Vol. 11, No. 4

INFECTION AND IMMUNITY, Apr. 1975, p. 873-875 Copyright 0 1975 American Society for Microbiology

Printed in U.S.A.

Detection of a Leukocytic Endogenous Mediator-Like Mediator of Serum Amino Acid and Zinc Depression During Various Infectious Illnesses ROBERT W. WANNEMACHER, JR.,* ROBERT S. PEKAREK,' ALBERT S. KLAINER,2 PETER J. BARTELLONI,3 HERBERT L. DUPONT,4 RICHARD B. HORNICK, AND WILLIAM R. BEISEL U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21701; Division of Infectious Diseases, Ohio State University Medical College, Columbus, Ohio 43210: and the Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, Maryland 21217

Received for publication 6 January 1975

Serum samples from patients with many different kinds of infection were shown to contain a substance characteristic of leukocytic endogenous mediator. Generalized bacterial infections produced more leukocytic endogenous mediator than did localized ones; viral infections produced very little. In typhoid fever, mediator concentrations increased before the onset of illness.

from stool filtrates; malaria induced by Plasmodium falciparum; and typhoid fever. Each individual was completely informed as to the purpose, nature, risk, and other details of each study before volunteering. Written and oral consent was obtained from each volunteer or patient. Details concerning the design and methods of these studies have been published elsewhere (1-5, 10, 11). Fasting blood samples were obtained during illness in the volunteers and patients. In addition, preexposure samples were obtained from volunteers with sandfly fever and sequential samples were obtained from those with typhoid fever. Individual serum samples were prepared for assay by membrane filtration (Millipore membrane filters). Half of each sample was also heated at 90 C for 30 min to inactivate any mediator present. Rats were prepared for assay by injecting them subcutaneously with 1 pCi of [1- 14C ]aminocyclopentane-1-carboxylic acid (cycloleucine) (New England Nuclear, Boston, Mass.) per g of body weight. Twentyfour hours later, one group of six rats was injected intraperitoneally with 1 ml of saline (controls), a second group of six rats was injected with 1 ml of serum, and a third group of six rats was injected with 1 ml of the heattreated serum (heat-treated controls). All rats were killed 4 h later; their serum zinc concentraPresent address: Human Nutrition Laboratory, Agricul- tions and liver content of [I 4C ]cycloleucine were tural Research Service, Grand Forks, N.D. 58201. Present address: Department of Medicine, University determined (7, 12). Between-group differences West Virginia Medical Center, Morgantown. W.Va 26506. in serum zinc and hepatic cycloleucine concen3Present address: Tripler Army Medical Center, APO San trations were derived for each serum specimen. Francisco, Calif. 96348. Serum obtained from healthy persons caused 4Present address: Division of Infectious Diseases, Univerbut significant decrease in serum zinc a slight 70025. Tex. sity of Texas Medical School, Houston,

A product of phagocytizing cells termed leukocytic endogenous mediator (LEM) appears to be responsible for initiating many infectionrelated metabolic responses in laboratory animals (6, 8, 9, 12). A LEM-like mediator has also been found (10) in the serum of volunteers during experimentally induced typhoid fever. The present studies were conducted in man to determine: (i) whether a LEM-like substance appeared in serum during other naturally occurring or experimentally induced infections; (ii) whether different infections released different amounts of LEM into serum; and (iii) whether LEM appeared in serum before the onset of clinical illness. Bioassays for LEM were performed on serum samples from patients with naturally occurring infections who were hospitalized at the Ohio State University Hospital, Columbus, Ohio. Other serum samples were assayed as minor ancillary portions of volunteer studies conducted primarily to improve diagnostic, protective, or therapeutic measures against infections. Sandfly fever was studied at the Institute. At the Maryland House of Correction, Jessup, Md., the following were investigated: diarrhea induced with Shigella flexneri 2a, noninvasive enterotoxin-producing Escherichia coli, or nonbacterial (presumably viral) agents derived I






and an increase in hepatic cycloleucine as compared with matched heat-treated control sera (Table 1). These findings in control subjects suggested that very small amounts of mediating substance were normally present in serum. Systemic bacterial or parasitic infections produced high levels of circulating mediator (Table 1). In relatively localized bacterial infections such as cellulitis, pyelonephritis, Staphylococcus aureus abscesses, or Shigella diarrhea, the concentration of circulating mediator was less than that observed with systemic bacterial infections, but in many patients it still was significantly increased above base line. A modest but definite increase in the mediator was also induced in patients with diarrhea due to an enterotoxin. In contrast, viral infections stimulated only small increases in serum concentration of mediator. When compared with sera from patients with generalized bacterial infections, three times as much serum from volunteers with sandfly fever was required to detect significant increases in mediator. Increases in mediator concentrations in viral infections were almost at the limits of detection.

In volunteers who developed typhoid fever, concentrations of circulating mediator became significantly increased before the onset of fever. These observations support the concept that increases in the serum concentration of LEM are among the earliest indicators of the presence of systemic infections. Although the present studies demonstrate the presence of a LEM-like mediator in the serum of patients with a wide variety of infectious diseases, they do not indicate whether the mediator is a single substance or a group of closely related substances. It remains possible that LEM is similar to or identical with endogenous pyrogen. In none of the attempts to purify LEM has it been possible to separate the metabolic effects of LEM from the pyrogenic activity of endogenous pyrogen (6). LITERATURE CITED 1. Clyde, D. F., R. M. Miller, A. R. Schwartz. and M. M. Levine. 1971. Treatment of falciparum malaria with sulfalene and trimethoprim. Am. J. Trop. Med. Hyg.

20:804-810. 2. Dolin, R., N. R. Blacklow, H. L. DuPont, S. Formal, R. F. Buscho, J. A. Kasel, R. P. Chames, R. Hornick, and R.

TABLE 1. Detection of significant concentration of the mediator of serum zinc depression and hepatic cycloleucine accumulation in serum obtained from patients with various infections Serum zinc depression

Hepatic cycloleucine accumulation


Mean changeb

(j'g/Ioo ml)


Mean changeb (ng/g of liver)

Diplococcus pneumoniae .7/7

- 13 + 4 -37 4 5

Simultaneous mixed gram-negative and gram-positive infections .5/5 Staphylococcus aureus abscess .3/3 Pyelonephritis .2/2 Cellulitis .1/2 Viral hepatitis .0/26 Infectious mononucleosis .0/8 Influenza.. 0/1 Escherichia coli (noninvasive) diarrhea 3/3 Shigella flexneri dysentery .3/6 Infectious nonbacterial diarrhea .0/9 Malaria .3/3 Sandfly fever (1 ml) .4/17 Sandfly fever (3 ml) .17/17 Typhoid fever .9/9

0/17 7/7

135 ± 31

-42 ± 4 -30 5 -35 ± 5 -13 ± 7 -1 i3 -2 ± 4 6 3 -34 ± 6 -22 ± 7 -10 ± 7 - 41 ± 5 -27 ± 4 -74 3 -52 ± 8

5/5 3/3 2/2 1/2 6/26 0/8 0/1 3/3 3/6 0/9 3/3 8/17 17/17 9/9






20± 6

120 ± 51 ± 50 ± 41 ± 23 i 16 ±

30 10 11 10 9 11

7 9 12 8 25 14 169± 25 83 ± 15

53 ± 40 ± 21 ± 123 ± 44 ±

a Ratio of the number of patients with significant amounts of circulating mediator to total number of patients. bMean + standard error of the difference between serum and heat-treated serum of all patients. Each individual sample was tested in six rats. Serum zinc concentrations were 128 + 2 and 125 + 3 jg/100 ml and hepatic cycloleucine content was 110 ± 10 and 115 ± 8 ng/g of liver (mean ± standard error) in rats injected with heat-treated serum and saline, respectively. Three injections of febrile serum given at 4-h intervals. Three injections of preexposure serum result in depression of serum zinc of -40 + 2 gg/100 ml and accumulation of hepatic cycloleucine of 89 ± 11 ng/g of liver. "

VOL. 11, 1975







M. Chanock. 1971. Transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates. J. Infect. Dis. 123:307-312. DuPont, H. L., S. B. Formal, R. B. Hornick, M. J. Synder, J. P. Libonati, D. G. Sheahan. E. H. LaBrec, and J. P. Kalas. 1971. Pathogenesis of Escherichia coli diarrhea. N. Engl. J. Med. 285:1-9. DuPont, H. L., R. B. Hornick, M. J. Snyder, J. P. Libonati, S. B. Formal, and E. J. Gangarosa. 1972. Immunity in shigellosis. II. Protection induced by oral live vaccine or primary infection. J. Infect. Dis. 125:12-16. Hornick, R. B., S. E. Greisman, T. E. Woodward, H. L. DuPont, A. T. Dawkins, and M. J. Snyder. 1970. Typhoid fever: pathogenesis and immunologic control. N. Engl. J. Med. 283:686-691. Kampschmidt, R. F., H. F. Upchurch, C. I. Eddington, and L. A. Pulliam. 1973. Multiple biological activities of a partially purified leukocytic endogenous mediator. Am. J. Physiol. 224:530-533. Pekarek, R. S.. W. R. Beisel, P. J. Bartelloni, and K. A. Bostian. 1972. Determination of serum zinc concentra-






875 tions in normal adult subjects by atomic absorption spectrophotometry. Am. J. Clin. Pathol. 57:506-510. Pekarek, R. S., and R. W. Wannemacher, Jr. 1972. A mediator for triggering non-specific host defense mechanisms. Army Sci. Conf. Proc. 111:129-140. Pekarek, R. S.. R. W. Wannemacher, Jr., F. E. Chapple III, M. C. Powanda, and W. R. Beisel. 1972. Further characterization and species specificity of leukocytic endogenous mediator (LEM). Proc. Soc. Exp. Biol. Med. 141:643-648. Wannemacher, R. W., Jr., H. L. DuPont, R. S. Pekarek, M. C. Powanda, A. Schwartz, R. B. Hornick, and W. R. Beisel. 1972. An endogenous mediator of depression of amino acids and trace metals in serum during typhoid fever. J. Infect. Dis. 126:77-86. Wannemacher, R. W., Jr., R. S. Pekarek, P. J. Bartelloni, R. T. Vollmer, and W. R. Beisel. 1972. Changes in individual plasma amino acids following experimentally induced sandfly fever virus infection. Metabolism 21:67-76. Wannemacher, R. W., Jr., R. S. Pekarek, and W. R. Beisel. 1972. Mediator of hepatic amino acid flux in infected rats. Proc. Soc. Exp. Biol. Med. 139:128-132.

Detection of a leukocytic endogenous mediator-like mediator of serum amino acid and zinc depression during various infectious illnesses.

Serum samples from patients with many different kinds of infection were shown to contain a substance characteristic of leukocytic endogenous mediator...
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