Podcasts about lcls

For review:1. Diplomats Work to De-escalate Israeli Response to Druze Village Rocket Attack.Israeli officials suggested that the response would be serious but not lead to an all-out conflagration (conflagration = an extensive fire which destroys a great deal of land or property).2. Spain to provide another Hawk Air Defense System to Ukraine. Spain will send another Hawk air defense system to Ukraine in September, Minister of Defense Margarita Robles told her Ukrainian counterpart (Rustem Umerov), last week.3. UK Ministry of Defence (MoD) will expand the ability to produce gun barrels by partnering with Sheffield Forgemasters. The initiative will help maintain the flow of L119 Light Guns and AS90 self-propelled howitzers into Ukraine.4.  Kyiv Post Opinion Article - Is Peace Coming to Ukraine? Opinion piece written by Timothy Ash on signs leading to potential peace talks before the end of the calendar year.5. Royal Australian Navy to receive new Landing Craft Light (LCL) vessels.The LCLs will transport up to 4.5 tons of payload or up to 30 personnel and will provide a ship-to-shore connector capability.6. US Navy creates a new position for overseeing investments in the surface and submarine industrial bases. US Navy Statement: “The Direct Reporting Program Manager for the Maritime Industrial Base (or the DRPM for the MIB) will play an instrumental role in realizing Secretary Del Toro's vision to engage in a whole-of-government effort to rebuild the Nation's comprehensive maritime power." The US Navy announced Frederick “Jay” Stefany as the DRPM. 7. US Army Fixed-Wing ISR Program Ready in 12-18 Months. 

Scientific Sense ® by Gill Eapen: Prof. Matthias Kling is Professor of Photon Science and Applied Physics at Stanford University and the Director of the Science, Research and Development (SRD) Division at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. Please subscribe to this channel: https://www.youtube.com/c/ScientificSense?sub_confirmation=1 --- Send in a voice message: https://podcasters.spotify.com/pod/show/scientificsense/message Support this podcast: https://podcasters.spotify.com/pod/show/scientificsense/support

Scientists at the SLAC National Accelerator Laboratory are putting the finishing touches on their LCLS-II laser, which will be 10,000 times brighter than the first LCLS laser.

Scientists at the SLAC National Accelerator Laboratory are putting the finishing touches on their LCLS-II laser, which will be 10,000 times brighter than the first LCLS laser.

Stretching nearly two miles under a freeway in Palo Alto, CA, the Linac Coherent Light Source (LCLS) is a huge X-ray laser with the power to capture images of molecules in motion. Now, a big upgrade is taking this SLAC Lab facility to the next level.

In this episode, I speak with Heidi Alexander, Deputy Director of the non-profit organization Lawyers Concerned for Lawyers.  Heidi helps manage LCLs operations and leads the organization’s law practice management program known as Mass LOMAP (the Law Office Management Assistance Program). Episode 32 Introduction In 2020, it has never been easier to run your own law practice.  On-line tools for marketing, accounting, document assembly and contact management have never been more affordable or easier to use.   But launching a law practice is not for the faint of heart. My guest Heidi Alexander talks about some of the challenges in launching and managing a small law firm and how Mass LOMAP is a great resource to help lawyers get started.  We also discuss how Heidi decided to pursue an alternative legal career and the challenges she had in making her own transition. Not long after Heidi graduated from law school, she became very interested in law firm management.  She completed a judicial clerkship, practiced for a year and then joined Mass LOMAP in 2012.  Since joining LOMAP, she has worked with solo practitioners and small firm attorneys to  develop healthy, sustainable, and productive law practices.  Heidi is an avid speaker on topics ranging from time management and productivity to legal technology, and an author of numerous articles and a book published by the American Bar Association's Law Practice Management Division, "Evernote as a Law Practice Tool".  This year she is a chair of the American Bar Association‘s TECHSHOW Conference. Additional Resources ABA Legal Technology Resource Center Law Technology Today  Law Sites Blog Mass LOMAP Lawyers Concerned for Lawyers SJC Lawyer Well-Being Report Leveraging Your Blog Posts and Saving Time with TextExpander

Welcome to Finance and Fury,  Past few Monday eps on Share concentration – and the holdings and influence that super funds have Today – talk about the legislation put into place and the plans going on now where you might end up renting an apartment from your super fund or bank The plan to help increase apartment supply – decline in prices = lower incentive for developers as a build to sell model – from slumping demand for apartment building New residential product: “build-to-rent” Won’t help Australia’s housing affordability stress, may make it worse, but it helps to achieve public policy objectives - widened housing diversity – for affordable housing close to city centres enhanced build standards – Avoid develop to sell disasters like Opal towers better-managed and secure form of rental housing – economies of scale from LCLs – which we will run through Look into other areas this has been implemented to see how well objectives met   What is it? This refers to apartment blocks built specifically to be rented, at market rates or ‘affordable rates’, and held in single ownership as long-term income-generating assets Policy came to public attention when Labor reforms to taxation around build-to-rent leading up to the election Aim - to increase the supply of rental dwellings through developing a ‘build to rent’ and a large corporate landlord (LCL) sector -  While these two sectors may share similarities, there is a subtle difference between them.   Build to rent - developers and their financiers build multi-unit buildings and, instead of selling the units, retain them to rent to tenant households. Rents may be set at market rents or, for affordable housing, an appropriate discount to market rents could be offered with appropriate government support to make up the funding gap.   ‘Build to rent’ is an established practice in both the UK and USA but it has not been taken up in Australia -   Australia’s tax settings, which were designed for a ‘build to sell’ model, as a major impediment, in particular land taxes and the inability to defer GST costs on construction materials makes retaining dwellings unprofitable. AHURI research identified a number of barriers for institutional investment in the Australian market, reducing the attractiveness of 'build to rent' for investment by the large banks, insurance companies and the superannuation funds.  In a nutshell – large financial companies will become the driver of investment in inner city apartments QLD – We have a program open for bids in May within 10km of CBD. Goldman Sachs is a major investor in one of the biggest build to rent companies in USA.   Large Corporate Landlords – Buy to rent model - financial institutions that acquire large numbers of dwellings and make them available to the rental market, or potentially at a discount to market rents for low-income tenants if appropriate government support is provided. LCLs don’t necessarily build new housing stock, they can purchase properties in the market or through mergers and amalgamations with other LCLs. Indeed the largest LCL in the USA, Mid-America Apartments, (99,939 apartments in 2017) LCLs can merge with build to rent developers who can help manage rental dwellings   Proponents claim LCLs and ‘build to rent’ schemes offer greater supply of rental housing, greater security of tenure for tenants, and better professionalism in tenancy management than small scale 'mum and dad' landlords. These models have also been criticised in other countries for maximising rent increases and for evicting tenants In one case, 60 families were threatened with eviction in Ireland in 2016 when the LCL that owned the residential complex had to sell over 200 houses to an internationally based financial institution in order to repay debts From an international flow of money – part of the profit shifting scheme which caused the Ireland property bubbe QLD - Deputy Premier and Treasurer Jackie Trad today invited industry to register their ideas on how to deliver a large-scale Build-to-Rent development within 10kms of the Brisbane CBD. “Delivering these affordable rentals will contribute to the Government’s Queensland Housing Strategy 2017-2027 target of over 1000 affordable homes by 2022” – says homes but means high-density high-rises   Why hasn’t it taken off here yet? - the tax treatment and returns in Australia make build-to-rent less viable. cost-effective variations of the build-to-rent model are being trialled, including student accommodation, co-living arrangements, or build-to-rent accommodation where the tenant has an option to buy their unit after a few years of renting – talked about in another ep constant rental income from tenants is a particularly appealing investment for institutions that seek reliable income, like super funds   What do the people implementing this want: under current conditions, even market-rate build-to-rent projects are barely viable – at least in Sydney. Australia’s urban housing markets are expensive to purchase land. a housing policy perspective – limitations on foreign purchases of residential property “withholding tax” decision that treats overseas-based institutional investment in rental property less favourably than investment in commercial property – Morrison doubled Since such global funds would likely lead the establishment of a new Australian build-to-rent asset class, revisiting the withholding tax changes could be a significant step in making build-to-rent a reality in Australia. Will build-to-rent make housing more affordable? No – at least not in the short term. While Labor has offered separate affordable housing initiatives, build-to-rent developments themselves will not necessarily deliver affordable housing. evidence from existing build-to-rent developments suggest that rent will be more expensive than traditional renting arrangements. JLL research based in the UK found that on average, the premiums on build-to-rent accommodation were 11 percent over the respective local rental markets.   Who will be doing this - The enduring owner might be, for instance, an insurance company, an Australian super fund, a foreign sovereign wealth fund, a private equity firm, or the building’s developer. Although new in Australia, build-to-rent is quite common in many other countries. Under its North American name, “multi-family housing” – 6.3m new apartments since 1992 A scattering of build-to-rent schemes is already underway or completed, mainly in inner Sydney and Melbourne. And they may prove to be the forerunners of a new Australian residential property sector – but that is far from guaranteed. In Australia, our private rental market is almost entirely owned by small-scale mum-and-dad investors, so this kind of housing would be a largely new departure from typical Australian real estate Another property bubble – main access from massive financial companies – now you are competing for the property with financial institutions Potential benefits – Done to manage the economy and help stimulate economic growth, expand the money supply through borrowings to help keep rates low May get better building standards – MAY – building to own long term versus build to sell – skip forward 100 years of this trend where companies own most buildings Increase demand for buying high-density residential property – Australians long term don’t want this (80%)   Proposal for policy - to enable an affordable housing element the government is looking at allocating sections of federal or state-owned redevelopment sites to community housing providers at discounted rates. this strategy was recently advocated by newly designated federal housing minister Michael Sukkar. urban renewal projects like Sydney’s Central-to-Eveleigh and Rozelle Bays – a pretty big chunk of Syd – fulfill the widely voiced demand that 30% of these developments should be affordable housing Being implemented to fulfil several important public policy objectives. This comes from recommendations/policies laid out in UNs SDG (sustainable development goal) 11.1 and 11.3, along with SDG9 and SDG17, which is about increasing global partnership of multinational companies’ cooperation with Governments. The IMF needs somewhere to pump their SDRs into so they are using the SDGs of the UN which will cost trillions in funding, along with side private companies like Goldman sacs buying all the residential property here (which they have been doing in the programs in the USA). So end game of this build to rent is that we now have to compete with the largest companies in the world when wanting to buy any new residential stock coming onto the market.   Who will live in this? – come back to this 9Global migration compact Thank you for listening, if you want to get in contact you can do so here  

Persis S. Drell Professor, Stanford University The Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory is the world's brightest source of hard X-ray laser light. Not only is this light a billion times brighter than any previous hard X-ray source, it also comes in strobe-like pulses just a few millionths of a billionth of a second long. This combination of high intensity and ultrafast shutter speed allows scientists to make stop-motion images of very fast processes at a very small scale—the scale of atoms and molecules. Dr. Drell will focus on the conception, construction, and start up of the LCLS, as well as some of the first experimental results, with a view to the new frontier of science that this remarkable tool has opened.

Dave Burgess       US History, author, workshop/seminar presenter (West Hills High School -- San Diego, CA)            Resource of the Week: www.newsela.com (Bethany Whinnem, Fairhaven High School) NewsELA provides high interest nonfiction articles about current events that are updated daily. There are several benefits of the website: 1. The same article can be adjusted to various reading levels. 2. At least one article each day has a four question quiz attached, which is tied to specific reading standards indicated by the anchor in the corner. (The questions are also worded differently depending on the reading level selected. 3. When students log in and create an account with the teacher code, it corrects and keeps track of the questions for each student. So, teachers can track problem areas for the class. 4. They usually send out a monthly progress email to the teachers. (email me your favorite resource talkswithteachers@gmail.com)    Segment I – Background and Inspiration   Tell your story. Where are you from and how long have you been teaching? What classes have you taught?  – Dave has taught US History at West Hills High School in San Diego, CA for 17 years. He came to the school first as a basketball coach and a teaching job soon ensued. He is currently on a one-year leave to promote his book, Teach Like a PIRATE: Increase Student Engagement, Boost Your Creativity, and Transform Your Life as an Educator.  Who has helped you in your journey to become a master teacher?   – Dave worked for three summers at the John Wooden basketball camp. To learn from such an extraordinary coach and man of character has benefitted him. Wooden's idea of success, the piece of mind arising from doing your best, has influences Dave's teaching. He tries to embed LCLs (life-changing lessons) into everything that he does in the classroom.   It is important for other teachers to know that we all have had setbacks in the classroom. Identify an instance in which you struggled as a teacher and explain what you learned from that experience. – Dave created a whole lesson around the War of the Worlds. He had the classroom in complete darkness and wanted students to listen to the radio broadcast. There were behavior problems and the students were disengaged. He was very discouraged. Through reflection he realized that the broadcast is flat-out boring. Good teaching is like a heat-seeking missile.  Any time it is off course is has the intuition to return to its proper direction. Through reflection we can get ourselves back on course.   What role does literacy play in a U.S. History class? – Dave believes there should be an emphasis on primary sources. Also, writing forces students to get clear about their thinking. Both enables students to become more critical as thinkers. What is one thing that you love about the classroom?  – Dave loves the day-to-day interactions and relationships with students. It is something that is missing right now while he is on this year-long book/workshop tour.  Segment II — Digging into the Teacher Bag of Goodies    What book do you recommend to a developing teacher?  -- Teach Like a PIRATE: Increase Student Engagement, Boost Your Creativity, and Transform Your Life as an Educator is about embracing the spirit of a pirate -- to be a maverick and sail into unchartered waters without guarantee of success. They also embrace diversity. Their crews reflect a broad range of abilities and backgrounds. There is also the whole idea of trying to hook students and draw them into content. One of the key lessons of the book is encouraging teachers to find what is unique about themselves and draw on those talents and energies and bring them into the classroom.  

(October 3, 2012) Joshua Turner talks about moving to the LCLS and discusses some of the basics about the technology behind the revolutionary facility.

(October 3, 2012) Cathy Knotts talks about the process through which individual researchers can submit proposals and work to get beam time at LCLS.

Bill Schlotter introduces the free-electron laser in the last session dedicated to the discussion of lightsource technologies. The series is geared toward scientists new to the field, as well as those considering new techniques. (October 23, 2011)

Das Epstein-Barr Virus (EBV) infiziert ruhende primäre humane B-Zellen und indu-ziert deren unbegrenzte Proliferation. Dieser Prozess der Wachstumstransformation stellt ein Modellsystem dar, das die pathogenen Mechanismen in der Tumorentsteh-ung widerspiegelt. Die Epstein-Barr Virus nukleären Antigene 3A und 3C (EBNA-3A und EBNA-3C) werden in Publikationen aus dem Zeitraum von 1993 bis 1996 als essentiell für den Prozess der B-Zellimmortalisierung eingestuft. In dieser Arbeit wurde mit einer neuen Technologie, der Maxi-EBV Methode, die Rolle der EBNA-3A und -3C Proteine erneut untersucht. Sowohl mit EBNA-3A negativen als auch mit EBNA-3C negativen Viren konnten erstmals Kulturen von infizierten B-Zellen etabliert werden. Während sich aus EBNA-3A negativen B-Zellkulturen Langzeitkulturen etablieren ließen, starben EBNA-3C negative B-Zellkulturen in der Regel nach 40-70 Tagen ab. Die Effizienz der B-Zellimmortalisierung von EBNA-3A negativen Viren war im Vergleich zur Wildtyp infizierten B-Zellen 24-fach, die der EBNA-3C negativen Viren 140-fach erniedrigt. Sowohl EBNA-3A negative, als auch EBNA-3C negative LCLs sind in ihrer Viabilität eingeschränkt, weisen jedoch unveränderte Zellteilungsraten auf. Die weitere Charakterisierung der EBNA-3A negativen LCLs ergab, dass diese eine Variante des viralen LMP1-Proteins exprimieren. Offen blieb, ob diese Variante das Auswachsen der EBNA-3A negativen B-Zellkulturen begünstigt hat. In der Folge wurden die EBNA-3A negativen LCLs zur Identifizierung von EBNA-3A-Zielgenen eingesetzt und zahlreiche aktivierte und reprimierte Kandidatengene identifiziert. Eines dieser Kandidatengene, Matrix-Metalloproteinase 7 (MMP-7), das durch EBNA-3A induziert wird, wurde im Rahmen dieser Arbeit validiert. Auch mit EBNA-3C negative Viren konnten wider Erwarten LCLs erzeugt werden, die für einen begrenzten Zeitraum in Kultur gehalten werden können. Aus dem Material eines Spenders war es auch möglich, EBNA-3C negative Langzeitkulturen zu etablieren. Die Mehrzahl der EBNA-3C negativen infizierten B-Zellkulturen durchlaufen jedoch zwischen Tag 40 und 70 eine Krise und sterben. Mit der Generierung eines konditionalen EBNA-3C Systems, durch Transfektion eines Tetrazyklin-regulierbaren EBNA-3C Expressionsvektors in frisch isolierte primäre B-Zellen und anschließender Infektion mit EBNA-3C negativen Viren, wurde ein neuer Weg geschaffen, um EBNA-3C-Funktionen zu untersuchen. Dieses 2-Schrittsystem kann nun im Prinzip für jede Virusmutante eingesetzt werden.

The Epstein-Barr virus (EBV) is associated with a number of human malignancies. Following primary infection, the virus persists lifelong in the infected host by latently infecting B cells and occasional cycles of reactivation, virus production and re-infection. Adoptively transferred EBV-specific T cells, generated by repeated stimulation with autologous lymphoblastoid cell lines (LCL) in vitro, are able to cure post-transplant lymphoproliferative disease (PTLD). However, the generation of these vaccines is labor and cost intensive precluding their general availability for all patients at risk. Novel insights into the mechanisms of protective antiviral immunity is expected to provide a better understanding of the pathogenesis of EBV-associated diseases and to facilitate the development of novel and generally available immunotherapeutic options. The aim of this work was to assess specificity and breadth of the EBV-specific T helper cell response, using two different experimental strategies. To define specificity, LCL-stimulated CD4+ T cell lines were established from 23 EBV-negative and -positive donors. The T cell lines generated from EBV-negative donors responded poorly against LCL and failed to show EBV-specificity. By contrast, all T cell lines established from healthy virus carriers were EBV-specific. Half of the lines from acutely EBV-infected patients with infectious mononucleosis (IM) were also EBV-specific, while the other half recognized EBV-positive and EBV-negative target cells. Unexpectedly, the EBV-specific T cell lines did not recognize latent antigens of EBV expressed in all LCL. Instead, these lines were specific for lytic cycle antigens predominantly derived from virion proteins. Several of the T cell lines recognized BNRF1, a viral tegument protein. Most T cell lines, however, recognized different virion antigens, suggesting that the family of virion proteins forms the immunodominant targets of the EBV-specific T helper cell response. Studies on the breadth of the EBV-specific T helper response demonstrated that all healthy virus carriers maintain CD4+ T cell memory to lytic cycle antigens. T cells specific for virion antigens recognized EBV-positive cells directly and, surprisingly, a much higher percentage of target cells than those expressing lytic cycle proteins. Antigen was efficiently transferred to bystander B cells by receptor-mediated uptake of released virions, resulting in recognition of target cells incubated with less than one virion per cell. T cell recognition did not require productive infection and occurred early after virus entry before latency was established. By secreting perforin and granzyme B upon antigen recognition, virion-specific T helper cells inhibited proliferation of LCLs and suppressed the outgrowth of LCLs following infection of primary B cells with EBV. These results established a novel role for virion-specific T helper cells in the control of EBV infection, and identify virion proteins as important immune targets. The findings have implications for the treatment of diseases associated with EBV and potentially other coated viruses infecting MHC class II-positive cells.

EBNA-2 is a multifunctional viral oncogene involved in the immortalisation of B-cells by EBV. EBNA-2 regulates transcription of viral and cellular genes in the proliferative phase of the viral life cycle, which in vitro results in the outgrowth of EBV positive B-cells into lymphoblastoid cell lines (LCLs). EBNA-2 transcriptional signalling is mediated by cellular DNA-binding proteins, such as RBP-J and PU.1, since EBNA-2 does not contain its own DNA-binding domain. In order to better characterise EBNA-2 signalling we conducted a mutational analysis of the viral LMP-1 promoter that is strongly induced by EBNA-2 in the EBV-immortalised B-cells. Our mutational analysis of the LMP-1 promoter confirmed that the PU.1 binding site is important for transactivation of the LMP-1 promoter by EBNA-2, whereas RBP-J binding to the LMP-1 promoter leads to repression and EBNA-2 binding to RBP-J is not required for transactivation. These results imply that EBNA-2 transactivates the LMP-1 promoter preferentially by an RBP-J independent mechanism. We further characterised EBNA-2 signalling by dissection of promoter targeting domains in the EBNA-2 protein. Two EBNA-2 mutants, the CR4del and WW mutant, preferentially activated RBP-J dependent and independent signalling indicating that EBNA-2 uses at least two separate signalling pathways. We introduced the characterised EBNA-2 mutants into the EBV genome and produced recombinant viruses carrying specific mutations in the EBNA-2 genes. Primary B-cells were infected with increasing titres of recombinant EBVs lacking the EBNA-2 ORF or carrying the WW or CR4del mutant. Viruses lacking the EBNA-2 ORF or carrying the WW mutant were not able to immortalise primary B-cells even at high viral titres. The CR4 region of EBNA-2 strongly influenced B-cell immortalisation efficiency and growth rate of the immortalised B-cells. These results indicate that EBNA-2 and the RBP-J signalling of EBNA-2 are absolutely essential for B-cell immortalisation by EBV. In contrast, the CR4 EBNA-2 region mediating RBP-J independent signalling is critical, but not absolutely essential for the process of EBV immortalisation.

EBV is a γ-herpes virus which is able to infect human resting B-cells and to transform them into permanently growing lymphoblastoid cell lines (LCLs). EBNA2 (Epstein-Barr virus nuclear antigen 2) is one of the first viral proteins expressed after in vitro infection and interacts with different cellular proteins like RBP-Jκ and PU.1. The EBNA2 protein acts as a transcriptional activator of the viral Latent Membrane Proteins 1 and 2 (LMP1 and LMP2) and the viral nuclear genes EBNA1, EBNA3A, -3B, -3C, EBNA-LP. Additionally EBNA2 is also able to transactivate cellular genes like CD21, CD23 or c-myc. To study the different EBNA2 target genes and the function of EBNA2 a LCL was established (ER/EB2-5 cells, Kempkes et al., 1995) harboring an estrogen-inducible EBNA2. In the presence of estrogen the ER/EBNA2 fusion protein (estrogen receptor binding domain) is located in the nucleus were EBNA2 can transactivate its target genes, whereas in the absence of estrogen the ER/EBNA2 fusion protein is kept in the cytoplasm and therefore inactive. The cells proliferate in the presence of estrogen and they arrest in the absence resulting in a phenotype similar to resting B-lymphocytes. By using the ER/EB2-5 cell line I could clearly show that the cell surface molecule CD83, belonging to the immunoglobuline superfamily (Zhou et al., 1992), is upregulated after the activation of EBNA2. By using a derivative ER/EB2-5 cell line that constitutively expressed LMP1 I could show that CD83 is still expressed even in the absence of functional EBNA2 suggesting that LMP1, the viral target gene of EBNA2, is responsible for the induction of CD83. Therefore I analysed the activation of the CD83 promoter by LMP1. LMP1 is a transmembrane protein with a short intracellular N-terminus, 6 hydrophobic transmembrane domains and a long intracellular C-terminus, containing C-terminal activator regions CTAR1, 2 and 3. The different CTAR regions are responsible for activating genes via NF-κB, ATF, AP1 and STAT signaling pathways. For the activation of its target genes LMP1 uses the same signaling molecules (TRAF, TRADD) as family members of the TNF-R family (CD40, TNF-R1, TNF-R2). The CD83 promoter was activated by LMP1 as shown by promoter luciferase reporter assays in 293-T cells. The induction was not observed in the absence of a NF-κB binding site in a CD83 promoter mutant. Furthermore LMP1 mutants which are mutated in the binding regions for TRAF2 (CTAR1) or TRADD (CTAR2) are not able to transactivate the CD83 promoter. By co-transfection of LMP1 and dominant/negative IκB the CD83 promoter could not be activated because of inactivation of NF-κB. These experiments clearly demonstrate that the CD83 promoter is transactivated by LMP1 via NF-κB. Additionally to the regulation of CD83 I was also interested in the functional role of CD83. Until now only little is known about the function of CD83. CD83 seems to have a specific role in the decision to single positive CD4+ T-cells in the thymus (Fujimoto et al., 2002). I have tested a possible co-stimulatory function of CD83 to CD4+ T-cells by retroviral expression of CD83 in non-professional antigen presenting cells (RCC). Indeed CD83 expression increased the CD4+ response in comparison to CD80 or GFP retroviral infected RCC cells. In mixed lymphocyte reactions this co-stimulatory effect could not be clearly demonstrated although a soluble CD83-Ig showed a small inhibitory influence. The identification of a CD83 ligand molecule could give new insights into the function of CD83. Therefore a CD83-Ig fusion protein as well as a CD83-tetramer construct were generated and used to screen for a potential ligand of CD83. First results showed that the CD83-Ig fusion protein and the CD83-tetramer construct bound to CD4+ and to CD8+ T-cells of isolated PBMCs as well as to activated T-cells in a culture of mixed T-cell populations.

Die gezielte Generierung antigenspezifischer T-Zellinien, zum Beispiel für den Einsatz in der adoptiven Immuntherapie, erfordert die Stimulation der T-Zellen durch Kokultivierung mit anderen Zellen, die das Antigen in einem geeigneten molekularen Kontext auf ihrer Oberfläche präsentieren. Für den Spezialfall der Stimulation Epstein- Barr-Virus-(EBV)-spezifischer T-Zellen existiert ein besonders effizientes System: EBV-immortalisierte B-Zellen, genannt lymphoblastoide Zellinien (LCLs). Solche LCLs sind leicht für jeden beliebigen humanen Spender herzustellen und proliferieren unbegrenzt. Sie exprimieren mehrere virale Proteine, die in vitro wie in vivo eine starke antivirale T-Zell-Antwort hervorrufen, und stimulieren effizient spezifische T-Zellen gegen diese EBV-Antigene. EBV wurde genetisch modifiziert, um ein neues System zur rationellen Generierung antigenpräsentierender Zellen für die T-Zell-Stimulation ermöglichen. Dieses System beruht auf rekombinanten EBV-Vektoren, mini-EBVs, in die das Gen für ein beliebiges Antigen eingebaut wurde. Mini-EBVs immortalisieren B-Zellen, und in den entstehenden Zelllinien, genannt mini-LCLs, wird das gewünschte Antigen exprimiert. Zudem können mini-LCLs im Gegensatz zu LCLs kein infektiöses EBV bilden und sind daher im Hinblick auf einen therapeutischen Einsatz als besonders sicher anzusehen. In dieser Arbeit sollte die Eignung von mini-LCLs gezeigt werden, effizient ein Fremdantigen zu präsentieren, um antigenspezifische T-Zellen zu restimulieren und expandieren. Schwerpunkt der Arbeiten bilden Untersuchungen mit einem mini-EBVVektor, der als Modellantigen das Gen für pp65 trägt, ein immundominantes T-Zell- Antigen aus dem humanen Cytomegalovirus. Die mini-EBV-Immortalisierung wurde so weit optimiert, daß schließlich bei acht von neun gesunden Normalspendern aus einer kleinen Blutprobe pp65mini-LCLs etabliert werden konnten. Sie waren frei von Wildtyp-EBV, exprimierten intrazellulär pp65 und auf ihrer Oberfläche essentielle Präsentations- und Kostimulationsmoleküle. Durch Restimulation mit der autologen pp65mini-LCL wurden T-Zellinien generiert. Detaillierte Zytotoxizitätsanalysen zeigten die HLA-restringierte, pp65-spezifische Zytotoxizität der pp65mini-LCL-stimulierten T-Zellinien aus vier von vier CMV-seropo-sitiven Spendern. Bei allen diesen T-Zellinien dominierte nach wiederholter Restimulation die pp65-spezifische über die EBV-spezifische Zytotoxizität. T-Zellinien aus EBV-seronegativen Spendern sowie Kontroll-T-Zellinien, die mit Kontroll-mini-LCLs ohne pp65-Expression stimuliert worden waren, zeigten dagegen lediglich eine EBVspezifische Zytotoxizität. Durch Färbung mit HLA:Peptid-Tetrameren wurde die Expansion pp65-epitopspezifischer T-Zellen in allen pp65mini-LCL-restimulierten T-Zellinien aus CMV-seropositiven Spendern nachgewiesen. Bei den drei pp65-T-Zellinien mit einer Kultivierungsdauer von mindestens 50 Tagen, die mit Tetrameren untersucht werden konnten, wurden 40% oder mehr pp65-epitopspezifische CD8+-T-Zellen nachgewiesen. Auch EBV-epitopspezifische Zellen waren nachweisbar, jedoch bestätigten die Tetramer- Analysen, daß durch Restimulation mit pp65mini-LCLs bevorzugt pp65-spezifische T-Zellen expandiert wurden. Der Vergleich mit den Resultaten verschiedener aktuell publizierter Protokolle zur invitro- Generierung pp65-spezifischer T-Zellen für die adoptive Immuntherapie zeigt, daß pp65mini-LCLs ein vorteilhaftes Werkzeug sein werden, um dieses Ziel zu erreichen. Die Erweiterung des mini-EBV-Systems auf andere therapierelevante Antigene ist die Aufgabe der Zukunft.