Effects of 1-year anti-TNF-α therapies on bone mineral density and bone biomarkers in rheumatoid arthritis and ankylosing spondylitis.

OBJECTIVES: Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) have been associated with generalized and localized bone loss. We conducted a comprehensive study using imaging (dual-energy X-ray absorptiometry, DXA) and laboratory biomarkers in order to determine bone health and to study the effects of anti-tumor necrosis factor (TNF) biologics in RA and AS. PATIENTS AND METHODS: Thirty-six RA and 17 AS patients undergoing 1-year etanercept (ETN) or certolizumab-pegol (CZP) therapy were studied. Bone density was assessed by DXA at baseline and after 12 months. Serum C-reactive protein (CRP), calcium, phosphate, parathyroid hormone (PTH), vitamin D3, osteocalcin, procollagen type I N-propeptide (P1NP), C-terminal telopeptide (ßCTX), osteoprotegerin, sclerostin (SOST), Dickkopf-1 (DKK-1), soluble receptor activator nuclear kappa B ligand (sRANKL), and cathepsin K (cathK) levels were determined at baseline and after 6 and 12 months. RESULTS: TNF-α inhibition was clinically effective. Anti-TNF-α halted further bone loss over 1 year. In general, anti-TNF therapy significantly increased P1NP, SOST levels, and the P1NP/ßCTX ratios, while decreased DKK-1 and CathK production at different time points in most patient subsets. In the full cohort and in RA, baseline and/or 12-month bone mineral density (BMD) at multiple sites exerted inverse relationships with CRP and ßCTX, and positive correlation with SOST. In AS, L2-4 BMD after 1-year biologic therapy inversely correlated with baseline ßCTX, while femoral neck BMD rather showed inverse correlations with CRP. CONCLUSIONS: Anti-TNF therapy slowed down generalized bone loss, in association with clinical improvements, in both diseases. TNF blockade may enhance bone formation and suppress joint destruction. Anti-TNF therapy may act inversely on DKK-1 and SOST. Independent predictors of BMD were SOST and ßCTX in RA, whilst CRP in AS.Key Points• One-year anti-TNF therapy halted generalized bone loss in association with clinical improvement in arthritides.• Anti-TNF therapy may inversely act on DKK-1 and SOST.• Independent predictors of BMD were SOST and ßCTX in RA, while CRP in AS.

[Experience with a rheumatoid arthritis biobank: analysis of biological samples and clinical data of 204 patients]. / Rheumatoid arthritises biobankkal szerzett tapasztalataink: 204 beteg biológiai mintáinak és klinikai adatainak összevetése.

INTRODUCTION: A biobank is a registry, which is suitable for the storage of biological samples (e.g. tissues, DNA, protein), genetical abnormalities and clinical data. Several biobanks have been created worldwide, which contribute to research and the better understanding of disease pathogenesis, genetical polymorphisms. Biobanking also helps to improve the efficacy of therapies. AIM: Our purpose was to create an internet-based biobank, in which laboratory test results, genetic alterations and related disorders of rheumatoid arthritis (RA) patients can be registered. This biobank would be able to make the research easier and it can help to improve our knowledge about diseases and it can inhibit loss of data. PATIENTS AND METHOD: We have biological samples from 204 RA patients and we have entered their data in the biobank which can be found on the website http://rheuma.biobank.eu . Statistical analysis was performed by SPSS20 statistical programme. RESULTS: By the creation of biobank that contains clinical data and biological samples of 204 RA patients, we have a database which can help to improve our knowledge about the disease and help to develop new treatment strategies. CONCLUSION: Biobanking is suitable to analyze blood samples and clinical data together. Orv. Hetil., 2017, 158(7), 270-277.

Evaluation of the immunogenicity of the 13-valent conjugated pneumococcal vaccine in rheumatoid arthritis patients treated with etanercept.

OBJECTIVES: To prospectively evaluate the immunogenicity of a 13-valent conjugated pneumococcal vaccine (PCV13) in rheumatoid arthritis (RA) patients undergoing etanercept therapy. METHODS: Twenty-two RA patients treated with etanercept (ETA) in combination with methotrexate (MTX) (n=15) or monotherapy (n=7) for at least one year were included. Altogether 24 osteoarthritis patients not receiving biological or MTX therapy, treating only NSAIDs or analgesics served as controls. All subjects were vaccinated with a single dose (0.5ml) of the PCV13. Pneumococcal antibody levels at baseline, 4 and 8weeks were assessed by a VaccZyme™ Anti-PCP IgG Enzyme Immunoassay Kit. Based on recommendations of the American Academy of Allergy, Asthma & Immunology, an at least two-fold increase in antibody level, as the protective antibody response (pAR) was an indicator of responsiveness (i.e., ratio of postvaccination and prevaccination antibody levels). The antibody levels and their ratios were analysed in a variety of different ways, vaccine safety parameters (fever, infections, changes in regular antirheumatic treatments) were assessed at baseline, 4 and 8weeks after vaccination. RESULTS: Four weeks after vaccination, the anti-pneumococcal antibody levels significantly increased in both groups. At week 8, antibody levels somewhat decreased in both groups, however, still remained significantly higher compared to baseline. Compared with postvaccination levels at 4 and 8weeks between two groups, the mean protective antibody levels were higher in control group (1st month P=0.016; 2nd month: P=0.039). Possible predictors of pAR were analysed by logistic regression model. In RA, increases of antibody levels at week 8 compared to baseline exerted a negative correlation with age, (Spearman's R=-0,431; P=0.045). There were no clinically significant side effects or reaction after administration of vaccine observed in any of these patients after the 2-month follow-up period, all patients medical condition were stable. CONCLUSIONS: In RA patients treated with ETA, vaccination with PCV13 is effective and safe, resulting in pAR one and two months after vaccination. Higher age at vaccination was identified as predictors of impaired pAR. The efficacy of vaccination may be more pronounced in younger RA patients. The vaccine is safe in RA patients on ETA.

Genetic association analysis in a clinically and histologically confirmed otosclerosis population confirms association with the TGFB1 gene but suggests an association of the RELN gene with a clinically indistinguishable otosclerosis-like phenotype.

BACKGROUND/HYPOTHESIS: Otosclerosis is a frequent cause of hearing impairment characterized by abnormal resorption and deposition of bone in the human otic capsule. It is a disease of complex etiopathogenesis that is caused by both environmental and genetic factors. The goal of this study is to replicate association for genes that were previously reported to be associated with otosclerosis. However, in this study, patients were used in which the presence of otosclerotic foci was confirmed by histologic investigation, in contrast to previous studies, that did not use histologic confirmation. METHODS: Case-control association study using 153 cases and 300 controls. Thirteen single nucleotide polymorphisms (SNPs) in 6 genes (COL1A1, TGFB1, BMP2, BMP4, AGT, and RELN) were genotyped. RESULTS: An association between TGFB1 (rs1800472) and otosclerosis was detected, confirming several previous reports. It is surprising that no association was found between RELN and otosclerosis because the current analysis had very reasonable power and the RELN association has been published before in different articles using several independent populations. CONCLUSION: Our findings strengthen the association of TGFB1 (rs1800472) with otosclerosis. The fact that other genes did not replicate could be due to different reasons like lack of power (BMP2 and BMP4) and possible false-positive initial association (COL1A1 and AGT). A plausible explanation for the lack of association for RELN is that RELN could be associated with a specific otosclerosis-like phenotype that is different from the histologically confirmed phenotype of the patients in this study, and that is clinically not distinguishable.

Diagnostic value of cone-beam CT in histologically confirmed otosclerosis.

This retrospective case review was performed with the aim to asses the value of cone-beam computed tomography (CBCT) in the preoperative diagnosis of otosclerosis. A total of 32 patients with histologically confirmed stapedial otosclerosis, who underwent unilateral stapedectomies were analyzed. Preoperative temporal bone CBCT scans were performed in all cases. CBCT imaging was characterized by a slice thickness of 0.3 mm and multiplanar image reconstruction. Histopathologic examination of the removed stapes footplates was performed in all cases. Findings of CBCT were categorized according to Marshall's grading system (from grade 0 to grade 3). Histopathologic results were correlated to multiplanar reconstructed CBCT scans, respectively. Histologically active foci of otosclerosis (n = 21) were identified by CBCT in all cases with a sensitivity of 100 %. However, CBCT was unable to detect histologically inactive otosclerosis (n = 11, sensitivity = 0 %). According to CBCT scans, no retrofenestral lesions were found and all positive cases were recruited into the grade 1 group indicating solely fenestral lesions at the anterior pole of stapes footplates. In conclusion, CBCT is a reliable imaging method with considerably lower radiation dose than high-resolution CT (HRCT) in the preoperative diagnosis of otosclerosis. These results indicate that CBCT has high sensitivity and specificity in the detection of hypodense lesions due to histologically active otosclerosis.

Effects of intranasal steroid treatment on the presence of biofilms in non-allergic patients with chronic rhinosinusitis with nasal polyposis.

Microbial biofilms have been implicated in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). Intranasal application of corticosteroids (INCS) is a reliable option in the management of CRSwNP. INCS medication has been suspected to influence the presence and thickness of microbial biofilms and inflammatory cell patterns in CRSwNP. Two series of identical nasal polyps obtained from non-allergic patients with CRSwNP (n = 56), who underwent endoscopic sinus surgery (ESS), were processed to hematoxylin-eosin (H.E.) and Gram staining, respectively. Patients were recruited into three groups. Group A (n = 21) consisted of patients with continuous preoperative INCS treatment. In group B (n = 17), patients were never treated by INCS, while in group C (n = 18) INCS medication was stopped at least 6 months before ESS. Biofilm positivity varied from 76.4 to 88.8% in different subject groups. These values and average thickness of biofilms did not reach statistically significant levels (Mann-Whitney's U probe, p > 0.05) in different patient groups. In contrast, microscopic pattern and numbers of predominant inflammatory cell populations displayed obvious differences according to INCS treatment (Mann-Whitney's U probe, p < 0.001). According to these observations, INCS treatment does not affect the presence and thickness of microbial biofilms in CRSwNP. In contrast, it has significant effects on the pattern of inflammatory cells infiltrating the subepithelial layer, which might result in beneficially altered extracellular matrix production and cytokine release.

Microbiological profile of adenoid hypertrophy correlates to clinical diagnosis in children.

OBJECTIVE: Adenoid hypertrophy is a common condition in childhood, which may be associated with recurring acute otitis media (RAOM), otitis media with effusion (OME), and obstructive sleep apnea syndrome (OSAS). These different clinical characteristics have some clinical overlap; however, they might be explained by distinct immunologic and infectious profiles and result in various histopathologic findings of adenoid specimens. METHODS: A total of 59 children with adenoid hypertrophy undergoing adenoidectomy were studied. Three series of identical adenoid specimens were processed to hematoxylin-eosin (H.E.) and Gram staining and to respiratory virus specific real-time PCR, respectively. RESULTS: According to the clinical characteristics, patients were recruited into three groups: RAOM (n = 25), OME (n = 19), and OSAS (n = 15). Bacterial biofilms were detected in 21 cases, while at least one of the studied respiratory viruses was detected in 52 specimens. RAOM cases were significantly associated with biofilm existence (n = 20, P < 0.001). In contrast, OME group was characterized by the absence of bacterial biofilm and by normal mucosa. Showing a statistically significant correlation, all OME cases were positive for human bocavirus (HBoV, P < 0.001). CONCLUSIONS: Bacterial biofilms might contribute to the damage of respiratory epithelium and recurring acute infections resulting in RAOM. In OME cases persisting respiratory viruses, mainly HBoV, can cause subsequent lymphoid hyperplasia leading to ventilation disorders and impaired immunoreactivity of the middle ear cleft.

Detection of otosclerosis-specific measles virus receptor (cd46) protein isoforms.

Genetic predisposition of otosclerosis has long been suspected, but unclarified. Unique coexpression pattern of measles virus receptor (CD46) splicing isoforms in the human otic capsule is assumed, since otosclerosis is a measles virus-associated organ-specific disease. In order to identify CD46 involved in the pathogenesis of otosclerosis, we used representative groups of histologically diagnosed otosclerotic, nonotosclerotic, and normal stapes footplates (n = 109). Consecutive histopathological examinations and CD46-specific Western blot analysis were performed. Normal and nonotosclerotic stapes footplates showed consistent expression of the conventional c, d, e, f, and l CD46 isoforms. In contrast, four novel isoforms (os1-4) translated as intact proteins were additionally detected in each otosclerotic specimen. The study herein presented provides evidence for the otosclerosis-associated expression pattern of CD46. This finding might explain the organ-specific, virus-associated and autoimmune-inflammatory pathogenesis of otosclerosis. Regarding our current knowledge, this is the first report that confirms the presence of four new disease-specific protein variants of CD46.

Activation of Src, Fyn and Yes non-receptor tyrosine kinases in keratinocytes expressing human papillomavirus (HPV) type 16 E7 oncoprotein.

BACKGROUND: The Src family tyrosine kinases (SFK) are cellular regulatory proteins that influence cell adhesion, proliferation, invasion and survival during tumor development. Elevated activity of Src was associated with increased cell proliferation and invasivity in human papillomavirus (HPV)-associated malignancies; therefore, transduced human foreskin keratinocytes (HFK) were used to investigate whether SFK activation is a downstream effect of papillomaviral oncoproteins. Activation of ubiquitously expressed SFKs, namely Src, Yes and Fyn, was investigated in both proliferating and differentiating keratinocytes. RESULTS: In proliferating keratinocytes, Src, Yes and Fyn mRNA levels were not affected by HPV 16 E6 or E7 oncoproteins, while at the protein level as detected by western blot, the presence of both E6 and E7 resulted in substantial increase in Src and Yes expression, but did not alter the high constitutive level of Fyn. Phospo-kinase array revealed that all ubiquitously expressed SFKs are activated by phosphorylation in the presence of HPV 16 E7 oncoprotein. Keratinocyte differentiation led to increased Yes mRNA and protein levels in all transduced cell lines, while it did not influence the Src transcription but resulted in elevated Src protein level in HPV16 E7 expressing lines. CONCLUSIONS: This study revealed that HPV 16 oncoproteins upregulate Src family kinases Src and Yes via posttranscriptional mechanisms. A further effect of HPV 16 E7 oncoprotein is to enhance the activating phosphorylation of SFKs expressed in keratinocytes.

No evidence for the expression of renin-angiotensin-aldosterone system in otosclerotic stapes footplates.

INTRODUCTION: Recent studies have reported genetic associations between with single nucleotide polymorphism (SNP) of the several genes of the renin-angiotensin-aldosterone (RAA) system in otosclerosis without the confirmation of RAA system expression in human stapes footplates. There are conflicting results. These results are conflicting because RAA system expression has been attributed exclusively to neural, vascular, and renal tissues, exclusively. MATERIALS AND METHODS: Ankylotic stapes footplates (n = 20), cortical bone fragments (n = 10), and human kidney tissue specimens (n = 10) were processed to hematoxylin-eosin (HE) staining and RAA system-specific immunofluorescent assay (IFA), respectively. RESULTS: Histologic diagnosis of otosclerosis was established in all ankylotic stapes footplates. Histologically active- (n = 13) and inactive (n = 7) foci of otosclerosis were consequently characterized by negative immunoreactions for renin, angiotensin converting enzyme (ACE), angiotensin-II (AT-II), and angiotensin-II receptor (AT-IIR), consequently. In cortical bones, a considerable RAA system expression was observed confirmed in the perivascular bone marrow progenitor cells. Kidney specimens, applied as positive controls, showed intense RAA system-specific immunoreactions. CONCLUSION: Concerning current observations, the 4 studied members of RAA system that did not display active expression were not expressed at protein level in otosclerotic stapes footplates. This phenomenon was independent from the histologic activity of otosclerosis. Between these conditions, the etiologic role of RAA system is questionable in the pathogenesis of otosclerosis.

The presence of CD209 expressing dendritic cells correlates with biofilm positivity in chronic rhinosinusitis with nasal polyposis.

Biofilm-positive cases of chronic rhinosinusitis with nasal polyposis (CRSwNP) may form a separate clinical entity, which is characterized by high recurrence rates and resistance against different therapeutic strategies. This can be explained by a special immunologic phenotype. Biofilm existence has been supposed to correlate with increased amount of dendritic cells that are responsible for antigen presentation in CRSwNP. A total of 20 patients with CRSwNP undergoing endoscopic sinus surgery (ESS) were analyzed. The negative control group consisted of ten patients undergoing septoplasty without CRSwNP. Three series of individual nasal polyps and control specimens were processed to hematoxylin-eosin (HE) and Gram staining and to CD209-specific immunofluorescent assay, respectively. Biofilm was detected in 13 of 20 patients (65 %) with CRSwNP and in none of the ten negative controls. The subepithelial layer of biofilm-positive nasal polyps displayed a statistically significant (p < 0.001) increase in the numbers of CD209-expressing dendritic cells compared to biofilm-negative specimens. It was found that biofilm detectability showed strong correlation to the architecture of respiratory mucosa and to the dominant inflammatory cell type of the subepithelial layer. Persisting bacterial biofilms may affect the type of antigen presentation and consecutive immune reactions in the subepithelial layer of nasal mucosa. This phenomenon may result in different inflammatory pathways with specific cytokine profile compared to biofilm-negative cases. Co-existence of bacterial biofilms and dominant pattern of dendritic cells suggest a biofilm-associated immunologic phenotype in CRSwNP. This can explain the mucosal changes, functional disorders and therapy resistance featuring CRSwNP.

Optical coherence tomography for biofilm detection in chronic rhinosinusitis with nasal polyposis.

Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a multifactorial disease that seems to be associated with the presence of microbial biofilms and corresponding subepithelial inflammatory reactions. Optical coherence tomography (OCT) might be applied to detect bacterial and fungal biofilms in patients with CRSwNP. A total of 27 patients with CRSwNP undergoing endoscopic sinus surgery (ESS) were analyzed. The negative control group consisted of six patients undergoing septoplasty for nasal obstruction without CRSwNP. The nasal polyps and inferior turbinate mucosa specimens applied as negative controls were processed to OCT analysis and H.E. and Gram staining. Biofilm was detected in 22 of 27 patients (81.5 %) with CRSwNP and in none of six negative controls. In our series, OCT scan showed an obvious association with the findings of H.E. and Gram staining and was allocated to be a good predictor of biofilm existence. On OCT images, biofilms were displayed as distinct superficial layers with high optical density. It was found that microscopic architecture of biofilms was strongly associated with the integrity of nasal mucosa and to the cellular pattern of subepithelial inflammatory reaction. This study confirmed the presence of microbial biofilms in patients with CRSwNP according to OCT scans and histological analysis. Since biofilms may affect the severity and recurrence rate of CRS treated by ESS they should be detected preoperatively. In conclusion, single application of OCT analysis or combination with conventional histological protocols provides a robust and reliable method for the detection of bacterial and fungal biofilms in CRSwNP. Level of evidence 3b, individual case-control study.

Low-frequency ultrasound for biofilm disruption in chronic rhinosinusitis with nasal polyposis: in vitro pilot study.

OBJECTIVES/HYPOTHESIS: Microbial biofilms have been implicated in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). Although biofilms are characterized by an extremely high resistance against chemical and physical agents, low-frequency ultrasound (LFU) treatment has been suspected to be an efficient and safe method for biofilm disruption. STUDY DESIGN: Basic science experimental study. METHODS: A total of 10 patients with CRSwNP undergoing endoscopic sinus surgery were analyzed. Two series of identical nasal polyps (n = 20) were processed to hematoxylin-eosin (HE) and Gram staining and to continuous-wave LFU treatment (5 minutes, 0.4 MHz, 37°C), respectively. RESULTS: Presence of microbial biofilms was confirmed in all patients with CRSwNP. HE staining showed a strong correlation with the results of Gram protocol in biofilm detection. In the LFU-treated group (n = 10), a significantly decreased inflammatory cell count was found in the subepithelial layer of nasal polyps (P < .001). In addition, bacterial biofilms were completely removed from the surface of the epithelial layer. Microscopic tissue injuries or significant temperature changes were not detected due to LFU treatment. CONCLUSIONS: Between in vitro conditions, LFU treatment appeared to be a reliable and microscopically safe method for the disruption of microbial biofilms in CRSwNP. These results may provide a basis for a prospective human study investigating the efficacy and safety of this therapeutic modality alone or in combination with antibiotics or topical steroids in biofilm-positive cases of CRSwNP.

The value of HRCT in stapes fixations corresponding to hearing thresholds and histologic findings.

OBJECTIVE: To estimate the correlations between high-resolution computed tomography (HRCT) scans, preoperative audiometric findings and histopathologic results in stapes ankylosis. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: A total of 57 patients with stapes ankylosis, who underwent unilateral stapedectomies were analyzed. INTERVENTIONS: Diagnostic and therapeutic. MAIN OUTCOME MEASURES: Preoperative HRCT examinations were performed in all cases. Findings of HRCT were categorized according to Marshall's grading system. Preoperative air-bone gaps (ABGs) and bone conduction (BC) thresholds were determined. Stapes footplates removed surgically were processed using hematoxylin and eosin staining. RESULTS: Active otosclerosis (n = 29) was demonstrated by HRCT with a sensitivity of 76.31%, whereas its sensitivity to establish inactive otosclerosis (n = 13, 61.9%) and nonotosclerotic stapes fixations (n = 15, 51.7%) was much lower. Nonotosclerotic stapes fixations were characterized by pure conductive hearing loss that was not associated with HRCT findings. HRCT grades showed statistically significant association with BC levels at the averages of 0.5-1-2 kHz frequencies in the group of ears with inactive otosclerosis, exclusively (p < 0.05). CONCLUSION: HRCT is a reliable imaging method in the preoperative diagnosis of different types of stapes fixations. Imaging findings should be evaluated together with clinical history and audiometric data for obtaining as precise diagnosis for stapes fixation as possible.

Expression of bone morphogenetic protein 2, 4, 5, and 7 correlates with histological activity of otosclerotic foci.

CONCLUSION: This study is the first to establish that bone morphogenetic protein 5 (BMP5) plays a role in the pathogenesis of otosclerosis. These results confirm that elevated expression levels of BMPs, members of the transforming growth factor (TGF)-ß superfamily, contribute to the pathologically increased bone turnover in early, active stages of otosclerosis. OBJECTIVES: Otosclerosis is a complex bone remodeling disorder of the otic capsule, which might be characterized by increased expression of different types of BMPs. TGF-ß and BMP are both members of the TGF-ß superfamily and play a critical role in bone resorption and new bone formation. It has been suggested that BMP and its receptors may be involved in the pathologically increased bone turnover observed in otosclerosis. METHODS: Fifty-one otosclerotic and 16 non-otosclerotic ankylotic stapes footplates were histologically analyzed: conventional hematoxylin-eosin staining and BMP2, 4, 5, and 7specific immunofluorescent assays were performed. Cortical bone fragments (n = 35) and incus specimens (n = 6) were used as negative controls. RESULTS: Active otosclerosis (n = 39) was characterized by increased expression of BMP2, 4, 5, and 7. Inactive cases of otosclerosis (n = 12) were characterized by negative immunoreaction for BMPs. Non-otosclerotic stapes specimens (n = 16) and negative controls (n = 41) showed negligible BMP expression. The BMP expression pattern showed a strong correlation with the histological activity of otosclerosis.

Tumor necrosis factor-α receptor expression correlates with mucosal changes and biofilm presence in chronic rhinosinusitis with nasal polyposis.

OBJECTIVES/HYPOTHESIS: Biofilms might play a potential role in the pathogenesis and high recurrence rate of chronic rhinosinusitis with nasal polyposis (CRSwNP). Biofilm persistence has been thought to correlate with epithelial damage, subepithelial inflammatory cell infiltration, and tumor necrosis factor-α receptor (TNFR) expression in CRSwNP. STUDY DESIGN: Case-control experimental study. METHODS: A total of 36 patients with CRSwNP undergoing endoscopic sinus surgery were analyzed. The negative control group consisted of eight patients undergoing septoplasty for nasal obstruction without CRSwNP. The nasal polyps and inferior turbinate mucosa samples applied as negative controls were processed by hematoxylin-eosin (HE) and Gram staining and TNFR-I and TNFR-II-specific immunofluorescent assay. RESULTS: Biofilm was detected in 29 of 36 patients with CRSwNP and in none of the eight negative controls. Staining by HE showed strong correlation with the results of Gram staining protocol. In the biofilm-positive cases, TNFR-I and TNFR-II displayed homogeneous pattern of significantly increased epithelial expression compared to the biofilm-negative nasal polyps. In cases of biofilm absence, the expression pattern of TNF-α receptors was characterized by increased TNFR-II-specific immunoreaction. It was found that biofilm detectability corresponded to the integrity of nasal epithelium and to the dominant inflammatory cell type of the subepithelial layer. CONCLUSIONS: Persisting biofilms might increase the epithelial sensitivity against TNF-α that result in epithelium destruction. Coexistence of biofilms and increased TNFR expression might explain the inflammatory mucosal changes, functional disorders, and therapy resistance featuring CRSwNP.

Controversies in RELN/reelin expression in otosclerosis.

Several studies have reported a potential genetic association between disease-specific single nucleotide polymorphism (SNPs) of RELN and otosclerosis and confirmed RELN expression in human stapes footplates. These are conflicting results, since RELN expression has been attributed exclusively to neural tissues and to odontoblasts. Otosclerosis is a disease of complex bone remodeling disorder, which is limited to the human otic capsule. Genetic predisposition has long been suspected, however, the pathogenesis remained unclear. Ankylotic stapes footplates (n = 85), cortical bone fragments (n = 4), hearing ossicles (n = 2) and human brain tissue specimens (n = 4) were processed to RELN-specific RT-PCR and reelin-specific immunofluorescent assay (IFA). The first group of ankylotic stapes footplates (n = 22) showed a consistent positive reaction against reelin by IFA; however, RELN-specific mRNA could not be detected in the second, RT-PCR group (n = 63). Brain specimens were characterized by robust expression of reelin (n = 2) and RELN-specific mRNA (n = 2). In case of bone-specific controls (n = 6), reelin/RELN expression was excluded obviously. Concerning current observations, RELN gene does not show active expression in adult stapes footplates. Since, the otic capsule surrounds a special neural structure (membranous labyrinth), reelin might play a coordinative role in the early embryonic stage of development. As being a part of the otic capsule, stapes footplate might be characterized by persisting reelin detectability without mRNA expression. Between these conditions, the etiologic role of RELN is questionable in the pathogenesis of otosclerosis.

No evidence for disturbed COL1A1 and A2 expression in otosclerosis.

Otosclerosis is a complex bone remodeling disorder of the human otic capsule that might be associated with various mutations of A1 and A2 alleles of type-I collagen. The study herein presented, investigates the possibilty of the genetic involvement of type-I collagen in the pathogenesis of histologically confirmed otosclerosis. A total of 55 ankylotic stapes footplates were analyzed. Cortical bone fragments (n = 30), incus (n = 3) and malleus (n = 2) specimens were employed as negative controls. Specimens were divided into two groups. The first group was processed using conventional H.E. hematoxylin-eosin (H.E.) staining and type-I collagen-specific immunofluorescent assay (IFA), while the second group was examined by COL1A1 and A2-specific RT-PCR. Otosclerotic- (n = 31) and non-otosclerotic stapes footplates (n = 9) as well as cortical bones (n = 20), incus (n = 2) and malleus specimens (n = 1) showed normal and quite similar A1 and A2 allele expression confirmed by IFA. RT-PCR analysis revealed normal and consistent mRNA expression of both alleles in each specimen. Expression levels and patterns of COL1A1/A2 alleles did not show significant correlation with the histological diagnosis of otosclerosis. Type-I collagen is a highly conserved structure protein, which plays a fundamental role in the integritiy of various connective tissues. Mutations of A1 and A2 alleles result in serious systemic disorders of the skeleton, tendons and skin. Since otosclerosis is an organ-specific disease, it is difficult to explain its genetic association with type-I collagen. In conclusion, we found no evidence supporting the putative link of COL1A1 and COL1A2 alleles with otosclerosis.

Biofilm detection in chronic rhinosinusitis by combined application of hematoxylin-eosin and gram staining.

The pathomechanism of chronic rhinosinusitis with nasal polyposis (CRS/NP) seems to be unclear. Bacterial-, fungal- and combined biofilms might play a potential role in the pathogenesis of various inflammatory diseases and recently in CRS/NP. A prospective, blinded observational study was performed to confirm that the combination of conventional hematoxylin-eosin (HE) and Gram staining protocols could be used to detect bacterial and fungal biofilms in patients with CRS/NP. A total of 50 patients with CRS/NP undergoing endoscopic sinus surgery (ESS) were analyzed. The negative control group consisted of 12 patients undergoing septoplasty for nasal obstruction without CRS/NP. The nasal polyps and inferior turbinate mucosa specimens applied as negative controls were processed to HE and Gram staining. Biofilm was detected in 44 of 50 patients with CRS/NP and in none of 12 negative controls. In our series, HE method showed an obvious correlation with the results of Gram staining and was allocated to be a good predictor of biofilm existence. It was found that the microscopic structure and thickness of biofilms were strongly associated with the integrity of nasal mucosa and with the characteristics of subepithelial cellular infiltration. This study confirmed the presence of bacterial and fungal biofilms on the surface of NPs obtained from patients with CRS. Since biofilms may affect the severity and recurrence rate of CRS treated by ESS they should be detected histologically. In conclusion, HE staining combined with Gram protocol is a robust and reliable method for the detection of bacterial and fungal biofilms in CRS/NP.