IHE ALTERATION OF BACTERIAL ENTtOXr3N BY hUMAN AND RABBIT SERUM By John Edward Stauch A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the University of Michigan 1957 Committee in charge: Assistant Professor Arthur Go Johnson, Chairman Associate Professor Murry Ro Abell Assistant Professor Isadore Ao Bernstein Assistant Professor Harold Jo Blumenthal Professor Walter Jo Nungester

ACKNWLEDGEMENTS The author wishes to express his appreciation to Dro Ao Go Johnson for his guidance and encouragement during the course of this investigationo Particular gratitude is expressed to my wife whose helpful encouragement and understanding has made this study possibleo This investigation was partially sponsored by the United States Public Health Service grant #E 15240 The author wishes to express his appreciation to the United States Air Force for the opportunity to attend the University of Michigan while on active duty for the purpose of completing the requirements for the degree of Doctor of Philosophyo ii

TABLE OF CONTENTS Page INTRODUCTION o0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 HISTORICAL o o o o o o o o 0 0 3 MATERIALS AND METhiOCD o o o o o 0 0 o 0 0 0 0 0 o o o 0 0 O 9 lo Production of Bacterial Endotoxin o o o o o o o o o o0 o 9 20 Preparation of O-polysaccharide o o. o 0 0 0 0 0 0 0 11 3o Preparation of SO typlosa Whole Cell Vaccine o o0 o o o 0 17 40 Preparation of Anti-O Rabbit Serum o o o o o o o o o o o 18 50 Normal Rabbit Serum 0 o o o o 0 0 0 0oo o o o o o o o o 22 60 Normal Human Serum o o o o o o o o o o o o o o o o o o o 22 70 Versene Treated Serum o o o o o o o o o o o o o o o o 22 80 Resin Treated Serum o o o o o o o o o o o o o o o o o o o 22 9o Zymosan Treated Serum o o o o o o o o o o o o o o o o o 23 lOo heat Inactivated Serum 0 o o o o o o o o o o o o o o 23 1lo Tolerant Serum o o o o o o o o o o o o o o o o o o o o o o 24 12o Fluoride Treated Serum o o o o o o o o o o o o o o o o o 24 13o Mercury Treated Serum o o o o o o o o o o o o o o o o o o 26 14o Fractionation of Serum 0 o o o o o o o o o o o o o o o o o 26 15 o Normal Rat Serum * o o o o o o o o o o o o o o o o28 16o Preparation of White Blood Cells and White Blood Cell Lysate 28 17o Standard Quantitative Precipitable'Antibody Nitrogen Qurvqs 30 18o Serum Incubation Test System o o o o 0 o o o o 42 EXPERDMENTAL RESULTS o o o o o o o o o o o o o o o 0 0 o 0o 0 45 1e Action of Normal Serum on Endotoxin o o o 0 0 0 0 0 0 0 45 2o Activity of Serum on Purified Lipopolysaccharide o o 0o o 51 3o The Influence of Time of Incubation on the Alteration of Endotoxin by Normal Serum o o 0 o o o 0 o o o o o o o o 0 53 iii

Page 4, The Influence of Temperature of Incubation on the Alteration of Endotoxin. 0 0o o * o e 0 0 < o ao 0 o o o o 53 5, The Effect of Volume of Serum on the Alteration of Endotoxin o 58 6, The Effect of Concentration of Endotoxin on the Altering Capacity of Human Serum, o. ~ < o o 0 0 o o o 0 58 7. Effect of pH on the Alteration of Endotoxin 0 *. 0 0 a. o. 61 8, The effect of Storage of Serum on Its Altering Capacity. * o 61 9. The Properdin System in Relation to the Alteration of Endotoxin by Serum........ *. * * 0 o o o o o o62 10. The Effect of Normal Rat Serum on Endotoxin * o o o o 0 * o 066 11, The Effect of Leucocytes and Leucocytic Extract on the Alteration of Endotoxin o. * * o * * *. o o. * * * o o o o 68 12. The Effect of Chemical Inhibitors *. * * 0 ~. * * * * * o 0 71 13, The Effect of Boiled Serum on the Alteration of Endotoxin o 0 72 14. The Effect of Specific Enzymes on Endotoxin * o o ~ o 0 o 0 72 15, The Effect of Serum on 0-polysaccharide 0 0 o 0 0 0 0 0 0 0 0 73 16. The Effect of Tolerant Serum on Endotoxin. o. o o 0 0 0 o74 17. The Effect of Serum Fractions on Endotoxin * * * o *o o o 0 83 18. Biological Activity of Blood Components Incubated with Endotoxin ~ 0 ~ 0 O o. o 0 o 0 ~ 0 o 0 o o o o o o 91'DISCUSSION.. o,0 0 0 0o. o o o o o o o o o98 SUMMARY ~ o. * 0 0 0 ~ o o 0 0 0 0 0 o o o o o o 0o 1 o106 APPENDIX *. *. * o * *. o. 0.. o a,. o o0 o 0 107 1. Reagent Solutions * *. ~. ~ * * o o o o. 107 BIBLIOGRAPHY *. * *. o a, o ~ 0 o 0 0 0 o a * o 112 iv

LIST OF TABLES Table Page lo Program of Injection of Antigen for the Production of Antiserum o 0 o o o o o o o o o 20 20 Program for Injecting Endotoxin to Render Rabbits Toleranto o o o o 0 o o o o o o o o 0 25 3o Preparation of Standard HC1 from Constant Boiling HC1 o o 0 o o o 0 o o o o 110 4o Results of Quantitative Precipitable Antibody Nitrogen Tetermination Employing S1 Anti-O Serum and B3 Endotoxin o o 0 o0 o o o o o 32-33 5o Assay of Endotoxin After Incubation with Normal Rabbit Serum o a o o o o.0 oo 0 46 60 Activity of Normal Rabbit Sera on Ultracentrifuge Fractions of B3 Endotoxin *, oo o o o o 0o47-48 70 Per Cent Recovery of Endotoxin after Incubation with Normal Human Serum o o o o o o o 50 80 Comparative Activity of Normal Sera in the Alteration of Either Purified Lipopolysaccharide or Boivin Complex o o o o o o o o o o o 52 9. Effect of Resin on Normal Serum in Alteration of Endotoxin oo o o o o o o o o o 63 10o Effect of Versene on Normal Serum in Alteration of Endotoxin o o o o o o o o o o o o o o 64 11o Effect of Zymosan Adsorption on Normal Serum in Alteration of Endotoxin 0 o o0 o o o o oo 65 12o Effect of Heat Treated Serum on the Alteration of Endotoxin o o o o o o o o o o o o o 0o o 67 v

Table Page 13, Effect of White Blood Cells and White Blood Cell Lysate on the Alteration of Endotoxin. o o o o o o o 69-70 14, The Activity of Normal Sera on O-polysaccharide Compared with the Activity of Serum on Endotoxin o o o o 0 75 150 The Effect of Ammonium Sulfate Fractions of Normal Rabbit Serum on the Alteration of Endotoxin o o o o o o o 84 16, The Activity of Serum Fractions Obtained by Alcoholic Fractionation on the Alteration of Endotoxino, o o o 87 vi

LIST OF FIGURES Figure Page lo Flow Sheet for Preparation of Boivin Antigen,, o 12 2. Fractionation of Boivin Antigen by Ultracentrifugation * * * *. o * o o 0* 0 o 0 0 0 o o * 13 3. Purification of Boivin Antigen by Ethanol and Ammonium Sulfate Fractionation o o o. o o. o 15 4. Quantitation of cells of S. tvYhosa by Turbidity o. 19 5o Flow Sheet for "Alcohol Fractionation of Serum o o. 27 60 Quantitative Precipitable Antibody Nitrogen Curve with S1 Antiserum and B3 S. tvDhosa Endotoxin, o o 34 7, Quantitative Precipitable Antibody Nitrogen Curve with S2 Antiserum and B3 S. tvphosa Endotoxin * o. 35 8. Quantitative Precipitable Antibody Nitrogen Curves for Ultracentrifuged Fractions of B3 Endotoxin o - o 36 9. Quantitative Precipitable Antibody Nitrogen Curves for B3 (10,000 x Go Fraction) and Boivin Antigen *. 37 10. Quantitative Precipitable,Mntobody Nitrogen Curve for S3 Antiserum and B5 Endotoxin (10,000 x Go Fraction )................. 38 11. Quantitative Precipitable Antibody Nitrogen Curve for S3 Antiserum and Purified S. tvphosa Lipopolysaccharide. o *.. a,. a.. o. o o. 39 12. Quantitative Precipitable Antibody Nitrogen Curves for S3 Antiserum and P1 0-polysaccharide, Purified Lipopolysaccharide and Boivin Antigen (10,000 x G. Fraction). o 0 0 0 0 o 0 0 0 0 0. 0 o o 40 13. 4uantitative Precipitable Antobody Nitrogen Curves for S4 Antiserum and P2 0-polysaccharide and B12 Endotoxin (10,000 x G. Fraction) o. o o o O 41 vii

Figure Page 14o Procedure for Studying Reaction Between Serum and Endotoxin 0 0 0 o 0o o o o o o0o o oo 43 15o Rabbit Serum Incubated with Endotoxin at 37~C for Various Lengths of Time o o o o o o o o o o o o o 54 16, Human Serum Incubated with Endotoxin at 370C for Various Lengths of Time 0 0 0 o o 0 0 0 o 0 0o 55 17. Human Serum Incubated with Endotoxin at Various Temperatures o o. * * oo o o0 0 0 0 o 0 56 18o The Influence of Various Amounts of Serum on the Alteration of a Constant Amount of Endotoxin o o o 57 190 The Influence of Various'Anounts of Endotoxin on its Alteration by a Constant'Amount of Human Serum 59 20. The Effect of pH of Normal Serum on the Alteration of Endotoxin o 0 o.* * o. o o 60 21. Per Cent of Endotoxin Recovered After Incubation at 37~C/4 hrs. with Sera of Tolerant Rabbits o o 76 220 Per Cent of Endotoxin Recovered after Incubation at 37~C/4 hrs. with Sera of Rabbits during the Production of Tolerance o o. o. o o o 77 230 The Effect of Normal Rabbit Sera, Bled and Tested with Endotoxin as Controls to the Tolerant Rabbits o0 0 0 0 0 0 0 o *o * 0 * 0 0, *, o 79 24. The Effect of Sera upon Endotoxin from Rabbits Being Rendered Tolerant to Endotoxin o o o o o 80 25, Comparison of the Effect of Normal Sera and Sera from Rabbits being Rendered Tolerant o o * o o 0 81 26, The Effect of pH of Zinc Ion Preciptated Protein on the Alteration of Endotoxin o o. o o * o o o o 89 27. Pyrexia in Rabbits caused by Injection of Various Blood Components incubated 4 HrSo/37~C with Endotoxin o o o. 0 * o * 0, o o 93 viii

Figures Page 28, Pyrexia in Rabbits injected with Saline and H21 Serum Supernates diluted to contain 0.1 ugo of Recovered Endotoxin. o.o,o. o e o. o. o 95 29. Pyrexia in Rabbits injected with Saline and H21 Albuminelpte Globulin Serum Fraction Supernates. diluted to contain 0.1 ugo of Recovered Endotoxin * * * * *. 9....... o 97 ix

INTRODUCTION Living and dead Salmonella t.yphsa cells and the endotoxin obtained therefrom provoke varied pathologic findings and physiological effects in the hosto The methods of destruction of these cells or detoxification of their endotoxin have stimulated much interesto Most of the progress attained in the study of the interaction of bacterial endotoxins with host substances has been made in elucidating the changes in the host following endotoxin administrationo Little has been done to show whether or not the host exerts any effect upon endotoxino Beeson (1) reported in 1946 that endotoxin injected intravenously into rabbits was rapidly removed from the circulation by the reticulo-endothelial system (RoEoSo)o Braude e al (2) in 1955 and Cremer and Watson (3) in 1957 augmented this information by demonstrating that the majority of the injected endotoxin was taken up by the RoEoSo and granulocytes within five minutes after injectiono Animals pretreated with cortisone were able to remove endotoxin from the circ culation at the same rateo Howeverg where as endotoxin disappeared from the RoEoSo after 10 hours in normal rabbitsp the endotoxin in cortisone treated rabbits was retained by the RoEoSo for periods up to ten dayso These studies demonstrated that mechanisms of the host were able to remove endotoxic substances from the circulations but no evidence was obtained that the endotoxin was changed after uptake by the RoEoSo Several in vitro studies have demonstrated that endotoxin could be 1

2 altered by normal serumo In 1955 Cluff (4) found that a beta globulin in serum was capable of complexing with Shiaella endotoxin thereby altering its immunological activityo Employing agar gel diffusion procedures Cluff was able to show that three separate zones of precipitate (indicating at least three antigens) were formed when ShJiella endotoxin was testedo However, only one diffuse zone of precipitate was produced when Shiqella endotoxin9 which has been incubated with normal human or rabbit serum9 was employed as the antigeno In 1956 Rowley (5) reported the presence of a serum phosphatase that was capable of splitting phosphate from endotoxino Rowley employed endotoxin of Escncrica coL labeled with p32 and found that after incubi ation with normal rat serum, labeled phosphate could be recovered by dialysiso The purpose of this investigation was to determine whether or not normal serum was capable of altering purified endotoxin of tYphosao Also, if normal serum was found to alter endotoxin, thenr the physical conditions affecting the alteration would be investigated; characterization of the alteration would be attempted; and serum would be fractionated by various methods in an attempt to determine the serum factor involved in the alterationo Inasmuch as this endotoxic substance is considered to be synonymous with the Oc-antigenu any change in the Oantigen after incubation with serum would be reflected in its precipitating ability with specific antiserumo

HISTORICAL With Koch's unequivocal proof of the role of bacteria in disease, the mode of action in the causation of symptoms of disease led to much speculationo Many interesting theories in cause of death were offeredo These included capillary plugging, production of anoxia in the host, utilization of the host's vital foods, red blood cell destruction and the production of bacterial poisonso Brieger's (6) search for bacterial poisons led to the discovery of ptomaines which at best were only capable of causing mild distresso Roux and Yersin (7) were the first to show that bacterial toxins existed and that they were found in the cell-free filtrates of cultures of the diptheria bacilluso Subsequent investigations revealed that only a few bacteria were capable of producing these toxins while the majority of t- he disease producing bacteria were not associated with bacterial toxinso The interest in toxins was spurred on by the revelation of von Behring and Wernicke (8) that toxins injected in sublethal doses protected the host against the classical diseaseo Another type of toxin was found by Koch and by Rowland and Macfadyen when the tubercle bacillus was ground upo This toxin was intimately associated with the bacterial cell and required disruption of the cell before it could be demonstratedo Hence, the two types of toxins were called exotoxins, those secreted by the bacterial cell, and endotoxins, those released when the bacterial cell was disruptedo The investigation of toxins has proceded along three lines, one, their mode of actin and fate in t he host, necond, the her immunogenic response and, last their compositiono The mode of action of the 3

4 lipoplysaccharide toxins of endotoxins is obscureo Recent work has answered many questions concerning their fate in the host9 their composition9 and the immunogenic response0 Endotoxins cause numerous physiological responses when injected into a hosto However, the pathologic changes are varied and inconsistanto Following a single intravenous injection into a rabbit a sufficient dose of endotoxin to cause death, small punctate hemmorrhages are found in the thymus9 abdominal lymph nodes9 lungs and intestinal wallo Also9 there may be small areas of necrosis in the liver9 spleen9 lymph nodes and myocardiumo The presence of small thrombi attached to the endothelium of the veins in the liver9 heart and brain may be encounteredo (9)0 Zenker's hyaline necrosis has been observed in striated muscles and fibrinoid was observed beneath the endothelial lining of the coronary arteries (l),o The pathologic alterations following two spaced injections of endotoxin into rabbits include bilateral renal cortical necrosiso This has been designated the generalized Shwartzman reaction (11)o Brunson, Thomas and Gamble (12) have shown the generalized Shwartzman to be due to the occlusion of the renal arterioles by fibrinoid causing a hemorrhagic infarcto More recently Thomas and coworkers (13) have shown that there is an increase in arteriolar permeability due to the liberation of epinephrine in the kidneyso Delauney and co-workers (4) were able to show that9 following an intravenous injection of endotoxin9 waves of vasoconstriction and dilation of the arterioles occured with increasing severity until deaTho The Capillary beds and venules were not notably affectedo Tachycardia

and a diminished systolic force were also noted9 but, the arterial pressure was not affectedo Numerous investigators as reviewed by Burrows (15) have shown that a hyperglycemia occurs one to two hours after injection of endotoxin, but hypoglycemia developes prior to deatho The production of fever of endotoxin has stimulated the interest of many workerso Bennett and Beeson (16) presented a comprehensive review of the pyrogenic effect of Gram-negative bacteria and their endotoxins9 and indicated that no single mechanism adequately accounts for the fever productiono Two main mechanisms are apparento Heat may be retained due to the vasoconstriction of the surface arterioles9 or in the case of humans where the peripheral vasoconstruction is little noted9 the fever may be caused by the over production of heato The results of work of numerous investigators indicate that endotoxin Deer se is not an active pyrogenic agents but that the endotoxin either forms a hemic complex which is pyrogenic or that it stimulates the tissues to elaborate an endogenous substance which is pyrogenico The main basis for this reasoning is that the onset of fever is delayed from 90 to 120 minutes after an intravenous injection of endotoxino Menkin (17) was able to show a correlation between the action of pyrexins in the host with a bacterial pyrogen but9 indicated that they were not the sameo Bennett and Beeson (189 19) were not able to transfer the pyrogenic activity from tissues of animals injected with endotoxino However9 the granulocytes and the exudate contained a heat labile fraction causing fever with a shortened latent periodo Atkins

6 and Wood (20) were able to show by means of passive transfer of serum from rabbits injected with typhoid vaccine that an "endogenous pyrogen" appeared in the host after 60 minutes and remained circulating for 120 minuteso Grant and Whalen (21) were able to demonstrate that the latent period for production of fever was approximately one hour in rabbits given endotoxino The latent period could be reduced to 20 minutes when rabbits were injected with endotoxin which was incubated at 37~C for three hours with blood or plasma, which indicated an endotoxin activation of some factor in bloodo Gerbrandy, Cranston, and Snell (22) reduced the latency of fever in humans by injection of endotoxin which had been incubated with plasma. More recently they found (23) that endotoxin which was incubated with blood rich in leucocytes caused an enhanced fever production and a shortened latent period, In contrast, a mixture of endotoxin and blood poor in leucocytes caused no demonstrable activity, Goodale et. gja (24) showed that endotoxin lost its ability to trigger pyrexia in humans when it was incubated with normal serumo Hegemann (25) demonstrated that both serum and plasma were capable of neutralizing the pyrogenic activity attributed to endotoxino He also showed that the leucocytosis stimulating effect of endotoxin was lost after incubation with either serum or plasma. Pillemer and co-workers (26) demonstrated that endotoxin injected intravenously into mice combined with properdin and caused a temporary decrease in the circulating properdin level, but after 24 hours the properdin level returned to a higher levelo

7 Braude and co-workers in 1955 (2) using Cr51-labeled endotoxin were able to show that endotoxin had a great affinity for reticuloendothelial tissues0 More recently Cremer and Watson (3) using fluorescein-tagged antibody demonstrated that large doses of endotoxin injected into a rabbit were rapidly adsorbed by blood platelets and absorbed by granulocytes, As a result of the accumulation of leucocytes in the lungs after the injection of endotoxin, leucopenia developed, Both of these methods showed that endotoxin was adsorbed by the histiocytes within twenty minutes after injection of the endotoxin and was no longer present there after ten hours. However, rabbits treated with cortisone retained the endotoxin in their phagocytes which were unable to destroy or dispose of it. Ribble and co-workers (27) using Cr51-labeled. o endotoxin demonstrated that cortisonetreated mice had an increased splenic uptake of endotoxin while the hepatic uptake was impaired. All of these studies showed that endotoxin could combine with or alter blood constituents. However, little investigation has been done relative to the possible change in endotoxin during any of the above processes. While this present study was in progress two investigators having made a different approach reported results showing alteration of endotoxin by normal serum. Cluff (4) found that normal rabbit serum, normal human serum or the beta globulin fraction of normal serum incubated with Shiqella endotoxin altered the immunological reaction of the endotoxin as shown by the gel diffusion techniqueso One diffuse band of precipitate developed when an incubated mixture of endotoxin and serum was used as the antigen while three distinct

8 more dense than the saline-endotoxin controlso Moreover, Cluff demonstrated that the serum of rabbits tolerant to S., marcescens had the same capacity for altering the immunological activity of endotoxino The fraction of serum to which this activity was attributed was found to be heat stable at 56~C for 30 minuteso In contract the findings of Goodale and co-workers (23) and Hegemann (25), Cluff was also able to show that incubation of endotoxin with normal rabbit serum caused a fever of greater magnitude than endotoxin aloneo However, the latent period was not appreciable shortenedo Rowley (5) using,o coli endotoxin labeled with 32 found that a heat labile fraction of normal human, rat9 mouse and rabbit sera were capable of liberating dialyzable P32from the endotoxino He also found the optimum pH for the reaction to be about 8o The activity was greatly decreased when divalent cations were removed from the serum by ethylene diamine tetra-acetic acido The purpose of this study was to investigate the effect of normal serum in producing immunochemical or biological changes in endotoxin jer se., The endotoxin of S.o t is synonymous with the somatic antigen9 and therefore, if a known amount of endotoxin was added to serum or saline and incubated, one can assay these mixtures by the quantitative. precipitin technique as adapted to measurement of antigenso Recovery of 100% of the edotoxin by the quantitative precipitin reaction would indicate no alteration, while recovery of less than 100% of the endotaxin would indicate some change in the endotoxin manifested by its loss of combining ability with antibodyo

EMATERIALS ND METHODS lo Production of Bacterial Endotoxin The endotoxin of o,tphsa 0-901 was employed throughout this entire study with one exception where rabbits were rendered tolerant to endotoxin by Serrati.a marcesc ns- endotoxin*o A culture of So tyDhosa 0-901 was obtained from the Walter Reed Army Medical Graduate Schoolo The organism was maintained in the departmental culture collection by periodic transfer on tryptacase soy agaro Before the organism was used in the production of endotoxin, it was plated out on SS medium to ascertain its purity and checked by Kliger iron agar cultures and gram stain reactionso A typical smooth colony was picked from the SS medium and cultured in 50 mlo of trypticase soy broth for 18 hours at 37OCo This culture was used as inoculum for seed cultures which were prepared by adding 3 mlo of incculUm to 25 mlo of trypticase soy broths and incubated at 37~C for 18 hourso Stainless steel trays (18"xl6lx6") were used for mass cultures They were fitted with tops made of paper layered with cotton and Reynolds wrap aluminum and sterilizedo Trypticase soy agar, 1800 mlo was placed in the rabbit trays which gave an agar depth of about i incho The trays were autoclaved, cooled, and incubated to check for sterilityo One seed culture was used as inoculum for each tray which was poured onto the agar at one corner and then the tray was tilted back and forth to distribute the inoculum over the surface of the agaro The mass cultures were propagated at 370C for 18 hoursfafter which, the cells were scraped from the surface with a rubber *Supplied by Difco Corporation 9

10 spatula and washed off of the agar with cold phosphate saline buffer pH 7o0o Endotoxin was prepared according to the method of Boivin (27) or Webster and co-workers (28) employing trichloracetic acid and ethanol fractionationso Figure lo shows the procedure followed in the preparation of lots Boivin 2 (B2), Boivin 3 (B3) and Boivin 5 (B5) endotoxino Using this procedure the yield of lyophilized endotoxin was 4305 mgo (B2), 414o5 mgo (B3) and 998 mgo (B5)o Because of the unwieldy nature of using rabbit trays for mass cultures a simplier method was sought to grow large numbers of cellso It was decided to employ the method of Tyrrell, MacDonald and Gerhardt (30) for concentrating and increasing the yield of cellso The precedure employs the principle of biphasic media, a solid agar base, trypticase soy agar, overlaid with trypticase soy broth with a ratio of 4:1o The culture media were prepared in two liter Erlenmeyer flasks and after inoculation with the organisms were grown at 370C for 48 hours with aeration on a New Brunswick rotary shaker at a setting of fiveo Using this method lots Bll B12 and B13 endotoxin were preparedo The results of plate counting showed that a yield of 10 times the number of cells was obtained as prepared in a flask containing only nutrient brotho The lyophilized endotoxin represented a yield of 452 mgo (Bll)g 100 mgo (B12)9 and 537 mgo (B13)o These yields show that the same ammount of endotoxin can be prepared from one flask of biphasic media (750 mlo) as from one rabbit tray utilizing solid medium (1800 mlo)o B3 and B5 endotoxins were further purified by two methodso Figure 2 indicates the first method employed using the ultracentrifugeo

11 Because of the polydisperse nature of endotoxin a constant sedimentable fraction was obtained in the following mannero Endotoxin suspended in distilled water was centrifuged at 10,000 x G for 2 hourso The supernate was then centrifuged at 20,000 x G for 2 hours and again the resultant supernate was centrifuged at 35,000 x G for 2 hourso This procedure yielded four fractions, the 10,000 x G, 20,000 x Go, 35,000 x Go, and supernate fraction. After lyophilization, the various fractions represented 60 mgo9 20 mngo 12 mgo and 5 mg respectivelyo Endotoxin, 500 mg., was further purified according to the method of Webster and coworkers (29) employingethanol fractionation in the presence of high salt concentrations ammonium sulfate fractionation, and ethanol fractionation without salto The various fractions obtained were designated lipopolysaccharide (5L1) to lipopolysaccharide (5L10)o 223 mgo of this purified lipopolysaccharide (5L10) was obtained0 Figure 3o represents the procedure employed in the purification of the Boivin antigeno B2, B39 B5, and 5L10 endotoxin were compared for biological activity with an endotoxin received from Walter Reed Army Medical Graduate Schoolo These endotoxins were tested for pyrogenicity, antigenicity, and toxicity by localized Shwartzman activity, and they compared favorable with the standard preparationo Bll B12, and B139 endotoxin were compared with B5 endotoxin and were equally favorableo 2o Pre paration. Q-Posaccha;r e The haptenic moiety of lipopolysaccharide was prepared by two methodse Both methods are described in detail by Staub and Combes (31)o The method of Mesrobeanu (32) was first employed which involved the acid

12 Mass Culture - 1800 ml ToSoAgar 18 hr/370C Cells harvested (scraped & washed off aaar) 40C Cell susDension centrifuaed 15,000 x G/30 min. Cells wshedtwice (ice Cold PBS* pH 7_0) Cells weihed wetweiht) 4 miro ice cold water,/qgm cells Equalvoume ice co2ldu O..5 TCA** Agitate for 3J i ouys/40C Centri.fue 15.00 x G/5 min. SuperMate plus 2 vol0 ice cold abso ethanol Mix well - let stand 18 hrso/-2~C _Centiuge. 2000 R__lA2~c/60 min. Dissolve:recipitate ita in cold difstA water ialize, aaint dist _ wate r42 C * PBS- phosphate buffered saline * TCA - trichloracetic acid Figure l1 Flow sheet for preparation of Boivin antigen

13 100 mqo Boivi'n antigen 10.000 x G/2 hrs. PelI Iet Su lerna te 20. 000 x G/2 hrso Pellet Su- ernate 35.000 x G/2 hrsO ellet ernate Lvo1hilize Yield 60 mg 20 mg 12 mg 5 mg Figure 2. Fractionation of Boivin Antigen by Ultracentrifugation

14 hydrolysis of the somatic antigen. 110 mgo of Bll endotoxin was suspended in 100 ml. of distilled water to which 5 ml. of 1N acetic acid was addedo This mixture was heated in a boiling water bath for 1 houro The mixture was allowed to cool and the resultant precipitate was removed by centrifugation at 2500 RPM for 30 minutes (International refrigerated centrifuge)o The O-polysaccharide was precipitated from the supernate upon addition of four volumes of absolute ethanol. The resulting precipitate was removed by centrifugation at 2500 RFM for 30 minutes, and dissolved in 11 gm, of distilled water. The O-polysaccharide was again reprecipitated with six volumes of absolute ethanol, Since Staub states that O-polysaccharide prepared by this procedure is contaminated by immunologically inactive substances, it was decided to further purify by Freeman's procedure (33) employing glacial acetic acid fractionationo After the O-polysaccharide was reprecipitated in 6 volumes of absolute ethanol and removed by centrifugation, it was dissolved in 11 gm, of distilled water. The O-polysaccharide was carried through three ethanol precipitations at 46% and 86% ethanol by weighto The 45% fractions were discardedo The final precipitate was dissolved in 11 gmo of distilled water and to this 86% glacial acetic acid was added by weights and the resultant precipitate was discardedo The O-polysaccharide was precipitated when the concentration of glacial acetic acid in the supernate was increased to 91.8% by weight. The precipitate was dissolved in 11 gmso distilled water and refractionated with glacial acetic acid at 86% and 91,8% by weight two more times, The glacial acetic acid was eliminated from the precipitate by reprecipitation

15 Part A 500 mgo B5 endotoxin in 250 mlo disto water plus 8705 gmo NaC1 plus 99o3 mloabs ethanol (rapid stirrinq) 2000 rpm/_30' Pellet ol mole fraction supernate Plus 247.7 mlo ethanol I 2000 rpm/ 30' 2 mole fraction Pellet'supernate. plus 535 ml. ethanol. 1 [_ o 0~3 mole fraction I Pellet 2000 rpm/30' supernate _L2000 rnm/30'-.....Iplus 1118 mlo ethanol Pellet o4 mole fraction supernate dialvsis with dist. water lvonhilization Part B 357 mg, or o3 mole fraction in 357 mlo..dist. water plus l405o qao ammonium sulfate 15.000 x G/15'..... Pellet 50% sato Isupernate i~~~~~~~~~~~' ~~plus 28 gmo.i'~~~~~~~~~~~~~ ~ ~(NH4) _S04 I.!5000 x.G/15p' supernate plus 28 gmo Pellet 60% sato (.H4) S0__. I i _....15o000 x G/G5' I I Pellet 70% sato supernate i i d 1 -:I —-J.... LI., _,...... Dialvsis with dist_ water lyophilization Part C 250 ma. of 70% sato fraction in 250 mlo disto water carried through ol9, o2, o3 and o4 mole fractions as in P rt A without aCl concntrat Pellets dialized lyophilized Figure 3o Pu'ification of Boivin Antigen by Ethanol and Ammonium Sul fate Fra-ctionation

16 twice with 6 volumes of absolute ethanol. The O-polysaccharide was twice washed with absolute alcohol and twice washed with ethero It was finally dried.n vacuo over phospherous pentoxideo The yield was 34 mgo per 110 mgo of endotoxino The second method of preparation of 0-polysaccharide that of Freeman (33) employing the direct hydrolysis of So tvphosa 0-901 cellso The organisms were grown in the biphasic media as previously described using six flaskso After the cells were twice washed with PBS pH 71l, 34 gmo wet weight of cells were obtainedo The cells were suspended in 100 mlo distilled watero To this 1 N acetic acid was added so that the final concentration was N/5 acetic acid, and the resultant mixture was refluxed for two hours in a boiling water bathe After cooling and centrifuging at 2000 rpm/30 minutes in the International refrigerated centrifuges the supernatant was concentrated at 50~C to 1/8 of its original volumeo It was then neutralized with sodium bicarbonate to a pH of 6o5 and 1 volume of absolute ethanol was addedo The resultant precipitate was discarded and the O-polysaccharide was precipitated by addition of 5 volumes of absolute ethanol to the supernateo The precipitate was removed by centrifugation and dissolved in a smaller volume of distilled watero The two alcoholic fractionations were repeated five more timeso After addition of the alcohol, the precipitate was allowed to develop by standing at 4~C for four to five hourso When the precipitate would not develop after the 5 volume fractionation, 1 to 2 mil of glacial acetic acid was added to aid the formation of the precipitateo After the sixth 5 volume precipitation, the partially purified O-polysaccharide

17 was dissolved in a small volume of distilled water and weighedo The O-polysaccharide was precipitated upon addition of 94% glacial acetic acid by weighto The precipitate was removed by centrifugation at 109000 x G for 30 minuteso The 94% glacial acetic acid fractionation was repeated 3 timeso Finally the glacial acetic acid was removed by reprecipitation with 6 volumes of absolute ethanolo The 0-polysaccharide was twice washed with absolute ethanol and twice washed with ethers and dried in vacuo over phosphorus pentoxideo The yield was 246 mgo per 34go cells wet weighto These two products were designated P1 and P2o They were tested for pyrogenicity, antigenicity, and toxicity by local Shwartzman activityo They caused no elevation of fever, produced no antibody and did not cause a localized Shwartzman reactiono Standard quantitiative precipitable antibody nitrogen curves were determined for further quantitationo 3o PreParation ofS t osa Who Cell Vaccine Vaccines of two types were prepared0 lAcetone'killed=dried (AKD) cells were preparedo The cells were grown by shake culture for 18 hours at 37~Co The cultures were centrifuged at 2~C for two hours at 2100 rpm in the International refrigerated centrifugeo The cells were twice washed with cold PBS pH 7o09 and finally packedo Half of the cells were treated with three volumes of acetone and allowed to stand over night at 2~Co The acetone was decanted and the cells were dried at 37~Co After grinding the cells by mortar and pestleg the yield was 200709 gramso To prepare the vaccine the AKD cells were suspended

18 in phosphate saline buffer pH 7o0o A stock concentration of 2 mgo per m1o was thusly preparedo This vaccine was used to stimulate the production of antibody in rabbitso The other half of the cell mass was heat killed at 560C for one houro After testing for sterility in nutrient and thioglycollate broths the organisms were diluted with phosphate saline solution pH 7o0 to a concentration of 1 x 109 cells per mlo Standardization was accomplished by the Klett colorimeter after checking the tubes against a direct count of So tvhosa cellso Figure 4o represents the standard curve for the McFarland tubes with a 540 unb filtero 2o9 liters of -this vaccine were prepared and tested with antiserum of high titer (ls25,600) by the agglutination procedureo This preparation is a standard antigen suspension for use in the agglutination reactiono 40 Prepration of t-.Q_ b Rbbit Serm Five batches of antisera were prepared employing 44 rabbitso The same general pattern was employed for each batch of antiserumo The antisera was prepared employing AKD cells of So t 0bhosa o090l according to the protocol in Table lo Rabbits were bled on the 7th and 8th day after the last immunizing doseo A total of 100 mlo of blood was taken from each rabbit by incising the marginal ear veino After the second bleeding the rabbits were given 50 m1o of 10% dextrose-saline intraperitoneally to restore blood volume and prevent death due to loss of bloodo The sera from all rabbits were titered by the agglutination reaction employing the standard heat killed o vt osa antigeno 005 ml of serum was serially diluted to 1g25,6000 with either PBS pH 7o0 or

19 0 150 CD I-. L l j/ 50 4 8 12 16 20 24 x 10I CELLS ML. FIGURE 4. QUANTITATION OF CELLS OF S. TYPHOSA BY TURBIDITY (540 MU)

20 TABLE I Program of Injection of Antigen for the Production of Antiserum Day Route Amto injected Bled Subcutaneous 100 Uo 1 Intraperitoneal 100 ugo 3 Intravenous 500 ugo 5 Intravenous 1 mgo 8 Intraperitoneal 1 mgo 9 Intravenous 2 mgo 10 Intravenous 2 mgo 17 Bled _ - _.. c.. 18 Bled 32 Intravenous 1 mgo 39 Bled 40 Bled ".,.1.1.1..1 -

21 veronal saline pH 7o4o To each tube 0o5 mlo of antigen suspension was added and the reaction was incubated at 520C for 18 hourso The last tube showing macroscopic evidence of agglutination just different from the control was designated the titer of the serumo All sera with a titer of lt3200 or less were pooled, frozen in a separatory funnel and thawedo There were three apparent layers in the funnel. Each layer was removed and tested with the 0-901 standard antigen by the aforementioned agglutination reactiono The bottom layer gave a titer of 1-129800; the second layer a titer of 1:1l600; and the third or top layer was l:800o The heavy, high-titer layer was pooled with the other sera. The rabbits were rested 2 weeks, bled and injected with 1 mgo of AKD 0-901 cells intravenouslyo On the 7th and 8th day after the booster dose the rabbits were bled again as previously described and the sera tested as described. The two pools of sera were combined into one lot and complement was inactivated by an unrelated antigen-antibody reaction using the bovine serum albumin and antibovine serum albumin system. The reaction occured while incubation at 370C for two hours and for 48 hours at 4~Co After this reaction occured and the resulting precipitate was removed by centrifugation9 the batch of antiserum was distributed in 20 ml. amounts and stored in the frozen state at -20C until usedo Agglutination titers and quantitative precipitable antibody nitrogen curves were established for future quantitation of endotoxino

22 5 Normal Rabbit Serum Normal rabbits were bled from the marginal ear veino The blood was allowed to clot and the serum removedo The serum was tested for the presence of specific O-agglutinins by the agglutination reactiono All rabbit serum used as normal rabbit serum had a titer of less than 1:10 since this was the serum dilution in the first tubeo These sera were used for incubation with endotoxin and fractionationo 60 Normal Human Serum Human donors in good health were bled by syringe from the cephalic veino The blood was allowed to clot and the serum was removedo The serum from all donors was tested for the presence of specific O-agglutinins as described be fore and only sera with a titer of less than lslO was used as normal human serumo These sera were used for incubation with endotoxin and fractionation, 75 Versene Treated Serum Versene treated sera were prepared by adding 0,05M and Oo5M per mio of tetrasodium ethylene diamine tetraacedic acid (EDTA) to normal rabbit and human serao EDTA was added to these sera in excess for the removal of magnesium ionso According to Levine and co-workers (34) only Oo0073M EDTA is necessary to chelate all the magnesium ions present in serumo These sera were used to incubate with endotoxin to see if magnesium ions are necessary for the alteration of endotoxino 8o Resin Treated Serum Resin treated sera was prepared by adding an equal volume of the sodium form of Amberlite IRC - 50 to fresh normal rabbit and human

23 sera and gently mixed for ten minuteso The treated serum was then pipetted from the resino Amberlite IRC - 50 was.converted to the salt phase according to the method described by Lepow and co-workers (35)0 This method of removing magnesium ions from serum was employed by Pillemer and co-workers (36)o Resin treated serum was prepared to support the evidence obtained from versene treated sera, that magnesium ions are not necessary for altering endotoxino 9o Zyvmoosan Treated Serum Zymosan, the insoluble polysaccharide obtained from yeast cell walls by extraction, was added to fresh normal human sera (3 mg. per mio)o The mixture was gently mixed periodically during the 30 minute incubation at 24~Co The zymosan was removed from the serum by centrifugation in the Serval centrifuge at 10,000 x G for 15 minuteso According to Pillemer and co-workers (37) zymosan complexes with properdin during this procedure and removes the properdin from serum (RP) upon centrifugationo These sera were used to incubate with endotoxin to determine whether or not properdin is involved in the alteration of endotoxino 10o Heat Inactivated Serum Fresh human and rabbit normal sera were inactivated by heating in the water bath at 56 C for 30 minuteso It has been well documented by numerous investigators that the heat labile components of complement are inactivated by this techniqueo Also Pillemer and co-workers (38) have shown that properdin is inactivated by:this procedure0 Heat treated sera were used for incubation with endotoxin to determine if complement

24 was involved in the alteration of endotoxin and to support the evidence that properdin was not involved in the alteration of endotoxino 11o Tolerant Serum Since it has been shown by numerous investigators that heterologous endotoxins can render rabbits tolerant to other endotoxins, rabbits were treated as shown in Table 2o with S. marcesens endotoxin which was supplied,by the Difco Corporation of Detroit, Michigano The sera of these animals were tested by the agglutination test employing S, typhosaO 0-901 standard antigen suspension and no anti-typhosa 0-901 agglutnin s were presents These sera were used to incubate with So typhsa endotoxin to determine whether or not they were capable of altering ito Sera were collected from tolerant rabbits one day, 8 days, 15 days, 22 days and 29 days after their last injection of endotoxin and used to study their activity on endotoxino In order to further investigate the serum activity in tolerance, sera were collected from four rabbits while tolerance was being producedo The sera were collected before the 1st injection, 4, 8, 129 24 hours after the first injection, 24 hours after the second injection, 24 hours after the third injection, 24 hours after the 7th injection, 24 hours after the 10th injection, 14 and 21 days after the 10th injectiono 120 Fluoride Treated Serum Sodium fluoride was added to normal human serum so that the final concentration was 0o01M or 0olM fluoride ions. In addition 0olM magnesium ions and 0olM fluoride ions were added to serumo These sera were

25 TABLE 2 Program for Injecting Endotoxin To Render Rabbits Tolerant Degrees Fo change in Day Amto injected intravenously temperature (R19) 1 1 ugo 3o3 2 2 ugo lo4 3 2 ugo 005 4 5 ugo 2o0 5 5 ugo lo6 6 5 ugo 1ol 7 | 5 ugo 07 8 10 ugo 2o9 9 10 ugo 0o7 10 1 ugo Oo3 11 Bled A__~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

26 used to test the phosphatase inhibiting activity of serum on the alteration of endotoxin. 13 Mercury Treated Serum Mercuric acetate was added to normal sera so that the final concentration was OoOlM or OolM mercuric ionso The precipitate that developed was removed by centrifugation at 15,000 x G for 15 minutes0 These sera were used to study the effect of enzyme inhibition in serum on the alteration of endotoxino 14o Fractionation of Serum Fresh normal human and rabbit sera were fractionated by three methodso These employed the use of ammonium sulfate, alcohol in the cold, a combination of these two, or zinc ion fractionation in the coldo Rabbit serum was fractionated in the cold utilizing ammonium sulfate concentrations of 50% and 100%o Fresh human and rabbit sera were fractionated utilizing alcohol in the cold as described by Deutsch (39)o Figure 5o illustrates the procedure employedo The supernate to prep cipitate A contains the albumins and alpha globulinso After dialysis in the cold, these were lyophilizedo Precipitate A contains the beta globulins, gamma globulins and conalbuminso The beta globulins (precipitate B) are removed by changing the pH to 5ol with o051t acetic acid and precipitated with 50% ethanol to a final concentration of 10% at-2~Co The gamma globulins are removed from the supernate to precipitate B by changing the pH from 5l1 to 7b4 with 0o5M sodium bicarbonate and precipitated upon addition of an equal volume of 50% ethanol at 6~Co, The supernate to precipitate C contains the conalbuminso

27 Fresh serum diluted 1:3 with distilled water lo pH 7o4, chilled to -6~C 2o add 50% ethanol (-150C) to concento of 25%, drop by drop s'pernate Precipitate A lo add 100 mlo ice cold water 2o adjo to pH 502 with cold 0005M acetic acid 3o let stand 10 minutes 4o check pH 5o add 50% ethanol (-150C) to concento of 10% 60 tempo -20C for human and -60C for rabbit serum supernate Precipitate B lo adjust to pH 7o4 with 0o5M NaHC03 2o add equal volume of 50% ethanol 3o tempo -6~C supernate Precipitate C Figure 50 Flow sheet for the alcohol fractionation of serum.

28 The various fractions were recovered by dialysis and lyophilizationo The albumin-alpha globulin fraction was further fractionated with ammonium sulfate first using 50% and 10G% saturation, and then 40%, 50%, 60%, 70%, 80%, 90% and 100% saturationo All of the various serum fractions were used for incubation with endotoxin in an attempt to locate the serum component which was responsible for alteration of endotoxino The third method of fractionation employed zine ions according to the method of Cohn et al (40)o After the pH of fresh serum was adjusted to pH 7o4 with lN HC1, it was cooled to -50C in the alcoholdry ice batho While the serum was being agitateds zinc acetate was added slowly so that the final concentration of zinc ions was Oo02MO The precipitate was allowed to develop for 15 minutes and was removed by centrifugation at 109000 x G for 20 minuteso The precipitate was solubilized in 0o05M EDTAo The solubilized precipitate and the supernate were dialyzed in the cold with distilled watero The fractions were recovered upon lyophilization, and were used to test their activity on endotoxino 15o Normal Rat Serum Blood was taken by cardiac puncture from six ratso After the serum was separated from the clots, the sera were pooledo Rat serum was used to test its ability to alter endotoxino 16o Preparation of White Blood Cells and hie Blood Cell Lsate White blood cells were collected by two methodso They were collected from the peritoneal exudate of rabbits according to the

29 method of Hirsh (4)o The rabbits were injected intraperitoneally with 250 mlo of saline containing 250 mgo of glycogeno Four hours after the injection the rabbits were sacrificed by air embolism, and 200 mil of citrate saline was injected intraperitoneallyo The abdomen was kneaded and opened to the right of the midlineo The exudate was pipetted off. 780 mlo of exudate was collected from four rabbitso The exudate was maintained at room temperature while a total and differential while blood cell count was madeo There were 203 x 106 cells per mlo or a total of lo9 x 109 cells, 95% of the cells were granulocytes with neutrophiles predominatingo The exudate was centrifuged at 1400 rpm for 10 minutes at room temperature (International #2, in 250 mlo bottles)o The fluid was carefully drained and any red blood cells present were lysed on the addition of 20 mil of hypotonic saline solution (lysis solution)o After 10 minutes at room temperature 20 mi. of hypertonic saline solution (neutralizing solution) was addedo The cell suspension was placed in centrifuge tubes and spun at 1200 rpm for 10 minutes at room temperatureo The cells were twice washed with citrate salineo The white blood cells were suspended in a buffered salt solution (intracellular salt solution) at a concentration of 1 x 109 cells per 5 mlo White blood cells were collected from human heparinized blood after centrifuging at 1200 rpm for 10 minuteso The buffy coat was removed and suspended in citrate saline solutiono By repeated washing most of the red blood cells were eliminatedo Those remaining were lysed as in the previous methodo After the cells were twice washed

30 with citrate saline9 they were suspended in intracellular salt solution at a concentration of 1 x 109 cells per 5 mlo To prepare a white blood cell lysate, rabbit and human cells suspended in a buffered salt solution were frozen 3 times in an acetonedry ice bath (-900C) for 10 minutes and just thawed in a 380C water batho The lysate was placed in 50 mlo centrifuge tubes and spun at 3000 rpm for 15 minuteso The supernatant was stored at -200Co The white blood cells and the white blood cell lysates were incubated with endotoxin and with serum and endotoxin in order to see if they were active in altering endotoxin or enhanced the activity of serum in altering endotoxino 170 Standard Quantitative Preciitae Antibod Nitroen Curve Inansmuch as endotoxin is synonymous with the 0-somatic antigens a standard quantitative precipitable antibody nitrogen curve can be preparedo Since endotoxin is a phosphorylated lipopolysaccharide, all the nitrogen precipitated will be antibody nitrogeno If a constant amount of anti~serum is added to various concentrations of endotoxin9 a curve can be plotted for the amounts of antibody nitrogen precipitatedo This curve then can be used for quantitation of unknown amounts of endotoxino One mio of high titered antiserum (112s,800 or higher by agglutination reaction) was added to various amounts of endotoxin by weight in PBS pH 7000 The system was incubated at 370C for two hours and at 4~C for 18 hourso The resultant precipitates were centrifuged in the cold and the supernates were tested to determine the zone of activity, such as the zone of antibody excess, equivalence or

31 antigen excesso To determine this activity the supernates were divided into two sampleso To one sample, 10 micrograms of endotoxin were added and to the other 1 drop of antiserumo After incubation the tubes were centrifuged and inspected for the presence of precipitateo If a precipitate occured in the tubes where endotoxin was added, this indicated the zone of antibody excesso If precipitates formed in both of the paired tubes, this was evidence of the equinve alence zone and when precipitates formed in only those tubes where antiserum was added, this would demonstrate antigen excesso The precipitates for the antibody nitrogen curve were washed twice with cold PBS pH 7o0, and then digested with 1 mlo of kjeldahl digestion mixture plus a few crystals of potassium sulfateo The digestion mixture was heated until it was colorlesso The digestion mixtures were chilled and diluted with distilled water, made alkaline with saturated sodium hydroxide, distilled into saturated boric acid solution, and titrated with N/70 hydrochloric acid using methyl purple as an indicatoro The milligrams of nitrogen can be calculated using the factor of 002 times the number of milliliters of acid usedo Table 40 shows the amounts of endotoxin employed, the results of the nitrogen determination, and the supernate analysiso The determinations were done in duplicate and the results averagedo The blank was subtracted from the average values and these were plottedo Figure 60 is the resultant curve using S1 antiserum and B3 endotoxino Once this curve has been established it was used as a reference for quantitating unknown amounts of endotoxin so long as the pre

32 TABLE 4 Results of Quantitative Precipitable Antibody Nitrogen Determination Employing Sl Anti-O Serum and B3 Endotoxin Antigen Antibody Antibody Average Tube Added Precipo Ao Precipo B Antibody N2 Mlo W/70 mgo M1o N/70 mgO HCI N2 HC1 N2 1o 0 o05 o010 o05 o010 010 2o 15 o185 o037 o205 o041 o039 3o 30 o35 o070 o335 o067 o069 4o 50 047 0094 o47 0094 p094 5o 75 o57 oll4 o58 oll6 o115 6. 100 o70 ol40 o105 1o41 1o41 7o 150 o90 o180 o83 o166 o173 8o 200 o95 o190 o90 1o80 ol85

33 Table 4 (Continued) Supernate Tested Supernate Tested for excess A0 for excess Bo Antibody Antigen Antibody Antigen 74 17 I- t ttj - t

z 200I 0 z oop 0 20 40 60 80 100 120 140 160 180 200 MICROGRAMS ENDOTOXIN FIGURE 6. QUANTITATIVE PRECIPITABLE ANTIBODY NITROGEN CURVE WITH SI ANTISERUM AND B3 S. TYPHOSA ENDOTOXIN

z 250 0 z2 0 I,z 150 - r <1: 100 - 050 20 40 60 80 100 120 140 160 180 200 MICROGRAMS ENDOTOXIN FIGURE 7. QUANTITATIVE PRECIPITABLE ANTIBODY NITROGEN CURVE WITH S2 ANTISERUM AND B3 S. TYPHOSA ENDOTOXIN

A. 10000 X G FRACTION A B. 20000 X G FRACTION B C. 35000 X G FRACTION 0 D. SUPERNATE FRACTION cm IZC) CD 0_ 50 100 150 200 MICROGRAMS ENDOTOXIN FIGURE 8. QUANTITATIVE PRECIPITABLE ANTIBODY NITROGEN CURVES FOR ULTRACENTRIFUGED FRACTIONS OF B3 ENDOTOXIN

A. 10000 X G FRACTION 140 B. BOIVIN ANTIGEN 120- -:1001 ~80 60- - 40 20 50 100 150 200 MICROGRAMS ENDOTOXIN FIGURE 9. QUANTITATIVE PRECIPITABLE ANTIBODY NITROGEN CURVES FOR B3 10,000 X G. FRACTION AND BOIVIN ANTIGEN

140 i I z 100 oo CD Cs 20 40 60 80 00 120 140 160 200 MICROGRAMS ENDOTOXIN AND B5 ENDOTOXIN (10000 X G. FRACTION) cD AND B5 ENDOTOXIN (10000 X G. FRACTION)

39 200 - - I| Q i 150 OC 0 0 OC 0 50.2 4.6 S I 2 3 MILLIGRAMS LIPOPOLYSACCHARIDE FIGURE II. QUANTITATIVE PRECIPITABLE ANTIBODY NITROGEN CURVE FOR S3 ANTISERUM AND PURIFIED S.TYPHOSA LIPOPOLYSACCHARIDE

LU A., 0o /.^B. Cz15 o |cD^ A LIPOPOLYSPACHARIDE co | B. B5 ENDOTOXIN C. Pl O-POLYSACCHARIDE 0 a: o I I IpI 1I 40 80 120 160 200 400 MICROGRAMS ANTIGEN FIGURE 12. COMPARATIVE QUANTITATIVE PRECIPITABLE ANTIBODY NITROGEN CURVES FOR S3 ANTISERUM AND PI O-POLYSACCHARIDE, PURIFIED LIPOPOLYSACCHARIDE AND B5 ENDOTOXIN (1OOOO X G. FRACTION)

200,, 0 A 150 10- z <IJfr~~~ 51Rn~ / <~~ AA. P2 O-POLYSACCHARID at V iB. B12 ENDOTOXIN c: 20 40 60 80 100 120 140 160 180 200 MICROGRAMS ANTIGEN FIGURE 13. QUANTITATIVE PRECIPITABLE ANTIBODY NITROGEN CURVES FOR S4 ANTISERUM WITH P2 O-POLYSACCHARIDE AND B12 ENDOTOXIN (10000 X G. FRACTION)

42 cipitin reactions occur in the zone of antibody excess, and as long as these two reagents, Sl antiserum and B3 endotoxin, were employed. If either of these reagents were changed, a new curve was madeo Accordingly curves were established for S1 antiserum and B3 endotoxin, S2 antiserum and B3 endotoxin, S2 antiserum and B3 endotoxin ultracentrifuge fractions, S2 antiserum and B5 endotoxin:-10,000 x G fraction, S3 antiserum and B5 endotoxin 10,000 x G fraction, S3 antiserum and purified lipopolysaccharide (5L10), S3 antiserum and 0-polysaccharide (P1), S4 antiserum and B12 endotoxin 10,000 x G fraction, and S4 antiserum and O-polysaccharide (P2). Figures 6. to 13 are the resultant curves. 18. Serum Incubation Test Svstem The system employed to test the effect of fresh normal sera, treated sera, serum fractions, white blood cells and white blood cell lysate on endotoxin was basically the same, Figure 14. represents diagramatically the procedure employed. In all tests a salineendotoxin control was run along with the test substance and endotoxin. The test substance, such as 2.5 ml. of normal serum was added to a known amount of endotoxin. The amount of endotoxin was 2 mgo in the initial tests but 1 mg. was later used as the standard amount. The mixture was frequently shaken during incubation at various temperatures for various lengths of time. The standard conditions selected in the test procedure was 370C and the time of incubation was 4 hours for rabbit serum and 6 hours for human serum. After incubation the mixture was subjected to ultracentrifugation at 35,000 x G for two

43 2 M. SERU G ENDOTOXIN* INCUBA1E 370C ULTRACENTRIFUGE 35 000 x G/2 HOURS PESLET SUPERNATE / 5 ML. VERONAL BUFFER ASSAY FOR ENDOTOXIN Figure 14, Procedure for Studying Reaction Between Serum and Endotoxin *Suspended in 0.5 mlo veronal buffer

44 hours, After this the supernate was carefully collected and the pellet was solubilized in 5 ml. of veronal buffered saline pH 7.4. Any insoluble material present was removed by centrifugation at either 4000 rpm for 5 minutes or 2000 rpm for 10 minuteso Aliquots of the solubilized pellet and the supernate were assayed for the presence of endotoxin. Inasmuch as this endotoxic substance is the 0-antigen, it will combine with its specific antibodyo Accordingly, the aliquots were added to 1 mle of antisera (S1, 52, S3 or S4) and the amount of endotoxin present was analysed by the quantitative precipitable antibody nitrogen procedureo The amount of endotoxin was determined by interpolation from the standard curveso The amount of endotoxin recovered in the saline-endotoxin control was designated 100%. Therefore, if 100% of the endotoxin was recovered after treatment with the test substance, then the endotoxin was not affectedo

EXPERIMENTAL RESULTS 1 o Action of Normal _erum on Endotoxino A, Normal Rabbit Serumo Initial serum incubation experiments employing endotoxin derived by the technique of Boivin (28) were unsatisfactory for assay by the quantitative precipitin technique because more endotoxin was recovered in both the saline-endotoxin control and serum-endotoxin mixture then was added in the testo These data, as well as a detailed tabular account of the assay, can be seen in Table 5o From these results it is apparent that the Boivin antigen suspension could not be quantitated by this procedure so that the amount of endotoxin recovered from saline equaled the amount of endotoxin put in the mixtureo It is known that endotoxin is a polydisperse mixture of molecules varying widely in molecular weighto Therefore in order to obtain a constant sedimentable fraction of endotoxin, it was submitted to differential ultracentrifugation as previously described (Figure 2o)0 After the quantitative pgrecipitable antibody nitrogen curves were prepared for each of the various fractions of endotoxin (Figure 80), 1 moo of each fraction was incubated with 205 mlo of normal serum at 370C for two hourso Table 6 shows the results of this experimento The amount of endotoxin recovered from saline controls was approximately 100% except:11 _-1 -........:_.,,,......^ - -.: a:: In this study the term "altered endotoxin" means that per cent of endotoxin which was unable to combine with antibody as compared to that amount of endotoxin precipitated by antibody in the saline controlo The term "recovered endotoxinw means that per cent of endotoxin precipitated by antibody as compared to the amount of endotoxin precipitated by antibody in the saline controlo 45

46 TABLE 5 Assay of Endotoxin after Incubation with Normal Rabbit Serum Sample Saline 7 2 mgo Serum / 2 mg, endotoxin endotoxin Assay of Assay of Pellet Supernate Pellet Supernate Mio of antibody 1 1 1 1 Mlo of sample.5 o2 o5 o2 Mlo of buffer lo5 18 15 10o8 Reaction occur in Abo excess $ Incubate 3f1?2 hrso, overnight 4~C Mlo of N/70 o765 o345 ~665 o715 HCL X 0.2 - ug. Abo N 153 79 133 143 Pgo of endotoxin* 116 38 92 100 X dilo factor** mg. of endotoxin 2,784.560 2o208 1590 Total recovered mgo of endotoxin 3o344 3o798 Per Cent Recovered 167 189 Incubation time, 2 hours at 37C * Interpolation from Figure 6. ** Supernate dilution factor 1:15, pellet dilution factor 1:25

47 TABLE 6 Activity of Normal Rabbit Sera on Ultracentrifuge Fractions of B3 Endotoxin* Mgo endotoxin recovered Endotoxin fraction. saline serum Pelo supo Pelo sup 10,000 x Go 0475 o525 o538 o322 20,000 x Go o263 o810 o263 o450 35,000 x Go 113 1o005 o175 o698 Supo to 35,000 x Go 163 lo380 o138 lo140 *Incubation for 2 hours at 37~C

48 Table 6 Continued Total endotoxin recovered Per cent recovered saline serum saline serum 1000 o860 100 86 lo073 o7l3 100 66 loll8.873 100 78 lo543 lo278 100 83

49 in the supernate fraction following 359000 x Go The amount of endotoxin recovered from the serum-endotoxin mixtures was in all cases less than that recovered in the saline controls, indicating some activity by the serum on the endotoxin which prevented it from combining with its specific antibodyo This experiment was repeated twice and similar results were obtainedo Despite the fact that alteration appeared less when using the fraction sedimenting at 10,000 x Go,, it was employed for all further studies inasmuch as it contained the bulk of the whole endotoxion and allowed 100% recovery when incubated with salineo Bo Normal Human Serum Thirteen human sera were prepared from blood of donors and incubated with B3 and B5 endotoxin at 370C for various lengths of time0 The results of the experiments are presented in Table 7o In this series of experiments incubation of endotoxin with saline resulted in essentially 100% recovered endotoxino A comparison was made of the amount of endotoxin recovered in the supernate to that recovered in the pelleto In saline controls the supernate~ pellet ratio was 0ol to 03 or in other words 70~90% of the endotoxin recovered was found in the pelleto However, incubation of endotoxin in serum resulted in a shift of the amount of endotoxin recoverable from the pellet to the supernate, with a supernate M pellet ratio of 2 to 3, in other words only 20-35% of that recovered was found in the pelleto The supernate: pellet ratios in this experiment are fairly consistent falling in the range of

50 TABLE 7 Per Cent Recovery of Endotoxin after Incubation with Normal Human Serum Time Mg. Ratio Per Cent Serum Incubated Endotoxin Supernate Endotoxin Hours/37C Recovered To Pellet Recovered Hi 2.642 1,3 62 H2 3.502 0.9 46 H3 3.423 0,95 46 H4 4.442 0,7 41 H5 4.352 0,25 33 H6 4.400 0,7 38 H7 4,445 0.8 43 H8 4 o337 1,2 33 H9 4.533 2,7 52 H10 4.428 9,0 41 Hl! 4.419 18,0 42 H14* 6.289 1.2 28 H160 6.650 2.0 62 * Diabetic Serum (child) 0 Serum Stored 20 Days 40C

51 1 to 30* These data also show that, in general, human sera were capable of altering a greater percentage of endotoxin than rabbit serao 2o A ctivity Serum n Purified Lipoplvsaccharide The endotoxin used in the previous experiment is the Boivin complex of polysaccharide, lipid and protein (29)0 The protein moiety initially present in this product can be removed by purification, employing the method of Webster e X (29)o This procedure results in a product that retains complete antigenic and serological activity, Accordingly, a sample of endotoxin (B5) was purified by this method as described in Materials and Methods< After the quantitative precipitable antibody nitrogen curve was prepared for the purified lipopolysaccharide, 1 mg, of this endotoxic substance (#5L10) was incubated with normal rabbit and human sera and assayed as previously deScribedo Table 8 gives the results of four such experimentso These data indicate that normal serum has the ability to alter purified, protein-free lipopolysaccharide, and that the extent of alteration is essentially the same as that observed with the 10,000 x Go fraction of the Boivin antigeno Because of the greater ease in preparation, the Boivin antigen was used in all further experiments, except where notedo *The two exceptions (H10 & Hll) can be explained When endotoxin was prepared in suspension, one large batch was made so that uniform results would be obtained, It was observed that constant freezing and thawing of such an endotoxin suspension caused a shift from the pellet to the supernate of the endotoxin when incubated in the saline controlo In these two cases such results with saline control were obtainedo For later experiments endotoxin suspensions were prepared in a large batch but stored in small quantitieso This eliminated the freezing and thawing effect on the supernate: pellet ratioo

52 TABLE 8 Comparative activity of Normal Sera in the Alteration of either Purified Lipopolysaccharide or Boivin Complex* Purified lipopolysaccharide Total recov- Total recovy Per cent reSerum sample ered saline ered serum covered serum R 90 1 o90 o735 62 H 18 o940 o512 54 R 86 lo00l o708 71 R 93 10190 o752 63 Boivin complex Total recov- Total recov- Per cent reSerum sample ered saline ered serum covered serum R 90 10097 o772 70 H 18 0910 0505 55 R 86 a R 93 c *Boivin complex (B5, 10,000 x Go fraction)

53 3. The Influence of Time of Incubation on the Alteration of Endotoxin b. Normal Serum In order to determine the effect of time of incubation on the loss of endotoxin, a sample of endotoxin was incubated with normal rabbit serum at 37~C for 0,5, 2, 4, 6, 12 and 18 hours. After assay for endotoxin it was found that the amount of loss of endotoxin was a function of time with a maximum alteration occuring after four hourso Figure 15 is the curve showing the percentage of endotoxin recoveredo Two human sera were also studied with reference to the incubation time on the alteration of endotoxin, One normal serum was fresh while the other had been stored for twenty days at 4~Co Figure 16 shows the results of these two experiments. The fresh normal serum had a maximum activity at six hours incubation when only 22% of the endotoxin was recovered. The stored serum at 18 hours incubation had altered 58% of the sample* The latter two curves do not represent a comparison of stored and fresh sera inasmuch as they were different serum samples, 4, Te Influence of Temperature of Incubation Q the Alteration of Endotoxin. H17 serum and endotoxin were incubated at various temperatures for six hours, and the data are presented in Figure 17o At 0~C and 40C no alteration occured, but as the temperature was increased, the amount of alteration increased from 37% alteration at 560~C to 98% at 65~Co H18 serum and endotoxin were incubated in a similar manner and like results were obtained except that 55% of the endotoxin was altered at 560Co

a 80 0 I \ ~6O a 60~ 0' 60- 20O w LJ 20 O - I I f f 2 4 6 8 10 12 14 16 18 HOURS OF INCUBATION FIGURE 15. RABBIT SERUM INCUBATED WITH ENDOTOXIN AT 37C FOR VARIOUS LENGTHS OF TIME

o 10OIt w \ A FRESH SERUM uo B. STORED SERUM ~ 80 LJ cc: I, I \ I^,, *, x 60 I ^^^~ —— ~^ —^ L 40 - C~20 Q. 2 4 6 8 10 12 14 16 18 HOURS OF INCUBATION FIGURE16, HUMAN SERUM (H9-FRESH AND H16-STORED 20 DAYS/4C) INCUBATED WITH ENDOTOXIN AT 37C FOR VARIOUS LENGTHS OF TIME

56 Q 8 LU =: 80LU. 0 20 ( 4. x 0\.-. 4 -I - 20 40 60. 80 DEGREES CENTIGRADE FIGURE17. HUMAN SERUM INCUBATED WITH ENDOTOXIN AT VARIOUS TEMPERATURES VARIOUS TEMPERATURES

57 I0 o I - - z6 B 60 0 i 40 LL eL 20 20 L A. R8 RABBIT SERUM ~C B. H16 HUMAN SERUM 2 4 6 8 ML. OF SERUM FIGURE 18. THE INFLUENCE OF VARIOUS AMOUNTS OF SERUM ON THE ALTERATION OF A CONSTANT AMOUNT OF ENDOTOXIN(I MG.)

58 Again, no endotoxin was recoverable at 65~Co Saline-endotoxin controls incubated at the same temperature resulted in 100% recovery of the endotoxino 50 Effect ius Aoun of Serum o Alteration of Constant Amount of Endotoxin, The effect of volume of rabbit and human serum incubated with a constant amount of endotoxin at 370C for six hours was investigatedo No activity of rabbit serum was observed until a concentration of 205 mlo was reached and it remained constant with increasing volumes of serumo However, as the concentration of human (H17) serum was increased, the amount of endotoxin altered increased until the maximum alteration of 48% occured with 6.25 mlo of serumo H21 serum and endotoxin was incubated in the same manner and the curve was essentially the same except that 55% of the endotoxin was altered with 6o25 mlo of serum, 60 Effect of Concentration of Endotoxin on the Altering Capacitv of Human Serumo In order to study the influence of a constant amount of human serum (2.5 mlo) on varying amounts of endotoxin, 0o5 mgo to 4 mgo of the latter were incubated at 37~C for six hourso Figure 19 presents the results and indicates that serum is capable of altering a greater percentage of endotoxin if a smaller amount of endotoxin is incubated with it, since 50% of the endotoxin was altered when 0o5 mgo was incubated and the percentage decreased to 18% when 4 mgo was usedo

59 LU,g 80 o or ui Lu C4o z / 20 t. I 2 3 4 MG. OF ENDOTOXIN FIGURE 19. THE INFLUENCE OF VARIOUS AMOUNTS OF ENDOTOXIN ON ITS ALTERATION BY A CONSTANT AMOUNT OF HUMAN SERUM (2.5 ML.)

LU I,00U c 80 \ 60 0 6) z8Ou, ~~~~~~~~~~~~~~~~~~~~o Q 20 I — z 40 4 5 6 7 8 9 10 pH OF MIXTURES FIGURE 20. THE EFFECT OF pH OF NORMAL SERUM ON THE ALTERATION OF ENDOTOXIN

617 Effect of j onthe Alteration of Endotoxin Samples of normal human serum were adjusted to pH 4o0 to 10o0 with olN HC1 and OolN NaOHo Saline controls were similarly adjustedo To each of these, 1 mgo of endotoxin was added and the mixture incubated for six hours at 37~Co The pH's of the supernates were rechecked after the mixtures were centrifuged at 35,000 x Go for two hours and the supernates removedo The saline-endotoxin controls showed 100% recovery of the endotoxino Figure 200 shows the per cent of endotoxin recovered as a function of pH of the supernates after centrifugation, At pH 3,7, 5.1 and 9o8 normal human serum exerted no effect, since essentially 100% of the endotoxin initially added was recoveredo This experiment was repeated with the same serum and the pattern was essentially the same with maximum alteration at pH 7olo 80 Th Effect of Storage f Serum on Its Endot n Alterin Caacit Human serum stored at 40C for various lengths of time was tested for capacity to alter endotoxino The results of this experiment showed that there was no loss in this activity when stored for 10 dayso At 18 days the altering capacity had diminished 18% and at 25 days it had diminished by 25%. A second experiment employed a human serum sample which originally altered 37% of the endotoxin, but after 4 months at 4~C the altering capacity was completely losto Human serum stored at -24~C for a period of at least 30 days did not show a loss of its endotoxin altering capacityo

Summary of Phenomenon of Ateration ndotox by Normal Seum It has been shown that normal human and rabbit sera are capable of altering endotoxin that has been purified either by ultracentrifugation at 10,000 x Go for two hours or according to the method of Webster et a1. The amount of alteration is manifested by the loss of ability by the endotoxin to combine with its specific antibodyo It was found that the amount of alteration of endotoxin by serum was a function of time of incubation temperature of incubation9 pH of the mixture, and concentration of serum and endotoxino The serum component responsible for the alteration was relatively stable when serum was stored at -240~C A slight loss in activity was observed on standing at 4 to 60C for several weekso The following experiments were designed to investigate the nature of the serum factor and to gain insight into its mechanism of action on endotoxino 90 ah Properdin Sys i elatin o temration of Edtoxin b2 Serumo Pillemer et al (26) found that endotoxin is capable of combining with properdin in the presence of complemento Magnesium ions (36) were found essential for this reactiono In order to determine whether serum properdin was responsible for the alteration of endotoxin observed in the experiments reported herein, resin (IRC050) treated serum and versene (EDTA)-treated serum were prepared as previously described and incubated with endotoxino Tables 9 and 10 show that not only did these sera have the same ability to alter endotoxin as untreated sera, but

63 TABLE 9 Effect o Resinon Normal Serum n Alleratio n of E ndotoxin Total Mgo Endotoxin Recovered Serum Used Untreated Treated Rabbit 8 o532 o554 Human 10 0428 o298 Human 16 o652 0481 *Resin- IRC-50, sodium form

64 TABLE 10 Effect of Versene on Normal Serum in Alteration of Endotoxin Total Mg9 Endotoxin Recovered Oo5M/Mli Oo5M/Mlo Serum Used Untreated Treated Treated Rabbit 7,758 o734 Human 4 o442 0409 o323 Human 6 o400 o463 o322 Human 16 o650 o743 o536 Saline 1l035 o982 o913 4~~~ lo035 - _S_.1...'.11-

65 TABLE 11 Effect of Zvosan Adsorption on Normal Serum.in Alteration of Endotoxin Total Mg0 Endotoxin Recovered Serum Used Untreated Treated Human 16 o457 0437 Human 17.502.494

66 they were capable of altering a slightly greater percentage in most cases, Properdin was removed from serum by treatment with zymosan as previously described, and its effect on endotoxin tested. Table 11 demonstrates that zymosan adsorbed sera had the same altering capacity as untreated sera. In addition, nine sera were heat treated to 56~C for 30 minutes to inactivate complement, and then, incubated at 370C with endotoxin, The data in Table 12 indicates that there was a partial loss of the sera's ability to alter endotoxin; however, two sera, R6 and R4, had an increased activity when they were heat treatedo For the most part it would appear that heat treatment of serum causes a partial inactivation of the serum component responsible for altering endotoxin, The mean diminished activity of the nine sera was found to be 11%i 4,6o Three sera heated to 65~C for thirty minutes and tested for endotoxin altering activity showed the following results. One serum had the same altering capacity as unheated serum while the other two showed a diminished activityo In one case there was a 20% loss in altering capacity and in the other there was a 40% losso 10o The Effect of Norma Rat Seum on Endotoxin Rats have been found to be highly refractory to the pyrogenic, Shwartzman, and lethal effects of endotoxino In addition, rat serum contains a high level of properdino It was therefore of interest to study the effect of their sera upon endotoxino Sera from young rats were pooled and incubated with endotoxin at 37~C for six hourso After assay for endotoxin, 100% of the endotoxin was recovered as in the saline controlso There was, however, a shift of the supernate:pellet

67 TABLE 12 _he Effect oQf Heat Trate d Serwum* a Alteration of Endotoxin Mgo of Endotoxin Mgoof Endotoxin Mgo of Endotoxin Recovered with Un- Recovered with Recovered in Saline Serum heated Serum Heat Treated Serum Control R6 o635 o460 o951 H2 o502 o688 o951 H3 o423 o638 o927 H4 o442 o414 10088 H6 400 o509 1o040 H7 o445 o638 10040 H8 o337 o442 lo035 H9 o533 o703 o035 H10 o428 o685 10043 Mean diminished activity 11%L 406 *Serum heated to 56~C/30 minutes but incubated with endotoxin at 37Co

68 ratio and essentially all of the endotoxin recovered was in the supernate in contrast to the saline controlo Serum from another group of older rats was similarly tested with endotoxino This sera did have a small amount of activity since only 89% of the endotoxin was recoveredo A similar supernate:pellet ratio shift was seen hereo 1llo, Effect of Leucocytes and Le ucocvsYtic Extract on ti Alteration of Endotoxin. Since Braude and co-workers (2), Cremer and Watson (3) and Beeson (1) were able to show that endotoxin injected in vivo was rapidly absorbed by reticulo-endothelial cells and granulocytes, it was decided to study the effect of leucocytes and leucocytic extracts either alone or added to serum, for endotoxin altering activityo Accordingly, rabbit and human leucocytes were incubated with a saline solution of endotoxin and with a mixture of serum and endotoxino Table 13 presents the results of this experimento The addition of leucocytes to the serum-endotoxin mixtures resulted in essentially no change in the serumQs altering ability when the mixtures were incubated at 37~Co However, the combination of leucocytes, serum and endotoxin when incubated at 40C resulted in 15% alteration in one case and 17% alteration in anothero This is of interest inasmuch as it had been shown previously (Figure 17) that serum incubated with endotoxin at 4~Co resulted in no loss of endotoxino Rabbit leucocytic lysate incubated at 37~C with saline-endotoxin solution in the absence of serum caused a 12% loss in one sample and a 31% loss in anothero However, when the leucocytic. extract was incubated

69 TABLE 13 The Effect of h Biood Cells and White Blood Lvsate on the A teratn of Endotoxin* Endotoxin Temperature of Mgo of Endotoxin Per Cent of Incubated in Incubation Recovered Endotoxin Recovered R6 Serum 37 o585 60 R6 Serum f wbcQ 37 o648 66 R6 Serum 4 lo027 100 R6 Serum / wbc 4 o810 83 Saline / wbc 37 o880 90 Saline / wbc 4 o785 80 H2 Serum 37 0495 49 H2 Serum $ wbc| 37 o533 53 H2 Serum 4 o927 93 H2 Serum / wbc 4 o860 85 Saline / wbc 37 l1007 100 Saline / wbc 4 o905 90 H3 Serum 37 o447 45 H3 Serum / wbcw 37 o433 44 Saline $ wbc 37 loOOO 100 Rabbit wbc Lysate g Q $ Saline 37 o925 88 Rabbit wbc Lysate / Saline 4 o080 8 Rabbit wbc Lysate $ Saline (Stored 6 month/-24C 4 o560 53 Continued next page

70 TABLE 13 Con"to Rabbit wbc Lysate / Saline 37 o697 69 Rabbit wbc Lysate / Saline 4 o168 16 Human wbc Lysate / Saline (Stored 6 month/-24C) 37 10140 100 Human wbc Lysate 4 ol68 17 / Saline (Stored 6 month/-24C) * All tests in each block run concurrently l lo8 x 104 cells added " 203 x 106 cells added " wbc Lysate equivalent to 5o0 x 108 cells

71 at 4~C with endotoxin and saline, there was essentially a complete loss of endotoxino One rabbit leucocytic extracts stored for 6 months at -240C and tested for its ability to cause a loss of endotoxin retained 47% activity ioe. only 53% endotoxin was recoverable. A human leucocytic extract also stored for 6 months at -24~C was still extremely active at 4~C incubation with only 17% recoverable endotoxino In contract, 100% of endotoxin was recoverable when this same mixture was incubated at 37~Co 120o e Effect Chemical Inhibitors. In an attempt to inhibit the reaction between serum and endotoxin, fluoride ions in Oo01M and OolM final concentration, fluoride ions and magnesium ions in OolM final concentration, mercuric ions in OoO1 M and Ool M final concentration, and para-chloromercuric benzoate in OolM final concentration were added to normal serum as previously described in materials and methodso The sera so treated were added to endotoxin and incubated at 37~C for 4 to 6 hourso Mercuric ions in a OolM concentration caused precipitation of the serum proteinso The resultant supernate was unable to alter endotoxin since 100% of endotoxin was recovered as in the saline-endotoxin controlo None of the other chemicals had any effect in reducing the serum altering capacity on endotoxino The fluoride ions in OoOlM and OolM concentration in serum resulted in 50% recovery of endotoxin while the serum without fluoride ions resulted in 62% recovery of endotoxino Serum containing o1lM fluoride ions and OolM magnesium ions was able to alter 24% of the endotoxin while the untreated serum resulted in 18% alteration of

72 endotoxino'hen paraachloromercuric benzoate was added to serum in OolM concentration and tested with endotoxin, it resulted in 84% recovery of endotoxin, and the untreated serum at the same time resulted in 87% recovered endotoxino 130 Ie Effect of Boiled Serum on tf e Alteration of Endotoxin0 Since boiling of serum results in inactivation of nearly all enzymes, human serum was boiled for two minutes in a water bath to determine if such a treatment would inhibit the serumgs effect on endotoxino The jelled serum was centrifuged at 35,000 x Go for 30 minutes and the resultant supernate was incubated with endotoxin at 37 C for 6 hourso After assay for endotoxin the results showed that boiling serum causes essentially a complete loss in ability to alter endotoxin since 95% of the endotoxin was recovered while the same unboiled serum resulted in only 60% of the endotoxin recoveredo 14o t Effect of Specific Enzmes on ndotoxi Because the reaction between serum and endotoxin appeared to be an enzymatic reaction, alpha amylase, lysozyme and wheat germ lipase were incubated with endotoxin to see if any of these enzymes were capable of altering endotoxino Alpha amylase was chosen because it is a constituent of animal sera and is known to degrade glucose polysaccharides of alpha 1-4 linkageso Lysozyme (43) was employed since it has been used for the preparation of bacterial protoplasts from Gram negative organisms by degradating the polysaccharide in their cell wallso Wheat germ lipase was used since Kirsch (44) obtained evidence that it could detoxify endotoxin when it was incubated with this enzyme0 Alpha

73 amylase was added to saline and endotoxin in vernal buffer pH 704 in 100,ugo 10 uggo and 1/sgo concentrationso The lysozyme and wheat germ lipase were added to saline and endotoxin in vernal buffer pH 7o4 in 01% concentration, and all tests were incubated at 37~0C for six hourso The alpha amylase had some effect on the endotoxin in that 84% of the endotoxin was recovered from the 100lgo concentration, 89% in the 10 ugo concentration and 92% in 1 9go concentration of alpha amylaseo The lysozyme and wheat germ lipase had no effect since the amount of endotoxin recovered was essentially the same as the saline control0 15o Tc Effect of Serum on polvsaccharideo Since the polysaccharide moiety of endotoxin is generally regarded as responsible for the serological activity of endotoxin, and since serum alters the precipitating activity of endotoxin, it was of interest to test the effect of normal serum on this haptenic substanceo 0polysaccharide preparations, PI and P29 were prepared as previously described and the quantitative precipitable antibody nitrogen curves were prepared (see Figures 12 and 13o)o The latter were characteristic of the curves found by Landy et al (45) using a purified O~polysaccharidecharid e c -pys harides P1 and P2, were incubated with normal rabbit and human sera in the same manner as when testing the activity with endotoxino At the same time the same serum was tested with endow toxino After centrifuging at 35,000 x Go for two hours a pellet did not form either in the O-polysaccharide-serum mixtures or in the 0-polysaccharide-saline mixtureso Despite increasing the force to 1549000 x Go for 2 hours, no 0-polysaccharide was deposited as a pelleto As a result

74 all of the precipitating activity was found in the supernateso Table 14 shows the results of these experiments and clearly illustrates that normal serum has no effect on the polysaccharide moiety of endotoxino 100 l g of alpha amylase in PBS pH 7o0 was incubated with P2 O0polysaccharide and saline, and this experiment resulted in 100% recovery of the hapteno 16o Effect of Tolerant.Se on Endotoxin. Since rabbits rendered tolerant to endotoxin are refractory to pyrexia and localized Shwartsman activity, it was of interest to see if the serum of such animals had any effect on endotoxino Accordingly, three rabbits were rendered tolerant to S. marcescens endotoxin as previously describedo The sera of these animals were collected 1, 8, 15, 22 and 29 days after the last injection of endotoxino The sera from these animals were not tested before tolerance was beguno The sera while fresh were incubated with endotoxin for 4 hours at 370Co Figure 21o represents the activity of the sera on endotoxino The sera collected one day after the last injection were unable to alter endotoxin, while the sera collected 8, 15 and 22 days after the last injection had increasing activity in altering endotoxin until it was in the normal rangeo Since these results suggested that the sera of tolerant rabbits are unable to alter endotoxin, it was of interest to see how the sera of rabbits would react as they were being rendered toleranto Accordingly, four rabbits were made tolerant as previously described, and their sera were obtained before the first injection, 4, 8, 12 and 24

75 TABLE 14 he Activity of Nolma Sera 0-Povsaccharide*.Com a gdi tthe A tivt of Serum on Endotoxin 1 mgo of O-Poly.- Per Cent mg. of Per Cent Experiment Sample saccharide Recovered Endotoxin Recoered 1 Saline P1 100 B5 100 H18 Serum P1 100 B5 56 2 Saline P1 100 B5 100 Rll Serum Pi 100 B5 73 H19 Serum Pi 100 B5 54 3 Saline P2 100 B12 100 H20 P2 100 B12 60 *Incubation at 37C for 6 hourso

100 R84 R83 80 R85 60 40 20 3 I I I i. I, i *. i I I 2 4 6 8 10 12 14 16 18 20 22 24 26 28 DAYS AFTER IOTH INJECTION OF ENDOTOXIN FIGURE 21. PER CENT OF ENDOTOXIN RECOVERED AFTER INCUBATION AT 37C/4HRS. WITH SERA OF TOLERANT RABBITS

R86 —R8 —- 90 R89 **0 % 70.P-.. LAST INJECTION \......... 100 60'*.o,.. S.*1 2 3\ 4 85 6 7 B 9 t 11 12 1 2 16 17 21 DAYS AFTER COMMENCING THE PRODUCTION OF TOLERANCE 60 -....... I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 DAYS AFTER COMMENCING THE PRODUCTION OF TOLERANCE FIGURE22. PER CENT OF ENDOTOXIN RECOVERED AFTER INCUBATION AT 37C/4HRS. WITH SERA OF RABBITS DURING THE PRODUCTION OF TOLERANCE

78 hours after the first injection, 24 hours after the second, third, fifth, seventh and tenth injections, and then, at weekly intervals for three weeks. These sera were incubated with endotoxin to study their effect upon ito Figure 22 shows the results of this experiment. The activity of the serum during the first three days while tolerance was being developed showed considerable variation, However, after the third day there was a definite pattern showing a loss in ability to alter endotoxin. But, in no case did any of the sera show a complete loss as was seen in the preliminary experiment. After tolerance was established, the serum collected at weekly intervals showed an increasing activity to alter endotoxin until in the normal ranges Since the frequent bleeding of the rabbits may have had some effect on the reaction under study, a third experiment was done in the same manner as before employing three normal rabbits not injected with endotoxin and three rabbits which were rendered tolerant to endotoxin, Both the normal rabbits and the injected rabbits were bled at the same time and both sera were tested with endotoxin at the same time, Figures 23, 24 and 25 represent the results obtained after the samples were assayed for endotoxino The sera of the normal and injected rabbits had essentially the same pattern of activity during the experimentO The sera collected during the first 24 hours were variable in their altering activityo The results of analysis of the sera collected on the second day are most interesting in that both groups caused a high amount of precipitable nitrogen to be recovered which would indicate upon interpolation fiom the standard curve that more endotoxin was present than

166 I I - j l-4 j.I' Io I L.,J * i 2 4 6 0 17 24 La a ~ I 20 R.,. DAYS' Z -- "P.........' - - - - - - - LtA - 20- R2-25 R126 - R127..... 2 4 6 8 I0 17 24 DAYS FIGURE 2.3. THE EFFECT OF NORMAL RABBIT SERA, BLED AND TESTED WITH ENDOTOXIN AS CONTROLS TO THE TOLERANT RABBITS

140 LAJ 100 80 06 ~ ~ ~ ~ TLRN T NOXR Z I /' 46 46 8.......t72 LL't2 4 6 8 10.,17 24 DAYS FIGURE 24. THE EFFECT OF SERA UPON ENDOTOXIN FROM RABBITS BEING RENDERED TOLERANT TO ENDOTOXIN

140 LUL rI.,.I iNM 2 4 6 8 10 17 24 c) a:: 8 IL L NORMAL RABBITS INJECTED RABBITS 2 4 6 8 10 17 24 DAYS FIGURE 25. COMPARISON OF THE EFFECT OF NORMAL SERA AND SERA FROM RABBITS BEING RENDERED TOLERANT(MEAN OF 3 RABBITS; EXCEPT 17TH DAY, MEAN OF 2 N. SERA; AND 24TH DAY, I N. SERA; R125&127 DIED)

82 was employed in the testo It is possible that a compensating serum protein may be present that was capable of complexing with endotoxin, which, when combined with antibody, was responsible for the high amount of recovered precipitable nitrogen. After the second day, as Figures 23 and 24 show, the altering activity of the sera returned within the expected range, then, became hyperactive, and as the bleedings became less frequent there was a leveling off in the normal rangeo Figure 25 which is the mean of the three rabbits in each group shows that the two graphs parallel quite wello These results indicate that frequent bleeding may effect the endotoxin altering effect of the serao Summarv of Nature of the Serum Factor and Mechanism of Action on Endotoxino The evidence gathered in these experiments indicate that the properdin system is not involved in this reaction on endotoxino A factor that is capable of altering endotoxin in the absence of serum to a greater degree at 40C than at 370C can be obtained from white blood cells, Of the known enzymatic chemical inhibitors employed only mercuric ions in 01M concentration caused a loss of the serum's altering capacity. However, boiling serum for two minutes also produced a similar effects Of the three enzymes tested with endotoxin only alpha amylase had any effect, but this was minimal and due reservation must be exercised in implying that this is the factor in serum responsible for the altering activityo It was shown that serum is not active on the O-polysaccharide produced by acid hydrolysis of the lipopolysaccharideo Although an initial experiment indicated that the sera

83 from rabbits rendered tolerant to endotoxin was inhibited in the alteration of endotoxin, later experiments indicated that this finding may not be the general picture, Inasmuch as normal and injected animals showed a similar pattern in the altering activity on endotoxin indicates that the serum is affected by the frequent bleedingso 17, The Effect of Serum Fractions on Endotoxin. Preliminary to the purification of the factor in serum involved in the alteration of endotoxin, serum was fractionated by several procedures in an attempt to place the factor in one of the major serum protein groups. A. Ammonium Sulfate Fractions of Serum. Rabbit serum was fractionated into two fractions by precipitation with 50% and 100% saturated ammonium sulfate. These fractions were reconstituted in the normal concentration calculated to be contained in 2.5 ml. of whole serum and incubated with one mg. of endotoxin at 4~C and 37~C for three hours. Table 15 shows the results of this experimento The fraction precipitated at 100% saturation when incubated at 40~C had no activity but had 16% altering activity when incubated at 37~C. The samples of the 100% precipitable fraction that were heat treated to 560~C and 650~C for 30 minutes and incubated at 37~C also had approximately the same amount of activity. The 50% precipitable fraction incubated at 4~C had essentially no activity. However, the samples that were incubated at 370~C were interesting in that instead of causing a loss in precipitable nitrogen they gave a considerable increased amounts which would indicate 148% of endotoxin present~ Also,

84 TABLE 15 The effect of aammoniu sulfate fractions of normal rabbit serum on the Alteration of Endotoxin Temperature of Total Recov- Ratio Per Cent endoSample incubation ered endotoxin SOP toxin recovered Saline 37,881 2 100 100% fraction 4 o878 1 100 100% fraction 37 o740 2 84 100%(56C/30 Min) 37.705 2 80 100%(65C/30 Min) 37.680 2 77 50% fraction 4.846 1 96 50% fraction 37 1,480 58 167 50%(56C/30 Min) 37 1 456 46 165 50%(65C/30 Min) 37 10007 9 114 Saline 37 10089 ol 100 40% fraction 37.794 o25 73 60% fraction 37 1,143 3 105 80% fraction 37 1,010,2 93 Supernate fract, 37 1,136,13 104

85 essentially all of the precipitable nitrogen was in the supernateo Human serum #21 was similarly fractionated into 50% and 100% amrm'nuim sulfate saturation fractionse After reconstitution as above, these fractions were incubated with 1 mg, of endotoxin at 37~C for 6 hours, Whole normal serum when tested with endotoxin, resulted in 45% alteration of the endotoxino In contrast, the 100% ammonium sulfate fraction caused a loss of 16% of the endotoxin whereas the 50% fraction caused a 30% loss in endotoxin, Also,. the supernate:pellet ratio for the 50% fraction was as normally seen with a ratio of 2 while the endotoxin-saline ratio was 0,1, Another rabbit serum was fractionated into four fractions with 40% ammonium sulfate saturation, 40-60% saturation, 60-80% saturation and supernate to 80% saturation, These fractions were incubated at 370C with 1 mg, of endotoxin for four hours. Table 15 gives the results of this experiment. The 40% fraction contained essentially all the activity causing a 27% alteration of endotoxin, however, the 80% fraction did cause a slight loss of endotoxin. The 60% fraction was of interest in that though this fraction resulted in 100% recovery of endotoxin it caused the shift of recovered endotoxin from the pellet to the supernate with a ratio of supernate pellet of 3,0 compared to the saline ratio of 0,1o Bo Alcoholic Fractions of Serum Serum fractions obtained by the alcoholic procedure in the cold as previously described were incubated with endotoxin at 37~C for 6 hourso The albumin-alpha globulin fraction was the only fraction that was able

86 to show any appreciable activity, though some of the other fractions showed a small amount of activityo Table 16 shows the results of this studyo H19 and H21 albumin-alpha globulin fractions resulted in 45% and 38% alteration of endotoxin and caused a complete shift of the endotoxin from the pellet to the supernate with essentially all the recovered endotoxin in the supernate, while the saline control had 90% of the endotoxin in the pelleto One human serum (H20) was capable of 40% alteration of endotoxin, while all of the alcoholic fractions obtained from the serum had no altering ability of endotoxino However, the albumin-alpha globulin fraction did cause a complete shift of the recovered endotoxin from the pellet to the supernate as seen with H19 and H21, It is possible that this serum suffered denaturation since the temperature rose from -6~C to f20C during the initial fractionation stepo The sera from ten rabbits were pooled and fractionated by the alcoholic procedure. The whole sera when incubated with endotoxin resulted in 18% alteration of the endotoxin's precipitating activityo The albumin-alpha globulin fraction resulted in 19% alteration of the precipitating activity with again a complete shift of the recovered endotoxin from the pellet to the supernateo The other alcoholic fractions had essentially no activity with a supernate:pellet ratio like the saline control of 0olo Co I Frac tions f Sre 0 Because of the possibility of altering protein by the alcoholic procedure, it was decided to employ the zinc ion fractionation of CohnO This procedure yields a precipitated fraction which is completely

87 TABLE 16 The Activity of Serum Fractions Obtained by Alcoholic Fractionation on the Alteration of Endotoxin Serum Endotoxin Per cent of endotoxin recovered v.hen incubated with used Saline Albumin Alpha Beta Gamma serum -.._________. globulin globulin globulin H19 B5 100 55 100 79 46* H20 B12 100 100 128 98 60 H21 B13 100 62 132 100 55 Pooled Rabbit B5 100 81 113 94 82 * not done concurrently

88 soluble and unaltered in the presence of heavy metal chelating agents (40)o The fractions were reconstituted to the normal serum concentration and were incubated with endotoxin at 370C for 6 hourso The zinc ion soluble fraction resulted in 94% of the endotoxin recovered while the zinc ion precipitated fraction resulted in 84% of the endotoxin recovered* When the fractions were reconstituted in j the normal concentration of serum and incubated with endotoxin, the zinc ion soluble fraction resulted in 100% recovery of endotoxin, while the zinc ion precipitable fraction resulted in 87% recoveryo The zinc ion precipitable fraction was reconstituted to 1/8 the normal serum concentration and incubated with endotoxino This resulted in 91% recovery of endotoxino Because human serum (H20) was capable of altering 40% of the endotoxin and the zinc ion precipitated fraction was only able to alter 16% of the endotoxin, it was through that the pH of the reaction should be studied since it might have some effect on the amount of endotoxin alteredo Accordingly, 17 mgo of zinc ion precipitated protein (1/8 normal serum concentration) was suspended in buffers ranging from pH 4.0 to pH 10o0 and were incubated with 1 mgo of endotoxin at 37~C for 6 hourso Saline controls and endotoxin were similarly suspended in these bufferso All of the saline controls resulted in 100% recovery of endotoxin with the same supernate:pellet ratio of Ool. Figure 26 represents the results obtained for the zinc ion precipitable fraction and show that at pH 7o0 there was 28% loss in precipitating activity of the endotoxin which is 2/3 of the altering capacity of the

W I00 I.O 02 4 5 6 7 8 9 10 pH OF MIXTURES FIGURE 26 THE EFFECT OF H OF ZINC ON PRECIPITAED PROTE(H20 ON THE ALTERATION OF ENDOTOXIN 4 5 6 7 8 9 10 pH OF MIXTURES FIGURE 26, THE EFFECT OF pH OF ZINC ION PRECIPITATED PROTEIN (H20 SERUM) ON THE ALTERATION OF ENDOTOXIN

90 normal serumo The tests carried out at the ther pH values had less activity as seen in the figure, hence, pH 700 was considered to be the optimum for the altering activity of the zinc ion precipitated fractions of serumo This result compares favorably with the pH study on serum (see 9o) Human serum 21 was similarly fractionated with zinc ions and the two fractions were reconstituted to the normal serum concentration in PBS pH 7o00 After the two fractions were incubated with endotoxin at 37~C for 6 hours, the zinc ion precipitated fraction resulted in 36% alteration of endotoxin while the zinc ion soluble fraction resulted in no activity since 99% of the endotoxin was recoveredo Do Mercuric Ion Fractons of Serum Since OolM mercuric ions caused a precipitation of the serum proteins and the resultant supernate was not active on endotoxin, H21 serum was treated with mercuric ions as previously describedo The resultant precipitate was solubilized in versene (EDTA), dialyzed and lyophilizedo The precipitate and soluble fractions were then tested for activity in altering endotoxino The mercuric ion soluble fraction resulted in 100% recovery of endotoxin, whereas the mercuric ion precipitable fraction resulted in 30% alteration of the endotoxino Summary of the Effect of Alteration of Endotoxin wj Serum Fraci The active factor responsible for the alteration of endotoxin was found in the fraction precipitated by 50% saturated am"monium sulfate and in the albumin-alpha globulin fraction by the alcoholic procedure0 Zinc ions at Oo02M concentration at pH 7,4 precipitates alpha, beta

91 and gamma globulins, beta-lipoprotein and copper protein, while the soluble fraction contains albumins, metal-combining globulin, glycoprotein, amylase, iodoprotein and esterase, The endotoxin altering factor was present in the precipitated fractiono Mercuric ion fractionation at pH 7o4 in OlM concentration precipitated all protein, and the endotoxin altering factor was present in the precipitable fraction, These results indicate that the active factor responsible for endotoxin alteration is an alpha globulino 1.80 Bioloqical activit f Blood Components Incubated with Endotoxin When serum or fractions of serum containing the active factor are incubated with endotoxin and assayed, there is generally a certain amount of endotoxin that is recovered in the supernateo It was of interest to see if the endotoxin that is recovered had been altered in any other manner since there is the possibility that endotoxin could be changed without altering its serological activityo The lipopolysaccharide could be degraded to phospholipid and 0-polysaccharide, and in this case the 0-polysaccharide would have equal precipitating activity as the complete antigeno It is generally considered that endotoxin must contain the complete molecule to be pyrogenico Therefore, this activity was used as a method to denote such a degradation of the molecule of endotoxin other than involving its precipitating ability0 Ao lae Pyroqenic Activity of Blood and Serum Incubated with Endotoxino Grant and Whalen (21) have reported that when endotoxin is

92 incubated with rabbit whole blood at 37~C for three to four hours and then injected into rabbits, there was a reduced latent period in the subsequent production of fevero Cranston et al (23) reported that when human blood rich in leucocytes is incubated with endotoxin and injected into humans, the latent period is reduced and the extent of fever is increased, while blood poor in leucocytes is incubated with endotoxin, the latent period is not affected and the elevation in body temperature is decreasedo Goodale and co-workers (24) and Hegemann (25) have reported that when normal human serum is incubated with endotoxin, there is a neutralization of the pyrogenic effect of endotoxin. In an attempt to reproduce these experiments with rabbits and to gain insight in the effects of blood components incubated with endotoxin 8 ml. of rabbit whole blood, blood rich in leucocytes (blood plus buffy coat), blood poor in leucocytes (blood minus buffy coat), normal rabbit serum and saline were each incubated with 4)pgo of So tvphosa endotoxin (B5, 10,000 x Go fraction) at 370C for four hourso Following this, 1 mlo (calculated to contain 0o5pgo of endotoxin of each sample) was injected into groups of two rabbits eacho The rectal temperatures were taken every 30 minutes until the fever returned to normalo Figure 27 represents the degrees of fever produced by these sampleso The graphs are the mean response of two rabbitso The whole blood gave the poorest fever response with only lo9~F and the latent period was not reducedo The blood rich in leucocytes had a normal latent period but had a slightly increased fever compared to blood poor in leucocytes which had a delayed latent period

./// BLORIw.B.C.LUe' 2 i. ~ LJ _ 3e. I... tiL2~;..:-. 0LA 2 -3 4 LL'': // BINCUBPOOR 4 W.BjC - \'\ CD I'.'0 SALINE +.5 UG. \" WHOLE BLOOD +.5 UG... ee~0' ~BLOOD POOR IN WBC.t5UG. —.. — SERUM +.5 UG.. ee 2 3 4 5 HOURS AFTER INJECTION FIGURE. 27 PYREXIA IN RABBITS CAUSED.3Y INJECTION OF VARIOUS BLOOD COMPONENTS INCUBATED 4 HR./37C WITH ENDOTOXIN

94 and a decreased fever response. The normal serum had a delayed latent period but gave the greatest fever response with 3o2~F and the most prolonged fever response, This is in contrast to the results of (21), (24) and (25). B. e Pvroqenic Activity of th Supernate after Serum Incubation with C. Mq, of Endotoxin. In order to determine whether the endotoxin that is present in the supernate of serum after incubation has been altered, it was decided to investigate its pyrogenic activity. 2,5 ml, of normal human serum was incubated with endotoxin, centrifuged at 35,000 x Go for two hours, and the pellet and supernate assayed for endotoxin, The supernates were diluted so that 1 ml. contained 0.1 g. of endotoxin, The saline-endotoxin supernate was similarly diluted. One ml, of these samples was injected I.V. into rabbits and the rectal temperatures recorded at 30 minute intervals, Figure 28 represents the degrees of fever produced by these samples. The results of this experiment shows that the endotoxin recovered in the supernate was not altered with respect to pyrogenic activity. C The Pyroqenic Activity of the Supernate after Albumin-Aha Globulin Incubation with Mq of Endotoxino In studying the effect of the albumin-alpha globulin fraction of serum on the alteration of endotoxin, it was observed that all of the endotoxin present was found in the supernate. To determine if this endotoxin was altered by the fraction, its pyrogenic activity was studied. As with the serum supernate in the previous experiment, the

R116 SALINE SUPERNATE.I UG. R117 SERUM SUPERNATE.IU_.._ R118 SERUM SUPERNATE.IUG___ c: w LL LL 0J U / %''so 7U /1!lp~ % 2 3 4 5 HOURS AFTER INJECTION FIGURE 28. PYREXIA IN RABBITS INJECTED WITH SALINE AND H21 SERUM SUPERNATES D(LUTED TO' CONTAIN.1 UG. OF RECOVERED ENDOTOXIN

96 saline-endotoxin supernate and the albumin alpha globulin supernate were diluted so that 1 ml. contained O01 ygo of recovered endotoxins and one ml. was injected I.V. into rabbitso Rectal temperatures were recorded at 30 minute intervals. Figure 29 represents the degrees of fever produced by these samples. The latent periods were not reduced by the albumin-alpha globulin supernates, but they caused a slightly increased and prolonged fever response than the saline control supernateo These results show that the endotoxin recoverable following incubation was not biologically or serologically altered by the albumin-alpha globulin fraction,

R141 SALINE SUP PJ~ ^*S. R R142 ALB-' >GLOB. SUP___ e0c 2s - / ^~ >~ ~R143 ALB.- cGLOB. SU.... w AJ / S"!/i \., I 2 3 4 HOURS AFTER INJECTION FIGURE 29 PYREXIA IN RABBITS INJECTED WITH SALINE AND H21 ALBUMIN- o GLOBULIN SERUM FRACTION SUPERNATES DILUTED TO CONTAIN,. UG. OF RECOVERED ENDOTOXIN

DISCUSSION It was found that normal human and rabbit sera were capable of altering the precipitating activity of endotoxin when incubated with it. The extent of alteration was found to be a function of time of incubation, temperature of incubation, pH, and concentration of serum and endotoxin, Along with these attributes, the fact that the endotoxin altering substance in serum deteriorates upon storage for four months at 4~C, and is inactivated upon boiling suggests that the reaction between serum and endotoxin may be an enzymatic oneo Rowley (5) working with the endotoxin of Eo coli found that normal serum from rats and other mammals was capable of splitting lipopolysaccharide as demonstrated by the liberation of labeled phosphorouso The amount of recovered P32 was found to be a function of temperature of incubation, time of incubation and concentration of serumo In contrast to this study, Rowley found the optimal pH to be 802 and that heating the serum to 56~C for twenty minutes completely inactivated the serum-splitting ability, Also, the addition of versene (EDTA) to serum reduced the splitting activity by sixty per cento Moreover, Rowley found that rat serum was more active than human or rabbit serum, while in this study human serum was most activet then rabbit, and rat serum had little if any activity. Rowley (5) distinguished between the properdin system and the phosphatase splitting activity by showing that human serum with the third component of complement removed (R3) had splitting activity 98

99 equal to the serum from which it was derivedo The properdin system was eliminated in this study on the alteration of endotoxin by removal of magnesium ions which are essential for the properdin system; the heat inactivation of complement; and the removal of properdin by zymosan adsorptiono The sera from which magnesium ions were removed were capable of altering as much as or slightly more endotoxin then the same untreated serao Heat treatment of serum to 56~C accomplishes inactivation of complement and properdino The sera so treated had a partial inactivation for the alteration of endotoxino The mean diminished serum activity for nine sera was 11%s 4o6 (95% probability) of the untreated serao The sera from which properdin was removed by adsorption with zymosan had equal endotoxin altering ability as the serum from which it was derivedo Two experiments which were done indicate that Rowley's splitting factor is not the primary action of the serum factor in this studyo Normal sera that were treated with OoO1M and OolM fluoride ions and a combination of OolM fluoride ions and OolM magnesium ions were not inhibited by the presence of these ions, and fluoride ions in general inhibit phosphatase activity, Rats are highly refractory to the Shwartzman reaction, pyrogenicity, and lethality of S~ tvphosa endotoxino Their sera showed little or no alteration of this endotoxin when incubated with it, while Rowley's splitting factor was most active in rat serumo Cluff (4) studied the immunological activity of Shiqella endotoxin following incubation with normal serumo Employing the agar

100 diffusion techniques to denote any change manifested by the normal serumendotoxin mixture. Cluff found that one diffuse zone of precipitate developed, while three distinct zones of precipitate developed in the saline-endotoxin control, The observed results indicate that each of the three different antigenic components of Shiqella endotoxin contains a substance common to all three which is capable of combining with a serum component, Such a complex, then, would result in one diffuse zone of precipitation instead of thethree zones seen in the salineendotoxin controls, Cluff also found that human serum was equally capable of altering the immunological reaction of endotoxin and that by heating the serum to 560C for thirty minutes prior to incubation with endotoxin did not change the capacity of serum for altering the endotoxin's immunological reactivityo Moreover, serum from rabbits made tolerant to Sn marcescens endotoxin was equally capable of altering the endotoxin's serological activityo Following zonal electrophoresis in starch of normal rabbit serum, it was found that the beta globulin fraction was responsible for this activity on endotoxino In thisstudy, in contrast to Cluff's procedure, various fractionation procedures were employed to determine the type of serum protein involved in the alteration of endotoxin, The results of these fractionations indicate that possibly an alpha globulin is associated with the endotoxin-altering activityo Hence, it is a different factor than Cluff's beta globulin altering componento Cluff's factor could cause an iincrease in recoverable preciptable nitrogen if an endotoxn-beta globulin-antibody complex was formedo There was some evidence of this

101 type of activity during the course of this studyo Three different beta globulin fractions obtained by alcoholic fractionation resulted in increased amounts of precipitable nitrogen recovered compared to the saline-endotoxin controls. Cluff's factor may also explain the results obtained from the sera of normal animals and animals being rendered tolerant on the second day (Figures 23-25) where increased amounts of precipitable nitrogen were obtainedo Perhaps, in compensating for the loss of blood volume, unusual levels of various serum proteins were produced, and a greater amount of this beta globulin which is able to combine with endotoxin was present on the second day. In contrast to Cluff's results, the alteration of endotoxin by serum in this study demonstrated a loss in precipitating ability of endotoxino Two experiments performed in this study indicate that the endotoxin altering component is different than Cluff's reported factor. Cluff performed his test employing approximately 1 mlo of normal rabbit serum which was incubated with endotoxin for only thirty minutes at 37~Co In contrast to Cluff's results, in this study essentially no endotoxin precipitating ability was lost when endotoxin was incubated for only thirty minutes at 37~C, and a volume of more than 1l25 mio of rabbit serum was required to demonstrate any loss of endotoxin precipitating ability, In other words under similar conditions, there was no demonstrable alteration of endotoxin in this studyo In testing the effect of concentration of endotoxin with a constant amount of serum, it was found that a greater percentage of

102 endotoxin lost its combining power when 0O5 mg. of endotoxin was used than when 4 mg. of endotoxin was used, Cluff employed 4 to 6 mgo of endotoxin in his test, Perhaps, in Cluff's work some of the endotoxin lost its combining ability, but since such an excess was employed, there was more than enough to present his observed results of one diffuse zone, Cluff did not investigate a quantitative effect on endotoxin er se, but only demonstrated that the endotoxin present was capable of combining with antibody, and as a result one diffuse zone of precipitate developedo Braude et al (2) and Cremer and Watson (3) were able to show that endotoxin injected into rabbits was adsorbed by reticulo-endothelial cells and granulocytes within five to ten minutes after injectiono The endotoxin was retained by these cells up to 10 hours, after which it could no longer be foundo In this study to determine whether or not leucocytes or leucocytic extract had any effect on endotoxin, these substances were tested with endotoxin. The results indicate that a factor is present in white blood cells that is capable of altering endotoxin in the absence of serum and that a greater extent of endotoxin is altered when incubated at 4~C than at 37~C, The rate of reaction for enzymes is in general slow at 4~C, but perhaps incubation of the mixture for 6 hours is long enough for the demonstrated activity to occuro Also, there is the possibility that a naturally occuring inhibitor is present in white blood cells that is able to limit the reaction at 37~C but is not inhibitory at 40C0 Moreover, the enzyme, if one is responsible for

103 the activity is more stable at 4~C than at 37~C, Whether or not this factor is able to operate in the host has not been studied and is yet to be determined. In this study the serum-endotoxin mixture and the saline-endotoxin mixture were subjected to ultracentrifugation for two hours at 35,000 x G. in order to gain insight into the change, if any, in molecular sizes The fact that endotoxin can be fractionated by differential ultracentrifugation into molecules of different size, is in keeping with the polydisperse nature of endotoxino The endotoxin used for this study was a fraction obtained by ultracentrifugation at 10,000 x Go for two hours. This fraction contains the largest molecules, and when 1 mg, of this fraction was incubated in saline and subsequently centrifuged at 35,000 x G for two hours and assayed, 90% of the endotoxin was found in the pellets However, when 1 mgo of this endotoxin was incubated with serum and similarly centrifuged and assayed, up to 90% of the endotoxin recovered was then found in the supernate instead of the pellet, Three experiments eliminated the specific density of serum as the cause for this shift from the pellet to supernate. Serum was mixed with 1 mgo of endotoxin and centrifuged at 35,000 x Go for two hours without incubation. Essentially 100% of the endotoxin was recovered and 90% of the endotoxin was found in the pellet like the saline controls Serum incubated with endotoxin at 40C for 6 hours also resulted in 100% recovery of the endotoxin with the same ratio of 90% in the pellet after centrifugationo Moreover, serum stored for four months at 4~C and incubated with 1 mgo of endotoxin

104 resulted in 100% recoverable endotoxin and the same ratio existed with 90% of the endotoxin in the pellet~ From this evidence one might conclude that serum changes the molecular size in the process of alteration of endotoxino This would indicate that serum degrades the molecules to a size so that they are too small to be sedimented at 35,000 x G. for two hours, but are still active with antibody while the endotoxin that is lost is degraded to a point where it will. not precipitate antibody, The polysaccharide moiety is generally considered to be responsible for the serological activity of endotoxino Therefore, since there was a loss in precipitating ability as well as a change in molecular size, it was thought that the serum component must be primarily directed against the polysaccharide moiety of endotoxino However, when normal sera were incubated with 0-polysaccharide, the polysaccharide moiety of endotoxin, there was no loss in precipitating ability of this haptenic substance* This indicates that the serum altering factor is not primarily directed against the O-polysaccharide serologically active site, but rather, it must degrade the molecule at some other linkageo Landy et (45) in studying the homogeneity of purified lipoplysaccharide found that it contained three serologically active components by the Oudin gel diffusion technique, while the hapten derived from it showed only one visible band. Also, 0-hapten was only capable of precipitating 75% ofre the total antibody precipitable by the lipopolysaccharideo Therefore, the immunological role of lipopolysaccharide in the precipitin reaction is not wholly confined to the serological activity

105 of the polysaccharide moietyo The observation of Landy et al (45) may, therefore, explain the lack of serum altering ability with the 0-hapten, It may be that the serum altering component is primarily associated with the two other serologically reactive components of lipopolysaccharide that are absent from the polysaccharide moiety. Since the endotoxin altering reaction by normal serum has been confined to in vitro studies, the role that this reaction may have in vivo on the destruction or detoxification of living So tvphosa cells, can only be postulated. Since endotoxin is rapidly removed from the circulation by the reticulo-endothelial (S,.E.) system, it would appear that the endotoxin would not be in contract with the serum altering factor long enough to have any effect. However, Cremer and Watson (3) reported that the endotoxin disappears from the Ro E, system after ten hours. Therefore, if the endotoxin returns to the blood and remains there for any period, perhaps, the serum altering factor may have importanceo If such a sequence occurs, it must yet be determinedo

SUMMARY Normal human, rabbit and rat sera were studied for their ability to alter endotoxin when incubated at 370C and assayed by the quantitative precipitin technique. It was found that human and rabbit serum were able to alter endotoxin as manifested by loss in precipitating ability with homologous antiserum, However, rat serum had little if any ability to alter the precipitating activity of endotoxino The ability of serum to alter endotoxin was found to be a function of time of incubation, temperature of incubation, pH, and concentration of serum and endotoxin. The altering component in serum was relatively stable when stored at 40C and was able to withstand heating to 560~C for thirty minutes. Evidence was presented that eliminated serum properdin as a possible factor responsible for the alteration of endotoxin in this reaction. Fractionation of human and rabbit serum indicates that the endotoxin altering factor is a protein and is associated with the serum globulins, White blood cells of human and rabbit contain a factor which in the absence of serum alters endotoxin to a greater extent when incubated at 4~C than at 37~C, 106

APPENDIX I Reagent Solutions The following reagent solutions were routinely used in this investigation. a. Posphate Buffered Sline Solution (PBS) h 7. NaHiP04 6,204 gm, Na2PO4 3.014 gmi NaCI 52,605 gmo Distilled water 6000 mlo pH adusted to 7.0 This saline was used as a general purpose saline solution in the washing of cells in the preparation of endotoxin, for resuspending the AKD cells in the preparation of the vaccine for antibody production, for resuspending the heat killed cells in preparation of the standard suspension antigen for the agglutination reaction, and as a diluent in the quantitative precipitin reaction. b. Veronal Buffered Saline Solution pH 7.4 5T5 diethyl barbituric acid 5.75 gm. Sodium 55 diethyl barbiturate 3.75 gnm NaCr 85.00 gm. Dist. water to make 2,000 ml. The acid was dissolved in 500 ml, of hot distilled water and solutions of other components are added. This prepared a stock solution. A working solution of 1 part of stock solution was added to 4 parts distilled water and was prepared fresh for use, It was employed as a diluent for endotoxin that was used for incubation with serum and serum fractions and as a diluent of serum for the agglu1307

108 tination reactiono c, Saline Glvcoqen Solution Saturated IsC1l solution in distilled water 26 ml, Glycogen 1 gmi Distilled water 974 ml. This saline solution was used to create an. jntraperitoneal inflammatiory condition in rabbits and 250 ml. was injected intraperitoneally to stimulate the accumulation of white blood cells in the exudateo do Citrated-saline Saturated NaCI solution in disto water 26 mli Sodium citrate(anhydrous) 4 gmo Distilled water 974 mlo This saline solution was injected (200 ml.) intraperitoneally in rabbits just prior to the harvest of peritoneal exudate. The citrate is in sufficient concentration to prevent the formation of a fibrin clot so that the white blood cells may be recovered, e, Lysis_ Solu ti'on Saturated NaCl solution (37g. NaC1/100 ml, dista water) 7 mil Sodium citrate(anhydrous) 1 gmn Distilled water 993 mlo Adj. to pH 5,0 with 1N HC1 This hypotonic saline solution was used to lyse any contaminating red blood cells in the white blood cell preparation. The white blood cells were suspended in 20 ml, of this saline and incubated at room temperature for ten minutes. f, Neutralizing Solution Saturated NaCl solution in distilled water 41 mli Sodium citrate (anhydrous) 7 gm. Distilled water 959 mlo

109 This hypertonic saline solution was used to neutralize the hypotonic saline used to lyse red blood cellso This solution (20 mlo) was added to the hypotonic suspension of white blood cellso g, Intracellular Salt Solution KH2PO4 7.5 gmi MgS04 2,46 gmi Sodium citrate (anhydrous) 3,85 gmin Saturated KC1 solution in dist. water 11,4 ml, Distilled water to make 1000 mlu This solution was used to suspend the purified white blood cells, White blood cells were suspended in a concentration of 1 x 109 cells per 5 ml. h. Kieldahl ligestion Mixture Conc. H SO0 9 lbo Saturated CuS04 solution in dist. water 40.0 ml. Selenium 4,4 gmi Heat the mixture until the components dissolve and turns from a green color to colorless, This solution is used to digest the washed antigen-antibody precipitates in the quantitative determination of precipitated antibody nitrogen. i, N/70 Hydrochloric Acid The barometric pressure was obtained from the weather office at Willow Run Airport and concentrated HC1 was added to 1000 mlo of distilled water as indicated in Table 3,

11-0 TABLE 3 Preparation of Standard HC1 from Constant Boiling HC1 (42) (After Foulk and Hollingsworth) Barometer Grams of acid weighed pressure Per Cent HC1 in in air, required for mm Hg. acid 1 liter of N/70 acid 770 20.197 2,577 760 20.221 2.574 750 20,245 2.571 740 20,269 2.568 730 20.293 2.565 The HC1 solution was titrated against 0.01N NaOH with methyl red methylene blue indicator and corrected to give N/70 HC1, After correction the N/70 HC1 was retitrated with 0.01N NaOH to ascertain the proper normality of the HC1 solution. This solution was used to titrate the ammonia distilled over the microkjeldahl procedure. This normality of acid gives a factor of 0,2 times the ml. of N/70 HC1 used to give mg, of protein nitrogen, j. Boric Acid Solution A saturated solution of boric acid in distilled water was prepared and 5 ml, was used to receive the ammonia distilled over in the microkjeldahl procedure.

ll k. Saturated Sodium Hvdroxide A saturated solution of sodium hydroxidein distilled water was prepared and the insoluble carbonate was removed by filtration. Five ml, of this solution was added to the Kjeldahl digestion mixture in the microKjeldahl aparatus to neutralize the sulfuric acid and liberate the ammonia so that it could be distilled over into the boric acid.

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