Ambrisentan, an endothelin receptor type A-selective antagonist, inhibits cancer cell migration, invasion, and metastasis.

Several studies reported a central role of the endothelin type A receptor (ETAR) in tumor progression leading to the formation of metastasis. Here, we investigated the in vitro and in vivo anti-tumor effects of the FDA-approved ETAR antagonist, Ambrisentan, which is currently used to treat patients with pulmonary arterial hypertension. In vitro, Ambrisentan inhibited both spontaneous and induced migration/invasion capacity of different tumor cells (COLO-357 metastatic pancreatic adenocarcinoma, OvCar3 ovarian carcinoma, MDA-MB-231 breast adenocarcinoma, and HL-60 promyelocytic leukemia). Whole transcriptome analysis using RNAseq indicated Ambrisentan's inhibitory effects on the whole transcriptome of resting and PAR2-activated COLO-357 cells, which tended to normalize to an unstimulated profile. Finally, in a pre-clinical murine model of metastatic breast cancer, treatment with Ambrisentan was effective in decreasing metastasis into the lungs and liver. Importantly, this was associated with a significant enhancement in animal survival. Taken together, our work suggests a new therapeutic application for Ambrisentan in the treatment of cancer metastasis.

GPCR-specific autoantibody signatures are associated with physiological and pathological immune homeostasis.

Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer's disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system.

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Plasma cells within granulomatous inflammation display signs pointing to autoreactivity and destruction in granulomatosis with polyangiitis.

INTRODUCTION: Plasma cells residing in inflamed tissues produce antibodies in chronic inflammatory and systemic autoimmune diseases. This study examined if plasma cells, located within inflamed nasal tissue in granulomatosis with polyangiitis (GPA), express features potentially associated with the autoimmune and destructive character of this disease. METHODS: Ig gene mutation patterns of individual tissue-derived plasma cells from GPA (n = 5) were analyzed, by using laser-assisted microdissection followed by semi-nested polymerase chain reaction (PCR). Signs of B-lymphocyte maturation (ectopic lymphoid structures, ELS) and survival (a proliferation-inducing ligand, APRIL; B-cell maturation antigen, BCMA; transmembrane-activator and calcium modulator and cyclophilin interactor, TACI; receptor activator of nuclear factor κB ligand, RANKL) were examined in nasal tissues or serum, respectively, by using immunohistochemistry/fluorescence and enzyme-linked immunosorbent assay, ELISA. RESULTS: Plasma-cell derived Ig genes (light- and heavy-chain pairs, n = 4; heavy chains, n = 33) resembled mutation patterns seen in other autoimmune diseases, predominantly displaying selection against replacement mutations within the framework region of Ig genes (10 of 15), which is responsible for structural integrity. Ectopic lymphoid structures were similar between GPA and a disease control (that is, unspecific chronic rhinosinusitis. However, histomorphologic features distinguishing GPA from rhinosinusitis (that is, neutrophilic microabscess and granuloma) expressed considerable amounts of membrane-associated and secreted APRIL, respectively. The latter was co-localized with CD138 and found in close proximity to cells expressing IgG, TACI, and BCMA. Interestingly, plasma cells strongly expressed receptor activator of nuclear factor κB ligand (RANKL), apart from fibroblast-like cells. CONCLUSIONS: Plasma cells within granulomatous inflammation appear to display features that might be required for autoreactivity and, possibly, RANKL-mediated destruction in GPA.

Granuloma in ANCA-associated vasculitides: another reason to distinguish between syndromes?

The 2012 renewed Chapel Hill Consensus Conference (CHCC) officially named three clinicopathological entities, i.e. granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA), and microscopic polyangiitis (MPA), as major variants of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV). Recent genetic and cohort studies revealed the need for further differentiation between the entities, for example regarding differences in outcome. As well as ANCA reactivity, upper and lower airway disease were found to be differentiating factors for AAV variants, improving prognostic ability regarding relapse prediction and associated clinical features. Extravascular granulomatosis, or "granuloma", which describes both clinically relevant granulomatous manifestations and histopathologically documented granulomatous inflammation, is characteristic of localized and systemic GPA, but not MPA. This review summarizes new knowledge regarding granuloma in the head and neck region of AAV, its histomorphological equivalents in the upper and lower respiratory tract, and evidence for a granulomatous phenotype of a persistent localized GPA variant. This comprises the development of disease activity and damage scores for extravascular lesions in the ear, nose, and throat (ENT) regions, and imaging techniques. In addition, findings linking extravascular manifestations to granulomatous inflammation are described. We hypothesize that, as for ANCA, necrotizing granulomatous inflammation and its clinical manifestations are discriminators, assisting subclassification of AAV and/or GPA subphenotypes which will be useful both for designing clinical trials and for treating patients successfully.

Hedgehog signaling in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant brain tumor in adults with a median survival of 14.6 mo under the best available treatment. New treatment strategies are therefore urgently required, for which a profound understanding of tumor biology is necessary. Much effort has been devoted to tumor-specific aberrant signaling processes. Recently it was discovered that the transcription factor Gli1, which is activated by hedgehog signaling, is a highly predictive marker in GBM, as determined by immunohistochemistry. To determine whether GBM cells have transcriptionally active Gli1, we performed experiments with reporter genes with cells isolated from surgically removed human tumors and cell lines. We also determined whether the hedgehog signaling inhibitor cyclopamine influences reporter gene expression and cell viability, and we determined the expression of Gli1, SHH and Patched1 by quantitative real-time RT-PCR. Reporter gene analysis of nine cultures and four cell lines demonstrated a significantly enhanced transcriptional activity in six tumor cell cultures and all cell lines. Analysis of cell viability in the presence of cyclopamine revealed a response of all cell cultures with the exception of one primary culture and one cell line, but only one cell line responded to cyclopamine with reduced hedgehog signaling activity. This indicates that the toxicity of cyclopamine toward GBM cells is independent from hedgehog signaling. Since no correlation between hedgehog activity and SHH, Gli1 and Patched1 mRNA levels was observed we conclude that other mechanisms aside from transcriptional regulation of these factors are responsible for hedgehog activity in tumor cells derived from GBM.

Lower numbers of FoxP3 and CCR4 co-expressing cells in an elevated subpopulation of CD4+CD25high regulatory T cells from Wegener's granulomatosis.

Defects in regulatory T (Treg) cells have been implicated in the pathogenesis of chronic inflammatory and autoimmune diseases, such as Wegener's granulomatosis (WG). This study aimed at evaluating numbers, phenotype and suppressive capacity of Treg cells in WG. Peripheral blood (PB) mononuclear cells from 22 WG-patients (17 active, 5 remission) and 22 sex- and age-matched healthy controls (HC) were examined for Treg cells by flow cytometry measuring CD4, CD25, transcription factor forkhead box P3 (FoxP3), chemokine receptor CCR4 and interferon receptor I (IFNRI). Suppressive function of CD4+CD25high Treg cells from 3 WG-patients and 3 HC was analysed using a carboxyfluoresceindiacetate-succinimidylester-based in vitro proliferation assay. Endonasal biopsies of 10 WG- and 5 sinusitis-patients were investigated for CD3+FoxP3+ cells, employing double immunohistochemistry. WG-patients displayed elevated numbers of CD4+CD25med T cells and of CD4+CD25high Treg cells. CD4+ T cells of WG-patients contained higher numbers of CCR4+ cells. However, CD4+CD25high Treg cells of WG-patients exhibited decreased numbers of cells co-expressing FoxP3 and CCR4. A low but significant increase of CD4+CD25highIFNRI+ Treg cells was detected in WG-patients. 9 days following stimulation with interferon (IFN)alpha + proteinase 3 (PR3), a reduced suppression of proliferation of responder T cells was observed for WG and proliferated CD4+CD25high Treg cells still showed downregulated co-expressions of FoxP3 and CCR4. Wegener's granuloma exhibited increased numbers of CD3+FoxP3+ cells. The results indicate upregulated numbers of Treg cells in PB and nasal mucosa as well as phenotypical and functional alterations of PB Treg cells in WG, some presumably mediated through PR3 and IFN-alpha.

Extrapyramidal symptoms in Wilson's disease are associated with olfactory dysfunction.

Wilson's disease is a rare autosomal recessive disorder characterized by the accumulation of copper, mainly in the liver and the brain. As copper accumulation in the brain leads to disturbances in basal ganglia function, neurological-type patients typically present with hypo- and hyperkinetic extrapyramidal symptoms, with Parkinsonism being very common. Although there are numerous reports on olfactory deficits in primary neurodegenerative disorders, olfactory function has not been investigated in metabolic disorders presenting with extrapyramidal features. Twenty-four patients with Wilson's disease participated in the investigation. All patients were treated pharmacologically. They comprised patients with liver disease alone (including mild enzyme elevation in asymptomatic individuals; n = 11) and/or neurological symptoms (n = 13) at the time of testing. Twenty-one patients underwent both [18F]fluoro-2-deoxy-D-glucose positron emission tomography ([18F]FDG-PET) and magnetic resonance imaging (MRI). The severity of extrapyramidal symptoms was judged using a clinical score system ranging from 0 (no symptoms) to 3 (severe symptoms). In all patients, psychophysical testing was performed using the Sniffin' Sticks, which involved tests for odor threshold, discrimination, and identification. Results from the present study revealed that Wilson's disease patients with neurological symptoms show a significant olfactory dysfunction compared to hepatic-type patients. Individuals who are more severely neurologically affected also present with a more pronounced olfactory deficit. Of interest, there was no significant effect of long-term treatment with penicillamine on olfactory function. Olfactory function did not correlate significantly with the presence of MRI visible lesions in the basal ganglia or with any regional glucose metabolism as measured by [18]F-FDG-PET. In conclusion, these findings indicate that the underlying pathological alterations with degeneration in the basal ganglia and neuronal loss in association with a marked increase of the copper content in this brain region play a role in the olfactory deficit.

Recovery of olfactory function following closed head injury or infections of the upper respiratory tract.

OBJECTIVE: To investigate the outcome of olfactory function in patients with olfactory loss following infections of the upper respiratory tract (post-URTI) or head trauma. DESIGN: Retrospective patient-based study. SETTING: Smell and Taste Outpatient Clinic at a university hospital. PATIENTS: A total of 361 patients (228 women, 133 men) were included. MAIN OUTCOME MEASURES: Olfactory function was assessed using the "Sniffin' Sticks" test battery, which result in a threshold, discrimination, and identification score. The mean interval between first and last visit was 14 months. RESULTS: In comparing the overall threshold, discrimination, and identification scores between the last and first visit, olfactory function improved in 26% of the patients whereas it decreased in 6%. The cause of olfactory impairment had a significant effect on the recovery rate of olfactory function. Within the post-URTI group (n = 262), 32% of the patients improved, but in the posttraumatic group (n = 99) only 10% improved. In patients with post-URTI olfactory loss, a negative correlation was found between age and recovery of olfactory function. In general, the factor "sex" had no significant effect on recovery of smell function. CONCLUSIONS: To our knowledge, the series of patients presented herein is the largest in the literature to date in which standardized testing methods were used to assess the progression of impaired olfaction. It showed that the rate of improvement of olfactory function was significantly higher in patients with post-URTI dysosmia compared with patients with posttraumatic dysosmia. During an observation period of approximately 1 year, more than 30% of patients with post-URTI olfactory loss experienced improvement, whereas only 10% of patients with posttraumatic olfactory loss experienced improvement. Furthermore, age plays a significant role in the recovery of olfactory function.

Beta-(1-->3)-D-glucan modulates DNA binding of nuclear factors kappaB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1beta/IL-1 receptor antagonist ratio.

BACKGROUND: Beta-1-->3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal beta-1-->3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1). RESULTS: Despite an activation of nuclear factor (NF) kappaB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1beta, IL-6, tumor necrosis factor alpha or interferon gamma induced by glucan phosphate. Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h: 5000 pg/ml), likely due to binding of NFkappaB to a consensus site in the IL-8 promoter. An increase in IL-1receptor antagonist (RA) production (peak at 24 h: 12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels. Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments. When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFkappaB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA. Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1beta and IL-6. As a consequence, glucan phosphate shifted the TSST-1-induced IL-1beta/IL-1RA ratio towards an anti-inflammatory phenotype. Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2. CONCLUSION: Thus, beta-1-->3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction. Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a beta-1-->3-D-glucan.

Reduced olfactory bulb volume in post-traumatic and post-infectious olfactory dysfunction.

The olfactory bulb is a highly plastic structure the volume of which partly reflects the degree of afferent neural activity. In this study, 22 patients with post-infectious olfactory deficit, nine participants with post-traumatic olfactory deficit, and 17 healthy controls underwent magnetic resonance volumetry of the olfactory bulb. Patients presented with significantly smaller olfactory bulb volumes than controls; significant correlations between olfactory function and bulb volume were observed. Patients with parosmia exhibited smaller olfactory bulb volumes than those without parosmia. Findings indicate that smell deficits leading to a reduced sensory input to the olfactory bulb result in structural changes at the level of the bulb. Reduced olfactory bulb volumes may also be considered to be characteristic of parosmia.

Cutting edge: neutrophil granulocyte serves as a vector for Leishmania entry into macrophages.

Macrophages (MF) are the final host cells for multiplication of the intracellular parasite Leishmania major (L. major). However, polymorphonuclear neutrophil granulocytes (PMN), not MF, are the first leukocytes that migrate to the site of infection and encounter the parasites. Our previous studies indicated that PMN phagocytose but do not kill L. major. Upon infection with Leishmania, apoptosis of human PMN is delayed and takes 2 days to occur. Infected PMN were found to secrete high levels of the chemokine MIP-1beta, which attracts MF. In this study, we investigated whether MF can ingest parasite-infected PMN. We observed that MF readily phagocytosed infected apoptotic PMN. Leishmania internalized by this indirect way survived and multiplied in MF. Moreover, ingestion of apoptotic infected PMN resulted in release of the anti-inflammatory cytokine TGF-beta by MF. These data indicate that Leishmania can misuse granulocytes as a "Trojan horse" to enter their final host cells "silently" and unrecognized.

Detection of presymptomatic Parkinson's disease: combining smell tests, transcranial sonography, and SPECT.

Olfactory loss is among the early signs of Parkinson's disease (PD). We investigated whether "idiopathic" olfactory dysfunction might relate to signs of nigral degeneration. Olfactory tests were combined with transcranial sonography of the substantia nigra and single photon emission computed tomography (SPECT) imaging. Thirty patients diagnosed with idiopathic olfactory loss participated. Eleven of these patients exhibited an increased echogenicity of the SN in the transcranial sonography. In 10 of these 11 patients, SPECT scans with (123)I-FP-CIT were performed. Median uptake ratios in the basal ganglia were pathological in 5 patients, 2 patients exhibited borderline findings, and 3 patients had normal results. Considering patients with idiopathic olfactory dysfunction, noninvasive transcranial sonography seems to be helpful in identifying patients potentially at risk to develop PD. Longitudinal follow-up studies are necessary to estimate the ratio of patients with dopaminergic cell loss in the basal ganglia who will develop PD in the future.

Frequency of proteinase 3 (PR3)-specific autoreactive T cells determined by cytokine flow cytometry in Wegener's granulomatosis.

OBJECTIVE: Previous studies have shown proliferation to Wegener's autoantigen, proteinase 3 (PR3). We tested immunogenicity of PR3-derived peptides and determined frequencies of PR3-specific T cells using cytokine flow cytometry in Wegener's granulomatois (WG). METHODS: Peripheral blood T-cell responses were measured after stimulation with previously described PR3-derived peptides. PBMC were stimulated with PR3-derived peptides or control stimuli for up to 10 days. Cells were stained with antibodies against CD4 or CD8, CD69 and intracellular TNF-alpha and analyzed by flow cytometry. PR3-specific T cells were counted as the percentage of CD69(+)TNF-alpha(+)double positive T cells after stimulation. RESULTS: Frequencies of PR3-specific peripheral blood T cells after short-term stimulation (

Heterogeneity of CD4 and CD8+ memory T cells in localized and generalized Wegener's granulomatosis.

Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin (CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO+ (positive for the short human isoform of CD45) memory T cells reverts to the CD45RA+ (positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls (n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5+ and CCR3+ cells within the CD4+CD45RO+ and CD8+CD45RO+ memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA+ T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO+ memory T cells, as well as on CD45RA+ T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4+ and CD8+ memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.

Peripheral blood and granuloma CD4(+)CD28(-) T cells are a major source of interferon-gamma and tumor necrosis factor-alpha in Wegener's granulomatosis.

To elucidate whether the fraction of CD28(-) T cells within the CD4(+) T-cell population is a major source of Th1-like and proinflammatory cytokine production driving Wegener's granulomatosis (WG) granuloma formation, we analyzed the phenotype and functional characteristics of peripheral blood CD4(+)CD28(-) T cells and of T cells in granulomatous lesions of 12 patients with active WG. Surface markers and intracytoplasmic cytokine and perforin expression were assessed by flow cytometry. Cytokine secretion was measured by enzyme-linked immunosorbent assay. Immunohistological studies demonstrated interferon-gamma and tumor necrosis factor-alpha cytokine positivity attributable to CD4(+)CD28(-) T cells in granulomatous lesions. Peripheral blood CD4(+)CD28(-) T cells expressed CD57, also found on natural killer cells, and intracytoplasmic perforin. They were generally CD25 (interleukin-2 receptor)-negative. CD18 (adhesion molecule beta(2)-integrin) was strongly up-regulated on CD4(+)CD28(-) T cells, whereas only a minority of CD4(+)CD28(+) T cells expressed CD18. CD4(+)CD28(-) T cells appeared as a major source of interferon-gamma and tumor necrosis factor-alpha. In contrast, CD4(+)CD28(+) T cells were able to produce and secrete a wider variety of cytokines including interleukin-2. One-quarter of CD4(+)CD28(+) T cells expressed the activation marker CD25, but they lacked perforin. Thus, CD4(+)CD28(-) T cells appeared more differentiated than CD4(+)CD28(+) T cells. They displayed Th1-like cytokine production and features suggestive of the capability of CD4(+) T-cell-mediated cytotoxicity. CD4(+)CD28(-) T cells may be recruited into granulomatous lesions from the blood via CD18 interaction, and may subsequently promote monocyte accumulation and granuloma formation through their cytokine secretion in WG.