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Nobel prize winning geneticist Barbara McClintock wrote, "Everything is one. There is no way to draw a line between things." This is certainly true in my own field of Neuroscience, where the study of brain and behavior integrates many areas of study and emphasizes the connection between mind and body. It is also true of my work here at Simmons. It is difficult to draw a line between my teaching, research, and mentoring efforts, in part because they are intertwined around a central theme of increasing the participation of women in the sciences. I traveled a nontraditional path in finding my passion and vocation, and I especially enjoy helping others explore their interests and career possibilities. Sometimes you'll be surprised where life takes you, and it is the life skills and education you acquire in college that can help you make the most of your opportunities. I love to teach, and here at Simmons I'm able to work closely with students as they become active participants in a learning process that occurs in the classroom, in the research lab, and in advising relationships and extracurricular activities.
Dr. Strowman teaches Biostatistics and Capstone Seminar III in the Doctor of Nursing Practice (DNP) program. She also provides statistical consultation on Capstone projects and Master's theses in the Nursing and Nutrition departments. Dr. Strowman joined the School of Nursing and Health Sciences faculty in 2008 after working for several years in the Academic Technology Department as a Statistical Software Consultant.
Professor Strowman has supervised several DNP Capstone projects including:
Shirong Luo has been a member of the philosophy department at Simmons College since 2006. Prior to his appointment at Simmons, he had been a visiting professor at Mount Holyoke College for two years (2004-2006). The first recipient of the Dean's Award for Exceptional Faculty Scholarship at Simmons College (2012), Dr. Luo has focused his research on Chinese philosophy of the classical period, comparative philosophy, and feminist ethics. He was an invited lecturer at the National Endowment for the Humanities Summer Seminar for College and University Teachers (2008).
Among the many courses Dr. Luo has taught at Simmons are Asian Philosophy, Philosophy of Religion, World Religions, Ancient Greek Philosophy, Philosophy and the Arts, Philosophy through Literature and Film, Philosophy of Human Nature, American Pragmatism, Biomedical Ethics, and MCC.
My areas of teaching include contemporary philosophy, ethics, logic, nineteenth-century philosophy, and philosophy of language. A recipient of the 2001 Simmons College Dean's Award for Excellence in Teaching, I have taught many different courses with the same objective of guiding students on the path of philosophy, the pursuit of wisdom. My mission as a teacher is to inspire students to think philosophically—to wonder, reflect, and reason methodically about the great problems. The synergy of my teaching and scholarship is reflected in my textbook as leading editor, World Ethics (CA: Wadsworth, 2003), which includes multicultural and feminist perspectives along with the European classics in one comprehensive anthology in ethics.
My scholarship is dedicated to the philosophy of language and I specialize in twentieth-century German philosopher, Martin Heidegger. In addition to professional presentations and journal articles that focus on Heidegger and compare his views with those of twentieth-century analytic philosophers W.V. Quine, Rudolf Carnap, and Ludwig Wittgenstein, I have published the following two co-translations of Heidegger's works on language: On the Essence of Language (NY: SUNY Press, 2004), which was nominated in the spring of 2005 for the German Translation Award presented by the American Translators Association; and Logic as the Question Concerning the Essence of Language (NY: SUNY Press, 2009), which was nominated for the Goethe Institute's 2010 Helen and Kurt Wolff Translator's Prize.
Candy Schwartz is a green-card-carrying resident alien, born in Toronto and raised in Montreal. She has a BA (in linguistics) and an MLS from McGill University, Montreal, and a PhD in information transfer from Syracuse University.
I am an Associate Professor of Biology and co-director of the Undergraduate Program in Public Health. I did my graduate training in Applied Microbiology at London University. I am also the co-director and founder of the Simmons Center for Hygiene and Health in Home and Community Settings.
My research looks at broad issues associated with the transmission of microbial pathogens in the indoor environment. The applied research in my lab allows undergraduate students to get involved in developing and piloting research methodologies and also, to get published in peer review journals.
I serve as a member of the Scientific Advisory Board of the International Forum on Home Hygiene and on the editorial board of the American Journal of Infection Control. This allows me to disseminate information on matters of hygiene and infection control and I am frequently quoted in the press.
My passion is for microbiology and public health and I am committed to the rigorous preparation of Simmons students for graduate programs and careers in this field. I am also deeply committed to two other related issues. One is finding strategies to inform the general public on aspects of microbiology and infection control that impact our daily lives. The other is the issue of keeping women in the STEM disciplines and strengthening the STEM pipeline for women from school through to postdoctoral careers.
As a college student I was drawn to economics because it taught me how to think about, and address the many challenges that ail our world. I became an educator because I wanted to share this knowledge and inspire my students to make a positive difference in the world.
Dr. Doherty received her PhD in Government with a concentration in Public Policy from Northeastern University after receiving a BA in Government from Clark University. As an undergrad, she had many different roles as a student, not unlike many of our students here at Simmons: captain of the Women's Basketball team, member of the Admissions Staff, and explorer of the many academic opportunities that a liberal arts college has to offer.
Currently, Dr. Doherty serves as Associate Dean for Academic Affairs for the College of Arts and Sciences where she supports the Dean of CAS around faculty and student academic needs. These include areas such as general education, academic support, and majors/program curriculum. She is also the director of the graduate Program in Public Policy as well as the 3+1 accelerated program in Public Policy.
A proud member of the Department of Political Science and International Relations, Dr. Doherty's teaching is centered around the American Political System, with a concentration on gender and politics, popular culture and public policy. She was honored to be named Professor of the Year by students in 2008 and received the Dean's Award for Excellence in Undergraduate Advising in 2009.
She is also the co-host of the Sports Profs, a weekly sports talk radio show on Simmons College internet Radio. Each week, Dr. Doherty with Dr. Daren Graves from the Education department talk sports as it intersects with race, gender, and class. "Real talk, with an academic twist." When not at Simmons, Dr. Doherty enjoys watching and playing all sports with her two partners in crime, husband Mark and son, Donovan. She also enjoys music, reading, and really bad television.
Chaoqun Ni got her Bachelor's and Master's degree in E-Commerce and Information System from Wuhan University, and Doctoral Degree in Information Science from Indiana University in Bloomington.
Chaoqun Ni's research has appeared in a variety of computer science, informatics, library, and scientific publications, including Nature, Scientometrics, Journal of Association for Information Science and Technology, and Simmons SLIS' Library and Information Science Research. In addition to receiving a Dean's Fellowship from the Department of Information & Library Science at Indiana University Bloomington, Ni received the Association for Information Science and Technology's New Leader Award in 2011, and the Association for Library and Information Science Education Doctoral Student Award in 2014.
Chaoqun Ni's Curriculum Vitae
With a background in Economic Development and Political Economy and a joint appointment in Economics and Women's and Gender Studies, Professor Biewener's most current research interests address the political economy of food – a fabulous way of combining her love of gardening and food with her keen interest in community economies.
In 2013 she completed a long-term, collaborative research project with Marie-Hélène Bacqué (Professor of Urban Studies, Université de Paris Ouest Nanterre) that traced the genealogy of the term "empowerment" in the fields of gender and development, urban policy and social work. Publications from this research include "Feminism and the Politics of Empowerment in International Development" (forthcoming in Air and Space Power Journal–Africa and Francophonie); L'empowerment, une pratique emancipatrice (Editions La Découverte, 2013; Spanish translation forthcoming in 2015); and "Different manifestations of the concept of empowerment. The politics of urban renewal in the United States and Great Britain" (International Journal of Urban and Regional Research, August 2012).
Prior research and publications have addressed community development and social economy projects in the United States and Canada, debates at the intersection of poststructuralist feminism and postmodern Marxism, and the French Socialist government's financial and industrial policies in the 1980s.
Currently Professor Biewener is an Associate Editor for Signs, Journal of Women in Culture and Society and she has served on the Editorial Board of Rethinking Marxism (1990-2001). In 2009 she co-organized (with Randy Albelda,
UMass-Boston) the 18th Annual Conference of the International Association for Feminist Economics, "Engendering Economic Policy," which was hosted by Simmons College.
Professor Biewener oversees the Economics Department Internship program, and was Coordinator for the interdisciplinary minor in Social Justice from 2004-2013.
Patricia White has been teaching in the nursing programs at Simmons College since 1987. She is currently the Director of the Doctor of Nursing Practice Program and Co coordinated the Adult and Geriatric Nurse Practitioner Program for twenty five years. She received her B.S. and M.S. Degrees from Boston College and completed her PhD from University of Rhode Island. She recently completed the Clinical Ethics residency in Nursing at Massachusetts General Hospital in Boston. She has maintained a clinical practice since 1983 and currently practices in primary care and in an NP owned geriatric primary care practice. She currently teaches Ethics and capstone courses in the DNP program and teaches adult and geriatric content in the FNP Program. She also teaches research in the MS program and is a clinical instructor in community health.
In addition to her teaching and practice, she has been involved in several research projects and enjoys working with students and colleagues on research related to Nurse Practitioner practice, patient outcomes in NP practices, end of life care, best teaching practices for accelerated students and DNP pedagogy and evaluation of the impact DNP alumni have on practice change and improving patient outcomes.
Dr. White has also written and presented on the topics of clinical consultation, polypharmacy, ethics and bereavement and DNP program pedagogy and program evaluation. She recently served for four years on the Board of Directors of the Leavitts Mill free health center in Buxton, Maine which is an NP owned free health clinic providing primary care to uninsured in rural Maine. She is active on the legislative committee of the MCNP and is the current PAC treasurer and is involved with the NONPF and is currently the co chair of the Faculty Development committee. She is a Fellow in the National Academies of Practice and she has recently been selected and inducted as a Fellow in the American Association of Nurse Practitioners (FAANP) June, 2014 at the AANP national conference in Nashville, Tennessee.
Lena Zuckerwise is a political theorist specializing in contemporary political thought and democratic politics. Her first book, which will be published in 2015, brings the work of Hannah Arendt into the company of several key debates in democratic theory. Professor Zuckerwise's writings on the subjects of post-colonial feminism, the philosopher Martha Nussbaum, and the theory of foundationalism will appear later this year in the Encyclopedia of Political Thought. Other interests include gender and feminist theory, and the politics of race in the United States. Professor Zuckerwise teaches courses in historical political thought; contemporary feminist theory; the politics of human rights; and modern and contemporary theories of justice. Prior to coming to Simmons, Professor Zuckerwise taught at Wellesley College and Mount Holyoke College in the political science and gender studies departments, respectively. She completed her Ph.D. in 2010 at the University of Massachusetts, Amherst, where she was the recipient of the Distinguished Teaching Award. Professor Zuckerwise lives in the city of Boston with her partner, three-year-old son, and corgi, Olive, who has befriended many of her students. In her spare time, she enjoys hiking and cooking.
Rebecca Koeniger-Donohue is a Professor of Practice in the Family Nurse Practitioner (FNP) program and Doctor of Nursing Practice (DNP) programs at Simmons School of Nursing and Health Sciences, in Boston, MA. Dr. Koeniger-Donohue has published numerous peer-reviewed articles, book chapters, as well as a case study review book for board certification. She gives frequent professional presentations, regionally and nationally, on both health information technology tools and Women's Health in Primary Care. She serves on the editorial board of the Journal of Clinical Nursing; an international journal based in the United Kingdom and is a fellow in both the American Academy of Nurse Practitioners (FAANP) and the interdisciplinary National Academy of Practice. She is dedicated to the global advancement of nurse practitioners and the high-quality health care they deliver.
Dr. Koeniger-Donohue maintains an active clinical NP practice and is an investigator in several applied clinical research projects.


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Crazy Substitute Teachers
Enjoy, NG4.03 / 5.00 2,637 Views
Vacuum Salesman
This dude and his Megazord vacuum cleaner4.01 / 5.00 2,901 Views
Markiplier Animated | Mark Of The Survey Corps
This took far too long to make...3.96 / 5.00 7,517 Views
YAY!!! Awesomeness
I liked the part where the guy won LOL! XD
Yum Indeed
This made me very hungry for some .swf files. So I tore my old laptop apart and started munching down on the tiny components. They were pretty tasty, however I still prefer my pringles.
Keep it up
- Chris
First submission?
That was pretty good for a first contribution. Concept and story were lacking, but that don't matter none right now.
Good job!
nice job
Very nice combo of tweening and fbf, it was really really good, I liked it. You might need a more concrete concept, but that was great.
Also, your music continues to play before through if you replay it, so you should have it stop and start over when the movie is replayed. A replay button would be nice, looks better than going back to the beginning.
I love this song, it was in Smash Kingdom too, I have a feeling it's going to start being in lots more flashes.

subset: commoncrawl


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|Publication number
|Publication type
|Application number
|Publication date
|Aug 25, 1998
|Filing date
|Apr 3, 1996
|Priority date
|Nov 9, 1995
|Fee status
|Also published as
|Publication number
|Georg Wilhelm Bornkamm, Dirk Eick, Bettina Kempkes, Nicola Maria Jochner, Lothar J. Strobl
|Original Assignee
|Gsf-Forschungszentrum Fur Umwelt Und Gesundheit Gmbh
|Export Citation
|BiBTeX, EndNote, RefMan
|Non-Patent Citations (10), Referenced by (99), Classifications (9), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
1. Field of the Invention
The present invention relates to a process for the preparation of human monoclonal antibodies and to their use.
2. Description of the Related Art
Antibodies are widely used in the treatment and therapy of humans. Antibodies are used in immunoprophylaxis, in the treatment of acute infections, and in the treatment of immunosuppressed patients. At the present time human antibodies are not to be prepared in a sufficient amount. A process of the generic type is set forth e. g. in Casali et al., (1986). Science 234, 476-479.
For this reason animal antibodies are frequently employed. One disadvantage of animal antibodies is that they cause interfering immune responses and loose their activity. Human antibodies can be obtained from human serum. This process, however, shows three fundamental disadvantages: suitable human serum is available only in a restricted amount, all of the purified antibodies are polyclonal, the treatment is very expensive.
Epstein-Barr virus (EBV) is a lymphotropic herpes virus causing infectious mononucleosis. The infectious mononucleosis, also called Pfeiffer's disease, is a lymphoproliferative disease leading to hyperplasia and hypertrophy of lymphatic tissue combined with characteristic blood count alterations. EBV infects primary resting B cells giving rise to lymphoblastoid cell lines (LCLs), which are characterized by unlimited proliferation in vitro, a process synonymously called immortalization or transformation. EBV is also associated with several human disease states including Burkitt's lymphoma (BL), nasopharyngeal carcinoma, lymphomas in immunocompromised individuals, and Hodgkin's disease (cf. reviews in Miller, 1990; Klein, 1994; Masucci and Ernberg, 1994).
In LCLs only a subset of viral genes is expressed, the expressed viral genes coding for six nuclear proteins, three membrane proteins, and two small nonpolyadenylated nuclear RNAs. The minimal set of genes required for transformation is unknown to date. The EBNA2 gene is deleted in the transformation-defective virus produced by the Burkitt lymphoma cell line P3HR1. In natural isolates it appears in two allelic forms (EBNA2A and EBNA2B) which share about 50% homology (Zimber et al., 1986). EBNA2, together with EBNA-LP, is the first viral gene expressed after infection of primary B cells and is a transcriptional activator of latent viral as well as latent cellular genes (CD21, CD23, and c-fgr) (Calender et al., 1987; Wang et al., 1987; Cordier et al., 1990; Ghosh and Kieff, 1990; Fahraeus et al., 1990; Abbot et al., 1990;
Knutson, 1990; Woisetschlaeger et al., 1991; Zimber-Strobl et al., 1991; Sung et al., 1991; Jin and Speck, 1992; Ling et al., 1993; Laux et al., 1994a; Meitinger et al., 1995).
EBNA2 has been shown to exert its transactivating function at least in part by binding to an ubiquitously expressed cellular gene, RBP-Jk, which binds to the DNA in a sequence specific manner (Zimber-Strobl et al., 1994; Henkel et al., 1994; Grossmann et al., 1994; Waltzer et al., 1994), and through interaction with transcription factors of the ets gene family (SPi-1, PU-1) (Laux et al., 1994b; and Johannsen et al., 1995). Even though the transformation of primary B cells in vitro is strictly dependent on EBNA2 (Cohen et al., 1989; Hammerschmidt and Sugden, 1989; Kempkes et al., 1995) EBNA2 appears not to be expressed in BL tumors (Rowe et al., 1986), Hodgkin's disease (Kanavaros et al., 1993), and nasopharyngeal carcinomas (Fahraeus et al., 1988; Young et al., 1988) thus questioning its role in the development of these disease states in vivo.
The aim of the present invention is to develop a process of the aforementioned type in a way that monoclonal human antibodies can be prepared in a sufficient amount and with high specificity.
This aim is achieved by means of the characteristic features of claims 1 and 12. The dependent claims describe advantageous embodiments of the present process. The antibodies prepared by the process of the present invention are employed preferably as therapeutic agents in a pharmaceutical preparation.
The present process makes it possible for the first time to produce human antibodies in cell culture in such amounts that allow for purification of the antibodies and for their medical utilization.
In the following the present invention is illustrated in more detail by preferred embodiments; however, the present invention is not meant to be limited to those. Further embodiments can be achieved by one skilled in the art without being inventive.
Thus, according to the present invention antibody-producing B cells are first infected with an Epstein-Barr virus or a naturally occurring as well as a genetically engineered derivative thereof. In a further embodiment of the present invention a vector is used which has at least those sequences of the Epstein-Barr virus necessary for immortalization of human B cells. Preferably primary human B cells are infected.
FIG. 1 shows a flow cytometeric analysis of estrogen-treated (white histograms) and estrogen-deprived (black histograms) lymphoblastoid cell lines expressing an EBNA2-estrogen binding domain fusion protein after staining for CD21, CD23, and surface IgM.
FIG. 2 shows the expression of CD21 and surface IgM in the presence (white histograms) and absence (black histograms) of EBNA2 activity in two Burkitt's lymphoma cell lines.
FIG. 3 shows the suppression of expression of the Ig μ heavy chain by EBNA2 in B lymphoma cells and in lymphoblastoid cells.
FIG. 4 demonstrates the co-regulation of Ig μ and c-myc expression in Burkitt's lymphoma cells expressing an EBNA2-estrogen binding domain fusion protein.
FIG. 5A shows a mini-EBV plasmid according to the present invention.
FIG. 5B shows a map of EBV strain B95.8.
FIG. 6 shows a restriction map of an ER/EBNA2 mini-EBV plasmid according to the present invention.
For a better understanding of the role of EBNA2 in the process of B cell transformation a conditional system has been developed wherein the function of the EBNA2 protein can be reversibly switched on and off. Using the conditional system developed by Picard et al. (1988) and Eilers et al. (1989) the hormone binding domain of the estrogen receptor has been fused to the N- or C-terminal of EBNA2 thus rendering the function of the EBNA2 protein dependent on the presence or absence of estrogen. As expected the chimeric EBNA2 proteins exhibit their transactivating function on viral and cellular genes only in the presence of estrogen.
According to the present invention it has been surprisingly found that the surface IgM expression and the transcription of the Ig μ locus is very efficiently decreased by the EBNA2 protein. EBNA2 thus functions as a negative regulator of the Ig μ transcription.
In the following the present invention is illustrated in more detail by the following exemplary embodiments using the Figures, wherein FIGS. 1 and 2 show by way of example the increase in immunoglobulin protein production resulting from switching off of EBNA2. The FIGS. 3 and 4 show the corresponding production of immunoglobulin RNA.
The present invention is based on the surprising finding that B cells infected by Epstein-Barr virus produce only minor amounts of immunoglobulin whereas the production is increased 100-fold following inactivation of EBNA2 function.
The present invention is described in more detail in the exemplary embodiments with respect to an IgM-producing B cell line. The present invention, however, shall not be limited to such an IgM-producing B cell line but according to the invention B cells expressing different immunoglobulin classes, e. g. IgG and IgA, can be immortalized.
The source of the B cells used is generally a human having a high titer of antibodies for the desired antigen. Cells are preferably obtained from the peripheral blood, and B cells are enriched according to standard methods, and infected with a virus derived from Epstein-Barr virus expressing a conditional EBNA2. During infection and proliferation of the B cells the EBNA2 function has to be in an activated state. The virus described by Kempkes et al. (1995), EMBO J. 14, 88-96, is particularly preferred for infection (cf. FIG. 5 and the subsequent description). For activation of the EBNA2 function it is necessary to add estrogen to the culture medium to ensure constitutive EBNA2 function. The immortalized B cells are expanded and diluted several times with medium containing estrogen until the desired number of cells is obtained. The cells are plated in a low cell density in 96-well flat bottom plates and again expanded. The cellular supernatant is tested for the desired specific antibodies. B cells producing the highly specific antibodies are used as the basis for the preparation of antibodies.
In these B cell clones the EBNA2 activity is switched off by washing out the estrogen. Other methods for estrogen removal can be also applied. Alternatively, the activity of the EBNA2 gene may also be controlled by using the hormone binding domain of the androgen receptor or another hormone binding domain, or on a transcriptional level by using inducible promoters for the EBNA2 gene. By switching off the EBNA2 function the immunoglobulin production within the B cells is increased steadily and after 2 days has been increased up to about 100-fold. 4 days, death of a large proportion of the cells occurs.
Another embodiment of the invention employs cell lines producing monoclonal antibodies immortalized with EBV or EBV-derived vectors, which are known in the art. Prior to the present invention, such cell lines produced antibodies only in amounts of less than 1 μg/ml. However, one embodiment of the present invention is a method for obtaining a dramatic increase in antibody production from such cells.
In this embodiment, human antibody-producing B cells are infected or transfected with an Epstein-Barr virus or a derivative thereof in a first culture medium. Said virus or derivative expresses at least a functional Epstein-Barr virus nuclear antigen 2 (EBNA2) protein. Expressing of at least said EBNA2 protein will produce immortalized B cells.
Said immortalized B cells are screened for specificity for a desired antigen to identify an immortalized B cell clone producing monoclonal antibodies specific for the desired antigen. Said identified B cell clone is propagated,in said first culture medium to obtain a desired amount of B cells.
A vector is introduced into the B cells which is capable of expressing a genetically altered dominantly negative form of an EBNA2 protein. The genetically altered form of said EBNA2 protein can inhibit activity of the wild type EBNA2 protein. There are several possibilities for introducing said genetically altered dominantly negative form of said EBNA2 protein. Either transfection or infection can be used. A vector capable of expressing said genetically altered dominantly negative EBNA2 protein can be transfected or infected into the human antibody-producing B cells, before or after infection with an Epstein-Barr virus or a derivative thereof, or into said propagated B cell clone producing monoclonal antibodies.
The wild type form of EBNA2 consists of 487 amino acids. A suitable genetically altered dominantly negative form of the EBNA2 protein is truncated at the carboxy terminus, and, in one embodiment, the deletion comprises amino acids 150-487. This truncated EBNA2 protein forms functionally inactive heterodimers with the functional EBNA2 protein thereby inhibiting the functional EBNA2 protein.
In a first variation of this embodiment, the nucleotide sequence encoding said genetically altered EBNA2 protein is operatively linked with a nucleotide sequence that provides inducible expression of said nucleotide sequence encoding said genetically altered EBNA2.
In a second variation of this embodiment, the nucleotide sequence encoding the genetically altered EBNA2 is fused, in functional connection, with a nucleotide sequence including a hormone-binding domain to produce a fusion gene encoding a fusion protein comprising said genetically altered EBNA2 fused to said hormone-binding domain. The genetically altered EBNA2 can be fused at its N- or C-terminus to said hormone-binding domain. This fusion protein inhibits functional EBNA2, as described above, when a hormone specific for the hormone-binding domain binds to the fusion protein, i.e., hormone binding activates the inhibitory function of the genetically altered dominantly negative EBNA2 protein. In preferred embodiments of the invention, said hormone-binding domain binds estrogen or an androgen.
When the hormone-binding domain is an estrogen-binding domain, for example, the identified B cell clone is propagated in the absence of estrogen, and a few days before harvesting monoclonal antibodies, the cells are treated with estrogen. By this estrogen treatment, said genetically altered EBNA2 protein is activated and thereby the endogenous functional EBNA2 protein present in the cells is inhibited. This process effects the production of monoclonal antibodies in considerably larger quantities than any process known in the art.
In the following the construction of a plasmid is described that is useful for the expression of conditional EBNA2 function. The plasmid includes the EBNA2 gene or a derivative thereof. The present invention shall, however, not be limited to the plasmid illustrated herein. According to the invention other vectors can be used by which EBNA2 function can be conditionally expressed, i.e., its function is at least partly switched on upon immortalization of the B cells and is at least partly switched off prior to recovery of the antibodies. Preferably EBNA2 function is substantially switched on for immortalization and is substantially switched off for antibody recovery.
The hormone binding domain of the estrogen receptor was fused to either the N- or the C-terminal end of the open reading frame (ORF) of EBNA2 resulting in plasmids ER/EBNA2 and EBNA2/ER. Both plasmids had the overall structure of the mini-EBV p554 expressing wild-type EBNA2, EBNA-LP, and the open reading frame BHRF1 (Hammerschmidt and Sugden, 1989). The N-terminal fusion construct was generated by introducing an EcoRI restriction site immediately 5' to the ORF of EBNA2 by in vitro mutagenesis. The gene fragment encoding the hormone binding domain was generated by the polymerase chain reaction (PCR) using HE14 (Kumar et al., 1986) as a template flanked by EcoRI recognition sites and ligated into the EcoRI site preceding the EBNA2 ORF. This PCR fragment included the Kozak's sequence provided by HE14 but excluded the stop codon of the estrogen receptor. A restriction map of a preferred ER/EBNA2 mini-EBV plasmid is shown in FIG. 6. The hormone binding domain starts at position 13565 (D in FIG. 6) and includes the complete receptor domain (DEF). The C-terminal fusion construct was generated by inserting BglII and EcoRI recognition sites immediately before the stop codon of EBNA2 by in vitro mutagenesis. A BamHI-EcoRI fragment of HE14 coding for the hormone binding site of the estrogen receptor was ligated into the BglII-EcoRI sites at the end of EBNA2. The subsequent cloning steps regenerated EBV strain B95.8 with the coordinates 13944-54364 (cf. Baer et al., 1984) with two alterations: the NotI repeats were deleted, and only two BamHI W repeats were inserted.
FIG. 5 illustrates the strategy for the expression of conditional EBNA2 mutants in growth-transformed B cells.
(A) FIG. 5 shows mini-EBV plasmids composed of cis-acting elements essential for the propagation of these plasmids in all phases of the Epstein-Barr virus life cycle. These mini-EBV plasmids carry the plasmid origin of replication (orip) required for the maintenance of the EBV plasmids in the target cells, the lytic origin of replication (oriLyt) which allows amplification to high copy number upon induction of the lytic replication of the virus, and the terminal repeats (TR) permitting packaging of the mini-EBV plasmids into virions. Mini-EBV plasmids are employed with a helper virus, such as P3HR1, that provides for amplification and packaging of the plasmids.
The hormone binding domain of the estrogen receptor (white boxes) was fused to either the C- or the N-terminus of the wild-type EBNA2 open reading frame (black boxes) leading to the EBNA2/ER and the ER/EBNA2 mutant respectively. The EBNA2 expression cassettes are flanked by BamHI restriction fragments C, W, Y, and H of the EBV genome representing in part the genomic organization of the DNA of EBV, and including the deletion (ΔP3HR1, open bar in B) also present in the non-transforming EBV strain P3HR1. EBNA2 as well as EBNA2 mutants can be transcribed from two alternative promotors localized in the C or W fragments.
The FIG. 5 (B) represents the map of EBV strain B95.8, showing the EBNAs and LMPs (latent membrane proteins) transcribed in growth-transformed B cells, and the localization of the promotors thereof with respect to the BamHI restriction map of this strain.
The estrogen dependent cell lines ER/EB2-3 and ER/EB2-5 are lymphoblastoid cell lines (LCLs) established by coinfection of primary B cells by P3HR1 virus and a mini-EBV plasmid expressing ER/EBNA2 and complementing the EBNA2 defect of P3HR1 virus in trans (Kempkes et al., 1995). Briefly, P3HR1 cells, which carry the P3HR1 virus, were transfected with mini-EBV plasmids and simultaneously induced for lytic cycle replication. Supernatants of the transfected cells containing mini-EBVs together with the P3HR1 helper virus were harvested and used to coinfect primary B cells.
The estrogen independent EB2-2 cell line was generated simultaneously using wild type EBNA2 to complement the EBNA2 defect of the P3HR1 virus. BL41 is an EBV-negative BL cell line bearing a t(8;14) translocation (Lenoir et al., 1985), BL41/P3HR1 and BL41/B95-8 are BL41 cells stably infected (converted) by P3HR1 or B95-8 virus respectively (Calender et al., 1987). BJAB is an EBV-negative B lymphoma cell line (Klein et al., 1974); BJAB/P3 is a BJAB cell line stably infected by P3HR1 virus. P3HR1 is a single cell clone of the EBV-positive B cell line Jijoye containing a t(8;14) translocation (Hinuma et al., 1967). Jijoye produces an EBNA2 competent virus, P3HR1 cells an EBNA2 defective virus (Bornkamm et al., 1982). The lymphoma cell lines expressing ER/EBNA2 are described by Kempkes et al. All cell lines were cultured in RPMI1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and streptomycin, and 1 mM pyruvate. β-estradiol (Merck, Darmstadt) was added to the cell culture medium at a final concentration of 1 μM.
For FACS analysis 1�106 cells were incubated with an excess of unlabeled monoclonal mouse antibodies directed against CD21 (BL13) and surface IgM (AF6) (Dianova, Hamburg). FITC (fluorescein) conjugated goat anti mouse F(ab)2 fragments (DAKO, Hamburg) were used as secondary antibody to stain positive cells. Dead cells were identified, and after propidium iodide staining (0.1 μg/ml) excluded from analysis. The samples were analyzed using a FACSSCAN device (Becton and Dickinson). 3-H thymidine incorporation assay was performed as described in Kempkes et al., 1995.
Northern blot analysis was performed as described in Eick and Bornkamm, 1989. The 1.2 kb EcoRI/EcoRI fragment and the constant region 0.9 kb EcoRI/SacI fragment described in Eick et al. (1985) were used as Ig μ gene probes.
The lymphoblastoidal cells transformed with the conditional EBNA construct described above were tested for expression of surface markers prior to and after removal of estrogen.
The FIG. 1 shows a flow cytometric analysis of estrogen treated (white histograms) and estrogen deprived (black histograms) ER/EB2-5 cells after staining for CD21, CD23, and surface IgM.
The FIG. 1 particularly shows the amount of IgM antibodies produced by an EBV immortalized B cell in the presence (white area) and in the absence (black area) of EBNA2 activity after elapse of two days. Comparable results were obtained for two other cell lines following immortalization of primary B cells with the above mentioned virus.
The FIG. 1 demonstrates that the expression of CD23 and to a lesser extent of CD21 was decreased by switching off the function of EBNA2 by removal of estrogen. At the same time, however, the expression of IgM was strongly increased. Increase of IgM expression and decrease of CD21 and CD23 expression were reversible since the expression of CD21 as well as CD23 increased, and the expression of IgM decreased upon following readdition of estrogen. Those results are not detailed herein.
Decrease of surface IgM may either be mediated by EBNA2 itself or by other viral targets located downstream (e. g. the viral latent membrane proteins) induced by EBNA2. To discriminate between these possibilities, the mini-EBV plasmid expressing the ER/EBNA2 fusion protein was stably introduced into the EBV-negative B cell lymphoma lines BL 41 and BJAB, and for comparison also into P3HR1 cells. In the stably transfected cell lines the activation of the TP1 or the LMP1 promotor was strictly dependent on the presence of estrogen indicating that the ER/EBNA2 protein is indeed functional in these cells. FACS analysis prior and after addition of estrogen revealed a strong decrease of expression of sIgM (surface IgM) after addition of estrogen (FIG. 2).
This leads to the conclusion that the decrease in expression of sIgM is a function of EBNA2 and not a function of any other viral target gene.
Northern blot analysis was carried out to detect the level at which IgM expression is downregulated by EBNA2. By including RNA of paired cell lines differing in the presence or absence of the wild-type EBNA2 gene the Northern blot analysis further provided an answer to the question whether the decrease in IgM is caused not only by the EBNA2-estrogen receptor fusion protein but also by wild-type EBNA2. The paired cell lines included P3HR1 producing an EBNA2 defective virus, and its parental cell line Jijoye having a functional EBNA2 gene (EBNA2B allele) and BL41 cells stably infected/converted by P3HR1 virus (BL41/P3HR1) or the transformation competent EBNA2 wild-type positive virus B95-8 having the EBNA2A allele. As becomes apparent from FIG. 3, EBNA2 downregulates the surface expression of IgM by decrease of the Ig μ RNA.
The FIG. 2 shows the amount of IgM antibodies produced in the presence (white area) and absence (black area) of EBNA2 activity in two Burkitt lymphoma cell lines after 2 days.
The FIG. 2 particularly demonstrates the suppression of cell surface IgM by EBNA2 in B lymphoma cell lines. Cells transfected with the mini-EBV plasmid expressing ER/EBNA2 (BJAB/K3, BL41/K3, and P3HR1/3B6) were cultured for two days in the presence (white histograms) or in the absence (black histograms) of 1 μM β-estradiol and then were analyzed for cell surface CD21 and expression of surface IgM by FACS analysis. 1�106 cells were incubated with excess of unlabeled mouse monoclonal antibodies recognizing human CD21 (BL13) or IgM (AF6). FITC conjugated goat anti mouse F(ab)2 fragments were used as secondary antibody to stain positive cells. Control cells were marked exclusively with secondary antibodies but showed no alterations following addition of estrogen (data not shown). Dead cells were identified, and after propidum iodide staining (0.1 μg/ml) excluded from analysis.
The FIGS. 3 and 4 represent correspondingly the amount of the equivalent immunoglobin RNAs.
The FIG. 3 particularly shows the suppression of the expression of the Ig μ heavy chain by EBNA2 in B lymphoma cells and in LCLs. Total cellular RNA was isolated from Jijoye, P3HR1, BL41, BL41/ P3HR1, BL41/ B95-8, and ER/EBNA2 transfected cells cultured for two days in the presence or in the absence of estrogen (A), or from LCL cell lines expressing the ER/EBNA2 protein (ER/EB2-3 and ER/EB2-5) or the wild-type EBNA2 protein (EB2-2), the cell lines having been treated with estrogen or deprived of estrogen for four days (B), isolated, and the cells were analyzed with respect to Ig μ heavy chain, Ig k light chain and GAPDH RNA by Northern blot analysis.
The FIG. 4 demonstrates the co-regulation of Ig μ and c-myc gene expression in ER/EBNA2 transfected BL cells. P3HR1/3B6 cells (A) were treated with estrogen (+) for the period indicated and tested for expression of Ig μ and c-myc RNA by Northern blot analysis. The ethidium bromide stained gels are shown at the bottom of the Figure. In estrogen dependent LCLs the Ig μ and c-myc expression are regulated oppositely to each other by estrogen (B).
By using the process of the present invention it is possible for the first time to provide human B cell derived antibodies in large amounts. For this purpose B cells are used which have been immortalized by EBV or a derivative of EBV and containing the EBNA2 gene in a conditional form. For immortalization of the B cells the EBNA2 gene is switched on, and for production of antibodies the EBNA2 gene is switched off.
The supernatants of cell cultures are harvested preferably between days 2 and 5, and the antibodies are purified. The latter are then available for therapeutic purposes. Processes for recovery of the antibodies are familiar from the state of the art, and can be readily applied to the present process by one skilled in the art.
The antibodies are preferably employed in those cases where animal antibodies or manipulated animal antibodies may lead to undesirable side effects in humans.
All publications referred to herein are hereby expressly incorporated by reference in their entirety as indicative of the level of skill in the art.
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|The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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|Musc Foundation For Research Development
|Human monoclonal antibodies and methods for producing the same
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|Biogen Idec MA Inc.
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|Jan 13, 2005
|Biogen Idec Ma Inc.
|Aglycosyl anti-cd154 (cd40 ligand) antibodies and uses thereof
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|Oct 5, 2006
|The General Hospital Corporation
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|Sep 18, 2008
|Dana-Farber Cancer Institute
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|Nov 6, 2008
|Zymogenetics, Inc.
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|Irm Llc
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|Regents Of The University Of Minnesota
|Bispecific targeting reagents
|Jun 27, 2012
|Jan 3, 2013
|Method for screening cells
|U.S. Classification
|435/70.21, 435/69.1, 435/325, 530/350, 435/320.1
|International Classification
|Cooperative Classification
|European Classification
|Jun 24, 1996
Owner name: GSF-FORSCHUNGSZENTRUM FUR UMWELT UND GESUNDHEIT GM
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Cloud AppsRodrigo Leon on February 22nd 2013
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chocolate mug cake (in 1 minute) ~ http://iambaker.net
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Popular Sons of Anarchy Quotes
Ashby: I couldn't do anything to help save the son, but I can do something to save the grandson.
Jax: Save him from what?
Ashby: The life of his father.
Hale: This is blackmail.
Salazar: Yeah. I got an app for that homes.
Truth is, I feel closer to SAMCRO then I do most folks in this house.Unser
Not the hair, man. Not the hair.Tig
Tara: How are you feeling?
Bobby: How do I look?
Tara: Like you've been shot.
Gemma: There's not much more this bitch can do to me.
Clay: Don't underestimate her. She's been demoted, she's desperate.
Stahl: Hey I'm in the room kids.
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Jax: I can't do what I need to do with [Clay] in place. I ride independently for a few years, then wait for him to step down.
Tara: A few years? We barely made it through the last few months.
That freaky little holy man liked to live close to the flame and God can only protect us so much.Venus Van Dam
Unser: Let's do this quietly.
Gemma: Not exactly my style, sweetheart.
If I stay in truth and I give it to people straight. No lies, no spin, no games. I always know that whatever happens is going to be the right thing, whether I like it or not.Nero
I'm not my father. I'm not weak.Jax
Donny: Who do you think has to clean that up?
Jimmy: You.


text:
We enjoyed an extremely relaxing 6 day break at Ponta dos Ganchos, our final destination in a wonderful round the world trip. It's a perfect place to unwind and relax. The location is beautiful with stunning views of the bay. The service was excellent throughout, with particular praise for the wonderful Nina, the guest relations manager, who helped us deal with the trauma of having had our luggage broken into en route to the hotel (including translating for us at the local police station). Our villa was superbly appointed. And we finished our stay with a wonderfully romantic dinner on the little island across from the hotel. We can't recommend this hotel highly enough.
- Official Description (provided by the hotel):
- Located on a private peninsula, surrounded by a fisherman village on Brazil's south coast, it is the country's most exclusive beach resort. Within a short distance from Rio de Janeiro, Sao Paulo or Buenos Aires, this romantic getaway delivers the dream: a ridge of Green Mountains runs down to the clear water of the Emerald Coast, where three little islands meet a stunning white sandy bay. Contemporary interiors in 25 beautiful bungalows mix comfort and refinement on a rustic style and the service that is second to none. The Restaurant lies within meters from the beach, provides a great wine list (with over 220 options); for lunch and dinner a diversified and sophisticated menu is on offer at any hour - the doors only shuts when the last guest leaves. If you are looking for something more romantic, cross a small bridge and get to one of the islands where you will find a gazebo that's specially decorated at night with flowers and candles for only one couple per night have dinner. There's an indoor pool, tennis, games room, snooker room, fitness centre, restaurant, bars, business centre, and even a private cinema. Massages, ecological trails, snorkeling, scuba diving, and boat trips are also on offer. Due to local landscape and in order to maintain calm and tranquility, we do not accept children under 18 years old. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Hotels.com, Orbitz, Hotwire, Priceline, Despegar.com, Odigeo, Agoda, Expedia and Travelocity so you can book your Ponta dos Ganchos Exclusive Resort reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Ponta Dos Ganchos Hotel Governador Celso Ramos
- Ponta Dos Ganchos Exclusive Resort Brazil/Governador Celso Ramos, Santa Catarina


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MacBook Pro 17" from 2007, is it worth?
Started May 29, 2011 | Discussions thread
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SwitttchBlade • New Member • Posts: 5
MacBook Pro 17" from 2007, is it worth?
May 29, 2011
I have a dead fan on my current working laptop, since i'm looking for replacement I found this :' MacBook Pro 2,1 Intel Core 2 Duo 2.33 GHz, RAM 3 GB, 500 GB HDD, VRAM 256 Mb ATI Radeon X1600 1680 x 1050.' for around 800$... Would this 5 years old Mac be capable of running todays Lightroom and Photoshop aplications for photo editing, or should I stay with some newer laptop pc's?
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Tuesday, 26 July 2016
Last updated 2 min ago
Mar 5 2007 | 1:46pm ET
Thales Fund Management, a New York-based registered investment advisor with $2 billion in assets under management, has launched the Leros Fund – a quantitatively managed 130/30 strategy.
The Leros Fund is modeled to maintain 100% net exposure to large & mid-cap U.S. equities with an optimized market-neutral long-short overlay, according to the firm. This overlay can vary between 130/30 and 160/60 depending on the market environment.
"Our alternative variable overlay approach takes the 130/30 investment strategy one step further, allowing us to better adapt to constantly changing stock market conditions," said
Marek Fludzinski, Thales founder and portfolio manager for the Leros Fund.
"With our considerable expertise in quantitative modeling, short selling and controlling portfolio volatility, we are confident that this fund will stand out from the crowd of new entrants into this strategy."
Jul 8 2016 | 7:01pm ET
Recent market volatility has investors seeking greater insight into alternative...
Jul 25 2016 | 6:29pm ET
If you are compelled by stories of entrepreneurial vision & drive, or simply...
Jul 22 2016 | 8:44pm ET
When the press reports extraordinarily strong relative or risk-adjusted returns...


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Apple Said to Build Big-Screen iPhones Next Month
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46:10 - Chetan Ahya, co-head of global economics and chief Asia economist at Morgan Stanley, discusses the Bank of Japan policy meeting, Japan's trade data and what we can expect from the Bank of England. He speaks to Bloomberg's Rishaad Salamat on "Trending Business." (Source: Bloomberg)


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...I am patient and kind. I care about the student's academic growth as well as their personal growth. I enjoy close interaction with my students and respect their individuality.I was born in Beijing China, I have taught many Chinese students and taught in after school programs in the U.S.
Cupertino, CAView Tutor
...I help students with basic sentences, paragraphs, essays, and other documents, though I don't teach tech writing itself. I can help you improve your writing structure as well as your method of beginning and achieving the desired results on your writing projects. English refers to help for nativ...
Sunnyvale, CAView Tutor
...I graduated from the University of Tokyo and have an M.S. from a U.S. university. I have tutored elementary to high school students and business professionals. I am familiar with AP requirements and Japanese language proficiency tests.
Cupertino, CAView Tutor
...I have a California Adult Ed Credential. I went to school at UC Berkeley and have an MBA as well. I love working with people from all over the world to help improve their communication and productivity.
Mountain View, CAView Tutor
...I've spent my life writing. I am a published poet and have just completed a memoir of my five years in Japan. I have written short stories and many, many essays.
San Jose, CAView Tutor


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When doing family history research, a vital part of the process is evaluating the results of your inquiry and sharing your information with others.
I ask myself the question, "What do I see?" Sometimes what I find is only a clue; other times, it's a gold mine. I record what I learn in my research log. At this point, based on the information I've gathered, I decide where I want to go and start with step one again.
FamilySearch.org encourages as you evaluate your information, consider the following questions:
- Did I find the information I was looking for?
- Is the information complete?
- Does the information conflict with other information I have?
- Is the source of the information credible?
Next, organize your records for easy access.
FamilySearch.org points out that there are "a number of computer programs that can help you organize your records on your home computer." If you are just starting, consider the following tips provided on the website:
- Keep pedigree charts numbered and arranged numerically.
- Keep family group records in alphabetical order by the husband's name.
- Keep notes, research logs and copies of documents behind the related family group record.
Editor's note: The original version of this story posted on April 27, 2013, failed to properly attribute all source materials, which violates our editorial policies. The story was revised on March 19, 2014, and attribution to original sources were added. A version of this column also appeared in the print edition of the Deseret News on August 8, 2013. The Deseret News demands accuracy in attribution and sourcing and considers any lapses to be a serious breach of ethics. The Deseret News is no longer publishing Barry J. Ewell's writings.
Barry J. Ewell is author of Family Treasures: 15 Lessons, Tips, and Tricks for Discovering your Family History and founder of MyGenShare.com, an online educational website for genealogy and family history.
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