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To examine functional features of CD4+CD8+ double-positive T-cells in patients with granulomatosis with polyangiitis (GPA) using phenotypic and transcriptomic analysis.Staining of cellular surface marker was performed using freshly collected whole blood. For intracellular cytokine staining freshly collected whole blood was stimulated with phorbol myristate acetate and ionomycin. Multicolor flow cytometric analysis was performed on a FACSCanto II cytometer using FACSDiva software. Lymphocytes were gated on CD3, CD4, and CD8 staining. FACS-sorted CD4+CD8+ double-positive T-cells of GPA-patients and HC (n=3 each) were subjected to transcriptional profiling using an Affymetrix Human Genome 2.0 microarray. Differently expressed genes were analysed using biological databases.Frequency of CD4+CD8+ double-positive T-cells was increased within the total CD3+ T-cell population in GPA, but no difference was detected between patients with active disease and remission. Percentages of interferon γ (Th1-type), interleukin 17 and interleukin 22 (Th17-type) producing CD4+CD8+ double-positive T-cells exceeded the percentage of interleukin 4 (Th2-type) producing cells. There were no significant differences in the percentages of the respective cytokine-positive CD4+CD8+ double-positive T-cells between GPA and HC. Up-regulated genes of CD4+CD8+ double-positive T-cells in GPA were enriched within Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to nuclear factor kappa-lightchain-enhancer of activated B cells signalling, toll-like receptor signalling, nucleotide-binding oligomerisation domain-like receptor signalling as well as major histocompatibility complex class-II antigen presentation.Employing a combined phenotypic and transcriptomic approach we disclosed a Th1/Th17 phenotype as well as innate and adaptive functions of CD4+CD8+ double-positive T-cells in GPA.

Patients with giant cell arteritis (GCA) refractory to standard immunosuppressive therapy may constitute a significant clinical problem with a high risk of glucocorticoid-related adverse effects.To evaluate efficacy and safety of cyclophosphamide for remission induction in GCA patients with persistent disease activity despite standard immunosuppressive treatment.Thirty-five individuals from 3 tertiary rheumatological centres treated for persistently active GCA unresponsive to treatment with glucocorticoids plus at least either methotrexate or azathioprine for a minimum of 3 months and unable to reduce daily glucocorticoid dose to10 mg prednisolone equivalent. We recorded signs of disease activity (clinical, laboratory, imaging); course of glucocorticoid doses during cyclophosphamide treatment and follow-up; relapse rate; treatment-related adverse events; and survival. Since all patients had been refractory to standard therapy, a matched control group could not be defined.Data from 31 patients completing cyclophosphamide treatment were available for analysis. Twenty-eight patients (90.3%) responded with improved disease activity and sustained reduction of daily prednisolone intake to10 mg (mean reduction -13.1 mg or -51.6%, p0.001). Twelve months later, doses7.5 or5 mg were achieved in 89.3% and 67.7% of these patients on maintenance immunosuppressive treatment, respectively. Relapses occurred in 12 patients after a median of 20.5 months. Survival over 5 years was similar to expected rates of the general population. Adverse events comprised transient leucopenia, infections and 1 case of haemorrhagic cystitis.Cyclophosphamide can be considered a therapeutic option with an acceptable safety profile for remission induction in GCA refractory to standard immunosuppressive treatment.

Granulomatosis with polyangiitis (GPA) frequently starts with an affection of the nasal and paranasal mucosa. Localised GPA of the nasal mucosa or persistent disease activity ('grumbling disease') is often encountered even under immunosuppressive therapy. Necessity for reconstructive surgery is common and careful scheduling to prevent failure and minimise revision rates is crucial. Therefore, reliable estimation of GPA activity in the upper airways using a score is mandatory for diagnosis, follow-up and scheduling reconstructive surgery.Fifty endoscopic, endonasal images of 45 patients with GPA were used. Twelve (4 German, 8 Mexican) experienced (n=7) and inexperienced (n=5) physicians assessed GPA-activity at two times (T1/T2) in dichotomy and in a grading approach (none, mild, moderate and high activity) using the novel ENT Activity Score (ENTAS). All documents were written in English.Estimation of activity in dichotomy (none vs. mild/moderate/high): Cohen's Kappa (κ) for intra-rater reliability T1/T2 in inexperienced and experienced physicians was κ=0.58 (agreement 85%) and κ=0.72 (agreement 91%). The inter-rater reliability (Fleiss's κ) T1/T2 for inexperienced and experienced physicians was κ=0.62/κ=0.59 and κ=0.50/κ=0.58 respectively. Estimation of activity in grading approach (none, mild, moderate, high): for inexperienced physicians the intra-rater reliability T1/T2 was κ=0.67 (agreement 56%) and the inter-rater reliability at T1/T2 was κ=0.29 (intraclass correlation coefficient, ICC=0.69) and κ=0.27 (ICC=0.59). For experienced physicians the intra-rater reliability T1/T2 was κ=0.80 (agreement 67%) and the inter-rater reliability at T1 and T2 was κ=0.41 (ICC=0.77) and κ=0.39 (ICC=0.75) respectively.Intra-rater reliability is high in decision in dichotomy and even in grading activity. There is no difference for experienced or inexperienced physicians. Inter-rater reliability is high in dichotomy, but low for activity grading. Thus, the ENTAS provides a reliable instrument for assessing, documenting and following GPA-related disease activity in the upper respiratory tract. The relationship of activity and following damage needs to be investigated in further studies.

To analyse whether a specific cytokine pattern is elicited in response to the autoantigen proteinase 3 (PR3) in active Wegener's granulomatosis (WG).Six-colour flow cytometry was used to analyse cytokine production and surface markers of the total CD4+ T-cell population ex vivo and in PR3-stimulated T-cell lines of patients with active PR3-ANCA-positive WG, PR3-ANCA-negative Churg-Strauss syndrome (CSS), and healthy controls (HC).The cytokine response of the total PB CD4+ T cell population was skewed towards distinct pro-inflammatory cytokine patterns in WG (Th1-type) and CSS (Th17, Th1-/Th2-type). Th2-type as well as Th17 cell populations including Th17/Th1, Th17/Th2 and Th22 cells were elicited in response to PR3 stimulation in WG. In contrast, CSS patients displayed a Th2-type dominated response following PR3 stimulation.These data suggest that the cytokine response of the total CD4+ T-cell population and PR3-specific cells is influenced by the underlying disorder.

Nasal S. aureus carrier rates are significantly higher in patients with Wegener's granulomatosis (WG) compared to healthy controls (HC), and nasal colonisation is a risk-factor for relapse. Antimicrobial peptides (AMP) are important defence molecules maintaining an intact barrier function. It is the aim of this study to see if there is a possible link between the nasal AMP pattern and S. aureus colonisation, a link which has not been investigated so far.ELISA was applied to quantify LL-37 and hBD-3 concentrations in nasal secretions (14 WG patients, 13 HC) with and without nasal S. aureus colonisation. Immunohistochemistry was used to detect the cellular sources of AMP in the nasal mucosa. Functional analyses of primary nasal epithelial cell cultures (NEC) of these groups stimulated with S. aureus were performed.LL-37 was found in significantly higher concentrations in colonised individuals (WG: p=0.001; HC: p=0.014).Using immunohistochemistry, local cellular sources for AMP could be demonstrated. After stimulation with S. aureus, significantly higher concentrations of LL-37 and hBD-3 could be detected in the supernatant of NEC of WG patients (LL-37: p=0.001; hBD-3: p=0.001) and HC (LL-37: p=0.019; hBD-3: p=0.001). HBD-3 concentrations were significantly lower in the supernatant of stimulated NEC of WG patients compared to the NEC of HC (p=0.032), and the dynamic range of the hBD-3 answer was significantly smaller in WG compared to HC (p=0.016).The dynamic response towards challenges with microbes is dysregulated in WG, and this might be one reason for higher S. aureus colonisation rates in WG.

Nasal colonisation with Staphylococcus aureus (S. aureus) has been implicated in Wegener's granulomatosis (WG) disease activity. In this study, the frequency of nasal colonisation with S. aureus in WG was compared to healthy and disease control groups for the first time. Moreover, endonasal activity was correlated to colonisation.Nasal carriage of S. aureus of a well-defined group of 89 patients with WG was compared to 40 patients with chronic rhinosinusitis with nasal polyps (CRS), 35 patients with rheumatoid arthritis (RA), 50 hospital staff members and 25 subjects without regular hospital contact and correlation analysis of nasal carriage and endonasal activity of WG was performed.WG patients showed significantly higher rates (72%) of nasal colonisation with S. aureus compared to CRS patients (28%) and healthy subjects without regular hospital contact (25%, 95%-CI), but not to RA patients (46%) and hospital staff members (58%). WG patients with nasal carriage of S. aureus had significantly higher endoscopically proven endonasal activity (p=0.01), significantly more often first manifestation of WG in the upper respiratory tract (p=0.02) and higher relapse-rates (p=0.052) than WG patients without such carriage.Endonasal activity in WG is associated with higher nasal S. aureus colonisation rates and subsequent higher relapse rates. The higher frequency of S. aureus colonisation could be a consequence of a recently shown mucosal barrier defect in WG and facilitate chronic inflammation and granuloma formation in the upper respiratory tract.

gammadelta T-cells are a numerically small subset of T-cells with distinct features. They recognise antigens that are not seen by other immune cells. At the functional level, gammadelta T-cells share some features with alphabeta T-cells but also exert functions that are otherwise performed by specialised subsets of alphabeta T-cells (e.g. IL-17 production, regulatory activity). We discuss the potential role of gammadelta T-cells in various clinical forms of vasculitis.

While remission is achieved in the majority of Wegener's granulomatosis (WG)-patients with cyclophosphamide, maintenance of remission remains a challenge due to the high rate of relapses. The purpose of this study was to evaluate the safety and efficacy of the combination of methotrexate (MTX) plus leflunomide (LEF) for the treatment of minor relapsing WG not warranting cyclophosphamide.Retrospective chart analyses of 51 WG-patients with non-life-threatening relapses under MTX or LEF maintenance monotherapy. Relapsing patients were subsequently treated with a combination therapy of MTX+LEF.Fifty-one WG patients with relapses under MTX (n=36) or LEF (n=15) maintenance monotherapy were identified. They were subsequently treated with MTX+LEF to reintroduce remission. Mean follow-up was 26.0 (3-93) months. MTX+LEF controlled relapsing WG in 43/51 (84%) patients: 28/51 achieved a Birmingham Vasculitis activity index (BVAS)=0 and 15/51 a response (BVAS reduction of= or). 8/51 patients did not respond to MTX+LEF (50% BVAS reduction) and were switched to cyclophosphamide and/or a biological for ongoing disease activity. Follow up showed a sustained remission (BVAS=03 months) in 14/51 patients, a minor relapse in 27/51, and a major relapse in 2/51 (subsequently switched to cyclophosphamide). Fifty adverse effects were observed. MTX+LEF therapy was discontinued in 18/51 patients because of adverse effects (main causes: gastro-intestinal complaints, hypertension, infections).Although side effects limited the overall performance of MTX+LEF, this combination, if tolerated well, remains an effective treatment in patients not warranting cyclophosphamide.

Wegener's granulomatosis (WG) usually starts as granulomatous disease of the respiratory tract (so-called localized WG) before it converts to systemic disease (generalized WG) with the emergence of proteinase 3-specific antineutrophil cytoplasmic autoantibodies (PR3-ANCA) and PR3-ANCA associated autoimmune vasculitis. So far, it remains unresolved how tolerance to "Wegener's autoantigen" PR3 is broken and the immune response to PR3 sustained. Further, the relationship between granulomatous lesions and systemic vasculitis is poorly understood. None of the ANCA-animal-models has reproduced granulomata typical of WG so far. A number of endogenous and exogenous factors (HLA-DPB1*0401/PTPN22*620W, respiratory epithelial barrier dysfunction? S. aureus, cPR3?) could favour initial formation of granulomata in the respiratory tract and break of tolerance. PR3 induces dendritic cell maturation via the protease activated receptor (PAR)-2 and evokes a strong Th1-type T-cell res-ponse in WG. Clusters of PR3+ cells (neutrophils/monocytes) surrounded by antigen-presenting cells, Th1-type CD4+CD28- effector memory T-cells, maturing B- and plasmacells are found in WG-granulomata of the upper respiratory tract. Thus, WG-granulomata might provide the necessary "proinflammatory environment" for the break of tolerance and display features of lymphoid-like tissue neoformation, in which autoimmunity to PR3 could be sustained. Subsequent PR3-ANCA associated systemic vasculitis gives rise to new inflammatory lesions in many other organs, thereby promoting a self-perpetuating pathology characterized by inflammation and autoimmunity to PR3.

Clinical measures of vasculitis activity (Birmingham vasculitis activity score = BVAS) and disease extent (Disease Extent Index = DEI), serological and immunological parameters were evaluated for the monitoring of hepatitis C virus (HCV)-associated cryoglobulinemic vasculitis (CV), treated with either cyclophosphamide or interferon-alpha 2b depending on disease severity.Serial serum samples of 15 patients with HCV-associated CV were analyzed, and BVAS, DEI, serological and immunological parameters were recorded at diagnosis and during therapy. Eight patients were treated with interferon-alpha 2b and 7 patients with cyclophosphamide.A complete or partial response of the CV was seen in both treatment groups. BVAS, complement factor C3c, cryoglobulinemia, and rheumatoid factor significantly decreased in both treatment groups during 6 months (p0.05). DEI decrease was significant in the cyclophosphamide group (p0.05), and there was a trend in the interferon-alpha 2b group (p = 0.06). BVAS and DEI were significantly positively correlated, and both parameters were significantly negatively correlated with C3c levels in both treatment groups (interferon-alpha 2b/cyclophosphamide: r = -0.89, p = 0.001 versus r = -0.87, p0.001, respectively) whereas other parameters were not, e.g. ESR and CRP.Patients with different degrees of disease severity, treated with either cyclophosphamide or interferon-alpha 2b depending on their disease activity, achieved remission of their CV. BVAS, DEI and C3c were especially useful in the follow-up of HCV-associated CV. C3c correlated with BVAS and DEI during therapy and provided additional information about vasculitis activity that was not reflected by other serological or immunological parameters, e.g. ESR or CRP.