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the appearance of antigens with cell passage number (1 to 5 weeks after fusion compared to 1 to 2 months after starting cocultivation). However, our cell fusion procedure never led to overt virus production. This means that the exact nature of this possible virus-related antigen has still to be determined. Acknowledgments Drs. H. Behrendt, G. van Zanen and G. Wagemaker are thanked for providing the human bone marrow sampies. This work was partly supported by a grant from the Dutch Organization for the Fight against Cancer, "the Queen Wilheimina Fund". References HaIes A (1977) A procedure for the fusion of ceIIs in suspension by means of polyethylene glycol. Somatic CeII Genet 3:227-230 - Joustra M, Lundgren H (1970) Preparation of freeze-dried monomeric and immunochemically IgG by a rapid and reproducible technique. In: Peeter H (ed) Protides of the biological fluid. Pergamon Oxford, p 511 - Nooter K (1979) Studies on the role of RNA tumour viruses in human leukaemia. Thesis, University of Leiden, pp 38-40 - Nooter K, Coolen J, Dubbes R, Zurcher C, Koch G, Bentvelzen P (1979) Type C virus antigen detection in co-cultures of human leukaemic bone marrow and dog ceIIs. J Gen Virol 45 : 711-721 - Szybalski W, Szybalska EH, Ragni G (1962) Genetic studies with human cell lines. Natl Cancer Inst Monogr 7: 75 529
Haematology and Blood 'ftansfusion Vol. 26 <strong>Modern</strong> Trends in Human Leukemia IV Edited by Neth, Gallo, Graf, Mannweiler, Winkler © Springer-Verlag Berlin Heidelberg 1981 ELISA for the Detection of Antigens Cross-Reacting with Primate C-Type Viral Proteins (p30, gp70) in Human Leukemic Sera* R. Hehlmann, H. Schetters, and V. Erfle A number of methods have been applied in the past to the serologie identifieation and characterization of C-type viral proteins. The most specifie and most sensitive system used has been the competition radioimmunoassay (RIA). This assay detects group specific as well as interspecies activity of the major viral proteins (Parks and Scolnick 1972; Strand and August 1973). Disadvantages of this assay inc1ude the necessity of radioactive isotopes, false positive results from contaminating proteases, and the limited stability of the radioactively labeled reagents. Recently, the enzyme immunoassay (EIA) technique has been developed (Engvall and Carlsson 1976). This technique has also proved to be highly specific and sensitive in detecting antigens and antibodies in a variety of systems (Voller et al. 1976). It avoids the biohazards of radioactivity and is simple and cheap. A further advantage is the stability of the coupled reagents. Low cost and simplicity of the EIA technique allow the screening of large numbers of sampies. We report here the application of the enzyme linked immunosorbent assay (ELISA) to the detection and quantification of the core protein p30 of the murine leukemia viruses (MuLV), the baboon endogenous virus (BaEV), and the simian sarcomavirus (SiSV) and of the envelope glycoprotein gp70 of SiSV. We further report attempts to detect with this technique the presence in human leukemic sera of antigens cross-reacting with primate viral structural proteins. * Conducted, in part, as study of the Süddeutsche Hämoblastosegruppe (SHG). A. Methods and Materials I. Antigens MuLV p30, BaEV p30, and SiSV p30 were purified by a two step electrofocusing procedure (Schetters et al. 1980). Homogeneity of the purified viral proteins was demonstrated by SDS gel electrophoresis. SiSV gp70 was the gift of Dr. R. Gallo, NCI. 11. Antibodies Antibodies against viral p30 proteins were prepared from immunized rabbits. The specificity of the antisera was controlled by precipitation of disrupted virus and subsequent gel electrophoretic analysis of the precipitated proteins. The activities of the sera were directed almost exc1usively against the respective p30s. Antiserum against SiSV gp 70 as the gift of Dr. R. Gallo, NCI. IgG was purified as described (Schetters et al. 1980). 111. Coupling Procedure 01 Peroxidase to IgG In essence the procedure of Nakane and Kawaoi (1974) as modified by Mesa-Tej ada et al. (1978) was followed which involves blocking of the amino groups of the peroxidase with phenylisocyanate, introduction of aldehyde groups with Na-periodate, and formation of a Schiff's base with the IgG by incubation of the IgG with the activated peroxidase. IV. ELISA Procedure In essence, the procedure as described by Schetters et al. (1980) was followed. Microtiter plates were coated with 200 ~l per weH of 5-10 ~g IgG/ml 50 mM carbonate-bicarbonate buffer, pH 9.6, at 37° for 2 h. After washing, the antigen of interest was added 530
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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
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Blood Monocyte. B lood Monocytes 1-
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and nonhistone proteins can serve a
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The findings of the evaluation of s
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Fig. 3. Tumor grawth of L 428 cells
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Cell biological and Immunological A
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consideration. Sanel (1973) obtaine
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endotoxin serum, were used. A parti
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monocytic leukemia cell line in cul
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B. Isolation, Characterization and
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don al hemopathies generally displa
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nitors. Blood Cells 6:595-607 - Min
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\I) ....I ....I IU Jl 200 r 100 90
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their haemopoietic cells' ability t
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normal normal ALL ALL ALL persons p
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64.5 { V)
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References Biermann E, Neth R, Gros
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ehambers, the growth pattern observ
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References Anderssen LC, 10kinen M,
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REH CELL UNE TO CALLb~Ö ~q~ V.M. c
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the macrophage series in which the
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Table 1. Production of factor depen
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Table 1. Characteristics of the adh
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Fig. 3. A human marrow culture at 6
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300 250 • HYPERPROLIFERATIVE PATT
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Fig. 9. Nonadherent population from
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The cobblestone areas were often no
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L (1976) Induction of colony format
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B. Materials and Methods J. Tissue
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Fig. 2. Ultrastructural appearance
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Table 1. Monoclonal antibody reacti
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al. 1979; Janossy et al. 1979) is e
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to which this is an exact replica o
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inger JL (1976) Distribution of la-
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I region antigens were found to be
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have been reported to be present on
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V. FunctionalAssays Assays for PHA
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Functional studies of UCHT1 separat
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40 ,...... (5 L.. C o u 30 .9 ~ .~
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with immunohistochemieal methods (L
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can be dissected by cloning. Utiliz
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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) (
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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
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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
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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
Page 489 and 490: Table 1. Transformation of 3T3 cell
Page 491 and 492: inserts in the genome of rapidly tr
Page 493 and 494: pulations, it seemed likely that th
Page 495 and 496: contains antigens which react with
Page 497 and 498: Haematology and Blood Transfusion V
Page 499 and 500: le of reacting in sensitive radioim
Page 503 and 504: gic approach. In: Hiatt HH, Watson
Page 505 and 506: 12 Eco R [ Fig. 1. Hybridization pa
Page 509 and 510: 10 - '1~ )( -~ S:! E A U I o 1 2
Page 511 and 512: Haematology and Blood 'fransfusion
Page 513 and 514: cultures. These cells did not conta
Page 515 and 516: indication of malignant T-cells in
Page 517 and 518: Fig. 3. Thin-section electron micro
Page 519 and 520: Table 3. Lack of detectable related
Page 521 and 522: J. HTLV Was Present in the Primary
Page 523 and 524: specific signals into nonspecific s
Page 525 and 526: Table 2. Distribution of HTLV-relat
Page 527 and 528: u 10 ~ 20 ~ :c 30 a 40 o Fig. 1. Ki
Page 531 and 532: SSV TrS-gp labeled effectively with
Page 535 and 536: 60 HFF/SSV cDNA CELL RNAs: 0 HF/SSV
Page 537: Antisera a Table 1. Immunofluoresce
Page 541 and 542: y competition RIA yielded virtually
Page 543 and 544: SiS'V' gp70: ANTIGEN, ANTI BODY, IM
Page 545 and 546: type-C virus p30 antigen in cells f
Page 547 and 548: c. Results The fractionated whole-b
Page 549 and 550: induced antibodies as well as exper
Page 551 and 552: - Fig. la-f. Retravirus particles p
Page 553 and 554: eplication of infecting murine leuk
Page 557 and 558: immunodeficiency, multic10nal lymph
Page 559 and 560: Bovine leukemia --, proteins of 499
Page 561 and 562: Human T- cell - -, characterization
Page 563 and 564: RNA -, of tumor virus 439 Rous sarc
Page 565 and 566: Haematology andBlood Transfusion Su
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