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.

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.

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.

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.

High numbers of tumour-infiltrating activated cytotoxic T lymphocytes, and frequent loss of HLA class I and II expression, are features of aggressive B cell lymphomas of the brain and testis.

Loss of both HLA class I and class II expression in B cell lymphomas is a mechanism of escape from a cytotoxic T lymphocyte (CTL) immune response and will therefore give a strong selective survival advantage in tumours expressing strong immunogenic antigens. We investigated loss of HLA expression using specific antibodies on tissue sections from 254 B cell lymphomas originating from nodal and different extranodal sites in relation to numbers of tumour-infiltrating T cells. Complete loss of HLA class I and II was observed in a minority of the nodal, stomach, and skin lymphomas but in the majority of the lymphomas originating from the testis and the CNS. Interestingly, relatively high percentages of activated CTLs were detected in both primary testicular and CNS lymphomas compared to lymphomas at other sites, with highest percentages in the testis (p < 0.0001). We conclude that loss of both HLA class I and II expression occurs very frequently in lymphomas originating from the testis and the CNS as compared to nodal and some other extranodal sites. The presence of high percentages of activated CTLs in the testicular and CNS lymphomas suggests that loss of HLA expression provides a strong growth advantage for lymphoma cells in these immune-privileged sites.

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.

Flow cytometric sorting of paraffin-embedded tumor tissues considerably improves molecular genetic analysis.

The characterization of genetic aberrations in paraffin-embedded tumor material is impaired by contaminating normal cells. In the present study on the genetic causes of loss of HLA expression in diffuse large B-cell lymphoma (DLBCL), we compared the efficacy of microdissection with flow cytometric sorting of tumor cells. Single-cell suspensions from paraffin-embedded material of 5 DLBCL cases were stained for CD79a and DNA content (propidium iodide). Fluorescent in situ hybridization (FISH) using HLA class II and chromosome 6 centromeric probes and loss of heterozygosity (LOH) analysis with 5 HLA-specific microsatellite markers were performed on microdissected and flow cytometry-sorted fractions. FISH confirmed considerable enrichment of the samples after flow cytometric sorting and disclosed tumor heterogeneity in 4 cases. Moreover, lymphomas with a so-called zebra LOH pattern in the microdissected material showed unambiguous LOH after flow cytometric sorting, revealing in 1 case a biologically relevant hemizygous deletion in the HLA region.