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i NK19, 0126 0%/j 0 Biological Aspect® of Cancer Ileee;arclz's . c,.4aewas C. irrrte, &a., Diremr Bmeriua, Rosnue B. ,]adcson Mmarial La6oratory, Bar HarBor, Maine; 3ciu,u'fic Diroor, Tobaao lnduaay Reaeardr Conrmiaee The following discussion of cancer research is frankly from the biological point of view and is therefore limited in scope and application. Principles and processes of biological interest will be chiefly considered. No attempt will be made to oover in detail the vast amount of published data on experiments which contribute to-definition or evaluation of the various broader subdivisions of the field. If the reader is interested in a more detailed perusal of c-epporting experimental evidence for general etate- mentg, he oan find a partial bibliography in the writer'e booklet on Genetica, Btiotagieat Individualify, and Chvuer.^_ From this legimcing he can, by covering the literature since 1954 and by tracing back from tho given references, obtain some idea of the tremendous vigor of preaentrday reseaech in this field and the extent and divereity of the experimental work already accomplished. Bankgronnd Researoh The history of the development of the contemporary major action in the $eld of experimental biological research on aanew presents an intereat- ing parallel with the last great critical battle in the war between the Staft. At (Iettyeburg the first contacts of the opposing forqee was largely a "blind" and unexpected meeting of the cavalry "antennae" of both armies. Each had fittle or no idea where the other was until actual contact occurred. From and around t,hie initial center of entanglement there developed, by the assembly of more and more unite of infantry and artiltery, a major alignment and a fierce and final engagement of long duration and decisive significance. In biological cancer research early experiments on animele, largely with mice, rate, and fowl, were 9cattered and chiefly observational. The technique of tumor traneplantation was one of the most important tools and was being extensively employed by workers under the Imperial ~ WoUsA tapu6lloetlam eeptem6n!!, tYbf. • Dr. UtW.u IovlOed by tEe naefd er 6Alear m esprou Ele vlem mlmvet proElem• m oenaer. e 9lrolmd Ldvanlty Pnq ML 40 l.-d d fb Nnlwd drw lnden4 V.L {q l1w b ne..f 1189

I H KE01 26061 442 LI7TLE 1 Cancer Fund in Great Britain and at the Rockefeller Institute, the Cancer Commission of Harvard Unive.-eity, and the Crocker Institute in this country. Continental European research was largely aimed at the discovery of infectious agents and the possible development of immunity. This point of view also activated a number of British and American investigators. This was the stage comparable in many ways to the "cavalry" contacts with the enemy. There was similar limitation of the type of a-eapons which could be used and there was also the same sort of isolation and . importance of individual effort or the work of smell units. It is, of course, impossible to single out all the factors disclosed by research at this stage, which later became the major elements in leading to further developments. Some, however, can be safely mentioned because their basic connection with existing and future research is clear and functional. In presenting them there is no intent to indicate relative "imporsance" for, until the final solution of <uany problems that are still unsolved or even unformulated, any rating would be unwise if not impossible. Dtsoooery ond Isobcion cLf 111eenable Agewte From the pioneer work of Rous and his associates, the existence of a filterable agent (virus) was dearly identified as the chief etiological factor in producing psreomsa in the domestic fowl. This was an exciting and stimulating discovery. From it there developed a still active series of experiments (Claude, Duran-Reynals, Porter, and others) which gave important information on the morphology, reproduction, and modifiability of the virus and of other similar agenta. Shope described a papilloma-fonming virus in the rabbit and thereby demonstrated that the mammslian cell can include a virus which actually allows an increased rate of cell division. The staff of the Jackson Laboratory, stimulated by the recognition of a strong matensl influence in the etiology of mouse mammary adesooar- einomes (Murray, Little), published in 1933 evidence of the general esistr enoe of this phenomenon in several high-tumor strainA. In the eame laboratory, Bittner detenained that the agent which caused this maternal in9nence was ordinarily transmitted in milk during nursing by the young. The agent was later shown to be preeent in the blood and in various body tissues of both males and females of "high-tumor" etrains (Woolley, Little). - Its selective distribution was later demonstrated by Hummel who found that it was absent from the placenta. The fact that it apparently remained inactive in the intact meles was also highly significant and suggestive, which encourages the theory that other latent virmses may be factors in the etiology of other types of neopleem. The filterable nature of the mammary-tumor inciter and its immuno- logicel properties, together with some evidence of its identification under the electron microec+ope, have further etrengthened the belief that viruses are possible etiologieal agents of general importance in neopUaeia. The work of Gross aud others on salivary-gland tumora and on leukemia in mice also sse..el ur N. flW..d Cawen lrtltW I

I HY,801 26062 BIO100iCAL ABFEC'r8 OF CANCER RESEARCH 44H falla in with the theory that virus phenomena are a field of continuing promise for the investigator. In pure genetic research certain similarities between viroid and genic qualities and behavior keep active the interest of research v'vologlata and biologists in intracellular activity in relation to mitosis and molecu'.ar organization. The development of the initial and early contacts with the tumorigenie filterable agents into a full-scale, broad, and vigorous research "front" is one phase of the major "engagement" in which those iuves- tigating the oause and control of neoplesia are and will be concerned. Genetic Theory qf Tissue Traneplantation I The initial objectives of tumor transplantation in animals were: (a) to propagate the chosen tumor in order to obtain a larger supply of tissue for continued study, (b) to investigate the host reaction to subcutaneous or intraperitoneal implants of auto-, homo•, and heteroplastic tissues with a vim to detsrmining the nature of the factors which determine suscepti- bility and nonsueceptibility. These two objectives were, o[course, pursued at the same time, with the emphasis of earlier work ah.ifting somewhat whimeically as tme experimental results suggested. Since, in most cases, little or nothing was known s~.R•ut the genetic nature ot the annhals used as hosts and since these animals differed funda- mentaAg and invisibly from one another in their genetic characteristics, their responea to inoculation was varied. Succeai;ve groups of animals inoculated gave "t,akes'• of the tumor from 0 to 100 percent. T're British sehool' and some Americans explained this variation eq a response to changes in °virulenco" in the tumor itself. Extensive papers were published dealing with euch changes both in untreated animals and in those which bed, before or after inoculation, received treatment with varioo4 eupposedlJ+ "preventive" or °therapeutic" agents. To add to the confusion of the unpredictability and of the irregularity of consecutive results, it was obaerved that temporary pLTeietenee or even growth of the tumor implant was foIlowed, in some aninnale, by its regrrs- eion and dieappearance even in "untreated" animals. A quantitative phase was thus added to the already obscune problem This was the eituat.lon in the 6rat decade of the Twentieth Century when th-s work of Tyszer begge, to point the way to an opportuaity for progress toward a more definite and satiefaotory level of experimentation. This waas the phase of bringing up heavier units after the "cavalry•' aontaot. Tysaer had access to a etrain of Japanese waltzing mice which had been used by Yerkes in extensive behavior atudW, and which were being produced for research purposes by a Mrs. Lambert in a town outeide of Bost9n. The Japanese mice were probably descended from the Asiatic mouse Mue badris+HCa and were undoubtedl',p consh!irabl,y inbred having been derived from an originally very restricted nucleus of animsls. They were amall and delicate, and latsr experience with their eueceptibili,ty to 1.te4naa,H.miriia

;i K101 2Ei063 i i infections suggests that Mrs. Lambert possessed an unusual amount of skill and patience to produce them in the numbers that she did. • These nrica developed "spontaneously" a number of matamary adeno- careinomas and an occasional Sbrosarcoma. Tyzzer found that such tumors, on transplantation to other mice of the lesmbert strain, grew in practically 100 percent of the inoculated animals. The same tumors failed to grow in all mice of other strains. This presented Tyner with an opportunity to study the reaction of first- and second-generation hybrids between the 100 percent susceptible and the nonsuscaptible strains. The crosses were made and a mammary adenocarcinoma JWA was used for inoculation. Ald the first generation hybrids grew the tumor. Surprisingly none of the 70-odd second-generation hybrids grew it. At this point the writer took over the experiment and repeated the crosses. As before, all F, hybrids were susceptible. Of 183 seoond-generstion hybrids, 3 grew the tumor and 180 were noneusceptible. This reaultt wes confusing and unorthodox genetically. In 1814-10 the writer explained the experimental findings on the theory that the Japfinean we3"g mice introduced into the cross from 12 to 14 independent Mendelian genes which were p,e+eut ia att fi, animaFa, thus producing suaceptibility, to the Japanese waltzer tumor tissue. In the second generation the random aasortment and recombination of these genes would not reproduce simultaneously ;he total of 12 to 14 required genps, eacept rarely, for most F, animals would be derived from germ cells lacking one or more of the needed genes and these animale wnuld be nonsusceptible. A later tsperiment using a Japanese waltzer fibmsarcoma gave an F, ratio, suggesting that here 4 to 6 genes were involved. This wss the Geneti.3 Theory of Mransplantaitaon. Soon after, Strong began working with transplanted tumors of various cownon (nonwaltffing') mouse strains. He obtained 3-factor, 2-factor. and finally 1-faetor ratios. Backcroas generations confirmed the genetic theory and it became established and is generally accepted. By the use of this genetic theory various important lines of investigation were opened up. Among them the following may be mentioned as examples. 1) Changes in the genetic nature of tumora themselves were detated and analysed. These were always in the directiou; of simplification by the inactivation or by the disappearance of one or more genes. The changes occurred suddenly tuo irregularly as far as controllable faatom were concerned (Bittner, Strong, Cloudman). 2) By selective inbreeding, "single factor" lines were established and the nature o4 the genes uv anelfzed (Saell, Gorer). As a result, the genetics of "histooompatzbility" has been, and is being, determined with great aocaracy. 8) The genetio theory has led to the recognition of "enhancing" effects in tumor and in normal tissue growth following transplantation; these enhancing effects are of basic importance to our inLreaeing knowledge of immunologiaal prooesees. /aart .t ub R.N.d CoM. Irs~.

I H H101 26064 BIOLOpICAL A8P8CT8 OF CANCER 8F$EARCH 445 i 4) The ose of known and controllable genetic stocks and of tbeir tumors has provided the animal material for the established program bioassay of chemotberspeutic agents. 6) The fact that the host apimal can sometimes modify the biologicel nature of tho transplanted tumor has been recognized and is being investi- gated (Barrett). 6) The problem of temporary growth followed by regression of the tumor he.s been sufficiently defined to make it practical to study and control some of the quantitative aspects of that proeess. 7) The successful application of the genetic theory of transplantation to normal as well as to neoplestio tissue hes provided an experimental method of analyzing strain and individual differences in biochemical composition and in growth phenomena of various organs and tissues as well as the comparison of normal with neoplastic tissue. 8) The rate at which the individual develops its specificity in various tissues and in organ systems can be studied and analyzed by inoculation of alien tissues at various chronological nge8. Transplantation has thus eorue to bu snot,her bra:d and active 8eld in which biological research will continue to expand and to deepen with fruitful results. It is a major "front" in the war against cancer. Development of Inbred St.aina In t909 Johannsen, a Danish botanist, described and explained the production and development of genetically homogeneous "pure lines" of besns by the continued pronese of inbreeding through oelt-fertilitation. He bed previously (1903) seen the possibility of such a technique but did not prove it experimentally until latsr. In that same year the writer, working under Dr. W. E. Castle at Harvard, had, by recombination of coat-cofor genes, produced dl7ute-brown (DBA) and pink-eyed dilute- brown p)BAp) mice. Brother X eiater matings of several linee of each of these varieties was etsrted at once. One of the dilute-brown hpmoge- neous lines survived and is now the DBA/t strain. It ia eleo the progenitor of the homogeneous DBAJ2 eubstaain. The proeeea of producing homogeneous genetic strains by eliminating hetero$ygoeity can be sueoesefully osrried out by unbroken brother )( sister or parent X offspring matings. Although the "purification" proceeds more slowly than under self-fertilization, the end result is the eeme. Strong, by similar methods, produced the albino A strain and the very valuable C8H agouti strain. The writer eimilerf,v produced C67BL and turaed over C88BL to MacDowell who completed its inbreeding. The C57BR strains A, B, end C were isolated from descendsnte of a brown segreg4te in C67BL by J. M. Murray. These strains, together with BdLBk: STaLI, AKIi, and otbers, form the foundation for the great majority of medical reseanoh being carried on with inbred mice: Duuning and Curtis, at the GTocker Institute;Xing, at the Wietar Institute; and Castle, at Harvard, produced and studied inbred rat straine. Eaton and Wright did the same for guinea pigs. Castle and, later, 8nwin inbred a.u.% n..a.un..roa

H NI01 26065 I i I 446 r.rMe rabbits also, though not eo closely as was the case with smaller rodents. At about the same time that the earlier inbred strains of mice were being developed, Slye was publishing a eeries of papers emphasizing the effecL of beredity on the production of various types of neoplaems among the mice in bar laboratory. Although the pedigreea of her experimental animals were complicated, she established beyond any doubt: (a) that cancer in general occurred much more frequently in successive generations of some families than it did in others; (6) that tumors of the sama type often occurred within certain families much more frequently than within others. The creation of inbred strains definitely proved that the tendency to produce a given type of tumor could be fixed and maintained in successive generations. This was also evidence that at least some of the etiological factors in tumorigenesis are specific for tumor type and can be separated from one another by genetic selection. Within a genetically homogeneous strain, transplants of normal or of neoplastic tissue from animal to animal are treated by the host as auto- tranaplanta. F, animals produ^.ed by or-Asing two inbred strains can grow, either simultaneously or separately, normal or neoplastic tissue from bctA parent.etraina. Some of the important lines of research opened up by the formation and utilisstion of inbred strains are as follows: 1) By the appearance with a high degree of fidelity of the same general types of morphological and physiological development among the in- dividuals and among successive generations of an inbred strain, the inveatigator knows in advance with a far greater degree of accuracy what to expect than would be possible under any other circumstances or with any other type of material. 2) By exhibiting definite characteristic types and degrees of hormonal aution within thp inbred strain, experimental manipulation of hormonal balance can give a new method for anal.ysing the possible role of hor- monal action in tumortgenesie or in other biological prorease.s. 3) The high degree of genetic uniformity in an inbred strain provides a "constant" biological unit for quantitation in assaying extraneous agenta, or in experimental procedures introduced by the investigator. 4) The reverse technique can be used aleo with great effectiveness; a single substance or experimental procedure can be tested against a num- ber of different genetic backgrounde of known origin end natiu-e. Use of these opportunities bae already resulted in exciting and stimulat- ing advances in almost every phase of experimental cancer research. 1lere is every reason to believe that they will continue to be an easential ,lement In hastening and extending discovery of new knowledge and will also be the beat obtainable tool for repeatiag, extending, and evaluating experimental procedures in biological and in medical research. The inbred strains and their hybrids used in conjunction with the other methods ard tenhniques descibed is tbus an invaluable "weapon" for future re- eeareh. jCOIMW W do NatlOtld dumR ItldlYtY

1 NK1012606G BIOIA(iICAL ABPSC'1'8 OF CANCER RESEARCH 447 7yseue-Ceafture Deoe[opments Although interesting and important discoveries in cell physiology and morphology have already resulted from the use of tissue-culture techniques, there remains a great number of exciting twa~aibiiities as yet uninvesti- gated or not su9iciently explored. Among these is the potential value of synthetic culture medin in analysis of cell composition and physiology. There should be experimental variation in chemical components not only of adequate synthetic media but a]so of those media which may reveal selective and significant survival differences, in the response of various genetically controlled cells or tissues. The possibilities in this field of research would seem to be almost unlimited. More extensive use of exposure in vitro of various genetically controlled tissues to diS'ereotsuspected or known carcinogens should, if followed by transplantation into hosts of known but differ•ent genetic types and into hosts of different chronological ages, reveal many new facts of impor- tance. These are the eorta of techniques chat should help ce to bring isto direct obserroatiwa ohanges in tissue response which will in turn aid in analyzing and in cventually understanding tho activities of cells and tis- sues in different types of msmmaLsn individo.als tnhere direst obasasntion is sot g+eeaible. . IrrstHaeion ffects and Isotopes From the more directly biological point of view, the ui.ilisation of gene~.ically controlled animals, tissues, and cells offers great opportunities bot.L in quslit3 :md quantity r.i irradiation problems. The work of W. L. and L. B. Russell has alreaay shown the practicsl:ty and fruitfulness of this approxob. Much of the wor:; with tbese agents will, however, be in their biopbysia.l and biochemical relationship. The writer is not sufficiently inforu4ed in either of these fields to comment intelligently on their possible details of development. If full advantage is taken of the known techniques of transplantation, inbreeding, and t5ssue culture, either singly or in combination, thp venra- tility and effectiveness of naaly:ing and interpreting irradiation eSeotg and the significance of isotope pathways i„ ,u.d.e'ur.lio ond morphogenetic processes will be greatly incresaed. Since be'n- and psr,ma =;rraiaat+'or: has been well identaBed as hn-ring mutagenic and carcinogenic properties, the rolationship betaeen the exaerimentp3 responses of somatic and germ ce119 should be further ex- plained by experiments comparing such irradistion effects on fJto two types of celle. Up to now the effects of irradiation on the germ cells of mamtaals have been expreased in mutations which are usually either lethal or which produce morphological abnormalities involving dieproportion or other depsrtures from the symmetry and balance of normal development. It is interesting that there la little if any evidence of Increased unoon- trolled neoplastio growth in the emblyos or young nrodueed by the combination or participation of mutated germ cells. Ye1. !q Ne. 0. rn.1 19fs

i HKI0126067 I 448 zrrm On the other hand, there are in somatic cells many casea of induced neopkaiia, which often occur after a long latent period. The biological significance of such prolonged latent periods is not understood and is not receiving oearly so much attention and investigation as it deserves. Compared with the vast number of cells receiving irradiation in any given area, the number that exhibit a neoplastic change is infinitesimal. If a direct, simple, single process is involved in this change, why do not alt or at least the great majority of exposed cells show it? The evidence seems to favor a hypothesis of a series or chain of intracellular changes which, in the vast majority of cells, is broken or obstructed before the sequence reaches the tumorigenic threshold. What the trigger mecha- ni4ms may be for each step and for each type of tissue are the sort of problems that will require patient and prolonged investigation to match the duration and finality of the processes being studied. Cell Cheoaistrr and Funetion Modern methods of analyzing the chemical composition of cells give real promise of adding knowledge of the differences betweea cRlls at various stages of differentiation, of various specific tissues, an;f even at different stages of functional activity in the same tissue. It would, however, be well to remember that when a chemical is isolated fmm the cell and is measured or ageayed, the osll itself is no longer dynamic or an integrated living system. This, of course, is not in the nature of a atitieiem. It is merely a naturol technical Gmitation which is made necessary by the definition of the type of the investigation itself. The same limitation applies to pathology where it will always be impossible to state definitely what the "next stage" of future development would have been hed the cell or tissue been left in the living bod~ instead of being fixed, stained, and sectioned. Studies of cell ehemistiy and cell function based on isolated material should thoniore be integrated with and eupplemented by studies on grrups of living cells of krlown and uniform genetic composition in oioo or in oilro. Farpert.nental Cancinogeneais Since the initial discovery of the prooess of experimental oaroinogenesis on the ears of rabbits, there have been literally thousands of ezperiments with different challenging agents, dosages, sites, strains, species, and ages of animals. Agents have been used alone, in combination, or in soguenoe. The excellent and comprehensive assembly and tabulation of tbese ea- periments by Hartwell w8l con'vince anyone of several facts. i) There is a very large number of chemical agents that ean prove to be carcinogenic in one or more types of animal material. 2) There is every sown to believe that the number of auch substances will be steadily and eigni& cantl jr innressed as time goes on. 3) The action of these substances ie now being recorded and assayed under such scattered and uncoordinated conditions that no integrated or progressively analvyaable program of prooedure, approach, or reduction of variables essts. s.o.d a do r.uead c~ t..o.r .

I r i I HKI0126068 sloLGaICAL ABPSCTB OF cAMCBn a88BAaca 449 There is naturally a question as to when and bow far one can extrapolate from animal research to man under any given set of experimental condi- tions. Some general comments in this conneetion may be helpful. The germ cells of higher mssmmstls appear, as a group, to poesee~a most of their baeic types of organization in common. The proeeas of matura- tion, ovulation, ferta7ization, embryonic developmental sequence, im- plantation, and parturition aro essentially similar in primates, carnivores, ungulatee, and rodents, which are the four orders commonly studied. The internal organization of chromosomes and the behavior of genes in the four orders aro closely parallel. It would eeem likely, therefore, that a high degree of extrapolation f' om one order to another is permissible in the field of germ-ceII and early developmental studies. Hormonal origins and behavior are also very much alike in the four orders. Until new evidence of divergence is found, one may compare with oonsiderable scientifio justification, experimental results in endocrin- ology with expectation of fairly olose conformity in interorder behavior. The functions and baei a utructure of the great organ eyetsrss-nervoue, digestive, excretory, circulatory, respiratory, skeletal, and reproductive- are essentially the same, although proportional, regional tissue develop- ment may vary considerably within an organ or organ system. The outer epithelial tissues show a considerable variation in structure and response to environmental stimuli. The sldn, hair, nw1s, and teeth are higWy specialized; the3e vary greatly in form and function not only between orders but also betn eon species, strains, individuals, and even between different parts of the same individual. Esperieiental re:tponse of these structures to various challenges or environmental faetors may be expected to be as specific, characteristic, and varied as is the type of animal selected for study. It is very important to remember this fact, for the speed and convenient experimental response of many of these easily accessible and observable tissues sre tempting qualities for the avid and impatient investigator. There is real danger of overextension and unwise generalization from highly restricted and specialized data. This is the main reason why in order to use such dara to fJieir full limit of f ustt}/'iabre extrapol,aeion. the de- wlopmend of a tabfe of alandard Quantitotim raeponase of peneteeallyeontrofled animal teasues to known chaflenges or expoetaw is an absofute pragquieife. Motlr such a foundation available, the value of such tissues for assay may prove to be unique and of last3ng importance. R'itAove it, observa- tions :;,ll renkain isolated, fragmentary, e.nd of doubtful usefulness in transfer, extension, or applicability to other work with the same or with different species. It may prove to be impractical to integrats work of this sort; but until the possibility is considered and some concerted e,nd continuing effort at definition and coordination is made, the total of eaparimental results will remain a hodgepodge of disconnected observa- tions of minimum value as knowledge exchangeable or utilizable between investigetme. It may be well, while waiting for the development of new and more wL a4n.ur.nk 1060 I

r HK10126069 wM$ "o refined methods, to review the commonly used methods for bioassay of chemical carcinogens and to attempt to determine and to define the variables encounteied in the present experimental approach to this problem. SwJaee paintdng is one of the more important and widely used methods of application. lnaedion is another. Inhalation (which is often ingestion) is a third. The following discussion applies primarily to the first two methods but also, at least in some of its phases, to experiments using inhalation or aerosol techniques. The first series (I) of six factore, noted below, involves pertinent controllable attributes of the animal or tissue used for assay. The second series (Q) of three variables deals with methodology to be considered in broad techniques of application. I. Host Reaponar A) The age factor should be evaluated, both by observation of the total animal and by study of the relative aging of the speciHcally challenged tissue. This can be done at prenatal, neonatal, and postnatal levels up to snd including senescence. Phyeiological and chronological age do not always develop pard paesu. B) The gen,eEic atn.dae of animals used should be considered as an aid in the analysis of the carcinogenic effects. Strain response to chailenge should be correlated with the known and reoorded morphological and physiological characteristics of the strain. C) The effects of application of the oxperimental :.gent should be studied on various areae of the akia or on selected target tissues and organs which develop in different ways atrudura[lp or at different rates (according to degree of differentiation, physiological or mitotic activity, repair rate, etc.). Such studies shonld lead to additionat information on host reaction not only in relation to laiatologdodl changes dure+ig mor- phogsnest,s, but in relation to certain genet+c influences which may predictably modify the development of the challenged structures (genes for brachyury, polydactyliem, short-eare, hairlessnese, et.o.). D) The evaluation of the factors of dispersion and of localisation of the agent used should a]eo be considered. This involves applica- tion to a defined structure (tail, ear, digit), which oan later be removed by surgery or isolated in part or sn toto by physical (temperature, radiation) or chemical (anesthesia, neutralization) means. E) Experimental manipulation of hormonal level should be intro- duced as another means of modifying internal balance and resulting host response. Such studies will oontribute not only to our knowledge of the generaC processes of earainogenesla but also to the possible anal,ysis of recognized ees (gonad, pituitary, adrenal) or developmental differences (thymus, thyroid, and above-mentioned glande), and the detection of new ones. F) Manipulation of vitamins and other ratttritional elementa as modifiers of the tissues obs]lenged should be a field of investigation. It is t.o.r .r H. n.d..d Ga... 1.ulue. I i r