Ototopical drops containing a novel antibacterial synthetic peptide: Safety and efficacy in adults with chronic suppurative otitis media.

OBJECTIVE: Chronic suppurative otitis media (CSOM) is a chronic infectious disease with worldwide prevalence that causes hearing loss and decreased quality of life. As current (antibiotic) treatments often unsuccessful and antibiotic resistance is emerging, alternative agents and/or strategies are urgently needed. We considered the synthetic antimicrobial and anti-biofilm peptide P60.4Ac to be an interesting candidate because it also displays anti-inflammatory activities including lipopolysaccharide-neutralizing activity. The aim of the present study was to investigate the safety and efficacy of ototopical drops containing P60.4Ac in adults with CSOM without cholesteatoma. METHODS: We conducted a range-finding study in 16 subjects followed by a randomized, double blinded, placebo-controlled, multicentre phase IIa study in 34 subjects. P60.4Ac-containing ototopical drops or placebo drops were applied twice a day for 2 weeks and adverse events (AEs) and medication use were recorded. Laboratory tests, swabs from the middle ear and throat for bacterial cultures, and audiometry were performed at intervals up to 10 weeks after therapy. Response to treatment was assessed by blinded symptom scoring on otoscopy. RESULTS: Application of P60.4Ac-containing ototopical drops (0.25-2.0 mg of peptide/ml) in the ear canal of patients suffering from CSOM was found to be safe and well-tolerated. The optimal dose (0.5 mg of peptide/ml) was selected for the subsequent phase IIa study. Safety evaluation revealed only a few AEs that were unlikely related to study treatment and all, except one, were of mild to moderate intensity. In addition to this excellent safety profile, P60.4Ac ototopical drops resulted in a treatment success in 47% of cases versus 6% in the placebo group. CONCLUSION: The efficacy/safety balance assessed in the present study provides a compelling justification for continued clinical development of P60.4Ac in therapy-resistant CSOM.

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms.

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.

An antimicrobial peptide modulates epithelial responses to bacterial products.

INTRODUCTION: Changes in the respiratory epithelium and chronic and recurrent infections are thought to play a central role in the pathogenesis of otitis media and sinusitis. The airway epithelium is the primary defense system of the respiratory tract. Bacterial cell membrane components like lipopolysaccharide (LPS) and lipoteichoic acid (LTA) can affect the mucociliary clearance function of the respiratory epithelium. P60.4-Ac is a synthetic antimicrobial peptide based on the structure of the cathelicidin LL-37 that neutralizes the pro-inflammatory activity of LPS and LTA. MATERIALS AND METHODS: Normal respiratory sinus epithelium was cultured at the air liquid interface. The cells were incubated with LPS or LTA in the presence or absence of P60.4-Ac. RESULTS: P60.4-Ac neutralized the LPS- and LTA- induced effect on air-liquid interface cultured epithelial cells. P60.4-Ac significantly inhibited the increase in the epithelial layer caused by LPS or LTA. CONCLUSION: These data demonstrate that P60.4-Ac might be of clinical benefit in the management of otitis media with effusion and sinusitis.

Transforming growth factor beta and wound healing in human cholesteatoma.

OBJECTIVE: Cholesteatoma is a nonmalignant, destructive lesion of the temporal bone that gradually expands and causes complications by the erosion of the adjacent bony structures. The consequences can be as severe as facial paralysis and intracranial complications. Until now, surgery has been the only treatment of choice. The pathogenesis of cholesteatoma remains controversial. Current concepts postulate that cholesteatoma may be a wound-healing process, although formal proof is lacking as of yet. Several reports provide evidence for the involvement of transforming growth factor (TGF)beta in both normal and abnormal wound healing. STUDY DESIGN: The expression of TGFbeta, the activated form of its intracellular effector, phosphorylated-Sma-Mad (pSmad)2, its natural inhibitor, Smad7, and target gene extra domain A-positive fibronectin (EDA-FN) were examined. METHODS: Quantitative immunohistochemical analysis was performed using an image analysis system. RESULTS: In 12 cholesteatoma and control samples, protein expressions showed consistent relationships among TGFbeta, nuclear pSmad2, and Smad7. We found concordant expressions of TGFbeta and nuclear pSmad2 in cholesteatoma epithelium and its control. Epithelial Smad7 expression was significantly reduced in cholesteatoma when compared with control epithelium (P = .04). In cholesteatoma extracellular matrix (ECM), a significantly increased TGFbeta, and nuclear pSmad2 was demonstrated (P < .01). Smad7 expression in the ECM was comparable in cholesteatoma and its control. EDA-FN deposition in cholesteatoma ECM was excessive, whereas EDA-FN expression was absent in controls. CONCLUSION: Our results confirm reports of in vitro experiments and support the concept that cholesteatoma behaves as a chronic wound healing process.

Characterization of mucosal biofilms on human adenoid tissues.

OBJECTIVES: To demonstrate the presence of mucosal biofilm in adenoid tissue using double staining for visualization of both the bacterial matrix and the bacterial cells. To identify bacterial species present on the surface of the studied adenoids. STUDY DESIGN: Prospective study. METHODS: A total of 39 specimens of adenoidectomy were removed from children with chronic and/or recurrent otitis media. The specimens were prepared for light microscopy using Gram staining, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Double staining was performed with CLSM to visualize both the bacteria and the glycocalyx matrix. Nine adenoids on which bacterial biofilms were visualized with CLSM were used for identification of bacterial species by 16S-DNA polymerase chain reaction (PCR) amplification and homology analysis. RESULTS: Of the 39 adenoids investigated, 22 (54%) showed evidence of mucosal biofilms. Gram staining, SEM and CLSM showed the presence of bacterial cells, organized in bacterial microcolonies. CLSM with double staining demonstrated mucosal biofilms by showing the presence of both bacteria and the glycocalyx. The use of 16S-DNA polymerase chain reaction (PCR) amplification and subsequent sequence analyses identified the presence of Corynebacterium argentoratense, Streptococcus salivarius, Micrococcus luteus, and Staphylococcus aureus. CONCLUSIONS: This study demonstrates that adenoid tissue in children with chronic or/and recurrent otitis media contains mucosal biofilms in 54% of the cases. The existence of living bacteria has been demonstrated. Further studies are required to describe the panel of bacteria that can be harbored in the biofilms present in adenoids and the mechanisms involved in the physiopathology of otitis prone children.

Survival signaling and terminal differentiation in cholesteatoma epithelium.

CONCLUSION: There is a strong indication that epithelial keratinocytes in cholesteatoma are protected against apoptosis. The late terminal differentiation program in cholesteatoma epithelium is disturbed. OBJECTIVES: Previously, minimal apoptosis has been demonstrated in cholesteatoma epithelium. The phosphoinositide 3-kinase/Akt/protein kinase B (PI3K/Akt/PKB) and the mitogen activated protein kinases (MAPK) signaling transduction pathways have been reported to protect epithelial cells against apoptosis. Both pathways have also been proven to regulate late terminal differentiation of keratinocytes. In cholesteatoma epithelium, MAPK activation has been shown to be associated with terminal differentiation. The purpose of this study was to investigate whether in human cholesteatoma epithelium protection against programmed cell death by means of PI3K/Akt survival signaling is present and associated with MAPK activation and terminal differentiation. MATERIALS AND METHODS: Fifteen human cholesteatoma and patient-matched retro-auricular skin samples were immunohistochemically stained for pAkt/PKB, phosphorylated extracellular regulated kinase1/2 (pERK1/2), phosphorylated JNK/SAPK, phosphorylated p38, involucrin and filaggrin. Positive cells were counted by computer-assisted digital image analysis. RESULTS: Protein expressions of pAkt/PKB, pERK1/2, pp38, and involucrin in cholesteatoma epithelium were significantly increased when compared with retro-auricular skin (p<0.01). Filaggrin expression was significantly decreased (p=0.03). The positive correlation was confirmed between both pERK1/2 and pp38, and involucrin (p < or = 0.05).

Demonstration of bacterial cells and glycocalyx in biofilms on human tonsils.

OBJECTIVES: To demonstrate mucosal biofilms in human tissue by direct visualization of bacteria and glycocalyx using confocal laser scanning microscopy with double fluorescent staining on tonsils and to compare the findings with the results of scanning electron microscopy analysis. DESIGN: Prospective study. SETTING: Tertiary university-based referral center. PATIENTS: Twenty-four tonsils were obtained from children with chronic or recurrent tonsillitis. INTERVENTIONS: Tonsils were prepared for analysis by scanning electronic microscopy and by confocal laser scanning microscopy. MAIN OUTCOME MEASURES: Double fluorescent staining for confocal laser scanning microscopy consisted of propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A staining to detect the glycocalyx matrix. Images were analyzed for characteristic biofilm morphologic features by 3 investigators who evaluated the images independently in a blinded retrospective manner. Consensus of all observers was required to demonstrate the presence of a biofilm in a specimen. RESULTS: Findings from analyses using scanning electronic microscopy suggested the presence of biofilm formations on tonsils by showing bacterial cells in microcolonies. Double-staining technique using confocal laser scanning microscopy showed bacterial cells and the glycocalyx matrix, providing visual evidence for the presence of biofilms on tonsils. CONCLUSION: Using a novel visualization approach in single sections of human mucosal tissue, the presence of biofilms was demonstrated on tonsils in most (17/24 [70.8%]) patients with tonsillitis.

Terminal differentiation and mitogen-activated protein kinase signaling in human cholesteatoma epithelium.

OBJECTIVES: To investigate whether--in cholesteatoma epithelium--terminal differentiation, resulting in high involucrin expression, is associated with mitogen-activated protein kinase (MAPK) signaling. BACKGROUND: Alterations in specific signal transduction pathways may explain abnormal differentiation of the keratinocytes in cholesteatoma. Signaling pathways used by eukaryotic cells to transduce extracellular signals into cellular responses converge on activated mitogen-activated protein kinases, mainly extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38. METHODS: Tissue samples were taken from 16 patients with acquired cholesteatoma. Histologic examination showed that 12 of the 16 cholesteatomas were inflamed. Immunohistochemical methods were used to determine expressions of involucrin and the activated form of p38, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase proteins. The results obtained from cholesteatoma tissue were compared with paired control samples from retroauricular skin. RESULTS: We demonstrated increased levels of involucrin and increased levels of the activated forms of p38 and ERK1/2 in cholesteatoma epithelium when compared with control samples. No abnormality was found in the activation and expression of JNK1/2. A positive correlation was found between p38, pERK1/2, and involucrin expression (p<0.05). CONCLUSION: Our results demonstrate that signaling via the mitogen-activated protein kinases ERK1/2 and p38 is increased in cholesteatoma epithelium when compared with control skin. The correlations between involucrin-and phosphorylated pERK1/2 expression and between involucrin-and phosphorylated p38 expression indicates that terminal differentiation in cholesteatoma epithelium proceeds via activation of these mitogen-activated protein kinase signaling pathways. We discussed whether this increased mitogen-activated protein kinase-driven terminal differentiation is probably part of a keratinocyte survival program or caused by an inflammation-induced cellular stress response.

Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application.

New peptides for lipopolysaccharide (LPS) and lipoteichoic acid (LTA) neutralization in upper respiratory tract infections were developed and evaluated in terms of efficacy and safety for application in humans. Based on the sequence of the human antimicrobial peptide LL-37 we developed and investigated length variants, substitution analogues and modifications to stabilize the peptides to prevent enzymatic degradation and to improve efficacy. The most promising peptide appears P60.4, a 24 amino acid peptide with similar efficacy as LL-37 in terms of LPS and LTA neutralization and lower pro-inflammatory activity. In addition, the acetylated and amidated version of this peptide shows no toxicity and displays higher or equal antimicrobial activity compared to LL-37.

Sustained extracellular signal-regulated kinase1/2 mitogen-activated protein kinase signalling is related to increased p21 expression in cholesteatoma epithelium.

CONCLUSION: These results show for the first time that the RAS/RAF/ERK1/2 MAPK signalling pathway is active and involved in p21-mediated cell cycle arrest in human cholesteatoma epithelium. OBJECTIVE: In a previous report we have demonstrated that the epithelium in human cholesteatoma is characterized by high p53-dependent p21 expression. The RAS/RAF/extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signalling pathway can induce p21 expression and subsequent cell cycle arrest via p53-dependent or -independent mechanisms. We designed the present study to investigate whether the RAS/RAF/ERK1/2 MAPK signalling pathway is involved in p53-dependent and p21-mediated cell cycle arrest in human cholesteatoma. MATERIAL AND METHODS: A total of 18 cholesteatoma samples and 18 paired control retro-auricular skin samples were immunohistochemically stained for p53, p21, phosphorylated ERK1/2 (pERK1/2) and total ERK1/2. Positive cells were counted by means of digital image analysis. Double-label fluorescence immunohistochemistry was performed to demonstrate co-expression of p21 and pERK1/2. RESULTS: Protein expression of p53, p21 and pERK1/2 differed significantly between cholesteatoma epithelium and retro-auricular skin (p <0.01). In cholesteatoma, co-expression of p21 and pERK1/2 was prominent, whereas in retro-auricular skin there was hardly any co-expression. Positive correlations were found between p53 and p21 (p =0.003) and between p21 and pERK1/2 (p =0.013).

Bacterial products increase expression of the human cathelicidin hCAP-18/LL-37 in cultured human sinus epithelial cells.

The respiratory epithelium plays a major role in the primary defense of the airways against infection. It has been demonstrated that bacterial products are involved in the induction of inflammatory reactions of the upper airways. Little is known about the effects of bacterial products on expression of the antimicrobial peptide hCAP-18/LL-37, the only human cathelicidin identified so far. The aim of this study was to investigate the effects of bacterial products from both gram-positive and gram-negative bacteria on the expression of hCAP-18/LL-37 by sinus epithelial cells using an air-exposed tissue culture model. Lipopolysaccharide and lipoteichoic acid both increased hCAP-18/LL-37 expression in cultured sinus epithelium as assessed by immunohistochemistry, where maximal stimulation occurred at 100 ng ml(-1) lipopolysaccharide or 10 microg ml(-1) lipoteichoic acid. The stimulatory effect of lipopolysaccharide and lipoteichoic acid was not restricted to expression of hCAP-18/LL-37, since also mucin expression and IL-8 release from cultured sinus epithelium cells were increased by lipopolysaccharide and lipoteichoic acid. This suggests that bacterial products may stimulate innate immunity in the upper airways.

Effects of bacterial toxins on air-exposed cultured human respiratory sinus epithelium.

The present study was designed to investigate the effects of the bacterial toxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on air-exposed cultured human respiratory sinus epithelium. The morphological changes, proliferation, and differentiation of sphenoid sinus mucosa were examined after incubation with different LPS or LTA concentrations. Air-exposed cultured sinus mucosa differentiated from pseudostratified respiratory epithelium to squamous ciliated epithelium with few goblet cells. High concentrations of bacterial toxins induced a significant increase in mucus production and a decrease in ciliated cells. Ki67 immunostaining showed an increased cell proliferation after incubation with moderate levels of LPS or LTA. High concentrations of bacterial toxins, on the other hand, induced a decreased proliferation. Involucrin expression was clearly altered by incubation with high levels of bacterial toxins, indicating an increased degree of terminal differentiation. These results indicate that the bacterial toxins LPS and LTA both induce comparable dose-dependent morphological changes in sinus epithelium.

Cholesteatoma epithelium is characterized by increased expression of Ki-67, p53 and p21, with minimal apoptosis.

OBJECTIVE: To investigate differences in cell proliferation, cell cycle arrest and apoptosis between cholesteatoma and control skin. MATERIAL AND METHODS: Immunohistochemical sections of 15 cholesteatoma and 15 paired control retro-auricular skin samples were examined for Ki-67, p53, p21 and active caspase 3, using image analysis, as well as for DNA fragmentation. RESULTS: The retro-auricular skin samples contained 5.7% +/- 3.6%, Ki-67-positive cells and showed a normal expression pattern. In the cholesteatoma epithelium 11.7% +/- 9.5% of the cells were Ki-67-positive and these cells were dominantly expressed in the basal and parabasal cell layers. Retro-auricular skin contained 5.8% +/- 5.4% p53-positive cells and 1.0% +/- 0.9%, p21-positive cells. In the cholesteatoma epithelium 17.8% +/- 12.3% of the cells were p53-positive and 14.3% +/- 11.6% were p21-positive The expression of Ki-67, p53 and p21 differed significantly between the two groups (p < 0.05). In the cholesteatoma epithelium a positive correlation was found between p53 and p21 expression (p = 0.016). Active caspase 3 positivity and DNA fragmentation were rarely seen in the cholesteatoma epithelium. CONCLUSION: Our results indicate that increased cell proliferation in cholesteatoma epithelium is accompanied by an increase in p53 and p21 protein levels, whilst apoptosis is minimal.

Air-exposed tissue culture of human middle ear epithelium and meatal epidermis: a method to study the advancing front of cholesteatoma.

The suitability of an air-exposed culture model consisting of a collagen matrix was investigated for constructing an advancing front (AF) of human middle ear epithelium (MEE) and meatal epidermis (ME). Three different culture settings were used: (i ) MEE; (ii) ME; and (iii) AF (MEE + ME). Small tissue biopsies were placed on a fibroblast-populated collagen matrix and grown at the air-liquid interface. After 3 weeks of culture, the MEE and ME outgrowth was differentiated. Light, scanning electron and transmission electron microscopy showed no visible differences compared to native MEE and ME. Cytokeratin 8 and cytokeratin 10 expressions were comparable to the expression seen in the native MEE and ME tissues. Proliferation, which was demonstrated by the expression of Ki-67, was present in the basal layers of cultured MEE and ME. A double layer of cells in which the ME covered the MEE formed the AF. In the AF, the MEE and ME showed the same morphological and immunohistochemical characteristics as in their native tissues. The results of the study show that this in vitro system is a well-defined model system offering the possibility to study the effects of external stimuli on the different epithelia of the AF involved in the pathogenesis of cholesteatoma.

Initial evaluation of the Clarion CII cochlear implant: speech perception and neural response imaging.

OBJECTIVE: To evaluate the new Clarion CII cochlear implant with the perimodiolar HiFocus electrode array, including both speech perception outcomes and the device's capabilities of measuring the electrically evoked compound action potential (eCAP) of the auditory nerve (Neural Response Imaging, NRI). DESIGN: The speech perception scores on CVC words without lip reading were monitored prospectively for the 10 postlingually deaf patients implanted with the Clarion CII device in the period July 2000 until May 2001 in the Leiden University Medical Center. Preoperative and postoperative NRI recordings were made, applying various combinations of monopolar stimulating and recording electrodes with the alternating polarity paradigm available in the test bench software. RESULTS: Nine patients preferred the CIS, one the PPS strategy, none the SAS strategy. With their favorite strategy they acquired significant open set speech understanding within a few weeks, resulting in an average CVC phoneme score of 84% (word score 66%) at the end of the study (follow-up 3 to 11 mo). In speech-shaped noise, the average phoneme recognition threshold (PRT) was reached at a signal to noise ratio just below 0 dB. The NRI recordings had clear N1 and P1 peaks if there was at least one contact between the stimulating and recording electrodes, necessitating just 15 sweeps for a reliable recording. We observed considerable inter-patient and inter-electrode variability, but for a given situation NRI input/output curves were stable over time. More apical contacts generally elicited larger eCAPs. Response amplitudes tended to peak at recording sites around apical and basal stimulating electrodes, suggesting a limited spread of excitation. Preliminary recordings with the forward masking paradigm were consistent with the ones with the alternating polarity scheme. CONCLUSIONS: The Clarion CII is a promising cochlear implant with which our first 10 patients have obtained excellent speech perception results. The NRI system yields high quality signals with a limited number of sweeps at a high sampling rate.