Patent Application: US-9498187-A

Abstract:
this invention relates to methods for modulating the effects of tnf and il - 1 , both the deleterious and the therapeutical effect of these cytokines . in case of deleterious effects , the invention provides methods for modulating the deleterious effect of tnf and / or il - 1 in mammals , by administering to a mammal sub - deleterious amounts of tnf and / or il - 1 . the tnf and / or il - 1 to be modulated may be endogenous , i . e . generated in the organism in severe cases in amounts deleterious to the organism , or exogenously administered to a patient in amounts which are potentially deleterious . the compositions of the invention comprise effective amounts of tnf and / or il - 1 with at least one pharmaceutically acceptable carrier . the invention also relates to methods for monitoring the modulation of the tnf effect in patients treated with tnf and / or il - 1 .

Description:
1 . identification of il - 1 as the leukocyte produced cytokine desensitizing cells to the cytotoxic effect of tnf crude preparations of leukocyte - produced cytokines were exposed to ph 2 . 0 . tnf is inactived by such treatment -- both with regard to its cytolytic function and to its ability to induce resistance to its own cytotoxicity . the tnf - free preparations were fractionated as shown in fig1 a and 1b and the desensitization activity copurified with il - 1 . desensitization activity was measured by applying the tested sample on human sv80 cells for a few hours and subsequently applying tnf and chi and measuring the extent of resulting cell death . further confirmation of the identity of the desensitizing cytokine as il - 1 was demonstrated by the neutralization of il - 1 with a monospecific antibody against il - 1 , as shown in fig2 . titration of the desensitization activity of semi - crude cytokine preparation produced by the u937 cells with ( x --- x ) or without ( x - x ) treatment of this preparation with antiserum raised against il - 1 , demonstrated that il - 1 can mediate the desensitizing effect (). desensitizing effect of il - 1 in cells of three different human cell lines : sv80 , hela , l132 was also demonstrated , illustrating the generality of this phenomenon , as shown in fig3 a - 3c . cytolytic and protective ( desensitization ) activities of the cytokines were determined in all the figures using sv - 80 cells which , for measurement of cytolytic activity , were incubated with serial dilutions of the tested cytokine for 12 hours together with 50 μg / ml chi ; for measuring protective activity , they were incubated for 4 hours with the tested cytokine and then for 12 hours with tnf , at the indicated concentrations , together with 50 μg / ml chi . the extent of cell killing was quantitated by the neutral - red uptake assay . a unit of cytolytic activity is defined as the concentration of the tested cytokine at which the amount of cells remaining viable was 50 % of those that remained viable on incubation with chi alone . a unit of protective activity is defined as that cytokine concentration protecting 50 % of the cells from killing by tnf . thus , the component of ph 2 . 0 - treated cytokine preparations inducing resistance to killing by tnf was identified as il - 1 , based on the following findings : a ) the protective activity and a typical activity of il - 1 ( thymocyte activation ) copurified , when crude preparations of u937 - produced cytokines were subjected to a series of fractionation steps , resulting in effective purification of il - 1 ( see fig1 a and 1b ). b ) monospecific antiserum to il - 1 neutralized the protective activity of such preparations ( see fig2 ). c ) il - 1 of different souces induced resistance to the cytotoxicity of tnf as effectively as did the crude preparations of cytokines ; some resistance was observed even on treatment with as little as 0 . 1 unit ( 3 pg ) of il - 1 per ml , as shown in fig2 . bacterial lipopolysaccharide , which may contaminate preparations of il - 1 , did not induce resistance even when applied at concentrations as high as 10 μg / ml . native purified il - 1 was prepared as follows : crude preparations of cytokines induced in the human histiocytic lymphoma cell line u937 ( 47 ) by 4 - beta - phorbol - 12 - myristate - 13 - acetate ( 5 ng / ml ) and sendai virus ( 48 ) were adsorbed to controlled - pore glass beads ( pg - 350 - 200 , sigma , st . louis , mo ). most of the protective activity and of laf activity , and only a minor part of the cytolytic activity and of ifn was recovered in the unbound material . it was concentrated by ultrafiltration on an amicon ym5 membrane and depleted of ifn - alpha and of all residual tnf by application on immunosorbent columns constructed of monoclonal antibodies against them . it was then dialyzed for 12 h against phosphate buffered saline , ph 2 . 0 . insoluble material was removed by centrifugation . following equilibration with 1m nacl , 10 mm sodium phosphate buffer , ph 7 . 4 , the protein was fractionated on an ultrogel aca 54 column ( 16 × 110 mm ) in 1m nacl , 10 mm sodium phosphate buffer , ph 7 . 4 , 0 . 1 mm ethylene diamine tetraacetic acid , 0 . 1 % polyethylene glycol ( m r 7000 - 9000 ) and 30 % ethylene glycol . fractions of 2 . 5 ml were collected and assayed for laf activity and for induction of resistance to tnf . the active fractions were pooled , concentrated , equilibrated with 20 mm sodium phosphate , ph 7 . 4 , and applied to a deae sephacel column ( 7 ml ) ( pharmacia , uppsala , sweden ) preequilibrated with the same buffer ( 49 ). both the protective and the laf activity were fully recovered in the material which remained unbound to the column . in analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis , that material was found to contain a major protein with an m r of about 17000 and only minor amounts of other proteins of lower m r . ( laf activity - lymphocyte activating factor activity ). 3 . interrelations between the effects of il - 1 and tnf on cell viability the protective effect of il - 1 , induced in human sv - 80 cells in the absence of chi , was comparable to that induced by tnf , as shown in fig3 a . however , as shown in fig9 a , unlike tnf , il - 1 has just a barely detectable cytolytic effect on these cells in the presence of chi . as illustrated in fig9 a under particular situations , i . e . when protein synthesis in the cell is blocked , il - 1 can have the reverse - potentiation effect on cell killing . at even minute concentrations , il - 1 potentiated the cytotoxicity of tnf , increasing the effectivity of cell killing by low concentrations of tnf to that elicited by as much as 25 - fold higher concentrations in the absence of il - 1 . in two other human cell lines , hela and l132 , il - 1 by itself was clearly cytolytic when applied together with chi , and this cytotoxicity appeared to be additive to that of tnf ( fig7 ). both in hela and in l132 cells , a protective effect of il - 1 , as well as of tnf , could be observed when these cytokines were applied in the absence of chi . ( fig3 b and 3c ). il - 1 or tnf provide protection not only against the cytotoxicity mediated in the presence of chi , but also against killing of cells in which synthesis of proteins has been suppressed by other agents . table ii shows the cytotoxicity of tnf against sv - 80 cells in the presence of the protein synthesis inhibitor , emetine and the rna synthesis inhibitor , actinomycin d . like chi , these two other inhibitors sensitized the sv - 80 cells to killing by tnf ; and as with chi the sensitization by both inhibitors is greatly reduced by pretreatment either with il - 1 or with tnf in the absence of the inhibitors . 4 . relation of the decrease in tnf receptors to increase in resistance against the cytotoxicity of tnf observed following treatment with il - 1 . cells treated with il - 1 as well as with tnf become more resistant to the cytolytic effect of tnf ( 50 ). as shown in fig4 the induction of resistance by il - 1 and by tnf followed a similar time course , save that with tnf resistance was reached earlier . resistance to cytolysis kept increasing until about 5 h following application of il - 1 ( fig4 ), even though the tnf receptor level had reached its lowest value already at about 1 h ( fig5 ). this slow development of resistance in treating sv - 80 cells with il - 1 can perhaps be related to the fact that in these cells il - 1 can also have the opposite effect . while inducing resistance to cytolysis when applied prior to the application of tnf . it is found to potentiate cytotoxicity when applied together with tnf ( 48 ). indeed , as shown in fig4 sv - 80 cells treated with il - 1 for less than an hour exhibited a higher vulnerability to the cytolytic effect of tnf did the untreated cells ( dashed line in fig4 ). an even more pronounced discrepancy between the effect of il - 1 on the receptors for tnf and on cell vulnerability to cytolysis by tnf could be observed in the recovery from those two il - 1 effects . as shown in fig8 removal of il - 1 from sv - 80 cells was followed by a quite rapid recovery of the receptors for tnf , but not of the vulnerability to its cytolytic effect . at 7 h following removal of il - 1 , tnf receptors were restored almost to their normal level while the resistance to killing by tnf remained unaltered . the maintenance of resistance to killing by the protein following removal of il - 1 from treated cells and the recovery of tnf receptors indicates that il - 1 induces some other changes which contribute to cell resistance to killing by tnf , in addition to its effect on the receptors to tnf . treatment of sv - 80 cells with il - 1 resulted in decreased expression of the receptors for tnf . in kinetic studies , tnf binding was found to decrease within a few minutes of il - 1 application , reaching its lowest level at about 1 h . thereafter , it increased slightly ; nevertheless even after 20 h in the presence of il - 1 , tnf binding was significantly lower than in untreated cells ( fig5 ). scatchard plot analysis of the binding shown in fig6 indicated that in both il - 1 treated and untreated cells , tnf binds to receptors of a homogeneous nature and that the affinity of the binding sites remains unaltered following il - 1 treatment , while their density greatly decreases ( 900 receptors / cell and kd of 9 . 7 × 10 - 11 m in cells treated for 4 h with 60 pg / ml of il - 1 , as compared to control values of 6200 receptors / cell and kd of 1 . 1 × 10 - 10 m ). in repeated examinations , variations in the level of receptors for tnf ( 2234 to 6960 binding sites / cell ) and some variation also in the estimated values of kd ( 9 . 6 × 10 - 11 m to 3 . 1 × 10 - 10 m ) were observed . yet in all experiments il - 1 , as well as pma , were found to affect only the number of binding sites for tnf and not their affinity . data from a representative example are shown in fig6 . ( pma - 4 - beta - phorbol - 12 - myristate - 13 - acetate ). the decrease in tnf receptors was temperature dependent . as shown in fig5 it could not be observed at 4 ° c ., even when il - 1 was added a few hours prior to the addition of tnf or at a great excess ( as much as 500 - fold ) of tnf . on the other hand , it appears that the effect of il - 1 was not dependent on protein synthesis . tnf and il - 1 were found not to compete directly for binding to their target cells . these findings indicate that the receptors to tnf and il - 1 are distinct molecules and that the expression of the receptors to tnf can be subject to regulation by il - 1 . as shown in table i and fig5 at 4 ° c . il - 1 , added to cells either together with radiolabelled tnf or a few hours prior to application of tnf , had no effect on binding of tnf . in contrast , at 37 ° c ., treatment of the human fibroblastoid sv80 cells and of foreskin fibroblasts ( fs - 11 ) with il - 1 resulted in a marked and rapid reduction of tnf binding . binding was maximally inhibited at 1 h following application of il - 1 and then slowly recovered . however , even 20 h following initiation of il - 1 treatment the binding was still markedly reduced . the effect of ifn - gamma on tnf receptors was also examined as shown in fig5 . as in other cells , ifn - gamma induced in the sv - 80 cells an increase in receptors for tnf which was initiated a few hours following the addition of ifn . cells in which the receptors for tnf had been increased by ifn also responded to il - 1 by a decrease in the number of tnf receptors , although not to the same low level as in cells which were not treated with ifn . quantitation of ifn - gamma receptors in the sv - 80 cells showed no alteration in their level after treatment of the cells by il - 1 or tnf . as shown in fig7 human foreskin fibroblasts were found to respond to the effect of il - 1 on tnf binding to an even greater extent than did sv - 80 cells . tnf receptors showed a more pronounced decrease and the effect could be observed at lower il - 1 concentrations . some decrease was induced in these cells by as little as 3 . 5 × 10 - 14 m il - 1 ( 0 . 02 laf u / ml ). decrease of tnf receptors was induced by il - 1 also in hela cells although less effectively than in the sv - 80 cells . on the other hand , no decrease in tnf receptors could be observed when u937 histiocytic lymphoma cells were treated with il - 1 . examining the binding of radiolabelled il - 1 to these different cell types , as shown in table i , suggested a correlation between the effect of il - 1 and the level of receptors to this protein . il - 1 binding was highest in the fs11 cells , lower in sv80 cells , even lower in hela cells , and below detectable levels in u937 cells . tnf did not compete with the labelled il - 1 for the binding . furthermore , treating the cells for 4 h with tnf ( at 17 ng / ml ) did not result in a decrease in their ability to bind il - 1 ( table i ). also the binding of radiolabelled rifn - gamma to the cells was not significantly changed following treatment by il - 1 or tnf . nine to thirteen weeks old balb / c mice were used in all experiments . tnf , il - 1 , lps , actinomycin - d ( act - d ) and d - galactosamine ( galn ) were solubilized in pbs and injected i . p . ; each in aliquots of 0 . 5 ml . for lethality determination tnf , il - 1 or lps were administered alone or 10 minutes following injection of act - d or galn ; for desensitization experiments mice were injected with tnf or il - 1 12 h prior to a challenge consisting of the same regimen as the lethality testing . following treatments the mice were continuously observed for a period of 72 h in order to determine the time of their death . in all cases , mice that survived 72 h appeared completely normal at that time . furthermore , part of these mice were observed for a week and found to show no sign of deterioration . all experiments were performed in duplicate with qualitatively the same results . two mice were examined for each experimental point , in each of the experiments . their actual survival time as well as the average of the survival time of the two mice used for each of the points are presented . act - d and galn sensitize mice to the lethal effect of tnf and il - 1 . in an attempt to elucidate the nature of the sensitizing effect of act - d and of galn , it was tested whether injection of tnf or il - 1 in the absence of these agents affects the response of mice to a subsequent injection of tnf or il - 1 together with act - d or galn . as shown in fig1 and 11 , mice injected with tnf + act - d 12 h after injection of tnf , survived the lethal effect of tnf + act - d for a longer time than mice which had not been pre - exposed to tnf alone . that protective effect was prominent in mice which were pretreated with a high dose of tnf ( 5 μg / mouse ) but could clearly be discerned even in mice which were pretreated with as little as 0 . 02 ug tnf . similarly , viability of the mice following injection with il - 1 + act - d was prolonged by prior injection with il - 1 . pretreatment with il - 1 also increased the ability of mice to survive a subsequent injection with tnf + act - d . inversely , mice pretreated with tnf showed an increased ability to survive the lethal effect of il - 1 + act - d . injecting mice with tnf and il - 1 together protected them from the lethal effect of tnf + act - d more effectively than their preinjection either with tnf or il - 1 alone , as exhibited by the rectangular in fig1 . mice injected with tnf or il - 1 were also protected from the lethal effect of subsequent injection of tnf or il - 1 in the presence of galn . that protection was even more effective than the protection against the lethal effect which these cytokines exert in the presence of act - d . at those concentrations of tnf and il - 1 applied in the experiment described in fig1 , death in sensitization with act - d was just delayed by a prior treatment with il - 1 or tnf while death occurring in sensitization with galn was actually prevented . galn - sensitized challenges were therefore chosen in examining the kinetics of the protective effect shown in fig1 . partial protection from the lethal effect of tnf galn , reflected in prolongation of survival time , could be observed already at 30 &# 39 ; after injection of tnf or il - 1 alone ( 10 μg and 0 . 4 μg / mouse , respectively ). one h after tnf / il - 1 injection , the mice were fully protected . they remained protected 12 h after the injection . twenty four h after pretreatment with tnf , one of the two mice died when injected with tnf + galn , suggesting some decrease in the protective effect . fourty eight h after pretreatment the protection had fully abated . the compositions of the invention can be used for antitumor , antibacterial , antiviral or antiparasitic treatment . effective amounts administered to mice are for pretreatment with il - 1 in the range of between 0 . 5 to 25 ng per one gr of body weight of mouse and subsequent treatment ( subcutaneous ) with tnf in the range of 0 . 1 to 10 ug / g body weight , or subsequent treatment ( intraperitional ) with tnf + actinomycin - d , 4 - 40 ng / g and 0 . 5 to 1 . 5 μg / g respectively . the amounts of active compound to be administered to humans will depend on various factors , such as the state of the patient , the symptoms to be treated , the severity of the affliction , the route of administration and the judgment of the prescribing physician . in the more severe cases , higher dosages of combinations of tnf and il - 1 may be considered , optionally together with interferon and / or sensitizing agent . administration of the il - 1 and tnf , with or without interferon , metabolic blocker , or chemotherapeutically active drug can be via any acceptable mode of administration . treatment via injection is preferred . known suitable modes of administration are intravenous injection , intraperitoneal , intramuscular or intralesional injection or infusion . local treatment may be considered in case of external infections . the compositions of the invention are prepared for administration by mixing the active materials , i . e . the il - 1 , tnf , ifns , metabolic blockers or the chemotherapeutically active drugs , with physiologically acceptable carrier , i . e . carriers which are non - toxic to recipients at the dosages and concentrations employed . for example , the carrier could be one or more of the following two materials : buffers , antioxidant , wetting or emulsifying agents , amino acids , polypeptides , proteins carbohydrates , chelating agents such as edta and other stabilizers and excipients . an example for a composition according to the invention is as follows : two sterilized glass vials , vial a containing sub - deleterious amounts of il - 1 dissolved in a physiological saline solution and vial b containing therapeutically effective amounts of tnf dissolved in physiological saline . the administration of vials a and b is carried out in accordance with written instructions which accompany the two vials , the instructions indicating to inject the contents of vial a and after a predetermined time interval to inject the contents of vial b . table i__________________________________________________________________________effects of il - 1 on the binding of tnf , of tnf on the binding of il - 1 , andof tnf and il - 1on the binding of ifn - gamma to various cells . sup . 125 i - tnf . sup . 125 i - il - 1 . sup . 125 i - ifn - gama competition pretreatment competition pretreatment pretreatment pretreatment with il - 1 with il - 1 with tnf with tnf with tnf with il - 1 -- ( 30 ng / ml ) ( 60 pg / ml ) -- ( 17 ng / ml ) ( 17 ng / ml ) -- ( 17 ng / ml ) ( 60 pg / ml ) __________________________________________________________________________sv80 2400 2200 800 400 400 400 1600 1600 1500fs11 1800 1800 100 900 900 900 1700 1800 1800hela 7900 7900 6400 200 n . d . n . d . * n . d . n . d . n . d . u937 3300 3400 3100 & lt ; 20 & lt ; 20 & lt ; 20 n . d . n . d . n . d . __________________________________________________________________________ * n . d . = not determined legend to table i : effect of il - 1 on the binding of tnf , of tnf on the binding of il - 1 and of tnf and il - 1 on the binding of ifn - gamma to various cells binding of 125 i - rtnf ( at 3 . 6 ng / ml ), 125 i - ril - 1 ( at 49 ng / ml ) and 125 i - rifn - gamma ( at 13 ng / ml ) to the indicated cells was determined at 40 ° c . as described below . competition of il - 1 and tnf with each other for binding to their receptors was examined by applying the nonlabelled cytokines , at the indicated concentrations , simultaneously with the labelled proteins to the binding asay . the effect of pretreatment with il - 1 or tnf was examined by applying the proteins on the cells at 37 ° c . for 4 h prior to application of the labelled cytokines . human tnf and ifn - gamma were radiolabelled as previously described ( 30 , 44 ); the first with the chloramine t reagent to specific radioactivity of 42 ci / g , and the second with the bolton and hunter reagent to specific radioactivity of 18 ci / g . il - 1 was radioiodinated with the chloramine t reagent ( 31 ) to specific radioactivity of 40 ci / g . recovery of bioactivity following the iodination , as estimated by measuring the protective effect of the protein against the cytotoxicity of tnf ( 25 ) was over 95 %. for determining binding of the radiolabelled proteins to sv80 ( 45 ), or hela ( 46 ) cells or to the fs11 strain of foreskin fibroblasts ( established in the laboratory ), these cells were seeded in growth medium ( eagle &# 39 ; s minimal essential medium containing 10 % fetal calf serum ) into 18 mm tissue culture plates , at a density of 2 . 5 × 10 5 cells / plate . following 24 h incubation at 37 ° c ., the plates were transferred to ice , the growth medium removed and the radiolabelled proteins applied , in duplicates , either alone or in the presence of 1000 - fold excess of the nonlabelled protein , in 150 ul growth medium also containing 20 mm hepes buffer and 15 mm sodium azide . the nonadherent u937 cells ( 47 ) were incubated with the labelled tnf in samples of 5 × 10 5 cells in tubes under otherwise identical conditions . following 2 h incubation , with constant agitation , at 4 ° c ., the fs11 and hela cells were rinsed 3 times with a buffer containing 140 mm nacl , 1 . 5 mm kh 2 po 4 , 8 mm na 2 hpo 4 , 2 . 7 mm kcl , 0 . 5 mm mgcl 2 , 0 . 9 mm cacl 2 , 0 . 5 % bovine serum albumin and 15 mm sodium azide ( pbs / bsa ). the cells were then detached in ca 2 + and mg 2 + free pbs containing 5 mm ethylene diamine tetraacetic acid and transferred to counting tubes for determining their associated label . sv80 cells were found to detach from the substrate in cold , therefore , following incubation with the labelled proteins they were transferred to tubes , washed 3 times by spinning , each time , for 10 mins , at 250 g and resuspending in 5 ml pbs / bsa and then transferred to counting tubes . u937 cells were washed in the same way . nonspecific binding of the radiolabelled tnf and il - 1 , observed in the presence of an excess of the nonlabelled cytokines were as follows : in sv80 cells 200 and 200 cpm , in fs11 cells 300 and 200 cpm , in hela cells 700 and 500 cpm and in the u937 cells 600 and 200 cpm for the labelled tnf and il - 1 respectively . non specific binding of ifn - gamma was 300 cpm in sv80 cells and 600 cpm in the fs11 cells . specific binding was calculated by subtracting the values of nonspecific binding from the binding observed with the labelled cytokines alone . intraduplicate variation in binding was in the range of 10 % of the average value . table ii______________________________________protective effect of il - 1 and oftnf against the cytotoxicity mediatedby tnf in presence of chi , emetine and actinomycin d . cell viability upon treatment with tnf ( 500 u / ml ) in ab - pretreatment with sence no pre - tnf il - 1sensitizing of tnf treatment ( 20 u / ml ) ( 2 u / ml ) agent od . sub . 540 od . sub . 540 % od . sub . 540 % od . sub . 540 % ______________________________________ -- 0 . 305 0 . 319 105 0 . 308 101 0 . 293 96cyclohexi - 0 . 226 0 . 020 9 0 . 195 86 0 . 163 72mide ( 50 μg / ml ) emetine 0 . 262 0 . 029 11 0 . 234 89 0 . 220 84 ( 10 μg / ml ) actino - 0 . 243 0 . 052 21 0 . 236 97 0 . 215 88mycin d ( 5 μg / ml ) ______________________________________ sv - 80 cells following 4 h pretreatment with il1 or tnf or without such pretreatment were tested for their vulnerability to the cytolytic effect of tnf applied for 12 h together with the indicated sensitizing agents . viability of the cells is presented as neutral red uptake ( od . sub . 540 ) and , in cultures with tnf , percentagewise as compared to the viability in cultures incubated with the sensitizing agent alone . table iii______________________________________decrease of tnf receptor expression by human peripheralblood leukocytes in response to il - 1 tnf binding to : il - 1 applied granulocytes mononuclear leukocytes ( u / ml ) ( lpm ) ______________________________________0 520 11620 . 01 510 n . d . 0 . 1 280 n . d . 1 305 100010 284 900100 236 700______________________________________ n . d . not determined granulocytes and mononuclear leukocytes were isolated from freshly collected blood by spinning through a &# 34 ; monopoly &# 34 ; cushion . they were then incubated for 1 hr at 37 ° c . in a dulbecco &# 39 ; s modified eagle &# 39 ; s medium containing 10 % fetal calf serum and il - 1 at the indicated concentration . binding the 125 i - labelled tnf to aliquots of 10 6 leukocytes was then determined . as shown in the table , a significant decrease in tnf receptor expression by granulocytes was found to be induced with as little as 0 . 1 u / ml ( laf activity ) of il - 1 . 1 . aggarwal , b . d . et al . j . biol . chem . 260 , 2345 - 2354 ( 1985 ). 3 . auron , p . e . et al . proc . natn . acad . sci . u . s . a . 81 , 7907 - 7911 ( 1984 ). 4 . cameron , p . et al . j . exp . med . 162 , 790 - 801 ( 1985 ). 7 . granger , g . a . & amp ; kolb , w . p . j . immunol . 101 , 111 - 120 ( 1968 ). 8 . ruddle , n . h . & amp ; waksman , b . h . j . exp . med . 128 , 1267 - 1279 ( 1968 ). 9 . carswell , e . a . et al . proc . natn . acad . sci . u . s . a . 72 , 3666 - 3670 ( 1975 ). 10 . wallach , d . in interferon 7 ( ed . gresser , i .) 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