A new addition to our range that includes Craftsman and Art Deco collections, the hand forged Gingko leaf design was used extensively during this period, especially at the beginning, coming as it does from earlier craftsman-style designs; so you can quite appropriately use this firescreen in an Art Deco or Craftsman setting whilst a burnished version would really shake-up a modern setting too!. These beautifully etched leaves are delightfully detailed and faceted to produce rich shadowing. Together with the elegant stalks and buds they produce a stunning piece of art work that will enhance your room and entrance your guests. The elegant Gingko leaf design is entirely unbroken, as the firescreen is opened using a Ginko leaf latch.
See inset photo for freestanding version.
Hand Forged Iron Custom Made Design The Art Deco fireplace screen door brings the elegant lines of the signature style of the 20s and 30s to your demure home decor. Custom iron works and designer styling for the center of your attention on nights when fires enliven your atmosphere.


Chalkboard design bundle (PSD templates, design elements, hi-res chalkboard backgrounds) - in this mega collection, you'll find over 200 resizable drag / drop elements for all kinds of designs. Also there are 10 hi-res background boards, 3 grunge overlay .png's and 4 already pre-designed chalkboards.
• 50 download links for free fonts in *.DOC file - list of awesome vintage / hand / script / press / hipster fonts. Of course you can use any hand-written fonts instead.


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Music, Vocal Performance
Master's Emphasis
The Department of Music offers a Master of Music degree, with a variety of emphasis areas, including performance, conducting, and music education. Within the area of performance, students may select a vocal emphasis. This degree is designed to prepare individuals for professional advancement or for entry into further study at the doctoral level.
Awards for full-time graduate assistantships are available on a competitive basis. Recipients of these positions will take active leadership roles in the department's ensembles, teaching and accompanying private lessons, and/or serving as an assistant to the ensemble director.
About Master's Program
In addition to classroom and studio work, students in the vocal performance graduate program are afforded multiple performance opportunities ranging from opera and oratorio roles to solo recitals and chamber music concerts. Graduate voice students will also receive hands-on opportunities to develop their teaching skills including applied lessons, coursework, and assisting ensemble directors.
The exceptional voice area faculty at Pittsburg State University are dedicated to the development of their craft. As teachers, they are continuously involved in research to stay current with pedagogical development. Additionally, the voice faculty are active performers both regionally and nationally, appearing with critically ensembles and companies.
The voice area at PSU strives to maintain a competitive yet supportive performance learning environment. The departmental faculty collaboratively support students as they pursue their degrees.
Professional Singer
Studio Teacher
Preparation for pursuing a doctoral degree or other advanced training
Requirements for full admission into this degree program:
Meet Pittsburg State University Entrance Requirements (see below)
Audition and Interview
Complete Diagnostic Exams in Music Theory and Music History
About Music Department
The Department of Music at Pittsburg State University is an accredited member of NASM, the National Association of Schools of Music.
Our department averages 100 music majors and 40 minors, and 200 non-music majors per semester, and is staffed by 13 full-time and 9 part-time faculty members. Please explore our website for complete information concerning our programs, degrees, faculty, scholarship auditions, upcoming performances, and other areas of interest.
Locate the Music Department in McCray Hall using Google Maps or the PSU Campus Map.
Music Homepage

redpajama_set_name: RedPajamaCommonCrawl


The album peaked at number 5 on the Billboard 200 and sold over 100,000 copies in the US. It debuted at number 3 on the Billboard 200 and was an international commercial success primarily due to the hit "I'm Yours".
In 2008, Mraz released his third studio album, We Sing. The song peaked at number 6 on the Billboard Hot 100, giving him his first top ten single, and spent a then-record 76 weeks on the Hot 100.
I had to give this One to Nigel Blackwell because not only does he use that gloriously misinterpreted quote from Sartre as his title (sadly, Jean-Paul wasn't quite being as misanthropic as Nigel and I might wish), but also because this track contains (among various other hilarious ramblings) perhaps the best Half Man Half Biscuit line ever... Other Top Tesco songs (I could have done a Top Ten, but I'm waiting for the sponsorship deal to kick in) came from Lily Allen (LDN), The Pogues (Rain Street*), King Krule (Easy Easy) and Glass Animals (Life Itself).
Teenage Winter is possibly my favourite Saint Etienne track. This song is about the death of pop music as a physical object for teenagers to cherish.
"I went for the one thing that would separate me from everybody else," she says. With help from her beach-culture-hip father, she was soon living the "Blue Crush" dream, taking fifth place in a statewide heat and earning endorsements from companies like Volcom and Nixon watches, for whom she recently shot a series of print ads.
Just like in the Kate Bosworth flick "Blue Crush," surfer chicks — Prettyman was chagrined to discover — experience a good deal of sexism.
"Because it doesn't matter how many times you go out or how good you are one day.
The ocean is always testing you." Life, too, seemed to be testing Prettyman.


Dead Island Definitive Edition boasts many improvements over the original 2011 release including improved lighting and shadow effects and increased texture resolutions to name but a few. Take a look at this comparison video which shows the differences across the same locations. Both versions are running on PC at max settings.
Dead Island Definitive Collection is available from May 31st on PC, Xbox One, PS4 and includes remastered Dead Island, Dead Island Riptide and the bonus arcade game Dead Island Retro Revenge.


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Home / Movies / The 'Rambo: Last Blood' Teaser Trailer, The Rock Is Very Much Excited For This Movie
The 'Rambo: Last Blood' Teaser Trailer, The Rock Is Very Much Excited For This Movie
By FizX on May 30, 2019@fizx
Sylvester Stallone is coming back in one of his long-standing roles. John Rambo, the Vietnam veteran who first grunted his way onscreen in 1982's First Blood, is back for one "last" fight.
That's what one could expect from the name, Rambo: Last Blood, it is the fifth movie of the franchise. And who was more excited ahead of this teaser trailer's arrival? Dwayne "The Rock" Johnson. He saw one of Stallone's behind-the-scenes Instagram clips and couldn't resist commenting: "Drop the spear, dive and get ready to cut his leg off". F*ck. Yes.
This is how it in action scene is set up. Presently we are underground in a cave, obviously it's very dusty and difficult to breathe but it's real … #rambo5
A post shared by Sly Stallone (@officialslystallone) on May 27, 2019 at 3:24am PDT
The Rock's watching the above teaser on repeat while hitting the gym, but what we see mostly plays up the already teased modern-day Western approach of this sequel. Rambo warns that destruction is coming for all of his enemies, and the movie's synopsis ensures that Rambo "teams up with a reporter to save a kidnapped girl from a vicious drug cartel in Mexico."
Related ItemsmovieramboRAMBO: LAST BLOODtrailer


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Plea on ancient grass trees
Bagged grass trees were mostly dead after the hot spell.
By Catherine Watson
BASS Coast Shire Council has entered the battle to preserve Grantville's magnificent grass tree forest by seeking an assurance that mining work orders protect Bass Coast's distinctive native vegetation and endangered fauna.
Community members who visited the Grantville Nature Conservation Reserve last weekend were shocked to see more than 250 grass trees, many pre-dating European arrival, had been removed and bagged and were now dying on the edge of a Sand Supplies pit.
The destruction of the grass trees was raised as an urgent motion at the first meeting of the new council on Wednesday.
Councillors voted unanimously in support of a six-part motion moved by Rochelle Halstead, the new councillor for Western Port ward, to try to save the grass trees that have already been removed.
The council support is significant as the community gears up to protect the remnant Western Port coastal forest from an influx of sand mining companies, encouraged by the State Government to secure sand for Melbourne's urban expansion. Proposed planning provisions would remove the right of communities, farmers and local government to object to sand mining.
"They're ripping the guts out of the country," Cr Michael Whelan said. "We can't continue to tear down buildings and put them into landfill and then tear up pristine bush to make new buildings. It doesn't make sense."
After the meeting, Mayor Brett Tessari said there was a limited amount the council could do in a legal sense.
"We need to continue to communicate with the owners of the sand mine. [Robbie Viglietti] has previously been very community minded. If we can continue to work with him, hopefully we can get a result that works for everyone. But we need the state to step up and lead on this."
The Gurdies resident Meryl Tobin, who has long campaigned to save the grass tree forest, called for urgent intervention by the Stater Government and the Minister for the Environment, Lily D'Ambrosio.
The Sand Supplies mine sits on a mining lease that carves into the Grantville Nature Conservation Reserve. Many of the work orders allowing mining in the Grantville forest corridor date back to the Kennett era and earlier.
The forest affected, running from the Adams Creek Nature Conservation Reserve (Lang Lang) to Grantville is the last significant stand of riparian coastal forest in the whole of Western Port and the only coastal forest remaining in the Bass Coast region.
Sand mine motion
That Council act as a matter of urgency to ensure the grass trees removed at the Grantville Sand Quarry site are handled and cared for in an appropriate manner to ensure they are kept healthy and able to be replanted at the earliest possible time.
That Council seek to have on record the details of the rehabilitation bond and work plan held and monitored by the Earth Resources Regulator for the Grantville Sand Quarry and assess whether the work order appropriately protects our region's distinctive native vegetation and endangered flora. That council seek to include as part of the review any presence of endangered populations of wildlife.
The Earth Resources Regulator confirm the rehabilitation bond being held for the Grantville Sand Quarry is sufficient to ensure full rehabilitation of the site
Request that rehabilitation work to any used and closed sections of the site begin immediately
Council establish through the relevant Victorian State Government Authority what assessments for any Aboriginal or Cultural Heritage Values of the Grantville Sand Quarry site have been undertaken to date and to liaise with the Bunurong Land Council to discuss the significance of the site and what further action is required.
Council seeks to confirm with the Victorian State Government that the ERR has demonstrated that it can effectively discharge its regulatory functions by appropriately balancing its commitment to the Minister for Resources under statements of expectations 2018-20 and the commissioner for better regulations, getting the ground work right to reduce the regulatory burden on operators by facilitating and streamlining approval and regulatory processes giving effect to the objective of the act, that risks to the environment are identified and eliminated as set out in the Auditor-General's inquiry into the determination of rehabilitation bonds for the mining in the state.
Save Western Port's Coastal Forest spokesman Tim O'Brien said multiple quarries operate under historic sand mining leases in the environmentally-sensitive Grantville forest corridor. Several of the mine operators have plans to expand their operations.
"It beggars belief that the State Labor Government seems prepared to put the tourism industry at risk in this important region, the gateway to Phillip Island and its world-famous Penguin Parade, the gateway to the spectacular Bunurong coastline, and an area renowned for its rural amenity and distinctive landscapes.
"Worse, that it is prepared to trade the extinction of fragile communities of endangered wildlife and ancient, rare, coastal flora, for carparks and bridges in Melbourne, is a gross failure of public policy.
"The question for Environment Minister D'Ambrosio is when is a conservation area actually for conserving, and a biolink corridor actually for protecting, and not just a resource to be ravaged by sand miners?"
Mr O'Brien said there should be no further expansion of any of the mines in the fragile forest corridor and a review of all existing work orders in sand mining leases.
"Where endangered populations of flora and fauna are identified, the Victorian government must begin a process of extinguishment of leases.
"At the end of the day, this mine is operating under a lease. The forest belt the miners are pillaging – the endangered flora and fauna, the ancient grass trees – belong to the people."
Catherine Watson is a member of Save Western Port's Coastal Forest.


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The role of IFN-γ in regulation of IFN-γ-inducible protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures
Maria Torvinen1,
Hinnah Campwala1 &
Iain Kilty1
Interferons play a critical role in regulating both the innate and adaptive immune responses. Previous reports have shown increased levels of IFN-γ, IFN-γ-inducing IL-12 and IFN-γ-inducible chemokine IP-10 in patients with chronic obstructive pulmonary disease (COPD).
The present study focuses on the regulation of the IP-10 secretion in co-cultures of lung epithelial cells and peripheral blood mononuclear cells (PBMCs).
No IP-10 secretion was detected in cells cultured alone, whereas a significant increase in IP-10 levels was observed in epithelial cell/PBMC co-cultures. Furthermore, the results show that interactions between lung epithelial cells, lymphocytes and monocytes are needed for basal IP-10 secretion. Interestingly, we have also shown that incubation with IL-12 can induce an IFN-γ independent increase in IP-10 levels in co-cultures. Furthermore, inhibition studies supported the suggestion that different intracellular pathways are responsible of IFN-γ and IL-12 mediated IP-10 secretion.
These studies demonstrate a novel diversity in IFN-γ/IL-12 pathways, showing that the IP-10 expression in co-cultures is regulated by multiple factors, such as intercellular interactions in addition to IFN-γ and IL-12 levels. These results may be valuable in designing novel strategies to antagonize IP-10 mediated immunological reactions and chemotactic effects on T cells.
Multiple inflammatory cells, mediators, and proteases are involved in the pathophysiology of COPD. It is characterized by chronic inflammation primarily in the small airways and lung parenchyma, with increased numbers of macrophages, neutrophils and T lymphocytes in comparison to healthy controls [1]. T helper (Th) lymphocytes can be classified into two types depending on the secreted cytokines. Th1 cells are mainly involved in cell-mediated inflammatory reactions and in development of chronic inflammatory conditions, whereas Th2 cells enhance antibody production by B cells and are prominent in the pathogenesis of allergic diseases [2, 3]. A bias towards a Th1 cell profile has been hypothesized in COPD, with Th1/T cytotoxic 1 (Tc1) pattern and increased Th1 cytokine levels [1].
Th1 cells secrete IL-2, IL-12, and IFN-γ, which has been shown to regulate Th mediated immune and allergic responses by inducing Th1 differentiation. IFN-γ secretion from natural killer (NK) cells and monocytes/macrophages is likely to be important in early host defence against infection, whereas T lymphocytes become the major source of IFN-γ in the adaptive immune response [2, 3].
IFN-γ-inducible protein 10 (IP-10) is induced by IFN-γ in many types of cells including monocytes and lung epithelial cells [4, 5]. IP-10, also named CXCL10, is a potent chemokine for activated T lymphocytes and regulates cell proliferation, apoptosis and adhesion molecule expression [6]. Previous studies have shown that physical interactions between cells grown in co-cultures induce IP-10 secretion; between endothelial cells (EnC)/monocytes [7], EnC/alloantigen-primed T cells [8], EnC/PBMCs [9], leucocytes/synoviocytes [10] as well as human bronchial epithelial cell (BEAS-2B)/eosinophils [11]. The increased IP-10 secretion in BEAS-2B/eosinophil co-cultures was regulated by p38 MAPK and NF-kappaB activities of BEAS-2B cells, at least partly via intercellular contact [11].
IP-10 binds to a G protein coupled receptor CXCR3 that is preferentially expressed on Th1 type cells, causing chemotaxis of these cells towards this chemokine [12]. CXCR3 is also expressed by many cell types including lung epithelial cells [13, 5, 14] and it has been shown to be involved in epithelial cell movement via p38 MAPK and PI3K dependent signalling pathways in human airway epithelial cells (HAEC) [15]. Furthermore, HAEC have also been shown to release IP-10 as well as express CXCR3, suggesting the potential for autocrine signalling [14].
IFN-γ-inducing cytokine IL-12 is produced by many cell types including monocytes/macrophages, and neutrophils. The major actions of IL-12 are on T cells, resulting in induction of Th1 differentiation, proliferation, IFN-γ production and increased cytotoxic activity. [16] Th1 cytokine phenotype has been demonstrated in peripheral blood [17] and in lung portions removed surgically from patients with COPD [18]. Furthermore, increased IL-12 levels have been shown in patients with COPD [19, 20]. Relative expression levels of IFN-γ in COPD patients are variable, with previous studies having shown an increase [19, 18], decrease [21] or no change [22] in IFN-γ secretion in COPD patients compared with controls. Enhanced IP-10 secretion [23, 18, 24] as well as expression of the IP-10 receptor CXCR3 [23] have been demonstrated in COPD. As shown by Saetta et al. (2002), most of the CXCR3 positive cells in peripheral airways in patients with COPD were CD8+ positive T cells and produced IFN-γ [23].
The present study focuses on the regulation of the IP-10 secretion. The aim was to investigate the pathways of IP-10 secretion in a in vitro system including the cell types most likely involved in the IP-10 secretion in the lung tissue of COPD patients. Although several studies have demonstrated an increased IP-10 secretion via intercellular contact, little is known of the regulation of the Th1 IFN-γ/IL-12 pathway upon intercellular interaction between lung epithelial cells and leucocytes. Since increased activity of the IFN-γ/IL-12 pathway as well as increased levels of IP-10 in COPD is most likely due to a complex interaction between lung epithelial cells and white blood cells, we decided to investigate the role of the IFN-γ/IL-12 pathway on IP-10 secretion upon the interaction of peripheral blood mononuclear cells with two human lung epithelial cell lines, A549 (alveolar epithelial cell line), Calu-3 (bronchial epithelial cell line) in addition to primary normal human bronchial epithelial (NHBE) cells.
Maintenance of human epithelial cell lines
Cells from a human bronchial epithelial cell line (Calu-3) and from a human alveolar epithelial cell line (A549) were used for the present studies. Both cell lines were cultured routinely at 37°C with 5% CO2 in Minimum essential medium (MEM) with Earle's Salts and L-Glutamine (Invitrogen) supplemented with 10% of heat-inactivated foetal calf serum (FCS) (PAA Laboratories), 1.5% sodium bicarbonate solution, (Sigma-Aldrich), 10 mM Sodium pyruvate solution (Sigma-Aldrich), 1× MEM non-essential amino acid solution (Sigma-Aldrich) and 1× Primocin (Autogen Bioclear) in cell culture polystyrene flasks with vent caps (Corning). The splitting of cell cultures was performed by replacing the medium with cell dissociation solution (Sigma-Aldrich). Both cell lines were used up to 32 passages.
Maintenance of normal human bronchial epithelial cells
Normal Human Bronchial Epithelial Cells (NHBEs) were cultured according to the manufacturer's instructions (Cambrex, Inc.). However, during the experiment and the co-culture conditions, the NHBEs were transferred to the Minimum essential medium (MEM) with Earle's Salts and L-Glutamine (Invitrogen) supplemented with 1% foetal calf serum (FCS) (PAA Laboratories).
Peripheral blood mononuclear cell (PBMC) Isolation
PBMCs were obtained from healthy non-smoking and smoking adult volunteers. Usage of human blood for the present studies was approved by the local ethical committee, and the informed consent of all participating subjects was obtained. The venous blood was collected into 50 ml centrifuge tubes each containing 5 ml of Hank's Balanced Salt Solution (HBSS) (Sigma-Aldrich) with 2.7% (w/v) Hepes (Sigma-Aldrich). The blood sample was diluted 1:1 with modified Dulbecco's phosphate buffered saline (PBS) without calcium chloride and magnesium chloride (Sigma-Aldrich). PBMCs were isolated with density centrifugation with ACCUSPIN™ System-HISTOPAQUE-1077 tubes (Sigma-Aldrich) at 400 g for 35 minutes at room temperature. Following centrifugation the layer containing the PBMCs (according the manufacturer's instructions) was collected, resuspended in PBS and centrifuged at 200 g for 10 minutes at room temperature. The supernatant was discarded and PBMC-rich pellet was resuspended in cell media (See above in Maintenance of Human Epithelial Cell Lines) with 1% FCS. A differential cell count was performed using a Beckman-Coulter Act5diff haematology analyzer to determine total cell number and the purity of the cell preparation. This method typically yields a cell suspension containing 80–95% of lymphocytes and 5–20% monocytes. The cells were resuspended in cell media with 1% FCS to 1 million white blood cells/ml and plated in 48 well cell culture polystyrene clusters (Corning) and cultured with or without A549 or Calu-3 cells.
Conditioned media and transwell studies
PBMCs and lung epithelial cells were cultured alone in cell media with 1% FCS for 18 hours. The cells were centrifuged at 200 g for 5 minutes and the supernatant was collected, filtered with sterile 0.22 um filters and frozen at -80°C. For the experiments, PBMCs or lung epithelial cells were resuspended in the conditioned media and cultured for 18 hours.
For transwell studies, lung epithelial cells were grown to 80% confluency (approximately 1 × 105) on 12 well transwell chambers (Corning). Subsequently lung epithelial cells and 5 × 105 PBMCs are co-cultured (1,5 ml/well) for 18 hours in transwell chambers separated by a filter (0.4 μM pore size) or not (as control), where-after the supernatant was collected for IP-10 and IFN-γ ELISA analysis.
Isolation of lymphocytes from PBMCs
After resuspension in 1% FCS cell media to 1 × 106 white blood cells/ml (see above in Peripheral Blood Mononuclear Cell (PBMC) Isolation) the PBMCs were plated cell culture polystyrene flasks for 1 hour in 37°C with 5% CO2. Since monocytes attach to the plastic whereas lymphocytes stay in suspension, the supernatant was collected after 1 hour and centrifuged at 200 g for 5 minutes. A differential cell count was performed using a Beckman-Coulter Act5diff haematology analyzer to determine total cell number and the purity of the cell preparation. This method typically yields a cell suspension containing 99–100% of lymphocytes.
Isolatation of monocytes from PBMCs with MACS
PBMCs are incubated with anti-human CD14 antibody conjugated to super-paramagnetic microbeads (Miltenyi Biotec). Labelled suspensions are passed through a depletion column in the magnetic field of a MACS separator (Miltenyi Biotec) according to the manufacturer's instructions. A differential cell count was performed using a Beckman-Coulter Act5diff haematology analyzer to determine total cell number and the purity of the cell preparation. This method typically yields a cell suspension containing 70–100% of monocytes with a contamination range between 0–30% of lymphocytes. Purity of 88%–100% of monocytes was set as acceptable range for the present studies with monocyte/lung epithelial cell co-culture studies.
Interferon and chemokine ELISA assays
Human IP-10 and IFN-γ levels were specifically quantified with human IP-10 CytoSets™ and human IFN-γ CytoSets™ assays (Biosource). The epithelial cell lines were grown into 80% confluency before the experiments whereas the PBMCs were cultured at the density of 1 × 106 cells/ml. The cultures were performed in 48 well clusters with 0.5 ml cell media (500 000 PBMCs/well) with or without A549, Calu-3 and NHBEs. The epithelial cell lines and PBMCs were cultured either alone or in co-culture in 48 well clusters for 18 hours in cell media (see above) with 1% FCS before the ELISA assay. Pretreatments were performed with an addition of human recombinant IL-12 (100 ng/ml) (eBioscience) or human recombinant IFN-γ (0.1–10 ng/ml) (eBioscience) for 18 hours. Potential inhibitors 100 nM p38 inhibitor BIRB796, 500 nM IKK-2 inhibitor V, (Calbiochem), 100 nM beclomethasone (SigmaAldrich), 1 μM PI3 kinase inhibitor (Novartis, characterised as PIK 93 in [25]), 100 nM PDE4 inhibitor Rolipram (SigmaAldrich)), 5 μg/ml human IFN-γ antibody (Serotec MCA1554XZ) and 10 μg/ml human CD40 ab from (Serotec, MCA1590XZ), were added one hour before addition of IL-12 or IFN-γ. The chosen concentration for the inhibitors were roughly 10× IC50 from present and previous studies. All ELISA assays were performed according to the manufacturer's instructions. Maxisorp 96 well microplates (Nunc) were used for the assays and Skanwasher 300 (Skatron Instrument) was used to wash the microplates with 0.01 M PBS with 0.05% Tween 20 (pH 7.4) as the wash buffer. The results were read with microplate reader (SpectraMax 250) at 450 nm.
All data are expressed as mean (ng/ml) ± SEM. All data were transformed into logarithmic data before the statistical analysis and compared with analysis of variance (ANOVA). The means of groups whose variances were determined to be significantly different were then compared by Bonferroni's multiple comparison test using GraphPad Prism (GraphPad Software Inc., San Diego, CA).
Basal and IFN-γ mediated IP-10 secretion from PBMC/lung epithelial cell co-cultures
IP-10 levels in cell culture medium collected after 18 hours from PBMCs, Calu-3, A549 and PBMC/lung epithelial cell co-cultures were measured with ELISA. When plated alone, very little secretion of IP-10 was detected from unstimulated PBMCs and lung epithelial cell lines (Figure 1. and 2.). However, significantly increased IP-10 secretion was detected in lung epithelial cell/PBMC co-cultures (Figure 1.). The secretion from A549/PBMC co-cultures was significantly higher than Calu-3/PBMC co-cultures (p < 0.01, 0.88 ± 0.19 ng/ml and 0.22 ± 0.07 ng/ml, respectively).
Basal and IFN-γ mediated secretion of IP-10 in lung epithelial cell/PBMC co-cultures. Data represent the mean ± SEM of 4 independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05 compared to without IFN-γ treatment; ¤¤¤p < 0.001, ¤¤p < 0.01, ¤p < 0.05 for each concentration of recombinant IFN-γ treatment in co-cultures compared to both PBMCS and respective lung epithelial cell lines cultured alone, ANOVA with Bonferroni's multiple comparison test.
IL-12 (100 ng/ml) mediated secretion of IP-10 in lung epithelial cell/PBMC co-cultures. Data represent the mean ± SEM of 7–14 independent experiments, **p < 0.01, ANOVA with Bonferroni's multiple comparison test.
Pretreatment with recombinant IFN-γ for 18 h induced a small dose-dependent increase in IP-10 secretion in PBMCs cultured alone, whereas no detectable levels of IP-10 were found in either Calu-3 or A549 cultured alone (Figure 1). However, IFN-γ (0.1–10 ng/ml) induced a significant dose dependent increase in IP-10 secretion in lung epithelial cell – PBMC co-cultures as shown in Figure 1.
IL-12 induces endogenous IFN-γ secretion in PBMC/A549 co-cultures
The presence of endogenous IFN-γ in supernatants collected after 18 hours from PBMCs, lung epithelial cell lines as well as in co-cultures was studied with ELISA. No detectable levels of IFN-γ were shown in either un-stimulated cells cultured alone or in co-cultures (Table 1). 18 hours incubation with recombinant IL-12 (100 ng/ml) did not induce any detectable secretion of endogenous IFN-γ in PBMCs, lung epithelial cell lines alone nor Calu-3/PBMC co-cultures (Table 1.) However, a significant increase in endogenous IFN-γ secretion was shown in A549/PBMC co-cultures after IL-12 treatment (Table 1). To establish the cell type in PBMCs interacting with the A549 cell line, secretion of IFN-γ was studied in lymphocyte/A549 and monocyte/A549 co-cultures. As shown in Table 1., lymphocytes exclusively interact with A549 resulting in a significant induction of IFN-γ secretion upon IL-12 stimulation.
Table 1 Secretion of IFN-γ in lung epithelial cell lines and PBMCs.
IL-12 induces IP-10 secretion in PBMC/lung epithelial cell co-cultures
18 hours preincubation with IL-12 did not modulate IP-10 secretion from cells cultured alone (Figure 2.). However, a significant increase in IP-10 secretion was observed in both Calu-3/PBMC and A549/PBMC co-cultures upon IL-12 pretreatment, as seen in Figure 2.
IL-12 and IFN-γ co-treatment in co-cultures
The effects of IL-12 (100 ng/ml) and IFN-γ (1 or 10 ng/ml) co-treatment on IP-10 secretion was studied in Calu-3/PBMC and A549/PBMC co-cultures. No additional increase in IP-10 secretion was observed with IL-12 and IFN-γ co-treatment in A549/PBMC co-cultures compared with IL-12 or IFN-γ treatment alone (IP-10 secretion 3.7 ± 0.5 ng/ml (IL-12 100 ng/ml), 3.5 ± 7 ng/ml (IFN-γ 1 ng/ml and 3.9 ± 0.7 ng/ml (IL-12 100 ng/ml+ IFN-γ 1 ng/ml)). However, in Calu-3/PBMC co-cultures, the secretion of IP-10 induced by IL-12 pretreatment was significantly lower compared with IFN-γ induced IP-10 secretion (IP-10 secretion 1.3 ± 0.2 ng/ml (IL-12 100 ng/ml), 2.6 ± 0.4 ng/ml (IFN-γ 1 ng/ml and 3.0 ± 0.7 ng/ml (IL-12 100 ng/ml+ IFN-γ 1 ng/ml), which might be explained by the absence of IL-12 mediated induction of endogenous IFN-γ secretion when compared with A549/PBMC co-cultures (See also Table 1).
Effects of IFN-γ antibody on IP-10 secretion
Treatment with 5 μg/ml IFN-γ antibody (ab) significantly inhibited the basal IP-10 secretion in both Calu-3/PBMC and A549/PBMC co-cultures (Figure 3.). The significant increase of IP-10 secretion in co-cultures mediated via recombinant (1–10 ng/ml) IFN-γ treatment was also inhibited by the IFN-γ ab treatment. However, the IL-12 induced increase in IP-10 levels was not inhibited by the IFN-γ ab, showing that at least a component of IL-12 mediated IP-10 increase is IFN-γ independent (Figure 3.).
Inhibition of IP-10 secretion in lung epithelial cell/PBMC co-cultures by human IFN-γ antibody.
Studies with conditioned media (CM) showed that lung epithelial cells are secreting a factor which augments IFN-γ mediated IP-10 secretion from PBMCs. PBMCs cultured with 10 ng/ml IFN-γ in CM from either Calu-3 or A549 cells induced a significant increase in IP-10 secretion compared with PBMCs cultured with IFN-γ (Figure 4.) The IP-10 is secreted by monocytes, since lymphocytes cultured with CM media from epithelial cells did not induce any IP-10 secretion (data not shown).
The effects of conditioned media (CM) on IP-10 secretion from cells cultured alone. The lung epithelial cells or PBMCs were cultured for 18 hours in CM simultaneously with either IFN-γ (10 ng/ml) or IL-12 (100 ng/ml) incubation. PBMCs cultured alone were used as control. Data represent the mean ± SEM of 4–6 independent experiments, *p < 0.05, **p < 0.01, ANOVA with Bonferroni's multiple comparison test.
Furthermore, a secreted factor from Calu-3 cells augments IL-12 mediated IP-10 secretion from PBMCs. PBMCs cultured with 100 ng/ml IL-12 in CM from Calu-3 but not from A549 cells induced significant increase in IP-10 secretion compared with PBMCs cultured with IL-12 (Figure 4). IP-10 is secreted by monocytes, since lymphocytes cultured with CM media from Calu-3 did not induce any IP-10 secretion (data not shown).
No detectable levels of IP-10 were secreted by lung epithelial cells cultured in CM from PBMCs with or without IL-12 or IFN-γ treatment (Figure 4). Moreover, IL-12 treatment did not induce any detectable IFN-γ secretion from either PBMCs or A549 cells cultured in CM from A549 cells or PBMCs, respectively (data not shown).
Transwell studies confirmed the results from conditioned media studies, as can be seen in Figure 5. The co-cultures were grown in transwell chambers separated (or not as control) by a filter. There is an increased IP-10 secretion in the presence of IFN-γ in co-cultures and a slight increase after IL-12 treatment (See Figure 5.). However, the basal, IFN-γ and IL-12 induced secretion of IP-10 in co-cultures is significantly decreased when separated with filter as compared to controls (Figure 5). These results confirm the results from conditioned media studies but also show that cell-cell interactions are likely to play an important role in IP-10 secretion in PBMCs/lung epithelial cell co-cultures.
Basal, IFN-γ and IL-12 mediated secretion of IP-10 in lung epithelial cell/PBMC co-cultures cultured in transwell chambers with separating filters. Data represent the mean ± SEM of 4 independent experiments. Control (□) values are shown as IP-10 secretion in lung epithelial cell/PBMC co-cultures cultured in transwell chambers without a separating filter.
However, endogenous IFN-γ secretion in lymphocyte/A549 co-cultures after IL-12 treatment was high (0.90 ± 0.45, mean (ng/ml) ± SEM, n = 3) even with separating filter, showing that although a co-culture of lymphocytes and A549 cells is necessary for the secretion of IFN-γ, no actual cell-cell contact is required.
Studies with monocyte or lymphocyte/lung epithelial cell co-cultures
Neither basal nor IFN-γ mediated secretion of IP-10 was observed in A549/lymphocyte or Calu-3/lymphocyte co-cultures (data not shown) Treatment with IL-12 did not increase IP-10 levels in lymphocyte-Calu-3 co-cultures and only modest IP-10 secretion was observed in lymphocyte/A549 co-cultures (0.006 ± 0.005 and 0.103 ± 0.209 ng/ml, respectively, n = 3).
Furthermore, low basal increase of IP-10 secretion was observed in both Calu-3/monocyte and A549/monocyte co-cultures (0.2 ± 0.1 and 0.3 ± 0.07 ng/ml, respectively, n = 5) compared with Calu-3/PBMCs and A549/PBMCs co-cultures (1.0 ± 0.3 and 3.0 ± 1.0 ng/ml, respectively, n = 5), showing that the interactions between all three cell types, monocytes, lung epithelial cells and lymphocytes, are crucial for the basal secretion of IP-10. However, treatment with recombinant IFN-γ increases IP-10 secretion in monocyte/lung epithelial cell co-cultures in the absence of lymphocytes (Calu-3/monocyte and A549/monocyte co-cultures (2.6 ± 0.5 and 2.7 ± 0.7 ng/ml, respectively, n = 5).
Inhibition of IP-10 secretion from PBMC/lung epithelial cell co-cultures
P38 inhibitor BIRB796, IKK-2 inhibitor V, beclomethasone, PDE4 inhibitor rolipram and PI3 kinase inhibitor strongly and significantly inhibited basal IP-10 secretion from PBMC and lung epithelial cell co-cultures (Table 2 and Figure 6.).
The dose dependent inhibition of IP-10 secretion by PI3 kinase inhibitor in Calu-3/PBMCs (A.) and A549/PBMCs (B.) co-cultures with or without IFN-γ and IL-12 treatment. The dose dependent inhibition by PI3 kinase inhibitor of IL-12 mediated IFN-γ secretion in A549/PBMCs co-cultures is seen in (C.) Data represent the mean ± SEM of 4–7 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA with Bonferroni's multiple comparison test.
Table 2 Inhibition of IP-10 secretion in co-cultures. The effects of 100 nM p38 inhibitor BIRB-796, 100 nM beclomethasone, 500 nM IKK-2 inhibitor V, 100 nM PDE4 inhibitor rolipram, 1 μM PI3 kinase inhibitor and human 10 μg/ml CD40 antibody on secretion of IP-10 from co-cultures.
The IFN-γ (10 ng/ml) mediated IP-10 secretion in both A549/PBMC co-cultures and Calu-3/PBMC co-cultures was dose dependently inhibited by the PI3 kinase inhibitor (Figure 6). In contrast, IL-12 mediated secretion of IP-10 in Calu-3/PBMC co-cultures was significantly inhibited by BIRB796, beclomethasone and rolipram (see Table 2). However there is a clear difference with the A549/PBMC co-culture, whereby IL-12 mediated IP-10 secretion was partially inhibited by beclomethasone and PI3 kinase inhibitor only (see Table 2). Human CD40 antibody (10 μg/ml) did not have any effects on IP-10 secretion in co-cultures (Table 2).
Inhibition of IFN-γ secretion from PBMC/A549 co-cultures
IL-12 mediated IFN-γ secretion in PBMC/A549 co-cultures was inhibited significantly by p38 inhibitor BIRB796, beclomethasone, and PI3 kinase inhibitor as seen in Table 3 and Figure 6.
Table 3 Inhibition of IFN-γ secretion in co-cultures. The effects of 100 nM p38 inhibitor BIRB-796, 100 nM beclomethasone, 500 nM IKK-2 inhibitor V, 100 nM PDE4 inhibitor rolipram, 1 μM PI3 kinase inhibitor and human 10 μg/ml CD40 antibody on secretion of IP-10 from co-cultures.
IP-10 secretion in PBMC/NHBE co-cultures
As shown in Figure 7. no basal IP-10 secretion was observed in PBMC/NHBE co-cultures. IFN-γ treatment significantly increased IP-10 secretion from NHBEs and PBMCs cultured alone. Interestingly, IFN-γ mediated IP-10 secretion was significantly increased in co-cultures compared to the PBMCs and NHBEs cultured alone in agreement with the A549/PBMC and Calu-3/PBMC co-cultures (See Figure 7).
Basal and IFN-γ mediated secretion of IP-10 in NHBE/PBMC co-cultures. Data represent the mean ± SEM of 4 independent experiments, ***p < 0.001, *p < 0.05 with ANOVA.
IP-10 was initially identified as IFN-γ inducible protein [26], which was shown to be a potent chemokine for Th1 cells. Its receptor CXCR3 is predominantly expressed by Th1 cells [18] but expression has also been shown in many other cell types including lung epithelial cells [5]. Increased levels of both IP-10 and CXCR3 have been shown in patients with COPD, and subsequently this chemokine has been suggested to be involved in the inflammatory process underlying COPD [23].
The aim of the present studies was to examine the effects of lung epithelial cells/PBMCs interaction on IP-10 secretion. We used PBMCs from both non-smoking and smoking volunteers since COPD is a smoking related disease. However, no differences were found in IP-10 secretion from PBMCs between non-smokers and smokers (results not shown). This is likely due to the fact that all volunteers used in the present study are healthy. However, at the present studies we characterize the complex interaction between PBMCs and lung epithelial cells on the regulation of IP-10 secretion by IFN-γ/IL-12 pathways. No basal secretion of IP-10 was observed in either cell type cultured alone, however, a significant increase of basal IP-10 secretion was observed in PBMC/lung epithelial cell co-cultures. The IP-10 secretion was found to be due to a specific interaction between monocytes and lung epithelial cells via cell-cell contact (Figure 8a), since no basal IP-10 secretion was detected in PBMC/lung epithelial cell transwell co-cultures. Surprisingly, no IP-10 secretion was observed in monocyte/lung epithelial cell co-cultures in the absence of lymphocytes. Since addition of recombinant IFN-γ could restore the elevated IP-10 secretion in monocyte/lung epithelial cell co-cultures, the significance of the lymphocytes in co-cultures is most likely to the source of endogenous IFN-γ (Figure 8b). A similar mechanism might also be involved in EnC/PBMC co-cultures studied by Raju et al. (2003) demonstrating that the basal secretion of IP-10 from EnC/PBMC co-cultures is IFN-γ dependent [9]. Therefore, it is likely that the increased amounts of leucocytes in lung tissue in COPD patients interact with several cell types including lung epithelial cells as well as endothelial cells in a similar manner increasing IP-10 secretion.
In summary, basal IP-10 secretion is induced by monocyte-epithelial cell interactions, with a presence of lymphocytes, most likely to provide a source of IFN-γ. The interaction of monocytes and lung epithelial cells are made by direct cell-cell contact. (A). Addition of recombinant IFN-γ induces strong IP-10 secretion in co-cultures even in absence of lymphocytes. Moreover, a secreted factor from lung epithelial cells augments the IFN-γ mediated secretion of IP-10 from monocytes (B). Addition of recombinant IL-12 induces IFN-γ independent IP-10 secretion in Calu-3/PBMC co-cultures which cannot be blocked by IFN-γ antibodies. Moreover, no detectable IFN-γ is present and Calu-3 cells secrete a factor which augments IP-10 secretion from monocytes in response to IL-12 (C). Addition of recombinant IL-12 induces IP-10 secretion both by inducing IFN-γ secretion from lymphocytes and by an IFN-γ independent pathway, which cannot be blocked by IFN-γ antibodies (D).
However, there are crucial differences in the IP-10 secretion from different types of co-cultured cells. In our study CD40 is not involved in the cell-cell interaction dependent basal IP-10 secretion, whereas CD40 has been reported to mediate IP-10 secretion in EnC/monocyte co-cultures [7]. Moreover, antibodies against ICAM, CD11b and CD18b have been used to show the importance of these proteins in leucocyte/synoviocyte IP-10 induction [10].
IP-10 is classically induced by IFN-γ, however, in the present studies no detectable basal secretion of IFN-γ was observed in the co-cultures. Nevertheless, antibodies against IFN-γ blocked the IP-10 secretion from co-cultures, suggesting that low levels of endogenous IFN-γ, undetectable with ELISA, are present in co-cultures The detection range for the IFN-γ ELISA is from ~0.015–1 ng/ml. The lowest detectable concentration would not be able to stimulate IP-10 secretion in PBMC cultures, since we did not detect any IP-10 secretion with 0.1 ng/ml IFN-γ (Figure 1). However, as shown in Figure 1, addition of 0.1 ng/ml IFN-γ strongly augments basal IP-10 secretion in Calu-3/PBMC co-cultures, which did not secrete any detectable levels of endogenous IFN-γ, suggesting that even a very low concentration of endogenous IFN-γ can induce strong IP-10 secretion when there are direct cellular interactions between monocyte and lung epithelial cells. The increasing concentrations (0.1–10 ng/ml) of IFN-γ resulted in a dose dependent increase in IP-10 secretion in co-cultures (Figure 1).
As previously described, IP-10 is specifically secreted by the monocytes in PBMCs. Interestingly, monocytes cultured in the conditioned media from either epithelial cell line, together with recombinant IFN-γ, induce significant increase in IP-10 secretion. These results suggest that a secreted factor from epithelial cell lines is at least partially responsible for the IFN-γ mediated IP-10 secretion in co-cultures. A recent study by Boulday et al. (2006) reported that vascular endothelial growth factor (VEGF) augments the IFN-γ mediated secretion of IP-10 in endothelial cells [27]. Interestingly, Koyama et al [28] show that A549 epithelial cells constitutively express high levels of VEGF and that this is augmented by IFN-γ. Whilst our studies confirm the high constitutive VEGF secretion (data not shown) neither human recombinant VEGF nor VEGF inhibitors had any effects on IP-10 secretion from monocytes. These data suggest that there are distinct soluble factors governing the IP-10 response in endothelial versus epithelial cells. The secreted factor from lung epithelial cells might be a growth factor, interleukin or interferon, since previous studies have shown an inducible expression of IP-10 in a wide variety of tissues and cells under the influence of stimuli including interferons, interleukins, lipopolysaccharide, tumor necrosis factor-α, platelet derived growth factor, and hypoxia [6].
IL-12 is a classic IFN-γ inducing cytokine, which induced secretion of endogenous IFN-γ in A549/PBMC co-cultures due to a specific interaction between lymphocytes and A549 cells IL-12 also induced an increase in IP-10 secretion in A549/PBMC co-cultures, potentially partly due to endogenous IFN-γ signalling. The IL-12 mediated induction of IFN-γ and IP-10 secretion in A549/PBMC co-cultures is via intercellular contact as this was only observed in co-cultures and not in transwells or conditioned media studies. Interestingly, IFN-γ antibody pre-treatment only partially inhibited IL-12 mediated IP-10 induction, suggesting that there may be both IFN-γ dependent and independent IP-10 induction pathways.
In contrast to A549/PBMC co-cultures, IL-12 significantly increased IP-10 secretion in Calu-3/PBMC co-cultures in the absence of any detectable increase in IFN-γ levels (Compare Figures 8c and 8d). Moreover, the IL-12 mediated IP-10 secretion was shown to be IFN-γ independent, since it could not be inhibited by the IFN-γ ab in Calu-3/PBMC co-cultures. This IL-12 mediated IP-10 secretion is likely to be mediated at least in part via a secreted factor from Calu-3 cells as it is maintained in conditioned media and transwell studies.
To further probe the signalling pathways involved in modulating IP-10 expression in the epithelial cell/PBMC co-cultures, we investigated the pharmacological effect of a number of signal transduction pathway inhibitors on this model. Present studies suggest that there are at least two pathways by which IP-10 can be induced which are either IFN-γ dependent or IL-12 dependent.
IFN-γ dependent IP-10 expression was sensitive to PI3K inhibitors and independent of signalling via IKK-2, p38 or PDE4. Interestingly, whilst corticosterioids are frequently prescribed for lung inflammation, they again did not modulate IFN-γ induced IP-10 expression in this system. As IFN-γ signals via a JAK-STAT1 pathway [2], resistance to these inhibitors would be expected, but the role of PI3K is very exciting. The PI3 kinase inhibitor PIK-93 used in the present studies targets several PI3 kinases and has high potency for the class I PI3 kinases p110α as well as p110γ. [25] The development of subtype specific inhibitors will help identify which subtype of PI3 kinase is responsible for the increased IP-10 expression in co-cultures. Consistent with these results, it has been reported that the non-selective PI3K inhibitor wortmanin can also inhibit IFN-g mediated IP-10 production from endothelial cells [27]. These studies suggest that the development of PI3K inhibitors may represent a novel anti-inflammatory treatment for COPD, as they will inhibit a pathway not modulated by current therapies.
In contrast, IL-12 mediated IP-10 induction was sensitive to each of the inhibitors tested, except antibodies to IFN-γ. This provides further evidence therefore, that there are at least two pathways for IP-10 induction, with the latter being dependent upon the classical inflammatory pathways, NFκB and p38 MAP kinase, as well as cAMP. Moreover, the IL-12 signalling cascade has previously been shown to be sensitive to dexamethasone, [29] and the present studies show that the IL-12 mediated induction of IP-10 in co-cultures is modulated by corticosteroids, which may contribute to the efficacy of these agents in treatment of respiratory inflammation.
The differences in IL-12 mediated IP-10 secretion between Calu-3/PBMC and A549/PBMC co-cultures were also evident in the inhibitor studies. Inhibition of IP-10 secretion in A549/PBMC co-cultures was only effectively inhibited by PI3K inhibitors and partially inhibited by dexamethasone. As these co-cultures were shown to endogenously express IFN-γ (Table 1), this would suggest that most of the drive to induce IP-10 was due to the IFN-γ JAK-STAT1 pathway, in addition to some residual signalling via a steroid sensitive pathway. In contrast, all inhibitors used in the present study strongly inhibited IP-10 secretion in Calu-3/PBMC co-cultures, suggesting IFN-γ signalling is not required for induction of IP-10. These differences might reflect the differences in bronchiolar vs alveolar lung epithelial tissue, which would have to be taken into account in design of novel inhibitors blocking the abnormally high IP-10 secretion in lung tissue of COPD patients.
In addition to the human lung epithelial cell lines, we also used the primary human epithelial cultures for the key experiments. In contrast to A549 and Calu-3, NHBEs cultured alone secrete IP-10 if pretreated with IFN-γ. Consistent with this result Sauty et al. reported that pre-treatment with IFN-γ induces IP-10 secretion in NHBEs but not in A549 cells [5]. However, in agreement with the results from A549/PBMCs and Calu-3/PBMCs co-cultures, significantly increased IFN-γ mediated IP-10 secretion was observed from NHBE/PBMC co-cultures compared with NHBEs or PBMCs cultured alone. This demonstrates a significant increase in IFN-γ mediated IP-10 secretion in PBMCs co-cultured with all lung epithelial cell lines as well as the primary bronchial epithelial cells used in the present study. These results indicate that PBMC-lung epithelial cell interactions are strongly promoting IP-10 secretion in response to IFN-γ, thereby attracting more lymphocytes to lung tissue and support the use of the A549 and CALU-3 cell lines as a model of the primary cell system. As example, application of cigarette smoke extract in the leucocyte-lung epithelial cell co-cultures or to the conditioned media is likely to provide an interesting additional in vitro model for COPD.
Since IP-10 is a potent chemoattractant for T cells, the suppression of the increased IP-10 levels in lung tissue of COPD patients may reduce the lung inflammation characteristic of this disease. Increasing IP-10 levels will cause a positive feedback loop attracting more T cells to the peripheral airways, in turn increasing IFN-γ secretion. Establishing a method to inhibit this positive feedback loop may be profitable in suppressing the inflammatory process underlying COPD. Barnes et al. (2004) suggests that T cell inhibitory strategies, such as the use of immunosuppressant's, might be effective in COPD, although side effects, such as increasing the risk of bacterial infection, is of particular concern. Inhibition of IFN-γ signaling may provide another approach [1]. As shown in the present study, basal IP-10 secretion in co-cultures is blocked with all inhibitors used, representing both current and experimental therapies for respiratory disease. However, in the presence of IFN-γ which is secreted by T cells in peripheral airways IP-10 secretion is only inhibited by inhibitors of PI3K. This in vitro model may represent the environment in the peripheral airways of COPD patients which contain a large number of Th1 T cells, and suggest that IP-10 mediated inflammation is not being addressed with current respiratory therapies such as corticosteroids in these patients. However, this pathway was modulated by non-isozyme selective PI3K inhibitors in this model. A number of Pharmaceutical companies are developing PI3K inhibitors and these results complement an emerging body of data that suggest they may also have utility in treating the inflammation associated with COPD [30].
IP-10 secretion is a potent chemokine for CD8 T cells and its expression is induced when circulating monocytes, T cells and epithelium are in close proximity. Moreover, expression of this chemokine is induced by signaling molecules such as IFN-γ and IL-12 known to be expressed in COPD. Therefore, it is tempting to speculate that therapies targeted at decreasing the levels of IP-10 in peripheral airways of COPD patients may have therapeutic benefit in the management of this disease. In the present studies we demonstrate a complex interaction between monocytes, lymphocytes and lung epithelial cells resulting in IP-10 secretion via multiple pathways. Furthermore, inhibition studies supported the suggestion that different intracellular pathways are responsible for IFN-γ and IL-12 mediated IP-10 secretion. These results may provide novel strategies for investigating means by which to modulate IP-10 mediated secretion and chemotactic effects on T cells.
CM:
Conditioned media
EnC:
IP-10 - IFN-γ:
inducible protein 10
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This study was supported by Pfizer Ltd. and Marie Curie Fellowship Association. We especially thank our phlebotomists Jenny Clayton, Jane Banks and Suzanne Williams (Pfizer Ltd., Sandwich, UK). We also thank Dr. Christelle Perros-Huguet (Pfizer Ltd., Sandwich, UK) for proofreading this manuscript. Laura Henderson and Dr. Susan Summerhill (Pfizer Ltd., Sandwich, UK) provided helpful support with the techniques used in the present studies.
Pfizer Global R&D, Dep. Allergy and Respiratory, 500, Pfizer Ltd, CT13 9NJ, Sandwich, Kent, UK
Maria Torvinen
, Hinnah Campwala
& Iain Kilty
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Correspondence to Iain Kilty.
All experiments performed in this study are supported by Pfizer Ltd. Authors declare that they do have no competing interests.
MT carried out experiments, performed statistical analysis, participated in the design of study and helped drafting of the manuscript; HC carried out experiments and performed statistical analysis, IK participated in the design of study and helped drafting of the manuscript. All contributors approved the final manuscript.
Torvinen, M., Campwala, H. & Kilty, I. The role of IFN-γ in regulation of IFN-γ-inducible protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures. Respir Res 8, 80 (2007) doi:10.1186/1465-9921-8-80
Lung Epithelial Cell
Rolipram
Normal Human Bronchial Epithelial


Below is an abstract of my doctoral dissertation.
Paul Cézanne: The railway painter.
In the foregoing paragraph, we saw that Cézanne, who painted The Ferry at Bonnières (summer of 1866) (Fig. 1), topicalizing the railway station, telegraph pole, and electric wires, was the first among impressionist painters to depict the steam railway. In other words, among impressionist painters, Cézanne had the most acute sensibility with regard to the steam railway, which was the typical subject of "modernité" (modernity) in those days.
Actually, Cézanne moved around France on a steam train from the 1860s to his later years. Camille Pissarro's letter to his son Lucien on January 20, 1896, shows that Cézanne took a train on the Paris-Aix line to travel between the two points.
Moreover, Aix, which was Cézanne's base throughout his life, is a railway town. In fact, the railway station (Fig. 2) near the main street of Aix connected three train lines. Chronologically, the railway company P. L. M. opened the train line from Aix to Rognac on October 10, 1856 (25 km) (Fig. 3, Fig. 4), from Aix to Meyrargues on January 31, 1870 (26 km), and from Aix to Marseille on October 15, 1877 (34 km) (2).
photographed by the author on August 26, 2006.
photographed by the author on August 23, 2006.
filmed by the author on August 24, 2006.
It is remarkable that Jas de Bouffan, Cézanne's residence in Aix, was situated near the train lines from Aix to Rognac and to Meyrargues. Therefore, in a span of about 40 years, whenever Cézanne stayed at this house, which contained his atelier, he must have been conscious of the steam locomotive that roared past his house.
Furthermore, it is important to note that, while he lived in Aix, Cézanne drew many railway subjects in various ways.
First, Cézanne sketched the railway cutting on the Aix-Rognac line as visible from his house garden about 100 meters away (Fig. 5) to create The Mont Sainte-Victoire and the Railway Cutting (c. 1870) (Fig. 6).
Second, in his preceding works, he painted not only the railway cutting but also the railway signal on the same line (Fig. 7): The Railway Cutting (1867–1868) (Fig. 8) and The Railway Cutting (1867–1870) (Fig. 9).
Third, The Railway Cutting (1867–1870) (Fig. 10) depicts the railroad as well as the railway cutting.
These paintings show his enthusiasm as he tried to topicalize the steam railway in his early years in Aix.
photographed by John Rewald around 1935.
In addition, Cézanne depicted the railway bridge on the Aix-Marseille line. In fact, he sketched the railway bridge that spans across the Arc River and the valley in a suburb of Aix (Fig. 11-Fig. 13), as shown in The Mont Sainte-Victoire and Large Pine (c. 1887) (Fig. 14), The Pine before the Arc Valley (1883–1885) (Fig. 15), The Viaduct of the Arc Valley (1883–1885) (Fig. 16), The Mont Sainte-Victoire (1892–1895) (Fig. 17), The Mont Sainte-Victoire with Viaduct (1885–1887) (Fig. 18), and many other paintings.
Cézanne also painted a steam locomotive on the Aix-Marseille line. Actually, he depicted the train passing through the railway bridge at Arc valley (Fig. 19), as shown in The Mont Sainte-Victoire Seen from Bellevue (1882–1885) (Fig. 20), The Mont Sainte-Victoire and Large Pine (1886–1887) (Fig. 21), and The Arc Valley (c. 1885) (Fig. 22).
photographed by the author on August 24, 2006.
filmed by the author on August 25, 2006.
Surprisingly, Cézanne even enjoyed taking an electric train on the Aix-Marseille line, including the railway bridge at Arc valley.
As we were finishing our luncheon, he dismissed the carriage and announced that he was going to Marseilles to see "the rest of the family." We went off to the tram and, during two hours of torrid heat, we chatted cheerfully. His radiant mien, healthy appearance, and carefree attitude delighted me (4).
From these facts, it is certain that, even in Aix, the steam (or even electric) railway had already become a part of Cézanne's daily life, and, as a painter of modern life, he was passionate about railway subjects such as the railway cutting, railway signal, railroad, railway bridge, and steam locomotive.
(3) Marcel Provence, Le Cours Mirabeau: trois siecles d'histoire 1651-1951, Aix-en-Provence: Éditions du Bastidon Antonelle, 1976, p. 82.
This is a revised edition of "New Viewpoint on Art: Cézanne and Steam Railway (3)" published in Nihon Art Journal, May/June, 2012.


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Savage Math
I recently dove into Savage Worlds, and I'm really liking what I'm seeing of the game. One thing that keeps tripping me up, though, is the math. As a hobbyist game designer, I always tend to look at the odds of success on various rolls, and Savage Worlds math is deceptive.
Take melee, for example. d6 is generally described as being "average" at a skill. (In Savage Worlds, your skill is determined by what die you roll, instead of a static bonus, so d4 is poor, d6 is average, d8 is good, d10 is great, and d12 is masterful). In melee, the number you need to hit with a roll of your fighting die is the other person's Parry score, which is half their Fighting die size +2. So for someone with a d6, they have a Parry of 5. That means that two equally skilled, average fighters only hit on a 5+… which is only 33% of the time. Ouch… that's a lot of whiffing for equally skilled characters.
But that's the deceptive part. You see, Savage Worlds PCs and special NPCs (collectively called "Wild Cards") also get a Wild Die. This is an extra die, always a d6, which they roll in addition to whatever skill or attribute they're rolling. You use the higher of the two die rolls and add modifiers afterward. Of course, figuring out that math by hand can be tricky, which is why I just plug the numbers into AnyDice.com, a fantastic resource for figuring out die math. For finding the highest of two rolled d6's, you plug in output [highest of 1d6 and 1d6], click "calculate" and then click "at least" and you get a graph like this:
The most interesting part is the numbers next to the graph. They show how likely you are to roll that number or higher (what anydice calls "at least"). So you have a 55.56% chance to roll at least a 5.
Exploding Dice
Savage Worlds' other trick up its sleeve which makes mathing possibilities even harder is exploding dice. Whenever you roll the maximum on a die, you grab another die of the same size, roll it, and add the result to the first die. That second die can explode the same way, and so on. So that d4-2 untrained check can roll a 4, and then roll another 4, and another, subtract 2, for a total of 10 on your d4-2. Kaboom.
To math up the possibilities there, we use AnyDice's explode feature. So, a Wild Card character rolling an untrained skill check would be calculated like this: output [highest of [explode 1d4]-2 and [explode d6]-2] (note that the -2 from being unskilled also applies to the wild die's roll, and in AnyDice, you need to put the -2 outside the exploded section, or the -2 gets applied to each newly rolled die… ask me how I know).
This gives us a graph that looks like this:
Technically there's no upper limit to what you can roll, but AnyDice only calculates 2 explosions, and that's unlikely enough that it runs into tiny percentages anyway.
So this is an interesting result. The chances of a Wild Card character succeeding on an untrained skill check is almost 1 in 3. That's not great, but it might be better than you were expecting, since 1d6-2 only gets a 4+ 16.67% of the time.
BUT… if you can get +2 from an edge or other beneficial circumstance, all of a sudden, you're looking at a 63% chance of success, untrained! That's pretty good! I mean, it's ain't a sure thing, but it happens more often than not.
And that's what I mean about the math in Savage Worlds being deceptive. You can't easily do the math in your head for any particular scenario, you have to play a lot and get a feeling for how often various rolls work… or you need some sort of crazy lookup table.
The Crazy Lookup Table
2+ 3+ 4+ 5+ 6+ 7+ 8+ 9+ 10+ 11+ 12+ 13+ 14+ 15+ 16+
untrained (d4-2) 63% 50% 32% 27% 19% 17% 13% 9% 4% 3% 2% 2% 1% 1% 0%
d4 96% 83% 63% 50% 32% 27% 19% 17% 13% 9% 4% 3% 2% 2% 1%
d6 97% 89% 75% 56% - 31% 26% 21% 16% 11% - 5% 5% 4% 3%
d8 98% 92% 81% 67% 48% 38% 25% 22% 18% 14% 10% 9% 7% 5% 3%
d10 98% 93% 85% 73% 58% 50% 40% 29% 18% 15% 12% 11% 9% 8% 6%
d12 99% 94% 88% 78% 65% 58% 50% 41% 31% 21% - 11% 10% 9% 8%
Numbers from here: https://anydice.com/program/23943
These are the chances of rolling the given number or higher, if your main die is what is shown on the left, and you're also rolling a d6 Wild Die. I rounded to the nearest whole number for legibility, and truncated the table at 16, because really, at that point, you're already succeeding with wild abandon.
While painstakingly copying the numbers from AnyDice, it took me a moment to notice that a skill of d6 can't roll a 6 or a 12. d4, d8, and d10 can roll their die size, because the wild die can explode to that amount. And it does explain why there's always a big dip in the percentage at the given's die's size, only the wild die can help get that number.
So, there you go. Now you know that when two Wild Cards with d6 fighting attack each other, they actually have a 56% chance of hitting. That's not that bad. And in fact, even a d4 fighting has a 50% chance to hit someone with a fighting skill of d6 (and thus a Parry of 5).
One of the impressive things about the exploding dice is just how long the tail is. An average d6 Wild Card still has a 5% chance to roll 13+.
You can definitely see that untrained is fairly bad, but even then you have almost a 1 in 3 chance of success a simple 4+. The lowly d4 has 63% chance of success on a 4+, and the "average" d6 succeeds 75% of the time. That's really good, actually.
I like high chances for success in an RPG. Failing half the time like some RPGs expect, means you can't actually count on any of your abilities. They're just a coin flip. Being "average" and having a 75% chance of success is just about perfect, though, in my opinion. Failure is definitely still quite likely, but it's somewhat unexpected when it happens. And that's exactly the way it should be. Failing all the time is not fun. It's the chance of failure that really makes the game interesting.
I'll touch on the percentages for Extras (non-Wild Cards) only briefly, mostly because I don't feel like typing out another gigantic table. You can see the results for yourself here:
https://anydice.com/program/23941
It ain't great. Chance of success isn't too hard to do in your head without the Wild Die, but here's the straight percents for hitting a generic 4+:
Untrained	19%
D4		    25%
D10		    70%
So, it takes an Extra with a d12 skill to be as good as a regular old d6 Wild Card. Ouch.
And that's what all I have for Savage Math right now. Pretty interesting stuff. It's super interesting how the exploding dice and the wild die really work together to make the math work for PCs (but don't count on your friendly Extras for much help!)
The d20 Is Special
There are a million RPGs out there, and they use a ton of different dice models for their main mechanics. D&D uses the d20, many use only d6s (Blades in the Dark, PbtA), some use only d10s (Warhammer, White Wolf)… but only one passes what I call "the T-shirt test"… that is, only one mechanic is popular enough to be put on a t-shirt, and that's the d20. Why is that?
As RPG designers, we like to look at probabilities. The d20 is very straight forward and honestly kind of boring - 20 sides, 5% chance for any side to come up, +1 to the roll is +5% chance to succeed. It's nice and regular, but it's not inspiring. And yet, it passes the T-shirt test, why? Well, I think there's a few reasons.
First, yes, it is the main mechanic of the 800 pound gorilla on RPGs, D&D. But I don't think that's the only reason it is popular. There are many rabid fans of other systems that don't go get their system's main dice on a t-shirt.
The key to thinking about what the t-shirt test means is thinking beyond the numbers. This is hard for us as designers, because we want to break everything down into probabilities to know how likely an action is to succeed at any point. But that's not what players are thinking when they grab their dice. They're thinking "Can I hit this dragon?" "Can I dodge this punch?" "Can I convince the starship captain to help us?" The d20's easy probabilities make it easy for a player to judge the likelihood of success, but nobody is doing the math when death is on the line.
And I think this is the one key reason why the d20 is seen as almost holy - special enough to be worn prominently on a t-shirt. In most games that use a d20 to determine results, it's not used for anything else. When you pick up a d20, you know something important is going down. The game is at a tipping point. This one die you hold in your hand holds the key to glorious victory or soul-crushing defeat.
And that's the second key reason why it's holy. There is only one. You don't roll 10, you roll one. In software development we call this difference pets vs cattle. You care more about your one pet than you do about a dozen cattle. When you roll 10d6, they're cattle - you don't care about any one die very much. When you roll a single d20, it's a pet.
Many times in your own games and games streamed online, you'll see players swap out their d20, or put their d20 in "jail" for rolling poorly. This is a pet. It's a singular die almost given a life of its own, in need of special attention and rituals. This is why it is on t-shirts.
You don't have to choose d20 for your game, and I totally understand why most indie game designers don't. It's hard to be seen as going along with the 800 pound gorilla. It's hard to choose a flat distribution. But think about the t-shirt factor of your choice. Maybe it's not a pet, but consider avoiding making it cattle.
Using Google Docs for RPG Books
I use Google Drive (or Google Docs or Google One, depending on what they call it this week) for basically everything. It's free, it auto-saves, it's the same no matter what computer I'm on, and it generally works great. This extends to tabletop RPG design. You could use expensive desktop programs like Microsoft Word, or Adobe Indesign… but they don't get you as much as you'd think, at least when you're starting out. Google Docs works great for writing game rules. It's basically just like writing in Microsoft Word, except it saves to the cloud, so you never have to worry about losing your work.
You can see an example of using Google Docs here. . This is a copy of the first page of my in-progress RPG. Not bad, right? Feel free to make a copy of this document and mess around with it to see how it works (go to File->Make a Copy).
Use Headings for section titles and sub sections. This lets you style all the section headings the same very easily. It's also used for creating an automatic table of contents later on. The easiest way to change a style is to set a line to a specific heading (see the dropdown in the toolbar labeled "Normal Text". Then, change the style of that line, and then right click the line and select "Update Heading to match". Then boom all your other headings will match that style. You can go to Format->Paragraph Styles to tweak your headings even more, to give them borders or background colors.
The only thing that I wish google docs supported that it doesn't is styling specific sections of text inside a paragraph. Applying a heading style applies it to the entire paragraph. You can't, for example, tag all skill names with a style and then update that style to update all instances of skill names (like making them italics or whatever). You also can't have more than 6 headings, and you can't name your styles. Maybe this will come later, but for now, it's not possible.
Please change the default fonts. While Arial is a perfectly fine font for reading, it looks incredibly generic. one great thing about Google Docs is that it has full access to Google's huge list of fonts at Google Fonts. Only a subset are in font list to start with, so go to the Font dropdown, and choose More Fonts… to get access to all of Google's fonts. There's fonts for basically anything you could ever want in there, so give yourself some time to look through all the options. If this is still not enough fonts, you can install the Extensis add-on to get even more fonts. Go to Add-Ons->Get Add-Ons, and search for Extensis. Follow the steps to install it. Now close out of Add-Ons. For every document where you want to use Extensis fonts, you have to "start" the add-on. Go into Add-Ons->Extensis->Start and you'll see a sidebar open up with a whole new world of fonts.
Don't go too crazy, choose a small number of fonts (2-4) to use in your text, and make sure they're used consistently, so that the reader can infer meaning just from what font is used. It's totally fine to just use one font for section headings, and one font for the body text.
It's probably a good idea to choose what size your book will be fairly early on. Things like images and tables are annoying to change up later (and in fact you might need completely different art for a different sized book). Google docs lets you change the "paper size" of the document. To do this, go to File->Page Setup and choose one of the paper sizes. At the time of writing, you can't simply choose a custom size, but there's a variety of popular sizes in there. If the one you want isn't there, choose a larger size and set the margins so the text fills the space you expect.
6x9 is a popular size for indie publishers these days. At almost exactly half the page area of 8.5x11, it'll double the number of pages in your book. This can be good if you have a book on the shorter end of things, to make it feel more substantial. At the same time, if this pushes your page count too high, it'll make the book feel like you're holding a pocket dictionary. Personally, I think 200-300 pages is ideal for most RPG books… it makes it feel like you're getting a good amount of text for your money, without feeling intimidating like a college textbook.
8.5x11 is the traditional "D&D" size, but please don't make your book full page-width. Such long lines are hard for humans to read. Instead, split your book into columns. In the last year or so, Google has rolled out column support for Docs. To change the whole document, make sure no text is selected, then go to Format->Columns and choose the one two or three column layout. Most 8.5x11 RPG books these days are two columns, but 1st and 2nd edition D&D used three columns, so if you want a really old school look, try that out. To get some text to span columns, select that text and go to Format->Columns again and choose single column. Thus you can have your chapter or major section titles span the full page. This is also a great way to break both columns in the middle of the page and still have text below them.
Speaking of breaks, you can insert a column break with Insert->Break->Column. This will end the current column (if it's the left/middle column, the text will flow to the next column, if it's the rightmost column, it'll flow to the next page).
Call Outs
The nice thing about two column layout is that it makes it easy to make call-out boxes. These are the boxes with a different color background that you can use to highlight some information or note it as an aside. Highlight the text, go to Format->Paragraph Style, turn on the borders lines, and set the background to a different color. Bam, call out. Call outs in single column layouts are a little trickier, but still doable. We actually cheat and force part of our text to be two-column. Select a paragraph of text, and use Format->Columns to make that part of the document two columns. At the end of the first column, use Insert->Break->Column Break to ensure that the text wraps correctly, then set borders and shading as above. It requires a bit more manual text management, but it's doable. You can even drag the column markers in the ruler at the top of the document to make them less than 50 / 50 on the page. If anyone finds a better way to do call-outs, let me know.
Everyone wants an awesome looking cover for their RPG. It grabs people the moment they open your PDF, and sets the tone for everything that follows. A huge part of a great cover is the artwork, which is beyond the scope of this post, but a cover is more than just artwork, you need your text in there as well. For this, Google Drawings works very well. It's a part of Google Drive, just go to New->More->Google Drawings. I'll write a separate post about google drawings, as I think it's a pretty great tool for designers.
Please folks, remove the borders on your tables. It'll immediately change them from looking like a document written in 1998, to something more modern. Tables are at once useful and a nightmare. They can convey a lot of information, but large tables can be intimidating and just look nasty. Discretion is advised. I like making every other row have a light gray background to help with readability, but there's no built-in way to do that with Google Docs. However, there is an add-on that'll do it for you. Go to Add-Ons->Get Add-On, and search for Table Formatter. Install that, and it'll give you a bunch of preformatted styles that it can apply to tables in a single click. It even lets you customize your own formats. Super useful.
There's basically two ways to share a google doc. You can either share a link to it directly, or you can create a PDF from it. I prefer the latter, because it feels more professional (and then I don't have to worry about whether or not I update that doc, if anyone is watching). Luckily, Google Drive is great for this, too, and you can share links to a PDF that anyone can open, even if they don't have a Google account.
Making a PDF of a Google Doc is trivial. Just go to File->Download As->PDF. This will save a copy of the document as a PDF on your local machine. To share this PDF, add it back to Google Drive. Go to your game design folder (you do have a game design folder, right?), and click the New button, and choose File Upload. Choose the PDF you just downloaded and click ok. Once it appears in your game design folder, right click the file and select "Get Shareable Link". Copy the link that pops up and that's it. Test out the link by opening an Incognito window and paste the link in, to make sure it opens the PDF directly.
Now here's where using google drive is better than just any old file hosting. You can actually update the file that opens from the link you just copied. So if you make a new version of the rules, you can create a new pdf and replace the old one. Find the pdf you uploaded in drive, but don't open it. Right click the file and click "Manage Versions". A small window will pop up, and allow you to upload a new version of the file, and now when people open the link, they'll get that version instead. Let me know in the comments if you have any other tricks for using Google Docs.
The Math of Cypher System
I finally took a look at Cypher System (Monte's Cook's newish generic system that powers Numenera and The Strange), since so many people had been suggesting it when I was looking at cyberpunk settings. And I just can't get past the math.
The core mechanic of Cypher System is the GM assigning a difficulty between 1 and 10, then the player adjusting that down with skill, effort, help, etc. The final difficulty is then multiplied by 3 to get a target number, and you have to roll over the target number on a d20.
Wait, what? Why are we multiplying by 3? That doesn't change the probabilities at all. In that system, you can never have a target number of 11. You get 9 or 12 from difficulty 3 or 4. There's no in between. 10 and 11 are useless rolls that hold no extra information or benefit over rolling a 9.
The probabilities are identical to just rolling 1d6 vs. the difficulty directly, with one minor difference - in Cypher system's way, you can fail at the difficulty 1 task 10% of the time (rolling a 1 or 2 on d20). The probabilities are otherwise exactly the same. Rolling a 3 on the d6 is exactly the same as rolling a 9 10 or 11 on a d20. A 6 is like an 18, etc. I guess the one other difference is that Cypher gives you extra benefits when you roll 17+ (tiers of critical hits).
What I really don't get is why there's even a concept of the difficulty number. The very first time you see difficulty number, it's included with a table of the target number that goes with it…. if you just chopped off the difficulty number column, you'd have the same system and you'd save yourself a step, plus you'd give yourself the ability to be more granular.
Just starting with a target number from 1-30 is a lot clearer odds when you know you're rolling a d20. What's more, reducing the difficulty by 1 (and thus the target number by 3) generally costs… 3 points from an attribute pool. Why are we not just directly subtracting those points from the target number? If they still wanted to restrict it to batches of 3, well, ok, but still, directly applying it to the target number seems a lot more straight forward.
Skill training can reduce your difficulty by one or two steps as well. But instead of having "skilled or expert climber" and knowing that reduces climbinb difficulty by 1 or 2 steps, you could just have "climb +3 or climb +6" and add that to your roll directly.
It's like this was all contorted to be different just for the sake of being different…. while at the same time losing a ton of granularity over a regular d20 system.
Cypher: The wall is climb difficulty 6.. ouch. Ok, so I am expert at climbing, so that's -2, my gear gives me a step, that's -1, and Bill will help belay me, that's another -1, so difficulty 2, multiplied by 3 is 6 (rolls d20).
d20: The wall is climb difficulty 18.. ouch. Ok, so I am expert at climbinb, so that's +6, my gear gives me +3, and Bill will help belay me, that's another +3, so +12 (rolls d20)
Not sure I see the purpose… at least in d20 you can hand out +1 for small modifiers, or +4 for large if you want.
The Best Cyberpunk Games
There are a ton of Cyberpunk RPGs around, and for better or worse, they all have their own unique spin on the genre, as well as mostly unique rules. Most of these are available on DriveThruRPG as a pdf. Please pay for them if you want them, their authors spent a ton of time working on them.
The Heavyweights
These are the most well-known names in the cyberpunk genra
Shadowrun. If you want Cyberpunk + Magic, this is The Game. The rules are a bit wonky, but it's the gorilla in the room for that genre. There's a ton of editions, I played 3rd, which was ok. I've heard the latest versions (4th and 5th) are a bit messed up, but have not played them. There are a couple competitors - see Entromancy under In Development, and Karma in the Dark under New Age.
Cyberpunk 2020 is the grandfather of the genre. It's pretty playable if you totally ignore netrunning (which you absolutely should). It's very combat-focused and deadly, and uses its own special system of d10s. It is the original, and if you love cyberpunk, you should definitely play it a few times. The flavor in the writing is just awesome.
Far Future
Cyberpunk is traditionally a near future dystopia, but the themes all work just as well in a far future, and many recent games go this route.
Stars Without Number is a interesting sci-fi system based on old school D&D (aka OSR / Old School Rennaisance). It has an expansion called Polychrome that makes it cyberpunk-compatible. Its unique system lets you generate entire worlds for use with your game. Its base book is free (which is still like 200 pages), and has gotten pretty good reviews.
Eclipse Phase is a sci-fi space-faring RPG based on the Altered Carbon books (and later TV show). Everyone has a cortical stack that can let them jump between bodies, and death is rarely permanent. A bit too high tech for my tastes, and the cortical stack takes a lot of the fear out of death, which IMO is critical for Cyberpunk.
The Generics
I love universal systems in theory, because you can do anything, but in practice, the extra step required to translate in-game things into generic ruless is kind of an annoyance.
GURPS is a universal roleplaying system and has an explicit cyberpunk book.
Hero System is much the same and also has a Cyberpunk book (though it's out of print).
These systems use more storytelling-driven systems that are significantly different for the players. Instead of rolling a shooting skill, you might roll to see what happens if you "Mix It Up". As an old RPGer used to his straightforward skills as descriptions of concrete abilities, these are a bit too different from traditional roleplaying for me, but a lot of people love them.
The Sprawl and The Veil are two PbtA games, but very different in feel. They're both less focused on combat than storytelling, so combat is pretty rules-light. The Sprawl is mission-based, which really divides the game down into bite-sized missions, which may be good for some people, but turned me off. The veil is very mental, existential, and explores emotions more than combat.
Remember Tomorrow is a rules-light system (I think it's like 40 pages and half of that is just descriptions).
Technoir is another rules-light system based on applying adjectives and verbs and uses d6 dice pools.
Karma in the Dark is a free magic + cyberpunk rules-light system.
The New Traditionalists
These are the ones that most closely resemble traditional RPGs with a "near future cyberpunk" feel. This is the list I'd probaby look at to actually play myself.
Ultramodern 5 and Neuropasta make 5th edition D&D into a cyberpunk-able game. Neuropasta is a bit more shiny happy star trek style than most cyberpunk settings, which is a bit of a turn off for me.
Interface Zero is a newer title with the most crunchy system outside of Shadowrun (it has like 300 pages which is about half rules and half setting) with what seems like a good amount of money behind it. It has three versions - pathfinder, fate, and savage worlds, so you can pick the ruleset you prefer. I haven't read through it much (mostly because it's so big), but it seems like fairly standard cyberpunk, maybe a bit further into the future than some "near future" ones (there are androids and "new humans"). My main nitpick is they make anthropomorphic animals (think Teenage Mutant Ninja Turtles) a standard race which is… just a bit much IMO.
Neomancer is a newly kickstartered standard cyberpunk feel with its own dice pool mechanic.
There are new RPGs coming out all the time, and that includes cyberpunk RPGs.
Entromancy is a novel and RPG work-in-progress. It's the only competitor to Shadowrun's magic + cyberpunk that I know of (and as far as I know, it's not anywhere near ready to play). It has a (funded!) kickstarter that will finish up in September of 2018.
Cyberpunk Red is the new version of Cyberpunk 2020, which currently has no release date or website, but was very briefly mentioned by 2020's author, Mike Pondsmith as being under development.
Identeco is another near-future standard cyberpunk game that finished up a successful kickstarter in September 2017. The book is not yet available as of August 2018, as far as I can tell.
If you haven't played Cyberpunk 2020, I highly recommend it. Even if some things are wonky, it's good to see the thing that launched an entire genre.
Interface Zero looks like a good modern option with good crunch.
Personally, I am not a fan of rules-light games, so if you're into PbtA etc, you'll have to make your own decision.
Shadowrun, obviously is the only choice for magic + cyberpunk right now, but it's not really my thing (not that I'd say no if someone was running it).
A post on reddit (no, I'm not going to link to it) managed to cram three different truly bad campaign ideas into one player, and I figured this would be a good subject to write on.
Here's a non-comprehensive list of really bad ideas that you should absolutely avoid in your campaign.
Evil Parties / PCs
Everyone eventually thinks they want to run a badass evil PC, or maybe all the players decide they want to run an all-evil party. This is a recipe for disaster for all but the best groups. Why? Because the whole foundation of D&D is predicated on the party trusting one another and working together. The party has to stay together, or there's no game. The game is about the party. And if people are betraying each other left and right, the players themselves will realize there's no logical reason for the party to stay together, and the game will just end with the dissolution of the party.
It can work if the players take a long view of their characters' relationships and the benefits they'll get from sticking with the party… but too often immature players will go for the short-sighted, stab-everyone-in-the-back (whether figuratively or literally) and make off with the loot.
The best way to avoid this is to just have a standing rule about no evil PCs. I've found that it's often good to extend that rule to Chaotic Neutral PCs, since that also often indicates the character will be selfish with no regard for the rest of the party.
For some reason, it's very common for one person in the party to want to betray everyone else. Often this goes with the evil PC, as above, but it doesn't have to. Again, this comes down the core principle of D&D in that your party has to work together and trust each other, or the game falls apart. The problem with betraying the party is that you're betraying the players, too. The players invest all this time and effort in the party, they've worked hard to build trust between the characters, and then that is just torn away. It can cause real-life arguments and fighting. It's just not worth it. If someone comes to you wanting to have a PC that will betray the party, just say no. If you really want to try it out, discuss it with the rest of the players first. Yes, this spoils the surprise, but if it keeps everyone friends at the end of the night, that's worth it.
Telling PCs How to Feel
A DM should never ever tell a player how their character feels. You can try to evoke emotion through your descriptions of the world, but you should never say "you feel scared". Because the player is going to immediately say, or a least think "The hell I do!" Players need to feel like they have control over their characters. And there's nothing more intimate about a character than what they think and feel. Unless there is some magical compulsion affecting their emotions, you should only describe what the characters experience with their senses, and let the player decide how their character reacts. And even magical compulsion should be avoided in all but the rarest of occasions, because players hate it. Most players would prefer their character be knocked out than forced to run away in fear.
This applies to normal social interactions, as well (though it's less severe if you get it wrong). Instead of saying "you believe what she's saying", instead say "she sounds like she's telling the truth". The difference is that one instructs the character how to feel, and the other gives the player information from the character's senses.
PC vs. PC Social Skills
The above also applies to PC vs. PC interaction. Social skill mechanics should never be used between PCs. One PC should never be allowed to roll a persuasion check to convince the other PC to do something. This is the same as telling the player how their character feels, except that it's even worse, because it's not the DM, it's another player doing it. If one PC wants to convince another PC of something, they have to roleplay it out, or at least talk to the other player and try to convince them the old fashioned way. Same goes for lying or hiding things from another player. You can't just have a PC roll and say it works. The tricked player will hate it, because the player knows what's going on, and you're taking away their ability to decide how their character would react.
Love, Lust, and Romance
Often times, players want their character to fall in love, either with an NPC or with another PC. This is generally a bad idea, because the game is not really built to support it, and it takes many people out of the comfort zone. If all the players are heavy roleplayers, then it can work, but you need to discuss it ahead of time. Most players do not come to the table with the maturity to describe how they woo a partner. This goes triple for PC to PC interaction. If any player wants to roleplay a romantic or sexual relationship with another PC, you must get the other player's consent before allowing it in the game. Otherwise it can make players extremely uncomfortable.
Usually, it's best just to avoid these situations. There's plenty to do in D&D without talking about sex or romance.
World Names
I had an interesting thought about naming things today. I had a name I liked for my planet (Athos), but a crappy name for the continent where my campaign takes place (Loroth). And it occurred to me that I don't actually need a name for the planet. Our own planet doesn't really have a name. The Earth just means "the dirt"… i.e., this thing right here that we're all standing on. Just like there's no name for our local star, it's just "The Sun". There's no real name for it, because there's only one. You don't need a name for something unless it's to disambiguate it from others. Similar "the moon".
So I used my world name for my continent, and just removed the reference to the world. Since this is a fantasy campaign, there's no space travel, and the people on the world probably wouldn't have a name for the planet. It's not something they really talk about… if they did, it would just be "the world". It doesn't need a name, there's only one.
Notably, if you have two moons or two suns, all of a sudden, you need names, because there's more than one, and you need to differentiate. I don't know if that would ever be the case for planets. Maybe in a binary planet system, but that's really just like a moon. Unless you can go there, it's still just a rock in the sky, and therefore easily differentiable from this rock you're standing on.