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Research Process
We invest bottom-up but worry in every direction.
The members of our research team work collaboratively and, as a group, constitute Marshfield's Portfolio Manager. This ensures that each client account is the recipient of our best collective thinking.
Our approach is consensus-based and highly disciplined, with the objective always being to understand what makes a company and its industry tick. The group process facilitates idea generation, allows for the application of a consistent investment philosophy, supports critical thinking, and encourages the flushing out of flaws in the investment thesis. We are skeptical, trained to air potential risks and to identify our own biases, and eager to understand the truth, whether that leads to buying a stock or consigning it to the scrapheap.
Integral to our process is the development of a "shopping list" of companies that we would buy at a designated price. This allows for good decisions to be made while under stress, as unless the fundamental investment thesis has changed, we are ready and willing to take action even in the face of chaotic external events.
The cornerstones of Marshfield's process are:
• Rigorous analysis, which entails developing a full understanding of the ecosystem in which the company exists. We read all relevant materials (journal articles, securities filings, etc.) about the company and its industry, talk extensively to management of both the subject company and that of its competitors, and conduct research into the economics underlying the industry. This often means talking to suppliers, customers, and regulators in order to arrive at a deep understanding of how the business works.
• Synthesis, in which we refine our theory of the company based upon our analysis, determine whether we need any additional information, and submit our conceptual case to unsparing critical review.
• Valuation of the company (free cash flow and/or book value, depending upon how cyclical the business is and how dependent on book value earnings are) is conducted collectively and using conservative assumptions. We stress test our valuation based upon a series of different scenarios, and set a target buy price that incorporates a substantial margin of safety.
• Continuous review of all holdings requires ongoing monitoring of the financial performance of each portfolio company as well as analysis of whether each company is executing as our conceptual case would suggest.
• Sales occur when we've made a mistake, when the fundamentals change, or when extreme overvaluation occurs.


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Joint Venture Secrets
Welcome To 2dollar Store all our products comes with Resale https://www.tradebit.com from here and sell it anywhere and make big profits or you can use it for your Personal Use.
Discover How You Too Can Launch Your Products To Millions Of Hungry Customers Through The Power Of Joint Ventures!
Full start to finish tried and tested joint venture guide.
Proven methods of the successful joint venture.
From your introduction and getting noticed, to your follow-up and securing your future and everything in between.
When I first started out in online business (almost seven years ago now) it always intrigued me to see marketers go out and pull a circulation of half a million potential views for their products just by getting themselves ten or fifteen deals with other marketers. Half a million targeted, almost guaranteed to be interested, ready to buy viewers, customers even. Wow. Imagine what that would do for your business.
(This is usually the point at which I pull out my calculator and start playing with numbers looking at the earning potential, I don't know any marketer that doesn't do the same, so feel free!)
So how did they do it?
Well that's what I wanted to know too, especially as I was on a budget and didn't have much cash to hand, meanwhile they were doing all this for free. So I went around and bought courses, bought info products, bugged people in the know with all sorts of questions.
The methods they were using all seemed suspiciously similar.
They'd say to me, it's easy. Go check out google (or yahoo, and what's now known as https://www.tradebit.com back in the day) and search for 'Online Business'. Then all you have to do is find the people who are successful, and e-mail them with your offer.
Something wasn't right here. What was it?
Think about it for a moment. If these marketers are really selling thousands of their 'How to Joint Venture' products, then how many people are contacting everyone on the first 10 pages of these search engines! They must be getting hammered with e-mails.
I gave it a shot anyway, here's what happened...
All of these list owners were replying to me with 'Look, you have a good offer, but since these products started popping up telling people to just use search engines and contact the successful, I've been inundated with more offers than I could handle in a lifetime'. (Oh and trust me when I say that was was one of the kinder responses stating the same point).
It became obvious to me what the problem was.
These marketers may have used these tactics when they first started out, but it just didn't work anymore. This method was saturated (and this was almost seven years ago) yet no one seems to correct themselves or come up with alternatives to such an outdated technique, and how can they afford not to when half a million or more circulation for a single product are at stake here?
Something had to change, and it did:
It took me seven years, it took me more than $38,000 to hone a new method for successful joint venturing, not to mention many many hundreds if not thousands of hours sitting in this very chair that I write to you from now, but finally, I did it, and now it's all yours. Check this out.. (Hint: I didn't even contact a single big name marketer to pull this off or pull random names from search engines to make this work)
* How To Find Real JV Prospects. We all know that a couple of simple ads here and there to large, well built, targeted lists can shove thousands, even tens of thousands of dollars into your pocket per mailing. We'll show you step by step, exactly how we go about securing no less than twenty of these large scale joint ventures every single time we launch a product with very little effort, whilst others having trouble securing even one.
* An Almost Endless Supply of Prospects. Overcome the hassles and ethical issues of having to cold call and drop in on prospects unannounced, only to be told they don't want to hear from you. There's a little known back door that we're sneaking through as you read this, pulling in scores of immensely profitable joint venture deals whilst the majority fight over the same 20 people.
* The one and only way to quickly get in on the ground floor with big joint ventures, even if it's your first day in online marketing. You may think you have nothing to offer in return for the circulation of your ad to tens of thousands of targeted buyers. You'd be wrong. Thankfully for us, only a few know of this powerful method, leaving you plenty of space to get in on this before the crowds flock to it.
* 12 Mistakes Of The Joint Venture Newbie. I'll show you the exact reasons that joint venture newbies get denied over and over again, when they could quite easily be circulating their products to many thousands, if only they knew what our decade + of experience tells us they were doing wrong.
* How to know before approaching anyone whether or not they're going to accept your offer. Don't waste your time by approaching people that have no interest in your deal. We have devised 7 easy to replicate methods over the years that will allow you to judge immediately whether your prospect is going to accept you or not.
* How to build and use a powerful information network. The future is always bright for your business' profits when you have a group of thirty or forty people with these valuable lists ready to promote for you on the launch of each of your products. What's more, they're easy to find. We'll show you exactly where. (Best of all this only takes ten minutes to do). Imagine the profits at having that type of promotion power at your disposal. It's all yours, right here.
* 5 ways to increase your acceptance rates by 85. Tailor your deal to your joint venture prospect and start seeing your ad sent to more of these hugely profitable targeted lists every single time you launch a new product. It's easy to hit the magic 85 mark if you have the right information. We'll show you how to get it for free in a mere three minutes of your valuable time.
* Learn to spot what your potential jv partner wants from your deal before you even meet. Imagine being able to present your prospect with everything they wanted as soon as you make first contact. Your response rates and acceptance rates will go through the roof, not to mention they'll start talking about you to their contacts, and we all know how powerful word of mouth is.
* 2 techniques to ensure that the joint ventures you make today, will be open to you every single time you launch a new product. Imagine succeeding in gaining just five separate joint ventures, each list containing 20,000+ hungry customers ready to buy your products. That's a real money maker, but it gets unimaginably profitable when you add another five of these lists every single time you launch a new product. We're doing that right now, as you read this, and we'll show you how.
* Learn the why's of Joint Venturing. It's time to stop buying products that only tell you how to do stuff that become useless when everything changes in a few months time. It's a waste of your time and money. This guide however will show you why everything works, benefiting you not just on the first read, but for life; allowing you to adapt to business situations that we all know change very rapidly.
Product Rights- Master Resale Rights
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To Your Great Sucess
our store http://2dollarstore.tradebit.com
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Thanksgiving Pregnancy Announcements are the best! Bringing new life into the world is not only a great and joyful time in your life but also a blessing. What better way to share this great news with your loved ones than on Thanksgiving? The whole family will be together and you'll all be toasting to what you're thankful for this year. And there isn't anything to be more thankful for than the promise of a new addition to the family. Use these creative ways to announce your pregnancy this Thanksgiving.
Use Baby Things
Drop hints here and there by using baby things. Adorable little baby accessories like toddler clothes, onesies, shoes and little spoons or a little pair of glasses. To be more creative, use thanksgiving themed things. This is going to be the most adorable way to announce your pregnancy. Find some creative way to incorporate this bundle of cuteness into your Thanksgiving meal.
Mini Fan and Big Sister Announcement
If you and your loved ones are sports fans, get a sports shirt for your baby with the team's logo on it. This is always a fun way to break the news! If you already have a child, then let him/ her break the news for a more wholesome family experience. Check the gallery for ideas.
Books – To Speak the Language of Love
Gift him a basket of baby books like What to Expect When You're Expecting. This is the most common way to break the news and always succeeds in sending a smile to his face.
More Thanksgiving Pregnancy Announcement Ideas
Pets are adorable because they're oblivious to what's going on. Let your pet deliver the good news for a more funny, yet creative announcement. You could also use simple, straightforward signs or cards and you can theme it around Thanksgiving.
There are many ways to break the news. It's a very exciting time. We hope these Thanksgiving Pregnancy Announcement Ideas rounded up in the gallery help in bringing extra joy to your family.From big brother announcement ideas to holding little baby shoes in the hand, there is too much to pick inspiration from
Source for Images: Facebook.com


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With the plethora of fresh vegetables available this time of the year in our own gardens, why not indulge in a healthy salad, bursting with colors, textures, flavors and nutrition?
Fresh corn is available in stores at the moment (choose organic over conventional since most of the corn grown today is genetically modified to feed cattles and farm animals).
One of my favorite addition for this healthy salad is mung Bean sprouts. It is one of the easiest thing to sprout. You can't mess it up regardless of the temperature, even if you don't rinse them twice a day as it is usually required for other types of seeds or beans. They also keep refrigerated for almost a week.
Mung beans sprouts are crunchy and produce a slight nutty flavor, making them a perfect addition to most salads and wraps.
They may seem small and plain but don't let this fool you. They pack a lot of nutrition in every bite. 1 cup of raw mung beans contains 3 grams of protein. It is an excellent source of folate (vitamin B for DNA and blood cells) and manganese, a mineral essential for a healthy metabolism and bone formation.
1-2 cups of mung bean sprouts
1 cup fresh corn kernels
1/3 cup of diced red onion
1/2 cup fresh or blanched green beans
1/2 diced avocado
1 grated carrots
2 Tbl pine nuts (optional)
2 Tbl grated coconut powder (unsweetened) optional
Fresh mint or coriander leaves
Salad dressing:
I usually don't buy salad dressing because I have been disappointed by most of them and because they always add all kind of weird stuff to make it thick. Since it only takes less than a minute to make it why not?
2 Tbl olive oil
1 Tbl balsamic vinegar
1 tsp lemon
1 Tbl nutritional yeast
salt, pepper to taste
By the way, this salad tastes even better if refrigerated over night.
I used the leftover to make a wrap with collard leaves from my garden. Talk about fresh!
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Own the City with your Friends! Convert civilians to your crowd, eat up smaller crowds than you and avoid the bigger ones! It's a Crowdy Crowd City!
Navigate the arrow through the gaps in each oncoming wave of barriers. If the arrow touches a barrier, it's game over. Although it looks easy this minimalist game is deceptively difficult. Can you make it to 60 seconds? Play now and find out!
There are no awards for this game!


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Austin's Grupo Fantasma performed at El Rey Theatre
Don't you hate it when people say: "oh man, you missed out…"? I do. But if you were not at El Rey Theatre on Sep. 23 to see Grupo Fantasma do its thing, well… sorry, but… you MISSED OUT!
Great band, music, venue, lighting, sound… the entire thing was on point. Below I'm sharing a few pictures from my phone, plus short videos. Enjoy, and the next time you hear this band is in town, be there!
Keep an eye on them at GrupoFantasma.com.


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Posted by admin on January 8, 2018 in Articles
["Give Me Liberty or Give Me Death"][Robert Russell]
The reason Patrick Henry recited the speech, "Give Me Liberty or Give Me Death," is to persuade the House that there is no other substitute but war. Henry starts off by recognizing the patriotism and abilities of the men who spoke before, and against him. Henry goes on to persuade his audience by use of rhetorical questions and comparisons to religious beliefs. Henry's speech was very motivations, and inspirational, as well as urgent and persuasive. Patrick Henry did a good job, and at the height of his speech, he convinces the House of Burgesses that the war needs to be fought. The first thing Henry did when giving his speech was he complimented his opponents, in order to gain their respect and confidence. Since the audience respects Henry, they will be more willing to listen to what it is he has to say. He then asks for forgiveness in advance for anything he says that might disregard or insult them, or anyone else. Henry rationalizes the basis for his speech, using a false dilemma to make the audience believe that there is only two options, when in fact, there are many more. The two options he gives them are freedom, and slavery. Henry then goes on and makes a reference to God and to America, as a way to connect to his audience. He makes the statement that he should not be held accountable for treason toward his country, for if he didn't debate this issue, he would be committing an act of disloyalty to the others in similar positions to him. When Henry says "we are apt to shut our eyes against the painful truth, and listen to the song of that siren till she transforms us into beasts", he is making reference to The Odyssey. Basically what he is saying is that people tend to push away problems, and head toward what is easy, and what they are told is right, when in fact, it could be the dishonest thing, and can in the end lead…


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I love to bake and create new recipes with Nutella and it seems you do to! It is one of the most popular ingredients on my blog! So to add to my ever growing list of Nutella recipes, I have created this Nutella Bueno Cheesecake for you to enjoy. I've made sure there's plenty of Nutella in the cheesecake filling so the flavour really comes through, then I've drizzled pure Nutella on top, followed by whipped cream and finally some yummy Kinder Bueno chocolates! It's also a really easy no bake recipe too, so if you've made any of my other no bake cheesecakes before then you'll know how simple this is. And if you haven't made one yet, then give this one a try and find out! If you like this recipe you'll love my Nutella Bueno Cupcakes and my Nutella Cheesecake Blondies too!
To make the base I used my food processor to whizz up the biscuits into crumbs. I mixed in the melted butter and pressed the mixture into the bottom of a 23cm springform tin. I put it in the fridge for 30 minutes to set.
For the cheesecake filling I used my electric hand whisk to mix together the cream cheese, Nutella and icing sugar. Once it was smooth with no lumps I added the double cream and whisked until it was very thick and held it's shape.
Then I smoothed the mixture into the tin on top of the biscuit base and put it in the fridge overnight to set.
The next day I removed it from the tin, and warmed the Nutella in the microwave. Then I piped it all over the cheesecake.
Then I whipped up the double cream with the icing sugar and vanilla, and piped it all around the edges of the cheesecake. To decorate, I placed the Kinder Bueno chocolates around the cheesecake on top of the whipped cream. I added some sprinkles too!
The Nutella Bueno Cheesecake was so yummy and full of Nutella flavour!
I had two slices in one day and my friends gobbled the rest up!
|Nutella Cake
|Cherry & Nutella Chocolate Cake
|Nutella Cheesecake Blondies
|Nutella Bueno Cupcakes
|Nutella Stuffed Cookies
|Raspberry & Nutella Bread Rolls
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Nutella Bueno Cheesecake (No Bake)
Creamy Nutella cheesecake decorated with whipped cream and Kinder Bueno pieces
For the biscuit base
- 300 g Digestive Biscuits
- 125 g Butter or baking spread melted
For the cheesecake filling
- 600 g Full fat cream cheese
- 125 g Icing sugar
- 300 g Nutella
- 300 ml Double cream
For the decoration
- 300 ml Double cream
- 1 tbsp Icing sugar
- 1 tsp Vanilla extract
- 75 g Nutella
- Kinder Bueno chocolates
- Sprinkles/Chopped hazelnuts optional
To make the base use a food processor to whizz up the biscuits into crumbs, or bash them with a rolling pin in a bowl
Mix in the melted butter and press the mixture into the bottom of a 23cm springform tin. Put it in the fridge for 30 minutes to set
For the cheesecake filling use a food mixer with a whisk attachment or an electric hand whisk to mix together the cream cheese, Nutella and icing sugar until smooth with no lumps
Add the double cream and whisk until it is very thick and holds it's shape
Smooth the mixture into the tin on top of the biscuit base, then put it in the fridge overnight, or for at least 4 hours, to set
Remove from the tin and put on your serving plate. Smooth the sides with a butter knife to create a neater finish
To decorate, warm the Nutella for 30-45 seconds in the microwave and pipe or drizzle it all over the cheesecake
Then whip up the double cream with the icing sugar and vanilla, and pipe it all around the edges of the cheesecake
Place the Kinder Bueno chocolates on top of the cream, then add some sprinkles or chopped hazelnuts if you like
Serve immediately, store any leftovers in the fridge and eat within 2 days
NB. this post is NOT sponsored by Nutella – I just love their product!
If you like this, check out more of my Cheesecake recipes!
Pin for later!
I'm linking this recipe up with Cook Blog Share hosted by Everyday Healthy Recipes and Bake Of The Week hosted by Casa Costello.


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Heavy rains lash Delhi-NCR
Heavy rains lashed Delhi and its nearby areas, with met department forecasting more rains to continue till Thursday.
IMD said that he sky will remain generally cloudy with light rain or drizzle expected.The department further added that winds over Delhi and NCR have changed to easterly and so intensity of rains will increase gradually.
Since the onset of monsoon on July 5, the national capital has recorded 54.2 mm rainfall.
The Met department has also forecasted heavy to very heavy rainfall at isolated places very likely over Kerala and Coastal Karnataka and heavy rainfall at isolated places over Tamilnadu, Puducherry, Lakshadweep, Karnataka, Coastal Andhra Pradesh, Rayalaseema, Konkan and Goa, Punjab, Haryana, Chandigarh & Delhi, West Uttar Pradesh and Odisha.
Further rough to very rough sea conditions are likely to prevail over southwest and central Arabian Sea, along and off Gujarat and Maharashtra coast, Maldives, Lakshadweep and Kerala coast.Squally weather is likely to prevail over north Andaman Sea and adjoining east central Bay of Bengal.
Fishermen are advised not to venture into these areas.


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Here is this week's playlist – songs picked by a reader from hundreds of suggestions on last week's callout. Thanks for taking part. Read more about how our weekly series works at the end of the piece.
This week's blog takes the form of an end-of-term celebration of readers recommend in three parts. We have the playlist, we have a few explanatory notes on the reasons for choosing certain of the songs – and then we finish with a rousing Christmas carol.
It'll never work of course, but I stubbornly persevered with the approach until it was too late to back out...
The A List
1. Trombone Shorty: Hurricane Season
2. Bonnie Raitt: Storm Warning
3. Pendulum: The Tempest
4. John Butler Trio: Gov Did Nothin'
5. Deep Purple: Stormbringer
6. Woody Guthrie: Great Dust Storm Disaster
7. AC/DC: Thunderstruck
8. The Spaniels: Stormy Weather
9. Max Lorenz sings Richard Wagner's: Wintersturme
10. Little Axe: Storm is Rising
11. Lovindeer: Wild Gilbert
12. The Imagined Village: Cold Hailey Rainy Night
The explanatory notes
Christmas is of course a time for giving, and this week I would like to think that I have distributed a reasonably generous helping of rock music. There's an argument that the genre sometimes gets overlooked on here, so there's maybe a lesson in readers gordonimmel and Silgen double-teaming the Deep Purple Stormbringer recommendation with detailed historical explanatory footnotes. If this is your type of tune, then be advised that Pendulum's The Tempest – as well as qualifying on the synonym stormfront – is about the heaviest number on the list.
I couldn't leave out the Bonnie Raitt tune, partly for its use of metaphor but mainly for the strikingly heartfelt nomination from PatLux; and I couldn't leave out the Spaniels' take on a bona fide classic, purely because they're called the Spaniels.
Wintersturme filled a chilly gap when it came to season-appropriate storms, and The Great Dust Storm Disaster catered for the opposite end of the temperature spectrum. In similar vein, the John Butler Trio's Gov Did Nothing identifies the righteous anger at the response of the US authorities to Hurricane Katrina in 2005, while Lovindeer is, as alexito puts it: "a hilarious, defiant Jamaican smash hit released in the aftermath of Hurricane Gilbert in 1988".
It's a genuinely uplifting tune. Which, ironically, leads us to:
The Christmas Carol
I'll say only this. There's a doff of the Santa hat to anyone that manages to crack the meter in this at their first attempt.
Don't fret ye Recommenders all, let nothing you dismay
The Christmas week hiatus plan, has now been put away*.
To seize the Yuletide spirit I'm endeavouring to play
Wi-ith band names by forcing them to rhyme
Some of the time,
And it's Trombone Shorty leading out the line.
From Bonnie Raitt to Pendulum, the nominations flowed
Which neatly laid foundations for the John Butler Tri-o.
But nothing with Deep Purple rhymed
So mid-line's where they go
Scansion progress is difficult, you see:
Woody Guth-rie
Dare I end this verse with AC (slash) DC?
Through upbeat stormy weather vibes, the Spaniels song prevailed
And pleased at getting A-listed, one bitch was Wagner tail.
With Little Axe's help, a shortened Yule Log was avail-
able, is there a rhyme for Lovindeer?
Santa's reindeer?
The Imagined Village, full of Christmas cheer.
Ho, Ho and indeed Ho!
New theme: how to join in
*In a change to a previous announcement the next theme will be announced at 8pm (GMT) on Thursday 21 December.
Here is a reminder of some of the guidelines for readers recommend:
- If you have a good idea for a theme, or you would like to volunteer to compile a playlist from readers' suggestions and write a blog about it, please email firstname.lastname@example.com.
- There is a wealth of data on RR, including the songs that are "zedded", at the new look Marconium. It also tells you the meaning of "zedded", "donds" and other strange words used by RR regulars.
- Many RR regulars also congregate at the 'Spill blog.


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text: Thursday February 7th 2019, His Excellency ZHAI Jun Ambassador of the People's Republic of China came to Châteauroux to celebrate the Chinese New Year (Year of the Earth Pig). He was greeted by Mr Ary BRUAND, President of the University of Orleans, Mr Stanislas PIERRET, diplomatic adviser to the Prefect of the Région Centre Val de Loire, Mr Gil Avérous, Mayor of Châteauroux Métropole, other representatives of the civil society and many students from Beijing International Studies University (BISU) and Beijing Sport University (BSU). He underlined the success of ongoing projects and the importance of the collaboration between these two universities of the city of Beijing and the University of Orléans (Campus of Châteauroux).


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Claudin-1 induced sealing of blood–brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis
- 2.4k Downloads
- Citations
In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood–brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.
KeywordsBlood–brain barrier Tight junction Claudin-1 Experimental autoimmune encephalomyelitis Vascular permeability
Under physiological conditions, the blood–brain barrier (BBB) maintains homeostasis of the central nervous system (CNS) by restricting uncontrolled diffusion of water soluble molecules from the blood into the CNS and by limiting immune cell trafficking into the CNS. The BBB is established at the level of CNS microvascular endothelial cells, which are highly specialized to achieve precise control over the substances that leave or enter the brain. Lack of fenestrae and an extremely low pinocytotic activity of BBB endothelial cells inhibit the transcellular passage of molecules across the barrier whereas tight junctions (TJ) between the brain endothelial cells restrict the paracellular diffusion of solutes and ions across the BBB [47]. BBB TJs are unique as in a belt-like region the opposing membranes of adjacent brain endothelial cells form continuous and highly interconnected TJ strands, which appear as a chain of fusion points at the ultrastructural level [10]. In freeze-fracture electron microscopy analysis the fusion points are visualized as strands of TJ particles [46]. At the BBB TJ particles are predominantly associated with the protoplasmic membrane leaflets (P-face) as usually observed in epithelial cells [46], whereas strands of TJs particles of peripheral endothelial cells are rather found associated with the extracellular fracture face (E-face) of the membrane bilayer. TJ strands or particles are composed of integral transmembrane proteins [16], of which occludin and the claudins have been identified to date [40, 41]. Although structurally unrelated, occludin and the claudins are both type II transmembrane proteins bearing 4 transmembrane domains. They are linked to the actin cytoskeleton by TJ associated cytoplasmic peripheral membrane proteins of the MAGUK (membrane associated with a guanylyl kinase-like domain) family, such as zonula occludens (ZO)-1, ZO-2 and ZO-3 [41, 45]. Occludin is highly expressed in TJ of the BBB but not in endothelial TJs of non-neuronal tissues [4, 22], while intact BBB TJs can form in the absence of occludin in vivo [38]. In contrast to occludin, claudins have been shown to be sufficient to induce TJ strands when transfected into fibroblasts [15]. The claudin family is comprised of 24 members, which form the backbone of TJs by establishing homophilic and heterophilic interactions via their extracellular loops [26]. The individual claudins display tissue specific expression patterns and differ in their individual properties in forming dynamic aqueous pores that allow size- and charge-selective paracellular diffusion [17]. Within each tissue, expression of a unique combination of claudins, therefore, determines the respective paracellular tightness of the TJs for different solutes. Claudin-5 has been demonstrated to be the endothelial cell-specific component of TJs strands [34] and is also expressed in BBB TJs [35]. Upon transfection into fibroblasts claudin-5 induces E-face associated TJs [34], whereas claudin-3, which is specifically expressed in TJ of CNS but not other endothelial cells [44, 47], induces P-face associated TJs [16]. This suggests a specific function for claudin-3 in BBB TJs, which is supported further by the recent observation that expression of claudin-3 correlates with BBB maturation in response to Wnt/β-catenin signaling during development [30]. Whereas the exact function of claudin-3 in BBB TJs remains to be investigated, an essential function for claudin-5 in sealing BBB TJs against small molecules up to 800 Da has been demonstrated by genetic deletion of claudin-5 in mice [35]. In addition to claudin-5 and claudin-3, expression of claudin-12 has also been described at the BBB [35]. Previous studies have shown immunostaining for claudin-1 in brain endothelium [31]. Later studies, however, which specifically excluded cross-reactivity of anti-claudin-1 antibodies with claudin-3 failed to detect any immunostaining for claudin-1 in CNS parenchymal microvessels or in primary mouse or human brain endothelial cells [2, 20, 43, 47]. Thus, the combined homophilic and heterophilic interactions between claudin-3, claudin-5, and probably also claudin-12, at the BBB seem to be responsible for the unique barrier function of the BBB TJs [26].
During neurological disorders such as multiple sclerosis (MS) or its animal correlate experimental autoimmune encephalomyelitis (EAE), focal loss of BBB integrity is observed. MS is an inflammatory demyelinating disease of the CNS, in which a large number of inflammatory cells gains access to the CNS parenchyma forming perivascular inflammatory lesions. Lesion formation in MS is known to be associated with focal BBB dysfunction as visualized by gadolinium-enhanced magnetic resonance imaging and occurs early in the pathogenesis of new lesions in MS [24]. Immunohistological analysis of post mortem brain samples of MS patients has identified BBB TJs as the anatomical route for BBB leakiness in MS [29]. In these studies, abnormal distributions of TJ proteins such as occludin and ZO-1 in brain vessels were found to correlate with enhanced BBB leakiness for serum proteins [25, 37]. EAE reliably models the inflammatory phase of MS and BBB alterations observed in EAE resemble those observed in MS [1]. We have previously observed the specific loss of claudin-3 immunostaining from those brain microvessels that were surrounded by inflammatory infiltrates [47], suggesting a direct role for inflammatory cells in disrupting BBB TJs. While much has been learned regarding the sequential steps of immune cell rolling or capture, adhesion and crawling on the BBB, the cellular pathways and the molecular cues mediating immune cell diapedesis across the BBB are only just being unraveled (summarized in [6, 19]). BBB TJs could be disrupted by the immune cells penetrating the BBB on a paracellular route through the endothelial cell–cell contacts or alternatively, immune cells might traverse the BBB on a transcellular route through the brain endothelial cell itself and thus indirectly alter BBB TJ architecture (summarized in [9]).
In the present study, we therefore aimed to delineate the role of BBB TJs in immune cell infiltration and focal BBB leakiness during EAE. Based on our previous observation of the specific loss of claudin-3 from BBB TJs, we hypothesized that in brain endothelial cells ectopic expression of claudin-1, which like claudin-3 induces P-face associated TJs upon transfection into fibroblasts [15], might seal BBB TJs and therefore reduce the paracellular component of immune cell diapedesis across the BBB and/or inflammation-induced BBB leakiness. This notion is supported by previous findings demonstrating that claudin-1 seals TJs in skin epithelial and lung endothelial cells [13, 14]. Furthermore, TJ strands induced by claudin-3 associate with those induced by claudin-1 suggesting that ectopic expression of claudin-1 in BBB TJs would productively integrate into the BBB TJ strands [16]. We, therefore, established transgenic mouse lines with tetracycline (TET)-regulated endothelial cell-specific expression of claudin-1. In two independent transgenic mouse lines, we observed TET-induced expression of claudin-1 in 30–50% of PECAM-1+ CNS microvessels. This partial expression of claudin-1 in BBB TJs sufficed to lead to a significant amelioration of chronic but not acute EAE in double transgenic Tie-2 tTA//TRE–claudin-1 C57BL/6 mice compared to single transgenic littermates.
Materials and methods
Transgene construction
Construction of the Tie-2 tTA vector and production of transgenic mice was described elsewhere [5]. The murine claudin-1 cDNA clone (genbank entry AI663222) was requested from the German Resource Center for Genome Research (RZPD) and sequenced to verify integrity of the open reading frame. The claudin-1 inducible construct was then created by ligating an XhoI/SacII fragment carrying the tet-response element (TRE from pUHD10-3; H. Bujard, Heidelberg), a SacII/BglI claudin-1 fragment, a BamHI/XbaI fragment containing an IRES and the EGFP open reading frame (from pIRES2-EGFP; Clontech, Palo Alto), and an XbaI/XhoI fragment containing the bovine growth hormone poly(A) signal into the XhoI site of pBluescript II. TRE-claudin-1 transgenic mice were then produced by pronuclear injection into fertilized mouse oocytes as described previously [5]. Six TRE-Claudin-1 transgenic founder animals were created from which five independent transgenic lines could be raised.
Transgenic mice were genotyped by PCR. The primers tTA-FW2 (GACGCCTTAGCCATTGAGATG) and tTA-REV2 (CAGTAGTAGGTGTTTCCCTTTCTTC) amplify a product of 550 bp in size from the DNA of Tie-2 tTA transgenic mice.
PCR using the primers CMV-IE FW (CCATAGAAGACACCGGGACC) and IRES REV2 (AAGCGGCTTCGGCCAGTAAC) amplifies a product of 700 bp in size from the DNA of TRE-Claudin-1 transgenic mice.
C57BL/6 mice were obtained from Harlan (Horst, The Netherlands). The Tie-2-tTA activator line #7770 was described before [5]. Double transgenic mice capable of TET-inducible expression of claudin-1 were obtained by crossing Tie2 tTA activator transgenic C57BL/6 mice with responder mice carrying a unidirectional construct for claudin–1. The experiments shown in the present study were performed with the TRE-claudin-1 mouse lines #23949 and #23974 backcrossed to the C57BL/6 background for at least 7 generations before being used in the experiments. Double transgenic (Tie-2 tTA//TRE–claudin-1) mice were obtained by breeding Tie-2-tTA activator with TRE-claudin-1 responder mice. Mice in matings were fed with Doxycycline supplemented chow (100 mg/kg; Provimi Kliba, Kaiseraugst, Switzerland) to suppress expression of claudin-1 in double transgenic pups during embryonic development and until weaning at postnatal day 21. Mice were housed in the animal facility of the University of Bern under specific pathogen free conditions in individually ventilated cages. All animal procedures were performed in accordance with the Swiss legislation on the protection of animals and approved by the respective government authorities.
Antibodies and reagents
The following primary antibodies were used at 10–20 μg/ml or as supernatants from hybridomas: rat anti-mouse JAM-A (BV11 and BV12, from E. Dejana, Milan, Italy), rat anti-mouse PECAM-1 (Mec13.3, BD Pharmingen, Heidelberg, Germany); rat anti-mouse CD45 (M1/9) and rat anti-human CD44 (Hermes-1 = 9B5, used as isotype control) (both from ATCC, Rockville, MA, USA), rabbit anti-mouse Claudin-5 (from H. Wolburg/H. Kalbacher, Tübingen, Germany). The following antibodies were obtained from Zymed, San Francisco, USA via LuBioScience GmbH, Lucerne, Switzerland: mouse anti-mouse Claudin-5 (35-2500), rabbit anti-mouse Claudin-1 (51-9000), rabbit anti-mouse Claudin-3 (34-1700), rabbit anti-mouse Claudin-5 (34-1600), rabbit anit-mouse Occludin (71-1500), rabbit anti-mouse ZO-1 (61-7300), rabbit anti-mouse ZO-2 (71-1400). Rabbit anti-human fibronectin (A0245, cross-reacts with mouse fibronectin) and rabbit anti-von Willebrand Factor were from DAKO Cytomation, Glostrup, Denmark.
The following secondary antibodies were used at 3–10 μg/ml: Alexa Fluor 488 goat anti-rat, Alexa Fluor 488 goat anti-rabbit (both from Molecular Probes via Invitrogen via LuBioScience GmbH, Lucerne, Switzerland), Cyanine Cy3 goat anti-rat, Cyanine Cy3 goat anti-rabbit, Cyanine Cy3 donkey anti-mouse (all from Jackson Immunoresearch via Milan Analytica AG, La Roche, Switzerland).
RNA isolation, cDNA synthesis, Gene chip array and qPCR of purified parenchymal mouse brain capillaries
Isolation of PECAM-1+ parenchymal mouse brain capillaries from 4-week-old female C57BL/6 mice (Harlan, Horst, The Netherlands) and RNA isolation from these capillary fragments, cDNA synthesis and gene chip microarray analysis (Mouse Genome 430 2.0 Array, Affymetrix UK, High Wycombe, UK) was performed exactly as described before [32]. qPCR was based on SYBR green (MasterMix Plus for SYBR Assay I Low ROX, Eurogentec, Seraing, Belgium) and carried out on an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Warrington, UK). To compare expression levels of claudin-1 with claudin-5, detection of the endogenous reference probe targeting the cDNA for s16 ribosomal protein was included in each experiment (5 experiments total) and set to 1.0 according to the comparative comparative ΔC T method (relative expression = \( 2^{{ - \Updelta C_{\text{T}} }} \), ΔC T value = average C T value of target−average C T value of endogenous reference). The following primer pairs were used: s16 ribosomal protein (Rps16, NM_013647): forward primer GATATTCGGGTCCGTGTGA, reverse primer TTGAGATGGACTGTCGGATG, length of PCR product 69 bp; claudin-5 (NM_013805): forward primer ACGGGAGGAGCGCTTTAC, reverse primer GTTGGCGAACCAGCAGAG, length of PCR product: 65 bp; claudin-1 (NM_016674): forward primer ACTCCTTGCTGAATCTGAACAGT, reverse primer GGACACAAAGATTGCGATCAG, length of PCR product: 91 bp.
Immunofluorescence stainings
Mice were anesthetized with Isofluorane (Baxter, Arovet AG, Zollikon, Switzerland) and perfused with cold phosphate buffered saline (PBS) through the left ventricle of the heart. Brains were removed, coronally cut into three pieces, embedded in Tissue-Tek (OCT compound, Sysmex Digitana AG, Horgen Switzerland) and snap frozen in an isopentane bath (2-Methylbutane, Grogg Chemie AG, Stettlen/Deisswil, Switzerland) at −80°C. Cryostat sections (6 μm) were air dried overnight before staining. Sections were fixed for 10 min with ethanol at 4°C, followed by 1 min in acetone at room temperature and rehydrated with Tris buffered saline (TBS). Sections were blocked with blocking solution containing 5% (w/v) skimmed milk, 0.3% (v/v) Triton X-100 (Fluka via Grogg Chemie AG, Stettlen/Deisswil, Switzerland) and 0.04% (w/v) NaN3 in TBS for 20 min. Primary antibodies were incubated in the same buffer, unless they were used as supernatants from hybridomas, for 1 h at room temperature. Following the washes with TBS, sections were incubated with secondary antibodies diluted in blocking solution for 1 h at room temperature. Sections were mounted in Mowiol (Calbiochem via Grogg Chemie AG, Stettlen/Deisswil, Switzerland). Sections were analyzed using a Nikon Eclipse E600 microscope connected to a Nikon Digital Camera DXM1200F with the Nikon ACT-1 Version 2.63 software (Nikon, Egg/ZH, Switzerland).
Induction of EAE in C57BL/6 mice
Active EAE was induced exactly as described before [8] by subcutaneous injection of 200 μg of myelin oligodendrocyte glycoprotein peptide (MOGaa35–55) emulsified in complete Freund's adjuvant (incomplete Freund's adjuvant (Santa Cruz, Switzerland) supplemented with 4 mg/ml attenuated Mycobacterium tuberculosis (H37 RA, DIFCO Laboratories, Detroit, MI) into 8-to 12-week-old female C57BL/6 wild-type, single, and double transgenic Tie-2 tTA//TRE–claudin-1 mice. 300 ng pertussis toxin from Bordetella pertussis (LuBioScience GmbH, Switzerland) per mouse was administered intraperitoneally at days 1 and 3 post-immunization (p.i.). Assessment of clinical disease activity was performed as described with the following disease scores: 0 = healthy, 0.5 = limp tail, 1 = hind leg paraparesis, 2 = hind leg paraplegia, and 3 = hind leg paraplegia with incontinence [8]. A total of 6 EAE experiments were performed comparing double and single transgenic mice of the Tie-2 tTA//TRE–claudin-1 lines 23949 and 23974. Graph Pad Prism 4.0 software was used to calculate area under the curve (AUC) values of EAE clinical scores and for subsequent statistical analysis utilizing Mann–Whitney test (non-parametric, unpaired).
In vivo BBB permeability assays
BBB leakiness for the small molecular tracert Hoechst nuclear stain (HOE 33258, 534D, Calbiochem-Novabiochem, Laufelfingen, Switzerland) and for endogenous fibronectin was assessed in single and double transgenic Tie-2 tTA//TRE–claudin-1 mice of lines 23949 and 23974 at day 50 post EAE induction. 100 μl of 2% Hoechst nuclear stain in PBS was injected intravenously into each mouse. 30 min after dye circulation, mice were decapitated, brains were removed, coronally cut into three pieces, which were embedded side by side in Tissue-Tek (OCT compound, Sysmex Digitana AG, Horgen Switzerland) and snap frozen in an isopentane bath (2-Methylbutane, Grogg Chemie AG, Stettlen/Deisswil, Switzerland) at −80°C. Brain cryosections of 4 double and 6 single transgenic mice were immunofluorescently labeled for CD45 and claudin-1. From each brain at least 10 cryosections (each showing 3 brain sections) were analyzed, with at least 70 μm between individual sections analyzed. Inflammatory cuffs in the brain parenchyma were identified by perivascular accumulation of CD45pos cells and subdivided into 4 leakiness groups according to claudin-1 expression and Hoechst leakage: claudin-1pos/Hoechstpos, claudin-1pos/Hoechstneg, claudin-1neg/Hoechstpos and claudin-1neg/Hoechstneg. The number of inflammatory cuffs was expressed either in percentage per brain or as a ratio of Hoechstpos to Hoechstneg cuffs and statistically analyzed with unpaired t test (Graph Pad Prism 4.0).
For quantification of endogenous fibronectin (FN) leakage across the BBB brain cryosections were performed as described above on day 50 p.i. of 6 double and 9 single transgenic Tie-2 tTA//TRE–claudin-1 mice (lines 23949 and 23974). Pairs of serial sections were immunofluorescently double-stained for CD45/claudin-1 and CD45/fibronectin, respectively. Per individual brain section-pairs were analyzed with at least 70 μm between them. Inflammatory cuffs in the brain parenchyma were identified by perivascular accumulation of CD45pos cells and subdivided into 4 fibronectin leakiness groups according to claudin-1 expression and fibronectin leakage: claudin-1pos/fibronectinpos, claudin-1pos/fibronectinneg, claudin-1neg/fibronectinpos or claudin-1neg/fibronectinneg. The number of inflammatory cuffs was expressed either in percentage per brain or as a ratio of fibronectinpos to fibronectinneg cuffs and statistically analyzed with unpaired t test (Graph Pad Prism 4.0).
Quantification of inflammatory cuffs
The number of CD45pos inflammatory cellular infiltrates surrounding CNS blood vessels ("inflammatory cuffs") was counted in a total of 10 double transgenic and 15 single transgenic Tie-2 tTA//TRE–claudin-1 mice from lines 23949 and 23974 at day 50 post-immunization and were classified based on their localization as leptomeningeal cuffs surrounding meningeal vessels, as periventricular when localized in direct vicinity to the CNS ventricles and as parenchymal cuffs when surrounding parenchymal blood vessels. The analysis was restricted to the brain as merging of parenchymal and meningeal inflammatory infiltrates in the spinal cord prohibited quantitation of individual inflammatory cuffs in spatial relation to individual blood vessels. The number of inflammatory cuffs per mouse was calculated as mean number of inflammatory cuffs per brain section and median number of inflammatory cuffs for all mice was statistically analyzed using the Mann–Whitney U test (Graph Pad Prism 4.0).
MOG specific T cell priming
In vitro antigen-recall assays were carried out with MOGaa35–55-immunized single, and double transgenic Tie-2 tTA//TRE–claudin-1 mice, as described before [42]. At day 10 post-immunization, mice were killed and draining lymph nodes were dissected. Single-cell suspensions were produced and cells were seeded in 96-well plates at 2 × 105 cells/well in RPMI 1640 supplemented with 10% FBS (GIBCO, LuBiosience, Lucerne, Switzerland), 10 U/mL penicillin/streptomycin, 2 mM l-glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate and 0.05 mM β-mercaptoethanol (Grogg Chemie AG). Increasing concentrations of MOGaa35–55 were used to test for antigen-specific T cell proliferation. T cell proliferation induced by cross-linking of CD3 and CD28 with 0.1 μg/ml of the respective antibodies (clones 145-2C11 and 37.51, Pharmingen BD Biosciences, Switzerland) was used as a positive control. T cell proliferation in medium without antigen or with 10 μg/ml of purified protein derivate of Mycobacterium tuberculosis (PPD, CFA-component) was used as negative and adjuvants control, respectively. All samples were plated in triplicates. [3H]Thymidine (1 μCi/mL) was added 16 h prior to harvesting the cultures on glass fiber filters using a cell harvester (Inotech, Dottikon, Switzerland) and incorporation of [3H]thymidine was measured by liquid scintillation counting.
Statistical analysis was performed with Graph Pad Prism 4.0, as described in the corresponding paragraphs.
Expression of tight junction molecules in CNS microvessels in C57BL/6 mice
Detection of tight junction proteins by immunofluorescence staining of the healthy brain
Parenchymal blood vessel endothelium
Meningeal blood vessel endothelium
Choroid plexus endothelium
Choroid plexus epithelium
Ependymal cells
Establishment and characterization of Tie2-tTA//TRE-claudin-1 double transgenic mice
Double transgenic Tie-2 tTA//TRE-claudin-1 C57BL/6 mice did not show any apparent phenotypic differences in the overall anatomy of their organs when compared to single transgenic and wild-type littermates. Histological analysis of PECAM-1+ blood vessels in tissue sections of the brain, spinal cord and the secondary lymphoid organs of double transgenic Tie-2 tTA//TRE-claudin-1 C57BL/6 mice revealed no obvious differences in the vascular architecture of these organs compared to the organs in single transgenic or wild-type littermates. Moreover, size and cellular composition of the secondary lymphoid organs was not altered in double transgenic Tie-2 tTA//TRE-claudin-1 C57BL/6 mice compared to single transgenic and wild-type littermates as determined by FACS analysis (data not shown). Interestingly, TET-induced claudin-1 was not expressed in high endothelial venules of double transgenic Tie-2 tTA//TRE-claudin-1 C57BL/6 mice and would, therefore, not interfere with lymphocyte recirculation to lymph nodes.
Ectopic TET-regulated claudin-1 ameliorates chronic but not acute experimental autoimmune encephalomyelitis (EAE)
TET-regulated claudin-1 does not influence immune cell infiltration into the CNS during EAE
TET-regulated claudin-1 reduces BBB permeability in chronic EAE
To investigate, if amelioration of chronic EAE in Tie-2 tTA//TRE-claudin-1 C57BL/6 mice correlated with reduced BBB leakiness, in vivo BBB permeability was examined using intravenous injection of Hoechst nuclear dye, which measures BBB permeability at a particular time point and with high sensitivity due to its small size of 540 Da. Further, staining for endogenous plasma fibronectin, which accumulates in the brain parenchyma over time during EAE, allows a retrospective view of BBB leakage.
Hoechst dye was injected intravenously into Tie-2 tTA//TRE-claudin-1 C57BL/6 mice and single transgenic littermates at day 50 after induction of EAE, after 30 min of dye circulation, brains were removed, and immunofluorescently stained for claudin-1 and for infiltrating leukocytes with the pan-leukocyte marker CD45. In both Tie-2 tTA//TRE-claudin-1 C57BL/6 mice and single transgenic littermates Hoechst dye leakage was restricted to vessels with concurrent perivascular accumulation of CD45+ leukocytes.
Increased BBB permeability is a characteristic hallmark of the pathological alterations observed in the CNS in MS and in its animal model EAE [27]. In fact, breakdown of the BBB identified by gadolinium-enhanced magnetic resonance imaging is the earliest detectable event in the development of most new CNS lesions in MS and is known to be associated with inflammation [24, 28]. Amelioration of clinical symptoms is observed, when CNS inflammation resolves and BBB function is restored, supporting the notion that lesion formation in MS is driven by inflammatory processes [28] and suggesting a causal relationship between inflammatory cell recruitment into the CNS and BBB dysfunction. Similarly, in EAE, loss of BBB integrity has been recognized as a fundamental event in pathogenesis and further the magnitude of BBB dysfunction has been correlated with disease severity [11]. The close association of inflammation-induced BBB dysfunction with immune cell entry into the CNS has, however, hampered attempts to elucidate the specific contribution of BBB dysfunction to MS pathogenesis.
Our present study provides evidence for the specific contribution of focal BBB leakiness to disease pathogenesis in EAE and identifies the BBB TJs as potentially critical structures for the observed BBB dysfunction during EAE. Specifically, sealing BBB TJs by ectopic expression of claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and therefore did not influence the onset nor the severity of the first clinical episode of EAE. However, expression of TET-regulated claudin-1 significantly reduced BBB leakiness for blood borne tracers or endogenous plasma proteins specifically in claudin-1 expressing vessels. Further, a reduced disease burden in the chronic phase of EAE was observed in Tie-2 tTA//TRE-claudin-1 mice compared to control littermates. Our study, which dissociates BBB leakiness from immune cell trafficking into the CNS during EAE, identifies the BBB TJs as critical structures regulating BBB permeability, and defines BBB dysfunction as a potentially crucial underlying event contributing to the disease burden in the chronic phase of MS.
Previous studies on post mortem MS brains have already suggested a relationship of BBB TJ abnormalities with BBB hyperpermeability in MS. In these studies, evaluation of a large number of brain vessels by immunofluorescence staining and semi-quantitative thick-section confocal analysis demonstrated TJ abnormalities manifested by beading, interruption or absence of junctional immunostaining or appearance of diffuse cytoplasmic staining for the TJ proteins ZO-1, occludin and junctional adhesion molecule-A (JAM-A) in up to 40% of the blood vessels in MS brain tissue from primary progressive and secondary progressive disease states [25, 29, 36, 37]. Aberrant TJ morphology observed in MS brains correlated with enhanced BBB leakiness for serum proteins, highlighting that BBB TJ disruption underlies BBB leakiness in MS [25, 37]. Based on these observations, these authors have proposed to consider BBB TJ pathology—besides inflammation and demyelination—a distinct entity of tissue injury in MS [37].
EAE reliably models the inflammatory phase of MS and a number of observations support the view that EAE equally well allows to model the BBB TJ pathology observed in MS. Mirroring the observations made in MS tissue samples, disrupted immunostaining for ZO-1 was observed in brain microvessels within areas of focal inflammatory lesions before the onset of clinical EAE and was maintained throughout the clinical disease course [1]. Similarly, we previously observed a focal loss of claudin-3 immunostaining from brain microvessels surrounded by inflammatory cells during EAE [47]. Alterations of the molecular composition of BBB TJs during EAE were found to be accompanied by an aberrant junctional morphology detected at the ultrastructural level, namely pronounced TJ convolution and thereby an apparent extension of the intercellular cleft revealing many kissing points [47].
Although the pathological alterations of BBB TJs described above are indicative of abnormal BBB TJ function as well, observation of altered distribution or lack of an individual TJ protein at the BBB in MS and EAE tissues may not suffice to define BBB TJ pathology or to conclude on deficient BBB TJ function in vivo. For instance, occludin has been shown to be expressed at high levels in brain endothelial cells [4, 22] yet the complete absence is compatible with the development of intact BBB TJs in vivo [38]. In contrast expression of specific occludin splice variants (summarized in [12] or changes in phosphorylation state may influence BBB TJ function. The latter is supported by the observation of occludin dephosphorylation in spinal cord microvessels in a rat model of EAE corresponding with the onset of CNS inflammation [33].
It has been suggested that during MS and EAE immune cells migrate across the BBB via a paracellular route through endothelial cell–cell junctions, thereby causing BBB leakiness and TJ pathology. This view is supported by the observation that mice deficient for the cell–cell contact molecule PECAM-1 develop aggravated EAE due to impaired BBB cell–cell contact integrity combined with increased inflammatory cell recruitment across the BBB into the CNS [18]. On the other hand, there is mounting evidence that immune cell diapedesis across the BBB during EAE might rather occur through the endothelial cell proper, leaving BBB TJs untouched (summarized in [9]). Therefore, alternative or more complex mechanisms of inflammation-induced BBB TJ pathology must be considered. It is conceivable that in the latter scenario soluble mediators released by immune cells might trigger BBB TJs alterations as implied by observations showing that at least in vitro Th17 cells can impair BBB integrity through the combined production of IL-17A and IL-22 leading to downregulation of occludin and to a lesser extent of ZO-1 in brain endothelial cells [23]. In addition, CD8 T cells were shown to initiate BBB TJ disruption in vivo through a non-apoptotic perforin-dependent mechanism [39].
In the present study, we hypothesized that experimental sealing of BBB TJs during CNS inflammation would allow us to determine the contribution of BBB TJs to immune cell diapedesis across the BBB as compared to inflammation-induced BBB leakiness during EAE. Our present study shows that TET-regulated endothelial cell-specific claudin-1 integrated into BBB TJs and significantly reduced BBB leakiness for both a small molecular tracer and endogenous plasma derived fibronectin. Interestingly, although TET-induced expression of claudin-1 could only be observed in 30–50% of brain vessels, claudin-1 induced sealing of BBB TJs during EAE was found to significantly ameliorate the chronic phase of EAE in two independent transgenic mouse lines. Our results, therefore, provide direct functional evidence identifying BBB TJs as the critical component in regulating barrier function of the BBB during EAE and confirming the notion that continued BBB TJ alterations are the basis of BBB dysfunction, which contributes to disease pathogenesis in this animal model of MS.
At the same time, we failed to find any difference in the overall number or size of inflammatory cuffs detected in the brains of Tie-2//TRE-claudin-1 double transgenic mice versus their single transgenic littermates demonstrating that claudin-1 induced sealing of BBB TJs did not influence immune cell recruitment across the BBB during EAE. Unimpeded extravasation of immune cells across the BBB at sites of claudin-1 expression, therefore, implies a limited contribution of the paracellular pathway in immune cell diapedesis across the BBB during EAE and suggests that transcellular immune cell trafficking across the brain endothelial cells proper suffices to fully establish inflammatory infiltrates in the CNS. Lack of any significant differences observed in the onset and severity of the first clinical episode of EAE between Tie-2//TRE-claudin-1 double transgenic mice and their single transgenic littermates suggests that the early phase of EAE pathogenesis is driven by immune cells infiltrating the CNS and might explain the therapeutic efficacy of drugs such as natalizumab in treating MS because of their inhibiting this process [7]. Although our study does not allow the elucidation of the molecular mechanisms initiating inflammation-induced BBB leakiness in EAE, it provides experimental in vivo evidence demonstrating that BBB dysfunction during EAE is due to BBB TJ disruption and identifies BBB TJ pathology as a specific pathogenetic entity contributing to overall disease pathogenesis in EAE.
As the degree of demyelination and/or axonal damage have been described as morphological correlates to increasing disability in MS and clinical severity of EAE [21], we undertook an effort to determine if ameliorated chronic EAE in Tie-2//TRE-claudin-1 C57BL/6 double transgenic mice was due to reduced CNS pathology. To this end, we performed preliminary double immunofluorescence stainings comparing macrophage infiltration and axonal injury in correlation to demyelination in brain and spinal cord sections of Tie-2//TRE-claudin-1 C57BL/6 double transgenic mice and single transgenic littermates during chronic EAE. Immunostaining for F4/80 showed indistinguishable levels of macrophage accumulation in the CNS of Tie-2//TRE-claudin-1 C57BL/6 double transgenic mice and single transgenic littermates. Similarly, immunostaining for MPB and Luxol Fast Blue staining revealed comparable degrees of demyelination in brain and spinal cord sections of Tie-2//TRE-claudin-1 C57BL/6 double transgenic mice and single transgenic littermates. Furthermore, similar degrees of enhanced neurofilament immunofluorescence in spinal cord tissue sections of Tie-2//TRE-claudin-1 C57BL/6 double transgenic mice and single transgenic littermates were indicative of comparable levels of axonal pathology. These observations suggest that claudin-1 induced sealing of BBB TJs does not readily translate into reduced CNS pathology but might lead to an improvement of the physiological conditions in the CNS of Tie-2//TRE-claudin-1 C57BL/6 double transgenic mice sustaining electrical activity and thus proper function of CNS neurons. Induced sealing of BBB TJs could reduce edema and radical formation, and/or improve ion homeostasis in the CNS, and thus lead to a neuroprotective outcome.
Persistent endothelial abnormalities and BBB leakage have been described in primary and secondary progressive MS [29] indicating the existence of the same pathogenic axis in patients. In light of the fact that progressing disability at later time points of the MS disease course is not prevented by the current anti-inflammatory treatments available [3], our present findings demonstrating a continuous contribution of BBB TJs pathology to EAE pathogenesis point to therapeutic sealing of BBB TJs as an attractive alternative for the restoration of BBB function and eventually CNS homeostasis. The challenges faced for developing such methods are our present lack of in depth knowledge on the precise molecular architecture of BBB TJs as well as our incomplete understanding of the function and regulation of expression of the individual TJ components in BBB TJs. The recent discovery of the Wnt/β-catenin signaling pathway involved in brain angiogenesis and claudin-3 expression may open such a novel avenue for therapeutic stabilization of BBB TJs during CNS inflammation.
Special thanks go to Dr. Silke Tauber for preparing the claudin-constructs and to Therese Périnat and Claudi Blatti for expert technical assistance. Furthermore, we wish to thank Dr. Axinia Döring for her tireless help in the daily clinical scoring of EAE mice, Nannette Rink for microinjection, Sorin Ciocan and Thomas Zeis for help with immunofluorescence stainings and Svetlozar Tsonev and Isabelle Oswald for state-of-the art mouse husbandry and care. This work was funded by the the Swiss National Foundation, the European Stroke Network (ESN, EU FP7 N° 201024 and N° 202213) and Integrating Project JUSTBRAIN (EU, FP7 N° HEALTH-2009-241861).
Conflict of interest
The authors declare that they have no conflict of interest.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
Supplementary material
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Open AccessThis is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


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I've never struggled with loneliness the way that I have been these past few weeks. Right now I'm in different spiritual, mental, and emotional places than the people around me are. Not that the place I'm in is better or worse…it's just different.
This morning at Mass when I walked in I had no clue what was going on. It hadn't started yet, but everyone was praying prayers I don't know yet and I was really struggling to follow along. Usually I don't have this problem, and today it was really discouraging.
I sat there thinking, "I don't fit anywhere. Not here in this place, and not there in the place I've come from."
So I looked up at my Christ on the cross and I prayed the only words that would come to mind: "I can't do this. Help me do this. Please."
When everyone got up to take Communion the lonely feeling persisted. I know it's only a few weeks away now, but I haven't taken Communion in a really long time and I miss it. I miss the feeling of being one with my fellow believers. I miss knowing that I'm partaking in Christ's body and blood.
Then the woman sitting next to me got up to receive the Eucharist. As she walked past me she stopped and touched my arm. When I looked up at her she took my face in her hands and just smiled at me. Then before the tears that had formed in my eyes slipped out, she was gone. I don't know if she knows the gift that that seemingly tiny gesture gave me.
I sat there and for the first time in weeks my soul was quiet enough to hear the still, small voice of the Lord. He said to me, "Don't you think the cross was a lonely place for Me? I have felt the feelings that you are feeling. You are not alone. I have surrounded you with people to love you and encourage you and pray for you. Not only that, but even if you were the last person on earth I wouldn't leave you by yourself. I will always be with you. Always."


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We hike with snowshoes through the beautiful valley of Utladalen - a landscape protection area on the border of the Jotunheimen Nationalpark. We pass several big waterfalls on our way.
Duration: 4-6 hours
Amazing experience of hiking on the snow in a landscape protection area with several big waterfalls along the way. We pass old wooden buildings, a maintained cultural landscape and cross the river Utla by crossing high bridges.


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Started by Nick, October 20, 2016, 18:02:39 pm
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Quote from: Nick on July 18, 2019, 14:16:19 pmSo the OBR now calculates that a no deal Brexit will create a £39 billion economic bit and would cause a U.K. recession (this is not the pessimistic version).Crazy crazy crazy, and Boris is waving around a kipper and again telling blatant lies.We are selling our Country down the river.
Quote from: DavidL on July 19, 2019, 01:58:22 amBased on IMF forecasts - a completely dicredited organisation, when it comes to economic predictions.
Quote from: rufus the dawg on July 22, 2019, 10:17:52 am........says the man without a plan.Have you found that plan yet or are you waiting for a diplomat to give some secret plans you can use!
Quote from: Nick on August 01, 2019, 17:12:44 pmSo the Government is now about to spend £2.1 Billion on preparing for a No deal Brexit that Boris recently advised was "a million to one". Madness i tell you. Could have just used this money from the magic money tree on healthcare/education etc.
Quote from: Nick on August 01, 2019, 19:38:14 pm- The Leave campaign said none of this stuff would ever happen. Imagine being slave-minded enough to still be sucking up the lies over 3 years later . Beyond comprehension
Quote from: Nick on August 01, 2019, 20:13:14 pmCheck the exchange rate, 2nd worst performing in the world for 2019. Really there are no good figures published anywhere for a no deal Brexit. I'm not saying that as a remainer but as a fact.
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In the Darkroom by Susan Faludi
The subject line of the email – from a father in Budapest to his daughter in Oregon – is 'Changes'. 'I've got some interesting news for you,' writes Steven. 'I have decided that I have had enough of impersonating a macho aggressive man that I have never been inside.' Thus Susan Faludi, an author known for her books on feminism and the collapse of traditional masculinity, learns that her father has undergone sex reassignment surgery in Thailand. At seventy-six, Steven has become Stefánie.
Faludi has barely spoken to her father in twenty-five years. She remembers him as an 'imperious patriarch' with a taste for violence, who cultivated the persona of a rugged outdoorsman when not tinkering in his basement workshop. But this email is so startling that she soon boards a plane to Budapest, with notebook in hand. When they hug at the airport, Stefánie's breasts poke into Susan's. As they walk to the car park, people stare at 'the dissonance between white heels and male-pattern baldness'. Back at Stefánie's fortress-like home in the hills of Buda, they begin the strained process of becoming reacquainted. And Faludi, a superb reporter and researcher, begins a quest to decode her father's many riddles that will eventually span ten years.
A Hungarian Jew born into a wealthy, cosmopolitan family, Steven survived World War II by hiding out and passing as a gentile. Nearly half a million Hungarian Jews died in the Holocaust; Steven lost twelve of his relatives. After the war, he moved to the United States, adopting a new identity as a suburban family man who 'aggressively celebrated Christmas and Easter'. Estranged from his parents, he rarely acknowledged his Jewish upbringing.
When Susan was seventeen, her parents split up. Her mother took out a restraining order against Steven; one night, he broke into their home and stabbed her lover repeatedly with a Swiss army knife. After convincing the authorities that he was 'saving' the family from a trespasser, he escaped serious punishment. Steven moved to Manhattan, where he ran a commercial photo studio, then returned to Hungary after the fall of communism. It was there he began dressing as a woman.
In the Darkroom blends memoir and reportage with historical forays into subjects as diverse as the origin of the term 'identity' and the creation of the modern Hungarian state. It is a deeply serious book, but hilarious, too. Much of the humour comes at the author's expense, as the noted feminist grapples with a father turned happy hausfrau. Stefánie's favourite attire at home is a crimson bathrobe with angel-wing sleeves, which always seems to be falling open. She barges into her daughter's room to show off a skimpy new negligee and is constantly demanding sartorial advice. 'You write about the disadvantages of being a woman,' she tells Faludi, author of Backlash: The undeclared war against women (1991). 'But I've only found advantages.'
Stefánie is one of the most vivid, infuriating characters I have encountered for some time. A proud Hungarian nationalist, she brushes off questions about her Jewish heritage. Although she cultivates the persona of a respectable matron (style-conscious, with a fondness for sachertorte and pearl earrings), she is the same bossy patriarch underneath. I felt sickened when I read of her past violence towards her family, but I admired Faludi's complete absence of self-pity when describing the events of her childhood. 'I became an agitator for women's equality,' she calmly tells us, 'in response to my father's fury over his own crumbling sense of himself as a man in command of his wife and children.'
Faludi researches the history of male-to-female surgery and interviews members of the Hungarian Tranny Club (including Lorelei, a retired police officer who owns a copy of Mein Kampf). She reflects on Hungarian nationalism, with its virulent strain of anti-Semitism, and the particular blend of 'self-pity and brutality' that characterises Hungarians' view of themselves as the 'losers of history'. In doing so, she connects her father's new identity to the social forces that shaped him. We begin to see the importance of clothes in Steven's life. As a teenager, during the war, he briefly got around in grey overalls, impersonating a Luftwaffe mechanic. He later mounted an audacious rescue mission in a so-called 'protected house' for Jews while dressed as a young fascist.
At the centre of all Faludi's investigations is a burning question: what prompted Steven to become Stefánie so late in life? Did he cross-dress while married? Does his desire for acceptance as a woman relate to his having felt rejected by his parents as a boy? Has he spent his whole life trying on identities? Is he happiest when acting a role?
In the Darkroom brims with wonderful scenes. One of the best describes Faludi's attempt to interview Stefánie's gynaecologist about her father's new body. As she sits there, notebook in hand, the doctor and his patient talk animatedly in Hungarian. Then the gynaecologist asks Faludi, 'Have you tried fertility treatments?' The pair, it transpires, have been discussing the 'problem' of this forty-nine-year-old feminist, who is childless by choice.
By the end, though, it is clear that Faludi's most touching achievement is the completeness of her portrait of her exasperating father. For Stefánie, it turns out, is a survivor of heroic proportions – a brave, stubborn shape-shifter in pearl earrings.


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MAZ in the mirror
Saw this on Facebook tonight and wish all parents could understand.
Parents that worry or complain about their body image will teach their children to have bad self-esteem. Daughters and sons.
I am so thankful to my parents. They taught me that health and time management are important, not looks.
They (mom) taught me the reason for exercise:
"You are grumpy, go ride your bike"
"You are grumpy, go climb a tree"
"You are grumpy, go run the neighborhood"
I began to understand that exercise was for people with anger issues....
And I now run about 5 days a week!
They (dad) taught me to be low maintenance:
"Stay out of your sister's way! We are late, let's go!"
2 older sisters, one bathroom...
no mirrortime for #3!
I can get ready in 15 minutes and never look in a mirror.
They (dad) taught me that cosmetics are.....hazardous.
"Do you smell cat spray? My gosh what is that smell?" Wheez wheez.....
Gasp "I can't breathe" choking, then
"Oh, you must be wearing perfume?"
"What is on your face? Are you pretending to be a clown?.....oh, you are wearing that stuff."....... makeup
Perfume and makeup trigger my migraines, close my lungs and induce a clown anxiety.
The only awareness I have is my crazy hair....thanks to my dad.
After bungee cords, spackle and a hot iron, I get in the car. Dad looks at me,
"You forgot to brush your hair this morning"
Seriously, my parents taught me that WHO you are is the most important thing and has nothing to do with physical appearance.
Proudly, I am an easily annoyed girl with entertaining hair that hasn't needed a mirror since the first year of life!
a day to worship, a day to hang with my special friend, a day to refocus, a day to not work....well WORKING on that....
learning something new everyday, guiding the boys education, and evenings with the guys
my day to feel domestic:
satisfaction in cleaning and organizing, successful school grading and planning, much wanted family time, and sometimes....I get to nurture and treat them like little boys again.
Although, I think I am out of practice....
Or not.
I was never the cutesy momma. More of the demented humor momma.
Today, Jack's snack was supposed to be a cute, smiling face but turns out it was more of a beat up, UFC face.
Is your mom a cabby in Tampa?
Jeff landed in Tampa this afternoon and ordered a cab. When the cab pulled up, he was afraid they had sent him the kind that take you from strip joint to strip joint.
It was pink and flashy!
He climbed in the back....
.......I did not ask why he was so willing to ride in an "exotic" cab, I just let him tell me the story......
The driver said "did he tell you this route is a flat $25 fee?"
Without looking up, Jeff said "no, he didn't. But I have been on this route before, so I know...."
The driver said "well, he should have told you...." As the cabby continued to talk, Jeff looked up
For my readers that don't know, Trish is my mother, Jeff's mother-in-law, and although my mom looks much better and skinnier, when Jeff sent me the above picture, the boys and I thought the cabby looked like mom and even held the steering wheel like mom!
Below is Memphis Trish:
A Secret Safe With Me
Last Wednesday, E and I found a secluded, "vacant" hallway to sit and gab. Our gabbing consisted of MAZ language, MAZ judging others and MAZ discussing her love for bourbon. E was totally innocent.
After half an hour of saying things that clearly no one at church should hear, someone in the dark, "vacant",
wide-opened door room 2 feet from us, cleared their throat.
Our eyes bugged out of our heads as we quickly fled our stoop, to avoid being seen by the person that heard my criminal words. We laughed until I cried and E enduced a lung virus that is still plaguing her today.
We recapped our conversation to see if anything was too incriminating. We never found out who was lurking in the dark room.
This Wednesday night we reminisced and laughed about the whole scene, again. We joked that our pastor, Sandy Willson, was probably praying in that room and heard my words.
E and I found a new hideaway tonight and we were discussing matters and cackling when the door opened and out walked Sandy Willson.
He shot a look our way that said "repent".
Or not....I'm not sure because I was distracted by the secret door that led to a secret back stairwell.
We found Sandy Willson's secret stairs!
A New Place To Live
Today, I was lost in a spreadsheet world. I am discovering that spreadsheets are like cleaning and rearranging the house.
A complete thrill!
For about 30 minutes, I worked in complete silence and I had tuned out the world, including the electrician that was on a ladder 2 feet from me.
He made a loud noise that sucked me back to reality in time to see a small metal part fly out of the ceiling and straight towards me. It landed in my tiny water cup.
For some reason, I found this to be an awesome skill. Involuntarily the word
came flying out of my mouth, at loud volume.
This startled the electrician and he grabbed the top of the ladder as his eyes widened.
I finished with "great shot!"
My 'awe' was contagious and he chuckled and smiled with true pleasure as he excitedly said, "did it go in?"
"Yes!" I held it up for him to see.
We smiled.
He went back to work and I quietly wandered back into my spreadsheet forest.
It was a great day of Harlem Globetrotter Skills and spreadsheet narcotics.
With a book in my lap
Saturday Dreamin'
I can hear the water lightly sloshing up on dry land.
The fall breeze marries well with the last of the summer sun on my face.
My brain works overtime, planning and organizing, while my senses enjoy the quiet nature, and my body naps.
Saturday reality
The January chill makes my bones cry as I crawl out from under my warm blankets. My brain plans and organizes while my senses are offended by the multiple cleaner smells, and my appendages move to the pulsating music, as they scrub every surface in the house.
Tue, Jan 7, 2014
I'm learning to "take a chill pill" and not lose my temper when the schoolwork is not complete.....because there is some sort of "fake/trickery" work turned in at least twice a week!
Max was thrilled that he used invisible ink. He waited for me to give him the "why the hell is this not answered" look, and then shoved the U V light at his answers.
He was delighted that he got me!
A student perk of homeschool!
Sunday Snippets
I told a story at church and listener laughed out loud, then she leaned in closer and quietly said...."you just made me pee"
It is dangerous to be funny in your 40s!
Uncontrollable Crying
In early 2002, on accident, baby Max pulled the soap dish and a few tiles off the wall of our main bathroom.
Here are before pictures of the bathroom.
Enjoy entertainment by baby Jack...
Under renovation
Life, time, travel, and finances delayed several advancements in redoing our main bathroom.
For 11 years, I have shared one bathroom with 3 boys.
Many visiting friends and family have had to awkwardly walk to our "not convenient but only" bathroom.
Several have actually peed in our backyard....2 grown men come to mind...you know who you are!
Today, January 1, 2014, found me uncontrollably crying....
Jeff is so talented!
Slow.....but he got mad skillz
I am overwhelmed and speechless!
It all works!
July 2017


text:
Yesterday, I couldn't find my keys. I said to myself, "Bea Bell, you'd lose your head if it wasn't attached." Well, I haven't lost my head yet but I'm about to lose my mind because people just never cease to amaze me. I don't mean that in a good way.
The other day, during the GOP debates, Texas Governor Rick Perry stumbled when trying to name the three agencies he would cut if elected president. The agency he could not remember was the Department of Energy, and the crowd went wild—at least the media crowd. The lead story for almost every news program that night, and for the days following, was Perry's memory lapse. On the television news show, Nightline, Political Director Amy Walter lamented, "Cats get nine lives…I don't know that candidates get that many."
I just don't get it. Have you ever tried to introduce someone you've known for years but your mind is a total blank when you start to say their name? Who hasn't walked into a room and suddenly can't remember why you walked in? I don't understand this need to have a president or a presidential candidate appear "presidential" or flawless. Of course, there are moral and ethical boundaries for everyone whether they're running for president or not, but to get fired up over a slip of memory is silly.
Herman Cain made a joke by asking, "How do you beat Obama? You beat him with a Cain!" Some bright reporter asked him to clarify as to whether this could be interpreted as promoting violence. Cain got testy, and rightly so, as he answered, "Herman Cain, C-A-I-N. Do I have to connect all the dots for you?"
These are the kind of things that drive me crazy. There are plenty of hard issues to focus on but we've become so pretentious that the slightest slip of the tongue is not only newsworthy, but could mean political death for a candidate.
I went to sleep last night and had the worst nightmare of my life caused either by those thoughts or the slice of Godfather's pizza I ate just before going to bed. I dreamed that it was 2012. The Republican and Democratic conventions were over. The candidates had been chosen and their running mates had been picked. Toward the middle and slightly to the left stood Barack Obama and Joe Bidden for the Democrats. Several steps from center and off to the right were Rick Perry and Herman Cain for the Republican Tea Party! Here's the nightmare part.
Perry and Cain won the presidential election (sob)! There was no particular strategy. Perry couldn't remember what platform he was running on, but he did recall the applause he got when defending the state of Texas as having executed 234 people, more than any other state in this nation. He also changed his campaign slogan to HANG 'EM HIGH! Of course Perry could empathize with Herman Cain's inability to remember the sexual harassments accusations against him, that's why he picked him as a running mate.
Perry managed to shift voter attention from jobs and the economy (how stupid) to the death penalty and smaller government. Naturally, the whole point of picking Cain was to siphon votes from Barrack Obama. It worked. Presto, the country wound up with 666 and the 999 plan! That woke me up.
After thinking about the dream, I promised myself three things: I'll never eat pizza before going to bed again, I'll never... Damn! I can't remember the second or the third thing—oops!
Happy musings.


text:
Coinciding with our launch across the East Coast, Boston and Vancouver, Collection 7 was our first themed call for art. In my martial arts faculty I train that it is the small issues that make up the large things, the entire is bigger than the parts of the sum. You will obtain the breadth and depth of information necessary to become a major school teacher, with specialisations in two areas chosen from the Arts program.
To achieve their Arts Award, younger individuals tackle challenges in an artwork form, participate in arts activities, experience arts occasions, get inspired by artists and share their arts expertise with others. This case underlines the distinctive challenges of working an impartial arts establishment inside a difficult political surroundings.
In 1877, he ready his first main piece of artwork, a life-measurement statue of a young man; it was this piece that would launch the start of both his recognition and his monetary wealth as a sculptor. Teachers and scholastics have discovered these seven and their normal order to be of nice utility.
The gallery is a program of the Transylvania Group Arts Council and has been in business for 18 years. So there you've it. Write …