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1 x 10 8 cells in 2 ml phosphate-buffered saline (PBS; pH 7.0) were added 1.0 mC sodium iodide 125 1, 200 fAl of lactoperoxidase (Sigma, 0.25 mg/ml), and 25 fAl of 0.03% hydrogen peroxide. The cells were incubated at room temperature for 10 min; during this period 25 f.ll of the peroxide solution was added twice. The re action was terminated by adding 8 ml 0.01 M cysteine and 0.01 M potassium iodide in PBS. The cells were washed thrice in Hanks balanced salt solution and placed in culture at 37°C in 5 ml minimal Eagle's medium. After 4 h the medium was discarded and the cells were washed and reincubated. The supernatant was harvested at 24 h. Cell suspensions showing less than 80 % viability were not used. 11. Irnmunoprecipitation To microtiter U-plate wells (Cooke Engineering Co., Alexandria, Virginia) prewashed with bovine serum albumin were added 20 f.ll of alloserum and 20 f.ll of the 125I-Iabeled supernatant. All tests were done in triplicate. The pI at es were shaken and allowed to stand for 1 hat 4°C. Coprecipitation was carried out by adding 1 00 ~d of Staphylococcal protein A (Enzyme Centre Incorporated, Boston, Mass.) to each weIl. The plates were shaken again, kept 15 min at 22°C, and then spun at 1800 r/min for 10 min. The supernatant liquid was gently sucked out of the wells, and the precipitates were washed three times in PBS (pH = 7 .2) and transferred to cuvettes. Radioactivity was counted in a Beckmann Biogamma II gamma spectrometer. 111. Gel Filtration Chromatography Gel filtration chromatography was conducted by applying 0.5 ml aliquots of culture supernatant on to 0.9 X 90 cm columns (Glenco Scientific, Inc.) of Bio-Gel A-1.5m (approximately 8% agarose, 200-400 mesh) equilibrated and eluted at 4°C with O.OIM ammonium acetate at a hydrostatic pressure of 20 cm water. IV. Isoelectric Focusing Solutions for isoelectric focusing were added to 0.75 ml of 40% ampholine (pH 3.5 to 10) and applied to an LKB 8100 column at 4°C to a total volume of 110 ml with sucrose gradient solution (Abraham and Bakerman 1977). V. LDS-Polyacrylamide Disc Gel Electrophoresis The pooled fractions or immunoprecipitates from these fractions were boiled with 1 % (weight/volume) lithium dodecyl sulfate (LDS)/O.OIM lithium phosphate buffer, pH 7.0, and then incubated further with this detergent at 37°C for 30 min. The resulting polypeptide subunits were resolved by LDS-polyacrylamide disc gel electrophoresis carried out according to Laemmli (1970) except for the substitution of LDS for sodium dodecyl sulfate in order to conduct the experiments at 4°C (Delepelaire and Chua 1979). VI. Cells Enriched T- or B-Iymphocyte preparations were obtained either by selective rosetting of T-Iymphocytes using sheep erythrocytes or by removing adherent B-Iymphocytes by absorption on a flask coated with affinity-purified go at antihuman Fab. The details of T - and B-cell enrichment procedures have been published earlier (Mohanakumar et al. 1979). VII. Preparation of Antiserum Heterologous anti-AMLSGA serum was prepared by injecting a monkey (M spesiosa) intravenously and intradermally (50 f.lg) three times, with aperiod of 14 days between immunizations (Mohanakumar et al. 1974). For intradermal injectjons the antigen suspended in PBS pH 7.2 was mixed with an equal volume of Freund's complete adjuvant (H37Ra). The antiserum described in this report was obtained 7 days after the third inoculation with antigen. VIII. Absorption of Antisera Immune and the preimmune sera were heat inactivated at 56°C for 30 min and then absorbed twice for 20 min at 4°C with an equal volume of cells (pooled normal human platelets, leukocytes, bone marrow cells, or myeloblasts). IX. Microcytotoxicity Assay A standard Amos modification of the microtechnique described by Mittal et al. (1968) was used for unfractionated cell preparations, and the incubation time with complement was extended to 2 h when enriched B- and T -lymphocytes were used as targets. Selected nontoxic rabbit complement was used throughout. D. Results I. Partial Characterization of Surface Compounds Leukernic myeloblasts radiolabeled with the lactoperoxidase iodination technique released labeled soluble compounds into supernatant 333
media. Following 24-h incubation, cells remained >90 % viable by trypan blue exclusion. Gel filtration chromatography in 8 % agarose of the labeled supernatant yielded two distinct peaks, coincident in radioactivity and protein concentration. The second peak was selectively reactive by coprecipitation with antileukemic antisera obtained from patients receiving immunotherapy with leukemie myeloblasts (Taub et al. 1978). Supernatant material derived from radiolabeled blasts and eluted in the second peak from the agarose column was applied to a DEAE cellulose column and yielded a single peak, coincident in radioactivity and protein levels. Carbohydrate conte nt of the eluted material was estimated at 10% by weight. This material retained immunologie activity with antileukemic alloantisera as measured by coprecipitation. 11. Development of Heteroantisera Antisera raised in monkeys to the compounds eluted from DEAE cellulose columns was tested for cytotoxie reactivity against leukemic and nonleukemic cells (Table 1). In complement-dependent cytotoxicity testing the monkey anti-AMLSGA was reactive with cells from leukemic patients but was unreactive with nonleukemic peripheral blood or bone marrow cells. Absorption of anti-AMLSGA with leukemie myeloblasts from patients with acute myeloblastie or chronie myelocytic leukemia removed all antileukemic activity. Ab- sorption with leukemic lymphoblasts or leukemic lymphocytes removed activity against ALL or CLL cells but did not remove antimyeloblast activity. Anti-AMLSGA was unreactive with B-lymphocytes, including those of an identieal twin to a patient with AML, and absorption with B-Iymphocytes did not re du ce antimyeloblast activity. In contrast, the rabbit anti-la was reactive with all nonleukemie B-Iymphoytes. Antileukemie activity was not reduced by absorption of anti-AMLSGA with the enriched mononuclear fractions of nonleukemie marrow or with neutrophils from nonleukemic patients. 111. Further Characterization of Surface Compounds The antileukemic heteroantisera were applied to the further isolation and characterization of compounds from the myeloblast cell surface. Compounds eluted from gel columns were applied to isoelectric focusing columns and fractions obtained were analyzed for reactivity by coprecipitation with the heteroantisera. A major reactive peak was obtained at pI 7.8. Analysis of the immune precipitate by LDS-PAGE yielded a homogeneous peak with molecular weight estimated between 70,000 and 80,000 daltons. IV. Testing' of Patients' Sera Serum sampies were tested for their ability to interfere with coprecipitation of soluble radio- Serum Target cells absorbed with AML CML ALL CLL (10)" (5) (5) (5) Unabsorbed +b + ± ± AMLc CML ALL + + CLL + + B-cells + + ± + T-cells B-cells (10) (5) Table 1. Reactivity of monkey anti-AML antiserum " Number of sampies tested b Plus (+) indicates all sampies were reactive. Minus (-) indicates all sampies were negative. Both signs (±) indicate weak positive reactions in some sampies C Abbreviations: AML, acute myeloblastic leukemia; CML, chronic myelocytic leukemia; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia 334
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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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Haematology and Blood Transfnsion V
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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
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 305 and 306: Haematology and Blood Transfusion V
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 343: Haematology and Blood Transfusion V
Page 347 and 348: nonleukemic eells (Greaves 1979). L
Page 351 and 352: Fig. 2. Cytocentrifuged smears of b
Page 353 and 354: Modification of amino-groups of hum
Page 355 and 356: disturb this balance in the directi
Page 357 and 358: The majority of human blood lymphoc
Page 359 and 360: of the EBV infected B cells. Howeve
Page 361 and 362: and immunoglobulins. J Immunol 120:
Page 363 and 364: " K 10 • j ~ ALL RNlL AL wtfh les
Page 365 and 366: tion akuter Leukämiezellen in "spo
Page 367 and 368: D. Results and Discussion J. Acute
Page 369 and 370: selectively responsive to different
Page 373 and 374: l1J ~ 100 ~ Q. ::) l1J Z 0 ~ z >- .
Page 375 and 376: Table 2. Growth inhibition of S49 e
Page 377 and 378: Reverse Infectious ASC/2X1Q6 transc
Page 381 and 382: munized animals to 1316 tumor cells
Page 383 and 384: Haematology and Blood Transfnsion V
Page 385 and 386: Fig. 3. Expression of retroviral gp
Page 387 and 388: pg70. J Exp Med 143: 151-166 - McGr
Page 389 and 390: Table 1. Expression of tumor antige
Page 391 and 392: Virological and Molecularbiological
Page 393 and 394: 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
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
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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
Page 463 and 464:
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
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
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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
Page 507 and 508:
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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
Page 519 and 520:
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
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:
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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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