MacDowell, E. Carleton

English

Cold Spring Harbor Laboratory Institutional Repository

'.JSECOND QUARTERLY PROGRESS REPORT OF RESEARCHcarried out byLONG ISLAND BIOLOGICAL ASSOCIATIONforTHE BIOLOGICAL DEPARBIENT, CHEMICAL CORPS, CAMP DETRICK^ ,f On Contract DA-l8-06lt-CML-2360January 1 - March 31, 1953RESEARCH STUDIES OF FACTORS liCUCING RESISTANCETO TRANSMISSIBLE MOUSE LEUKEMIAbyE. C« MacDowell.  i E. C. MacDOrtELL/■ /fc; - ■ .In the period between January 1st and March 30th 1953, seven experimentshave been performed in the Camp Detrick series, numbered CD? - CD13. The natureof the problems, the biological materials and the basic procedures have beendescribed in the previous report.CD?.The attempt to obtain the protective material in the supernatant after asaline suspension of line leukemic cells had been gently agitated (see CD5)was repeated using 3% Knox's special gelatin solution in the suspending medium.This necessitated the use of a non-refrigerated centrifuge of lower speed andshaking the material at room temperature instead of in an ice bath. To obtaina more nearly cell-free supernatant, the first supernatant was recentrifuged.But even so, this supernatant II was probably not cell-free. The sediment fromthe second run contained enough cells to kill the mice so treated about fourdays ahead of the controls in the challenging dose given after two days. Ofthe l5 mice treated with this supernatant II, lii died with, the controls and onesurvived. In experiment CDS three out of 25 survived, although the deaths ofthe others, mostly before the controls, proved that potent leukemic cells hadbeen present in the supernatant. In this experiment (CD?) the coincidence withthe control deaths seems to imply that there were no potent cells in the supernatant II, but it is entirely possible that enough cells were present tobalance out the effect of some resistance to the challenging dose. Since allof the h5 controls in the challenging dose of it"® x standard in these two experiments have died, the survival of four in liO strongly suggests an effect ofthe treatment,CD6.This experiment returned to the question of whether repeated treatmentswith heated (li6o C/llj min.) line ly^ cells will prolong the duration of theresistance (see CD6). In the earlier experiment, the challenging dose wasgiven on the ninth day after the third treatment. Since this proved too soon^the time was extended to lU days in the present case. Four groups of five micewere given three daily treatments with heated line cells, and five littermates of each of these groups were given a single treatment. In three of thesesets of litter mates, all receiving a single treatment died with the controlsbut two of the five mice receiving three treatments in each of these setseither survived or lived twice as long as the controls. In the fourth set,however, two of the single treatment mice survived as well as two of those receiving three treatments — none of the controls in the challenging dose survived, Three treatments compared with one evidently do prolong the resistantperiod, but they do not yield as strong or as enduring resistance as thatfollowing a single treatment to which a very few unheated cells have been added.CD9.Will the protective action of supernatants be increased by disrupting theleukemic cells? Leukemic cells may be disrupted by suspension in distilledwater and they may be mechanically torn by forcibly squirting a suspension froma syringe (without needle) held firmly against a flat surface. Both of thesemeans were employed in this experiment. The original suspension of line I^jleukemic cells was made with saline; this was replaced by sterile distilledwater, and every 3 ml separately was "squirted" for 100 shots. The supernatantfrom this after 2000 x G, 30 min. was recentrifuged twice, each time as before,and the final supernatant injected into lii mice and challenged in two days.Part of the squirted material was not centrifuged, but heated (1^6° C/lk min.)to prevent any remaining leukemic cells from producing leukemia, and so toreveal whether the resistance inducing substance was still operative in thewhole material. The sediment from the first centrifuging after "squirting" wasresuspended in H2O and injected into 15 mice as a check on the presence ofpotent leukemic cells and a similar check was made with the saline supernatantfrom the preliminary run (2000 x G/Uo min.) after four hours at room temperature.In spite of the distilled water and squirting, the final supernatant gavethe same kind of result as previously (out of lii, 2 survived - 3 lived longerthan any control); but the heated material, which in saline and not "squirted"would have protected all, actually did protect only h out of 11. Sediment fromthe first centrifuging after squirting failed to produce leukemia, and only 1in 15 survived challenged in lit days, whereas had a very few potent leukemiccells been present and been resisted, complete resistance would have been expected. Thus, rather than increasing the protective effect of the supernatant,the distilled water plus the squirting seemed to be responsible for a reductionin the protective action of the whole material. The failure of the unheatedsediment to produce any leukemia and to protect only 1 in 15 from the challengedose in lU days indicates that the rough treatment of the cells had effectivelydestroyed them. A side light from this experiment was the discovery thatcentrifuging 2000 x O/UO min, left in the supernatant so many leukemic cells,that even after 1| hours at room temperature the preliminary saline supernatantkilled the h mice inoculated as though inoculated with a dose of ^ -6 qj. ^ -7^that is, U to l6 times the dose used for challenging (1;"® x standard). Hencethe importance of recentrifuging, and in the future, the use of higher speedand longer time, as well.CDIO and CPU.Will heating line I^j leukemic cells (146° C/lh min.) liberate protectivematerial into the medium?The effectiveness of line leukemia in producing resistance after thismild heating is firmly established. One possible action of this gentle heatmight be to change cell-wall permeability. Accordingly, supernatants after thestandard heating procedure were tested for resistance in these two experiments.The centrifugation was carried out in a zero-degree room using 3250 x G/60 min.The first supernatants were centrifuged before injection into the mice. Thesediment from the first run was resuspended and injected.In these experiments the leukemic cells were suspended in standard concentration, whereas in previous experiments the supernatants were obtained fr<»nsuspensions diluted to lA standard Thus, although the mice have beengiven about 1 ml of the supernatants throughout, the concentration in these twoexperiments was about four times greater.The results give U in 10, and $ in 10 surviving the challenging dose of0U" in ii8 hours. In relation to the concentration of the dose of supernatantthese results are in agreement with previous ones, and give little, if ar^r,indication that the heating increased the effectiveness of the supernatants.However, the more intense and repeated centrifuging give these results greatersignificance than any previous ones, in that the supernatants were more nearlycell-free.It may be noted that the challenge dose for CDll was less than for CDIO,because the controls lived somewhat longer and one survived. The delay ofdeaths after supernatant treatment gives similar evidence. The greater survival after treatment with "white layer" in CDll may be interpreted as partlythe result of a larger dose of this material and partly the result of thesmaller challenge dose.CD12.'With this experiment attention is turned to the possibility that the protective material provided by line 1^ leukemic cells is not stored in or onthese cells, but is a cell product. In this case, instead of attempting to extract by various methods, the procedure wouH be to maintain the life of thecells for as long as possible in vitro before testing supernatants.In vivo, leukemic cells, after h6° C/lU min., in all probability being ofthe same genetic constitution as the hosts, continued to metabolize. The U8hours during -which resistance has been found to increase might well be an indexof the accumulation of some metabolites, Llth this, as well as practical considerations in mind, a period of 12 hours was chosen for the incubation (37° C)of the leukemic cell suspension, before the recentrifuged supernatant was testedis yivo (B). If cells with leukemic potency survived, the sediment might killthe hosts or, in case their numbers were very small, might yield firm resistance. So the sediment after incubation was in part inoculated into 10 mice asa test of surviving leukemic potency (F), in part heated li6° C/lk min. to inactivate the possibly surviving leukemic cells and permit a test of the survival of the capacity to induce resistance (C). 'Ihis experiment also includeda group of 10 mice treated with the recentrifuged supernatant removed from theoriginal saline suspension of cells after refrigerated shaking for 30 min. andbefore incubation (A), The challenging dose for this group was controlled by10 mice (D), and another 10 mice controlled the challenging dose used for themice given the incubated supernatant and the incubated and heated sediment (E),Under the given conditions, 12 hours incubation at 37° C proved to destroy boththe capacity to transmit leukemia and the capacity to induce resistance. Theonly resistant mice in the experiment were two of the ten given preliminarysupernatant before incubation (A) and one of these after 23 days came down witha modified distribution of leukemic invasions centering in the hypodermis, themuscles of the abdominal wall and the mesentaries leaving the spleen small.This is a condition characteristic of cases in which struggle between inducedresistance and leukemic growth is not quite completely settled and leukemiccells finally, in some cases after a delay of months, get the upper hand,especially in the hypodermis. The only group in this experiment that survivedwas that of the 10 unchallenged mice given the unheated sediment afterincubation (F),CD13,With 12 hours incubation at 37° C proving too long, a very simple experiment was set up to test how long potent leukemia cells would survive. After2 hours incubation, lO/lO died but the interval before death dorresponded tO adilution of x Standard. After h hours incubation 9/lO died at intervalsexpected from a dilution of x Standard. After 6 hours incubationh/lO died at intervals corresponding to dilution of x Standard, Withnutritive media and rotation during incubation, the survival of leukemic cellsmay be extended, but even four hours should give enough time for experimentsto indicate the probable success or failure of this approach. No detailedprotocol for this purely technical experiment is included..7Exp, CD? - CSH 2170 lb 2081Treatment with supernatant II from shaken line lb suspended in 3% gelatin-saline »Challenged in 2 days with lb U""®^/7/53 IDs50 ApMa - 2t20 From first killing of spleen donors to lasttreatment. Room temperature for all materials." 12>20 28 cc lb Std. •- spleen wt. x 2 - suspended in 3% gelatin insaline shaken on mechanical shaker set at 90,7 cc to each of it test tubes balanced in pairs, in plastic jackets,12i30 - 1;00 To International Clinical centrifuge - maximum speed 60G x G,6 cc removed from each tube to new tubes.I;l5 - l:it5 Recentrifuged5 CO from each of 3 tubes. 1.0 cc to each of 15 mice.Ith tube failed to upright at end of run so most of contents lost. Usedremainder "sediment" from all tubes to treat 5 mice.1/9/53 Challenge Dilutions made with chilled saline; all vials kept in crushedice; U-8 made in two lots from U-6. First lot used to inoculate first 10treated mice and their 10 litter mate controls, before second lot wasmixed and used for the second 10 treated mice and their litter mate controls .8CD'^ - ResultsInterval before death - Days after lb 1+-8Box 6 6+ 7- 7 7+ 8- 8 8+ 5- 9 9+ 9 i /5? 10- 10 10 1/2... 11A]_ Supernat^ II 5Ao Controls 2. 3 1Bi Supernat. II 2 1 1 1B2 Controls 2 1 1 1Ci Supernat, II 1 2 1C2 Controls 1 3 1negTotals Supernat. 3 1 8 1 1negControls 1 1 8 2 2 1Di'Sedinent II" I4 1D2 Controls U 1Total Controls 1 1 12 2 3 1U"® controls CDS 9 56 2 1 2CD8 CSH 2175 lb 2081i - Heat series 129Spleens from CSH 2168 lb 208l - standard saline suspension -ml saline = spleen -weight In g x 2.1/13/53 - 6 ml standard, In 3 ml lots - 5 mln In 2l|0 C - lii mln in 16® C.treat 20 micel/lh/53 - 6 ml standardtreat same 20 mice1/15/53 - 12 ml standard "treat same 20 mice and 20 llttermates1/29/53 - Challenged all liO mice and 10 untreated controls~dose= x Std,Results given by sets of llttermates.Intervals before death In l/h daysSet of litter mates !  8th'  « n •9th•  • •10th•  » •j ... 19thf  • • •1 SurvivedA 3 treatments 1 1  1[ m m m2/51 treatment 1 1  1 2/5B 3 treatments 1 2 2/51 treatment 2 1 0/50  3 treatments 2 1 2/51 treatment 3 1 1 0/5D  3 treatments 1 1 1 1 1/51 treatment 1 3  1 0/5Totals3 treatments 361- 1 - 1 1 7/201 treatment 7521 3 - - - 1 2/20;Controls 1  1 5 3same ages, not samelitters as above10CD?. C5H 2179 lb 2085Will suspension in distilled water and disruption of cells by "squirting"yield more resistance from supernatant?5 spleens from CSH 2175 lb 208ii - standard suspension - saline nl •2x spleen wt, in g.? ml Std, + 27 cc saline filtered through cotton*Centrifuged (l5^ C) - 3600 RM/UO min (2000 x G) Supernatant inoc* h ME)Sediment resuspended in H2O■'Squirted"' 100 shots for each 3 cc separatelyHalf of material heated ^6° C/l5 min - treated 1 ml 11 d-cf (0)Half of material centrifuged - 2000 x G/30 min.Supernatant recentrifuged 2000 x G/30 min.Supernatant 2nd recentrifuged 2000 x G/30 min, inoc, lii d'd".9 cc (A)Sediment I resuspended in H2O - inoc. 1 ml 15 dV (B)2/5/53 Challenged A and C (D s controls) 2085 - U"®2/17/53 Challenged B (F c control - unstaggered litters) 2087 - U"®Inter-vals before death in l/h daysTireatmentA, Supernatant9th i 10th \ 11th•  • • « •3 13 112th113th«  • •119thC. Heated sediment 2 3 2'iID, Controls on A & C ' 7i8 U ijB. Unheated sediment 3!li 6 2 jNo leuk, in A d.then challenged!F, Controls for • ^ 3 1 1| 1B challenged 1  smalli j  mouseE, Preliminary 3! 11isaline supernatant j iNo challenge | \1'  1recoveringaccident?Survived2/lUlj/110/201/160/10oA11CDIO.Does heating increase the protective effect of supernatants?15 ml, line Standard suspension, heated C/lk min (3 ml per heatingvial)7.I4 ml into each of two lusteroid tubes - 3250 x G/60 min.Supernatant recentrifuged - 3250 x G/60 min*Supernatant II -.9 ml to 9 cfcf; 1,0 cc to 1 (A)Sediment I from one tube resuspended - ,25 ml to 10 </</ (B)White layer of sediment I (upper) from other tube, in highly concentrateddose to 10 cfcT (C)All mice challenged in I1.8 hours with line Ijj, U"® x Std,; controls (D)CDll.Procedure as for CDIO, The only differences vjere in the greater amount ofstandard suspension and the slightly larger dose of the supernatant - 1,1 ml,and larger dose of the "white layer" material, which was taken from a centrifuge tube containing 10 ml instead of 7.U ml as in Exp, CDIO,Time of death in lA days after challenge with U*"®9th 10th 11th 12th Delayed , Survived«  • • ^ ..|A, SupernatantCDIOCDllB, Sediment (entire)CDIOCDllC, "White layer" of sed.CDIOCDllD, Controls onCDIOCDll1 13:3 2ilI1116 120 d1h/105/1010/109/10U/io9/100/101/101  n12CD12. CSH 2193 lb 2091CSH 219U lb 2091potency and the capacity to induce resistance survive afterline lb leukemic cells are incubated - 37° C/l2 hours?21; ml Standard suspension 2090, shaken in ice bath, setting "100" -30 min. Centrifuged in refrigerated room 32$0 x G - 60 min.Supernatant recentrifuged (as before) - 1-00 ml to lO^fd* (A), challenged inUS hours. Challenge control (D).Sediment resuspended - to 370 C incubator, 12 hours,Centrifuged - 3230 x G/60 min.Supernatant recentrifuged (as before) 1,0 cc to 10 dW" (B)Sediment resuspended - part - heated - U6° C/lk min - ,25 cc to 10 (C)(B) and (G) challenged in 1;8 hours - control on challenge = (E)Another part of resuspended sediment - ,25 cc to 10 cAf - no challenge (F)Time of death in l/h daysTreatmentA, Supernatantbefore incubationD, Controls on AchallengeB, Supernatant afterincubationC, Sediment afterincubation andIi6° C/ll; min,E, Controls on Band C challengeF, Sediment afterincubation -unchallenged9th*  t10th•  * »U 1 1jI  8 13 3 31; 1; 13 k 311th • Delayed23 dSurvived1/100/90/100/100/1010/10

Second quarterly progress report of research carried out by Long Island Biological Association for the Biological Department, Chemical Corps, Camp Detrick

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Second quarterly progress report of research carried out by Long Island Biological Association for the Biological Department, Chemical Corps, Camp Detrick

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