[CANCER RESEARCH 41, 2598-2555, June 1981]0008-5472/81 /0041-OOOOS02.00

Kinetics of the Acute-Phase Reaction in Rats after Tumor

Transplantation

Mohamed Abd-el-Fattah, Reiner, Scherer, Fouad Mounir Fouad, and Gerhard Ruhenstroth-Bauer'

Max-Planck Institut fürBiochemie, Abteilung fürExperimentelle Medizin, D-8033 Martinsried bei München,Federal Republic of Germany

ABSTRACT

Transplantation of Yoshida sarcoma (solid type) and Zajdelaascites hepatoma tumors in rats induces a biphasic change inthe concentration of the following five acute-phase proteins:a-1-acid glycoprotein; a-1-antitrypsin; haptoglobin; hemo-

pexin; and ceruloplasmin. These proteins and other plasmaproteins were quantitated by two-dimensional immunoelectro-

phoresis relative to normal serum concentrations. The elevationof most of these acute-phase proteins was greater in the

second phase, during which serum levels increased continuously as the tumor burden increased until the animals died.The increase in haptoglobin concentration during the secondphase was much higher in rats bearing Yoshida sarcoma thanin rats bearing Zajdela tumors. Rats receiving irradiated tumorcells showed neither tumor growth nor second-phase proteinchanges. Significant increases in uptake of 3H-amino acids by

isolated perfused livers of tumor-bearing rats provided evi

dence for an increase in the hepatic synthesis rates of theacute-phase proteins. Removal of the solid tumor resulted in agradual decrease of acute-phase protein concentrations withconcomitant increase in serum albumin concentration. Thesealterations in serum acute-phase proteins during tumor growth

and after removal of the tumor may make their use attractiveas biological markers of the response of the tumor-bearing

animal to its tumor.

INTRODUCTION

Acute-phase proteins are a group of glycoproteins, the con

centration of which increases in plasma in response to a widevariety of stimuli, such as acute and chronic inflammation (11,28), tissue injury (2, 8, 13), and tumor growth (10, 29). Themechanism by which these stimuli induce increased acute-

phase protein synthesis by the liver is not well known and maybe influenced by a variety of factors (4, 6, 14, 17). Theapplication of 2-dimensional immunoelectrophoresis has madeit possible to evaluate the complex changes of plasma acute-phase reactants evoked by subjecting experimental animals tovarious noxious stimuli (20, 25).

In malignant neoplasms, the plasma protein composition maybe altered qualitatively and quantitatively by the effect of thetumor and the host reaction to it. Many attempts have beenmade to detect specificity in the pattern of plasma proteinchanges in order to provide additional parameters for thediagnostic and prognostic evaluation of the clinical stages ofcancer. Scherer et al. (24) demonstrated different patterns ofcorrelation between the concentration of 20 individual plasma

' To whom requests for reprints should be addressed, at Max-Planck Institut

FürBiochemie, Abteilung FürExperimentelle Medizin, D-8033 Martinsried beiMünchen,Federal Republic of Germany.

Received December 27, 1979; accepted December 30, 1980.

proteins and erythrocyte sedimentation rate in patients withneoplasms and other diseases. Such changes in the concentration of the proteins studied were found to be specific to therespective diseases.

In the present study, the kinetics of alterations in plasmaconcentration of several acute-phase proteins were investigated after transplantation of tumors in rats.

MATERIALS AND METHODS

Animals. Specific-pathogen-free male Wistar rats (Mus-Rat-tus, Brunnthal, Federal Republic of Germany) weighing 125 to150 g were maintained on laboratory chow and water adlibitum. In each experiment, 100 rats were randomly subdividedinto control and experimental groups. Each group contained atleast 6 rats.

Tumor Origin. An original stock of rats bearing Yoshidasarcoma (solid type) and Zajdela ascites hepatoma wasgenerously donated by Prof. Dr. Gericke, Laboratorium fürKrebsforschung (Hoechst AG, Federal Republic of Germany).The tumors have since been routinely transplanted in ourlaboratories.

Transplantation of Tumor Cells. Yoshida sarcoma (solidtype) tumors were removed after about 10 days when the tumordiameter reached 3 to 5 cm. The tumors were dissected insterile 0.9% NaCI solution to remove connective and necrotictissue. Pieces of this tumor material (2- to 3-mm diameter) wereimplanted s.c. into experimental rats. Solid tumor growth wasdetected by manual palpation of the implanted zone. Positivedetection was possible at the fifth day after inoculation whenthe tumor size reached a diameter of about 1.5 cm. Shamoperations were carried out on a control group of rats.

Zajdela ascites hepatoma were isolated by sterile syringepuncture from peritoneal fluid at 6 to 7 days after inoculationwhen maximal ascitic fluid had developed. The experimentalrats were directly inoculated i.p. with 0.5 ml of the extractedfluid containing about 106 Zajdela cells. A control group of rats

was similarly inoculated i.p. with sterile pyrogen-free 0.9%NaCI solution.

All rats inoculated with either Yoshida sarcoma or Zajdelaascites hepatoma developed tumors. No weight loss was observed in the tumor-bearing rats during the duration of the

experiments. Under these conditions, all of the inoculated ratsdied within 10 days after tumor implantation.

Tumor Growth. Tumor growth was followed daily from Days4 to 9 by caliper measurement of 2 perpendicular diameters.Solid tumors were also excised and weighed. Ascitic tumorcells were counted. The mean values of both tumor weightsand number of Zajdela ascitic cells were plotted as a functionof time after transplantation. The Yoshida sarcoma showed nometastasis, whereas metastatic Zajdela cells were found inparatracheal lymph nodes.

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Acute-Phase Protein Changes in Tumor-bearing Rats

Kinetics of Changes in Acute-Phase Protein Concentration

after Tumor Transplantation in Rats. Blood was collectedfrom the tumor-bearing animal at Days 1,2,3, 4, 6, and 8 after

inoculation. On each of these days, one group of rats bearingeither Zajdela hepatoma or Yoshida sarcoma was sacrificed,and the sera of each group was pooled. A standard pool ofsera was collected from a large number of rats immediatelyafter transplantation of solid tumor or i.p. injection of ascitictumor cells (zero time). Blood was collected from groups ofcontrol animals at the beginning and at the end of the experiment. Serum samples were also collected at similar time intervals from' groups of rats inoculated previously with irradiated

tumor cells. Tumor cells were irradiated with a total dose of3000 R/30 min, at 280 kV and 10 mA, at a 30-cm distancewith a 1-mm copper filter, utilizing a Simens-Stabilipan X-ray

machine.Total serum protein was estimated by the method of Lowry

ef al. (16) prior to quantitative 2-dimensional immunoelectro-

phoresis.Immunoelectrophoresis. The method of 2-dimensional ¡m-

munoelectrophoresis, as described by Laurel! (15) and modified by Clarke and Freeman (5), was applied for the quantitativedetermination of rat serum proteins using the modified DesagaUGI apparatus (Desaga, Heidelberg, Federal Republic of Germany). Polyvalent rabbit antiserum to whole rat serum proteinswas purchased from Dakopatts A/S (Copenhagen, Denmark).Quantitation of the immunoprecipitated peaks was done usingdigital planimeter (Digiplan AM02; Kontron GmbH, Munich,Federal Republic of Germany) after defined enlargement byprojection. For quantitative comparison of various serum protein concentrations, the individual peak areas within the elec-

tropherogram of the standard pool of control rat sera and thesera of other experimental animals were measured. At least 30immunologically distinct rat serum proteins were demonstratedwithin the immunoelectrophoretic pattern. Fifteen of these proteins were identified on the basis of their electrophoretic mobility, specific staining methods, and other physicochemicalparameters or biochemical functions (1 ). For example, preal-bumin and albumin were identified according to their electrophoretic mobility. Further proof for the identity of albumin wasobtained by reacting the rat serum with monospecific anti-rat

albumin (Nordic Immunological Laboratories, Tilburg, TheNetherlands). In addition to electrophoretic mobility, a and ßlipoproteins were stained with Oil Red O. Haptoglobin andhemopexin were stained with benzidine. Ceruloplasmin wasidentified by staining with p-phenylenediamine, and cholines-terase was identified by indoxyl acetate staining. Transferrinwas identified by autoradiography utilizing its iron-binding capacity. C3 complement component was detected by partialconversion of ß,Cglobulin to ß,Aglobulin after repeated freezing and thawing of rat serum as well as after prolonged storageof the serum at 4°and by reacting against specific antiserumto rat C3 (Nordic Immunological Laboratories), a,AT2 was

identified according to its affinity to bind trypsin. Antiprotease-

enzyme complexes with the subsequent cathodal shift of theprecipitated peak were identified by their immunoelectrophoretic pattern. t^AT identity was further confirmed using anti-rat

aiAT provided by Dr. A. Koj (Institute of Molecular Biology,Krakow, Poland). The identity of rat AAG was characterized

2 The abbreviations used are: a,AT, a-1-antitrypsin; AAG, o-1-acid glycopro-

tein.

according to its electrophoretic mobility and glycoprotein staining. The microheterogeneity of the protein, indicated by theasymmetrical peak shape, is in agreement with the molecularheterogeneity of AAG, as isolated by Gordon and Dykes (9).AAG identity was further confirmed using anti-rat AAG provided

by Dr. M. Billingham (I.C.I., Cheshire, England). For detailedprocedures of identifications, see Ref. 1. A major advantage of2-dimensional immunoelectrophoresis is its ability to resolve,identify, and quantify (relative to control values) many serumproteins simultaneously. Also, the technique demonstratesclearly the physiological and pathological changes in the complex mixture of serum proteins. It should be noted that ratplasma proteins are less well characterized than are humanproteins and that monospecific antisera for most of individualrat plasma proteins are not available.

Liver Perfusion. Livers were removed from control animalsor from tumor-bearing animals on the sixth day after tumortransplantation. The perfusing medium was L-15 containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (0.05 M)which was oxygenated for at least 30 min before the operation.The liver was perfused by recirculation of 100 ml of medium.The portal flow rate was maintained at 10 ml/min at 37°.

During the entire period of perfusion, the pH of the perfusionmedium was kept constant at 7.4. The oxygen flow was maintained to achieve a constant gaseous O2 and CO2 content inthe medium of about 95 and 5%, respectively. Fifteen min afterstarting the perfusion, 10 juCi 3H-radioactive amino acid mixture(high specific tritiated radioactive amino acid mixture; Amer-

sham, England) was added to the perfusion medium. Samples(2 ml) were taken at zero time and hourly thereafter for 4 hr fordetermination of total radioactivity. Another 4 ml of the perfu-

sate were removed and dialyzed for 4 days in 0.9% NaCIsolution at 4°, followed by radioactive counting. After thewithdrawal of the 6-ml sample of the perfusate, another 6 ml ofL-15 medium containing 10 /¿Ci3H-amino acid mixture were

added at each time interval. The perfusion lasted for 4 hr, and40 juCi of 3H-amino acid mixture were added to the perfusion

medium during the entire period of perfusion. The sampleswere mixed with Triton X-100 (5 ml) and toluene/PPO solution(0.6%, 10 ml) and counted in an SL 30 liquid scintillationspectrometer (Intertechnique, Paris, France).

RESULTS

Serum Proteins of Tumor-bearing Rats. By means of 2-

dimensional immunoelectrophoresis, we were able to determine quantitatively and simultaneously 30 rat serum proteinswith high degree of resolution. Fifteen of these proteins wereidentified by specific staining methods and other physicochemical parameters or biochemical functions (Fig. 1). The technique is highly reproducible for normal control rat sera. Distinctchanges in the plasma protein pattern were precisely measuredupon examination of the sera of tumor-bearing rats.

Kinetics of Changes in the Concentrations of the Acute-

Phase Proteins. From Charts 1 and 2, it is clear that there isa biphasic change in the concentration of most of the plasmaproteins determined in the sera of both groups of rats bearingZajdela ascites hepatoma and Yoshida sarcoma. One day aftertransplantation, the concentrations of the following acute-phase reactants, AAG, haptoglobin, aiAT, hemopexin, cerulo-

plasmin, and Peak X, were substantially elevated. A maximum

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M. Abd-el-Fattah et al.

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Flg. 1. Crossed 2-dimensional ¡mmunoelectrophoresis of control rat serum. Proteins were precipitated with antiserum to whole rat serum proteins. 1. prealbumin;2, albumin; 3, a-lipoprotein; 4, a-1-macroglobulin; 5, AAG; 6, oiAT; 7, cholinesterase; 8, ceruloplasmin; 9, hemopexin; 70, haptoglobin; J 7, C3; 72, C3c (the productof C3 when split); 13, transferrin; 14, /8-lipoprotein; X, unidentified peak.

was obtained on the second day for most of these proteins(Figs. 2A and 3^). Their levels then declined gradually to alevel slightly higher than normal. A concomitant decrease wasobserved in the levels of prealbumin, albumin, cholinesterase,and transferrin during the first 48 hr, after which their concentrations began to increase again.

The second phase of response started 4 days after transplantation of both tumors and was characterized by a secondpronounced increase in the concentration of most of the acute-

phase proteins at the sixth day (Figs. 26 and 3B). The rise intheir levels continued at different rates as the tumor loadincreased until the rats died.

In both groups of experimental rats bearing the differenttypes of tumors, the concentration of ceruloplasmin reachedits maximum during the first 2 days. However, its level did notchange significantly during the second phase of response.Interestingly, the concentration of haptoglobin decreasedsharply at the fourth day in Zajdela tumor-bearing rats. There

after, its serum level increased again, but the percentage ofincrease was much lower than the very substantial rise demonstrated in Yoshida tumor-bearing rats during the same time

intervals (Charts 1 and 2). The concentration of prealbumin,albumin, cholinesterase, and transferrin showed further decreases at different rates in the sera of experimental rats ascompared to the control groups. The percentage of decreasewas more pronounced in this second phase, especially foralbumin in rats bearing Yoshida sarcoma. Analysis of the serafrom rats inoculated with irradiated tumor cells showed a similarincrease in the concentration of the estimated acute-phase

Chart 1. Time course of increase and decrease in rat serum protein levelsafter ¡.p.injection of Zajdela ascites hepatoma. AG, AAG; Alb, albumin; AT, c^AT;PA, prealbumin; HG, haptoglobin; TI, transferrin; Hpx, hemopexin; CE, cholinesterase; Cep, ceruloplasmin; pX, unidentified peak. The mean standard deviation of the percentage of increase or decrease for each protein is: AG. 24; AT,2.3; HG, 12.1; Hpx, 3.6; Cep, 9.9; Alb. 2.0; PA, 5.2; Tf, 2.2; CE, 2.9; pX, 7.3.

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Acute-Phase Protein Changes in Tumor-bearing Rats

Chart 2. Time course of increase and decrease in rat serum protein levelsafter s.c. implantation of Yoshida sarcoma (solid type). Abbreviations are as inChart 1. The mean standard deviation of the percentage of increase or decreasefor each protein is: AG, 22.4; AT, 9.7; HG, 26.0; Hpx, 6.1; Cep, 6.6; Alb, 1.3;PA, 2.5; Tf, 3.1 ; CE, 7.8; pX, 6.5.

proteins reaching a maximum on the second day after inoculation, decreasing gradually to normal levels on the third day,and remaining unchanged until the end of the experiment.

Correlation of Tumor Growth with Elevation of Acute-Phase Proteins. Chart 3 shows the increase in both the numberof Zajdela hepatoma cells and tumor weights of Yoshida sarcoma (solid type), respectively, after tumor transplantation.Tumor growth was least on the fourth day, after which bothtumors grew fast and continuously until the death of experimental animals within 10 days after transplantation. Basically,a linear correlation could be demonstrated when the linearregression of the number of ascitic tumor cells (Chart 4A) ortumor weights of Yoshida sarcoma (Chart 4ß) was plottedversus elevation of each of the acute-phase reactants duringthe second phase.

Release of 3HAmino Acid-Labeled Proteins by Perfused

Livers. As shown in Chart 5, the perfused livers from controlrats and rats bearing Zajdela hepatoma and Yoshida sarcomawere active in plasma protein synthesis over a 4-hr period ofperfusion. Also, the livers from tumor-bearing rats incorporatedcomparatively more of the 3Hamino acid mixture into the protein

fraction of their perfusates.Total Serum Proteins. Slight hypoproteinemia was observed

in the experimental rats during the different time intervals ofthe experiments as compared to the control rats. The meanvalues of the total protein concentration of the pooled serafrom control, and Yoshida and Zajdela tumor-bearing rats were6.10 ±0.15 (S.D.), 5.78 ±0.22, and 5.71 ±0.31 g/100 ml,respectively. These differences were insignificant in case ofthe rats bearing Yoshida tumors and slightly significant (p <0.02) in the group bearing Zajdela tumors.

DISCUSSION

Numerous investigators are involved in studying the systemicaction of the tumor on the organism. Several studies documented serum protein changes in tumor-bearing animals anddifferent serum acute-phase protein patterns in cancer patients(12, 21, 30-32). In the present study, we found that both

tumors, Yoshida sarcoma and Zajdela ascites hepatoma, induced a biphasic response after transplantation which can befollowed quantitatively by measuring the changes evoked inthe plasma proteins, especially the acute-phase proteins. Thehigh resolving power of the 2-dimensional immunoelectropho-

resis aided these studies. The primary phase following tumorimplantation is most likely associated with the initial stress ofinjury together with the tissue necrosis which takes placefollowing tumor transplantation, since the serum levels of mostof the acute-phase proteins returned to a level only slightlyhigher than normal on the third day. The duration of the acute-

phase protein changes during this phase is in agreement withprevious results obtained after local inflammatory stimulationwith carrageenan (23).

In the second phase of the response, the concentration ofthe acute-phase proteins increased continuously but at differ

ent rates as the tumor burden increased. The pronounced risein the blood levels of the major acute-phase proteins, AAG,

aiAT, haptoglobin, ceruloplasmin, and hemopexin, could bethe result of de novo synthesis by the liver in response to the

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M. Abd-el-Fattah et al.

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Chart 4. A, linear regression of the number of Zajdela ascites hepatoma cells versus the percentage of increase of each acute-phase reactant. Values ofcorrelation coefficients (r) are: AG, 0.99; AT, 0.95; HG, 0.98; Hpx, 0.99; Cep, 0.95; pX, 0.98. B, linear regression of the tumor weights of Yoshida sarcoma (solidtumor) versus the percentage of increase of each acute-phase reactant. Values of correlation coefficients (r) are: AG, 0.98; AT, 0.94; HG, 0.92; Hpx, 0.95; Cep,0.94; pX, 0.98. Abbreviations as in Chart 1.

tumor. These changes occurred primarily in the liver, as incases of various forms of inflammation (18, 19, 22). Furthermore, the rats inoculated with irradiated tumor cells showed nochanges in the concentration of plasma proteins during thesecond phase and no tumor growth. This demonstrates clearlythat the second-phase response in tumor-bearing rats is pri

marily associated with tumor growth. The level of albumin,prealbumin, transferrin, and cholinesterase was depressedduring the 2 phases of the inflammatory reaction induced byboth types of tumors. Hypoalbuminemia is a characteristicaspect of the acute-phase reaction which invariably accompanies the rise in the acute-phase proteins without producing an

osmotic imbalance (3). It is likely that the greater haptoglobinconcentrations in animals with the Yoshida solid tumor are dueto breakdown of hemorrhagic necroses which are unlikely toaccompany the ascites tumor. Such a difference in haptoglobinconcentration during the second phase of response couldserve as a parameter for differentiation between these 2 typesof tumors. It should be mentioned here that ceruloplasmin wasthe acute-phase protein which responded least to both typesof tumors in rats, while Ungar-Waron ef al. (27) recommended

its use as a reliable biochemical marker for neoplastic activityin rabbits bearing carcinoma VX-2.

The correlation between the increase in both the concentration of individual acute-phase reactants and tumor growthduring the second phase is linear, as demonstrated in Chart 4,for animals bearing Zajdela hepatoma and Yoshida sarcoma,respectively.

The pathophysiological mechanism of increased synthesis ofindividual acute-phase proteins is not fully understood, butseveral evidences indicate that many humoral factors or prod-

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Chart 5. Incorporation of radioactivity from 40 /il [3H)amino acid mixture into

proteins of the perfusate during 4 hr of isolated liver perfusion.

ucts from the proliferating tumor cells themselves could stimulate the host liver for the de novo synthesis (7, 26, 27). Theincreased incorporation of 3H-radioactive amino acid mixture

by the isolated perfused livers from rats bearing Yoshida sarcoma and Zajdela hepatoma during the entire period of perfusion is consistent with both increased synthesis and more rapidprotein turnover rates since the total serum protein concentrations are virtually identical. The results of Shiraska and Fujji(26) also demonstrated the induced high activity of livers from

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Acute-Phase Protein Changes in Tumor-bearing Rats

rats bearing Yoshida sarcoma (solid type) or AH 130 (solidtype). They observed increased thymidine incorporation intoDNA and an approximately 100-fold increase in de novo synthesis of DNA-synthesizing enzymes.

The slight reduction of total serum protein concentration,despite increased protein synthesis, may be due to the depletion from the blood and accumulation into the ascitic fluidexúdate (21). Analysis of'the ascitic fluid developed in rats

bearing Zajdela ascites hepatoma at the sixth day after inoculation, by 2-dimensional immunoelectrophoresis, revealed the

presence of albumin in a relatively higher concentration thanthe other individual plasma proteins. Interestingly, removal ofthe Yoshida sarcoma solid tumor at the fifth day after transplantation under aseptic conditions resulted in a gradual decrease in the concentration of most of the acute-phase proteins. Three days after removal of the solid tumor, the serumalbumin concentration increased appreciably, whereas thelevel of the acute-phase proteins declined continuously. Thesealterations in serum acute-phase proteins during tumor growth

and after removal of the tumor may possibly make their useattractive as biological markers of tumor-host responsiveness.

The biological activity of some of these acute-phase proteinsis well defined (12, 25), such as hemoglobin binding by hap-toglobin, transport of copper by ceruloplasmin, antiproteaseactivity of a,AT. Hemopexin scavenges circulating heme fromcirculation. As yet, no definite major biological function of AAGhas been provided. However, this acute-phase protein may beinvolved in the healing of wounds and tissue repair and mayalso represent a natural inhibitor of lysosomal enzymes (25).

In conclusion, our present study shows that the levels of thefollowing acute-phase proteins, namely, AAG, haptoglobin,

a,AT, ceruloplasmin, hemopexin, and Peak X, increased in theblood of rats bearing tumors of different types and at differentsites. Similarly, their blood concentrations increased continuously as the tumor load increased. Accordingly, the determination of the blood levels of these acute-phase proteins couldfurnish a useful biochemical parameter of clinical value inmonitoring tumor growth.

ACKNOWLEDGMENTS

We are indebted to Professor Dr. F. Deinhardt (Max von Pettenkofer-lnstitutfürHygiene und Medizinische Mikrobiologie der Ludwig-Maximilians Universität,Munich, Germany) for reading the paper and providing helpful comments. Alsowe would like to thank Dr. M. Billingham (Biology Department. PharmaceuticalDivision, I.C.I., Cheshire. England) for providing us with anti-rat AAG, and Dr. A.Koj (Institute of Molecular Biology, Jagiellonian University, Krakow, Poland) forproviding us with anti-rat aiAT.

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M. Abd-el-Fattah et al.

Fig. 2. CrossedProteins are identified as in Fig. 1

inoculation.

2554 CANCER RESEARCH VOL. 41

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13

13 x

10

Fig. 3. Crossed 2-dimensional immunoelectrophoresis of serum from rats bearing Yoshida sarcoma (solid tumor) at 2 days (a) and 6 days (t>)after tumortransplantation. Proteins are identified as in Fig. 1.

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1981;41:2548-2555. Cancer Res   Mohamed Abd-el-Fattah, Reiner Scherer, Fouad Mounir Fouad, et al.   TransplantationKinetics of the Acute-Phase Reaction in Rats after Tumor

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