, immunocytochemistry and western blot. Inhibition of Cx43 expression using siRNA increased Bcl-2 protein levels in SCC25 (tongue squamous cell carcinoma) cells, while forced Cx43 expression reduced Bcl-2 levels and supported paclitaxel cytotoxicity in FaDu (hypopharynx squamous cell carcinoma) cells. In vitro results were in line with protein expression and clinicopathological features tested in tissue microarray samples of HNSCC patients. Our data demonstrate that elevated Cx43 and reduced Bcl-2 levels may indicate HNSCC sensitivity to taxane-based treatments. On the contrary, silencing of the Cx43 gene GJA1 (gap junction protein alpha-1) can result in increased Bcl-2 expression and reduced paclitaxel efficiency. Clinical tumor-based analysis also confirmed the inverse correlation between Cx43 and Bcl-2 expression.

Cochlear implantation using posterior suprameatal approach.

When cochlear implantation was first introduced, the mastoidectomy and posterior tympanotomy approach was the most frequently used technique to gain access to the middle ear and the cochlea. Since 2000, several authors have routinely used a non-mastoidectomy nonposterior tympanotomy technique, which has undergone several modifications. Alternative surgical techniques for cochlear implantation have recently been introduced, such as endomeatal-alone or suprameatal-alone and combined posterior tympanotomy/endomeatal approaches. The goal of this study was to describe another modification of this less invasive technique to perform cochlear implantation. Cochlear implantations were performed between January 1, 2002, and December 31, 2012, in 220 patients through the posterior suprameatal approach. In reviewing our experiences, we have concluded that this approach, which eliminates the need for mastoidectomy, is considered safe, time-efficient, and minimally invasive. The possible pitfalls and the microsurgical relevance of anatomic structures of this technique are discussed in detail. Using this technique, not all classical anatomic orientation points are identified. However, certain landmarks predict the depth and the three-dimensional location of invisible anatomic structures.

Expression of PD-L1 on Immune Cells Shows Better Prognosis in Laryngeal, Oropharygeal, and Hypopharyngeal Cancer.

Despite great enthusiasm towards immunotherapy, reliable biomarkers are still lacking. The importance of subsets based on human papillomavirus (HPV) status is supported by a growing body of evidence. However, role of other possible subgroups such as anatomic localization of primary tumor remains controversial. Our objective was to investigate immune cell infiltrate and checkpoint inhibitor proteins in above-mentioned head and neck cancer subsets. Archival tumor samples of 106 laryngeal, oropharyngeal, and hypopharyngeal cancer patients were stained with PD-L1, PD-L2, PD-1, and CTLA-4 antibodies. Proportion of tumor-infiltrating lymphocytes was assessed as well. In HPV-negative tumors, PD-L1 immune cell positivity was associated with better disease-specific survival. PD-L1 expression on immune cells correlated with improved disease-specific survival in laryngeal tumors. Furthermore, PD-L1 immune cell positivity correlated with CTLA-4 expression on immune cells and it was accompanied by high tumor-infiltrating lymphocyte rate. PD-L1 expression on tumor cells and PD-1 status showed strong correlation in all patients and in oropharyngeal and laryngeal localization, but not in hypopharynx. HPV-negative oropharyngeal cancers showed negative PD-L1 status on tumor cells. CTLA-4 positivity was observed in 49.5% and 20.6% on immune cells and on tumor cells, respectively. We concluded that PD-L1 expression on immune cells indicates better prognosis in laryngeal squamous cell carcinoma and in HPV-negative head and neck squamous cell carcinoma. We have not found any essential differences between anatomic subgroups. A possibly distinct role of hypopharyngeal localization regarding immune activity requires further clarification.

PD-1, PD-L1 and CTLA-4 in pregnancy-related - and in early-onset breast cancer: A comparative study.

PURPOSE: We aimed to compare the immunohistochemical expression of PD-1, PD-L1 and CTLA-4 of pregnancy-related breast cancer (PRBC) and early onset non-PRBC (YWBC), and their prognosis prediction potential was correlated to that of conventional clinicopathological factors. METHODS: Twenty-one PRBC cases were paired with 21 YWBC in this matched case-control study. Immune-checkpoint markers (ICM) were evaluated with immunohistochemistry (IHC) on whole slides using the following antibodies: PD-1 (NAT-105), PD-L1 (28-8) and CTLA-4 (F-8). IHC score was defined as the percentage of positive cells, assessed separately among tumor cells, intratumoral lymphocytes and peritumoral lymphocytes. RESULTS: The optimal threshold of PD-L1 expression of tumor cells occurred at 10% for overall survival (OS, AUC = 0.847, p = 0.009), and at 1% for disease-free survival (DFS, AUC = 0.795, p = 0.010). For PD-L1 expression on intratumoral lymphocytes, the optimal cut-off was 1% (AUC = 0.763, p = 0.048). Considering PD-1, PD-L1 and CTLA-4 expression, no significant difference occurred between PRBC and YWBC (p > 0.05 for all comparisons). PD-1, PD-L1 expressed on peritumoral lymphocytes and CTLA-4 failed, but PD-L1 expressed on tumor cells and on intratumoral lymphocytes was suitable to distinguish patient cohorts with different OS and DFS (p ≤ 0.011 for all comparisons). Higher PD-L1 expression was associated with poor prognosis. PD-L1 expressed on tumor cells represented an independent association with OS (p = 0.023) and DFS (p = 0.032). CONCLUSIONS: Our results suggest that PRBC and YWBC do not differ in the expression of PD-1, PD-L1 and CTLA-4. However, our findings emphasize the relevance of PD-L1 expression in early-onset breast cancer, as an independent negative predictor of prognosis.

p16INK4 expression is of prognostic and predictive value in oropharyngeal cancers independent of human papillomavirus status: a Hungarian study.

Head and neck cancer treatment protocols still lack well-established biomarkers of prognostic and predictive value. It is well known that human papillomavirus (HPV)-related and non-HPV-related oropharyngeal cancers are distinct entities concerning tumor biology and clinical outcome. However, there is an ongoing debate whether tumor suppressor p16INK4 status alone or both p16INK4 and HPV detection should be used in clinical settings. The aim of this study was to investigate p16INK4-immunolabelled and HPV-induced rates and determine their clinical significance in 110 primary head and neck squamous cell carcinomas. The expression of p16INK4 protein was assessed with immunohistochemistry, while high-risk HPV detection was performed using DNA PCR method. P16INK4 immunolabelling was detected in 17.3% of all tumor samples, and in 38.1% of oropharyngeal malignancies. Oropharyngeal, p16INK4-immunolabelled tumors showed an improved disease-specific survival compared to the non-p16INK4-immunolabelled group (median survival: 30.3 vs. 8.8 months, p < 0.001 with the log-rank test). Furthermore, 56% of p16INK4-immunolabelled cases were tested positive for HPV-DNA. The HPV-induced group presented better disease-specific survival compared to the non-HPV-induced cases (median survival: 25.9 vs. 9.5 months, p = 0.024 with the log-rank test). Improved response rates to neoadjuvant chemotherapy were observed both in p16INK4-immunolabelled and p16INK4- immunolabelled/HPV DNA- containing groups (Fisher's exact test: p = 0.025 and p = 0.009). In conclusion, p16INK4 immunohistochemistry proved to be a reliable and affordable tool for prognostic and predictive testing of head and neck squamous cell cancers. The p16INK4 immunopositivity status alone was confirmed to be an equally precise indicator of clinical outcome as p16INK4/HPV DNA PCR double testing.

Copy number gain of PIK3CA and MET is associated with poor prognosis in head and neck squamous cell carcinoma.

The incidence of head and neck squamous cell carcinomas is still growing, and the long-term prognosis of advanced disease remains poor. Only a fraction of head and neck cancers are sensitive to the EGFR-inhibitor cetuximab, which is the only registered targeted therapy available today. In several cancers, gene copy number alterations of MET and PIK3CA have been found to be prognostic and predictive for therapy response. The aim of this study was to systematically analyze in head and neck cancers the pathological characteristics and prognostic significance of copy number changes of MET and PIK3CA genes. MET and PIK3CA copy numbers were analyzed by fluorescence in situ hybridization in tumor samples of 152 patients. Expression of EGFR, p16, and Ki67 was studied by immunohistochemistry. High polysomy of PIK3CA (chromosome 3) was found in 20 % of cases and amplification in 4.5 %. Regarding MET, 35 % of cases showed low or high polysomy of the gene (chromosome 7), while no intra-chromosomal amplification of MET was detected. PIK3CA copy number gain (high polysomy or amplification) was significantly associated with shorter disease-specific survival, larger tumor volume, and lower p16 expression. MET copy number gain (low or high polysomy) in tumors was significantly associated with shorter disease-specific survival and lower level of EGFR. PIK3CA and MET may play an important role in oncogenesis of certain specific subtypes of head and neck cancer. There is an urgent need for the development of novel targeted therapies against these tumors associated with poor prognosis.

The Potential Prognostic Value of Connexin 43 Expression in Head and Neck Squamous Cell Carcinomas.

Gap juctions are transmembrane communication channels known to be involved in the control of cell proliferation by mediating the exchange of ions and small molecules between cells. Gap junctions are composed of connexon hemichannels made up of 6 connexin proteins, which abnormal expression and functions have been linked to tumor progression and poorer prognosis. Here, we studied the prognostic impact of the most prevalent connexin isotype, connexin 43 (Cx43) in head and neck squamous cell carcinomas (HNSCC). Tissue microarrays made from tumor samples of 90 HNSCC patients were immunostained for Cx43 and cell cycle regulation-related biomarkers including p53, Ki67, p16, aurora A, geminin, and p21 proteins. Scoring and histopathologic evaluation were performed in digital slides. A 4-tier scoring distinguishing the percentage of positively stained tumor cells was used including score 1: <5%, score 2: 6% to 20%, score 3: 21% to 60%, and score 4: >60%. For statistics, Kaplan-Meier curves with log-rank tests, Cox-regression, and Pearson χ/Fisher exact tests were used.A significant positive correlation was found between Cx43 expression and disease-specific survival of patients (P=0.004). The rate of p21 protein-positive tumor cells also proved to be a significant positive prognostic marker (P=0.014). Cx43 levels also showed a significant positive correlation with p53 expression (P=0.036). However, there was no statistical association between Cx43 levels and the rest of the markers tested neither with T, N, or M stage.In conclusion, our data suggest that reduced Cx43 expression and low p21 protein levels may have a significant negative impact on HNSCC prognosis.

The soluble guanylate cyclase activator cinaciguat prevents cardiac dysfunction in a rat model of type-1 diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) leads to the development of diabetic cardiomyopathy, which is associated with altered nitric oxide (NO)--soluble guanylate cyclase (sGC)--cyclic guanosine monophosphate (cGMP) signalling. Cardioprotective effects of elevated intracellular cGMP-levels have been described in different heart diseases. In the current study we aimed at investigating the effects of pharmacological activation of sGC in diabetic cardiomyopathy. METHODS: Type-1 DM was induced in rats by streptozotocin. Animals were treated either with the sGC activator cinaciguat (10 mg/kg/day) or with placebo orally for 8 weeks. Left ventricular (LV) pressure-volume (P-V) analysis was used to assess cardiac performance. Additionally, gene expression (qRT-PCR) and protein expression analysis (western blot) were performed. Cardiac structure, markers of fibrotic remodelling and DNA damage were examined by histology, immunohistochemistry and TUNEL assay, respectively. RESULTS: DM was associated with deteriorated cGMP signalling in the myocardium (elevated phosphodiesterase-5 expression, lower cGMP-level and impaired PKG activity). Cardiomyocyte hypertrophy, fibrotic remodelling and DNA fragmentation were present in DM that was associated with impaired LV contractility (preload recruitable stroke work (PRSW): 49.5 ± 3.3 vs. 83.0 ± 5.5 mmHg, P < 0.05) and diastolic function (time constant of LV pressure decay (Tau): 17.3 ± 0.8 vs. 10.3 ± 0.3 ms, P < 0.05). Cinaciguat treatment effectively prevented DM related molecular, histological alterations and significantly improved systolic (PRSW: 66.8 ± 3.6 mmHg) and diastolic (Tau: 14.9 ± 0.6 ms) function. CONCLUSIONS: Cinaciguat prevented structural, molecular alterations and improved cardiac performance of the diabetic heart. Pharmacological activation of sGC might represent a new therapy approach for diabetic cardiomyopathy.

Strain and strain rate by speckle-tracking echocardiography correlate with pressure-volume loop-derived contractility indices in a rat model of athlete's heart.

Contractile function is considered to be precisely measurable only by invasive hemodynamics. We aimed to correlate strain values measured by speckle-tracking echocardiography (STE) with sensitive contractility parameters of pressure-volume (P-V) analysis in a rat model of exercise-induced left ventricular (LV) hypertrophy. LV hypertrophy was induced in rats by swim training and was compared with untrained controls. Echocardiography was performed using a 13-MHz linear transducer to obtain LV long- and short-axis recordings for STE analysis (GE EchoPAC). Global longitudinal (GLS) and circumferential strain (GCS) and longitudinal (LSr) and circumferential systolic strain rate (CSr) were measured. LV P-V analysis was performed using a pressure-conductance microcatheter, and load-independent contractility indices [slope of the end-systolic P-V relationship (ESPVR), preload recruitable stroke work (PRSW), and maximal dP/dt-end-diastolic volume relationship (dP/dtmax-EDV)] were calculated. Trained rats had increased LV mass index (trained vs. control; 2.76 ± 0.07 vs. 2.14 ± 0.05 g/kg, P < 0.001). P-V loop-derived contractility parameters were significantly improved in the trained group (ESPVR: 3.58 ± 0.22 vs. 2.51 ± 0.11 mmHg/µl; PRSW: 131 ± 4 vs. 104 ± 2 mmHg, P < 0.01). Strain and strain rate parameters were also supernormal in trained rats (GLS: -18.8 ± 0.3 vs. -15.8 ± 0.4%; LSr: -5.0 ± 0.2 vs. -4.1 ± 0.1 Hz; GCS: -18.9 ± 0.8 vs. -14.9 ± 0.6%; CSr: -4.9 ± 0.2 vs. -3.8 ± 0.2 Hz, P < 0.01). ESPVR correlated with GLS (r = -0.71) and LSr (r = -0.53) and robustly with GCS (r = -0.83) and CSr (r = -0.75, all P < 0.05). PRSW was strongly related to GLS (r = -0.64) and LSr (r = -0.71, both P < 0.01). STE can be a feasible and useful method for animal experiments. In our rat model, strain and strain rate parameters closely reflected the improvement in intrinsic contractile function induced by exercise training.

Cardiac effects of acute exhaustive exercise in a rat model.

BACKGROUND: The role of physical exercise in the prevention and treatment of cardiovascular diseases has been well described, however, elevations in cardionecrotic biomarkers after prolonged exercise (i.e. ultramarathon running) were observed. We aimed at understanding the biochemical, molecular biological, structural and functional alterations in the heart after exhaustive exercise in a rat model. METHODS: Rats of the exercise group were forced to swim for 3h with 5% body weight (workload) attached to the tail, control rats were taken into the water for 5min. After a 2-hour recovery period we performed left ventricular (LV) pressure-volume analysis to investigate LV function and mechanoenergetics. Additionally, blood and myocardium samples were harvested for biochemical and histological examinations. Gene expression changes were detected by qRT-PCR. RESULTS: When compared to controls, elevated plasma levels of cardiac troponin T and creatine kinase were detected after exhaustive exercise. Histological analysis showed sporadic fragmentation of myocardial structure and leukocyte infiltration in the exercised group. We observed increased end-systolic volume, decreased ejection fraction, impaired contractility (preload recruitable stroke work) and mechanoenergetics (ventriculoarterial coupling, mechanical efficiency) of LV after exercise. Myocardial expression of major antioxidant enzymes was increased along with increased myocardial nitro-oxidative stress. Bax/Bcl-2 ratio and TUNEL staining showed enhanced apoptotic signaling. Exhaustive exercise also resulted in the dysregulation of the matrix metalloproteinase system. CONCLUSIONS: Excessive physical activity has an adverse effect on the heart. The observed functional impairment is associated with increased nitro-oxidative stress, enhanced apoptotic signaling and dysregulation of the matrix metalloproteinase system after exhaustive exercise.

Rat model of exercise-induced cardiac hypertrophy: hemodynamic characterization using left ventricular pressure-volume analysis.

Long-term exercise training is associated with characteristic structural and functional changes of the myocardium, termed athlete's heart. Several research groups investigated exercise training-induced left ventricular (LV) hypertrophy in animal models; however, only sporadic data exist about detailed hemodynamics. We aimed to provide functional characterization of exercise-induced cardiac hypertrophy in a rat model using the in vivo method of LV pressure-volume (P-V) analysis. After inducing LV hypertrophy by swim training, we assessed LV morphometry by echocardiography and performed LV P-V analysis using a pressure-conductance microcatheter to investigate in vivo cardiac function. Echocardiography showed LV hypertrophy (LV mass index: 2.41 ± 0.09 vs. 2.03 ± 0.08 g/kg, P < 0.01), which was confirmed by heart weight data and histomorphometry. Invasive hemodynamic measurements showed unaltered heart rate, arterial pressure, and LV end-diastolic volume along with decreased LV end-systolic volume, thus increased stroke volume and ejection fraction (73.7 ± 0.8 vs. 64.1 ± 1.5%, P < 0.01) in trained versus untrained control rats. The P-V loop-derived sensitive, load-independent contractility indexes, such as slope of end-systolic P-V relationship or preload recruitable stroke work (77.0 ± 6.8 vs. 54.3 ± 4.8 mmHg, P = 0.01) were found to be significantly increased. The observed improvement of ventriculoarterial coupling (0.37 ± 0.02 vs. 0.65 ± 0.08, P < 0.01), along with increased LV stroke work and mechanical efficiency, reflects improved mechanoenergetics of exercise-induced cardiac hypertrophy. Despite the significant hypertrophy, we observed unaltered LV stiffness (slope of end-diastolic P-V relationship: 0.043 ± 0.007 vs. 0.040 ± 0.006 mmHg/µl) and improved LV active relaxation (τ: 10.1 ± 0.6 vs. 11.9 ± 0.2 ms, P < 0.01). According to our knowledge, this is the first study that provides characterization of functional changes and hemodynamic relations in exercise-induced cardiac hypertrophy.