Using blinking optical tweezers to study cell rheology during initial cell-particle contact.

Phagocytosis is an important part of innate immunity and describes the engulfment of bacteria and other extracellular objects on the micrometer scale. The protrusion of the cell membrane around the bacteria during this process is driven by a reorganization of the actin cortex. The process has been studied on the molecular level to great extent during the past decades. However, a deep, fundamental understanding of the mechanics of the process is still lacking, in particular because of a lack of techniques that give access to binding dynamics below the optical resolution limit and cellular viscoelasticity at the same time. In this work, we propose a technique to characterize the mechanical properties of cells in a highly localized manner and apply it to investigate the early stages of phagocytosis. The technique can simultaneously resolve the contact region between a cell and an external object (in our application, a phagocytic target) even below the optical resolution limit. We used immunoglobulin-G-coated microparticles with a size of 2 µm as a model system and attached the particles to the macrophages with holographic optical tweezers. By switching the trap on and off, we were able to measure the rheological properties of the cells in a time-resolved manner during the first few minutes after attachment. The measured viscoelastic cellular response is consistent with power law rheology. The contact radius between particle and cell increased on a timescale of ∼30 s and converged after a few minutes. Although the binding dynamics are not affected by cytochalasin D, we observed an increase of the cellular compliance and a significant fluidization of the cortex after addition of cytochalasin D treatment. Furthermore, we report upper boundaries for the length- and timescale, at which cortical actin has been hypothesized to depolymerize during early phagocytosis.

Broccoli sprout supplementation in patients with advanced pancreatic cancer is difficult despite positive effects-results from the POUDER pilot study.

Pancreatic ductal adenocarcinoma is a highly aggressive malignancy with short survival and limited therapeutic options. Broccoli sulforaphane is a promising new treatment due to the results of recent epidemiological, experimental and patient studies. Upon approval from the ethics committee and registration at ClinicalTrials.gov, 40 patients with palliative chemotherapy were placed into a placebo and treatment group in an unblinded fashion. Fifteen capsules with pulverized broccoli sprouts containing 90 mg/508 µmol sulforaphane and 180 mg/411 µmol glucoraphanin or methylcellulose were administered daily for up to 1 year. Twenty-nine patients were included in the treatment group and 11 patients were in the placebo group; these patients were followed for up to 1 year. The patient characteristics, overall survival and feasibility were assessed. Compared to those of the placebo group, the mean death rate was lower in the treatment group during the first 6 months after intake (day 30: 0%/18%, day 90: 0%/25%, and day 180: 25%/43%), and Kaplan-Meier analysis revealed a higher survival rate. There was a high drop-out rate (72% in the treatment group and 55% in the placebo group) after 1 year. We concluded from the Karnofsky index that the broccoli sprouts did not impact patient's self-care and overall abilities severely. The intake of 15 capsules daily was difficult for some patients, and the broccoli sprouts sometimes increased digestive problems, nausea and emesis. We did not obtain statistically significant results (p = 0.291 for the endpoint at day 180), but the knowledge about the feasibility is the basis for the development of new sulforaphane drugs.

Phenomenon of Endothelial Antibody Capture: Principles and Potential for Locoregional Targeting of Hepatic Tumors.

The systemic drug circulation represents a source of adverse effects during tumor targeting. We studied the binding efficacy of endothelium-specific antibodies after a very short contact with an antigen target, along with assessing the intravascular capture and targeting potential of these antibodies after locoregional injection. Fast-binding anti-CD 146 (clone ME-9F1) and anti-CD31 (clone 390) antibodies were selected based on histological analysis of their binding activity. The efficacy of antibody capture by hepatic endothelium under different conditions was analyzed using an isolated liver perfusion model. The local enrichment of R-phycoerythrin and 125 I-conjugated antibody was studied in vivo in two hepatic tumor models using biodistribution, scintigraphic imaging, and fluorescence microscopy. Upon injection into the tumor-feeding artery, the antibody was immediately captured in the microvasculature during the first passage. At doses not exceeding the saturation level of endothelial epitopes, the capture efficacy was almost 90%. We showed that the efficacy of endothelial capture is controlled by factors such as antibody affinity, number of binding sites on the endothelium, and microvascular flow rate. The targeting potential of endothelial capture was experimentally proven in vivo using scintigraphic imaging and biodistribution analysis after locoregional intra-arterial injection of 125 I-labeled antibodies in hepatic tumor models. Conclusion: The unique phenomenon of endothelial capture can broadly prevent systemic circulation of the antibody or antibody-drug conjugates applied by intravascular injection and may have specific relevance for targeting of hepatic tumors.

Intraductal papillary mucinous neoplasm of the pancreas rapidly xenografts in chicken eggs and predicts aggressiveness.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas has a high risk of progressing to invasive pancreatic ductal adenocarcinoma (PDA), but experimental models for IPMN are largely missing. New experimental systems for the molecular characterization of IPMN and for personalized prognosis and treatment options for IPMN are urgently needed. We analyzed the potential use of fertilized chicken eggs for the culture of freshly resected IPMN tissue. We transplanted 49 freshly resected IPMN tissues into eggs and compared the growth characteristics to IPMN tissues transplanted into mice; this was followed by an analysis of histology, morphology, and marker expression. Of the IPMN tissues transplanted into eggs, 63% formed tumor xenografts within 4 days, while none of the 12 IPMN tissues transplanted into immunodeficient mice engrafted. In the eggs, the grafting efficiency of high-grade (n = 14) and intermediate-grade (n = 17) dysplasia was 77% and was significantly higher than the 39% grafting efficiency of low-grade dysplasia (n = 18). According to mucinous expression, 46 IPMN tissues were classified into gastric (n = 6), intestinal (n = 3), oncocytic (n = 23), and pancreatobiliary (n = 14) subtypes. The grafting efficiency was highest for the pancreatobiliary subtype (86%), followed by the oncocytic (70%), gastric (33%) and intestinal (33%) subtypes. The morphology and expression patterns of mucins, progression markers and pancreatic ductal markers were comparable between the primary IPMN tissues and their xenograft copies. The individual tumor environment was largely maintained during subtransplantation, as evaluated upon passage 6. This new IPMN model may facilitate experimental studies and treatment decisions for the optimal personalized management of IPMN.

Simultaneous measurement of the Young's modulus and the Poisson ratio of thin elastic layers.

The behavior of cells and tissue is greatly influenced by the mechanical properties of their environment. For studies on the interactions between cells and soft matrices, especially those applying traction force microscopy the characterization of the mechanical properties of thin substrate layers is essential. Various techniques to measure the elastic modulus are available. Methods to accurately measure the Poisson ratio of such substrates are rare and often imply either a combination of multiple techniques or additional equipment which is not needed for the actual biological studies. Here we describe a novel technique to measure both parameters, the Youngs's modulus and the Poisson ratio in a single experiment. The technique requires only a standard inverted epifluorescence microscope. As a model system, we chose cross-linked polyacrylamide and poly-N-isopropylacrylamide hydrogels which are known to obey Hooke's law. We place millimeter-sized steel spheres on the substrates which indent the surface. The data are evaluated using a previously published model which takes finite thickness effects of the substrate layer into account. We demonstrate experimentally for the first time that the application of the model allows the simultaneous determination of both the Young's modulus and the Poisson ratio. Since the method is easy to adapt and comes without the need of special equipment, we envision the technique to become a standard tool for the characterization of substrates for a wide range of investigations of cell and tissue behavior in various mechanical environments as well as other samples, including biological materials.

Enhancing Nitric Oxide Bioavailability via Exogen Nitric Oxide Synthase and L-Arginine Attenuates Ischemia-Reperfusion-Induced Microcirculatory Alterations.

BACKGROUND: Nitric oxide (NO) is an important cytoprotective agent against ischemia and reperfusion injury (IRI). Enhancing NO bioavailability via exogen NO synthases (NOSs) and L-arginine promotes conversation to NO, circumventing the problem of nonfunctioning NOSs under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of endothelial, inducible and neuronal NOS, and L-arginine on reperfusion-induced microcirculatory alterations and hemodynamic adverse effects in the microvasculature of skeletal muscle. METHODS: Vascular pedicle isolated rat cremaster model was used that underwent 2 hours of warm ischemia followed by 1 hour of reperfusion. At 30 minutes before ischemia, normal saline (control group with/without ischemia), endothelial-, inducible-, and neuronal NOSs (2 IE) and L-arginine (50 mg/kg BW) were administered systemically (IV). Ischemia-reperfusion-induced microcirculatory alterations were measured after 1 hour of reperfusion. Mean arterial blood pressure and heart frequency were measured throughout the experiment to determine hemodynamic adverse effects. RESULTS: The isoforms of NOSs and L-arginine attenuated ischemia-reperfusion-induced vasoconstriction, improved red blood cell velocity, capillary flow, and leukocyte adherence to the endothelium wall. Hemodynamics was stable throughout the experiment. CONCLUSIONS: Enhancing NO bioavailability via exogen application of NOSs and L-arginine significantly attenuated ischemia-reperfusion-induced microcirculatory alterations in the microvasculature of skeletal muscle. Significant hemodynamic adverse effects were not present, thus demonstrating this approach might be useful for therapeutic intervention. This "pharmacologic preconditioning" could be an easy and effective interventional strategy to uphold conversation of L-arginine to NO under ischemic conditions.

Clinical presentation and long-term outcome of 144 patients with microscopic polyangiitis in a monocentric German cohort.

OBJECTIVE: To evaluate the clinical presentation and long-term outcome of a vasculitis centre cohort of patients with microscopic polyangiitis (MPA) with respect to organ manifestations, treatment, chronic damage and mortality. METHODS: We performed a retrospective chart review at our vasculitis referral centre. MPA patients admitted between 1991 and 2013 classified by a modified European Medicines Agency algorithm were diagnosed and treated according to a standardized interdisciplinary approach. RESULTS: Comprehensive data from standardized interdisciplinary workups was available for 144 patients (median follow-up 72 months). The overall standardized mortality ratio was 1.40 (95% CI 0.91, 2.07; P = 0.13). We observed a higher mortality [hazard ratio (HR) 4.04 (95% CI 1.21, 13.45), P = 0.02] in 17 patients with MPA-associated fibrosing interstitial lung disease (ILD) and 56 patients with peripheral nervous system involvement [HR 5.26 (95% CI 1.10, 25.14), P = 0.04] at disease onset. One hundred and fifteen patients (79.9%) responded to the initial treatment. Sixty-one (42.3%) achieved complete remission and 54 (37.5%) achieved partial remission. Twenty (13.9%) showed a refractory disease course. CONCLUSION: MPA patients at our tertiary rheumatology referral centre seemed to have a less severe phenotype resulting in a less severe disease course and better outcome than reported in other cohorts. Fibrosing ILD was significantly associated with mortality in this cohort.

Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity.

Late diagnosis contributes to pancreatic cancer (PaCa) dismal prognosis, urging for reliable, early detection. Serum-exosome protein and/or miRNA markers might be suitable candidates, which we controlled for patients with PaCa. Protein markers were selected according to expression in exosomes of PaCa cell line culture supernatants, but not healthy donors' serum-exosomes. miRNA was selected according to abundant recovery in microarrays of patients with PaCa, but not healthy donors' serum-exosomes and exosome-depleted serum. According to these preselections, serum-exosomes were tested by flow cytometry for the PaCa-initiating cell (PaCIC) markers CD44v6, Tspan8, EpCAM, MET and CD104. Serum-exosomes and exosome-depleted serum was tested for miR-1246, miR-4644, miR-3976 and miR-4306 recovery by qRT-PCR. The majority (95%) of patients with PaCa (131) and patients with nonPa-malignancies reacted with a panel of anti-CD44v6, -Tspan8, -EpCAM and -CD104. Serum-exosomes of healthy donors' and patients with nonmalignant diseases were not reactive. Recovery was tumor grading and staging independent including early stages. The selected miR-1246, miR-4644, miR-3976 and miR-4306 were significantly upregulated in 83% of PaCa serum-exosomes, but rarely in control groups. These miRNA were also elevated in exosome-depleted serum of patients with PaCa, but at a low level. Concomitant evaluation of PaCIC and miRNA serum-exosome marker panels significantly improved sensitivity (1.00, CI: 0.95-1) with a specificity of 0.80 (CI: 0.67-0.90) for PaCa versus all others groups and of 0.93 (CI: 0.81-0.98) excluding nonPa-malignancies. Thus, the concomitant evaluation of PaCIC and PaCa-related miRNA marker panels awaits retrospective analyses of larger cohorts, as it should allow for a highly sensitive, minimally-invasive PaCa diagnostics.

Genetically distinct subsets within ANCA-associated vasculitis.

BACKGROUND: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis. METHODS: A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria. RESULTS: We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti-proteinase 3 ANCA was associated with HLA-DP and the genes encoding α(1)-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P=6.2×10(-89), P=5.6×10(-12,) and P=2.6×10(-7), respectively). Anti-myeloperoxidase ANCA was associated with HLA-DQ (P=2.1×10(-8)). CONCLUSIONS: This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA-associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA-associated vasculitis and myeloperoxidase ANCA-associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.).

Pathogenesis of anti-neutrophil cytoplasmic antibody-associated vasculitis: challenges and solutions 2014.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) with specificity for proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA) are a defining feature of ANCA-associated vasculitides (AAV). They play a pivotal role in disease pathophysiology and have strongly improved early diagnosis and treatment of these infrequent, but potentially fatal diseases. Neutrophils and their products are major players in initiating the autoimmune response and tissue destruction in vasculitic as well as granulomatous inflammation. This review highlights recent findings on old and novel players (ANCA, neutrophils, neutrophil extracellular traps, fibroblasts, immune cells and complement) and puts them into context with the current understanding of disease mechanisms in AAV.

Doubled prevalence rates of ANCA-associated vasculitides and giant cell arteritis between 1994 and 2006 in northern Germany.

OBJECTIVES: The aim of this study was to investigate the period prevalences of ANCA-associated vasculitides (AAV), including granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), eosinophilic GPA (EGPA)/Churg-Strauss and GCA, in an urban and rural population in northern Germany in 2006 and to compare the data with our previous study performed in 1994. METHODS: We identified of all patients with AAV or GCA via questionnaires to all hospital departments, physicians, health insurance providers, pension funds, reference laboratories for autoimmune diseases and death registries in Luebeck (city) and the rural region of Segeberg (population 468 962) between January and December 2006. The type of vasculitis, gender, year of birth, postal code and death were documented and re-evaluated. RESULTS: One-hundred and fifty patients were identified, indicating a prevalence of 320 per million inhabitants for the complete catchment area (95% CI 285, 355). GCA was more prevalent than AAV: 171 (146, 197) vs 149 (126, 174). GCA and AAV have almost doubled since 1994. GCA increased from 240 (164, 315) to 440 (399, 481) per million in the population ≥ 50 years of age and AAV increased from 74 to 149 cases per million. GCA and AAV were more prevalent in the urban compared with the rural region. CONCLUSION: The prevalence rates of AAV and GCA almost doubled from 1994 to 2006 for this region with a stable population and using an identical study design. Increased awareness has led to an earlier diagnosis of systemic vasculitis and improved activity-adapted treatment mostly based on randomized controlled trials has led to longer survival. Aspects such as environmental factors and exposure to certain substances need further research.

Corrigendum: Establishment of tumor treating fields combined with mild hyperthermia as novel supporting therapy for pancreatic cancer.

[This corrects the article DOI: 10.3389/fonc.2021.738801.].

Nominally identical microplastic models differ greatly in their particle-cell interactions.

Due to the abundance of microplastics in the environment, research about its possible adverse effects is increasing exponentially. Most studies investigating the effect of microplastics on cells still rely on commercially available polystyrene microspheres. However, the choice of these model microplastic particles can affect the outcome of the studies, as even nominally identical model microplastics may interact differently with cells due to different surface properties such as the surface charge. Here, we show that nominally identical polystyrene microspheres from eight different manufacturers significantly differ in their ζ-potential, which is the electrical potential of a particle in a medium at its slipping plane. The ζ-potential of the polystyrene particles is additionally altered after environmental exposure. We developed a microfluidic microscopy platform to demonstrate that the ζ-potential determines particle-cell adhesion strength. Furthermore, we find that due to this effect, the ζ-potential also strongly determines the internalization of the microplastic particles into cells. Therefore, the ζ-potential can act as a proxy of microplastic-cell interactions and may govern adverse effects reported in various organisms exposed to microplastics.

A vasculitis centre based management strategy leads to improved outcome in eosinophilic granulomatosis and polyangiitis (Churg-Strauss, EGPA): monocentric experiences in 150 patients.

OBJECTIVE: To evaluate a vasculitis centre based management strategy for eosinophilic granulomatosis and polyangiitis (Churg-Strauss, EGPA). METHODS: A retrospective cohort study at a vasculitis referral centre was performed. All EGPA patients admitted from 1990 to 2009 were included. A structured interdisciplinary work-up for proof of diagnosis, Disease Extent Index and Birmingham Vasculitis Activity Score was performed. Immunosuppressive therapy was initiated and regularly adapted. Treatment targets were induction and maintenance of remission according to definitions given by the European League Against Rheumatism and the European Vasculitis Study Group. Outcomes were mortality, rate of remission, relapses, adverse events and prednisolone-dose. RESULTS: Out of 269 patients with suspected EGPA 150 fulfilled the inclusion criteria. Of those, 104 had more than one follow-up visit resulting in a mean follow up of 53±4.9 months. By using additional data sources the follow-up concerning survival was extended to 92±5 month. Severe organ manifestations occurred at heart (46%), kidney (18%) and lungs (10%). Cyclophosphamide was used in 107 patients (71%). The prednisolone-doses of all patients were within the targeted range (i.e. ≤7.5 mg) in 69% of the total follow-up time; the median dose at end of follow-up was 5mg/d. The 10-year survival rate was 89% resulting in mortality comparable to the general population (SMR 1.29). Only patients with cardiac failure associated with EGPA had an increased mortality (SMR 3.06). CONCLUSIONS: Regular re-evaluation and target-orientated adaption of therapy may lead to normalization of life expectancy and attenuation of disease progression. Continued centre based interdisciplinary treatment should be standard of care.

Cartilage destruction in granulomatosis with polyangiitis (Wegener's granulomatosis) is mediated by human fibroblasts after transplantation into immunodeficient mice.

A key feature of granulomatosis with polyangiitis (GPA; or Wegener's granulomatosis) is the granulomatous inflammation of the upper respiratory tract, which leads to the subsequent destruction of adjacent tissues. The aim of our work was to study the histopathological and cellular components of tissue destruction of human GPA tissue transplanted into immunodeficient mice. Biopsy specimens from patients with active GPA (n = 10) or sinusitis (controls, n = 6) were s.c. co-implanted with healthy allogeneic human nasal cartilage into immunodeficient pfp/rag2(-/-) mice. Transplants were examined for their destructive capability of the allografted human cartilage. In addition, nasal fibroblasts from patients with GPA (n = 8) and control healthy nasal fibroblasts (n = 5) were cultured, and cell proliferation and apoptosis were quantified. mRNA and protein levels of matrix metalloproteinases and cytokines were evaluated at baseline and after proinflammatory stimulation. GPA implants showed massive destruction of the co-implanted human cartilage, whereas cartilage destruction was only marginal in control samples. Destruction was mediated by human fibroblasts and could be inhibited by corticoid treatment. The up-regulated production of matrix metalloproteinases 1, 3, and 13 and cytokines IL-6 and IL-8 was found in vivo and in vitro. Although proliferation of isolated fibroblasts was comparable between GPA and controls, GPA samples showed a significant delay of apoptosis. The destruction of nasal cartilage in GPA is mainly mediated by fibroblasts that can be blocked by corticosteroids, and this tissue destruction is not dependent on the influx of leukocytes.

Progranulin antibodies in autoimmune diseases.

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis.

Toll-like receptor TLR2 and TLR9 ligation triggers neutrophil activation in granulomatosis with polyangiitis.

OBJECTIVE: The aim of the study was to characterize the expression of TLR2, TLR4 and TLR9 in PMNs of patients with granulomatosis with polyangiitis (GPA) and to elucidate the role of these receptors in GPA with respect to neutrophil activation. METHODS: The expression of TLR2, TLR4 and TLR9 was determined on ex vivo PMNs in whole blood samples of GPA patients (n = 35) and healthy controls (HCs) (n = 24). Isolated PMNs were stimulated in vitro with TLR agonists and assessed for degranulation, membrane proteinase 3 (mPR3) expression, soluble l-selectin shedding and cytokine production (IL-8) in five GPA patients and five HCs. The priming effects of TLR2 and TLR9 ligation were assessed by measurement of serine protease activity after stimulation with PR3-ANCA. RESULTS: There were no significant differences in the ex vivo expression of TLRs on PMNs in HCs and GPA patients. Stimulation of TLR4 and TLR9 induced MPO release, stimulation with TLR2, TLR4 and TLR9 ligands elicited IL-8 production and stimulation of TLR2 and TLR9 led to an upregulation in mPR3 expression on PMNs with no significant differences between GPA and HC after 1 or 24 h stimulation. Priming of PMNs with TLR2 and TLR9 ligands induced degranulation after subsequent stimulation with PR3-ANCA, which was comparable to priming with TNF-α. CONCLUSION: Expression of TLR2, TLR4 and TLR9 in PMNs and the TLR-induced activation of PMNs was comparable in GPA and HC. mPR3 upregulation by TLR2 and TLR9 stimulation and the priming effect of TLR ligands on PMNs may have a potential implication for triggering disease activity during infection in GPA.

Orbital masses in granulomatosis with polyangiitis are associated with a refractory course and a high burden of local damage.

OBJECTIVES: To identify and characterize patients with orbital masses in a monocentric cohort of 1142 GPA patients followed up from 1990 until the end of 2010 with regard to disease stage, local orbital inflammation, course of disease and outcome and to assess the efficacy of immunosuppressive treatment. METHODS: All GPA patients fulfilling ACR criteria or Chapel Hill Consensus Conference definitions or who had localized GPA and who developed orbital masses were evaluated regarding the course and outcome of the orbital masses (assessed by MRI, ophthalmologist and ENT specialist), all other clinical manifestations, disease stages, ANCA status, immunosuppression and its side effects and surgical procedures. RESULTS: Of 1142 GPA patients 58 developed orbital masses during a median follow-up of 101.5 months (range 23-255 months). Forty patients fulfilled the inclusion criteria and had complete clinical assessments [44% females, median age 43 (20-74) years, 85% ANCA positive]. Seventy-five per cent (29/40) had systemic disease when orbital masses occurred; both orbits were affected in 30%. Seventy-two per cent had evidence of infiltration from paranasal sinuses. Under highly potent immunosuppression (mostly CYC and glucocorticoids), 41% were refractory, 24% had unchanged activity, 24% showed a response and 8.1% had complete remission. Forty-four per cent had relapses of orbital masses. Seventy-two per cent developed visual impairment, 19% suffered blindness. Blindness was associated with a longer time to remission and a relapsing and refractory course. CONCLUSION: Orbital masses are a rare manifestation of GPA and are characterized by a refractory course and by a high rate of local damage. Patients with a refractory or relapsing course are at higher risk of developing blindness.

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.

Autophagy mediates survival of pancreatic tumour-initiating cells in a hypoxic microenvironment.

Involvement of dysregulated autophagy in cancer growth and progression has been shown in different tumour entities, including pancreatic ductal adenocarcinoma (PDA). PDA is an extremely aggressive tumour characterized by a small population of highly therapy-resistant cancer stem cells (CSCs) capable of self-renewal and migration. We examined whether autophagy might be involved in the survival of CSCs despite nutrition and oxygen deprivation typical for the hypoxic tumour microenvironment of PDA. Immunohistochemistry revealed that markers for hypoxia, CSCs and autophagy are co-expressed in patient-derived tissue of PDA. Hypoxia starvation (H/S) enhanced clonogenic survival and migration of established pancreatic cancer cells with stem-like properties (CSC(high)), while pancreatic tumour cells with fewer stem cell markers (CSC(low)) did not survive these conditions. Electron microscopy revealed more advanced autophagic vesicles in CSC(high) cells, which exhibited higher expression of autophagy-related genes under normoxic conditions and relative to CSC(low) cells, as found by RT-PCR and western blot analysis. LC3 was already fully converted to the active LC3-II form in both cell lines, as evaluated by western blot and detection of accumulated GFP-LC3 protein by fluorescence microscopy. H/S increased formation of autophagic and acid vesicles, as well as expression of autophagy-related genes, to a higher extent in CSC(high) cells. Modulation of autophagy by inhibitors and activators resensitized CSC(high) to apoptosis and diminished clonogenicity, spheroid formation, expression of CSC-related genes, migratory activity and tumourigenicity in mice. Our data suggest that enhanced autophagy levels may enable survival of CSC(high) cells under H/S. Interference with autophagy-activating or -inhibiting drugs disturbs the fine-tuned physiological balance of enhanced autophagy in CSC and switches survival signalling to suicide.