Studies in experimental endotoxemia in man

Studies in Experimental

Endotoxemia

in Man ROBERT

M.~LLODART,M.D.,

ILSE HAWTHORNE,B.S.,

Baltimore,

From the Surgical Research Laboratories and the Clinical Shock Unit, University of Maryland School of Medicine, Baltimore, Maryland. This work was supported by the U.S. Army Medical Research and Develofiment Command, Department of the Army, under Research Contract No. DA-49-193-MD2229 and U.S.P.H.S. Grant No. HE09341-02.

RAM-NEGATIVE septic shock is believed to be due to the release of endotoxin from the bacterial cell wall. The behavior of endotoxin in different species varies considerably. In man, the clinical study of septic shock is complicated by pre-existing and co-existing diseases, treatment, and lack of precision regarding diagnosis. There is no suitable clinical test for determining the presence of endotoxin in the blood. Some understanding of the mechanism of action of endotoxin has been obtained through studies in animals. The following study demonstrated the effects of small doses of gram-negative bacterial endotoxin in healthy volunteers under controlled conditions. The study was designed to test some observations made in experiments \?-ith animals and to confirm or reject them in man as well as to generate new hypotheses. AND

METHODS

Healthy male volunteers between the ages of eighteen and forty-five were fully informed of the nature of the studies about to be undertaken. Under close clinical supervision an intravenous catheter was placed into the antecubital veins with a very slow drip of dextrose and water. Base-line observations of blood pressure, pulse, and rectal temperature were made and when these stabilized, a base-line normal blood sample was drawn. The patient was then given 0.01 pg./kg. of Escherichia coli 0127B8 endotoxin intravenously. Subjects were observed at regular intervals and blood Volunteers.

Vol.

113.

May

1967

SAFUH ATTAR,M.D.,

samples were taken at half hour and then at one hour intervals throughout a five hour period of observation. The procedure was well tolerated and repeated at weekly intervals for three doses. Preparation of Endotoxin. E. coli 0127B8 endotoxin diluted to 0.5 pg./ml. was sterilized by boiling for fifteen minutes. It was then assayed for pyrogenic activity in rabbits which were given a dose of 1 pg./kg. intravenously after having been acclimated overnight to a rectal thermometer. Base-line and three hour rectal temperatures were determined. Endotoxin in the proper activity range produced an elevation of 2 to 3 degrees after three hours. If the elevation in temperature was in excess of that or if the activity was insufficient, a new preparation was made. Prior to each use the endotoxin was sterile tested for aerobic and anaerobic organisms. Immunologic Studies. The phagocytic index of the patient’s polymorphonuclear neutrophils was determined against E. coli 0127B8. White blood cell and differential counts were taken. The number of bacteria necessary to supply 500 coliform organisms to each polymorphonuclear neutrophil was calculated and 1 ml. of a suspension of E. coli 0127B8 containing this number of bacteria was prepared and added to 1 ml. of blood containing 8 units of heparin. The mixture was incubated for one hour and blood smears were made. Thirty to fifty consecutive neutrophils were examined and the average number of bacteria within their borders was taken as the phagocytic index. There was a linear relationship between the log of the number of bacteria per polymorphonuclear neutrophil and the phagocytic index in any given blood specimen. An error of f 10 per cent was found when a single sample was studied repeatedly on the same day. Serum $0 Per Cent Hemolytic Com@ement Titer. The serum 50 per cent hemolytic complement titer was measured according to the method of Mayer [I]. Measurement of the third component of complement was accomplished using zymosan-adsorbed reagent as described by Mayer [2].

G

MATERIAL

AND

Murylmd

599

Ollodart,

600 TABLE FEVER

I

INDEXES

-Dose First

Subjects

Responsive* D.I. B.R. H.A. P.F. (splenectomy)

Hawthorne,

of EndotoxinSecond

Third

13.5 8.3 9.1

7.3 3.4

5.0 3.1

9.3

a.7

5.8

5.3 7.4 3.0 6.3

3.7 7.0 2.7

2.6 4.7 1.3

Nonresponsivet H.E. O.L. OS. O.L.D.

* Patient had a shaking chill and appeared ill. t Patient had no shaking chill, did not look ill, but felt effects to some degree.

Serum Bactericidal Antibody Titer. Serum bactericidal antibody against E. coli 0127B8 was studied according to the method of Landy, Michael, and Witby [3]. Excess complement was added to the system in the form of precolostral calf serum which was tested for sterility, complement activity, and lack of independent bactericidal activity prior to its use. Logarithmic dilutions of serum were used. Bacteria in tubes were counted and plotted on a log scale to determine the 50 per cent kill serum concentration which was taken as the titer. Bentonite Flocculation. Bentonite flocculation was performed according to the method of Wolf, Ward, and Landy [4] using bentonite coated with E. coli 0127B8 endotoxin.

a

1 fever

TABLE WHITE

BLOOD

PHAGOCYTIC

CELL

II

COUNT

CHANGES

DIFFERENTIAL

AFTER

AND

ENDOTOXIN

ADMINISTRATION

Dose

First Reactor

Drop in Drop in White PolymorBlood Cell phonuclear Count Leukocytes

Drop in Phagocytic Index

4/4

214

4/4

l/4

o/4

l/4

Reactor Nonreactor Third

3/4 0

214 O/4

w4 O/4

Reactor Nonreactor

2/4 0

l/4 O/4

214 O/4

Nonreactor Second

and Attar

Microcirculatory Studies. A Zeiss stereobinocular operating microscope with a green filter was positioned to observe the bulbar conjunctiva of the volunteer. Base-line and frequent studies after injection of endotoxin were made. A particular field was picked and always observed. Measurement of vessel diameter was made by means of the scale in the optical system and observations were made for change in vessel diameter, shape, sticking of white blood cells along the edges of vessels as evidenced by irregularity in the lumen [f], evidence of agglutination of red cells, evidence of relative stasis of flow in the vessels, and edema as evidenced by a blurring of the field. Platelets. Number of platelets was determined by direct counting methods using phase microscopy. RESULTS

Of eight subjects studied, Clinical Response. four had relatively severe initial clinical responses and four did not. A relatively severe initial response was defined as shaking chill with a five hour fever index greater than 8. All subjects with a shaking chill had a fever index over 8, whereas all those without a shaking chill had one below 8. (Table I.) Initial symptoms would reach a plateau by approximately two hours and then would begin to subside while the fever continued to rise reaching a peak between two and four hours. The fever would then begin to resolve. There was occasional nausea but no emesis, occasional headache, and a feeling of malaise with hypersensitivity of the skin and scalp. Four hours after endotoxin was administered most of the subjects were feeling relatively well. On the second and third weeks, when the subjects received subsequent doses of endotoxin, the symptoms generally would be lessened. No shaking chills were observed after the first dose. The fevers were not as high and were delayed in onset. There was an increase in some subjects of nonspecific symptoms consisting of malaise, headache, skin hypersensitivity, and muscular and joint aches. White Blood Cell Count Dizerential and PhagoTable II illustrates data on these cytic Index.

parameters. A drop in white blood cell count greater than 20 per cent was considered significant. Of the initially responsive subjects, all had a drop in white blood cell count greater than 20 per cent. One initially unresponsive subject had a drop in white blood cell count which was significant. Three of the four subjects who were initially unresponsive had no significant drop in white blood cell count. American Journal of Surgery

Experimental

601

Endotoxemia

FIG. 1. Experimental data in an initially responsive subject. In two of five subjects with an initial drop in white blood cell count, the polymorphonuclear leukocytes made up a significant element of this drop. In three of the subjects with a significant drop, it appeared that both elements dropped since the differential did not change. A significant drop in the phagocytic index was noted in all four initially responsive subjects. One initially unresponsive subject also showed a drop in phagocytic index, whereas three of the four initially unresponsive subjects had no phagocytic index drop. After the second and third doses of endotoxin the drops in white blood cell count and phagocytic index diminished. Figure 1 illustrates this in a reactive subject. Of five subjects with an initial drop in white blood cell count, only three had evidence of it after the second dose and then to a lesser degree. After the third dose in only two patients was there a drop in white blood cell count and this was less than it had been after the first two doses. It appears that the drop in white blood cell count diminished as relative tolerance was acquired. In the same manner, of five subjects with an initial drop in phagocytic index, only two showed it after the third dose and in these cases it was less than it had been initially. Thus, the drop in phagocytic index also decreases as relative tolerance is acquired. Serum 50 Per Cent Hemolytic Complement Titers. There was no drop in the 50 per cent hemolytic complement titers and third comVol. 113, May

1967

ponent of complement titers in any subject as a result of this dose of endotoxin. There was, however, a gradual rise in complement titer in seven of the eight subjects so that after the third dose, titers ranged from 120 to 200 per cent of the original base-line level. If a dose of endotoxin was given three or four days after the second dose, the complement titer and phagocytic index could be driven higher than if a period of seven or eight days elapsed before subsequent doses were given. Figure 2 shows n= Endodoxin given

Dll. Phogocyltc 500 Index 400 % of Bore 300

I2545

12345

'

012345 A

7

01 IA

5 IO

FIG. 2. Experimental data in an initially responsive subject.

602

Ollodart, TABLE

BACTERICIDAL

ANTIBODY

Hawthorne,

antibody titers. Titers beginning at less than 1,000 would often reach levels of 300,000 or more by the time the three weekly doses of endotoxin had been given. Bactericidal antibody titers seem to parallel the bentoniteagglutinating titer but seem to be much more sensitive. The behavior of the titer in the splenectomized subject who was late in acquiring a tolerance is illustrated in Figure 3. As can be seen, no significant rise in bactericidal antibody titer was obtained. In this patient no agglutinating antibodies were found throughout the course of study. Bactericidal Antibody in Splenectomized Subjects. Two other splenectomized subjects were studied for bactericidal antibody after administration of doses of endotoxin. (Table III.) These patients were not studied as a part of this experiment and consequently only serum specimens were available for analysis. In both of these subjects no significant rise in bactericidal antibody occurred as a result of the injection of endotoxin, whereas all nonsplenectomized persons studied showed a significant rise beginning after the fourth day. It would appear that the person with splenectomy is unable to respond to the intravenous administration of endotoxin with the production of significant increases in bactericidal antibody. Platelets. Table IV illustrates the data on platelet counts in four subjects, three of whom evidenced a drop in platelet count after receiving endotoxin. This drop was less after each subsequent dose, as illustrated in Figure 4. Microcirculatory Studies. The microcirculatory changes illustrated in Figure 5A and B and tabulated in Table v behaved as two distinct groups, one related to vasospasm and the other to cellular adhesiveness and stasis.

III

TITRATION

AFTER

ENDOTOXIN

ADMINISTRATION

-Days

after Administration

Patients Same Day

Three

Reactors D.I. 440 350 B.R. 400 680 Nonreactors M.C. 4,000 2,800 O.L. 1,700 420 H.E. 255 275 0,s. 250 180 Splenectomized reactors 100 O.R. 95 P.F. 98 88 1,400 C.A. 900

of First DoseEighteen or TwentySeven Four

1,300 18,000

100,000 60,900

6,000 1,120 240,000

11,000 15,000 12,100 300,000

95 76 820

+

32 210 50

NOTE: Bentonite antibodies not detectable until the fifth day after endotoxin administration and thereafter parallel bactericidal antibodies.

such complement changes in a typical subject with an initial reaction. Bentonite Flocculation Titer. No antibodies capable of agglutinating endotoxin-coated bentonite were found in serum prior to the introduction of endotoxin. Antibodies began to appear on the fifth day after the initial dose of endotoxin and they gradually increased. Bactericidal Bactericidal Antibody Titer. antibody titers are found in normal serum prior to the introduction of endotoxin. The titer remains relatively stable until the fourth day after the initial dose of endotoxin when it begins a rapid climb at the same time bentoniteagglutinating antibodies first appear. Table III illustrates the behavior of the bactericidal TABLE PLATELET

COUNTS*

First

Subject (base-line)

(3 hr.)

Reactor P.B. W.H.

225,000 140 ) 000

160,000 140,000

Nonreactor R.O. P.O.

180,000 160,000

80,000 70,000

AFTER

and Attar

IV

ENDOTOXIN

Endotoxin -Second-(base-line)

ADMINISTRATION

Dose Third-------(3 hr.)

(3 hr.)

(base-line)

130,000

90,000

250,000

200,000

180,000 110,000

125,000 75,000

110,000

100 ) 000

* Given in cells per cu. mm. American Journal of Surgery

Experimental Endotoxemia

iN3

PF (SPLEENECTOMYI

FIG. 3. Experimental

data in the splenectomized

All four subjects observed for microcirculatory changes had arteriolar contraction after the first dose; this contraction preceded the chill when chill did occur. It occurred without a chill in two of the four patients. The arteriolar contraction became less after the second and third dose and thus appeared to decrease as tolerance was acquired. After the second dose only one of three subjects studied had arteriolar contraction, and after the third dose none of the

FIG. 4. Typical Vol. 113. May

1967

changes

in platelet

subject.

three subjects demonstrated arteriolar contraction. All subjects showed clumping and stasis of red blood cells. In contrast to the vasospastic phenomenon, this clumping became more severe and occurred earlier after each subsequent dose of endotoxin. The appearance of a wavy shape to the lumen of the vessels is an indication of coating of white blood cells along the walls with the

count

after endotoxin

administration.

604

Ollodart,

Hawthorne,

B

A FIG. 5. Microcirculation

and Attar

before (A) and after (B) endotoxin.

red blood cells flowing centrally [5]. This phenomenon was demonstrated in all subjects, sometimes as early as five minutes after the administration of endotoxin. The phenomenon did not seem to change with subsequent doses. It occurred before the drop in the white blood cell count. Complete stasis in blood vessels was related to red blood cell clumping. An end point of observation was taken as one in which clumping in all vessels occurred and stasis occurred in some vessels. The second time patients received endotoxin, stasis and clumping in all vessels occurred earlier and was more severe in nature. The third time it occurred just as severely and sometimes more so than the second time. It seems that clumping and stasis are phenomena which are inversely related to the acquisition of tolerance. They seem to be more severe after the second and third dose when tolerance is present and when antibody titers, phagocytic activity, and hemolytic complement titers are at higher levels. COMMENTS

Experimental endotoxemia in man has revealed a fairly characteristic pattern of initial febrile reactivity followed by increasing tolerance coincidental with the acquisition of “immunity,” as defined by elevated amounts of antibodies and phagocytic activity against E. coli. The subjects fell into two groups, four patients showing relatively severe initial reactivity and four showing a relatively mild reactivity. Severe reactivity was accompanied

by a fever index greater than 8 with a shaking chill. The white blood cell count dropped as did the phagocytic index against E. coli, and vasoconstriction was present. Moderate reactivity consisted of a fever index of less than 8, no shaking chill, no drop in white blood cell count or phagocytic index, but some vasoconstriction. After repeated doses of endotoxin at weekly all patients acquired tolerance. intervals, Whereas the primary response had consisted of a fever index greater than 8, shaking chills, drops in white blood cell count and phagocytic index, and vasoconstriction, the factors involved in relative tolerance were as follows: decline of fever (fever index less than 8), no shaking chill, increase in white blood cell count, increase in phagocytosis, no vasoconstriction, increase of cellular sludging and stasis, increase in complement and antibodies. All the fever indexes declined to less than 8 by the time of administration of the second and third dose, except in the patients with splenectomy. No shaking chills were observed when fever indexes fell below this level. The drop in white blood cell count declined or disappeared as did the drop in phagocytic index. Instead of a fall in white blood cell count there tended to be an immediate rise after the dose of endotoxin ; vasoconstriction was inclined to disappear. Fifty per cent hemolytic titers of complement tended to rise as did the bactericidal and bentonite antibody titer against E. coli 0127B8. Coincidental with tolerance to the initial effects of endotoxin, an increase appeared in American

Jcurnal

of Surgery

Experimental

MICROCIRCULATION -~

-

__--

------First W.H. P.B. ~~~ ~~_____

Reaction

CHANGES

. R.O.

P.O.

605

Endotoxemia

TABLEV AF'TER ENDOTOXIS

ADMINISTRATION

Endotoxin Dose-------------Second----W.H. P.B. R.O. P.O.

\:asoconstriction

GO*

20

60

30

30

-

-

White blood cell coating

30

10

30

5

10

30

10

~--~ _~ W.H.

--_‘rilird P.B.

$5

Red blood cell clumping in all vessels

I”0

20

60

10

10

30

10

Stasis

120

180

60

20

30

30

10

5 10

360

290

180

120

180

240

180

210

Velocity ment

improve-

* Minutes

after administration

of endotoxin

_ I’.0

1.5

5

l-5

1.5

15

7

300

120

when changes are first seen.

intravascular cellular sludging after the second and third doses of endotoxin with increased stasis in the microcirculation. While the patients no longer had shaking chills and did not appear as ill as after the first dose, they complained of an increase in rather vague symptoms of generalized aching, hypersensitivity of the skin and scalp, and in some cases aching joints. These symptoms resolved by the end of the day. It has always been assumed that as the fever response to endotoxin decreased, all other harmful sequelae of endotoxemia would also decrease. Subjective responses cannot be obtained in animals and no reports of an increase in sludging and stasis coincidental with tolerance is in the literature. These studies imply that the reduction in fever does not necessarily mean that all of the potentially harmful sequelae of endotoxemia are also reduced. As initial responsiveness decreases, a secondary syndrome appears which is characterized by increase of sludging and stasis in the microcirculation, accompanied by vague symptoms, and coincidental with the acquisition of what appears to be a higher degree of immunity against the organism from which the endotoxin has been obtained, at least in terms of antibody, phagocytic activity, and 50 per cent hemolytic complement titers. The possible role of this secondary syndrome in the pathology of endotoxemia remains to be elucidated. It may represent a hypersensitivity to the bacterial material. There has been some controversy in the literature concerning whether endotoxin causes a fall in white blood cell count, as has been Vol. 113. May 1967

~..._~ R.O.

described with larger doses in animals [6]. The present study may have clarified this issue in that severe initial reaction with high fever and chills is shown to be accompanied by a drop in white blood cell count, whereas a relatively mild initial reaction is not. In addition to the decrease in white blood cell count, there is a decreased ability of the neutrophils to phagocytize bacteria. Since efficient phagocytosis of bacteria is dependent on opsonization by serum antibody and complement [7], it might be postulated that the drop in phagocytic index is due to a neutralization of these opsonins by endotoxin in the blood. Experiments in our laboratory with incubation of heparinized blood with endotoxin in doses spanning the maximal possible blood level in our volunteers, as calculated by their estimated blood volumes, have indicated that there is no comparable drop in phagocytic index until doses of endotoxin 1,000 times the maximal possible circulating levels in our volunteers are reached. This would imply that the drop in phagocytic index is mediated by some additional in vivo phenomenon not reproduced in heparinized blood in the test tube. It would not seem reasonable, therefore, to suppose that the drop in phagocytic index is due to simple neutralization of natural antibodies. Lee [8,9] has presented evidence for changes in the coagulation mechanisms as a result of the administration of endotoxin with resultant intravascular deposition of small amounts of fibrin which are cleared by the phagocytic cells. He postulates that while the phagocytic cells are clearing the fibrin, they are unable to clear subsequent colloidal material.

606

Ollodart,

Hawthorne,

A similar explanation might apply for the reduced phagocytic index we have observed in neutrophils. Complement was studied in man because of reports that serum complement drops after endotoxemia in animals [IO]. No falls in complement were noted in this study although this does not rule out the possibility of a fall in complement with larger doses. Certainly all the sequelae of endotoxemia reported in this paper occurred without a detectable fall in hemolytic complement titer. Complement did rise steadily throughout the course of treatment, presumably as a part of the response to repeated doses of endotoxin. What role, if any, complement plays in the gradual acquisition of tolerance to the initial reactivity and what role it may play in the secondary syndrome of increased blood sludging remain to be deduced. If cellular aggregation in vivo represents a form of immune adherence, complement may play a role. It has been hypothesized that serum antibody may play a role in the development of tolerance to endotoxin. These studies have indicated that serum bactericidal antibody does indeed rise as tolerance is acquired. Nevertheless, in splenectomized patients who eventually do acquire tolerance comparable to that in nonsplenectomized patients, although one or two doses later [ll], no rise is shown in bactericidal antibody coincidental with this belated acquisition of tolerance. This would imply that this particular antibody is not necessary for tolerance. Greisman, Carozza, and Hills have transferred pyrogenic tolerance to endotoxin with plasma from tolerant subjects [12]. If this tolerance is due to an antibody, it does not appear to be one which has been measured to date. Either the passively transferred plasma factor is not an antibody or it is an antibody against an antigen for which we are not testing. This theoretic antibody may be directed at an antigenic determinant on endotoxin which is different than the polysaccharide antigenic determinant against which the bactericidal antibody is directed [3]. Utilization of endotoxins deprived of their polysaccharide 0 antigenic determinants may help solve this problem. Coagulation and platelet count studies were undertaken because Herring and associates [13] showed that endotoxin was taken up by the platelets in the buffy coat, rather than by the white cells. In addition, the work of Lee

and Attar

[8,9] concerning intravascular deposition of fibrin must stimulate a very active interest in coagulation changes as possible basic mechanisms in the pathogenesis of endotoxemia. In preliminary studies the platelet count fell in three of four of the persons studied. There is also an indication of increased coagulability of blood in three of four patients studied by thromboelastography. Farr and associates [14,15] have indicated that virtually all the manifestations of endotoxemia can be produced in a rabbit using bovine serum albumin as an immunizing antigen. They believe that introduction of endotoxin into an animal mimics the introduction of bovine serum albumin into a partially immune rabbit. As the immunity increases, the fever observed in the partially immune animal tends to disappear. The present studies have indicated that two phenomena must be observed with repeated doses of endotoxin. The first is the acquisition of febrile tolerance to the initial reactivity which does not appear to be completely dependent upon bactericidal antibody against the 0 antigens of endotoxin, at least in splenectomized patients [ll]. The presence of some other antibody will have to be determined if the immunity theory for the acquisition of tolerance is to remain valid. On the other hand, increased sludging in the microcirculation occurs at the same time that the high antibody titers evolve. This phenomenon may be related to the secondary clinical sydrome described. It is worth considering the differences noted in this study compared to those of a previous study on patients in hypovolemic shock [16] because of suggestions that endotoxemia is operative in all forms of shock [17]. Patients in hypovolemic shock had decreased phagocytic indexes against E. coli as well as decreased complement levels and serum bactericidal titers. This was followed by elevated phagocytic indexes and complement titers after recovery from shock. The present study indicated that E. coli endotoxemia produced no decreases in complement but did decrease phagocytic indexes. A “recovery” rise in phagocytic index and complement did occur. These differences do not rule out endotoxemia as an occurrence in hypovolemic shock, however, since the doses of endotoxin used in this study were quite small and decreases noted in patients with hypovolemia could have been caused by abAmerican

Journal of Surgery

Experimental sorption of larger doses or by treatment fluids and blood.

Endotoxemia

with 4.

STJMMARY

Intravenous administration of E. coli endotoxin to healthy volunteers demonstrated a difference in host reactivity to endotoxin. Half of the subjects showed shaking chill accompanied by a fall in white blood cell counts and phagocytosis of E. coli by the granulocytes. The other half tolerated the initial dose and had no chill and generally no significant drop in white cell number of phagocytic activity. All volunteers showed tolerance to repeated doses accompanied by increased phagocytic activity and rising serum antibody titers and complement titers. Vasoconstriction disappeared with tolerance but microcirculatory sludging increased, implying a possible immune mechanism for this sludging. Drops in platelet counts and hypercoagulability after endotoxin administration tended to diminish with tolerance. Serum antibody increases did not seem to be necessary for tolerance in patients with splencctomy. ilcknozuledgment: We would like to thank the medical and nursing staff of the University of Maryland Clinical Shock Unit and Dr. R. Adams Cowley, Director of the Unit for their aid in this study. We are also grateful for the guidance of Dr. Sheldon R. Greisman of the Division of Infectious Diseases of the Department of Medicine and for the technical assistance of C. Seitz, T. Houtz, and J. Crook.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14. 15.

REFERENCES

1. MAYER, M. M. Experimental Immunochemistry, 2nd ed., p. 149. Edited by Kabat, M. and Mayer, M. Springfield, Ill., 1961. Charles C Thomas. 2. Ibid., p. 162. 3. LANDY, M., MICHAEL, J. G., and WITBY, J. T. A bacteriocidal method for measurement in nor-

Vol. 113, May 1967

16.

17.

tin;

mal herum of antibody to gram negative bacteria. J. Burt., 83: 631, 1962. WOLF, S. M., WARD, S. B., and LANIX, M. Serologic properties of bentonite particles coated with microbial polysaccharides Proc. Sec. Expcr. Sol. is Med., 114: 530, 1963. KNSELY, M., BLOCK, E., ELLIOT, T. S., and WARNER, L. Sludged blood. Tr. Anz. Thernp. sot., .49: 95, 1950. MECHAXIC, R. C., FREI, E., LANDY, M., and SMITII, W. A. Quantitative studies of human leukocyte and febrile response to single and repeated doses of purified bacterial endotoxin. J. C&z. Imest., 41: 162, 1962. ECKER, E., WEISBERGER, A. S., and PILLEMER, L. The opsonins of normal and immune sera. I. Methods: a comparison of the effects of normal and immune opsonins on staphylococcus aureus. .1. Inamunol., 43: 277. 1942. LEE, T. Reticuloendothelial clearance of circulatory fibrin in the pathogenesis of the generalized schwartzman reaction. J. EsP. YV?d., 115: 106.5, 1962. LEE, T. Antigen antibody reactions in the pathogenesis of bilateral renal cortical necrosis. J. Exp. Med., 117: 365, 1963. GILBERT, V. E. and BRAUDE, A. Reduction of serum complement in rabbits after injection of endotoxin. J. Exper. Med.. 116: 477, 1962. GREISMAN, S. E., YOUNG, E. J., and CAROZZA, F. A. Immunosuppression and endotoxin tolerance. Clin. Res., 14: 332, 1966. GREISMAN, S. E., CAROZZA, F. A., and HILLS, J. D. Mechanisms of endotoxin tolerance K relationship between tolerance and reticuloendothelio system phagocytic activities in the rabbit. J. Exper. Med., 663, 1963. HERRING, W. B., HERROS, J. C., WALKER, R. I., and PALMER, J. G. Distribution and clearance of circulating endotoxin. J. Cl&. Invest., 42: 79, 1963. FARR, R. S. Fever as a manifestation of experimental allergy. J. AZZeryy, 30: 268, 1959. FARR. R. S., CLARK. S. H.. PROFFETT. 7. E.. and CA&PBEL~, D. H.‘Some humoral a&cts ok the development of tolerance to bacterial pyrogens in rabbits. Am. J. Physiol., 177: 269, 19.54. OI.LODART. R. M. and MANSBERGER. A. R. The effect of hypovolemic shock on bacterial defense. Bn?. J. Surg., 110: 302, 1965. FINE, J, The Bacterial Factor in Traumatic Shock. Springfield, Ill., 1954. Charles C Thomas.