Endotypes of Chronic Rhinosinusitis with Primary and Recurring Nasal Polyps in the Latvian Population.

Chronic rhinosinusitis (CRS) is a complex syndrome with various inflammatory mechanisms resulting in different patterns of inflammation that correlate with the clinical phenotypes of CRS. Our aim was to use detected IL-1, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, Ki 67, HBD-2, HBD-3, and LL-37 to classify specific inflammatory endotypes in chronic rhinosinusitis with the tissue of nasal polyps (CRSwNP). Samples from 35 individuals with primary and recurrent CRSwNP were taken during surgery. The tissues were stained for the previously mentioned biomarkers immunohistochemically. A hierarchical cluster analysis was performed. The clinical parameters were compared between clusters. Five clusters had significantly different biomarkers between groups. There were no significant differences in the clinical parameters, except for the Lund-Mackay score, which was significantly higher in cluster 4 compared to that of cluster 1 (p = 0.024). Five endotypes of (CRSwNP) are characterized by different combinations of type 1, type 2, and type 3 tissue inflammation patterns. In the Latvian population, endotypes associated with neutrophilic inflammation or a combination of neutrophilic inflammation and type 2 inflammation are predominant. Increased proliferation marker Ki 67 values are not associated with more severe inflammation in the tissue samples of chronic rhinosinusitis with nasal polyps.

Comparison of Tissue Factors in the Ontogenetic Aspects of Human Cholesteatoma.

BACKGROUND: An acquired cholesteatoma is a benign but locally aggressive lesion in the middle ear. It is characterized by chronic inflammation and the destruction of surrounding bone. Therefore, the aim of this study was to compare defensins HßD-2 and HßD-4; pro- and anti-inflammatory cytokines IL-1α and IL-10; proliferation marker Ki-67; transcription factor NF-κß; angiogenetic factor VEGF; Sonic hedgehog gene protein SHH; and remodeling factors MMP-2, MMP-9, TIMP-2, and TIMP-4 in adult and pediatric cholesteatoma tissue, and to compare these groups with control skin tissue. METHODS: The study included 25 cholesteatoma tissue material samples from children, 25 from adults, and 7 deep external ear canal skin samples from cadavers. The tissues were stained immunohistochemically and evaluated using semi-quantitative methods. Nonparametric tests, such as the Kruskal-Wallis test and Spearman rank correlation, were used. RESULTS: There were no statistically discernible differences between the adult and children groups when comparing the relative numbers of factor-positive cells. CONCLUSIONS: There are no histopathological differences between adult and children cholesteatoma tissues.

Assessment of professional singers using laryngeal, respiratory, and airflow measurements.

PURPOSE: In the pedagogy of classical vocal singing, it can be difficult to determine the human voice fach, especially for the voice of aspiring vocalists. Hence, an objective metric-based system for the determination of the human voice is needed. In the present study, we investigated the anthropological and aerodynamic parameters for 60 professional singers with a professionally confirmed singing range. METHODS: Amongst the 60 included professional singers, there were ten participants each for sopranos, mezzo-sopranos, altos (female vocal fach), and tenors, baritones, basses (male vocal fach). Airflow measurements were recorded using spirometry whilst anthropological measurements were taken using CT scans. Appropriate statistical analyses were done using the Mann-Whitney U test and Kruskal Wallis H test with post-hoc tests and Bonferroni correction. p < 0.05 was considered statistically significant. RESULTS: Soprano singers, who have the highest pitch, were found to be the shortest and least heavy, whilst basses, who have the lowest pitch, were found to be tallest and heaviest amongst the study participants. Furthermore, sopranos had the smallest lung volumes while the basses had the largest lung volumes (raw spirometry measures). However, when normalized ratios were considered, no differences were observed. Finally, laryngeal size showed sexual dimorphism due to developmental changes. CONCLUSIONS: A mix of anthropological and aerodynamic measurements may be useful to assist singers and vocal pedagogues to assess and determine voice types before the beginning of their vocal studies.

Identifying the Microbiome of the Adenoid Surface of Children Suffering from Otitis Media with Effusion and Children without Middle Ear Effusion Using 16S rRNA Genetic Sequencing.

BACKGROUND: The upper respiratory tract harbors diverse communities of commensal, symbiotic, and pathogenic organisms, originating from both the oral and nasopharyngeal microbiota. Among the primary sites of microbial colonization in the upper airways are the adenoids. Alterations in the adenoid microbiota have been implicated in the development of various conditions, including secretory otitis media. AIM: This study aims to employ 16S rRNA genetic sequencing to identify the most common bacteria present on the surface of adenoids in children with otitis media with effusion and compare them with children without pathologies in the tympanic cavity. Additionally, we seek to determine and compare the bacterial diversity in these two study groups. MATERIALS AND METHODS: A total of nineteen samples from the adenoid surfaces were collected, comprising two groups: thirteen samples from children without middle ear effusion and six samples from children with secretory otitis media. The libraries of the V3-V4 hypervariable region of the bacterial 16S rRNA gene was made and sequenced using MiSeq platform. RESULTS: The most prevalent phyla observed in both groups were Proteobacteria, Firmicutes, and Bacteroidetes. The most common bacterial genera identified in both groups were Haemophilus, Streptococcus, Moraxella, Fusobacterium, and Bordetella, with Fusobacterium and Moraxella being more prevalent in the groups that had no middle ear effusion, while Haemophulus and Streptococcus were more prevalent in the otitis media with effusion group, although not in a statistically significant way. Statistical analysis shows a trend towards bacterial composition and beta diversity being similar between the study groups; however, due to the limited sample size and unevenness between groups, we should approach this data with caution. CONCLUSION: The lack of prolific difference in bacterial composition between the study groups suggests that the role of the adenoid microbiome in the development of otitis media with effusion may be less significant.

Antimicrobial and Defense Proteins in Chronic Rhinosinusitis with Nasal Polyps.

Background and Objectives: Chronic rhinosinusitis with nasal polyps (CRSwNP) presently remains a difficult disease to manage. Antimicrobial and defense proteins are important factors that could help characterize the role of microorganisms in CRSwNP pathogenesis, as the concept of microbial dysbiosis in CRS is still being considered. Our aim is to investigate the complex appearance, relative distribution and interlinks of human ß defensin 2 (HBD-2), human ß defensin 3 (HBD-3), human ß defensin 4 (HBD-4), and cathelicidin LL 37 (LL 37) in chronic rhinosinusitis with nasal polyps (CRSwNP)-affected human nasal mucosa. Materials and Methods: The study group consisted of 48 samples from patients with CRSwNP. Samples were collected during functional endoscopic sinus surgery. The control group consisted of 17 normal healthy nasal mucosa samples gathered during routine septoplasty. ß-defensin-2, ß-defensin-3, ß-defensin-4 and cathelicidin LL 37 in tissue were detected via immunohistochemical analysis. Results: HBD-2, HBD-3 and LL 37 were significantly decreased in epithelial cells in both primary and recurrent nasal polyp samples (p < 0.001) in comparison to control samples. HBD-2 was decreased in the subepithelial connective tissue of primary nasal polyp samples when compared to both recurrent polyp (p = 0.050) and control (p = 0.033) samples. In subepithelial connective tissue, significantly more HBD-3-positive structures were observed in primary nasal polyp samples (p = 0.049) than in control samples. In primary polyp samples, moderate correlations between connective tissue HBD-3 and connective (R = 0.584, p = 0.001) and epithelial tissue LL 37 (R = 0.556, p = 0.002) were observed. Conclusions: Decreased HBD-2, HBD-3 and LL 37 concentrations in the epithelium suggest a dysfunction of the epithelial barrier in patients with nasal polyps. Decreased subepithelial connective tissue HBD-2 suggests different responses to nasal microbiota in patients with primary nasal polyps compared to recurrent nasal polyps. Increased HBD-3 in subepithelial connective tissue suggests a possible role of this antimicrobial peptide in the pathogenesis of primary nasal polyps.

Morphopathogenesis of Adult Acquired Cholesteatoma.

Background and Objectives. The aim of this study was to compare the distribution of proliferation markers (Ki-67, NF-κß), tissue-remodeling factors (MMP-2, MMP-9, TIMP-2, TIMP-4), vascular endothelial growth factor (VEGF), interleukins (IL-1 and IL-10), human beta defensins (HßD-2 and HßD-4) and Sonic hedgehog gene protein in cholesteatoma and control skin. Methods. Nineteen patient cholesteatoma tissues and seven control skin materials from cadavers were included in the study and stained immunohistochemically. Results. Statistically discernible differences were found between the following: the Ki-67 in the matrix and the Ki-67 in the skin epithelium (p = 0.000); the Ki-67 in the perimatrix and the Ki-67 in the connective tissue (p = 0.010); the NF-κß in the cholesteatoma matrix and the NF-κß in the epithelium (p = 0.001); the MMP-9 in the matrix and the MMP-9 in the epithelium (p = 0.008); the HßD-2 in the perimatrix and the HßD-2 in the connective tissue (p = 0.004); and the Shh in the cholesteatoma's perimatrix and the Shh in the skin's connective tissue (p = 0.000). Conclusion. The elevation of Ki-67 and NF-κß suggests the induction of cellular proliferation in the cholesteatoma. Intercorrelations between VEGF, NF-κß and TIMP-2 induce neo-angiogenesis in adult cholesteatoma. The similarity in the expression of IL-1 and IL-10 suggests the dysregulation of the local immune status in cholesteatoma. The overexpression of the Sonic hedgehog gene protein in the cholesteatoma proves the selective local stimulation of perimatrix development.

Characterization of Cytokines and Proliferation Marker Ki-67 in Chronic Rhinosinusitis with Recurring Nasal Polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammation of the mucosa of the nose and paranasal sinuses with the presence of polyps, affecting between 2.7% and 4.4% of the population. Cytokine analysis has become important in research on inflammatory mechanisms in CRSwNP. Therefore, our aim is to investigate the complex appearance, relative distribution, and interlinks of IL-1, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, and Ki-67 in CRSwNP. METHODS: Samples of nasal polyps were obtained from 19 patients with previously diagnosed CRSwNP and the recurrence of polyps after previous surgeries. The control group consisted of samples from 17 otherwise healthy individuals with isolated nasal septum deviations. Tissues were stained for previously mentioned cytokines and Ki-67 immunohistochemically. RESULTS: Polyp samples showed an increased presence of cytokines in subepithelial connective tissue and a decreased appearance in epithelium when compared to controls. There were several very strong, strong, and moderate correlations among factors. CONCLUSIONS: IL-6 strongly correlates with other cytokines as well as with the proliferation marker Ki-67, which suggests significant stimulation of this regulatory cytokine and its possible involvement in the pathogenesis of recurrent nasal polyps. IL-4, IL-7, IL-10, and IL-12 correlate with Ki-67, which suggests the possible involvement of these cytokines in tissue cell proliferation in the case of recurrent nasal polyps.

An Overview of Adenoid Microbiome Using 16S rRNA Gene Sequencing-Based Metagenomic Analysis.

Background and Objectives: the upper respiratory tract harbors the highest bacterial density in the whole respiratory system. Adenoids, which are located in the nasopharynx, are a major site of bacterial colonies in the upper airways. Our goal was to use culture-independent molecular techniques to identify the breadth of bacterial diversity in the adenoid vegetations of children suffering from chronic rhinosinusitis and obstructive sleep apnea. Materials and methods: in total, 21 adenoid samples were investigated using amplification and sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene. Results: among the most common bacterial species found were Veillonella atypica, Fusobactrium nucelatum, Shaalia odontolytica, and Moraxella catarrhalis. Veillonella atypica and Fusbacteriumnucelatum dominated the microbiome in all 21 samples, attributing to more than 60% of all detected genetic material. Conclusions: since both Veillonella atypica and Fusobacterium nucleatum are, predominantly, oral cavity and dental microorganisms, our findings may suggest oral microbiome migration deeper into the oropharynx and nasopharynx where these bacteria colonize adenoid vegetations.

Remodeling Factors, Transcription Factors and Angiogenetic Factors in Cholesteatoma in Ontogenetic Aspect.

Introduction: The main goal of our study was to describe the transcription factor (NF-κß), angiogenetic factor (VEGF), and remodeling markers (MMP-9 and TIMP-4) of the cholesteatoma tissue compared to control skin tissue. There are still uncertainties how transcription, angiogenetic and remodeling factors affect the cholesteatoma course. Materials and Methods: Eight cholesteatoma tissue specimens were retrieved from children, seven - from adults, seven skin controls - from cadavers. Obtained material immunohistochemically were stained for NF-κß, MMP-9, TIMP-4, VEGF. Non-parametric statistic methods were used. Results: A statistically significant higher numbers of NF-κß and TIMP-4 immunoreactive cells in the cholesteatoma compared to control group. A very strong positive correlation between MMP-9 and TIMP-4 was seen in the patient group. A strong positive correlation - between MMP-9 in matrix and MMP-9, VEGF in perimatrix, between TIMP-4 in matrix and TIMP-4 in perimatrix, NF-κß in the matrix and VEGF; between TIMP-4 in perimatrix and NF-κß in the matrix. Conclusions: Correlation between MMP-9 and TIMP-4 suggests that TIMP-4 in cholesteatoma tissue intercorrelates to MMP-9. TIMP-4 likely regulates the development of cholesteatoma. Disbalance between MMPs and TIMPs affects NF-κß and causes uncontrolled cell proliferation and immune response in this tumor. There is a lack of VEGF strong expression in cholesteatoma perimatrix.

Distribution and Appearance of Ki-67, IL-1α, IL-10, and PGP 9.5 in Reinke's Oedema-Affected Larynx Tissue Compared with Control Tissue.

Smoking, laryngopharyngeal reflux, and vocal fold abuse can promote the development of Reinke's oedema, leading to vocal fold dysfunction and injury. The aim of the work was to investigate the appearance and distribution of proliferation marker Ki-67 (Ki-67), interleukin 10 (IL-10), interleukin 1 alpha (IL-1α), and protein gene peptide 9.5 (PGP 9.5) in Reinke's oedema-affected larynx tissue. METHODS: A routine histological and immunohistochemical Reinke's oedema and control group patient analysis was conducted. We used the biotin-streptavidin biochemical method to detect Ki-67, IL-10, IL-1α, and PGP 9.5 The semiquantitative grading method was used to evaluate immunoreactive cells' appearance and local distribution. A Mann-Whitney U test and Spearman's rank coefficient were performed. RESULTS: A low positive correlation between IL-1α epithelial and subepithelial immunoreactive cells in the patient group was found. Mann-Whitney U tests revealed significant patient and control group immunoreactive marker differences. All examined markers showed a higher number of immunoreactive structures in the patient group. CONCLUSIONS: Intensive proliferation of the surface epithelium was observed in patient tissues. The notable increase in IL-10 positive structures indicates the dominant anti-inflammatory tissue response. An increased number of IL-1α structures in the larynx epithelium and subepithelium in the patient group is linked to inflammation, proliferation, and tissue remodelling. The PGP 9.5 expression increase is involved in the morphopathogenesis of Reinke's oedema.

Singer's Nodules: Investigating the Etiopathogenetic Markers Progressing Their Pathogenesis and Clinical Manifestations.

Vocal nodules (or Singer's nodules) are benign vocal cord structures which are commonly encountered by clinicians. Though phonetic trauma/abuse is thought to be the main cause of the development of vocal nodules, the exact etiopathogenesis remains unknown. Hence, we compared the immunohistochemical markers for proliferation (Ki-67), apoptosis (TUNEL), growth (EGFR), ischemia (VEGF), inflammation (IL-1α and 10), and immunoreactive innervation (PGP 9.5), in vocal nodule tissue samples obtained from 10 females (17-56 years) and vocal cord tissue from seven controls. A statistically significant increase in Ki-67, TUNEL, EGFR, VEGF and IL-1α expression was noted (p < 0.05) between nodule tissue and control tissue in both epithelial and subepithelial layers. However, the difference was non-significant for both IL-10 and PGP 9.5 (p > 0.05). All markers demonstrated moderate to strong positive correlations, except for IL-10. These findings suggest increased cellular growth and proliferation in vocal nodules coupled with a persistent presence of inflammatory and ischemic environment. Furthermore, global prevalence of apoptotic cells and decreased anti-inflammatory cytokines highlight the presence of underlying complex mechanisms in the etiopathogenesis of vocal nodules, with age having a negligible impact on the marker levels. Our results could potentially further our knowledge in understanding the effects of different treatment modalities available at the cellular level.

Complex Evaluation of Tissue Factors in Pediatric Cholesteatoma.

The aim of this study was to describe the appearance and distribution of tissue remodeling markers (MMP-2, MMP-9, TIMP-2, TIMP-4), Sonic hedgehog gene protein (Shh), pro- and anti-inflammatory cytokines (IL-1, IL-10), transcription factor (NF-κß), proliferation marker (Ki-67), angiogenetic factor (VEGF), tissue defensins (HßD-2, HßD-4) of the pediatric cholesteatoma. Sixteen cholesteatoma samples were obtained from children, eleven skin controls from cadavers. Tissues were stained for MMP-2, MMP-9, TIMP-2, TIMP-4, Shh, IL-1, IL-10, NF-κß, Ki-67, VEGF, HßD-2, HßD-4. Non-parametric statistic, Mann-Whitney, and Spearman's coefficient was used. A statistically significant difference was seen between Shh and HßD-2 in perimatrix and control connective tissue, between NF-κß in cholesteatoma and control skin, and between HßD-4 in matrix and skin epithelium. Complex intercorrelations between MMPs, NF-κß and VEGF cause the intensification of angiogenesis in cholesteatoma. The persistent increase in Shh gene protein expression in cholesteatoma perimatrix suggests the stimulation of the cholesteatoma growth in children. Similar expression of IL-1 and IL-10 and their intercorrelation, proves there is a balance between pro- and anti-inflammatory cytokines. NF-κß, and not Ki-67, seems to be the main inducer of cellular proliferation. The main antimicrobial protection is provided by HßD-2.

Characterization of Cytokines and Proliferation Marker Ki67 in Chronic Rhinosinusitis with Nasal Polyps: A Pilot Study.

Background and Objectives: Chronic rhinosinusitis (CRS) is a condition that affects as much as 10.9% of the population and, along with presence of nasal polyps, is associated with significant morbidity and decreased quality of life. Studies on molecular pathways that have been activated in nasal polyp tissue are mainly based on cytokine concentration detection. Therefore, our aim is to investigate the complex appearance, relative distribution and interlinks of IL-1, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12 and Ki 67 in chronic rhinosinusitis with nasal polyps (CRSwNP) affected human nasal mucosa. Materials and Methods: Samples of nasal polyps were obtained from 12 patients with previously diagnosed CRSwNP and no prior surgery. Control group consisted of samples from 17 otherwise healthy individuals with isolated nasal septum deviation. Tissues were stained for IL-1, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12 and Ki67 immunohistochemically. Non-parametric statistic, Mann-Whitney U test and Spearman's rank correlation coefficient were used. Results: All factors, except connective tissue cytokine IL-10 and proliferation marker Ki-67, had increased presence in connective tissue and decreased presence in epithelium of nasal polyps when compared to controls. Very strong and strong positive correlations between factors were observed. Conclusions: Decreased appearance of IL-1α, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12 positive structures in the nasal epithelium with selective increase of IL-1α and IL-12 in nasal subepithelial connective tissue characterize the cytokine endotype with dysfunctional epithelial barrier and local stimulation of immune response in the connective tissue in case of chronic rhinosinusitis with polyps. Decrease of IL-6 in both-epithelium and connective tissue with strong correlation between it and IL-7 and IL-10 in connective tissue suggests significant stimulation of this regulatory cytokine and, possibly, the important role in pathogenesis of the development in nasal polyps. Correlations between Ki67 and cytokines indicate possible involvement of IL-4, IL-7 and IL-12 in regulation of cellular proliferation.

Cytokines, Proliferation Markers, Antimicrobial Factors and Neuropeptide-Containing Innervation in Human Nasal Mucosa after Rhinoseptoplasty Procedure.

The nasal cavity lined by nasal mucosa, is a significant part of respiratory system of human. However, there are no studies aimed to detect a molecular phenotype of healthy and normal functioning nasal mucosa, obtained after rhinoseptoplasty procedure, to understand its physiology and growth and inflammation processes. Thus, our aim is to identify human healthy nasal mucosa cytokines, neuropeptide-containing innervation and cell proliferation markers to form a control group for further tissue investigation of human nasal polyposis as the next step of our research. The study included surgery materials from 17 healthy humans. Biotin-streptavidin immunohistochemistry was performed for detection of tissue PGP9.5, Ki-67, ß-Defensin 2, IL-1, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12. Results were evaluated semi-quantitatively and by Friedman ANOVA and Spearman rang correlation tests. All factors were more widely expressed by superficial epithelium than by glandular one. Abundance of ILs-8, -10 and -12 positive cells was detected in comparison with moderate to numerous distributions of IL-1, IL-6 and ß-Defensin 2. Moderate number of PGP 9.5-containing nerve fibers and only few to moderate Ki-67 positive cells were found in healthy nasal mucosa. We revealed statistically significant difference between Ki-67 and ILs-4, -6, -7, -8, -10, -12 both in healthy nasal mucosa superficial and glandular epithelium. From nasal epithelia, commonly the surface one displays more cytokines and ß-Defensin 2 in comparison to the glandular one. Numerous to abundant expression of ILs-4, -6, -7, -8, -10, -12 and ß-Defensin 2 in nasal superficial and glandular epithelia proves probably these factors' role into the common immune response of tissue and stimulation of immune cell differentiation.

Deep neck infections: review of 263 cases.

OBJECTIVES: In patients hospitalized due to deep neck infections (DNIs), to investigate the association between demographic parameters, etiology, and localization of abscesses and/or phlegmons, complications, comorbidities, treatment, and bacterial cultures. METHODS: We analyzed data of 263 patients that were hospitalized from January 1, 2012, to December 31, 2015, due to deep neck space phlegmons and/or abscesses. We performed statistical analysis with the SPSS 22.0 software; statistical significance was set p<0.05. RESULTS: Among the analyzed patients, dental infections were the most frequent, followed by acute phlegmonous pharyngitis. Submandibular space abscesses or phlegmons of the neck were the most frequent complications of these infections. Re-operation was performed in 19.8% of cases, and complications developed in 11% (mostly airway obstruction). DNI complications were less common in smokers than in non-smokers (OR=0.038, p=0.025). DISCUSSION: In our study, dental infections accounted for 70.6% of DNI cases. Thus, our study explains why odontogenic DNIs are the most common DNI type studied in the literature. Poor oral health and odontogenic infections should not be underestimated because they can lead to uncommon but lethal diseases such as descending necrotizing mediastinitis, which requires aggressive surgical treatment and is associated with a mortality rate of 10%-40% despite treatment. The complication developed in 11.4% of cases. In conclusion, oral health and hygiene contribute to DNI development.