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cells tend to "hover" elosely over the stromal layers. Phase contrast observations immediately following fee ding of the cultures reveal that even with gentle agitation of the culture vessel, large numbers of nonadherent cells remain elose to the stromallayer. Possibly the viscosity of the growth medium enhances this effect. Therefore, we believe that the numbers of nonadherent cells harvested in the aliquots of spent medium may not accurately reflect the numbers of nonadherent cells present in the cultures. More vigorous agitation of the culture vessel results in some destruction of the delicate stromal microenvironment and cessation of hematopoiesis. We are presently attempting to improve our methods of quantitation. Erythroid (BFU-E) (Testa and Dexter 1977) and megakaryocytic (CFU-M) (Williams et al. 1978) progenitors are also produced in long-term murine cultures. The persistence for at least 16 weeks of pre-T cells in murine cultures has also been recently reported (Jones-Villeneuve et al. 1980). To date we have not attempted to assay for such populations in our cultures of human marrow. However, we have established three permanent, polyelonal cell lines of Epstein-Barr virus transformed B-Iymphoblastoid cells from marrow cultures of different nonleukemic specimens. All three arose from what appeared to be hematopoietically exhausted stromal layers, one developing at 3 months, one at 4 months, and one at 6 months after initiation of the cultures. All were derived from cultures in Fischer's medium with 25 % HoS and hydrocortisone. In the ca se of the 6-month-old stromallayer, no cobblestone areas remained, and no nonadherent cells had been recovered for 2 months prior to the appearance of the B cells. Moore and Sheridan (1979) reported the conversion of 10% of their human cultures to a lymphoblastoid morphology by the 6th week of culture. We are at present uncertain as to wh ether our observations represent the generation of B cells from immature precursors in these old cultures or the delayed outgrowth of long-lived EBV-transformed mature B cells. Given the complexity of the stromal microenvironment, it is quite possible that small numbers of mature B cells could have survived undetected. Methods for the culture of human bone marrow are essential for furt hering our understanding of normal hematopoiesis and of hematopoietic disease states as weIl as the effects of therapeutic regimens on marrow subpopulations. Although there are still many improvements to be made in the application of the liquid phase murine culture system to human marrow, we believe the modifications we have described allow for the routine establishment of such cultures in a reproducibly successful way. Acknowledgments This work was supported by research contract N01-CP-91044 from the National Cancer Institute, National Institute of Health, U.S. Dept. of Health, Education, and Welfare (DHEW). Suzanne Gartner is the holder of a predoctoral traineeship in a Cancer Biology Training Program supported by training grant CA-09302 awarded by the National Cancer Institute, DHEW. We thank Dr. J ames B. D. Mark for providing the rib specimens, Dr. Stuart Jamieson for providing the normal sternal marrow aspirates, and Drs. Peter Greenberg, Richard Hornes, Robert Feiner, and Ms. Betty Reitsma for providing the leukemic marrow specimens. We are also grateful to Ms. Glenda Garrelts for the nonspecific esterase tests, Dr. Werner Henle for the EBNA determinations, and Dr. Joel Greenberger for helpful discussions. References Allen TD, Dexter TM (1976) Cellular Interrelationships during in vitro Granulopoiesis. Differentiation 6: 191-194 - Bradley TR, Metcalf D (1966) The growth of mouse bone marrow cells in vitro. Aust J Exp Biol Med Sci 44:287-300 - Böyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest [Suppl 21] 77: - Dexter TM, Lajtha LG (1974) Proliferation of hematopoietic stern cells in vitro. Br J Hematol 28: 525-530 - Dexter TM, Testa NG (1976) Differentiation and proliferation of hematopoietic cells in culture. In: Methods in cell biology, vol 14. Academic Press, New York, pp 387-405 - Dexter TM, Allen TD, Lajtha LG, Schofield R, Lord BI (1973) Stimulation of differentiation and proliferation of hematopoietic cells in vitro. J Cell PhysioI82:461-470-DexterTM,AllenTD,Lajtha LG (1977) Conditions controlling the proliferation of hematopoietic stern cells in vitro. J Cell Physiol 91 :335-344 - Epstein AL, Kaplan HS (1974) Biology of the human malignant lymphomas. 1. Establishment in continuous cell cuIture and heterotransplantation of diffuse histiocytic lymphomas. Cancer 34: 1851-1872 - Fibach E, Gerassi E, Sachs 287
L (1976) Induction of colony formation in vitro by human lymphocytes. Nature 259: 127-129 - Gartner S, Kaplan HS (1980) Long-term culture of human bone marrow cells. Proc Natl Acad Sei USA 77:4756-4759 - Greenberger JS (1978) Sensitivity of corticosteroid-dependent insulin-resistant lipogenesis in marrow preadipocytes of obese diabetic (db/db) mice. Nature 275 :752-754 - Greenberger JS (1979) Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro. In Vitro 15: 823-828 - Greenberger JS, Sakakeeny MA, Berndtson K (to be published) Longevity of hematopoiesis in mouse long-term bone marrow cultures demonstrates significant mouse genotypic variation. J Supramol Struct - Golde D, Cline M (1973) Growth of human bone marrow in liquid culture. Blood 41 :45-57 -Jones-Villeneuve EV, Rusthoven JJ, Miller RG, Phillips RA (1980) Differentiation of Thy I-bearing cells from progenitors in long-term bone marrow cultures. J Immunol 124:597-601 - Kaplan HS, Goodenow RS, Gartner S, Bieber MM (1979) Biology and virology of the human malignant lymphomas: 1st Milford D. Schultz Lecture. Cancer 43: 1-24 - Metcalf D, Warner NL, Nossal GJV, Miller JFAP, Shortman K, Rabellino F (1975a) Growth of B lymphocyte colonies in vitro from mouse lymphoid organs. Nature 255: 630-632 - Metcalf D, MacDonald HP, Odartchenko N, Sordat LB (1975b) Growth of mouse megakaryocyte colonies in vitro. Proc Natl Acad Sei USA 72:1744-1748 - Moore MAS, Sheridan APC (1979) Pluripotent stern cell replication in continuous human, prosimian and murine bone marrow culture. Blood Cells 5:297-311 - Moore MAS, Sheridan APC, Allen TD, Dexter TM (1979) Prolonged hematopoiesis in a primate bone marrow culture system: characteristics of stern cell production and the hematopoietic microenvironment. Blood 54:775-793 - Pike B, Robinson W (1970) Human bone marrow colony growth in agar-gel. J Cell Physiol 76:77-84 - Pluznik DH, Sachs L (1966) The induction of clones of normal mast cells by a substance from conditioned medium. Exp Cell Res 43: 553-563 - Sredni B, Kalechman Y, Michlin H, Rozenszajn LA (1976) Development of colonies in vitro of mitogen-stimulated mouse T lymphocytes. Nature 259: 130-132 - Stephenson JR, Axelrad AA, McLeod DL, Shreeve MM (1971) Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci USA 68: 1542-1546 - Testa NG, Dexter TM (1977) Long-term production of erythroid precursors (BFU) in bone marrow cultures. Differentiation 9: 193-195 - Williams N, J ackson H, Sheridan APC, Murphy MJ, EIste A, Moore MAS (1978) Regulation of megakaryopoiesis in long-term murine bone marrow cultures. Blood 51 :245-255 - Yam LT, Li CY, Crosby WH (1971) Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 55:283-290 288
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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
Page 35 and 36:
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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Page 109 and 110:
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
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 257 and 258: Haematology and Blood Transfusion V
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 and 286: 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: The cobblestone areas were often no
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
Page 339 and 340: complete identity of gp 100 from no
Page 341 and 342: Table 1. Reaction of single anti se
Page 345 and 346: media. Following 24-h incubation, c
Page 347 and 348: 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
Page 411 and 412:
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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-
Page 423 and 424:
Page 425 and 426:
= 0.5 o E Q. o ILI 0.4 (J) « ILI .
Page 427 and 428:
Table 1. Oncogenic potential of avi
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100 III GI c: o "8 80 > w
Page 431 and 432:
Expt. No. Infecting virus Proportio
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Haematology and Blood 'fransfusion
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sequences, provided its gag portion
Page 437 and 438:
77:3009-3013 - Hu SSF, Moscoviei C,
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RI 2.2 md and Hind III 2.6 md leuke
Page 441 and 442:
Page 443 and 444:
RELATIONSHIP OF ENDOGENOUS AND EXOG
Page 445 and 446:
E ........ E Cl. U E :::l .... Q) (
Page 447 and 448:
References Astrin S (1978) Endogeno
Page 449 and 450:
the same animal were examined (e.g.
Page 451 and 452:
I) 'IQ a \0 - CL -- Fig. 2. Restrie
Page 453 and 454:
~ ..................... ~ Cell 3' 5
Page 455 and 456:
a :Sa,C! + . ~ 111 i' ( " '~ .J. b
Page 457 and 458:
Table 1 Sampie Tissue TS fragment"
Page 459 and 460:
a b Sac I / rep ._-_.-_ K J nl rep
Page 461 and 462:
Haematology and Blood 'fiansfusion
Page 463 and 464:
Osteopetrosis and osteosarcomas occ
Page 465 and 466:
Lymphoma cellline Table 1. T and B
Page 467 and 468:
Table 2. In vivo growth and oneogen
Page 469 and 470:
Page 471 and 472:
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
Page 477 and 478:
indieate a shifting of target eell
Page 479 and 480:
Table 2. Expression of Jl and Sourc
Page 481 and 482:
Page 483 and 484:
~ i ,..1---------,,':::::::::::::::
Page 485 and 486:
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
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inserts in the genome of rapidly tr
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pulations, it seemed likely that th
Page 495 and 496:
contains antigens which react with
Page 497 and 498:
Page 499 and 500:
le of reacting in sensitive radioim
Page 501 and 502:
Page 503 and 504:
gic approach. In: Hiatt HH, Watson
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12 Eco R [ Fig. 1. Hybridization pa
Page 507 and 508:
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
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u 10 ~ 20 ~ :c 30 a 40 o Fig. 1. Ki
Page 529 and 530:
Page 531 and 532:
SSV TrS-gp labeled effectively with
Page 533 and 534:
Page 535 and 536:
60 HFF/SSV cDNA CELL RNAs: 0 HF/SSV
Page 537 and 538:
Antisera a Table 1. Immunofluoresce
Page 539 and 540:
Haematology and Blood 'ftansfusion
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 555 and 556:
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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