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leukaemia IH. Springer, Berlin Heidelberg New York, pp 223-229 - Dexter TM, Allen TD, Lajtha LG (1977a) Conditions controlling the proliferation of hemopoietic stern cells in vitro. J Cell Physiol 91 :335-344 - Dexter TM, Scott D, Teich NM (1977b) Infection of bone marrow cell proliferation, differentiation and leukaemogenic capacity. Cell 12:355-364 - Dexter TM, Allen TD, Lajtha LG, Krizsa F, Testa NG, Moore MAS (1978). In vitro analysis of self-renewal and committment of hemopoietic stern cells. In: Clarkson B, Marks PA, Till JE (eds) Differentiation of normal and neoplastic hemopoietic cells. Cold Spring Harbor Press, New York, pp 63-80 - Dexter TM, Allen TD, Scott D, Teich NM (1979) Isolation and characterisation of abipotential hemopoietic cell line. Nature 277 :471-474 - Dexter TM, Spooncer E, Toksoz D, Lajtha LG (to be published a) The role of cells and their products in the regulation of in vitro stern cell proliferation and granulocyte development. J Supramol Struct - Dexter TM, Allen TD, Teich NM (to be published b) Production of 'normal' stern cell lines following treatment of long-term marrow cultures with Friend murine leukaemia viruses. - Dexter TM, Garland J, Scott D, Scolnick E, Metcalf D (1980c) Growth of factor dependent hemopoietic precursor celllines. J Exp Med 152:1036-1047 - Fibach E, Landau T, Sachs L (1972) Normal differentiation of myeloid leukaemic cells induced by a differentiation inducing protein. Nature 237: 276-279 - Greenberger JS, Gans PJ, Davisson PB, Maloney WC (1979) In vitro induction of continuous acute promyelocyte leukaemia cell lines by Friend or Abelson murine leukaemia viruses. Blood 53 :987 - Ichikawa Y (1969) Differentiation of a cell line of myeloid leukaemia. J Cell Physiol 74:223-234 - Lord BI, Mori KJ, WrightEG, LajthaLG (1976) An inhibitor of stern cell proliferation in normal bone marrow. Br J Haematol, 34:441-445 - Lord BI, Mori KJ, Wright EG, Lajtha LG (1977) A stimulator of stern cell proliferation in regenerating bone marrow. Biomed Express (Paris) 27:223-226 - Metcalf D (1977) Hemaopoietic colonies. Springer, Berlin Heidelberg New York, p 277 - Metcalf D, Moore MAS, Warner NL (1969) Colony formation in vitro by myelomonocytic leukaemic cells. J Natl Cancer Inst 43:983-1001 - Teich NM, Dexter TM (1978) Effects of murine leukaemia virus infection on differentiation of hemapoietic cells in vitro. In: Clarkson B, Marks PA, Till JE (eds) Differentiation of normal and neoplastic hemapoietic cells. Cold Spring Harbor Press, New York, pp 657-670 - Teich NM, Dexter TM (1979) Interaction between murine leukaemia viruses and differentiating hemopoietic cells. In: Oncogenic viruses and host cell genes. Academic Press, New York, pp 263-276 - Teich N, Boss M, Dexter TM (1979) Infection of mouse bone marrow cells with Abelson murine leukaemia virus and establishment of producer celliines. In: Neth R, Callo RC, Hofschneider PH, Mannweiler K (eds) <strong>Modern</strong> trends in human leukaemia IH. Springer, Berlin Heidelberg New York, pp 487-490 - Testa NG, Dexter TM, Scott D, Teich NM (1980) Malignant myelomonocytic cells after in vitro infection of marrow cells with Friend leukaemia virus. Br J Cancer 41: 33-39 275
Haematology and Blood Transfusion Vol. 26 <strong>Modern</strong> Trends in Human Leukemia IV Edited by Neth, Gallo, Graf, Mannweiler, Winkler © Springer-Verlag Berlin Heidelberg 1981 Long-Term Culture of Normal and Leukemic Human Bone Marrow S. Gartner and H. S. Kaplan A. Introduction Clonal culture systems using semisolid media for the detection of granulocyte-macrophage (Bradley and Metcalf 1966; Pike and Robinson 1970; Pluznik and Sachs 1966), erythroid (Stephenson et al. 1971), lymphoid (Fibach et al. 1976; Metcalf et al. 1975a; Sredni et al. 1976) and megakaryocytic (Metcalf et al. 1975b) progenitors have contributed greatly to investigations of hematopoiesis. Unfortunately, such systems possess two major limitations: (1) observations are restricted to relatively short time intervals and (2) the interaction between different kinds of cells cannot easily be studied. More recently, liquid culture systems have also been investigated. Golde and Cline (1973) described the short-term liquid culture of human marrow using an in vitro diffusion chamber in which cells grew both in suspension and on a dialysis membrane. Proliferation and maturation of granulocytes and macrophages in these chambers persisted for 4 weeks. Dexter et al. (1973) reported the development of a liquid system for the cocultivation of mouse thymus and bone marrow in which CFUcs (granulocyte-macrophage progenitor cells) were genera ted for at least 10 weeks and CFUss (pluripotent stern cells) were present for 14 days. The same group (Dexter and Lajtha 1974; Dexter and Testa 1976; Dexter et al. 1977) later developed a method for the long-term culture of mouse bone marrow cells alone in liquid medium. Such cultures produce both CFUcs and CFUss for several months. Hematopoiesis in this system is dependent upon the presence of a marrow-derived adherent population consisting of three cell types: phagocytic mononuc1ear cells, endothelial cells, and giant lipid-laden adipocytes. Initially, only certain lots of horse serum had the ability to stimulate the growth of these essential adipocytes. Greenberger (1978) reported that "deficient" lots of horse serum could be reconstituted with corticosteroids. Long-term liquid culture systems for the cultivation of human bone marrow cells are urgently needed. Moore and Sheridan (1979) reported the establishment of human marrow cultures using conditions similar to those described by Dexter et al., but CFUc pro duction was limited to 6-8 weeks. More recently, Moore et al. (1979) reported sustained longterm hematopoiesis in liquid cultures of marrow from a subhuman primate, the tree shrew (Tupaia glis). We now present additional information concerning a recently described method (Gartner and Kaplan 1980) for the long-term culture of human marrow cell populations based on modifications of the murine system. B. Materials and Methods J. Media Fischer's complete growth medium consisted of Fischer's medium (Gibco *-320-1735) supplemented with 10- 7 M hydrocortisone sodium succinate (Upjohn) and either 25 % horse serum (HoS; Flow Labs), 25% fetal bovine serum (FBS; Microbiological Associates), or 12.5% each of HoS and FBS. McCoy's complete growth medium consisted of the following: McCoy's 5a medium, modified, (Gibco #430-1500), 10- 7 M hydrocortisone, 1 % sodium bicarbonate solution (Gibco #670-5080), 1 % MEM sodium pyruvate solution (Gibco 320- 1360), 1 % MEM vitamin solution (Gibco #320- 1120), 0.8% MEM amino acids solution (Gibco 276
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Haematology and Blood Transfusion 2
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Dr. Rolf Neth, Universitäts-Kinder
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Participants of the Meeting Aaronso
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Kurth, R, Friedrich-Miescher-Labora
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Wilsede Scholarship Holders (Grante
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Preface Gut ist eine Lehrart, wo ma
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Personal and scientific discussion
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Acknowledgement We should like to t
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Wilsede, June 21 1978 Memorial Trib
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limiting benign lymphoproliferative
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this most unlikely, however (for re
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longer subject to the same control
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Bregula U, Wiener F, Harris H (1971
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fresh lymph node biopsies from ten
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Fig. 3. Binucleate mitosis in an ob
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y Fig. 4. Schematic diagram of post
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than that among patients with Stage
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Tests of binding affinity, agglutin
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N Engl J Med 297: 963-968 - Green I
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Clinical Aspects
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"ring chromosome". "Mar" is marker,
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191 chromosomally abnormal patients
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tumors of both African and non-Afri
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Haematology and Blood Transfusion V
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Retrospective group 26/93 evolved l
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Treatment
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advantage in terms of duration of f
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leukaemia. An analysis of 168 patie
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11. Treatment Program Chemotherapy
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efractory congestive heart failure
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period (Sequence II) was gene rally
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children with a multiple drug proto
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DAYS 1 5 10 15 20 25 30 35 , I I I
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Biologically Drug Outcome fit" sens
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may counterbalance the potential be
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Ht (0--0) 50 40 30 20 10 0 Platelet
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10 7 ,-----------------------------
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11. Haematological and Biochemical
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0\ 0\ INTERFERON ... 10 3 rl E "'
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("pre-B" phenotype) has been descri
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human bone marrow cells exhibit the
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10 9.,0 MBKCC .. .,4 B prot:oool 1'
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demann W, Büehner T, Arlin Z, Gee
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-...l 00 a-tLDRENS CAt«:ER S TU>Y
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prognostic characteristics under in
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significantly higher in HR} (14.5%,
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normal or elevated immunoglobulin l
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References Andreeff M, Darzynkiewic
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R GROUP 1 ( 1970 - 1971 ) 17 PTS. R
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Table 3. Influence of initial featu
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LIJ (f) a.. < -1 LIJ Q: u. 0 >- I-
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me nt indicated that additional the
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42:2123-2134 - Chessells JM, Cornbl
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Baum et al. 1979). This study was b
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1. 0 '--'1--" 0.9 e: o .- VI VI E C
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e: o e: 1. 0 ,.--...-- 1'"'\ ......
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In conclusion, we can state that th
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Haematology and Blood Transfnsion V
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INDUCTION VCR + PRED + ASNASE (wk 3
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SCHED.INT 1 MTX • ! Adria MTX •
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~ looh---------ooooc>----o---c>----
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Consoll- Irra .tlon t) .tlon Indu
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Highrisk Standard risk Table 1. Cli
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Cumulo/ive comp/el9 remission 1.0 .
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10° 8 = 6 ö 4 Non T Cell .~ :ö
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% SURVIVAL 100 ~ ~II 0 80 60 40 - -
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a) Percentage of donor cell mitoses
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Table 5. Results of bone marrow tra
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...... w N Table 7. Clinical result
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agent had to be added and that in t
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C. Chronic Myelogenous Leukemia The
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Patients with a suitable donor rece
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No. Recurrent Leukaemia Other death
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followed by dialysis against isoton
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HS, HL- Heme >_ 40 0 HS I ;:. =====
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knowing at present. These genes may
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esistance to antifolates. In Vitro
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a DNA probe specific for the gene t
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translocation between chromosomes 9
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Table 1. Subdivision of 1827 Myelo-
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NORMAL CELL ®
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some change of the primary type, in
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F. Transformation of Mouse Bone Mar
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were carried out with 3H-MTX in the
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the lysozyme cDNA prepared from ovi
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et al. 1976). The BJAB cellline in
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dalton bands corresponding to light
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1979). The EBV-carrying cell lines
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producer lines (DAUDI and P3HR-1),
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G, Giovanella B, Westman A, Stehlin
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Dc;bt CT + i J.I9 Raji; 1. i .. 100
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c. Discussion During infectious mon
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C. Nonepisomal Viral DNA We have us
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A 260 3.0 A B 1670 70 N2 2.0 c: E .
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vivo. Nature 260:302-306 - Kirk JTO
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Fig. 1. a Polyacrylamide gelelectro
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1979). The lymphocyte subpopulation
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lysin 0 antigens are found, and mix
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Pope JH (1978) Long-term T cell-med
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Charaeteristie Morphologie maturati
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after TPA-induction (Table 3). The
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PA (1978) Reactivity of a rabbit an
Page 235 and 236: Blood Monocyte. B lood Monocytes 1-
Page 237 and 238: Haematology and Blood Transfusion V
Page 239 and 240: and nonhistone proteins can serve a
Page 243 and 244: The findings of the evaluation of s
Page 245 and 246: Fig. 3. Tumor grawth of L 428 cells
Page 247 and 248: Cell biological and Immunological A
Page 249 and 250: consideration. Sanel (1973) obtaine
Page 251 and 252: endotoxin serum, were used. A parti
Page 253 and 254: monocytic leukemia cell line in cul
Page 255 and 256: B. Isolation, Characterization and
Page 259 and 260: don al hemopathies generally displa
Page 261 and 262: nitors. Blood Cells 6:595-607 - Min
Page 263 and 264: \I) ....I ....I IU Jl 200 r 100 90
Page 265 and 266: their haemopoietic cells' ability t
Page 267 and 268: normal normal ALL ALL ALL persons p
Page 269 and 270: 64.5 { V)
Page 271 and 272: References Biermann E, Neth R, Gros
Page 273 and 274: ehambers, the growth pattern observ
Page 275 and 276: References Anderssen LC, 10kinen M,
Page 277 and 278: REH CELL UNE TO CALLb~Ö ~q~ V.M. c
Page 281 and 282: the macrophage series in which the
Page 285: Table 1. Production of factor depen
Page 289 and 290: Table 1. Characteristics of the adh
Page 291 and 292: Fig. 3. A human marrow culture at 6
Page 293 and 294: 300 250 • HYPERPROLIFERATIVE PATT
Page 295 and 296: Fig. 9. Nonadherent population from
Page 297 and 298: The cobblestone areas were often no
Page 299 and 300: L (1976) Induction of colony format
Page 301 and 302: B. Materials and Methods J. Tissue
Page 303 and 304: Fig. 2. Ultrastructural appearance
Page 309 and 310: Table 1. Monoclonal antibody reacti
Page 311 and 312: al. 1979; Janossy et al. 1979) is e
Page 313 and 314: to which this is an exact replica o
Page 315 and 316: inger JL (1976) Distribution of la-
Page 317 and 318: I region antigens were found to be
Page 319 and 320: have been reported to be present on
Page 321 and 322: V. FunctionalAssays Assays for PHA
Page 323 and 324: Functional studies of UCHT1 separat
Page 327 and 328: 40 ,...... (5 L.. C o u 30 .9 ~ .~
Page 329 and 330: with immunohistochemieal methods (L
Page 333 and 334: can be dissected by cloning. Utiliz
Page 335 and 336: Table 1. Origin and marker profile
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complete identity of gp 100 from no
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Table 1. Reaction of single anti se
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media. Following 24-h incubation, c
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nonleukemic eells (Greaves 1979). L
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Fig. 2. Cytocentrifuged smears of b
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Modification of amino-groups of hum
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disturb this balance in the directi
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The majority of human blood lymphoc
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of the EBV infected B cells. Howeve
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and immunoglobulins. J Immunol 120:
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" K 10 • j ~ ALL RNlL AL wtfh les
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tion akuter Leukämiezellen in "spo
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D. Results and Discussion J. Acute
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selectively responsive to different
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l1J ~ 100 ~ Q. ::) l1J Z 0 ~ z >- .
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Table 2. Growth inhibition of S49 e
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Reverse Infectious ASC/2X1Q6 transc
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munized animals to 1316 tumor cells
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Fig. 3. Expression of retroviral gp
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pg70. J Exp Med 143: 151-166 - McGr
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Table 1. Expression of tumor antige
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Virological and Molecularbiological
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Table 1. Oncogenic properties of re
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also Fig. 1). It followed that the
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whether this sequence is related to
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zed in infected cells argue that th
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a specific viral transforming prote
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detect viral RNA as the only charac
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Eisen H (1980) Myeloproliferate vir
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p,60- -34K - 1 2 3 4 Fig. 1. In vit
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.. p - • a'T ... I .t'l Fig. 4. D
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Fig. 2. Photomicrographs of NY68 in
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DEAE (o(lJmn 'liI GI -2 2 o e s 1
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vity was deterrnined. For endogenou
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pr 180 - - - P 12/15 - A B c D E F
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85,- 66- -27 - -66 40- 27- -1'9 19-
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= 0.5 o E Q. o ILI 0.4 (J) « ILI .
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Table 1. Oncogenic potential of avi
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100 III GI c: o "8 80 > w
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Expt. No. Infecting virus Proportio
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Haematology and Blood 'fransfusion
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sequences, provided its gag portion
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77:3009-3013 - Hu SSF, Moscoviei C,
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RI 2.2 md and Hind III 2.6 md leuke
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RELATIONSHIP OF ENDOGENOUS AND EXOG
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E ........ E Cl. U E :::l .... Q) (
Page 447 and 448:
References Astrin S (1978) Endogeno
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the same animal were examined (e.g.
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I) 'IQ a \0 - CL -- Fig. 2. Restrie
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~ ..................... ~ Cell 3' 5
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a :Sa,C! + . ~ 111 i' ( " '~ .J. b
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Table 1 Sampie Tissue TS fragment"
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a b Sac I / rep ._-_.-_ K J nl rep
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Haematology and Blood 'fiansfusion
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Osteopetrosis and osteosarcomas occ
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Lymphoma cellline Table 1. T and B
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Table 2. In vivo growth and oneogen
Page 469 and 470:
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Table 1. Transforming aetivity of c
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Table 3. Transformation aetivity of
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- Shoemaker C, Goff S, Gilboa E, Pa
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indieate a shifting of target eell
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Table 2. Expression of Jl and Sourc
Page 481 and 482:
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~ i ,..1---------,,':::::::::::::::
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fragment of pBR322 DNA, a 3.0-kbp S
Page 487 and 488:
Mak TW (1979) Proc Natl Acad Sci US
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Table 1. Transformation of 3T3 cell
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inserts in the genome of rapidly tr
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pulations, it seemed likely that th
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contains antigens which react with
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le of reacting in sensitive radioim
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gic approach. In: Hiatt HH, Watson
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12 Eco R [ Fig. 1. Hybridization pa
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10 - '1~ )( -~ S:! E A U I o 1 2
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Haematology and Blood 'fransfusion
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cultures. These cells did not conta
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indication of malignant T-cells in
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Fig. 3. Thin-section electron micro
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Table 3. Lack of detectable related
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J. HTLV Was Present in the Primary
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specific signals into nonspecific s
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Table 2. Distribution of HTLV-relat
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u 10 ~ 20 ~ :c 30 a 40 o Fig. 1. Ki
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SSV TrS-gp labeled effectively with
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60 HFF/SSV cDNA CELL RNAs: 0 HF/SSV
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Antisera a Table 1. Immunofluoresce
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Haematology and Blood 'ftansfusion
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y competition RIA yielded virtually
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SiS'V' gp70: ANTIGEN, ANTI BODY, IM
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type-C virus p30 antigen in cells f
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c. Results The fractionated whole-b
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induced antibodies as well as exper
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- Fig. la-f. Retravirus particles p
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eplication of infecting murine leuk
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immunodeficiency, multic10nal lymph
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Bovine leukemia --, proteins of 499
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Human T- cell - -, characterization
Page 563 and 564:
RNA -, of tumor virus 439 Rous sarc
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Haematology andBlood Transfusion Su
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