Edwin Tamashiro

Cigarette smoke condensate inhibits transepithelial chloride transport and ciliary beat frequency

Although the pathophysiology leading to rhinosinusitis is complex, evidence indicates that decreased mucociliary clearance (MCC) is a major contributing feature. Normal respiratory epithelial MCC is an important host defense mechanism that is dependent on proper ciliary beating and the biological properties of the airway surface liquid (ASL). The role that tobacco smoke exposure plays as an inhibitor of MCC has yet to be elucidated. The present study investigates the consequences of cigarette smoke exposure on ciliary function and transepithelial chloride (Cl−) secretion, a major determinant of ASL.

Cigarette smoke exposure impairs respiratory epithelial ciliogenesis.

Background Cigarette smoke exposure is considered an important negative prognostic factor for chronic rhinosinusitis (CRS) patients. However, there is no clear mechanistic evidence implicating cigarette smoke exposure in the poor clinical evolution of the disease or in the maintenance of the inflammatory state characterizing CRS. This study aimed to evaluate the effects of cigarette smoke exposure on respiratory cilia differentiation. Methods Mouse nasal septal epithelium cultures grown at an air-liquid interface were used as a model of respiratory epithelium. After 5 days of cell growth, cultures were exposed to air on the apical surface. Additionally, cigarette smoke condensate (CSC; the particulate phase of tobacco smoke) or cigarette smoke extract (CSE; the volatile phase) were diluted in the basolateral compartment in different concentrations. After 15 days of continuous exposure, scanning electron microscopy and immunofluorescence for type IV tubulin were used to determine presence and maturation of cilia. Transepithelial resistance was also recorded to evaluate confluence and physiological barrier integrity. Results CSC and CSE impair ciliogenesis in a dose-dependent manner with notable effects in concentrations higher than 30 μg/mL, yielding >70% nonciliation and shorter cilia compared with control. No statistical difference on transepithelial resistance was evident. Conclusion CSC and CSE exposure negatively impacts ciliogenesis of respiratory cells at concentrations not effecting transepithelial resistance. The impairment on ciliogenesis reduces the mucociliary clearance apparatus after injury and/or infection and may explain the poor response to therapy for CRS patients exposed to tobacco smoke.

Effects of an LL-37-derived antimicrobial peptide in an animal model of biofilm Pseudomonas sinusitis

Background LL-37, an innate immunity protein expressed within sinonasal mucosa, has in vitro antibacterial and antifungal properties as well as efficacy against preformed Pseudomonas aeruginosa biofilms. We hypothesize that a 24 amino acid peptide derivative of LL-37 will show efficacy against biofilm-forming P. aeruginosa in an established animal model of sinusitis. Methods Five groups of six New Zealand rabbits were each infected with P. aeruginosa (PAO-1) and fitted with irrigating catheters 7 days later. Each group was instilled with either one of three different concentrations of peptide, a positive control of topical tobramycin, or the carrier solution without the peptide once a day for 10 days. Nasal diluent was collected throughout the irrigation period to assess for persistence or resolution of infection by determining colony-forming units (CFU). At study end, sinus mucosa was harvested for histological assessment of inflammation and SEM evaluation for ciliary integrity and presence of biofilms. Results Topical tobramycin at 400× minimum inhibitory concentration and 2.5 mg/mL of peptide were effective in significantly lowering CFUs after 10 days of irrigation. Histological evaluation showed increased signs of inflammation in a dose-dependent manner within mucosa and bone of the groups receiving the peptide. SEM analysis showed ciliary loss in a dose-dependent manner. Biofilms were present in all groups except for the highest concentration of peptide and tobramycin. Conclusion High concentrations of LL-37-derived peptide showed in vivo ability to eradicate Pseudomonas biofilms and decrease bacterial counts. However, increasing concentrations of peptide showed proinflammatory and ciliotoxic effects on sinus mucosa.

An in vitro model of Pseudomonas aeruginosa biofilms on viable airway epithelial cell monolayers.

Background Chronic rhinosinusitis (CRS) that is refractory to medical or surgical intervention may involve a particularly resistant form of infection known as a bacterial biofilm. Bacterial biofilms are three-dimensional aggregates of bacteria that often are recalcitrant to antibiotics secondary to physical barrier characteristics. To date, all studies investigating biofilms in CRS have been descriptive in either human or animal tissue. To better understand the interactions of bacterial biofilms with respiratory epithelium, we describe an in vitro model of biofilm sinusitis by establishing mature biofilms on airway epithelial air-liquid interface cultures. Methods Airway epithelial cell cultures were grown on collagen-coated semipermeable support membranes as an air-liquid interface on tissue culture inserts. Confluent air-liquid interface cultures were inoculated with the biofilm-forming PAO-1 strain of Pseudomonas aeruginosa and compared with cultures inoculated with two mutant strains (sad-31 and sad-36) unable to form biofilms. Inoculated tissue transwells were incubated for 20 hours, allowing for biofilm growth. The semipermeable membranes were then harvested and imaged with confocal laser scanning microscopy and scanning electron microscopy. Results Microscopic analysis revealed the formation of biofilm-forming towers in the PAO-1 inoculated wells. The bacterial biofilms were supported by a viable airway epithelial cell surface monolayer. Conclusion This study shows a reliable method for analysis of in vitro interactions of bacterial biofilms and airway epithelium. The experimental manipulation of this air-liquid interface model will help explore novel treatment approaches for bacterial biofilm-associated CRS.

High rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease.

Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases.

Prognosis of acute invasive fungal rhinosinusitis related to underlying disease.

BACKGROUND Acute invasive fungal rhinosinusitis (AIFRS) is a rare disease with high morbidity and mortality rates. The objective of this study was to correlate the initial clinical features of AIFRS to the prognosis after surgery. METHODS Thirty-two patients with AIFRS were evaluated retrospectively. The correlation of underlying disease, fungus isolated, and extent of the disease to the clinical outcome of AIFRS was also evaluated. RESULTS The most common underlying disease was hematological malignancy and aplasia (n=20). Aspergillus (n=13) and Mucoraceae (n=11) were the main fungi found in AIFRS. Mucosal biopsy confirmed fungal invasion to the nasal mucosa in all cases. Computed tomography and endoscopic findings showed a predominance of unilateral disease, with various stages of nasal involvement. All patients underwent surgical debridement and systemic antifungal therapy immediately after diagnosis. Sixteen patients died (50%) due to AIFRS. A poor prognosis was related to the extensiveness of AIFRS and to the underlying disease (patients with aplastic anemia and diabetes had the worst outcomes), but not to the fungus isolated. CONCLUSIONS Early medical and surgical treatment is essential to improve the prognosis of AIFRS patients. A poorer prognosis was associated with underlying disease and extensiveness of AIFRS, but not to the fungus isolated.

Peptidoglycan from the gut microbiota governs the lifespan of circulating phagocytes at homeostasis.

Maintenance of myeloid cell homeostasis requires continuous turnover of phagocytes from the bloodstream, yet whether environmental signals influence phagocyte longevity in the absence of inflammation remains unknown. Here, we show that the gut microbiota regulates the steady-state cellular lifespan of neutrophils and inflammatory monocytes, the 2 most abundant circulating myeloid cells and key contributors to inflammatory responses. Treatment of mice with broad-spectrum antibiotics, or with the gut-restricted aminoglycoside neomycin alone, accelerated phagocyte turnover and increased the rates of their spontaneous apoptosis. Metagenomic analyses revealed that neomycin altered the abundance of intestinal bacteria bearing γ-d-glutamyl-meso-diaminopimelic acid, a ligand for the intracellular peptidoglycan sensor Nod1. Accordingly, signaling through Nod1 was both necessary and sufficient to mediate the stimulatory influence of the flora on myeloid cell longevity. Stimulation of Nod1 signaling increased the frequency of lymphocytes in the murine intestine producing the proinflammatory cytokine interleukin 17A (IL-17A), and liberation of IL-17A was required for transmission of Nod1-dependent signals to circulating phagocytes. Together, these results define a mechanism through which intestinal microbes govern a central component of myeloid homeostasis and suggest perturbations of commensal communities can influence steady-state regulation of cell fate.

In vivo effects of citric acid/zwitterionic surfactant cleansing solution on rabbit sinus mucosa.

Background Chronic rhinosinusitis that is refractory to medical or surgical intervention may involve a particularly resistant form of infection known as a bacterial biofilm that is recalcitrant to antibiotics secondary to physical barrier characteristics. Recently, a novel sinus cleansing solution, citric acid/zwitterionic surfactant (CAZS) was shown to be extremely effective in disrupting biofilms in vitro. The purpose of this study was to determine the effects of CAZS on sinonasal epithelium in vivo compared with normal saline. Methods Indwelling catheters were placed into the right maxillary sinus of New Zealand white rabbits. CAZS solution or normal saline (10 mL) was instilled at a rate of 20 mL/minute into the sinus followed by aspiration. Rabbits were killed 1, 3, and 6 days after treatment. Mucosa from both maxillary sinuses was harvested and evaluated for physiological activity (ciliary beating) as well as morphological integrity of the epithelium by scanning electron microscopy. Results One day after treatment, beating cilia was evident with morphological analysis shown intact epithelium with 80–85% denudation of cilia compared with saline. Three days after treatment, ciliary activity was again noted with morphological evidence of persistent denuded cilia. By day 6 after treatment, the epithelium had regenerated cilia over the apical surface. Throughout the recovery period beating cilia was evident in CAZS-treated sinuses. Conclusion This study shows that although CAZS acutely denudes respiratory cilia, the remaining cilia are active. Additionally, the epithelial barrier appears intact with active ciliogenesis, and reciliation of the mucosal surface occurring 6 days after treatment.

Involvement of the parabrachial nucleus in the pressor response to chemoreflex activation in awake rats

Activation of the chemoreflex with potassium cyanide (KCN, 40 microg/rat, i.v.) in awake rats produces pressor and bradycardic responses as well as a tachypneic response. In the present study, we evaluated the involvement of the periaqueductal gray matter (PAG) and the parabrachial nucleus (PBN) in the neural pathways of the cardiovascular responses to chemoreflex activation. The cardiovascular responses to chemoreflex activation were evaluated before and after bilateral microinjection of 2% lidocaine, a local anesthetic, into the PBN or PAG in order to block in a reversible manner the neuronal activity and axonal conduction of fibers of passage in these areas. The data show that the pressor response to chemoreflex activation 3 min after bilateral microinjection of lidocaine into the dorsolateral aspect of the PBN was significantly reduced in comparison to the control response (32 +/- 5 vs. 48 +/- 4 mm Hg, n = 7), with no significant changes in the bradycardic responses. The effect of lidocaine was reversible since the pressor response was back to control levels 15 min after microinjection of this anesthetic. Bilateral microinjections of lidocaine into the dorsolateral (n = 11) or lateral (n = 8) columns of the PAG in distinct groups of rats produced no significant changes in the pressor or bradycardic responses of the chemoreflex. These data indicate that the PBN is part of the neuronal pathways involved in the sympathoexcitatory component of the chemoreflex while the PAG is not.

Implications of bacterial biofilms in chronic rhinosinusitis

The recognition of sessile form of bacteria with particular features, known as biofilm, has given new insights to the understanding of pathogenesis of several chronic diseases, including Chronic Rhinosinusitis (CRS). In this article we review the main characteristics of biofilms, describe the current methods used to demonstrate biofilms in chronic rhinosinusitis and discuss the future directions of research in the field.