text:
Æterna Zentaris (NASDAQ:AEZS)
Q2 2013 Earnings Call
August 09, 2013 8:30 am ET
Paul Burroughs - Director of Communications
David Alan Dodd - Chief Executive Officer, President and Director
Dennis Turpin - Chief Financial Officer and Senior Vice President
Boris Peaker - Oppenheimer & Co. Inc., Research Division
Philippa Flint - Bloom Burton & Co., Research Division
Good morning, my name is Matthew, and I will be your conference operator today. At this time, I'd like to welcome everyone to the Aeterna Zentaris Second Quarter 2013 Financial and Operating Results. [Operator Instructions] Paul Burroughs, Director of Communications, you may begin your conference.
Paul Burroughs
Thank you. Good morning, everyone. Welcome to our 2013 second quarter financial and operating results conference call. With me today are David Dodd, President and CEO; Paul Blake, Chief Medical Officer; and Dennis Turpin, Chief Financial Officer.
Please take note that during this call, we'll be making forward-looking statements regarding future events and the performance of Aeterna Zentaris that involve risks and uncertainties that could cause actual events and results to differ materially. These risks are described in further detail in the company's press releases and reports filed with the U.S. and Canadian securities regulatory authorities. These forward-looking statements represent the company's judgment as of today, Friday, August 19 (sic) [August 9], 2013, and the company disclaims any intent or obligation to update these forward-looking statements unless we're required to do so by applicable law or by securities regulatory authority. However, we may choose to update, and if we do so, we will disseminate the updates to the investing public.
It's now my pleasure to introduce the President and CEO of Aeterna Zentaris, David Dodd.
David Alan Dodd
Thank you, Paul, and good morning, everyone, and thank you for your interest. Been very exciting and significant progress made in the company over the last several months and I'm very happy to be here today to provide you an update and then to answer any questions you may have.
Having been here now slightly more than 100 days, I can tell you this organization is primarily and solely committed to progress and success. Employees are all committed to building value for shareholders and showing that we can be a competitive organization in the marketplace as a specialty biopharmaceutical company focused on oncology and endocrinology.
Over the last past several months, we focused internally on our organizational effectiveness and been reviewing the organization on how we might result in a stronger, more competitive organization better utilizing the resources we have. We continue to do that and, as changes and announcements may occur, we'll announce those going forward.
We've also been very active in the area of business development, a topic I had spoken about since joining the company. And there, we have been quite busy looking at opportunities that we can bring in, products that will enable us to begin to immediately generate revenues and cash and move the company into more operating status. Those activities continue and we have several promising opportunities under evaluation right now.
Within the areas of our primary and major development programs, our zoptarelin doxorubicin, or previously known as AEZS-108, or I would refer to as our ZoptEC trial, our Phase III program in endometrial cancer. There we are focused on providing the most advanced LHRH receptor targeting product in clinical development.
We've opened 11 sites to date, 4 in the U.S., 7 in Israel. We are ironing out our last regulatory and contracting details for the Canadian sites. We have been successful in the VHP, voluntary harmonization procedure, process in the EU with now 2 national approvals, in Austria and the U.K., just occurring within the last week. We recently announced the first patient dosing, formally kicking off this program. We're now concentrated on establishing our solid infrastructure to enhance and increase the patient enrollment. We have 2 additional patients who are scheduled for dosing right now.
We have previously announced the signing of the co-development and profit-sharing agreement with Ergomed as our CRO, who will contribute 30% of the cost of this trial up to a maximum of $10 million. I just want to remind you that the clinical program compares zoptarelin doxorubicin to doxorubicin alone as second-line treatment for women with endometrial cancer resistant to platinum/taxane-based chemotherapy. We will have approximately 500 patients in 100 to 125 sites located in North America, Europe and other countries such as Israel.
As you know, endometrial cancer is a very important and critical unmet medical need, very large market opportunity. It's the most invasive gynecologic cancer in women, with 95,000 new cases expected in North America and Europe just this year alone, with about 1 out of 5, or 20%, experiencing recurrent disease. There's no approved drug product in the U.S. for this. We hold worldwide rights to our compound and yet we're continuing to have discussions with potential partnerships and that will continue to be an option that we will consider seriously.
Remember that our Phase II trials, I'd like to comment on those, with this exciting compound in multiple cancers. The encouraging final data for dose-finding [ph] portion of our Phase I and II trial in prostate cancer, they were reported at the ASCO meeting in June. The results demonstrated promising antitumor activity in heavily pre-treated patients with castration- and taxane-resistant prostate cancer. The drug was well tolerated.
The Phase II portion is ongoing with 8 patients recruited so far. This trial is expected to involve up to 37 patients.
In addition, the Phase II trial in triple-negative breast cancer, currently 7 patients are enrolled in this trial. That's as an update from 6 at the last conference call.
Our Phase II trial in bladder cancer with 2 sites open, University of Pennsylvania and Miami. So this compound, which we're very excited about, continues on its successful path towards clinical development.
I'd like to comment on macimorelin acetate, or AEZS-130. That is our old diagnostic for adult growth hormone deficiency. Now let me just comment that this is, as you'll recall, it's an orphan-drug status. It's a drug that, when ingested, induces the secretion of growth hormone, which enables, through a testing manner, for a clinician to conduct a confirmatory test of growth hormone deficiency. This is very critical because, should one decide to then treat with growth hormone, we're talking about an annual expense of $10,000 or more. So it's very important to have such a confirmatory measure out in the market. Today there is no such product approved.
We have finalized our clinical portion of the NDA submission. We're very excited about that. We have finalized our commercial supply agreement, which had been an open issue the last time we spoke. And we're about to complete the quality agreement, which will then enable us to go forward with the NDA submission. We anticipate this to occur and be completed by the end of the summer.
I'll remind you that the advantages of our product versus the current test that's used, which is the insulin tolerance test. Recall that the insulin tolerance test, or ITT, is not indicated for this and yet it's been the only product that's available that has been able to be used.
Our product is an oral administration. The competitor is delivered peritoneally, either IV or IM. Ours is a very simple process: one ingest -- one fasts overnight, one ingests the drug, pass [indiscernible] over a 75-minute period depending on the physician's decision to do 1, 2 or 3 blood draws and the patient goes back to work.
Under an ITT, you have a situation where it's contraindicated for several populations because it induces hypoglycemia. For the people with coronary artery disease, they're contraindicated, those with a history of seizures and also the elderly. This includes the population that, in fact, ought to be administered this test. It requires close medical monitoring and supervision. It can take up to 4 hours, full half day, to complete this test, which literally then knocks out an entire day of work.
Ours is safe and well tolerated and it comes in at a much less billing cost, we anticipate, than one will see with the ITT.
There's a good market opportunity here for this drug. There are 40,000 diagnostic tests annually today for adult growth hormone deficiency, just alone in the U.S., and 36,000 in Europe annually.
More importantly is the opportunity to recognize that people who suffer traumatic brain injuries, which are approximately 215,000 adults in the U.S. annually, 1 out of 5 of those end up being classified as a moderate to severe injury. Those patients are recommended to have a test for adult -- for growth hormone deficiency at periods 3, 6 and 12 months post-injury and all. That provides a tremendous expansion opportunity for the use of macimorelin acetate, adding up to another 120,000 additional tests.
This does not require additional clinicals, this is within the initial indication. It really is a challenge, however, to go out through medical marketing and make sure that, that medical community becomes aware that there is now a confirmatory opportunity with this drug of ours.
We also will go forward in the near future with laying out plans to do trials to validate the dosing for the pediatric population. In doing this, we open up an opportunity that literally adds another 160,000 annual test opportunity for this product.
So you can see this drug has an exciting opportunity just within the area of growth hormone deficiency. The commercialization and launch plans are being finalized and, as we've mentioned before, we plan to go forward with this on our own.
In addition, macimorelin acetate is in an investigator-initiated, placebo-controlled trial for cancer-induced cachexia. This is an ongoing Phase II trial funded the DeBakey VA Center, under a CRADA between our company and that center. This trial will involve 18 to 26 patients and, to date, 7 patients have completed the protocol.
Cachexia is an involuntary weight loss of at least 5% of the pre-illness body weight over a period of the previous 6 months. What's more important is that cancer-induced cachexia affects about 1 million cancer patients in North America annually, representing about 20% of all cancer deaths, and there's no approved treatment for this condition. As you might imagine with these numbers, this is a very exciting opportunity for us to be pursuing and all.
Let me now mention our product AEZS-120, which is novel oral tumor vaccine candidate for prostate cancer. We previously announced that we have filed a CTA, or the Clinical Trial Application, in Denmark to be able to start this trial. That was done in May. All the material for clinical use has been produced, has been released. We've fully completed the preclinical program. And I'm happy to report that we have also now received the regulatory and ethics committee approvals in Denmark to proceed.
The initiation of the Phase I trial will start very soon. We're waiting the last element, which is approval from the Danish environmental authorities, in order to proceed. As soon as that's done, we'll move into the clinical phase of the development of this exacting compound.
Cetrotide, which as we had -- previously had mentioned, we are focused on the transferring of the manufacturing rights or the manufacturing of that to Merck Serono. That's on track. We will expect that will be completed by October 1 of this year. And I remind you that, in doing that, we will receive a $3.2 million one time payment. That would occur around the November time frame. And we'll also continue to receive payments to assist Merck Serono throughout a 36-month transition period, where we will assist them in the ongoing manufacturing and analytical work in support of that.
Finally, I just want to mention that we're very proud that the class action lawsuit, which had previously been filed against the company and certain of our officers in the U.S. District Court for the Southern District of New York, was entirely dismissed with prejudice and without leave to amend. No payment was made by the company to the plaintiff or his counsel in connection with this lawsuit.
So let me just summarize and say that times are very exciting at Aeterna Zentaris now. We're making significant progress on moving forward. Of the 5 milestones we have spoken of the last call we had, we achieved 4 of them. The one outstanding indeed, was the fact that we have not yet submitted the NDA for macimorelin acetate but that will be done in the near future and we anticipate that, that will be done by the end of this summer. And we'll continue to do several things: Keep you updated on our critical programs; keep you updated on our milestones; and we'll be absolutely transparent on whether or not we meet them or not; and as we -- if we miss them, we'll let you know and we'll let you know why and when we will meet them.
With that, I would like to turn our -- the call over to our CFO, Dennis Turpin, for a report on our financial activities and progress. Dennis?
Dennis Turpin
Thank you, David. First, let me update you on our cash position.
Cash and cash equivalent totaled $25.3 million as of June 30, 2013. Including net proceeds from our recent offering completed on July 30, 2013, we had a pro forma cash of $32.3 million, which enables us to continue to move our key product candidates through the pipeline.
Regarding our burn rate. Cash flow used in our operating activities were $9.2 million for the quarter ended June 30, 2013, compared to $6.5 million of cash flows used during the same period in 2012. The increase in our cash used in operating activities is mainly related to higher disbursement with respect to our SG&A expenses. This is related to the departure of our former CEO, to an increase in trade and other receivables related to the Cetrotide business and other current assets, partially offset by lower trade accounts payable settlements. We expect net cash used in operating activity to decrease in the second half of 2013 as compared to the first half of the year.
Taking into account our Cetrotide transactions, our co-development and revenue-sharing agreement with Ergomed, as well as the initiation of Phase III trial with AEZS-108 in endometrial cancer, the discontinuance also of perifosine Phase III program and the recent change to our management, we continue to expect that our overall operating burn in 2013 will be close to $26 million.
Now more details about our results of the second quarter. Revenues were $30.1 million for the quarter compared to $7.5 million for the same period in 2012. The increase is largely attributable to the acceleration of the recognition of previously deferred revenues received in connection with the 2008 sale of the Cetrotide royalty stream.
R&D costs were $5.3 million for the quarter, as compared to $5.2 million for the same period in 2012. SG&A were $5.8 million (sic) [$5.9] million for the quarter, compared to $3.6 million for the same period last year. The increase is mainly related to the termination benefits granted to the company's former CEO and to the related noncash, share-based compensation costs.
Net income was $9.3 million as compared to $4.5 million for the same period in 2012. This significant increase is due largely to the significant increase in royalty revenues, which is noncash, partly offset by lower net finance income, which is also noncash, as well as by higher SG&A.
Thank you for your attention. Now, David.
David Alan Dodd
Thank you, Dennis. Now every one of my colleagues and I will be happy to answer your questions and close it up. I'll turn the call over to the operator for instructions. Thank you.
Question-and-Answer Session
[Operator Instructions] Your first question comes from the line of Boris Peaker with Oppenheimer.
Boris Peaker - Oppenheimer & Co. Inc., Research Division
My question is for David. I just kind of want to ask a global question. Because you've been here for, as you mentioned, over 100 days and I'm sure you're familiarized with the program. I just want to get a sense of, with all the programs that you guys have ongoing, which ones do you view as high priority in making capital allocations decisions? And which ones could be potentially delayed in a context where you might not be able to advance everything at the speed that you may desire?
David Alan Dodd
Thank you, Boris. The ones I spoke of are the high priority ones, all of which we expect to fund. So the -- starting in the probably order of activity, not necessarily order of potential market value, but the filing of the NDA and the subsequent commercialization of macimorelin acetate is extremely important. We believe that's an exciting opportunity. It's a good drug. It's a drug that has several legs on it from evaluation initially, to be able to enable clinicians to conduct a confirmatory test and diagnosis of growth hormone deficiency, but then moving in to it being a treatment for a huge problem of cancer-induced cachexia. So that is a project that we're fully committed to and excited about. Following that is our ZoptEC trial, basically the development of zoptarelin doxorubicin. It's a very exciting compound, built on a concept that, if it's validated, then it's a great opportunity to be able to treat many cancers that to date have not been successfully treatable. And from a valuation that is an enormous opportunity because of the magnitude of the need out there. So we're fully committed to that and excited about that and into this trial now and gung ho and not looking back. I'll also say that AEZS-120, although early, is our most exciting compound, especially from my perspective, having been in the vaccine industry before, to look at a product that is a true vaccine, not a immunotherapy, that is built around a carrier that is very well characterized, defined, has been in about 350 million, 400 million doses so far, this salmonella carrier. So from a characterization and sort of a regulatory standpoint, it's a very well-known, non-scary type of carrier and yet to have a product that could go out there and actually be a vaccine for prostate cancer opens up a tremendous opportunity and would be a very important contribution to health care worldwide. So these 3 programs are our priorities as a company. And I would say, following that are our activities, which -- and almost in addition to that because they're done simultaneously, is the activity we have underway to identify on-the-market products that we can bring forward and begin to generate revenues and cash as an operating company. We realize that is critical for us to achieve, obviously to contribute to reducing our burn rate and, perhaps more importantly, to begin to define and brand this company as an operating company and as a basically specialty biopharmaceutical company. So we're quite active in that regard, also.
Boris Peaker - Oppenheimer & Co. Inc., Research Division
Now for macimorelin, just curious what you think a reasonable price is? I just want to get a sense of the market and how much that could offset some of your burn once it does come to market.
David Alan Dodd
Well I don't talk about the price and partly because we haven't finalized that. But even if we had, I would not because I wouldn't tip my hand. I would say this, that between -- if one looks at -- now the insulin tolerance test is not indicated as a confirmatory test. There simply -- there's nothing out there and yet it's used today and people would prefer -- in our concept testing and market research, it's clear the endocrinologist have a much greater interest in the profile of our product concept versus what they have available today. It's a tight market. There are approximately 30 centers that do a growth hormone deficiency diagnosis in the United States. So one does not need to have masses of people to sell this. And as you know, endocrinologists as a specialty, it's not a large number of people. But even more so, the target centers come down to about 30, so it's a tight group in which to commercialize. I would give you this guidance, though, that if one looks at what the current cost or sales price of an insulin tolerance test is to an office versus their billing cost, you'll see a spread of fivefold, which then suggested to us that, with a preferred features and benefits profile, we have quite a bit of room to be able to go in there on a pricing and yet still come in at a lower billing cost and so be able, from a reimbursement standpoint, provide significant value to the marketplace while also delivering to a physician a safer, quicker, less-involved type of test. And for the patient, we have recently spoken with people who just hate going through the 4-hour period of that parenteral IV process and all and so they look forward to something they can simply fast and take orally and then have their test done. So we see it as a niche market but as a real market opportunity. And I know you'd like me to say we're considering a certain price we're just not. We'll announce that when we launch the product and I'm sure between now and then there'll be -- in each quarter we'll give a little more guidance.
Boris Peaker - Oppenheimer & Co. Inc., Research Division
Well, in terms of -- when you said that the cost -- the billing for the insulin intolerance, what is that right now?
David Alan Dodd
Well, depending on -- it's in the thousands of dollars. That's the billing. Yes, the billing is in the thousands of dollars. The direct cost is in the hundreds. So you get an idea there.
[Operator Instructions] Your next question comes from the line of Philippa Flint with Bloom Burton.
Philippa Flint - Bloom Burton & Co., Research Division
A question in regards to your planned commercialization of macimorelin. Can you talk a little bit about your plans for hiring, timing costs and how many reps you plan to launch with once you get approval?
David Alan Dodd
Sure. Philippa, I'll intentionally not be specific for competitive reasons but I'll give you the guidance. If there are 30 core centers we're going to be targeting and there's less than 5,000 endocrinologists, in total, we'll be targeting, it does not require a lot of sales reps. Some of our other discussions we're having now also are built around the concept of -- to ensure that we have more than one product in the bag of those reps when they're going out there so that they're spending their time efficiently as sales reps. In general, I would use -- if you're building a model, I would use a cost of $200,000 a year for a sales rep. That would -- that should cover the cost of that. There are not -- samples will not be utilized, so the largest out-of-the-pocket expense one normally has in launching a pharmaceutical product, we will not have to deal with. And so it will be other associated costs, so you could add to that a cost of around $50,000, and that's probably still a little high but that would be a good, fully loaded cost of the reps and all. And you could work your own thoughts in terms of a workload model to see how many would it take to do combination of 30 centers in an intense manner and also to deal with less than 5,000 targeted endocrinologists and all. So the overall is a pretty tight budget and a very focused but tight commercialization plan.
Philippa Flint - Bloom Burton & Co., Research Division
Okay, that's great. Quick question for Dennis. In terms of the Cetrotide recognition, can we expect -- I think it's about $11 million remaining to be recognized? Is that going to all come in Q3?
Dennis Turpin
Yes, we expect that the remaining deferred revenue, the balance, will be recognized as of September 30. Almost -- in fact, it will be October 1. And the amount -- do you want to know the amount of deferred revenues?
Philippa Flint - Bloom Burton & Co., Research Division
That would be great. I think it's about $11 million. If you could confirm?
Dennis Turpin
Yes, it's short-term, $11.8 million.
Philippa Flint - Bloom Burton & Co., Research Division
Okay. So you'll see that in Q3?
Dennis Turpin
Yes. And Philippa, just one more precision. We do not expect to have any more deferred revenues to recognize related to Cetrotide after September 30. And we do not expect also to have manufacturing and cost of sales related to Cetrotide after September 30.
Philippa Flint - Bloom Burton & Co., Research Division
And you'll just have the agreement -- revenues from your transfer agreement?
Dennis Turpin
Exactly. We'll have some service to be provided to our partner.
[Operator Instructions] We have no further questions at this time. I'll turn the call back over to Mr. David Dodd for any closing remarks.
David Alan Dodd
Thank you. We appreciate everyone's interest in the company. And again, we look forward to keeping you updated as we continue to progress the developments here and we'll make the announcements accordingly. Thank you.
This concludes today's conference call. You may now disconnect.
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|[GT2005] Cocoon GetTogether 2005 is over... But wait! There's more!
|Mon, 10 Oct 2005 09:52:30 GMT
Hi all, Many thanks to all speakers that have made the program friday so nice.. And of course, all the attendees that were there at the Hackathon days, the dinners and the presentation day. I had fun. And now.. Goodies! You can relive the full GT day at the following page, which holds all slides, the booklet and the audio recordings: http://www.cocoongt.org/Slides-and-recordings.html I hope you enjoy them! ---- Kind regards, Arjé Cahn Hippo Oosteinde 11 1017WT Amsterdam The Netherlands Tel +31 (0)20 5224466 ------------------------------------------------------------- firstname.lastname@example.com / www.hippo.nl --------------------------------------------------------------
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On the Role of Mast Cells in Chemokine-Induced Leukocyte Recruitment
- Thomasina Little
- 2 years ago
- Views:
1 0 On the Role of Mast Cells in Chemokine-Induced Leukocyte Recruitment Yusheng Wang Academic Thesis With permission from the Medical Faculty at Lund University for the presentation of this PhD thesis in a public forum in the CRC, Entrance 72, Malmö University Hospital, Malmö, on Thursday, 26 th October 2006 at 1 pm. Faculty opponent: Johan D Söderholm, MD, PhD, Associate Professor, Linköping University Supervisor: Henrik Thorlacius, MD, PhD, Associate Professor Malmö 2006 Department of Clinical Sciences, Surgery Research Unit, Malmö University Hospital
2 monocytes and lymphocytes while CXC chemokine mainly attract neutrophils. However, the mechanisms of
3 To: Qing
4 ISSN ISBN Lund University, Faculty of Medicine Doctoral Dissertation Series 2006:113 Printed by Media-Tryck, Lund University Lund, 2006
6 1 On the role of mast cells in chemokine-induced leukocyte recruitment CONTENTS 1. LIST OF ORIGINAL PAPERS 3 2. ABBREVIATIONS 4 3. INTRODUCTION 5 4. GENERAL BACKGROUND Leukocyte recruitment Leukocyte recruitment is a multistep process Leukocyte rolling is a precondition in leukocyte recruitment Endothelial cells and activation Cell adhesion molecules Selectins and leukocyte rolling Integrins and ICAM-1 in firm adhesion PECAM-1 and transmigration Pro-inflammatory mediators TNF Chemokines Mast cells Role of mast cells in disease Distribution and subtypes of mast cells Activation of mast cells Mediators of mast cells Glucocorticoids AIMS OF THE THESIS 24
7 Yusheng Wang 2 6. MATERIALS AND METHODS Animals Cells Bone marrow mast cells (BMMC) Neutrophil isolation Endothelial cells Intravital microscopy Histology Cell adhesion assay Flow cytometry RT-PCR ELISA Statistic analysis RESULTS AND DISCUSSION SUMMARY (SWEDISH) CONCLUSIONS ACKNOWLEDGMENTS REFERENCES PAPERS 59
8 3 On the role of mast cells in chemokine-induced leukocyte recruitment 1. LIST OF ORIGINAL PAPERS This thesis is based on the following papers: I. Qing Liu, Yusheng Wang, Henrik Thorlacius. Dexamethasone inhibits tumor necrosis factor- -induced expression of macrophage inflammatory protein-2 and adhesion of neutrophils to endothelial cells. Biochemical and Biophysical Research Communications (2000) 271: II. Xiaowei Zhang, Qing Liu, Yusheng Wang, Henrik Thorlacius. CXC chemokines, MIP-2 and KC, induce P-selectin-dependent neutrophil rolling and extravascular migration in vivo. British Journal of Pharmacology (2001) 133: III. Ming Xiu Wan*, Yusheng Wang*, Qing Liu, Rene Schramm, Henrik Thorlacius. CC chemokines induce P-selectin-dependent neutrophil rolling and recruitment in vivo: intermediary role of mast cells. British Journal of Pharmacology (2003) 138: *These authors contributed equally to this work IV. Yusheng Wang, Henrik Thorlacius. Mast cell-derived tumor necrosis factor- mediates macrophage inflammatory protein-2-induced recruitment of neutrophils in mice. British Journal of Pharmacology (2005) 145:
9 Yusheng Wang 4 2. ABBREVIATIONS BMMC CD CMP48/80 ELC FITC fmlp GRO- HCC ICAM IL i.p. IP-10 KC MCP-1 MDC MIG MIP-1 MIP-2 NAP-2 PBS PECAM-1 PMNL RANTES RT-PCR SDF-1 TARC TNF- bone marrow mast cell cluster of differentiation compound 48/80 EBI1-ligand chemokine fluorescein isothiocyanate-conjugated N-formyl-methionyl-leucyl-phenylalanine growth-related oncogene- hemofitrate CC chemokine intercellular adhesion molecule interleukin intraperitoneal IFN- -inducible protein-10 cytokine-induced neutrophil chemoattractant monocyte chemotactic protein-1 macrophage-derived chemokine monokine induced by IFN- macrophage inflammatory protein-1 macrophage inflammatory protein-2 neutrophil-activating protein-2 phosphate buffered saline platelet endothelial cell adhesion molecule-1 polymorphonuclear leukocyte regulated upon activation, normal T cell expressed and secreted reverse-transcription polymerase chain reaction stromal-derived factor-1 thymus and activation-regulated chemokine tumor necrosis factor-
10 5 On the role of mast cells in chemokine-induced leukocyte recruitment 3. INTRODUCTION Inflammation is a tissue reaction to microbial invasion or non-self substances and vital to the survival of all complex organisms but plays also a key role in numerous inflammatory diseases. The inflammatory reaction is characterized by classical signs of pain (dolor), heat (calor), redness (rubor), swelling (tumor), and loss of function (functio laesa). All these clinical signs of inflammation are reflections of a series of overlapping changes in the tissue microcirculation, including vasodilatation, plasma leakage and leukocyte recruitment into the inflammatory focus. Indeed, leukocyte recruitment is a central event in the pathogenesis of most types of inflammatory disease. Diseases characterized by exaggerated inflammation are an increasingly important cause of morbidity and mortality in human medicine. A more sophisticated understanding and capability to control these complex inflammatory mechanisms may offer an opportunity to improve the treatment of patients suffering from pathological inflammation. Mast cells are tissue resident cells capable of secreting numerous powerful mediators and their close proximity to external surfaces, such as the skin and mucosal epithelium in the respiratory and gastrointestinal tract as well as the tissue blood vessels make them suitable for regulating the inflammatory process and of key interest in pathophysiological research. However, the detailed role of mast cells in regulating complex interactions between tissue cells and leukocytes in the blood remain elusive. The purpose of this work was to better define the role of mast cells in initiating and mediating leukocyte recruitment in vivo.
11 Yusheng Wang 6 4. GENERAL BACKGROUND 4.1. Leukocyte recruitment Leukocyte recruitment is a multistep process Tissue infiltration of leukocytes from the circulating blood into the extravascular space is a key feature of the inflammatory response. Leukocyte recruitment is mainly composed of leukocyte tethering, rolling, adhesion and transmigration (Blankenberg et al 2003). These leukocyte-endothelium interactions are supported by specific cell adhesion molecules expressed on different cells. Initially, leukocyte rolling is enabled by relatively weak and reversible adhesions and mediated by the selectin family of cell adhesion molecules. Firm adhesion of leukocytes to endothelial cells is mainly mediated by 2-integins (CD18) binding to members of the immunoglobulin supergene family. The last step named transmigration is largely supported by PECAM-1 (CD31) (Ebnet et al 1999). Figure 1. Leukocyte recruitment is composed of the leukocyte rolling, adhesion and transmigration Rolling (Selectin mediated) Adhesion (CD18 mediated) Transmigration (Chemokine gradient)
12 7 On the role of mast cells in chemokine-induced leukocyte recruitment Leukocyte rolling is a precondition in leukocyte recruitment Leukocyte rolling is the initial step and has been demonstrated to be a precondition for the subsequent firm adhesion and transmigration (Lindbom et al 1992, Mansson et al 2000). This rolling interaction is mediated by the selectin family of adhesion molecules. One investigation showed that inhibition of the selectins abolished not only leukocyte rolling but also firm adhesion and extravascular recruitment (Lindbom et al 1992). Another study reported that leukocyte adhesion and transmigration was reduced when leukocyte rolling was inhibited by enzymatic removal of L-selectin (von Andrian et al 1992). Notably, a key study demonstrated that leukocyte rolling abolished by an anti-p-selectin antibody can completely inhibit leukocyte adhesion and recruitment in TNF- activated tissue (Mansson et al 2000). One important aspect of leukocyte rolling is the velocity reduction, which facilitates detection of chemoattractant molecules either exposed on the endothelial cell lining or leaking between adjacent endothelial cells from the inflamed tissue (Jung et al 1999). Chemokines are chemoattractant cytokines, which directly activate a rolling leukocyte and convert it to a firmly adherent one. Although circulating leukocytes are main targets of most chemokines, an increasing literature has shown that also other cells, such as tissue resident macrophages and mast cells, express chemokine receptors and may mediate indirect effect of relevance to leukocyte recruitment in vivo. Nonetheless, it is pivotal to this work to note that leukocyte rolling is a prerequisite for leukocyte recruitment and that leukocyte rolling is dependent on endothelial cell activation and selectin expression Endothelial cells and activation The vascular endothelium is a single-cell layer that forms a continuous lining for the large container that holds the circulating blood. One main function of endothelial cells is to provide a homeostatic barrier between blood and tissues preventing thrombosis formation. However, as outlined herein, endothelial cell
13 Yusheng Wang 8 activation plays a pivotal role in initiating and supporting leukocyte recruitment. Various agonists, including physical factors, invading microbes, complement factors and cytokines, such as TNF- may activate endothelial cells. In leukocyte recruitment, the concept of endothelial cell activation must be considered with the following event, in which endothelial cells begin to express P- and E-selectin in order to initiate leukocyte rolling. Since leukocyte rolling is a critical precondition in leukocyte recruitment (Lindbom et al 1992, von Andrian et al 1992; Mansson et al 2000), it is obvious that endothelial cell activation is also of critical importance in the leukocyte recruitment process, i.e. without endothelial cell activation, no expression of selectins on endothelial cells and no rolling adhesive interaction. Thus, activation of endothelial cells exerts a key role in leukocyte recruitment by supporting leukocyte rolling. TNF- is a powerful inducer of leukocyte recruitment and has the capacity to induce all steps in the extravasation process of leukocytes, including leukocyte rolling (endothelial cell activation including P-selectin expression) (Mansson et al 2000). As we know, activation and trafficking of leukocytes are regulated by chemokines (Zlotnik et al 1999). However, it is not known whether chemokines can induce all steps in the extravasation process of leukocytes in vivo. In particular, it is unknown whether chemokines can induce leukocyte rolling, which would require endothelial cell activation as mentioned above. The literature on the expression of chemokine receptors on endothelial cells is complex and contradictory. Several authors reported that endothelial cells do not express chemokine receptors and, thus chemokines should be unable to activate endothelial cells directly in vitro (Schonbeck et al 1995, Petzelbauer et al 1995; Gupta et al 1998). But another author indicated endothelial cells do express CXC chemokines receptors mrna but the endothelial cells didn t respond to CXC chemokine, such as IL-8 and GRO- (Murdoch et al 1999). As the activation of endothelial cells is a prerequisite in leukocytes rolling, it is necessary to known if chemokines can
14 9 On the role of mast cells in chemokine-induced leukocyte recruitment directly activate endothelial cells or not and how chemokines may contribute to leukocyte-endothelium reactions in vivo. Considering this, one would not expect endothelial cells to express chemokine receptors, for example CXCR2, in the case of MIP-2- and KC-induced leukocyte recruitment. Alternatively, CXC chemokines may stimulate an intermediate tissue cell that both express CXCR2 and is capable of secreting pro-inflammatory mediators with the capacity to stimulate endothelial cell activation and P-selectin expression in order to initiate and support leukocyte rolling. Herein, we hypothesized that mast cells may fulfill these criteria in the inflammatory process. 4.2 Cell adhesion molecules According to the definition of the Gene Ontology Consortium, cell adhesion molecules are molecules expressed on the surface of a cell that mediate adhesion of the cell to other cells or to the extracellular matrix (Blankenberg et al 2003). These proteins are playing numerous crucial functions at the interface of a cell and its environment, whether this environment is another cell, from a similar or different origin or extracellular matrix or even sometimes soluble elements. Adhesion molecules are widely distributed and virtual every cell expresses cell adhesion molecules. Some adhesion molecules are the biochemical fundament in leukocyte recruitment and are listed in Table The selectins and leukocyte rolling The selectin family of adhesion molecules contains three members, L-, P- and E- selectin. L- referes to leukocyte; P- to platelet and E- to endothelial cell (Carios et al 1994, Vestweber et al 1999). All selectins have five common structures: (1) the selectin-like function of NH 2 -terminal domain; (2) an epidermal growth factor (EGF)-like part; (3) 2-9 fragments of short consensus repeat; (4) a transmembrane domain; (5) a cytoplasmic tail (Lasky 1995, Bevilacqua et al 1994, McEver 1994). Each selectin may recognize and bind to a very restricted number of high-affinity
15 Yusheng Wang 10 ligands individually in vivo (see Table 1.). P-selectin glycoprotein ligand (PSGL)- 1 has been shown to be the dominant physiologic ligand for P-selectin. In fact, all Table 1. The main cell adhesion molecules involved in leukocyte recruitment Adhesion molecules Ligands Functions Distribution Selectins L-selectin GlyCAM-1; CD34 Rolling Leukocyte E-selectin ESL-1; PSGL-1 Rolling Endothelial cell P-selectin PSGL-1 Rolling Endothelial cell Selectin-ligands ESL-1 E-selectin Rolling Leukocyte PSGL-1 P-selectin Rolling Leukocyte Immunoglobulin superfamily ICAM-1 L 2; M 2 Firm adhesion Endothelial cell ICAM-2 L 2 M 2 Firm adhesion Endothelial cell ICAM-3 D 2 Firm adhesion Endothelial cell PECAM-1 PECAM-1 Transmigration Endothelial cell Leukocyte Integrins L 2 (CD11a/CD18) ICAM-1; ICAM-2 Firm adhesion Leukocyte M 2 (CD11b/CD18) ICAM-1; ICAM-2 Firm adhesion Leukocyte D 2 (CD11c/CD18) ICAM-3 Firm adhesion Leukocyte P-selectin-dependent leukocyte rolling in vivo is mediated by PSGL-1. PSGL-1 can also bind to E-selectin but with much lower affinity than to P-selectin and the physiologic role of PSGL-1/E-selectin interactions remains to be demonstrated. A specific E-selectin ligand (ESL-1) has also been identified but its function is not yet well known (Blankenberg et al 2003).
16 11 On the role of mast cells in chemokine-induced leukocyte recruitment The molecular weight of L-selectin is dependent on the level of glycosylation from 74K (lymphocyte) to 110K (neutrophil). It is only expressed in hematopoietic cells as a constitutive component of the surface of the leukocyte. L- selectin may mediate not only binding to an inducible vascular ligand(s) but also to yet undefined ligands at sites of inflammation but mainly is recognized for its important role in the recirculation of lymphocytes through peripheral lymphoid tissues (Baggiolini et al 1999). P-selectin is a 140K protein and stored in Weibel-Palade bodies in endothelial cells and in -granules of platelets (Hsu-Lin et al 1984, McEver et al 1989). Following activation by thrombogenic or inflammatory mediators, -granules or Weibel-Plalade bodies containing P-selectin fused with the plasma membrane, thereby rapidly exposing P-selectin on the cell surface within 2 min. P-selectin expression is generally short-lived reaching its peak after only 5-10 min, declining to baseline after 3h, which is consistent with its role in mediating early leukocyteendothelial cell interactions. However, besides the preformed P-selectin stored in Weibel-Palade bodies, additional synthesis and expression of P-selectin at the gene level may be mobilized within 2h by cytokine stimulation (IL-1, TNF- ) (Blann et al 2004). Indeed, in vivo studies of P-selectin function suggest that it may also be important at later time points as a cytokine-induced adhesion molecule (Weller et al 1992, Robinson et al 1999, Kanwar et al 1999, Mansson. 2000). The molecular weight of E-selectin is nearly 115K. Only endothelial cells can express E-selectin when stimulated with TNF-, IL-1 or lipopolysarccharide (LPS) (Bevilacqua et al 1987, 1989). In resting endothelial cells, there is no E-selectin expression. Generally, it takes one hour for initial expression of E-selectin in endothelial cells and after 4-6 hours maximum expression is reached. E-selectin expression may be sustained for hours. It has been suggested that E-selectin plays a redundant role together with P-selectin in cytokine-activated tissues (Tang et al 1996), however, more recent data suggest that P-selectin is the dominant
17 Yusheng Wang 12 rolling receptor in inflamed venules and that E-selectin may be more important for events downstream of leukocyte rolling, such as an intermediate signaling function in between rolling and firm adhesion. Nontheless, expression of E- selectin may also be used as a marker of endothelial cell activation. Figure. 2. Selectin structure. The lectin domain, epidermal growth factor (EGF)- like domain, short consensus repeat (SCR) fragment, Transmembrane (TM) domain, and cytoplasm tail Integrins and ICAM-1 in leukocyte firm adhesion When rolling leukocytes are activated by chemokines, integrins are activated and upregulated, resulting in firm adhesion between the leukocyte and endothelial cells. Integrins are heterodimeric transmembrane glycoproteins formed by the non-covalent association of one -subunit and one -subunit. The integrins are
18 13 On the role of mast cells in chemokine-induced leukocyte recruitment expressed in a large variety of cells where they regulate many diverse functions, including growth, differentiation, motility and polarity (Kishimoto et al 1999) but only integrins relevant to the present work on leukocyte recruitment will be mentioned here. Integrins have exerted various states of activation, i.e. in resting cells they are usually in a non-adhesive conformation that provides low affinity for ligands. Changes in cell activity may trigger signals that modify their conformation to develop a high affinity. Activated integrins mediate firm adhesion by recognizing members of the immunoglobulin gene superfamily expressed on the endothelial cell surface, such as ICAM-1, ICAM-2 and VCAM-1 (Table 1). 2 (CD18) or leukocyte integrins are restricted to leukocytes and as adhesion receptors they are fundamental to both innate and adaptive immunity. Thus, the integrins involved in leukocyte firm adhesion to endothelial cells are mainly of the 2-integrin (CD18) subfamily, such as L 2 (LFA-1 or CD11a/CD18); M 2 (Mac-1 or CD11b/CD18); D 2 (p150, 95 or CD11c/CD18). Moreover, 4 1 (VLA-4) expressed on lymphocytes and activated neutrophils (Blankenberg 2003) appear to play a role in the recruitment process by interacting with VCAM-1 on endothelial cells. Intercellular adhesion molecules (ICAMs) belong to the immunoglobin (Ig) superfamily, which are membrane glycoprotein receptors containing a variable number of extracellular Ig-like domains. ICAM-1 is widely expressed at a basal level and can be up-regulated by pro-inflammatory cytokines on endothelial cells while ICAM-2 is present in high levels in resting endothelial cell and not inducible PECAM-1 and transmigration Transendothelial migration of leukocytes is the last step of recruitment process of leukocytes by which the leukocyte squeezes in an amoeboid fashion across the endothelial cell barrier and moves to the site of maximal inflammation guided by concentration gradients of chemoattractant molecules. This transendothelial passage is believed to occur along the junctions between tightly apposed
19 Yusheng Wang 14 endothelial cells. Some authors believe that there is a transcellular pathway through which leukocytes may directly migrate through endothelial cells (Engelhardt et al 2004). Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) belongs also to the Ig-like subfamily and exerts a unique role in the transmigration of leukocytes. It is expressed abundantly by endothelial cells and largely concentrated to the junctions between adjacent endothelial cells. PECAM- 1 is also distributed on the surface of most leukocytes as well as platelets. Blockade of PECAM-1 interferes with the infiltration of leukocytes without affecting adhesion of leukocytes to endothelial surface. During leukocyte transmigration, PECAM-1 on leukocytes and endothelial cells interacts in a homophilic manner (Muller et al 1999; 2003). 4.3 Pro-inflammatory mediators TNF- TNF- is a powerful regulator of acute inflammation and leukocyte recruitment in numerous diseases. It is mainly produced by macrophages activated by LPS in vivo. But also epithelial cells and mast cells have ability to secrete TNF- as well. TNF- has two active forms including a secreted and membrane-bound form. TNF- receptors are present on virtually all cell types of body implicating that TNF- exerts widely different biological actions although it is mainly recognized for its potent pro-inflammatory and pro-apoptotic effects. Another well-known function is that TNF- can induce necrosis in certain tumor cells for which it was once named tumor necrosis factor. In the context of leukocyte recruitment, one of the key effects of TNF- major effect is to activate cells to express adhesion molecules and chemokines. Thus, TNF- alone has the ability to induce the whole leukocyte recruitment process, including leukocyte rolling, adhesion and transmigration in vivo (Smart et al 1994, Tessier et al 1997, McColl et al 1999). Indeed, TNF- plays a fundamental role in multiple inflammatory diseases including rheumatoid arthritis, Crohn s disease, autoimmune disorders and
20 15 On the role of mast cells in chemokine-induced leukocyte recruitment surgical injury (van Deventer 1997, Klinkert et 1997, Ksontini et al 1998, Moore et al 2000). It has been shown that TNF- can promote leukocyte-endothelial cell interactions by increasing expression of adhesion molecules, including P- and E- selectin as well as ICAM-1 and VCAM-1 on endothelial cells (Bevilacqua 1987, Ley et al 1995, Pober et al 1986, Haraldsen et al 1996) Chemokines Chemokines are a kind of cytokines stimulating leukocyte chemotaxis, i.e. chemoattractant cytokines. Chemokines are mainly involved in leukocyte recruitment although it is clear from a growing body of literature that chemokines are also important players in processes, such as angiogenesis, growth and adaptive immune functions (Rollins 1997). Tissue resident macrophages, mast cells, and recruited leukocytes as well as fibroblasts, endothelial cells or epithelial cells of the site of inflammation or infection can express and release large amounts of chemokines. Potent stimulators of chemokine formation include mainly proinflammatory cytokines, such as IL-1, TNF-, interferon- (IFN- ) and hematopoietic growth factors as well as bacterial toxins, including LPS (Baggiolini 1999). From a structural perspective, chemokines constitute a family of small peptides (8-12K) and according to their primary structure (amino acid residual sequence) and number of amino acid residuals between the last two N- terminal cysteines, chemokines are divided into two large subfamilies, i.e. CC and CXC chemokines. CC and CXC chemokines can attract different kinds of leukocytes individually. In vitro, CC chemokine mainly attracts monocytes, lymphocytes but not neutrophils (Bonecchi et al 1999, McColl et al 1993, Cheng et al 2001). The CXC chemokines are also subdivided according to the presence of the ELR motif (glutamic acid-leucine-arginine) preceding CXC in the N- terminus. The ELR-CXC chemokines predominantly act on the neutrophil while non-elr-cxc chemokines are more specific for lymphocytes. The classic CXC chemokine is IL-8 which is secreted by various cells and acts as a main attractant
21 Yusheng Wang 16 of neutrophils. In mice, MIP-2 (macrophage inflammatory protein-2) and KC (cytokine-induced neutrophil chemoattractant) are produced by macrophages activated by LPS and perform chemotactic functions on neutrophils similar to IL- 8. In lack of an identical match to IL-8 in mice as yet, both MIP-2 and KC are defined as homologues to human IL-8 (Huang et al 1992, Tekamp-Olson et al 1990). Chemokine receptors are seven-transmembrane-domain proteins that signal through GTP-binding proteins (Baggiolini et al 1999). There are mainly two CXC chemokine receptors expressed in neutrophils, those are CXCR1 and CXCR2. CXCR1 can bind IL-8 but has low affinity to other ELR-CXC chemokines. CXCR2 binds mainly to ELR-CXC chemokines. So, in mice, the function of CXC chemokines, such as MIP-2 and KC are mediated via binding to CXCR2 (Huber et al 1991, Cacalano et al 1994, Jones et at 1997). Other CXC receptors such as CXCR3 are only expressed on activated T lymphocytes while CXCR4 appear widely on most hematopoietic cells and even some tissue cells (Baggiolini et al 1999). CC chemokine receptors are predominately expressed on monocytes and lymphocytes (Baggiolini et al 1999). It has been reported that mast cells can express different kinds of chemokine receptors (Table 2.) (Schramm and Thorlacius 2004, Juremalm et al 2005). Up to date, CXC chemokine-dependent leukocyte infiltration has been implicated as a fundamental part of the pathogenesis of several important clinical conditions, such as endotoxemia-induced lung injury (Schmal et al 1996), glomerulonephritis (Feng et al 1995) and bacteria meningitis (Diab et al 1999). However, the detailed mechanism of action of CXC chemokine-induced leukocyte-endothelial cell interactions remains elusive. To understand the molecular details of CXC chemokine-provoked tissue infiltration of leukocytes in vivo would not only deepen our understanding of the pathophysiology but may also provide mechanistic information that may form a basis for development of new and more Table 2. Chemokine receptors expressed in mast cells
22 17 On the role of mast cells in chemokine-induced leukocyte recruitment Receptors CCR1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 Ligands MIP-1, RANTES, MCP-2, HCC-1. HCC-2 MCP-1, MCP-2, MCP-3, MCP-4, MCP-5 MIP-1, RANTES, MCP-2, MCP-3; Eotaxin, HCC-1, HCC-2 RANTES, TARC, MDC MIP-1, MIP-1, RANTES MIP-3 ELC, SLC CXCR1 CXCR2 CXCR3 CXCR4 IL-8 GRO-, NAP-2, IL-8 MIG, IP-10, I-TAC SDF-1 CX3CR Fraktalkine effective therapies directed against inflammatory diseases. On the other hand, CC chemokines, such as MIP-1 (macrophage inflammatory protein-1 ) and MCP-1 (monocyte chemotactic protein-1) predominantly stimulate chemotaxis of lymphocytes and monocytes but not neutrophils. However, the majority of these studies on CC chemokines have been performed in vitro systems comprising only endothelial cells and subpopulations of leukocytes (Bonecchi et al 1999, McColl et al 1993, Cheng et al 2001). In fact, there is growing body of data supporting that CC chemokines may also be involved in neutrophil trafficking in vivo as well (Appelberg 1992, Shanley et al 1995, Matsukawa et al 1999, Diab et al 1999). It has been reported that treatment with antibodies to MIP-1 and MCP-1 may reduce extravascular neutrophil recruitment in pathological inflammatory models
23 Yusheng Wang 18 (Shanley et al 1995, Matsukawa et al 1999). Moreover, MIP-1 deficient mice exhibit clear-cut defects in neutrophil responses in complex inflammatory reactions and experiment in CCR1 gene-targeted mice implicate a role of CCR1 in neutrophil trafficking (Domachowske et al 2000). Nonetheless, it is not known whether CC chemokines can activate endothelial cells in vivo and thereby support selectin expression and leukocyte rolling, which is a precondition for subsequent leukocyte recruitment (Lindbom et al 1992, von Andrian et al 1992, Mansson et al 2000). 4.4 Mast cells Role of mast cells in disease Mast cells are multifunctional and tissue residual cells. Mast cells are predominantly localized along the tissue microvasculature and have a unique capacity to coordinate several aspects of acute inflammation, such as vascular leakage, expression of adhesion molecules and leukocyte infiltration (Schramm and Thorlacius 2004), which is classically manifested as anaphylaxis, urticaria, angioedema, and acute exacerbations of asthma. The contribution of mast cells in allergic disease and many chronic inflammations, such as rheumatoid arthritis, multiple sclerosis is well known. Moreover, it has been reported that mast celldeficient mice were more susceptible to experimentally induced lung bacterial infections than wild-type mice and provided evidence that mast cell-derived TNF- is an important cytokine in the clearance of bacteria by inducing the influx of neutrophils into the infected sites (Malaviya et al 1996). Thus, mast cells may be of key value in innate immune responses to bacteria and ischemia/reperfusion as well as in adaptive immunity to parasites. On top of that, mast cells have been implicated in wound healing, collagen turnover and fibrogenesis (Boyce 2004).
24 19 On the role of mast cells in chemokine-induced leukocyte recruitment Distribution and subtypes of mast cells Mast cell development requires intact function of the membrane-bound growth factor termed stem cell factor (SCF) and its receptor, the tyrosine kinase Kit. Mice lacking function in either SCF or Kit are profoundly deficient in all subtypes of mast cells. WBB6F1 mice, which were used in the present work, lack mast cells due to a mutation in the Kit oncogene of SCF receptors. Mast cells originate from hematopoietic CD34+ c-kit+ progenitor cells in the bone marrow. When released and migrated into the extravascular space, mast cells develop in situ from these progenitors into two distinct mast cell subpopulations dependent on the microenvironments of tissues (Boyce 2004). Distribution of mast cells is diversified in different organs and tissues as well as in between different species. For instance, mast cells can not be found in the mouse liver but are abundant in the dog liver (Gersch et al 2002). Mature tissue mast cells or connective tissue mast cells (CT) are commonly found in connective tissues while mucosal mast cells or reactive mast cells are abundant in the gastrointestinal tract during helminthes infection (Boyce 2004). Alcian blue safranin staining may distinguish connective tissue type mast cells (CTMC) from mucosal tissue mast cells (MMC). The CTMC are stained in red and MMC turn blue. The mast cells of skeletal muscle shows red staining. (Gersch et al 2002). Some major characters of two mouse mast cells are listed as (Table 3) (Feger et al 2002; Shimizu et al 2001) Activation of mast cells The classical way of mast cell activation is immunological and IgE-mediated. The receptors named Fc RI on the mast cell surface are aggregated by the binding of IgE and multivalent antigen or allergens. Receptor cross-linking may activate intracellular signal transduction including activation of serine proteases, tyrosine phosphorylation of multiple cellular substrates, increase of c-amp and Ca 2+ levels.
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text: Alright so I have some questions making the transfer here. I know that since I don't have access to the database on my free forum, there is not much I can transfer over. My question is, is there anything I can add/transfer without doing it manually? (probably not) For instance, I think I have to manually enter the users details (only have 15). Does this mean they can automatically log in with the previous info once the xenforo forums are up and running? Additionally, I was looking for this information but couldn't find it. I am trying to enable Full Friendly URLs but I am having some difficulty. When I enable this option, every link on the xenforo forum is redirecting back to my main site. Is there some edit I have to make to the .htaccess file (which .htaccess would this be, the one at root or under the forums)? Ok last thing is....my current forums at located at http://forums.animeinspirations.com/ is there some sort of redirect I can enable so that everything is pointed to my xenoform installation? (Currrently at http://animeinspirations.com/forums)


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Axis Theatre explores territory all its own in Rip! A Winkle in Time
As Vancouver's long-time physical-theatre troupe gets ready to debut Rip! A Winkle in Time, its cofounder looks to retire.
It says something about the form's wild possibilities that the head of the grandaddy of all Vancouver physical theatre is still hesitant about defining the genre after 39 years of creating it.
"Physical theatre—it's so evolving," says Axis Theatre's artistic director Wayne Specht, smiling as he sits with codirector Kathryn Bracht before a rehearsal for the company's Rip! A Winkle in Time at the CBC studios downtown. "It is an attractive, descriptive couple of words. It does set the imagination on fire. To this day, I don't really know what it means, other than to say it encourages us to explore the physicality of our world."
It's not that Specht hasn't had a lot of opportunity lately to reflect on his movement- and gesture-driven form: after helping to produce 55 original works of theatre, the influential artistic director and performer has announced that Rip! will be his final big show before he retires next year. But it's an apt way to sign off—and a vivid example of the diverse forms that physical theatre and Axis itself have been able to work in over the years. A clever, and totally unexpected, riff on the old legend of Rip Van Winkle, the production integrates everything from acting and text to acrobatics, dance, slapstick, silent film, music, and even puppeteering. Specht's final show brings in a lot of the various threads he's explored at Axis over his career.
It's a long way from the company's early work, after it was founded amid the burgeoning Vancouver arts scene of 1975. Specht, who studied at the National Theatre School and the École Internationale de Théâtre Jacques Lecoq in Paris, hooked up with some other young theatre grads here (including Wendy Gorling, Lin Bennett, and Elizabeth Murray-Byers) to start the Axis Mime Theatre. He jokes that it was the natural outcome of "like mimes" getting together. But Specht and his colleagues were soon pushing the form, collaborating with other companies in town like Carousel Theatre and Green Thumb Theatre, and eventually performing their first work using text, Dennis Foon's Heracles, in 1978. The popularity of that piece, and the explosion of the art form, led to Axis organizing Mime Fest '80. "There were so many pretenders, we said, 'Let's find out what it is,' " Specht explains.
From there, Axis launched the huge Beaux Gestes festival in 1986, when it also put on thousands of performances at Expo. "I got to bring in international actors, have a big symposium, and it had a big impact on our local community," says Specht.
Meanwhile, Axis was pushing beyond mime into other forms of physical theatre. "The influences became broader because myself and others were going out—it was so key to find innovations in the world," Specht says. Part of that widening reach was Specht's devotion (both personal and financial) to collaboration; in its almost four-decade history, the company's worked with everyone from Australia's Legs on the Wall (Flying Blind) to the Arts Club (Don Quixote) and Pi Theatre (Disposing of the Dead).
Which brings us to Axis's most famous project—and one of the biggest success stories on the local theatre scene. Out of the hothouse of movement-based performance forms that the company was working in grew a little show called The Number 14. It was a series of vignettes about bus-riding that worked in masks—equal parts commedia dell'arte and Monty Python. The Touchstone Theatre coproduction (created by Specht and Roy Surette) opened at the Firehall Arts Centre in 1992 for a modest two-week run.
"We said, 'Gee, a lot of people still want to see this,' " recalls Specht, who immediately booked a gig opening in the new year at the Waterfront Theatre on Granville Island. The rest is history: it enjoyed a two-decade run, travelling through Canada, Europe, Asia, and the Middle East, with two visits to the New Victory Theater on Broadway, earning a 1998 New York Drama Desk nomination.
Axis presents scores of shows for young audiences at schools each year, but Rip! will actually be its first adult-oriented full-length play since The Number 14. "I made a choice years ago that I would never do a Number 15," Specht says with a smile.
Rip! is exploring territory all its own. Playwright KC Brown came to Axis with the idea for the play, but the company has been helping develop it from the early stages, tapping the skills of a cast that ranges from puppeteer-actor Tara Travis to veteran thesp Simon Webb to clowning- and circus-trained Annette Devick, who's worked for Cirque du Soleil.
"This is more of a play play in its structure, and yet it is not linear," Specht says, comparing the work to The Number 14. "The theme of what if you fell asleep for 100 years and came back just really resonates so much with this play. There will be expectations from our audience: 'Oh, they'll show him operating a washing machine for the first time.' No, no, no." Specht explains Brown's twist on the age-old story plays with time, flipping back and forth between what may or may not be the present and the gold-rush era, and it's been a challenge for him to find ways for the audience to navigate its brain-teasing tricks.
Actor Stefano Giulianetti says the key is how Specht stages the opening of Rip!. He points out that just as The Number 14 opened with a "strange, wordless mask piece" that gave way to a more text-based vignette, Rip! opens with a stylized, speech-free piece about the ephemeral character of the moon. "Then two characters from the real world come in and you say, 'Okay, it's not a weirdo moon piece,' " he says with a laugh.
"Wayne, working over so many years, knows how to bring an audience onboard," explains Giulianetti, who has appeared in several Axis shows, including more than 500 presentations of The Number 14. "He knows how to tell a story. Art is abstract, but when it's made clear to you it becomes interesting and accessible.
"He knows that a stylized performance, if it's done gently, the audience will let you get away with murder. You can change the style as long as you establish that anything is possible. That's why it starts with the moon."
Like the best physical theatre, Rip! is about surprises, and Giulianetti, like Specht, is reticent to give away some of its moments of magic. Suffice it to say, from our glimpse of rehearsal, there's a fortuneteller who warns a man that his lifeline eerily disappears, a saloon-style barkeep, a life-size marionette dog, and talk of the ghosts of frozen men.
Before he heads onto the makeshift stage, Giulianetti, one of the many young actors in this town whom Specht's helped to flourish, dares to come closest to defining physical theatre.
"Here, you don't rehearse, like, 'My line, your line'—words come kind of last," he explains of the Axis process. "We did at one point go through a whole scene without words. Words will augment it, but the goal is to tell a story without words. Anybody should be able to watch this show or any Axis show with earplugs in or not knowing the language—and that's the one thing that connects physical performance."
Axis Theatre's Rip! A Winkle in Time is at the Waterfront Theatre from Thursday (May 1) to May 17.


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WiiChuck Adapter (Arduino Compatible)
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The WiiChuck Adapter is a small PCB that is designed to be inserted into the connector of a Nintendo Wii Nunchuck to provide access to all 4 wires of the remote. The power, ground and two-wire interface of the Nunchuck are all broken out to a 4-pin 0.1" pitch header. Compared with standard WiiChuck, the DFRobot WiiChuck has extra 4 pins which allow you connect it to two devices.
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Fri February 3, 2017 - 6:00 PM
Orlando, FL, 2017
with:In Hearts Wake ...less...more
House of Blues Orlando
1490 East Lake Buena Vista Dr
Orlando, FL
Doors - 6:00pm Show - 7:00pm The concert venue is general admission standing. This event is all ages. For accessible seating the guest must purchase a g.a. standing ticket and arrive to the venue at 20 minutes before door...See More