Signaling cascades associated with apoptosis contribute to cell death after focal cerebral ischemia. Cytochrome c release from mitochondria and the subsequent activation of caspases 9 and 3 are critical steps. Recently, a novel mitochondrial protein, apoptosis-inducing factor (AIF), has been implicated in caspase-independent programmed cell death following its translocation to the nucleus. We, therefore, addressed the question whether AIF also plays a role in cell death after focal cerebral ischemia. We detected AIF relocation from mitochondria to nucleus in primary cultured rat neurons 4 and 8 hours after 4 hours of oxygen/glucose deprivation. In ischemic mouse brain, AIF was detected within the nucleus 1 hour after reperfusion after 45 minutes occlusion of the middle cerebral artery. AIF translocation preceded cell death, occurred before or at the time when cytochrome c was released from mitochondria, and was evident within cells showing apoptosis-related DNA fragmentation. From these findings, we infer that AIF may be involved in neuronal cell death after focal cerebral ischemia and that caspase-independent signaling pathways downstream of mitochondria may play a role in apoptotic-like cell death after experimental stroke.

Pharmacological studies using bradykinin B 2 receptor antagonists suggest that bradykinin, an early mediator of inflammation and the main metabolite of the kallikrein-kinin system, is involved in secondary brain damage after cerebral ischemia. However, the time-course of bradykinin production and kinin receptor expression as well as the conclusive role of bradykinin B 2 receptors for brain damage after experimental stroke have not been elucidated so far. C57/Bl6 mice were subjected to 45 mins of middle cerebral artery occlusion (MCAO) and 2, 4, 8, 24, and 48 h later brains were removed for the analysis of tissue bradykinin concentration and kinin B 2 receptor mRNA and protein expression. Brain edema, infarct volume, functional outcome, and long-term survival were assessed in WT and B 2 À/À mice 24 h or 7 days after MCAO. Tissue bradykinin was maximally increased 12 h after ischemia (three-fold), while kinin B 2 receptor mRNA upregulation peaked 24 to 48 h after MCAO (10-to 12-fold versus naïve brain tissue). Immunohistochemistry revealed that kinin B 2 receptors were constitutively and widely expressed in mouse brain, were upregulated 2 h after ischemia in cells showing signs of ischemic damage, and remained upregulated in the penumbra up to 24 h after ischemia. B 2 À/À mice had improved motor function (Po0.05), smaller infarct volumes (À38%; Po0.01), developed less brain edema (À87%; Po0.05), and survived longer (Po0.01) as compared with wild-type controls. The current results show that bradykinin is produced in the brain, kinin B 2 receptors are upregulated on dying cells, and B 2 receptors are involved in cell death and brain edema formation after experimental stroke.

BackgroundNew therapeutic approaches with biologic agents such as anti-cytokine antibodies are currently on trial for the treatment of asthma, rhinosinusitis or allergic diseases necessitating patient selection by biomarkers. Allergic rhinitis (AR), affecting about 20 % of the Canadian population, is an inflammatory disease characterised by a disequilibrium of T-lymphocytes and tissue eosinophilia. Aim of the present study was to describe distinct cytokine patterns in nasal secretion between seasonal and perennial AR (SAR/PAR), and healthy controls by comparing cytokines regulating T-cells or stimulating inflammatory cells, and chemokines.MethodsNasal secretions of 44 participants suffering from SAR, 45 participants with PAR and 48 healthy controls were gained using the cotton wool method, and analysed for IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, GM-CSF, G-CSF, IFN-γ, MCP-1, MIP-1α, MIP-1β, eotaxin, and RANTES by Bio-Plex Cytokine Assay as well as for ECP and tryptase by UniCAP-FEIA.ResultsParticipants with SAR or PAR presented elevated levels of tryptase, ECP, MCP-1, and MIP-1β, while values of GM-CSF, G-CSF, IL-1β, and IL-6 did not differ from the controls. Increased levels of IL-5, eotaxin, MIP-1α, and IL-17 and decreased levels of IFN-γ, IL-12 and IL-10 were found in SAR only. RANTES was elevated in SAR in comparison to PAR. Interestingly, we found reduced levels of IL-4 in PAR and of IL-13 in SAR.ConclusionsElevated levels of proinflammatory cytokines were seen in both disease entities. They were, however, more pronounced in SAR, indicating a higher degree of inflammation. This study suggests a downregulation of TH1 and Treg-lymphocytes and an upregulation of TH17 in SAR. Moreover, the results display a prominent role of eosinophils and mast cells in AR. The observed distinct cytokine profiles in nasal secretion may prove useful as a diagnostic tool helping to match patients to antibody therapies.

Non-allergic rhinitis with eosinophilia syndrome (NARES) is an eosinophilic inflammation of the nasal mucosa without evidence of an allergy or other nasal pathologies. Patients complain about perennial symptoms like nasal obstruction, rhinorrhea, itchiness and sneezing of the nose sometimes accompanied by hyposmia. The aim of the study was to better characterize NARES patients using immunoassay-biochip technology to examine serum and nasal secretion. Sera and nasal secretion of patients with NARES (perennial nasal symptoms, no evidence of acute or chronic rhinosinusitis with or without polyps, negative SX1-Screening test and/or negative skin prick test, eosinophilic cationic protein in nasal secretion >200 ng/ml) were tested by immunoassay-biochip technology (ImmunoCAP(®) ISAC, Phadia). 112 different allergen components from 51 allergen sources were tested on the chip. Furthermore, serum and nasal secretion were tested for specific IgE to Staphylococcus aureus enterotoxin TSST-1 by fluorescence-enzyme-immunoassay (UniCAP(®), Phadia). Unrecognized systemic sensitization could be ruled out by negative ISAC results in sera of all patients. Testing of nasal secretion for allergen-specific IgE by ISAC chip technology was negative as well in all cases. In one patient, a systemic sensitization to Staphylococcus aureus superantigen TSST-1 was detectable but no allergen-specific IgE to TSST-1 was measurable in nasal secretion of any patient. The results demonstrate that NARES is not associated with local allergy (entopy) nor with a local inflammation driven by Staphylococcus aureus enterotoxin TSST-1. Further studies are necessary to better understand the underlying mechanisms of NARES.

Background:Dermatophagoides pteronyssinus is one of the most important perennial allergen sources worldwide. Molecular diagnostics using the commercially available major allergens (Der p 1 and Der p 2) in combination with Der p 10 do not detect house dust mite (HDM) sensitization in a number of cases when used alone. The objective was to evaluate the IgE reactivity profiles of these patients using an experimental immunoassay biochip. Methods: Sera of HDM-allergic patients (positive skin prick test, CAP class ≥1 for allergen extract, and positive intranasal provocation) were tested for IgE antibodies against Der p 1, Der p 2, and Der p 10 by ImmunoCAP fluorescence enzyme immunoassay. Negatively tested sera were examined by an experimental chip containing 13 microarrayed HDM allergens. Results: Of 97 patients tested, 16 showed negative results to Der p 1, Der p 2, and Der p 10. MeDALL chip evaluation revealed 5 patients monosensitized to Der p 23, and 11 patients were negative for all HDM MeDALL chip components. Seven sera were available for further testing, and 3 of them showed IgE reactivity to dot-blotted nDer p 1, and 2 reacted with high-molecular weight components (>100 kDa) in nitrocellulose-blotted HDM extract when tested with ¹²⁵I-labeled anti-IgE in a RAST-based assay. The HDM extract-specific IgE levels of the 11 patients were <3.9 kU/l. Conclusions: Recombinant allergen-based IgE serology is of great value when conventional IgE diagnostics fails. Der p 23 is an important HDM allergen, especially when major allergens are negative. Therefore, it would be desirable to have Der p 23 commercially available. Further research concerning the prevalence and clinical significance of different HDM allergens is needed.

BackgroundBeing one of the most common nasal diseases, chronic rhinosinusitis (CRS) is subdivided into CRS with nasal polyps (NP) and CRS without nasal polyps (CRSsNP). CRSsNP presents itself with a TH1 milieu and neutrophil infiltration, while NP is characterised by a mixed TH1/TH2 profile and an influx of predominantly eosinophils, plasma cells and mast cells. For the purpose of discovering disease-specific cytokine profiles, the present study compares levels of mediators and cytokines in nasal secretions between CRSsNP, NP, and healthy controls.MethodsThe study included 45 participants suffering from NP, 48 suffering from CRSsNP and 48 healthy controls. Allergic rhinitis constituted an exclusion criterion. Nasal secretions, sampled using the cotton wool method, were analysed for IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, IL-8, GM-CSF, G-CSF, IFN-γ, MCP-1, MIP-1α, MIP-1β, eotaxin, and RANTES, and for ECP and tryptase, using Bio-Plex Cytokine assay or ELISA, respectively.ResultsElevated levels of IL-5, IL-17, G-CSF, MCP-1, MIP-1α, MIP-1β, ECP, and tryptase, as well as decreased levels of IL-10, IL-12, IL-13, and IFN-γ were detected in NP. CRSsNP presented increased levels of RANTES and MIP-1β while IL-13 was decreased. No differences between the three groups were found for IL-4, IL-8, GM-CSF, and eotaxin.ConclusionsThe present work suggests a disequilibrium of TH1 and TH2, together with a down-regulation of regulatory T lymphocytes and up-regulated TH17 in NP. Moreover, elevated levels of diverse mediators represent the activation of various inflammatory cells in this disease entity. The inflammation in CRSsNP, however, is only weakly depicted in nasal secretions. Therefore, cytokines in nasal secretions may provide helpful information for differential diagnosis.

Background: Patients with nonallergic rhinitis with eosinophilia syndrome (NARES) show typical symptoms of persistent allergic rhinitis (PAR). The aim of the present study was to compare nasal cytokine patterns between NARES and PAR. Methods: Nasal secretions of 31 patients suffering from NARES, 20 patients with PAR to house dust mite and 21 healthy controls were collected using the cotton wool method and analyzed for interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) by Bio-Plex Cytokine Assay as well as eosinophil cationic protein (ECP) and tryptase by UniCAP-FEIA. Results: NARES and PAR presented elevated levels of tryptase, while ECP was markedly increased solely in NARES compared to both the controls and PAR. Elevated levels of IL-1β, IL-17, IFN-γ, TNF-α and MCP-1 were found in NARES compared to the controls as well as PAR. MIP-1β was elevated in NARES and PAR, while IL-4, IL-6 and G-CSF showed increased levels in NARES, and IL- 5 was elevated in PAR only. Conclusions: In patients with NARES and PAR, eosinophils and mast cells appear to be the pivotal cells of inflammation, reflected by high levels of tryptase and ECP as well as IL-5 and GM-CSF as factors for eosinophil migration and survival. The elevated levels of proinflammatory cytokines in NARES may indicate the chronic, self-perpetuating process of inflammation in NARES which seems to be more pronounced than in PAR. IL-17 might be a factor for neutrophilic infiltration or be responsible for remodeling processes in NARES.

Allergic rhinitis (AR), nasal polyps (NP) as well as chronic rhinosinusitis (CRS) are all known to be associated with eosinophilic infiltration and elevated numbers of mast cells (MC) within the mucosa. Both cell types and their markers eosinophilic cationic protein (ECP) and tryptase are utilized in the diagnosis and management of chronic sino-nasal diseases. Mucosal cytology samples were gathered by cytobrush, histological samples were obtained from the inferior turbinate. In both sample sets, the number of eosinophils and MC was determined. Their corresponding markers ECP and tryptase were quantified from nasal discharge. Patients were grouped with reference to their main diagnosis: AR (n = 34), NP (n = 25), CRS (n = 27) and controls (n = 34). Eosinophil counts from cytobrush and ECP levels were significantly elevated in NP compared to all other groups-31- and 13-fold over control, respectively. However, histologic review did not reveal any difference in eosinophil count among groups. Tryptase was significantly elevated threefold in AR versus CRS and controls. No correlation to cytological and histological MC counts could be found. ECP levels in nasal discharge as well as eosinophil counts can provide useful information with regard to the diagnosis. Likewise, tryptase concentrations can do. The presented data show that the measurement of markers in nasal discharge is superior in differentiating among diagnosis groups. Given that the collection of nasal secretions is more comfortable for patients than the more invasive techniques, we recommend first line ECP and tryptase testing performed on nasal secretions.