THE RELATIONSHIPS BETWEEN SERUM COMPLEMENT ACTIVITY AND THE DEVELOPMENT OF ALLERGIC LESIONS IN RABBITS

Total serum hemolytic complement activity is consistently reduced in animals undergoing accelerated "serum sickness". The time of maximum reduction in complement titer invariably coincides with the time of complete antigen disappearance. This temporal relationship supports the belief that the reduction in complement titer is brought about by in vivo antigen-antibody combination. The intravenous administration of a single daily large dose of BGG-precipitated complexes produces a marked but temporary reduction in total complement hemolytic activity. On the other hand, multiple injections of soluble BGG immune complexes prepared in antigen excess failed to even temporarily alter complement titers. Animals undergoing the development of accelerated "serum sickness" treated by either type immune complex showed a significantly reduced incidence of arterial lesions. Apparently, this inhibiting effect on the development of arterial lesions was independent of variations in total complement hemolytic activity.     

CHANGES IN THE TITRE OF SERUM OPSONINS AND PHAGOCYTIC PROPERTIES OF MOUSE PERITONEAL MACROPHAGES FOLLOWING INJECTION OF ENDOTOXIN

A study has been made of the changes in the opsonic properties of mouse serum and bactericidal activity of mouse peritoneal macrophages at different times following the injection of various doses of lipopolysaccharide. It has been found that changes in the percentage of bacteria phagocytosed and killed within the mouse peritoneal cavity in a given time could be correlated with changes in the opsonic titre of the serum. Macrophages harvested from the peritoneal cavities of mice injected with endotoxin appear to be more efficient in phagocytosing bacteria in the presence of serum opsonins than macrophages obtained from normal mice. The relative importance of these changes in determining an animal's resistance to infection is discussed.     

DEFICIENCY OF THE SIXTH COMPONENT OF COMPLEMENT IN RABBITS WITH AN INHERITED COMPLEMENT DEFECT

A strain of rabbits with an inherited complement deficiency was shown to lack the sixth component of the hemolytic complement system. A method was elaborated for the partial purification of this component from normal rabbit serum. Upon injection of partially purified rabbit C'6 into C'6-deficient animals, an antibody was obtained which specifically inhibited the hemolytic activity of C'6. The data suggest that C'6-deficient serum either lacks the C'6 molecule or contains it in a chemically modified and inactive form.     

THE ROLE OF SERUM COMPLEMENT IN CHEMOTAXIS OF LEUKOCYTES IN VITRO

By the use of chambers containing two compartments with an interposed micropore filter, chemotaxis of polymorphonuclear leukocytes (PMN's) in vitro was studied employing various agents that fixed serum complement (C'). Antigen-antibody complexes, zymosan, and aggregated human gamma globulin, in the presence of fresh rabbit, guinea pig, or mouse serum resulted in the migration of PMN's through the micropore filter. Pepsin-degraded rabbit antibody or unaltered duck serum containing antibody did not exhibit such activity after addition of antigen. Heating of the serum before treatment or the presence of EDTA prevented the generation of the chemotactic factor. The chemotactic factor could not be generated in whole serum from rabbits genetically deficient in C'. However, the defect in this rabbit serum could be corrected by addition of rabbit or human C'6. Serum of B10·D2 mice deficient in hemolytic C' also yielded poor chemotactic activity. Interaction of the first four reacting components of guinea pig C' did not result in significant chemotactic activity unless guinea pig euglobulin with heat labile components was also present. In rabbit serum, C'5 and C'6, when "activated" by interaction with the first four reacting components, behaved like a protein-protein complex and exhibited marked chemotactic activity. By employing conditions favoring dissociation of the complex, the individual components were isolated and shown to be chemotactically inactive. Upon recombination of the two components, however, activity reappeared. Using another approach, the C'5–C'6 complex was isolated intact, and shown to be chemotactically active while other fractions not containing these components were not active. It is postulated that the C'5–C'6 complex is the active chemotactic factor generated in serum after the addition of C'-fixing agents.     

SERUM SICKNESS IN RABBITS : I. MANIFESTATIONS OF SERUM SICKNESS

1. The injection of a single large dose of normal horse serum into rabbits results in the appearance 3 to 8 days later of erythematous and edematous reactions on the ears in 68.9 per cent of the animals. 2. The injections may be given by any of several routes and reactions appear when the site of injection is definitely distant from the ears. 3. Injections of various antisera into rabbits cause the appearance of similar reactions. 4. These reactions can be considered as manifestations of serum sickness in rabbits.     

THE IMMUNE RESPONSE OF RABBITS TOLERANT TO BOVINE SERUM ALBUMIN TO THE INJECTION OF OTHER HETEROLOGOUS SERUM ALBUMINS

Immunological tolerance produced in rabbits by neonatal injections of BSA can be terminated by a series of injections of certain heterologous serum albumins which cross-react with BSA. Injections of albumins distantly related to BSA were more effective in terminating the tolerant state than injections of albumins closely related to BSA. It was concluded from results obtained with several heterologous albumins that immunological tolerance to BSA is directed to both the over-all antigenic or physical-chemical composition of the protein and the individual determinant groups present on the protein. Several possible mechanisms were given to explain the ability of cross-reacting albumins to terminate the tolerant state of BSA-tolerant rabbits. A possible relationship between the termination of tolerance in BSA-tolerant rabbits injected with cross-reacting albumins and autoimmunily was discussed. It was also suggested that the relative ease with which tolerance could be established to heterologous serum proteins in comparison to bacterial antigens is the result of the close serological and physical-chemical relationship of the heterologous serum proteins to the serum proteins of the rabbit. Rabbits injected with 500 mg of BSA during the first 5 days of life failed to form antibody capable of either eliciting an immune elimination of an injection of I* BSA given 3 to 4 months later or complexing with the circulating I* BSA.     

PROTECTIVE ANTIBODIES IN THE SERUM OF SYPHILITIC RABBITS

1. When an emulsion containing virulent Treponema pallidum is added to serum from normal rabbits and from untreated immune syphilitic rabbits that have been infected with a homologous strain of T. pallidum the mixture incubated at 37°C., and injected intracutaneously into normal rabbits, typical syphilitic lesions commonly develop at the sites of inoculation of the normal serum-spirochete mixture, while at the sites of inoculation of immune serum-spirochete mixtures usually either no lesion develops or else the incubation period of the resulting lesions is shorter and the lesions remain smaller than those produced by normal serum-spirochete mixtures. 2. In a series of preliminary experiments, of 56 areas inoculated with serum-spirochete mixtures, in 42 the suppressive action of the syphilitic serum was manifest, in 10 areas questionable evidence of protection was noted, and in 4 areas there was no evidence that the syphilitic serum had exerted a suppressive or protective action. 3. The protective action of syphilitic serum seems to have been lessened by heating to 56°C. 4. The results of the protection test in three other series of experiments were as follows: (a) Of 12 areas in 6 rabbits inoculated with normal serum-spirochete mixtures typical syphilitic lesions developed, while in the same number of areas inoculated with immune serum-spirochete mixtures there was complete or partial suppression of lesions in all. (b) Of 45 areas inoculated with serum from 10 different immune syphilitic rabbits, definite evidence of protection was observed in 37, questionable evidence in 5, and no evidence of protection in 3. (c) Of 8 areas in 4 rabbits inoculated with immune serum-spirochete mixtures no lesions developed during the period of observation, while of 8 areas in the same rabbits inoculated with one of two normal serum-spirochete mixtures typical syphilitic lesions developed in each.     

A METHOD OF SERUM TREATMENT OF PNEUMOCOCCIC SEPTICEMIA IN RABBITS

The treatment of pneumococcic septicemia in the rabbits by large doses of immune serum is detrimental, since the serum causes the formation of large clumps of bacteria in the blood which are taken out chiefly by the vessels of the lungs in which they accumulate and impede the circulation. The large doses of serum are also detrimental when they follow upon small ones through which the small clumps formed are deposited in the spleen, liver, and other organs. In this instance, the large amount of serum leads to the destruction of the pneumococci under conditions which promote an intoxication. The precise mechanism of this action is not known. The treatment of pneumococcic septicemia in rabbits by small repeated doses of immune serum can be successfully carried out. The number of pneumococci capable of being brought to destruction through phagocytosis in the organs in this way is very great. Not all the rabbits treated with small repeated doses of the serum survive. Those that succumb do so not to a general infection but to a pneumococcus meningitis. The explanation of this phenomenon is simple. When the number of pneumococci originally inoculated is very great a small number penetrate into the subdural space. Those in this space do not come under the influence of the serum, hence they are not agglutinated and prepared for phagocytosis, whence they multiply and set up a fatal meningitis. The activity of the immune serum administered in this way against virulent pneumococci is so great that a revision of our notions in the limit of powers of the anti-infectious sera seems necessary. It is patent that the problem is not simply a relation between quantity of immune bodies and number of bacteria. It is more complex than that conception indicates. The factor of the leucocytes and the degree of their possible activities under the conditions of the experiment come into play. Hereafter, in defining the mode and power of action of anti-infectious sera the condition of cooperation of the body-forces will have to be more strictly considered.     

THE ANTIBODY RESPONSE IN THE HUMAN BEING AFTER INJECTION WITH NORMAL HORSE SERUM

After the injection of normal horse serum in the human being, serum sickness occurs even more regularly than in cases treated with the various immune sera, but this is not accompanied by the production, to any notable degree, of circulating antibodies of the various types that are regularly to be demonstrated after the administration of immune serum and its resulting serum sickness. Since normal horse serum therefore appears to be weakly antigenic, and immune serum highly antigenic for the human being, one must assume that this difference is the result of some alteration in its antigenic characteristics produced during the course of the immunization or of its preparation for use; or that the specific antibody which is responsible for the phenomenon of serum sickness has not yet been identified; or that this phenomenon is not in any way dependent on the presence of the various known antibodies to normal horse serum.     

A CYCLICAL APPEARANCE OF ANTIBODY-PRODUCING CELLS AFTER A SINGLE INJECTION OF SERUM PROTEIN ANTIGEN

After a single intravenous injection of rabbits with aggregated HuIgG, IgM- and IgG-plaque-forming cells (PFC) in both the spleens and peripheral blood of rabbits peaked 5, 13, and 21 days after injection, while almost no PFC could be detected on days 8 and 16. The available data suggest that the secondary peaks of PFC (days 13 and 21) resulted from stimulation of memory cells by persisting antigen that was localized in the germinal centers in the spleen. No such persistence of antigen occurred in the lymph nodes, and these lymphoid tissues did not exhibit secondary peaks of PFC. The identical kinetic patterns for IgM- and IgG-PFC indicate that the major portion of IgG-PFC did not result from IgM-secreting cells switching to IgG synthesis and secretion. The present data suggest that the antibody produced and present at the site of interaction between committed cells and antigen is responsible for the regulation of antibody synthesis to persisting antigens. Possible cellular events involved in both the regulation and an apparent synchronous appearance of antibody producing cells in the spleens of rabbits were presented.     

VARIATIONS IN THE COMPLEMENT ACTIVITY AND FIXABILITY OF GUINEA PIG SERUM

The following conclusions may be drawn from the foregoing series of experiments. The complementary activity varies within a definite limit in different specimens of guinea pig serum. With sera which stood in contact with the clot for twenty hours, the strongest and weakest were in the ratio of 0.015 cubic centimeter to 0.04 cubic centimeter. The former was 2.66 times stronger than the latter. The variation observed with the same series of sera after forty-six hours was still more striking. The strongest was 0.013 cubic centimeter, and the weakest, 0.06 cubic centimeter, that is, the former was 4.6 times stronger than the latter. These findings agree with those made by Massol and Grysez. The variations were not so marked with the majority of sera. It is noteworthy that a large number of the sera gained in the complementary activity when remaining in contact with the clot for forty-six hours, while some sera became weakened during the same length of time. The amount of serum fixed by given constant quantities of syphilitic serum and antigen varies much more markedly than the variations in their complementary activity. One serum failed altogether to be fixed. On the other hand, one sample of serum was so easily fixable that 0.24 cubic centimeter (corresponding to 9.6 complement units of this specimen) disappeared, while the average quantity fixed was only 0.098 cubic centimeter (corresponding to 4.64 complement units). The normal standard of fixability was shown in about 50 per cent. of the specimens examined. If the zone of normal fixability is enlarged in both directions by one unit, the percentage of normal fixability would become 65.8. There is no definite relationship between the complementary activity and the fixability of a given specimen of guinea pig serum. The facts derived from our present experiments, especially in regard to the exceptions in the fixative quality of this serum, demand the utmost precaution from those intending to employ it for diagnostic purposes, as, for example, in the Wassermann reaction. No quantitative work is possible with the complement fixation reaction unless the experimenter is capable of determining the fixability of the serum in use. One of us (Noguchi) has long realized this source of error, and in order to reduce it he has advised the employment of a mixture of sera from more than two guinea pigs.     

THE INTERFERENCE OF INACTIVE SERUM AND EGG-WHITE IN THE PHENOMENON OF COMPLEMENT FIXATIONS

The fixing property of a specific precipitate and of syphilitic serum in the presence of certain antigenic lipoids, can be removed by adding certain non-complementary proteins of blood serum or hen''s egg. This disappearance of the complementary activity in the syphilis reaction, as well as in the true Bordet-Gengou reaction, is a phenomenon which incidentally accompanies the fixation of certain serum constituents, some of which possess a complementary activity. The presence or absence of the complementary property in these protein components does not influence fixation. Whether the disappearance of the complementary activity during the phenomenon of so-called fixation is due to a mechanical precipitation of the molecules through absorption or whether it is due to a physico-chemical alteration of the active molecules, is unknown. It is more probable that a chemical interaction takes place in the case of the syphilis reaction. Certain sera, for example, those derived from man and goat, show a low fixability. It is interesting to note that the fixability is gradually diminished when these sera and egg-white are heated to a temperature above 56° C., and totally disappears at 85° C. The coagulation of proteins with absolute alcohol or by boiling, destroys their interfering property. The fact that the fixation is not selectively directed towards complement, has a very important meaning for exact serology. The one-sided accuracy as to the complementary unity is no longer sufficient for quantitative work. Both the complementary and the volumetric unity of a serum serving as the source of complement should be taken into consideration. Besides, the fixability of the sera of various species of animals must also be considered. From these facts a formula may be derived for deciding the degree of suitableness of a serum. see PDF for Equation X is the degree of suitableness; K, the species constant for the fixability; P, the complementary activity; and V, the volume of serum. It will be seen that the suitableness is proportional to the fixability constant and the complementary unity, and inversely proportional to the volume of serum employed. As to what species yields the largest value for X, we refer the reader to our studies published elsewhere.     

PRODUCTION OF EXPERIMENTAL OSTEOMYELITIS IN RABBITS BY INTRAVENOUS INJECTION OF STAPHYLOCOCCUS AUREUS

1. The conditions under which a certain strain of staphylococcus (OH 172) causes in rabbits the development of bone inflammation have been described. 2. The virulence of the strain for rabbits was markedly raised by passage through this animal species, and especially after the culture had been recovered from a bone abscess. 3. The results indicate that it is possible to produce consistently inflammation of the bones of rabbits by the mere intravenous injection of a suitable strain of staphylococcus, without resorting to any elaborate operative technique designed to localize the organisms in the bones. It appears also that the inflammatory process so produced bears a close resemblance to staphylococcal osteomyelitis as occurring in human beings.     

PRECIPITIN RESPONSE IN THE BLOOD OF RABBITS FOLLOWING SUBARACHNOID INJECTIONS OF HORSE SERUM

1. Rabbits which have received a single dose of normal horse serum in the subarachnoid space produce precipitins in the blood in greater abundance, of higher titer, and persisting longer than those in control rabbits which have received a similar injection intravenously. 2. Repeated subarachnoid injections of normal horse serum in rabbits induce precipitins in the blood early. These may appear in high titer as soon as 1 week after the initial injection, whereas in rabbits similarly treated intravenously no precipitins are found at this time. They may appear a few days afterward and reach a high titer. 3. No anaphylactic manifestations occurred in rabbits treated repeatedly with subarachnoid injections of normal horse serum when the precipitin content of the blood was high. 4. Anaphylactins, as determined by passive transfer of anaphylaxis, were demonstrated in sera with high precipitin content. 5. These experiments may explain clinical evidences of anaphylaxis, observed when an initial intravenous injection of horse serum followed a series of intraspinal injections of such serum.     

UREA TOLERANCE AFTER UNILATERAL NEPHRECTOMY IN RABBITS

The method of study has an objective somewhat different from, and lacking the precision of, the ratio of Addis and the urea clearance of Van Slyke. It serves, however, to demonstrate that although for a month after unilateral nephrectomy the remaining kidney shows diminished capacity to hold blood urea within the normal range, nevertheless after 4 months this function is maintained in essentially the same degree as if both kidneys were present. This does not imply that all activities of the kidney remaining after unilateral nephrectomy are potentially augmented, but the data offered justify the conclusion that as the remaining kidney undergoes enlargement its functional capacity increases and the process represents a genuine hypertrophy in the critical sense.     

SUBCUTANEOUS REACTION OF RABBITS TO HORSE SERUM

1. Anaphylaxis or allergy of rabbits against horse serum can be proved by subcutaneous test. 2. The test is best made in the following way. The skin of the animal, preferably of the abdomen or flank, is shaved. (This should be done a few hours before the injection.) The injection is made by means of a small hypodermic syringe and intradermally. An effort was made not to inject the serum under the skin. Those injections were considered most favorable by which the serum remained as a small bleb in the skin proper. Undiluted horse serum was used for most of the experiments. The amount injected varied from 0.0I cubic centimeter to I cubic centimeter. The reaction seemed as definite after 0.0I cubic centimeter as after a larger quantity. See PDF for Structure 3. The specific reaction appears in from twelve to twenty-four hours after the test is made and reaches its maximum in from twenty-four to thirty-six hours. It consists of a local swelling extending from 0.5 to 2 centimeters from the point of inoculation. The skin involved in the raised area is usually red and hotter than the surrounding skin. Macroscopically and microscopically the reacting area has the appearance of a local acute inflammation. 4. The altered reactivity (allergy) or hyper-susceptibility (anaphylaxis) sets in usually in from ten to fifteen days after the first injection of horse serum, and lasts at least three months. Individual rabbits show marked variation from the average time of the development of anaphylaxis. 5. The appearance of precipitines against horse serum in the blood of rabbits appears nearly synchronously with the allergic condition. 6. After large injections of serum the allergic rabbits still react subcutaneously. A suppression of allergy which would correspond to the so-called anti-anaphylaxis could not be proved. 7. Also in regard to the offspring of injected rabbits the subcutaneous test was not positive. The young of these rabbits did not develop a more active allergy than the young of normal rabbits. 8. Neither the injection of considerable quantities of horse serum nor the development of a marked local reaction in the skin after intradermal inoculations of horse serum in a sensitized rabbit is accompanied or followed by greater variations in the number or types of leucocytes in the circulating blood than is found in control animals.     

THE INDUCTION OF AUTOIMMUNITY IN RABBITS FOLLOWING INJECTION OF HETEROLOGOUS OR ALTERED HOMOLOGOUS THYROGLOBULIN

Experimental autoimmunity was produced in rabbits following injection of altered homologous thyroglobulin. The thyroglobulin was altered by coupling to chemically defined haptens and by heating. With some preparations antibody to native thyroglobulin as well as thyroid lesions were produced. Injections of thyroglobulin coupled to the diazonium derivatives of arsanilic acid and sulfanilic acid were effective when given in either soluble form or incorporated into incomplete Freund's adjuvant) while injections of the same preparations precipitated by alum had relatively little effect on production of antibody or induction of lesions. The injection of native thyroglobulin in soluble form, incorporated into incomplete adjuvant or precipitated by alum usually resulted in production of little or no antibody and only rarely in the formation of lesions. The injection of a heterologous thyroglobulin into rabbits resulted in the production of antibody reacting with both the heterologous and rabbit thyroglobulin, but no thyroid lesions were observed.