Modeling the Measurements of Cochlear Microcirculation and Hearing Function after Loud Noise

Objective: Recent findings support the crucial role of microcirculatory disturbance and ischemia for hearing impairment especially after noise-induced hearing loss (NIHL). The aim of this study was to establish an animal model for in vivo analysis of cochlear microcirculation and hearing function after a loud noise to allow precise measurements of both parameters in vivo. Study Design: Randomized controlled trial. Setting: Animal study. Subjects and Methods: After assessment of normacusis (0 minutes) using evoked auditory brainstem responses (ABRs), noise (106-dB sound pressure level [SPL]) was applied to both ears in 6 guinea pigs for 30 minutes while unexposed animals served as controls. In vivo fluorescence microscopy of the stria vascularis capillaries was performed after surgical exposure of 1 cochlea. ABR measurements were derived from the contralateral ear. Results: After noise exposure, red blood cell velocity was reduced significantly by 24.3% (120 minutes) and further decreased to 44.5% at the end of the observation (210 minutes) in contrast to stable control measurements. Vessel diameters were not affected in both groups. A gradual decrease of segmental blood flow became significant (38.1%) after 150 minutes compared with controls. Hearing thresholds shifted significantly from 20.0 ± 5.5 dB SPL (0 minutes) to 32.5 ± 4.2dB SPL (60 minutes) only in animals exposed to loud noise. Conclusion: With regard to novel treatments targeting the stria vascularis in NIHL, this standardized model allows us to analyze in detail cochlear microcirculation and hearing function in vivo

Tracheal vascular response to hypertonic and hypotonic solutions

One of the major roles of the upper respiratory mucosa is to humidify inspired air. This function requires the coordinated activity of respiratory epithelium and mucosal vasculature. It has been difficult to study this relationship in vivo. In the present study, we investigated the effects of osmolarity on the vessel diameter of mucosal vessels via a specially constructed chamber that allows direct visualization of the rat trachea microvasculature. With use of an anesthetized and instrumented rat preparation, the luminal surface of the dorsal wall of the trachea was superfused with physiological solutions at 37°C. The osmolarities were varied by removing or adding NACl or mannitol (200, 290, and 500 mosM). The mucosal vessels dilated when the airway surface was superfused with hypertonic solutions and constricted when superfused with hypotonic solution. The largest changes occurred in the arterioles (51 ± 5.6 μm diam), which constricted by 10 ± 2.18 μm (P = 0.0001) when exposed to a 200 mosM solution and dilated by 11 ± 1.55 μm (P = 0.0001) when exposed to a 500 mosM NaCl-enriched solution. Smaller changes of similar pattern were seen in venules. The changes in vessel diameter were readily reversible upon replacement of the hypo- or hypertonic solutions by an isotonic solution. We conclude that increase or decrease of solution osmolarity on the epithelial surface of the trachea can regulate diameter of mucosal blood vessels

Inhaled steroids with and without regular salmeterol for asthma: serious adverse events.

BACKGROUND: Epidemiological evidence has suggested a link between use of beta₂-agonists and increased asthma mortality. Much debate has surrounded possible causal links for this association, and whether regular (daily) long-acting beta₂-agonists (LABAs) are safe, particularly when used in combination with inhaled corticosteroids (ICSs). This is an update of a Cochrane Review that now includes data from two large trials including 11,679 adults and 6208 children; both were mandated by the US Food and Drug Administration (FDA).  OBJECTIVES: To assess risks of mortality and non-fatal serious adverse events (SAEs) in trials that randomised participants with chronic asthma to regular salmeterol and ICS versus the same dose of ICS. SEARCH METHODS: We identified randomised trials using the Cochrane Airways Group Specialised Register of trials. We checked websites of clinical trials registers for unpublished trial data. We also checked FDA submissions in relation to salmeterol. The date of the most recent search was 10 October 2018. SELECTION CRITERIA: We included parallel-design randomised trials involving adults, children, or both with asthma of any severity who were randomised to treatment with regular salmeterol and ICS (in separate or combined inhalers) versus the same dose of ICS of at least 12 weeks in duration. DATA COLLECTION AND ANALYSIS: We conducted the review according to standard procedures expected by Cochrane. We obtained unpublished data on mortality and SAEs from the sponsors, from ClinicalTrials.gov, and from FDA submissions. We assessed our confidence in the evidence according to current GRADE recommendations. MAIN RESULTS: We have included in this review 41 studies (27,951 participants) in adults and adolescents, along with eight studies (8453 participants) in children. We judged that the overall risk of bias was low for all-cause events, and we obtained data on SAEs from all study authors. All except 542 adults (and none of the children) were given salmeterol and fluticasone in the same (combination) inhaler.DeathsEleven of a total of 14,233 adults taking regular salmeterol and ICS died, as did 13 of 13,718 taking regular ICS at the same dose. The pooled Peto odds ratio (OR) was 0.80 (95% confidence interval (CI) 0.36 to 1.78; participants = 27,951; studies = 41; I² = 0%; moderate-certainty evidence). In other words, for every 1000 adults treated for 25 weeks, one death occurred among those on ICS alone, and the corresponding risk among those taking salmeterol and ICS was also one death (95% CI 0 to 2 deaths).No children died, and no adults or children died of asthma, so we remain uncertain about mortality in children and about asthma mortality in any age group.Non-fatal serious adverse eventsA total of 332 adults receiving regular salmeterol with ICS experienced a non-fatal SAE of any cause, compared to 282 adults receiving regular ICS. The pooled Peto OR was 1.14 (95% CI 0.97 to 1.33; participants = 27,951; studies = 41; I² = 0%; moderate-certainty evidence). For every 1000 adults treated for 25 weeks, 21 adults on ICS alone had an SAE, and the corresponding risk for those on salmeterol and ICS was 23 adults (95% CI 20 to 27).Sixty-five of 4229 children given regular salmeterol with ICS suffered an SAE of any cause, compared to 62 of 4224 children given regular ICS. The pooled Peto OR was 1.04 (95% CI 0.73 to 1.48; participants = 8453; studies = 8; I² = 0%; moderate-certainty evidence). For every 1000 children treated for 23 weeks, 15 children on ICS alone had an SAE, and the corresponding risk for those on salmeterol and ICS was 15 children (95% CI 11 to 22).Asthma-related serious adverse eventsEighty and 67 adults in each group, respectively, experienced an asthma-related non-fatal SAE. The pooled Peto OR was 1.15 (95% CI 0.83 to 1.59; participants = 27,951; studies = 41; I² = 0%; low-certainty evidence). For every 1000 adults treated for 25 weeks, five receiving ICS alone had an asthma-related SAE, and the corresponding risk among those on salmeterol and ICS was six adults (95% CI 4 to 8).Twenty-nine children taking salmeterol and ICS and 23 children taking ICS alone reported asthma-related events. The pooled Peto OR was 1.25 (95% CI 0.72 to 2.16; participants = 8453; studies = 8; I² = 0%; moderate-certainty evidence). For every 1000 children treated for 23 weeks, five receiving an ICS alone had an asthma-related SAE, and the corresponding risk among those receiving salmeterol and ICS was seven children (95% CI 4 to 12). AUTHORS' CONCLUSIONS: We did not find a difference in the risk of death or serious adverse events in either adults or children. However, trial authors reported no asthma deaths among 27,951 adults or 8453 children randomised to regular salmeterol and ICS or ICS alone over an average of six months. Therefore, the risk of dying from asthma on either treatment was very low, but we remain uncertain about whether the risk of dying from asthma is altered by adding salmeterol to ICS.Inclusion of new trials has increased the precision of the estimates for non-fatal SAEs of any cause. We can now say that the worst-case estimate is that at least 152 adults and 139 children must be treated with combination salmeterol and ICS for six months for one additional person to be admitted to the hospital (compared to treatment with ICS alone). These possible risks still have to be weighed against the benefits experienced by people who take combination treatment.However more than 90% of prescribed treatment was taken in the new trials, so the effects observed may be different from those seen with salmeterol in combination with ICS in daily practice

Serious asthma events with fluticasone plus salmeterol versus fluticasone alone

Background: the safe and appropriate use of long-acting beta-agonists (LABAs) for the treatment of asthma has been widely debated. In two large clinical trials, investigators found a potential risk of serious asthma-related events associated with LABAs. This study was designed to evaluate the risk of administering the LABA salmeterol in combination with an inhaled glucocorticoid, fluticasone propionate. Methods: in this multicenter, randomized, double-blind trial, adolescent and adult patients (age, ≥12 years) with persistent asthma were assigned to receive either fluticasone with salmeterol or fluticasone alone for 26 weeks. All the patients had a history of a severe asthma exacerbation in the year before randomization but not during the previous month. Patients were excluded from the trial if they had a history of life-threatening or unstable asthma. The primary safety end point was the first serious asthma-related event (death, endotracheal intubation, or hospitalization). Noninferiority of fluticasone-salmeterol to fluticasone alone was defined as an upper boundary of the 95% confidence interval for the risk of the primary safety end point of less than 2.0. The efficacy end point was the first severe asthma exacerbation. Results: of 11,679 patients who were enrolled, 67 had 74 serious asthma-related events, with 36 events in 34 patients in the fluticasone-salmeterol group and 38 events in 33 patients in the fluticasone-only group. The hazard ratio for a serious asthma-related event in the fluticasone-salmeterol group was 1.03 (95% confidence interval [CI], 0.64 to 1.66), and noninferiority was achieved (P=0.003). There were no asthma-related deaths; 2 patients in the fluticasone-only group underwent asthma-related intubation. The risk of a severe asthma exacerbation was 21% lower in the fluticasone-salmeterol group than in the fluticasone-only group (hazard ratio, 0.79; 95% CI, 0.70 to 0.89), with at least one severe asthma exacerbation occurring in 480 of 5834 patients (8%) in the fluticasone-salmeterol group, as compared with 597 of 5845 patients (10%) in the fluticasone-only group (P<0.001). Conclusions: patients who received salmeterol in a fixed-dose combination with fluticasone did not have a significantly higher risk of serious asthma-related events than did those who received fluticasone alone. Patients receiving fluticasone-salmeterol had fewer severe asthma exacerbations than did those in the fluticasone-only group. (AUSTRI ClinicalTrials.gov number, NCT01475721)

Visualizing B cell capture of cognate antigen from follicular dendritic cells

The prominent display of opsonized antigen by follicular dendritic cells (FDCs) has long favored the view that they serve as antigen-presenting cells for B cells. Surprisingly, however, although B cell capture of antigen from macrophages and dendritic cells has been visualized, acquisition from FDCs has not been directly observed. Using two-photon microscopy, we visualized B cell capture of cognate antigen from FDCs. B cell CXCR5 expression was required, and encounter with FDC-associated antigen could be detected for >1 wk after immunization. B cell–FDC contact times were often brief but occasionally persisted for >30 min, and B cells sometimes acquired antigen together with FDC surface proteins. These observations establish that FDCs can serve as sites of B cell antigen capture, with their prolonged display time ensuring that even rare B cells have the chance of antigen encounter, and they suggest possible information transfer from antigen-presenting cell to B cell

CD83 increases MHC II and CD86 on dendritic cells by opposing IL-10–driven MARCH1-mediated ubiquitination and degradation

By opposing IL-10–driven, MARCH1-mediated ubiquitination and degradation of MHC class II, CD83 may boost the immunogenicity of dendritic cells

In vivo confocal microscopy and histopathology of the conjunctiva in trachomatous scarring and normal tissue: a systematic comparison.

AIM: To compare in vivo confocal microscopy (IVCM) with the histopathological examination of tissue and cellular changes in normal and diseased conjunctiva. METHODS: Participants underwent clinical examination and IVCM of the tarsal conjunctiva. A biopsy of the upper tarsal conjunctiva was collected and stained with tinctorial stains and by immunohistochemical staining for CD45 and CD83. Connective tissue scarring, inflammatory cell density and the presence of dendritiform cells were quantitatively assessed in a masked manner by both IVCM and histological assessments for comparative analysis. RESULTS: Thirty-four participants with severe trachomatous conjunctival scarring and 33 participants with healthy conjunctiva were recruited. The IVCM connective tissue scarring score was strongly associated with the histological grading of scarring (p<0.001). There was limited evidence of an association between the IVCM inflammatory cell infiltrate and the histological inflammatory cell grade (p=0.05). We did not find any evidence to support the hypothesis that dendritiform cells seen with IVCM are mature, conventional dendritic cells. CONCLUSIONS: The results show that IVCM can be used to robustly quantitate connective tissue scarring and also has a role in measuring the inflammatory cell infiltrate. The discordance between IVCM dendritiform cells and immunohistochemical dendritic cells may be a result of study limitations or may be because these dendritiform structures represent another cell type, such as fibroblasts, rather than dendritic cells

Gender Differences in Myogenic Regulation along the Vascular Tree of the Gerbil Cochlea

Regulation of cochlear blood flow is critical for hearing due to its exquisite sensitivity to ischemia and oxidative stress. Many forms of hearing loss such as sensorineural hearing loss and presbyacusis may involve or be aggravated by blood flow disorders. Animal experiments and clinical outcomes further suggest that there is a gender preference in hearing loss, with males being more susceptible. Autoregulation of cochlear blood flow has been demonstrated in some animal models in vivo, suggesting that similar to the brain, blood vessels supplying the cochlea have the ability to control flow within normal limits, despite variations in systemic blood pressure. Here, we investigated myogenic regulation in the cochlear blood supply of the Mongolian gerbil, a widely used animal model in hearing research. The cochlear blood supply originates at the basilar artery, followed by the anterior inferior cerebellar artery, and inside the inner ear, by the spiral modiolar artery and the radiating arterioles that supply the capillary beds of the spiral ligament and stria vascularis. Arteries from male and female gerbils were isolated and pressurized using a concentric pipette system. Diameter changes in response to increasing luminal pressures were recorded by laser scanning microscopy. Our results show that cochlear vessels from male and female gerbils exhibit myogenic regulation but with important differences. Whereas in male gerbils, both spiral modiolar arteries and radiating arterioles exhibited pressure-dependent tone, in females, only radiating arterioles had this property. Male spiral modiolar arteries responded more to L-NNA than female spiral modiolar arteries, suggesting that NO-dependent mechanisms play a bigger role in the myogenic regulation of male than female gerbil cochlear vessels

Functional Characterization of the HuR:CD83 mRNA Interaction

Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1–3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein∶RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway