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

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

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

Ex Vivo Expansion of Human CD8+ T Cells Using Autologous CD4+ T Cell Help

Background: Using in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model. Methods/Principal Findings: We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells. Conclusions: We have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro

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 lifethreatening 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 asthmarelated 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 asthmarelated 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&lt;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.

Treatment response with mepolizumab in severe eosinophilic asthma patients with previous omalizumab treatment

International audienc

Mucin Production in the Middle Ear in Response to Lipopolysaccharides

OBJECTIVE: This study examined the response of middle ear tissue to establish the lowest dose of lipopolysaccharide to induce mucin production in a rat otitis media model. METHODS: Twenty-six male Sprague-Dawley rats' eustachian tubes were obstructed before transtympanic inoculation of the bulla tympanica with 35 μL of Krebs Ringer or 1, 10, 100, or 1000 μg/mL lipopolysaccharide. After 7 days the effusion and a lavage were collected for mucin ELISA measurement, and tissue was collected for histologic evaluation. RESULTS: Mucin secretion was significantly increased in the 100 μg/mL 51.20 ± 13.6 μg/mL (SE) and 1000 μg/mL 69.42 ± 8.57 μg/mL groups when compared with the Krebs Ringer control group 1.84 ± 0.28 μg/mL ( P < 0.05). Histologic evaluation shows goblet cell metaplasia and hyperplasia in the middle ear epithelium in the 1000 and 100 μg/mL groups. CONCLUSIONS: The histology and ELISA results suggest that a middle ear effusion is generated with a dose of lipopolysaccharide as low as 100 μg/mL

Oral corticosteroid dose changes and impact on peripheral blood eosinophil counts in patients with severe eosinophilic asthma: A post hoc analysis

Background: An inverse relationship between oral corticosteroid (OCS) dose and peripheral blood eosinophil (PBE) count is widely recognized in patients with severe eosinophilic asthma; however, there are limited data available to quantify this relationship. This post hoc analysis of the SIRIUS study (NCT01691508) examined the impact of weekly incremental OCS dose reductions on PBE counts during the 3-8-week optimization phase of the study. Methods: SIRIUS was a randomized, double-blind study involving patients with severe asthma (≥12 years old), which included an initial OCS dose optimization phase prior to randomization. Regression analysis assuming a linear relationship between change in OCS dose and change in log (PBE count) during the optimization phase was used to estimate the changes in PBE count following specific decreases in OCS dose. Results: All 135 patients from the SIRIUS intent-to-treat population were included in this analysis. During the optimization period, 44% (60/135) of patients reduced their OCS dose, with an increase in geometric mean PBE count of 110 cells/μL (200 to 310 cells/μL; geometric mean ratio from beginning to end of the optimization phase: 1.52) recorded in these patients. The model estimated that reduction of daily OCS dose by 5 mg/day led to a 41% increase in PBE count (mean ratio to beginning of optimization phase: 1.41 [95% confidence interval (CI); 1.22, 1.63]). Conclusion: These data confirmed and quantified the inverse association between OCS dose and PBE count. These insights will help to inform clinicians when tapering OCS doses in patients with severe eosinophilic asthma