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We're very happy to have Amanda as a member of our Racepoint Global team! Get to know our one of our newest Marketing team members in this intimate Q&A.
1. How would you explain what you do for work in 3 sentences or less to your 7 year old niece?
My team works together to help find the right people to like and use our companies. We attract these people mainly online through social media, TV, print, mobile etc.
2. What is your favorite website, blog or online community?
The Humans of NY blog. I like it on FB and follow it on Instagram so I never miss a post. I also recommend the HONY book that came out last fall!
3. What is the last book you read?
I am currently reading The Hobbit. I'm a huge LOTR fan but could never get through the books when I was younger-the recent "The Hobbit" movies inspired me to finally read the book. Although there are a lot of differences between the movies and the book, they're both great- and I especially don't mind the addition of Legolas in the most recent movie…
4. What do you have as your desktop wallpaper right now?
A red panda. I know cliché… 'Amanda' likes pandas.
5. What is your favorite drink?
Black tea, hot or cold.
6. What is your favorite YouTube video of all time? (Please provide the link)
If after this video you can't say you are obsessed with corgis, something is wrong.
7. What is your favorite lunch meat?
I love pretty much all lunch meats, but my favorite would be a toss-up between roast beef and salami.
8. Do you have any nicknames?
Panda, Pando, Mando, Mandy. Depends on the friend or family member. My RPG crew seems to enjoy panda J
9. When you were a kid, what did you want to be when you grew up?
A marine biologist. I never had an affinity for science in school, but to me a marine biologist was someone who swam with dolphins and fish for a living.
10. Name three places you want to visit before you kick the bucket.
Japan, China and South Africa. I could choose so many more though!


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Enhance your home's scent story with Air Wick® Life Scents™ Summer Delights Scented Oil. Air Wick® Life Scents™ Collection is the first constantly changing fragrance to create a welcoming atmosphere in your home. Each Air Wick® refill provides up to 45 days* of continuously diffused and carefully blended fragrance throughout your home, office, or any space. Air Wick® Scented Oil Warmers also allow you to control the fragrance level as desired, while enhancing your décor with a sleek look. *Based on low setting. Remove warmer from wall outlet. remove refill from warmer and screw cap onto empty fragrance bottle. Unscrew cap from new fragrance bottle and insert into warmer until "click" can be heard. Plug UPRIGHT into outlet. Rotate plug if needed. UNIT WILL LEAK IF PLUGGED IN SIDEWAYS OR UPSIDE DOWN. WHEN UNPLUGGED, KEEP UNIT UPRIGHT. Adjust fragrance intensity by rotating dial on top of warmer. Largest mark = max. setting. IMPORTANT: Risk of electric shock. This is not a toy and is not intended for use by children. For adult use only. DO NOT Put fingers or hands into product. For safe use, plug only into properly functioning 120-volt electrical wall outlet where product is ventilated and cannot contact bed covering or other material. DO NOT use with extension cords. If using of a GFCI outlet, test on a regular basis. DO NOT immerse in water. DO NOT plug anything in above it. DO NOT use air freshener with Mega fragrance bottle in vertical receptacles. DO NOT place fragrance oil bottle or cap on finished wood surfaces or place any object within 12" (30 cm) above warmer. DO NOT place in an area where the unit may be susceptible to hard knocks. DO NOT operate with wet hands or metal objects. DO NOT obstruct or block air flow from unit. DO NOT use in small, confined pet areas without adequate ventilation. DO NOT place near a source of heat or direct sunlight.
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• Life Scents is a multi-layered fragrance that constantly changes
• Summer Delights contains scents of white flowers, melon and vanilla
• User can control fragrance level
• Continuously diffuses for up to 45 days on low setting
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Ephesus Tour --- Full Day Tour---Half Day TOUR DETAILS:
Private Guided Historical - Cultural - Ancient Sightseeing Tours to Ephesus Efes from Kusadasi Turkey by Taxi - Cab - Chauffeured driven Car.
Optionally, you may ask your guide for cultural demonstration of Turkish handicrafts and do a little shopping to spoil yourself after an intense day.
Private Ancient Ephesus Efes Tours - Excursions :
Destination to Kusadasi :20 km
How you go there : takes 30 minutes from kusadasi by Taxi or Minibus
Charge of the Entrance :7 .- Euro per Adulth person.
Information :Guidebook and Map of about Ephesus Efes - Turkey, is avaiable to find in Kusadasi or at Place .(if wanted)
Duration :Half Day Tour 3 hours.------Full Day Tour 7 hours.
Available: All Year.
Departure: Includes all transportation to and from Kusadasi Port or izmir Port.
100% Satisfaction Guarantee
No extra hidden charge
We would like to introduce you local culturel aspect of Ephesus by showing as handmade Carpet & Rug Art Weawing Teaching-Village of Turkmen.And Ephesus Ceramic Center
Note: The temperature can get quite hot during the summer months so don't forget to bring a hat, sunglasses, water and of course your camera.
History :About the History of Ephesus EfesEPHESUS,once the most important commercial center western Anatolia,is 18 km far from Kusadasi.The city was established as aport on the mounth of the river Cayter and was one of the foremost cities of the world for its being on a strategic trade route in Anatolia.


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Asbestos Management Services & Consultants
Looking for Asbestos Survey Companies? We are industry leading consultants, protecting you and the environment since the turn of the millennia. With offices based in Oxfordshire, Northamptonshire, Yorkshire and Kent, Amiantus can provide accredited surveyors to carry out Asbestos Management or Refurbishment & Demolition Asbestos Surveys throughout the UK. All our surveys and sampling techniques for Asbestos Containing Materials are carried out in accordance with the requirements of the Health and Safety Executive Guidance document HSG 264 Asbestos: The Survey Guide. All samples obtained are analysed in our onsite laboratory which is UKAS accredited to ISO17025 for Polarised Microscopy Analysis for asbestos in bulk samples. On completion of your survey a bound, full colour report will be issued along with a pdf copy. Each report will include an asbestos register, risk assessments, recommended actions and floor plans. The report will assist you in forming the basis of an effective plan to manage asbestos. Successful management of Asbestos Containing Materials is based on effective identification, inspection and the implementation of an effective management program. Working alongside your own staff, we can review your existing management plan and risk assessments and update your records with any noted deterioration, or further asbestos which has been revealed since the original survey.


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- From mobile
- Conscallhome
- Dec 06, 2016
- by anonymous
- #967880
Misrepresentation of so-called phone service. I used it one month, saw it was a prepaid discount scam with inflated usage values. They also said i have a contract and haven't paid last 2 payments so i can not terminate my contract. It is bad enough that families are hurting without scammers adding to their pain.Add comment


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Nerve & Muscle Physiology Jeff Ericksen, MD –VCU Health Systems PM&R
Topics * Relevant anatomy Cell functions for signal transmission –Transport, resting potential, action potential generation & propagation –Neuromuscular transmission –Muscle transduction Volume Conductor theory
Acknowledgements Electrodiagnostic Medicine by Daniel Dumitru, MD –Chapter 1: Nerve and Muscle Anatomy and Physiology Superb text covering all aspects of EMG/NCS
Cell membrane Necessary for life as we know it Border role for cell –Separates intracellular from extracellular milleau Allows ion and protein concentration gradients to exist –Creates electric charge gradients
Cell membrane Provides structure for cell Modulates cell interaction with environment –Mechanical, hormone-receptor Controls material flow into/out of cell –Nutrition/waste management
3 Key Membrane Components Lipids 45-49% –phospholipids, cholesterol & glycolipids = amphipathic molecules Polar = hydrophilic vs. nonpolar = hydrophobic Proteins 45-49% Carbohydrates 2-10%
Lipid characteristics Membrane phospholipids have polar head group with 2 nonpolar tails In water - nonpolar tail groups form an inside excluding water 2 arrangements possible –Micelle = tails inside, heads face out –Bilayer
Lipid bilayer or fluid mosaic model Phospholipid sheet with tails aligned in center, heads facing out for a head-tail-head sandwich –No H 2 O at center, 75 Angstroms Model as 2-D liquid with 2 degrees of freedom of motion for lipid –Long axis rotation –Lateral diffusion
Proteins in membrane provide cell functions 2 membrane protein types –Transmembrane = integral - across whole layer, amphipathic Hydrophobic midportion acts with lipid layer tails Hydrophilic section faces intra/extra environment –Peripheral proteins - inside or outside of bilayer
Membrane transport Lipid soluble molecules cross readily but large water soluble molecules need transport across bilayer –Transport proteins - specific for ion or molecule to cross Channel proteins - span bilayer, large center, allow ion/molecule passage based on size Carrier proteins - binding with specific material, conformational change then crossing membrane
Membrane transport Diffusion –Driven by kinetic energy of random motion –Thru lipids or proteins –Follows concentration gradient Active transport –Needs energy source –Fights concentration or energy gradient
Simple vs. Facilitated diffusion Simple –Crosses membrane bilayer or channel without binding –Increases with kinetic energy + lipid solubility + concentration gradient –Protein channels specific for ions, often gated by cell functions Facilitated –Transmemb proteins –Needs protein binding, conformational change –Speed of transport limited by conformational change
Membrane transport Carrier proteins Energy Channel protein Simple diffusion Facilitated diffusion DiffusionActive transport
Acting on semi-permeable membrane allows the cell to maintain a high intracellular concentration vs. extracellular fluid Requires active process as diffusion would eventually equilibrate concentrations across membrane
Active transport Transmembrane carrier protein uses ATP energy to pump ions against concentration gradient to develop transmembrane resting potential
Resting membrane potential Excitable cells can generate and conduct action potentials over distances Intracellular space carries potential difference of 60-90 mV, inside with negative charge excess relative to outside
Resting membrane potential created by semi-permeable membrane and ions Intracellular –Na 50 –K 400 –Cl 52 Extracellular –440 –20 –560
http://www.bioanim.com/Cell TissueHumanBody6/O3chann els/ionCloudPoints1ws.wrl
Nernst used thermodynamics in 1888 to determine work done by membrane Work to move ion against concentration gradient is opposite to work to move against electrochemical gradient Can calculate contributions from different ions –K = -75 mV, Na = +55 mV
Nomenclature Polarized membrane: Intracellular potential is negative relative to extracellular space Depolarization = less polarization of the membrane -80mV -> +20mV Hyperpolarization = more polarization of membrane -80mV -> -100mV
Na influx with K efflux Na driven by negative charge excess inside + concentration gradient K driven by concentration gradient If continued, would lose resting potential
Na - K ATP dependent pump Plasma membrane structure uses active transport 2 K in for 3 Na out actively Thus 3 Na must diffuse in for 2 K out
Membrane potential from Goldman-Hodgkin-Katz equation Resting potential mostly from K contributions If sudden Na permeability change, potential approaches Nernst Na potential rapidly –Action potential!
Voltage dependent ion channels Ion flow across through membrane channels is initiated by membrane potential changes If potential exceeds a threshold, rapid increase in Na permeability followed by later K permeability increase
Voltage dependent ion channels Extracellular Na activation gate with intracellular inactivation gate and slow K activation gait Conformational changes due to membrane potential changes influence ion permeability
Voltage gated channels
Channels and voltage influence If resting potential depolarized by 15-20 mV, then activation gate opened with 5000x increase in Na permeability followed by inactivation gate closure 1 msec later Slow K activation gate opens when Na inactivation gate closes to restore charge distribution, slight hyperpolarization
http://www.bioanim.com/Cell TissueHumanBody6/O3chann els/naChan1ws.wrl
Refractory periods Absolute = state when activation gait cannot be reopened with a strong depolarization current, the membrane potential is relatively more positive Relative = state when activation gait can be reopened by strong depolarizing current as membrane potential returns to equilibrium state
Action potential timing
Action potential propagation Na + charge influx spreads longtiduinally down path of least resistance to induce depolarization in adjacent membrane, some transmembrane spread As + charge builds up, attracts intracellular - charges and they are neutralized by new ICF + charges
AP propagation Less electrochemical hold of ECF + charges which migrate and allow depolarization of membrane further Process is repeated down axon until end is reached AP is identical to AP from upstream nerve area, all or none event
Nerve membrane modeling Capacitor = charge storage device, separate poles separated by a nonconducting material or dielectric –Hydrophobic center to lipid bilayer is good dielectric, allows membrane to function well as a capacitor
Nerve membrane modeling Resistor = direct path to current flow but with some impedance Nerve axon has both transmembrane resistance as well as longitudinal resistance
Current spread Membrane capacitor model suggests transmembrane resistance is high, hence current flows more longitudinally vs. transmembrane capacitance flow or ionic channel resistance flow
Slow process Longitudinal AP spread requires sequential depol. to threshold, membrane capacitor discharge and then alteration of proteins to turn on Na activation channels. This process can be slow. Hence unmyelinated nerve conducts slowly = 10-15 m/sec.
Need velocity to interact with environment! longitudinal resistance will speed – diameter will resistance Eliminate need to fire all surrounding tissue will velocity of conduction –Insulate nerve to prevent leakage, spread out the gated Na channels Myelin & Nodes of Ranvier
Myelin All peripheral nerve axons surrounded by plasma membrane of a Schwann cell –Single layer of membrane = unmyelinated nerve, multiple layers = myelinated nerve –Gap between Schwann cell covers = node of Ranvier
Myelinated axons Outer myelin sheath + axon plasma membrane = axolemma covering axoplasm Schwann cell membrane has lipid sphingomyelin, highly insulating No Na channels under myelin, only at nodes. K channels under myelin in perinodal area
Current conduction with myelin insulation AP at node, Na charge influx and current spreads longitudinally down axon Minimal leak between nodes, reduced by 5000 vs. unmyelinated nerve –Charge separation, reduced protein leak channels & increased membrane resistance account for this
Current conduction Circuit is closed by efflux of ionic current at node Na ions accumulate beneath node, reduces electrochemical pull on ECF Na above node, they migrate back to upstream node to close loop Above tends to increase + charge inside membrane or depolarize to give AP
AP generation at node Nodes contain high # Na channels which open with depolarization –Na influx starts process again
Myelin effects Conduction velocity increases Current and action potential jumps from node to node = saltatory conduction Optimal internodal length is 100x axon diameter Optimal myelin/axon ratio is 60/40
Neuromuscular junction, transducing the electrical signal to mechanical force
Multiple branches from large motor axons
What happens if varying myelin and diameter in branches?
NMJ anatomy Presynaptic –Terminal axon sprout Mitochodria Synaptic vesicles = ACH –Presynaptic membrane Postsynaptic –Motor endplate Single muscle fiber Mitochondria Ribosomes Pinocytotic vesicles Postsynaptic membrane – ACH receptors
NMJ Electrochemical conduction Considerable slowing in smaller diam less myelinated branches AP depolarizes terminal axon, Na conductance increases –Calcium conductance also dramatically increased –Influx Ca ++ in terminal axon Possibly facilitates fusion of ACH vesicles with presynaptic membrane
Electrochemical conduction…. Vesicular fusion with presynaptic membrane Open to synaptic cleft, release quantum of ACH –100 vesicles per AP in mammals, 10k ACH per vesicle Ca ++ stays in terminal axon 200 ms, keeps axon readily excitable for repeat stimulation
ACH release Rapid diffusion across cleft in.5 msec timing, bind receptors –Large transmembrane proteins with ACH site and ion channel –Ligand activated vs. voltage activated ACH binding induces conformational change in ion channel –1 ms opening of cation specific channel = Na, K, Ca, repels anions with charge
Postsynaptic ion channel opening with ACH binding Predominant influx is Na, K blocked by electrochem gradient, Ca concentration gradient not that large Na influx locally depolarizes muscle membrane= endplate potential reversal which is not propagated = EPP –Single packet of ACH from vesicle gives MEPP
Muscle action potential Generated if sufficient ACH released to cause postsynaptic membrane to reach threshold, muscle membrane depolarized and propagated impulse follows Muscle AP travels along muscle membrane = sarcolemma –Similar to nerve, increased Na permeability in + feedback loop
T-tubules Small volume favors K accumulation during repolarization after AP, tends to make membrane easy to depolarize again Penetrate into muscle to spread AP into fiber High surface area of T-tubules increases capacitance qualities and slows conduction in muscle
Excitation-Contraction AP in T-tubule induces Ca ++ release in SR terminal cisternae, exposure for 1/30 sec, then reuptake via pump Ca ++ bind to troponin C, induces conformational change of troponin complex and influences tropomyosin to actin relationship - mechanical force
The End!
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By Jimmie Earls
Sun Sports Writer
After only a couple of weeks and a few home games into the volleyball season, Washington County High School's decision to offer students free admission to home athletic events has resulted in a significant increase in the number of students attending games.
Something the school noticed on the first night students used the pass was students repeatedly entering and exiting during the event. The school has remedied that situation by charging students who re-enter after being admitted the first time for free.
"That's just a safety issue," said WCHS principal Leon Smith. "We don't need to have kids running in and out of the parking lot."
Smith was quick to add his appreciation for what athletic director Chad Willis and Washington County Heartland Youth Coalition member Jill Settles have done to get the pass program in place.
Willis has stated that he noticed a "dramatic increase" in the amount of student participation. Several parents of WCHS students have also voiced their appreciation of the pass program.
It has been so much of an early success that the athletic director at North Hardin High School contacted Willis about the program and how to get it started, much in the same manner Willis inquired about it from Green County's athletic director.
The real test will come with the start of basketball season, when several games are played during the week, to see if WCHS students show up consistently for multiple basketball games during the week.
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- Oplysninger
- Anmeldelser
Please note: This tour is seasonal and runs annually in March and April. The dates are subject to change.
- Sæsontilbud
- Sug atmosfæren til dig på en vandretur
- Frokost inkluderet
- Få det store overblik over byen
- Oplev fantastisk udsigt fra luften
Take a break from sightseeing to enjoy a Japanese-style lunch in Western Tokyo, known as the Tama area.
Then re-board your coach and head to the Tokyo Tower Main Observatory to take in panoramic city views from the nearly 500-foot-tall (150-meter) tower. On a clear day, you can even see Mt Fuji in the distance.
This tour will take you to either Chidorigafuchi and Kitanomaru Park or Shinjuku Gyoen. Chidorigafuchi and Kitanomaru Park, where more than 1 million visitors arrive each year to view some 260 blossoming trees. Following the promenade at a section of the Imperial Palace, stroll through the tunnel of blossoms and observe reflections of the trees and bright blue rowboats on the water. Near the northern end of Kitanomaru Park, you'll get great photo ops of the cherry trees that grow near the oldest remaining gate of the Edo Castle compound, which dates to 1636. Alternatively, depending on blooming conditions and time of season, your guide accompanies you to Shinjuku Gyoen, a large park that was originally laid out in 1772 as part of the residence of a feudal lord. The current configuration of the gardens was completed in 1906 for the Imperial Palace and rebuilt after the destruction of World War II. The garden features 1,300 cherry trees of 65 different varieties and is a favorite spot for hanami.
After roughly an hour at either location, say farewell to your guide at Tokyo or Shinjuku Station and make your own way to your next destination.
Seneste billeder af denne tur
Vælg en måned for at se kalenderen over tilgængelighed og afgangstider.
Klik på linket nedenfor for at se pris og ledige pladser på din foretrukne rejsedato. Vores priser opdateres konstant for at sikre, at du altid får den lavest mulige pris.
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Yderligere information
Cherry Blossom Viewing and Tokyo Tower Tour
- Professional guide
- Entrance fees (exceptions apply)
- Lunch
- Hotel pickup and drop-off
- Food and drinks, unless specified
- Gratuities (optional)
- Confirmation will be received within 48 hours of booking, subject to availability
- A maximum of 9 people per booking
- Minimum numbers apply. There is a possibility of cancellation after confirmation if there are not enough passengers to meet requirements. In the event of this occurring, you will be offered an alternative or full refund. In such cases, you will be notified at least 4 days in advance
- If cherry blossoms fail to bloom on the day, the tour may be cancelled and you will get full refund. In such cases, you will be notified at least 4 days in advance
- The arrival time and order of itinerary may change due to traffic conditions, or other operational concerns on the day. In such cases, sections of the itinerary may be shortened, or omitted from the tour. No refunds will be provided
- Vegetarian option is available, please advise at time of booking if required
- Children 4–5 years old can participate in the tour are complimentary when accompanied by adults, but may not occupy their own seats. In addition, children are subject to a JPY 400 admission fee at Tokyo Tower
- If the cherry blossoms at Chidorigafuchi and Kitanomaru Park or Shinjuku Gyoen are not in bloom on the day, the tour may visit other popular cherry blossom viewing sites
- Please provide the information of your accommodation(s) before your travel date at checkout
Hvis du afbestiller mellem 3 og 6 dag(e) før den planlagte afgang, er der et afbestillingsgebyr på 50%.
Hvis du afbestiller 2 dag(e) før den planlagte afgang, er der et afbestillingsgebyr på 100%.


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Take advantage of the opportunity to own this multi unit home in West Warwick, RI. Four bedrooms, two full baths, approximately 2000 sq ft of living space on two floors. Spacious kitchen and some wood floors plus a partial basement and garage. Take a look and see what you can accomplish here. You can live in one unit and rent the others or rent them all. Submit your best offer now.
The following documents are available for 21 Levalley St West Warwick, RI 02893:
If Seller has approved bid terms, contract processing has started. Otherwise new auction period will start shortly.
Learn about the specific closing process for this property.Take a Look


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Understanding the mechanisms of immune cell migration to multiple sclerosis lesions offers significant therapeutic potential. This study focused on the chemokines CXCL12 (SDF-1) and CXCL13 (BCA-1), both of which regulate B cell migration in lymphoid tissues. We report that immunohistologically CXCL12 was constitutively expressed in CNS parenchyma on blood vessel walls. In both active and chronic inactive multiple sclerosis lesions CXCL12 protein was elevated and detected on astrocytes and blood vessels. Quantitative PCR demonstrated that CXCL13 was produced in actively demyelinating multiple sclerosis lesions, but not in chronic inactive lesions or in the CNS of subjects who had no neurological disease. CXCL13 protein was localized in perivascular infiltrates and scattered infiltrating cells in lesion parenchyma. In the CSF of relapsing–remitting multiple sclerosis patients, both CXCL12 and CXCL13 were elevated. CXCL13, but not CXCL12, levels correlated strongly with intrathecal immunoglobulin production as well as the presence of B cells, plasma blasts and T cells. About 20% of CSF CD4+ cells and almost all B cells expressed the CXCL13 receptor CXCR5. In vitro, CXCL13 was produced by monocytes and at much higher levels by macrophages. CXCL13 mRNA and protein expression was induced by TNFα and IL-1β but inhibited by IL-4 and IFNγ. Together, CXCL12 and CXCL13 are elevated in active multiple sclerosis lesions and CXCL12 also in inactive lesions. The consequences of CXCL12 up-regulation could be manifold. CXCL12 localization on blood vessels indicates a possible role in leucocyte extravasation, and CXCL12 may contribute to plasma cell persistence since its receptor CXCR4 is retained during plasma cell differentiation. CXCL12 may contribute to axonal damage as it can become a neurotoxic mediator of cleavage by metalloproteases, which are present in multiple sclerosis lesions. The strong linkage of CXCL13 to immune cells and immunoglobulin levels in CSF suggests that this is one of the factors that attract and maintain B and T cells in inflamed CNS lesions. Therefore, both CXCL13 and CXCR5 may be promising therapeutic targets in multiple sclerosis.
Multiple sclerosis is considered to be an immunopathological disease in which activated T cells enter the CNS and trigger an inflammatory cascade that leads to recruitment of other immune cells. Modulation of immune cell migration to the CNS offers great therapeutic potential (Kivisakk et al., 2001; Miller et al., 2003). Migration of immune cells is largely regulated by chemokines, which comprise a family of >50 molecules (Nelson and Krensky, 2001). The possibility of treating inflammatory diseases by targeting chemokines or their receptors has strongly stimulated research efforts in this field.
Chemokines are loosely divided into two functional classes: inflammatory chemokines that govern leucocyte trafficking to inflamed tissues 'on demand', and homeostatic chemokines that regulate constitutive trafficking, mainly, to lymphoid sites. Previous studies on chemokines in multiple sclerosis lesions focused largely on inflammatory chemokines. For example, the CCR1 and CCR5 ligand RANTES (CCL5), the CCR5 ligand MIP-1β (CCL4), the CXCR3 ligand IP-10 (CXCL10) and the CCR2 ligand MCP-1 (CCL2) have been associated with lesion development in multiple sclerosis (Simpson et al., 1998, 2000; Balashov et al., 1999; Sorensen et al., 1999; Trebst et al., 2001). These chemokines attract mainly monocytes and T cells.
However, some of the most prominent immunological abnormalities in multiple sclerosis are based on B cells: patients typically show intrathecal immunoglobulin (Ig) production, which presents as oligoclonal bands and a polyspecific Ig response. Oligoclonal Ig may persist in the same patient for many years (Walsh and Tourtellotte, 1986). Clonal expansion and somatic hypermutation of B cells (Qin et al., 1998; Colombo et al., 2000, 2003; Owens et al., 2003) and also plasma cells (Ritchie et al., 2004) have been observed in the CSF and in multiple sclerosis brain lesions (Owens et al., 1998; Baranzini et al., 1999). Lymphoid follicle-like structures have recently been described in the meninges of two out of three patients with secondary progressive (SP) multiple sclerosis (Serafini et al., 2004). Thus the currently available evidence indicates that B cells, plasma blasts or plasma cells migrate to the CNS of multiple sclerosis patients and persist there for many years, perhaps lifelong (reviewed, Uccelli et al., 2005). This long-term persistence of B cells might be promoted by local production of the B cell survival factor BAFF by astrocytes (Krumbholz et al., 2005). B-lymphocyte and plasma cell clonal expansion were observed in monosymptomatic optic neuritis CSF (Haubold et al., 2004), which supports the implication of B cell autoimmunity in multiple sclerosis, since it was observed at the first detectable phase of disease. The cellular composition of the CSF of patients shows considerable interindividual variation, but the pattern tends to be stable within the same patient and a high B cell/monocyte ratio may be associated with more rapid disease progression (Cepok et al., 2001). In vitro human B cells migrate across the brain endothelium more rapidly than autologous T cells (Alter et al., 2003), but almost nothing is known about chemokine regulation of B cell migration to the inflamed CNS (Ransohoff et al., 2003).
To address mechanisms of B cell trafficking to the CNS we analysed CXCL12 (SDF-1) and CXCL13 (BCA-1) in multiple sclerosis lesions. These two chemokines mediate germinal centre organization in lymphoid tissue (Allen et al., 2004).
The receptor for CXCL13, CXCR5, is expressed on virtually all B cells, a subset of T cells in blood and lymphatic tissue (Kim et al., 2001), and is transiently induced on T cells upon activation (Langenkamp et al., 2003). Recently, CXCL13 has been associated with inflammatory as well as homeostatic functions in the settings of neolymphoid development in the thyroid (Aust et al., 2004), in Helicobacter pylori-induced mucosa-associated lymphoid tissue and gastric lymphoma (Mazzucchelli et al., 1999), Sjoegren's syndrome (Salomonsson et al., 2002), rheumatoid arthritis (Shi et al., 2001) and ulcerative colitis (Carlsen et al., 2004).
CXCL12 is a potent chemoattractant for different immune cells including monocytes, T cells, B cells and plasma cells, and it regulates haematopoiesis. Beyond this, CXCL12 has complex functions in neurobiology. It is crucial for neuronal guidance in development (Lazarini et al., 2003; Klein and Rubin, 2004) and also expressed in mature brain (Stumm et al., 2002), but its function in the adult CNS is unclear.
In the present study we determined the presence of CXCL12 and CXCL13 in actively demyelinating and chronic inactive lesions. Because we found that CXCL13 is localized in infiltrating immune cells in active multiple sclerosis lesions, the regulation of the production of this chemokine by immune cells was analysed and found to be unusual. The CSF levels of these two chemokines were elevated and a linkage to intrathecal Ig production and immune cell subsets was analysed.
Materials and methods
Samples from patients and controls
A total of 14 snap-frozen CNS lesions from 6 multiple sclerosis patients were analysed. Patient 1 (two lesions analysed) had a rapidly progressive disease, which started at age 23 years and the patient died 3 years later. The patient did not receive immunomodulatory treatment within his last 2 years. Patient 2 (two lesions) had SP disease with a 20 year history and an onset at age 27 years. Starting 3 years before she died anti-CD4 therapy was given for 6 months followed by cyclophosphamide. Patient 3 (one lesion) had an SP course with a 17 year history with only short remissions and an onset age 39 years. Patient 4 (one lesion) had an SP course with a history of 8 years and an onset at age 21 years. Patients 3 and 4 did not have immunosuppressive therapy in the 2 years before death. Samples from Patients 1–4 were determined using a B cell monoclonal antibody (mAb) panel to contain B cells in perivenular cuffs and scattered in the parenchyma throughout these lesions. Patient 5 with a relapsing–remitting course has been described [Patient 2 in (Babbe et al., 2000)]. The two tissue blocks from this patient were active and probably in the early stage of lesion development as the lesions were removed 2 weeks after clinical manifestation of multiple sclerosis (Babbe et al., 2000). Scattered CD20+ B cells and CD138+ plasma cells/plasma blasts were present, but no lymph follicles were seen. Numerous CD14+ monocytes and CD68+ macrophages were present perivascularly throughout the whole lesion area and CD68+ cells were also abundant in the lesion parenchyma (Theil et al., 2005). This patient was untreated at the time of biopsy. The lesions from Patients 1–5 were classified as actively or recently demyelinating based on the presence of oil red O-positive macrophages and perivascular infiltrates. Patient 6 had an SP course with a history of 24 years and an onset at age 26. From this Patient, 6 blocks representing chronic inactive lesions were analysed. This patient had received mitoxantrone, the last time 11 months before he died. A total of 15 blocks from 3 adults without clinical or histological evidence of brain disease who died following accidents were also examined.
CSF samples were obtained from 30 patients with relapsing–remitting (RR) multiple sclerosis, 8 patients with primary progressive (PP) multiple sclerosis and 14 patients with SP multiple sclerosis. For comparison, CSF was also obtained from 28 patients with other inflammatory neurological diseases (OIND), including neuroborreliosis, viral inflammatory CNS disease (meningitis, encephalitis, zoster oticus and FSME), intracerebral abscess and mycoplasma meningitis. In addition, 14 patients with other non-inflammatory neurological disease (NIND) including headache, back pain, normal pressure hydrocephalus or seizure disorder, whose CSF did not show any evidence of inflammation such as intrathecal Ig production, presence of oligoclonal Ig, elevated cell numbers, or blood–brain barrier (BBB) disruption, were analysed. A disturbance of the BBB was diagnosed if the ratio of albumin in CSF and serum (Qalb) exceeded 7.4 × 10−3. Serum samples were obtained from 7 healthy blood donors, 8 of the NIND patients and 13 of the RR-multiple sclerosis patients. This study was approved by an Ethical Committee of the University of Munich.
Cell culture
Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by density gradient centrifugation. Monocytes were isolated by negative selection with immunomagnetic beads (Miltenyi, Bergisch Gladbach, Germany) and differentiated into macrophages for 9 days with M-CSF (20 ng/ml) as described (Nardelli et al., 2001). PBMC, monocytes and macrophages were cultured in RPMI supplemented with 5% FCS (PAN Biotech, Aidenbach, Germany). The following cytokines were added for 24 h: 25 ng/ml TNFα (Roche, Mannheim, Germany), 50 ng/ml IL-1β (R&D Systems, Wiesbaden, Germany), 100 U/ml IFNγ (Roche), 1000 U/ml IL-4 (PromoCell, Heidelberg, Germany), and subsequently supernatants were collected. RNA was isolated and cDNA was prepared using RNeasy Columns with a DNase digestion step (Qiagen, Hilden, Germany) and M-MLV reverse transcriptase (Promega, Mannheim, Germany).
Quantitative PCR
Frozen sections from tissue samples were collected in tubes, RNA was isolated and cDNA was prepared. A total of 14 lesions from the 6 patients described above and 15 samples from the three controls were analysed. Quantitative PCR was performed on the ABI 5700 (Applied Biosystems, Darmstadt, Germany) using the qPCR Core kit and uracyl-N-glycosylase for carry-over prevention (both Eurogentec, Seraing, Belgium). The reaction volume was 25 μl containing 25–50 ng RNA converted to cDNA. For all reactions the annealing temperature was 60°C. For detection of CXCL13 the following oligonucleotides were designed using Primer Express software (Applied Biosystems) and tested not to amplify genomic DNA up to 100 ng per reaction: forward (exon 1) CATCTCGACATCTCTGCTTCTCAT, reverse (exon 2) TCTCTTGGACACATCTACACCTCAA, probe FAM-AGCCTCTCTCCAGTCCAAGGTGTTCTGG-TAMRA.
Two housekeeping genes, cyclophilin A (peptidyl-prolyl isomerase A, PPIA) and GAPDH (both Applied Biosystems), were used.
The efficiency of these quantitative PCRs was determined as 2, meaning that the number of PCR products doubled at each cycle within the exponential phase of the PCR. The SD of the quantitative PCR experiments was calculated by the formula
Enzyme-linked immunosorbent assay (ELISA) of CSF, serum and cell culture supernatant was performed on Maxisorp 96 well plates (Nunc, Wiesbaden, Germany) using DuoSet ELISA development kits for CXCL13 (R&D Systems). The sensitivity of this ELISA was ≤10 pg/ml. Samples from patients and controls were analysed blind. The SDF-1α Quantikine Immunoassay kit and SDF-1β ELISA antibody pair (monoclonal capture antibody MAB350, polyclonal biotinylated detection antibody BAF351) were used according to manufacturer's instructions (all from R&D Systems).
Analysis of CSF, blood and serum by standard diagnostic methods and flow cytometry
Paired CSF and serum samples were examined as described (Cepok et al., 2001, 2005). In brief, CSF and serum were analysed for protein, albumin and IgG by nephelometry and for oligoclonal bands by isoelectric focusing and IgG immunoblotting. Total CSF cells were enumerated in a counting chamber and further characterized by flow cytometry using mAbs recognizing CD3, CD14, CD19 and CD138. Paired CSF and blood samples were analysed as described (Kivisakk et al., 2003) using anti-CD4 PerCP, anti-CD45RO APC, anti-CD19 FITC (all from BD Biosciences), anti-CXCR5 PE (R&D Systems) and isotype matched control mAbs (BD Biosciences).
A total of eight lesions from Patients 5 and 6 were analysed. Cryostat sections were air-dried, fixed in acetone or formalin, and incubated with 1.5% methanolic hydrogen peroxide for 10 min and with 5% normal rabbit or goat serum in 1% BSA for 30 min. Monoclonal anti-human CXCL13 MAB801 (18 μg/ml), anti-CXCL12 MAB310, or anti-CXCL12 MAB350 (8 μg/ml; all R&D Systems) was applied overnight at 4°C. CXCL13 immunostaining was detected with biotinylated rabbit anti-mouse antibody (1 : 500) followed by peroxidase-conjugated streptavidin (1 : 300 for 30 min), or directly with peroxidase-conjugated goat anti-mouse antibodies (1 : 100). CXCL12 immunostaining was visualized using the PAP or SABC system according to manufacturer's instructions (all secondary reagents by DAKO, Hamburg, Germany). Sections were developed with diaminobenzidine as chromogenic substrate for up to 10 min and lightly counterstained with haematoxylin. Negative controls included omission of the first mAb and control Igs. Tonsils and adenoids were used as positive controls and to set up the staining protocol.
Localization of CXCL12 in healthy brain and in active and inactive multiple sclerosis lesions
CXCL12 was detected on blood vessels in each of the analysed brains from subjects without neurological disease (Fig. 1A). In multiple sclerosis lesions CXCL12 was also seen on astrocytes. Immunolabelling of blood vessel walls and astrocytes was seen in both active and chronic inactive lesions (Fig. 1B and C). Occasionally CXCL12 was also detected on a few cells in perivascular infiltrates. Both monoclonal antibodies against CXCL12 gave similar staining patterns, although MAB350 staining was more sensitive and clearer (Fig. 1).
CXCL13 is locally produced within actively demyelinating multiple sclerosis lesions
Immunohistological analysis localized CXCL13 within the perivascular infiltrates in actively demyelinating lesions (Fig. 1D). CXCL13 immunostaining was found also on extracellular matrix in these lesions (Fig. 1D and E). A subset of infiltrating cells in the parenchyma outside the perivascular area was labelled (Fig. 1E), but no CXCL13 was detected in any of the multiple sclerosis CNS samples outside the inflammatory lesions (data not shown). The six chronic inactive lesions contained scattered CD68+ macrophages. No cells displaying CXCL13 were detected in chronic inactive lesions or in brain from subjects without neurological disease.
This differential expression of CXCL13 in active versus inactive multiple sclerosis lesions was also detected by quantitative PCR. Eight actively demyelinating lesions from multiple sclerosis Patients 1–5, six inactive lesions from Patient 6, and 15 brain specimens from three control subjects were analysed by quantitative PCR to assess the presence of CXCL13. Expression of CXCL13 was elevated in actively demyelinating multiple sclerosis lesions (Fig. 2). Data were normalized to two housekeeping genes, GAPDH (Fig. 2) and PPIA (data not shown). Elevated levels of CXCL13 expression in actively demyelinating multiple sclerosis tissues were detected by both approaches. In contrast to the actively demyelinating multiple sclerosis lesions, CXCL13 transcripts were not elevated in chronic lesions. In six chronic lesions the transcript level of CXCL13 was noted with a median of 0.001% GAPDH, which is about the same level as we observed in normal CNS (Fig. 2).
CXCL12 and CXCL13 are elevated in CSF of multiple sclerosis patients
The two isoforms of CXCL12 (SDF-1α and SDF-1β) were analysed separately by ELISA. SDF-1β was not found in CSF samples from eight multiple sclerosis and five NIND patients with a detection limit of 40 pg/ml. In contrast, SDF-1α was found to be a constitutive component of CSF (median 223 pg/ml in NIND, Fig. 3A). In RR-multiple sclerosis patients CXCL12 levels were significantly higher than in NIND patients (Fig. 3A). OIND patients displayed even higher CXCL12 CSF levels than multiple sclerosis patients (Fig. 3A). Among these OIND patients those with a disturbed BBB showed a more extensive elevation of CXCL12 (Fig. 3A).
CXCL13 was only detected in 1 out of 14 CSF samples from NIND patients, but was >30 pg/ml in the CSF of 17 out of 30 RR-multiple sclerosis patients. In contrast, the CXDL13 serum levels were in a similar range in RR-multiple sclerosis patients (median 51 pg/ml) and control subjects (56 pg/ml). In most of the multiple sclerosis patients with detectable CXCL13 in the CSF, this value was even higher than the corresponding serum level indicating the intrathecal production of this chemokine.
The CXCL12 and CXCL13 levels were also analysed in patients with PP- and SP-multiple sclerosis. Two out of eight patients with PP-multiple sclerosis and 4 out of 14 with SP-multiple sclerosis had elevated levels of CXCL13 in the CSF. Patients with PP- and SP-multiple sclerosis tended to have higher CXCL12 levels in the CSF than NIND patients (Fig. 3A).
The majority of the multiple sclerosis patients (RR-multiple sclerosis: 26/30; PP-multiple sclerosis: 6/8; SP-multiple sclerosis: 13/14) displayed no evidence in the CSF of a disturbed BBB, which is in accordance with the well-characterized features of CSF from multiple sclerosis patients. Only these multiple sclerosis patients without a disturbance of the BBB were included in this analysis (Fig. 3) to exclude a potential effect of pathological transfer of CXCL12 or CXCL13 from blood to CSF. The samples from multiple sclerosis patients with a disturbance of the BBB as judged by CSF analysis had the following chemokine levels for CXCL12: RR-multiple sclerosis 120, 995, 1207 and 1272 pg/ml; PP-multiple sclerosis 607 and 668 pg/ml; SP-multiple sclerosis 1229 pg/ml, and for CXCL13: RR-multiple sclerosis <10, 35, 93 and 487 pg/ml; PP-multiple sclerosis (two patients) <10 pg/ml; SP-multiple sclerosis 13 pg/ml.
Overall, CXCL13 but not CXCL12 levels showed a striking heterogeneity. In each group of multiple sclerosis patients there was a subset of patients with low or undetectable level of CXCL13 and another subset with high levels of CXCL13 in the CSF. The CXCL12 levels followed a Gaussian distribution in all subgroups, but not the CXCL13 levels. Therefore parametric tests also could be used to compare the CXCL12 levels. The t-test (P = 0.005 for NIND versus RR-multiple sclerosis patients) gave essentially the same result as shown by the Mann–Whitney rank sum test (Fig. 3A).
Among the OIND patients there were great variations in the levels of CXCL13. Both OIND patients without a disrupted BBB and high CXCL13 levels had neuroborreliosis. Not only neuroborreliosis, but also other inflammatory diseases of the CNS were associated with high levels of CXCL13 in the CSF. Eight OIND patients had a CXCL13 level in the CSF >1000 pg/ml. Five of them had acute neuroborreliosis, one a viral encephalitis, one a cerebral abscess and one was diagnosed with a cranial neuritis distinct from neuroborreliosis, since he was repeatedly negative by PCR and antibody testing. Patients with a BBB disturbance had higher CXCL13 levels in the CSF than those without (Fig. 3B). We assume that the higher level of CXCL13 in the CSF of the subgroup of OIND patients with a disturbed BBB due to the higher intensity of CNS inflammation, a more pronounced meningeal involvement, and possibly transfer from blood.
CXCL13 in the CSF of multiple sclerosis patients correlates with intrathecal Ig production
The QIgG (IgGCSF/IgGserum) reflecting intrathecal IgG production in the absence of BBB damage correlated strongly with the CXCL13 levels in RR-multiple sclerosis patients (P < 0.001, Fig. 4H). In contrast, neither the Qalb (R = 0.26, P = 0.17) nor the serum CXCL13 concentrations (R = 0.04, P = 0.9) correlated with the CSF CXCL13 levels. To exclude the possibility of a pathological transfer from blood, only multiple sclerosis patients without CSF signs of a disturbance of the BBB were included for this analysis. In contrast to CXCL13, the CXCL12 levels in the CSF did not correlate with the intrathecal Ig production (Fig. 4C).
CXCL13 in the CSF of multiple sclerosis patients correlates with the presence of B cells, plasma blasts and T cells
We then analysed the relationship between immune subsets and levels of CXCL12 and CXCL13 in the CSF of RR-multiple sclerosis patients. As previously, only CSF samples from multiple sclerosis patients without CSF signs of a BBB disturbance were included. Numbers of CD3+ T cells, CD14+ monocytes, CD19+ B cells and CD19+CD138+ plasma blasts were determined in the same CSF samples. Whereas the vast majority of CSF cells, ∼70–90%, consisted of T cells, the levels of the other immune subsets showed considerable differences. The number of B cells ranged from 11.2 to 3892 cells/ml, that of plasma blasts from 0 to 1657 cells/ml and that of monocytes from 55.7 to 1901 cells/ml.
The levels of CXCL13 correlated strongly and significantly with the number of all B cells, plasma blasts and T cells in the CSF of multiple sclerosis patients (Fig. 4). The number of monocytes also correlated with the CXCL13 level, but less strongly (Fig. 4J). Multiple sclerosis patients with a higher B cell/monocyte ratio had more elevated CXCL13 levels in the CSF. The B cell/monocyte ratio correlated significantly with the CXCL13 level (P = 0.002; R = 0.6). In contrast to CXCL13, the CXCL12 levels in the CSF did not show a significant correlation with numbers of T cells, B cells, plasma blasts or monocytes in the CSF (Fig. 4A–E).
We analysed CSF immune cell subsets, QIgG, CXCL12 and CXCL13 levels also in OIND patients. These patients had more pronounced inflammatory changes than the multiple sclerosis patients, as indicated by a higher percentage of OIND patients with a disturbed BBB, and higher cell numbers in the CSF (median RR-multiple sclerosis 11 cells/μl, OIND 60 cells/μl). The specific findings in the OIND group were as follows. Both CXCL12 and CXCL13 levels showed a strong positive correlation with B cells, plasma blasts and T cells: CD19+ cells (CXCL12: P = 0.02, R = 0.59/CXCL13: P < 0.001, R = 0.84), CD19+CD138+cells (P < 0.001, R = 0.75/P < 0.001, R = 0.79) and CD3+ cells (P < 0.001, R = 0.82/P < 0.001, R = 0.76). For CD14+ cells, the positive correlation was obvious for CXCL12 (P = 0.006, R = 0.67), but the association with CXCL13 was less pronounced (P = 0.047, R = 0.53) (CXCL12: 15 samples from 10 patients; 10 samples with a disturbed BBB; CXCL13: 14 samples from 9 patients; 9 samples with disturbed BBB). Further we noted also a correlation between CXCL12 and QIgG (P < 0.001, R = 0.64; 37 samples from 25 patients; 26 samples with disturbed BBB), and CXCL13 with QIgG (P < 0.001, R = 0.76; 38 samples from 26 patients; 26 samples with disturbed BBB).
Expression of CXCR5 by T cells and B cells in the CSF
CXCR5 expression was analysed by flow cytometry on parallel samples from CSF and blood. Virtually all CSF and blood B cells expressed the CXCL13 receptor CXCR5 and ∼20% of the CD4+ cells in blood or CSF expressed CXCR5 (Fig. 5). The CSF is known to be enriched for CD4+CD45RO+ cells. Also for this immune cell subset a similar percentage of CXCR5+ cells was observed in CSF and blood (Fig. 5). Patients with or without inflammation in the CNS did not show a striking difference in terms of percentage of CXCR5+ immune cell subsets. The CXCR5 expression in CSF compared with blood was slightly reduced, most obviously for the CD4+CD45RO+ and CD19+ subsets. Whether this reflects a ligand-induced receptor modulation remains to be determined.
Production of CXCL13 by blood-derived cells and regulation by cytokines
Since CXCL13 protein was localized to infiltrating immune cells in actively demyelinating multiple sclerosis lesions, we asked whether the CXCL13 production by PBMC was regulated by inflammatory cytokines and during macrophage differentiation. Analysis of subsets of PBMC indicated that the CXCL13 transcribing cells were within the CD14+ monocyte fraction (Fig. 6 and data not shown). The regulation of CXCL13 production by immune cells was studied in a total of seven experiments, two with PBMC, two with purified monocytes and three with differentiated macrophages. Figure 6 shows representative experiments. Purified monocytes and macrophages were stimulated with the indicated cytokines. The amount of CXCL13 secreted was measured by ELISA. In parallel the level of CXCL13 transcripts was determined by quantitative PCR. The production of CXCL13 was enhanced by IL-1β and TNFα, whereas IL-4 and IFNγ inhibited the spontaneous and induced CXCL13 production (Fig. 6). Similar results as with purified monocytes were obtained with PBMC (data not shown). Differentiation of monocytes to macrophages with M-CSF greatly enhanced the spontaneous CXCL13 expression. These macrophages constitutively produced a high amount of CXCL13 that was not further enhanced by TNFα and IL-1β. Again, an inhibition with IL-4 and IFNγ was observed. The CXCL13 protein release measured by ELISA mirrored the transcriptional regulation analysed by quantitative PCR (Fig. 6).
In order to gain further insight into mechanisms of B cell trafficking into the inflamed CNS, the chemokines CXCL12 and CXCL13 were analysed. Both chemokines mediate together the dark zone and light zone organization of germinal centres in lymphatic tissue (Allen et al., 2004). We report that CXCL12 is elevated in both active and inactive multiple sclerosis lesions, whereas CXCL13 was detectable only in active lesions. Levels of both CXCL12 and CXCL13 were elevated in the CSF. CXCL13, but not CXCL12, correlated strongly with intrathecal Ig synthesis, presence of T cells, B cells and plasma blasts.
We detected CXCL12 on blood vessel walls in the grey and white matter of subjects without neurological disease. Previous studies indicated that neurons and endothelial cells are the main producers of CXCL12 in the healthy adult CNS (Klein and Rubin, 2004). In multiple sclerosis lesions this chemokine was strongly displayed on astrocytes and blood vessels. The up-regulation of CXCL12 in multiple sclerosis lesions might be due to two different pathological alterations. First, the inflammation could induce enhanced production of CXCL12, since this chemokine is prominent in inflammatory skin (Pablos et al., 1999) and on the synovium in rheumatoid arthritis, where CXCL12 production was enhanced by CD40-stimlation (Nanki et al., 2000). IL-1 and TNF-α stimulate CXCL12 production by human astrocytes (Ambrosini et al., 2005). Neuroinflammation results in elevated levels of CXCL12 in the CSF [this study and (Pashenkov et al., 2003)]. The higher CSF level of CXCL12 in OIND patients with a disrupted BBB as compared with multiple sclerosis patients might be related to a more pronounced neuroinflammation in these cases. A second mechanism of CXCL12 induction in multiple sclerosis lesions could be based on a hypoxia-like metabolic state. Hypoxia and the hypoxia-induced transcription factor HIF-1 stimulate the production of CXCL12 (Hitchon et al., 2002; Ceradini et al., 2004). CXCL12 is up-regulated in the ischaemic penumbra following stroke and there localized on astrocytes (Hill et al., 2004). Interestingly, a hypoxia-like tissue injury was recently considered to be a component of multiple sclerosis lesions (Graumann et al., 2003; Lassmann et al., 2003). This hypoxia-like metabolic state in multiple sclerosis lesions can principally be induced via two ways, firstly by defective microcirculation due to inflammatory damage of the vascular wall or secondly by toxic metabolites that interfere with mitochondrial energy metabolism (Lassmann, 2003).
The consequences of up-regulated CXCL12 could be manifold. CXCL12 is a chemoattractant for T cells, monocytes, B cells, and also plasma cells and could contribute to the maintenance of immune cells in the CNS. CXCL12 is, in particular, important for the maintenance of plasma cells, since B cells lose most of their chemokine receptors during differentiation to plasma cells, but keep CXCR4, the receptor for CXCL12 (Hargreaves et al., 2001). The effects of CXCL12, however, exceed regulation of migration, e.g. it induces the production of TNF, IL-1 and CCL5/RANTES in astrocytes, which might promote inflammation and tissue destruction (Han et al., 2001). CXCL12 can induce neuronal apoptosis in certain conditions (Hesselgesser et al., 1998) and CXCL12 immunoreactivity has been linked to AIDS-related dementia (Langford et al., 2002). On the other hand, CXCL12 can promote survival of neurons (reviewed, Lazarini et al., 2003). Interestingly, cleavage by metalloproteinase (MMP)-2 converts CXCL12 into a neurotoxic form (Zhang et al., 2003). MMPs including MMP-2 were found to be present at elevated levels in post-mortem multiple sclerosis tissue (reviewed, Yong et al., 2001). Therefore, the elevated level of CXCL12 in multiple sclerosis lesions we describe here could contribute to the neuronal damage and axonal loss observed in multiple sclerosis (Kornek and Lassmann, 1999).
The cellular localization and the expression pattern of CXCL13 were very different from that of CXCL12. CXCL13 was undetectable in the normal CNS, up-regulated in active multiple sclerosis lesions, but not in chronic inactive lesions. This differential expression of CXCL13 in active versus inactive lesions was seen by both quantitative PCR and immunohistology of multiple sclerosis lesions. Also our CSF analysis supported this conclusion: CXCL13 levels were linked to inflammatory activity, in particular intrathecal Ig production as well as accumulation of B cells and T cells in the CSF of multiple sclerosis patients. This observation has implications for the still largely unknown mechanisms for recruitment of B cells into the inflamed CNS in multiple sclerosis, and extends the biology of CXCL13/CXCR5 beyond homeostatic trafficking to the regulation of chronic inflammation in the CNS. CXCL13 was also elevated in the CSF of OIND patients. Our results confirm a recent report about elevation of CXCL13 in neuroborreliosis (Rupprecht et al., 2005). In addition, we show that a high CXCL13 level can also be observed in non-neuroborreliosis patients, in particular in those with a disturbed BBB. Our PCR analysis of a total of 14 multiple sclerosis lesions and the CSF examinations indicated that intracerebral CXCL13 production is a feature of acute inflammation in the CNS.
Our immunohistological staining localized CXCL13 to infiltrating immune cells in early active multiple sclerosis lesions with highly abundant CD68+ macrophages in the parenchyma and around blood vessels (Theil et al., 2005). In the perivascular cuffs CXCL13+ staining could be seen between the infiltrating cells and infiltrating cells were labelled in the parenchyma. Additionally, CXCL13 staining was also seen on extracellular matrix within these lesions. This is in line with two other studies, one that co-localized CXCL13 in fibronectin+ fibrils in ulcerative colitis and rheumatoid arthritis (Carlsen et al., 2004) and another one that described CXCL13 in the wall of a blood vessel in a chronic active multiple sclerosis lesion, but not directly to any cells (Corcione et al., 2004). The localization of CXCL13 to infiltrating cells we have described here, probably monocytes/macrophages, in actively demyelinating multiple sclerosis lesions is in line with two other studies, one which localized CXCL13 to monocytes/macrophages in rheumatoid arthritis and ulcerative colitis (Carlsen et al., 2004) and another which found up-regulation of CXCL13 in an animal model of Lyme borreliosis and detected CXCL13 on myeloid cells (Narayan et al., 2005). This inspired us to analyse how this chemokine, classified as homeostatic, can be regulated by inflammatory cytokines and we identified four cytokines that affect its production in a complex way. The monocyte fraction of PBMC produced CXCL13 after stimulation with IL-1β and TNFα, whereas both IFNγ and IL-4 inhibited this. These findings were unexpected as IL-4 and IFNγ frequently have opposing effects on monocyte and macrophage activation. In harmony with a recent report (Carlsen et al., 2004) we observed in vitro that differentiation of monocytes to macrophages greatly enhanced their expression of CXCL13. It is presently unclear how the contrasting effects of IL-4 and IFN-γ on the one hand versus TNF-α, IL-1β, and macrophage differentiation on the other hand affect the CSF level of CXCL13.
A recent study reported immunostaining of CXCL13 in lymphoid follicle-like structures in the cerebral meninges in two out of three patients with SP-multiple sclerosis but not in patients with RR-multiple sclerosis and PP-multiple sclerosis (Serafini et al., 2004). CXCL13 was not detected by immunohistochemistry in the parenchyma of chronic active or chronic inactive multiple sclerosis lesions (Serafini et al., 2004), which is in line with our analysis of six chronic inactive lesions by histology and quantitative PCR. We observed CXCL13 immunostaining in a full blown early active lesion; no such lesion was included in a study that failed to detect CXCL13 on infiltrating cells within the lesions (Serafini et al., 2004). Our study detected a differential expression of CXCL13 in actively demyelinating and inactive lesions by combining quantitative PCR and immunostaining.
Overall, a picture is emerging of two different sites of CXCL13 production in the multiple sclerosis CNS. In lesions with strong inflammation the infiltrating macrophages could be a source of this chemokine, whereas in some patients with an SP-multiple sclerosis, CXCL13 might be produced in follicle-like structures in the meninges. Our study shows that the CXCL13 level in the CSF is linked to the degree of inflammation. Only a few SP-multiple sclerosis and PP-multiple sclerosis patients had considerable levels of CXCL13 in the CSF, whereas in the majority of CP patients very little or no detectable CXCL13 was found in the CSF. It remains to be established whether follicle-like structures, which were described in the meninges of two out of three patients with SP-multiple sclerosis (Serafini et al., 2004), are linked to a high level of CXCL13 in the CSF.
To analyse the potential functional relevance of the intracerebrally produced CXCL13, we examined expression of its receptor CXCR5 by CSF cells. In CSF virtually all B cells and ∼20% of the CD4+ T cells or of the CD4+CD45RO+ T cells expressed CXCR5, the receptor of CXCL13. The expression of CXCR5 was observed at similar levels in blood and CSF cells. This resembles previous observations on the chemokine receptors CCR1-6 (Ransohoff, 2002) and CCR7 (Kivisakk et al., 2004), whereas CXCR3 was present at higher levels on CSF cells than on the corresponding population in blood (Kivisakk et al., 2002).
We went on to analyse whether the intracerebral production of CXCL13 could be linked to recruitment or persistence of a particular immune cell subset. The CXCL13 level showed a strong correlation with the intrathecal Ig-production and also a significant correlation with the presence of B cells and plasma blasts, which are the main B cell effector subset during the course of multiple sclerosis (Cepok et al., 2005). This is the first report that correlates the number of B cells and plasma blasts in CSF to a particular chemokine and suggests that CXCL13 is important in directing B cells to the inflamed CNS.
Remarkably, the CXCL13 level in the CSF also correlated very strongly with the presence of T cells in the CSF. Whereas the role of CXCL13 and its receptor CXCR5 on B cells in the development of lymph follicles is well established (Ansel et al., 2000), far less is known about functions of CXCR5 on T cells. CXCR5-expressing T cells are functionally heterogeneous (Kim et al., 2001). In tonsils, a subset of CXCR5+ T cells are specifically localized in germinal centres and function as follicular B helper T cells (Moser and Ebert, 2003). CXCR5 expressing T cells in blood are, unlike the CXCR5+ T cells in the germinal centres, in a resting state and need to be activated to help B cells (Kim et al., 2001). In vitro CXCR5 is transiently induced on T cells after polyclonal activation (Langenkamp et al., 2003). The functional properties of CXCR5+ T cells in the CSF have yet to be defined.
In addition to its regulation of cell migration, CXCL13 is known to have several other effects, including a stimulatory cytokine loop: CXCL13 up-regulates membrane LTα1β2 on B cells, promoting the development of follicular dendritic cells and thus the production of CXCL13 itself (Ansel et al., 2000). This raises the possibility that CXCL13 in the multiple sclerosis lesion is not only involved in immune cell recruitment, but also in the regulation of the cytokine environment.
In summary, we have demonstrated elevated levels of CXCL12 in active as well as inactive lesions. CXCL12 might be involved in the maintenance of immune cells, in particular plasma cells, in the CNS. CXCL13 was up-regulated in active, but not inactive lesions. The intracerebral CXCL13 production strongly correlated with intrathecal Ig production, as well as the number of B cells, plasma blasts and T cells in CSF. Thus, CXCL13 is identified as a chemokine that is involved in recruitment of B cells and T cells to the inflamed CNS. Therefore, targeting CXCL13–CXCR5 interactions may be a promising novel therapeutic approach in multiple sclerosis.
We thank D. Zech for technical assistance, Dr S. Wagenpfeil for help with the statistical analyses, and Dr A. Flügel and Dr D. Jenne for comments on the manuscript. This work was supported by the DFG (SFB 571, GRK 688, He2386/5-1 and 5-2), the DMSG, the Verein zur Förderung der Therapieforschung für MS-Kranke and the USA National Institutes of Health (PO1 NS38667 to R.M.R.). The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation.
Author notes
1Department of Neuroimmunology, Max Planck Institute of Neurobiology, Martinsried, 2Institute for Clinical Neuroimmunology, 3Department of Neurology, Ludwig Maximilians University, Munich, 4Department of Neurology, Heinrich Heine University, Düsseldorf, Germany, 5Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA and 6NeuroResource, Institute of Neurology, University College London, London, UK


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text: Have you ever heard of background checks, starter kits, and similar BS? Well, if you want to work at home, and you're looking for programs or opportunities to join, you most probably have encountered with those. Never, ever pay anything for the ability to work at home. No matter what they say, or how well it's presented, that's a scam, living from those upfront or operating costs. Period. Remember, they're the ones who pay you, and not the other way around. Now, what's your input on this? Please share


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How Do I Know If My Rash Is Scabies
Scabies pictures can still leave someone confused when they are trying to identify their own rash. This is because scabies rashes can also resemble eczema, viral rashes as well as allergic responses to a chemical or food allergy.
Because the scabies mite can't be seen by the naked eye you can't see it like chiggers. Also rashes can be due to a reaction to an antibiotic, virus, contact dermatitis to a chemical or food allergy reaction. Many rashes appear to look similar having little red pimples, welts, patches and being extremely itchy.
If you hear that others in your family, class or work place have an itchy rash, then you know that there is a contagious rash going around. Scabies rashes may not appear right away and can take a few weeks to manifest. You may have been in contact with someone who was contagious and they also weren't aware of this issue.
Scabies pictures vary because not everyone will get little pimples in the webs of their fingers or on wrist areas. Also the elderly can get Norwegian scabies which are not common for younger people. Sometimes a dermatologist can see a thin line which indicates the burrow of the female scabies mite where she lays the eggs. Extreme itchiness at night is a hallmark sign of the scabies rash but it can also be a common problem for atopic eczema and nummular eczema.
Because scabies is a parasite it is important to treat it with a cream prescribed by your dermatologist or doctor so that it will stop the reproduction on your own body as well as no longer be contagious to other people.
If you live with other people, they suggest everyone also use the cream in the household so that the mites won't just jump from person to person and keep the cycle going.
A lot of the anxiety of rashes involves not knowing what the rash is. Scabies pictures on the internet can be helpful but look also at the description and see if you may be having an allergic reaction to something else in your environment. Some dermatologist will do a skin scraping Allergy House to check for the mites or have a biopsy done to rule it out.
The most common treatment used for killing scabies in the United States is to prescribe the elimite cream which is applied before bed and left on over night. Some dermatologists will recommend a second application a week later to get anything residual left. In India, neem oil is used which is actually a contraceptive and said to stop the eggs from hatching. Linens and bed blankets should be washed in very hot water and placed in hot dryer. Clothing recently worn should be bagged and washed days later after the mites have suffocated.
Despite the stressfulness of a scabies rash, remember that you may be itching also for psychological reasons and that the rash won't stop the minute you apply the cream. It can take a few weeks to resolve.
By: Dee Cohen
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Acute and Critical Care Clinical Nurse Specialists
Synergy for Best Practices
By (author) American Association of Critical-Care Nurses (AACN)
Edited by Mary G. McKinley
Format: Paperback
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Acute and Critical Care Clinical Nurse Specialists by American Association of Critical-Care Nurses (AACN)
Book Description"Acute and Critical Care Clinical Nurse Specialists: Best Practices" presents the knowledge and tools the CNS needs to provide the best standards of practice and performance. It focuses on the role of the CNS in acute and critical care, emphasizing the relationship between critical care nursing, the patient, and the environment of care. Divided into 3 main sections, the framework of the book is based on the Scope and Standards of Practice for the Clinical Nurse Specialist in Acute and Critical Care and the AACN's synergy model. This book delivers up-to-date information for today's health care along with practical tools for the CNS in acute and critical care settings.
Buy Acute and Critical Care Clinical Nurse Specialists book by American Association of Critical-Care Nurses (AACN) from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9781416001560
(235mm x 191mm x 15mm)
Pages: 288
Imprint: Saunders
Publisher: Elsevier - Health Sciences Division
Publish Date: 4-Jul-2007
Country of Publication: United States
Books By Author American Association of Critical-Care Nurses (AACN)
Aacn Procedure Manual for High Acuity, Progressive, and Critical Care, Paperback (January 2017)
Preceded by: AACN procedure manual for critical care / edited by Debra Lynn-McHale Wiegand. 6th ed. c2011.
AACN Essentials of Progressive Care Nursing, Paperback (June 2014)
Covers all the details on the care of adult progressive care patients and their families. This book includes essential information to successfully prepare for PCCN certification in progressive care and more.
AACN Essentials of Critical Care Nursing, Paperback (June 2014)
The authoritative, evidence-based coverage needed to safely and competently care for adult, critically ill patients and their families - or prepare for the critical care nursing certification exam
AACN Advanced Critical Care Nursing, Hardback (June 2008)» View all books by American Association of Critical-Care Nurses (AACN)
A resource to help you oversee or care for critical care patients in a practice setting. This book addresses serious and potentially life-threatening patient conditions.
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Arts, Performing Arts, TV & FilmFollow
|Performing Arts
Movies for your ears.
January 15th, 2015
Episode 60 of 91 episodes
Who knows... maybe it's true. Written by Diane McCorry. Performed by Phoebe Tyers and Dennis Pacheco, with Nick Mykins, T.J. Mannix, and Chris Dwane. Produced by Jonathan Mitchell.
You'll need to sign in to write a comment.
Welcome to the daily audio workout for your head.


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With over 10 years experience within the design, manufacture and installation of cabinetry within the domestic and healthcare sectors I now provide a professional kitchen installation service to suit all budgets. In addition to this I can provide interior and exterior...
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I am an indentured Carpenter with 30 years experience within the building trade. I pride myself on supplying quality work and service to every one of my customers Throughout my career I have gained a vast knowledge of construction methods and am sympathetic to traditional...
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Broadoak Floors & Doors offer a wide variety of quality products at very competitive prices to mainstream DIY stores. Products range form solid timber, engineered timber, laminate and LVT floors, to quality veneered and moulded doors. We are very competitive and offer...
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General builder, experienced with new builds, bespoke and contemporary. Any work or projects considered.
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ENADA NADH Is Back
The NEW ENADA nadh product has now been improved by having a GRAS(Generally Recognized as Safe) status enteric coating which improves theproduct more naturally than previously.
The new ENADA nadh helps promote optimal absorption, allowing individualsto achieve maximum cellular energy benefits; furthermore it contains no:sugar, starch, salicylates, wheat, gluten, corn, egg, soy, casein, caffeine,yeast, dairy, preservatives, artificial colors, or flavors. From studiesconducted by Dr. Birkmayer and associates stabilized NADH enhances theactivity in organs that develop a high metabolic turnover, like the brain, thecirculation system, and muscles.(*)
Also known as coenzyme 1 (Co-E1), NADH is a highly specialized form of theB-vitamin niacin that is found in all living cells. It plays a key role in theenergy production of cells and neurotransmitter processes. Stabilized, intactNADH appears to work by entering "fatigued" cells to promote cellular energyproduction in the form of ATP (adenosine triphosphate), a nucleotide thatfacilitates intracellular energy transfer.(*)
Birkmayer USA is announcing the introduction of 5mg ENADA NADH which isavailable end of June 2008. Other nadh products sold by Birkmayer USA are theCo-E1 NADH brand of 5mg Enteric coated and also the Sublingual Co-E1 NADH 10mgand 20mg tablets. The sublingual nadh products are ONLY sold and distributedby Birkmayer USA and are a popular product. The products will be distributedin health food stores and also online at http://www.enada.com.
Note: All present Enada nadh brand products presently on the market arebeing recalled to their distributor by the trademark owner due to low quality,low nadh content and low absorption of the tablets which damages the Enadabrand name.
(*)These statements have not been evaluated by the Food and DrugAdministration. This product is not intended to diagnose, treat, cure orprevent any disease.
About Birkmayer USA
Founded in 2003, Birkmayer USA is a nutrition company that held theexclusive Enada nadh distribution rights from Dr. Birkmayer during 2003 to2005 (before his bankruptcy). Birkmayer USA worked with Dr. Birkmayer to buildthe Enada name in the USA and has continued to distribute a high-qualityworking nadh product under the brand CO-E1 NADH. Birkmayer USA is seen as theauthority on nadh in the USA and analyzes all nadh products frequently. Forany further information, visit http://www.enadh.com , http://www.co-e1.com .
This release was issued through eReleases(TM). For more information,visit http://www.ereleases.com.Contact: Bill Armstrong Birkmayer USA 760-438-2755
SOURCE Birkmayer USA
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Market Segment
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New Products
New Products / Solutions
Since 2016 to date, Armstrong Ceiling Solutions have launched several new products, which will absolutely upgrade your design inspiration!
View Details
Latest News
Armstrong Ceilings Summer Launch Event. Shanghai
On August 26th, Armstrong Ceilings, the world leader of commercial ceiling solutions, unveiled its 2016 summer launch season at Shanghai, No.8 creative industry park.
View Details
SolutionsReverberation Simulator
Education Ceiling Solutions
Healthcare Ceiling Solutions
Office Ceiling Solutions
Case Study: Beijing Metro Line6
Trust & Innovation


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Child Boys Elvis Costume [36104]
Child Deluxe Gladiator Costume [00325]
Child Dragon Slayer Costume [00276]
Child Indian Boy Costume [3710]
Child Deluxe Black Knight Costume [00389]
1 Review
Good Costume
Good costume, although we were disappointed in the hat as it's soft and has to be shaped. Gave up & bought a separate one.
Sizing - chaps were very long but easily shortened. Better than being too short!!!
Still felt it was a good costume & value for money. :-)
Reviewed by Elizabeth from Runcorn on 12 June 2016


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Nothing is more exciting than a FOOD FESTIVAL! Finally,this initiative by Madea Creative House will have you Navi Mumbaikars gorging on food, fun and entertainment this Valentines week!
Register @ http://www.madea.in/navimumbai-food-festival-2016/
Entry Fees- It's FREE and always will be.
For More Details Kindly Contact:
Ph: 022 65550077 | 022 65070077


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We have moved!
Hall Aitken is excited to announce that from 23 January 2017 our Glasgow team has moved. Read more.
Who we are and what we deliver
We work with our clients to improve the delivery of services, save costs and prove impact. We do this mainly through strategic planning, management support and evaluation. We have a strong reputation with the international network of social value practitioners, through Social Value UK and Social Value International. We build an organisation's Contract Readiness to have a business with market credibility, financial strength, internal delivery skills and skills to bid for, win and deliver contracts effectively.
Clear - but never over-simplified
Why use three paragraphs when three sentences would do the same job? And why spend time on clever graphs and charts which show our computing skills - and little else? By using Plain English and Clear Graphics, we get to the heart of the matter.
Practical - but never ordinary
When you're choosing a partner, you're obviously going to demand robust research skills and sound methods. That's a given. However, what sets Hall Aitken apart is our insistence on applying real world experience to every project, to illuminate the process and the findings.
Innovative - but never rash
New tools. Fresh approaches. Workshops. Focus groups. Skills transfers. For all the novel ideas we bring to a project, there's always a clear focus on the end result. In other words, style never triumphs over substance.
Hall Aitken has been awarded a place on Grants and Programme Services Framework agreement for the supply of Evaluation services.
A flexible disgnostic and planning tool. Read more
News in brief
We have moved!
Hall Aitken is excited to announce that from 23 January 2017 our Glasgow team has moved.
If you would like to receive our regular newsletter containing Hall Aitken news and events, other interesting and useful news, top tips and the latest funding news please follow the link below.
We will never share your details with anyone else.
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Hall Aitken Research Director, Denis Donoghue revisits our 2007 Outer Hebrides Population study for BBC Alba report on depopulation in the Western Isles
Denis Donoghue, Research Director at Hall Aitken, in interviewed for a BBC Alba report on the depopulation of the Western Isles.


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We hope you have had a wonderful break and are ready (or getting ready) for spring semester!
Please remember that proposals for the Iowa Writing Center Consortium conference are due on January 27, 2017, by 11:59 pm! The CFP is attached here. Please feel free to share this CFP with anyone who may not be on our mailing list.
We will also have a limited number of poster slots available for a poster session, so if you (or your tutors) would like to participate in a poster session, please email Sue Pagnac separately. At this time, each college can present 2 posters for the poster session.
Registration information will be out soon.


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Join FREE | Login
+ Celebrations
+ How-To
+ Deals
Poppin' Up Happy Birthday Cake
Key Lime Pie Poke Cake
Coconut Cake with White Chocolate Frosting
Happy Birthday Marshmallow Cupcakes
Strawberries and Cream Cake
Treasure Chest Cake
Making dessert for a sporting celebration is child's play thanks to cake mix and ready-to-spread frosting!
Create an attractive display by covering a piece of sturdy cardboard with wrapping paper, then plastic food wrap. Stretch and secure with tape. Or cover cardboard with foil or cooking parchment paper.
*For all chocolate and devil's food cake mixes, use only 1 cup of the water.
© 2016 ®/TM General Mills All Rights Reserved


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We recently wrote about watching a directory using the new APIs introduced as part of Java 7. In this post let me throw some light on another class added to the java.nio package as part of Java 7 update- java.nio.file.Files. Another piece of … [Continue reading]
Java HelloWorld vs Scala HelloWorld
As a first step into learning Scala and as one who is familiar with Java, let us compare the customary Helloworld programs in Java and Scala. You might already know that to run a Java program, there must be a public class with a main method that … [Continue reading]
Introduction to Java Agents
In this article we will discuss about Java Agents. Java Agents are software components that provide instrumentation capabilities to an application. In the context of agents, instrumentation provides the capability of re-defining the content of class … [Continue reading]
Implementing WatchService API in Java 7 to monitor the directory changes
In Java 7 there were quite a lot of new things added to the File NIO package (java.nio)- there was a new java.nio.file package and java.nio.file.attribute package. Main highlights of the java.nio.file package are the following classes: Path: Its … [Continue reading]
Merge Sort implementation using Fork-Join Framework in Java 7
In our previous post we went through the basics of Fork-Join Framework introduced as part of Java 7. In this post lets apply the same Fork-Join to implement Merge sort algorithm. The pseudo code for Merge sort can be found here. also read: Java … [Continue reading]
A simple Location and Weather mashup using Gaelyk Framework
In our sample Gaelyk application here we stopped at just obtaining the location information. In this post lets update that application to fetch the Weather information as well. also read: Groovy Tutorials Creating JSON using Groovy Developing … [Continue reading]
Simple introduction to Fork-Join Framework in Java 7
Fork-Join Framework was added as part of Java 7 which makes use of the ExecutorService interface to distribute tasks to the worker threads in the thread pool. The difference in Fork-Join is that the ideal worker threads can steal queued subtasks from … [Continue reading]
Handling/Avoiding Null's in Java using Guava versus Scala
There was a discussion about nulls in the latest episode of JavaPosse. Having been inspired from that discussion and with an urge to check out Google Guava library, I came across a way to handle Null's in Guava which is very similar to the way Scala … [Continue reading]
Developing Groovy based web application and deploying to Google App Engine
We have seen how to develop a ZK based application and deploy it to OpenShift. Google has also a Paas offering called Google App Engine(GAE) which supports Java, Python and Dart applications. Gaelyk is a simple Groovy based toolkit to develop and … [Continue reading]
HelloWorld with Netbeans 7.2 beta and Scala 2.9
Here is the step by step procedure to set up the scala(2.9) environment with Netbeans(7.2) on a Windows Vista system 1. Install Netbeans 7.2 Beta if not already installed from here. 2. Download the Scala installable from here. Install Scala on … [Continue reading]
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