A HOMOHEMOLYTIC SYSTEM FOR THE SERUM DIAGNOSIS OF SYPHILIS

The elimination of the foreign complement and corpuscles from the test for the serodiagnosis of syphilis has been attempted, and the results so far obtained are very satisfactory. Instead of using guinea pig complement, fresh human serum was utilized for the source of complement for the production of hemolysis upon the human corpuscles in the presence of an adequate amount of the specific anti-human amboceptor (prepared in rabbits). Usually 0.1 cc. of fresh human serum contains enough complement to hemolyze 1 cc. of a 1 per cent suspension of human corpuscles, but the amount of anti-human amboceptor required in this combination is about five to seven times that necessary when guinea pig complement (0.04 cc.) is used. It has also been shown that when a given human serum contains insufficient complement, an adequate quantity (0.1 cc. is usually enough) of another fresh negative serum may be added to supplement it. However, one rarely encounters this group of sera. Inactivated human sera can also be examined by utilizing human complement from another source (the serum must be fresh, active, and negative). The only drawback to the present method is the comparatively large amount of anti-human hemolytic amboceptor required. It is estimated that 30 to 40 cc. of the anti-human hemolytic immune serum, from one rabbit, of high potency—say 0.005 cc.—would be enough to examine about 3,000 to 4,000 cases (0.01 cc. for each case), whereas if guinea pig complement were used the same amount would cover about 15,000 tests (0.002 cc. for each case). In a large hospital or in the Army there should be no difficulty in preparing any amount of the anti-human hemolytic amboceptor. For example, material for 100,000 tests could be prepared within 1 month in less than 100 rabbits. The amboceptor serum can be used in the fluid state, or, if the titer is high, impregnated into filter papers. Special attention should be called to the fact that to obtain a powerful anti-human hemolytic amboceptor five to six intraperitoneal injections of corpuscles, thoroughly washed (until there is no trace of serum in the supernatant fluid), in doses of 5, 7, 10, 10, 10, and possibly another 10 cc. of the concentrated suspension (restored to the. original volume of the blood) are required. The bleeding may be done by the 9th or 10th day. The animals may be kept after bleeding for the production of more amboceptor by subsequent injections of the washed corpuscles. Finally, it should be emphasized that only the acetone-insoluble fraction of tissue lipoids of required standards (Noguchi) should be used when utilizing the human complement in the fixation test.     

BARIUM SULPHATE ABSORPTION AND THE SERUM DIAGNOSIS OF SYPHILIS

The so-called syphilitic antibodies can be removed from a serum by means of absorption with barium sulphate. The removal is due either to an adsorption or a mechanical absorption. The activity of the syphilitic antibodies is thereby unimpaired. The readiness with which the absorption is accomplished with barium sulphate varies considerably with different syphilitic sera. That barium sulphate exerts the same absorbing effect upon non-syphilitic serum components is made evident by the interfering property which the latter manifest in the absorption experiment of the syphilitic antibodies. The selective removal of the serum components, other than the syphilitic antibodies, by means of barium sulphate absorption is, therefore, impossible. On the other hand, a partial removal of these components, with but little removal of the syphilitic antibodies, may be effected when the content of a given serum is poor in syphilitic antibodies and comparatively rich in the indifferent serum components. But this is impossible if the conditions are reversed. The main reasons why some negative syphilitic sera may be so modified by the barium sulphate treatment as to give positive reactions, are explained below, but these apply only to those methods in which inactivated serum is employed. The inactivation reduces the antibody content to about one-fourth to one-fifth of the original. When the serum is very rich in antibodies, this does not affect the result of the fixation test. But when the amount of the antibodies is small, the process of inactivation creates conditions quite unexpected. It may produce such a condition that a given amount of the serum contains, after inactivation, only one or two antibody units, while the other serum components remain undiminished. Here one must not lose sight of the vital fact that these apparently indifferent serum constituents are not at all indifferent in the fixation processes. They may possess affinities which are similar to those of complement for the fixing combination of syphilitic serum and antigen. Speaking quantitatively, one unit of the syphilitic antibodies plus antigen will fix 0.1 cubic centimeter of guinea pig complement, but this unit can also be saturated and blocked by nearly the same amount of the seemingly indifferent serum component of the serum to be tested. Moreover, the regular amount of inactivated serum used in the Wassermann system is 0.2 cubic centimeter, a quantity sufficient to saturate two units of the fixing combination. Fortunately, this self-saturation of the syphilitic antibody-antigen combination by the other serum components is not constant in occurrence, owing to the wide variations of the fixability of the serum components of man. Here the benefit of Wechselmann''s procedure becomes obvious. By removing a surplus of the fixable indifferent serum components by means of barium sulphate, the serum is made to react positively, or more strongly than before the treatment with barium sulphate. This masking of the positive reaction through the self-saturation is liable to occur in any system in which inactivated serum is recommended. Another equally important factor in masking the positive reaction in a serum in which the antibodies are poor, is the presence in considerable amount of natural antisheep amboceptor in human serum. It is a plain and simple fact that an excess of hemolytic amboceptor renders a positive reaction feeble or completely negative. As we have shown in our present investigation, a hemolytic amboceptor can be removed from the serum by means of absorption with barium sulphate. Thus it is easy to understand why Wechselmann found that barium sulphate absorption improves the reaction in the original Wassermann system. The treatment of syphilitic serum with this salt can have a twofold benefit in the case of the original method of Wassermann; namely, the removal of certain interfering serum components and the removal or diminishing of the natural antisheep amboceptor present in the syphilitic serum. In the method of Noguchi, there is no necessity for applying the barium sulphate absorption. Noguchi recommends the use of unheated serum, hence the absolute amount of the serum employed is only one-half of the absolute amount of complement. Eventually an old serum may be anticomplementary and need inactivation, but if the result is doubtful in this instance, a fresh serum from the same patient may be secured and subjected to reëxamination. In this method there is no danger of introducing an amount of hemolytic amboceptor which is both unknown and uncontrollable, for the reason that human serum is usually devoid of isolysin (antihuman hemolytic amboceptor), and, if the latter is present, it never reaches the strength which shows any effect upon the hemolytic system employed. We conclude, therefore, that the barium sulphate absorption is to be recommended for the original method of Wassermann under certain conditions, but that it is unnecessary for the antihuman hemolytic system of Noguchi.     

THE ABSORPTION OF FAT IN PARTIALLY,AND IN COMPLETELY DEPANCREATIZED DOGS

It was found that dogs with a subcutaneous transplant secreting and discharging pancreatic juice externally absorbed no more fat than dogs in which the pancreatic remnant was undergoing rapid atrophy and sclerosis. Hence, the condition of the pancreatic tissue remaining in the body does not influence the amount of fat absorbed by the intestine. The absorption of fat by the intestinal mucous membrane is always markedly disturbed when the pancreatic secretion is excluded from the intestine. After the complete removal of all pancreatic tissue from an animal, the absorption of considerable amounts of fat can still take place.     

ALLERGIC IRRITABILITY : THE FORMATION OF ANTI-SHEEP HEMOLYTIC AMBOCEPTOR IN THE NORMAL AND TUBERCULOUS GUINEA PIG.

The guinea pig infected with virulent tubercle bacilli develops much more anti-sheep amboceptor than do controls when given like amounts of sheep red blood corpuscles. The curve of antibody production in the guinea pig when treated with sheep red blood corpuscles shows a departure from curves previously determined in other animals. These facts were ascertained as part of an effort to learn mbre of the functional nature of the inheritable factors controlling natural resistance to disease. The nature of some of the problems involved is outlined, and the limited bearing of the experiments on these is discussed.     

A STUDY OF THE PROTECTIVE ACTION OF SNAKE VENOM UPON BLOOD CORPUSCLES

Washed blood corpuscles of certain species of animals in a concentration of about ₅ per cent suspended in salt solution containing above 4 per cent of cobra venom undergo changes in their resistance to certain physical and chemical agents. They become non-haemolyzable by water, ether, saponin, and quite strong solutions of lecithin, provided always that the excess of venom has not been entirely removed. On the other hand, certain acids and alkalis, excepting ammonia, lake the venomized corpuscles more easily than they lake normal corpuscles. Venom solutions of 2 per cent and less exert no protective property upon blood corpuscles, but they induce changes in the corpuscles whereby they are rendered more easily laked by the same physical and chemical agents.     

DIFFERENT AMOUNTS OF TRANSFORMED ATOXYL PRODUCED BY INCUBATING ONE PER CENT. AND TEN PER CENT. ATOXYL IN BLOOD

1. 10 per cent. atoxyl in blood incubated at 37° C. for 1 hour gives rise to a solution that is much more than ten times as toxic as a 1 per cent. solution of atoxyl similarly incubated. 2. When the comparison is made after incubation for 3 hours instead of for 1 hour, the toxicity of the 10 per cent. solution is but slightly greater than ten times that of the 1 per cent., provided the red blood corpuscles are not removed from the dilutions. 3. If the corpuscles are removed from both the 10 per cent. and the 1 per cent. atoxyl solutions immediately after incubation at 37° for 1 to 3 hours, the dilutions of the 10 per cent. atoxyl are much more than ten times as toxic as the corresponding dilutions of the 1 per cent. atoxyl. 4. After incubation with atoxyl at 37° for 1 to 3 hours, red blood corpuscles left at room temperature in dilutions made from the 10 per cent. and 1 per cent. solutions in blood increase markedly the toxicity of the dilutions made from the 1 per cent. atoxyl, but increase very slightly the toxicity of the dilutions made from the 10 per cent. atoxyl. 5. If one desires to produce a large amount of transformed atoxyl by incubating atoxyl in blood at 37° for 1 to 3 hours, strong solutions of atoxyl should be chosen in preference to weaker solutions.     

THE COMPARATIVE MERITS OF VARIOUS COMPLEMENTS AND AMBOCEPTORS IN THE SERUM DIAGNOSIS OF SYPHILIS

1. The maximum activity of an antihuman hemolytic amboceptor may be obtained by employing the homologous or heterologous complement, according to the variable relations existing between the species furnishing the amboceptor and the one supplying the complement. Thus, some amboceptors are best reactivated by the complement of the same species, while others may act most strongly when reactivated with the complements of certain suitable heterologous species. 2. From the above it is clear that the complementary activity of a given serum may be very variable according to the varieties of amboceptors employed. In expressing the complementary activity of a serum, the species of the host of the amboceptor must always be stated. Thus, one serum may have many different complementary titers according to the amboceptors used. A similar variation in the titers of the amboceptors occurs when a variety of complements are employed. 3. Certain species of animals (pig and sheep) yield sera which are comparatively poor in reactivating most varieties of antihuman amboceptors. The complements of these species deteriorate rapidly. 4. The serum of chicken contained but little complement for the amboceptors derived from the mammalia, while the amboceptor from the chicken was only poorly, or not at all, reactivable by the complements contained in the mammalian sera. The serum of pig was the only variety which reactivated this amboceptor in a fair degree. 5. For the fixation tests guinea pig complement is the most favorable. This complement is also the most active and durable of those which have been studied. The complements of pig and sheep are quite fixable, but their weakness and rapid deterioration render them unsuitable for fixation purposes. Rabbit complement is quite active but is not easily fixable. Goat complement is, as already stated, difficult to fix, in spite of its strong complementary activity. The other complements are unsuitable because of their feeble complementary activity. 6. For fixation tests the antihuman amboceptors produced in the rabbit and guinea pig are suitable. They are, moreover, very active and do not cause the phenomenon of non-fixation. The amboceptors from other animals are unsuitable, as we cannot find a complement which strongly reactivates them. The amboceptor from the goat is unsuitable because of the danger of masking the fixation phenomenon by subsequent hemolysis. 7. In summing up, we arrive at the conclusion that the rabbit is the best animal for producing antihuman amboceptor, and the guinea pig for supplying complement. The guinea pig produces a good amboceptor, but its small size renders it second in choice. So far, no other animals have been found useful for the fixation experiments.     

THE DETERMINATION OF THE LENGTH OF LIFE OF TRANSFUSED BLOOD CORPUSCLES IN MAN

1. It is possible in mixtures of corpuscles of different groups to separate the corpuscles practically quantitatively by treating with a serum that agglutinates the corpuscles of one kind, leaving the others unagglutinated. 2. After a recipient has been transfused with blood of a group other than his own, specimens of his blood treated with a serum that will agglutinate his own corpuscles but not the transfused corpuscles show unagglutinated corpuscles in large numbers. 3. These unagglutinated corpuscles which appear in the recipien''s blood after such a transfusion are the transfused corpuscles and their count is a quantitative indicator of the amount of transfused blood still in the recipient''s circulation. 4. The life of the transfused corpuscle is long; it has been found to extend for 30 days and more. The beneficial results of transfusion are without doubt not due primarily to a stimulating effect on the bone marrow, but, it is reasonable to assume, to the functioning of the transfused blood corpuscles.     

THE RELATION OF THE SPLEEN TO BLOOD DESTRUCTION AND REGENERATION AND TO HEMOLYTIC JAUNDICE : II. THE RELATION OF HEMOGLOBINEMIA TO HEMOGLOBINURIA AND JAUNDICE IN NORMAL AND SPLENECTOMIZED ANIMALS.

The results of this study may be stated as follows. 1. Rapid injection of more than 0.06 of a gram per kilo of hemoglobin intravenously into a normal animal is followed by the appearance of hemoglobin in the urine (pelvis of kidney) within eight to ten minutes. 2. After rapid injection of more than 0.012 of a gram per kilo per minute of hemoglobin, 16 to 36 per cent. of the total amount, if this equals 0.25 of a gram per kilo, is eliminated in the urine and is accompanied by choluria. 3. If the injection of not more than 0.35 of a gram per kilo is made slowly (less than 0.01 of a gram per kilo per minute), the amount eliminated in the urine is only 2.33 to 9.5 per cent. of the total amount injected, and choluria does not occur. 4. The concentration of free hemoglobin in the blood which constitutes the threshold value of the kidneys for hemoglobin is approximately 0.06 of a gram of hemoglobin per kilo of body weight. When about this concentration is reached, hemoglobin appears in the urine. 5. The amount of hemoglobin per kilo of body weight which, after rapid injection, may be retained without jaundice, is approximately 0.18 of a gram. When 0.22 or 0.23 of a gram is retained bile pigments appear in the urine. The threshold of the liver for jaundice in point of hemoglobin saturation lies, therefore, between 0.18 and 0.22 of a gram per kilo of body weight. With slow injections a greater amount may be retained without choluria. 6. The absence of the spleen does not alter greatly the percentage of hemoglobin eliminated by the kidney, nor does it raise the threshold of the liver for jaundice. 7. In the presence of jaundice, either hemolytic or obstructive, the amount of hemoglobin retained by splenectomized animals is slightly diminished and that eliminated by the kidneys is correspondingly increased. Upon these data may be based the following explanation of the mechanism by which free hemoglobin is removed from the blood serum. Hemoglobin is not removed by the kidney until its concentration in the blood serum reaches a certain level (0.06 of a gram of free hemoglobin per kilo of body weight). This constitutes the threshold value of the kidneys for hemoglobin and when it is reached hemoglobin appears in the urine. When the concentration is lower, hemoglobinuria ceases; at the same time, however, the liver, and possibly other tissues, take up hemoglobin as soon as mere traces are present in the serum and they continue this removal whether the renal threshold is exceeded or not. The two processes go on simultaneously, the rate of removal, when the renal threshold is exceeded, being for the kidneys 17 to 36 per cent., and for the liver and other tissues 64 to 83 per cent, of the total amount introduced. The hemoglobin which is removed by the liver is transformed into bile pigments. If the amount reaching the liver is small and is received slowly, the amount of bile formed is not increased above the excretory capacity of the liver, and it is removed by the bile passages without the occurrence of choluria. This is shown in our experiments in which injections of hemoglobin were made more slowly than 0.01 of a gram per kilo per minute. On the other hand, if the hemoglobin is taken up by the liver rapidly and in large amounts, the bile capillaries are overtaxed and the bile cannot be rapidly removed, but is reabsorbed into the blood, and choluria develops. If this theory is correct we have an explanation of those instances of blood destruction in man characterized by jaundice, but not accompanied by hemoglobinuria. In a slow, gradual destruction of the red blood cells, the liver removes the hemoglobin from the serum so rapidly that the concentration of hemoglobin in the serum does not reach the threshold value of the kidneys and hemoglobinuria, therefore, cannot occur. The constant absorption of large amounts of hemoglobin by the liver and the increase in bile formation which results does, however, overtax the bile passages and jaundice occurs. In the same way may be explained the continuance of jaundice after the disappearance of a transient hemoglobinuria. A rapid destruction of a large amount of blood raises the concentration of hemoglobin in the serum so quickly that the threshold value of the kidney is quickly exceeded and hemoglobin appears in large amounts in the urine. When an amount of hemoglobin sufficient to reduce the concentration of the serum below the threshold value of the kidney has been removed, a considerable amount of hemoglobin may still remain in the serum, and it is the slow elimination of this through the liver that causes the choluria to continue. The demonstration that the absence of the spleen has no important influence on the elimination of hemoglobin by the kidney, on its transformation into bile pigments, or on the removal of such pigments, is of interest in connection with an observation made in the first paper of this series. This was concerning the frequent failure of jaundice to follow the administration of hemolytic serum during the early period following splenectomy. Among the possible explanations was the suggestion that the spleen is in some way concerned in the disintegration of free hemoglobin or in the elaboration of its derivatives. The present investigations demonstrate that such an explanation is without experimental basis, though it does not controvert the possibility of the spleen being concerned in liberating hemoglobin from the red cells and suggests that the failure of jaundice is due to some other factor or factors. Evidence to indicate that the changes in the blood that follow splenectomy are important factors is offered in the third paper of this series.     

THE PERMEABILITY OF THE LINING OF THE LOWER RESPIRATORY TRACT FOR ANTIBODIES

The experimental results indicate that the walls of the lower respiratory tract are relatively as impermeable for antibodies as they are for serum proteins. The kind of serum employed does not influence this fact. Antibodies contained in homologous (rabbit) serum failed to enter the blood stream when injected into the trachea in greater concentration than those contained in heterologous (guinea pig and cow) sera. That a little serum and its contained substances are absorbed after intratracheal injection has been shown both by the experiments dealing with sensitization and anaphylaxis previously reported and by the experiments recorded in this paper. Besredka''s assertion that the laryngotracheal method is an efficient one for the administration of serum in the treatment of disease has not been borne out by the experimental evidence here presented. From the results one would conclude that it is one of the least efficient routes for administration. In contrast to the relative impermeability for proteins of the mucous membranes of the lower respiratory tract is the relative permeability of the endothelium of the peritoneal cavity. Antibody is there absorbed readily, whether it be in heterologous or in homologous serum. The indications are that a considerable portion of the antibody injected into the peritoneal cavity reaches the blood stream in 2 or 3 hours.     

ALLERGIC IRRITABILITY : III. THE INFLUENCE OF CHRONIC INFECTIONS AND OF TRYPAN BLUE ON THE FORMATION OF SPECIFIC ANTIBODIES.

1. The allergic irritability of the guinea pig (capacity of the animal to react to antigenic substances) is increased by infection with Bacillus abortus and a streptococcus, by the dead tubercle bacillus, and by intensive treatment with trypan blue, respectively. The effect of these influences, while definite, is less pronounced than that previously found for infection with the tubercle bacillus. The production of anti-sheep hemolytic amboceptor was used as the test reaction in these cases. 2. The allergic irritability of the guinea pig with reference to anti-typhoid agglutinin is increased by infection with the tubercle bacillus. 3. The allergic irritability of the rabbit with reference to anti-sheep hemolytic amboceptor is increased by an infection of suitable severity with the tubercle bacillus. 4. In the guinea pig the curve of antibody production is complex. Its peculiarities are developed during the production of antityphoid agglutinins as well as that of anti-sheep hemolytic amboceptor. In the latter case injections of antigen subsequent to the first give rise to a curve of production unchanged in form but somewhat affected in the time relations. 5. The effects of infection with Bacillus tuberculosis on allergic irritability with reference to anti-sheep hemolytic amboceptor are operative throughout a course of immunizing treatments. The successive increases due to the cumulative effect of repeated doses of the antigen are developed on a higher level. The end-result is that the animal with increased irritability furnishes more antibody not only in response to the initial injection of antigen as previously described, but an absolute increase over the amount attainable by a comparable number of treatments in series. That portion of the final result contributed by the increase in allergic irritability appears to be no less, and may even in instances be somewhat more than that due to the earlier doses of the specific antigen.     

ON ALBUMINOLYSINS AND THEIR RELATION TO PRECIPITIN REACTIONS

The writer believes that the experiments above recorded have demonstrated the following points: 1. As has been previously shown by other workers, complement is fixed by the precipitate formed in the reaction between unformed proteid and its homologous antiserum, and it is the precipitate only which fixes, the supernatant fluid being devoid of fixing power except to a slight degree in tubes very rich in antigen. This latter fixation is probably due to a redissolving of the precipitin-precipitinogen complex which, as Gay has shown, does not lose its fixing power on resolution. The fixation of complement by mixtures in which antigen is so slight in amount that no precipitate is formed may be explained by analogy with colloidal reactions in which combination of colloids takes place without precipitation unless definite quantitative relations are maintained. 2. In contrast to the results above described, precipitating mixtures of bacterial antigen and antiserum show complement fixation both by the precipitate and by the supernatant fluid. 3. The complement fixation by the supernatant fluid of such bacterial precipitin reactions, with the precipitate removed, is, in the presence of washed bacteria, equal to that exerted by the same amount of the original serum. The complement fixation of the precipitates, here, as in serum-antiserum tests, is proportionate to the bulk of the precipitate. 4. The precipitin body is not removed from a bacterial antiserum by treatment with washed whole bacteria, whereas the cell sensitizer or amboceptor is removed by such treatment. The writer believes that the work recorded above justifies Gengou''s conclusion that an albuminolytic sensitizer is formed in response to immunization with unformed proteids. This sensitizer, or amboceptor, is, however, distinct and independent of the sensitizer that reacts specifically with either bacterial or other cells. It would appear that immunization with unformed proteids calls forth only the formation of these albuminolysins, while immunization with cells, in our case the typhoid bacillus, calls forth both the cytolytic and the albuminolytic sensitizers. The albuminolytic sensitizers are not carried down mechanically with the precipitates in the precipitin reactions. We see no reason for not considering them identical with the precipitins themselves, bodies which combine with the dissolved antigens and, as in other colloidal reactions, lead to precipitation when definite quantitative relations are observed. The undiminished precipitating value of a bacterial antiserum after considerable amounts of bacterial amboceptor have been absorbed out of it, would point against the identification of agglutinins with precipitins. However, this question must be subjected to further study. It is thus clear that only the cytolytic sensitizer can enter into combination with the washed bacterial cell, while it is probable that both cytolytic and albuminolytic sensitizer may combine with the dissolved ingredients of a bacterial filtrate. Whether or not they combine with the same constituent of such an antigen cannot be decided by our experiments.     

HEMOLYSINS OF VEGETABLE ORIGIN

1. The sap of Cotyledon scheideckeri possesses hemolysins for the red corpuscles of different animals. 2. The hemolysins of vegetable sap can be bound by erythrocytes and cannot be separated. 3. A definite quantity of erythrocytes is able to extract from the sap only a part of the hemolysins it contains. 4. The quantity of hemolysins in the sap of different plants is subjected to the same fluctuations as that of bacterial agglutinins, precipitins, and hemagglutinins. 5. As the hemolysins are bound by erythrocytes, the hemolysis can take place not only at 37° but also at 15–16°. 6. The thermostability of the hemolysins varies from one individual plant to another. 7. In many cases the vegetable sap loses its hemolytic properties at a certain temperature and recovers them at a higher one. 8. The vegetable sap is unable to produce complete hemolysis of erythrocytes. 9. But the sap of many plants acquires the power to dissolve red corpuscles completely after I hour of heating at 134° and 144°. 10. Erythrocytes modified by Cotyledon sap cannot be dissolved completely even by distilled water. 11. The agglutination of the erythrocytes and their hemolysis are conditioned, probably, by different substances. 12. The hemolytic amboceptor and the hemolysin of Cotyledon scheideckeri can be bound with the same receptor of the erythrocytes.     

QUALITATIVE DIFFERENCES IN THE ANTIGENIC COMPOSITION OF INFLUENZA A VIRUS STRAINS

A study of the PR8, Christie, Talmey, W.S., and swine strains of influenza A virus by means of antibody absorption tests revealed the following findings: 1. Serum antibody could be specifically absorbed with allantoic fluid containing influenza virus or, more effectively, with concentrated suspensions of virus obtained from allantoic fluid by high-speed centrifugation or by the red cell adsorption and elution technique. Normal allantoic fluid, or the centrifugalized sediment therefrom, failed to absorb antibodies. Influenza B virus (Lee) caused no detectable absorption of antibody from antisera directed against influenza A virus strains, but it specifically absorbed antibody from Lee antisera. 2. The neutralizing, agglutination-inhibiting, and complement-fixing anti-bodies in ferret antisera were completely absorbed only by the homologous virus strain, even though 2 absorptions were carried out with large amounts of heterologous virus strains. 3. PR8 virus appeared to have the broadest range of specific antigenic components for it completely absorbed the heterologous antibodies in Christie and W.S. antisera and left only those antibodies which reacted with the respective homologous strains. The other virus strains (Christie, Talmey, W.S., swine) were more specific in the absorption of heterologous antibodies and completely removed only those antibodies which reacted with the absorbing virus. 4. The absorption tests revealed a higher degree of specificity and individuality of the virus strains than the various cross reactions previously reported. The strain specificity of PR8 virus was equally manifest in absorption tests with ferret sera and with human sera following vaccination. 5. The amount of homologous antibody remaining in a PR8 ferret serum after absorption with PR8 virus, obtained by the red cell adsorption and elution method, varied inversely as the concentration of virus used for absorption. A given concentration of virus, however, absorbed a greater percentage of neutralizing antibodies than either agglutination-inhibiting or complement-fixing antibodies.     

ABSORPTION OF ARSENIC FOLLOWING INTRA-MUSCULAR INJECTIONS OF SALVARSAN AND NEOSALVARSAN

Intramuscular injection of salvarsan and neosalvarsan in rabbits always produces necrosis of the muscles. A much more intense reaction is produced by salvarsan than by neosalvarsan. The rate of absorption of arsenic following intramuscular injections of salvarsan is very slow, while following intramuscular injections of neosalvarsan between 75 and 85 per cent of the arsenic is absorbed during the first week. The subsequent absorption is quite slow.     

STUDIES IN EDEMA : VI. THE INFLUENCE OF ADRENALIN ON ABSORPTION FROM THE PERITONEAL CAVITY,WITH SOME REMARKS ON THE INFLUENCE OF CALCIUM CHLORIDE ON ABSORPTION.

1. Adrenalin injected intraperitoneally increases the rapidity of absorption of fluid from the peritoneal cavity, independently of whether the solution to be absorbed is hypotonic or hypertonic or is approximately isotonic with the blood serum. The intravenous injection of adrenalin also increases the absorption of fluid, but not so markedly as does the intraperitoneal injection. 2. Adrenalin injected either intraperitoneally or intravenously increases the quantity of sodium chloride absorbed. The relative absorption of sodium chloride—the movement from the peritoneal cavity of sodium chloride, as compared with the movement of water—is slightly increased when 0.85 per cent. of sodium chloride solution and adrenalin are injected intraperitoneally; but it is diminished when adrenalin is injected intravenously, or when 1.5 per cent. sodium chloride solution and adrenalin are injected. When distilled water has been injected intraperitoneally, adrenalin decreases the relative amount of sodium chloride in the peritoneal fluid—a fact that is evidently related to the increased elimination of sodium chloride through the kidneys under the influence of adrenalin. 3. When 0.85 per cent. sodium chloride solution is injected into the peritoneal cavity, the blood becomes diluted after two hours and a half. When adrenalin is also injected, the dilution of the blood is less marked, in spite of the increased absorption under the influence of adrenalin. When distilled water is injected into the peritoneal cavity, the blood is diluted equally in control and adrenalin experiments. When 1.5 per cent. sodium chloride solution is injected, the dilution of the blood is very slight, and in adrenalin experiments it is the same as in control experiments or very slightly greater than in control experiments. 4. The increase of absorption from the peritoneal cavity caused by the injection of adrenalin is not due to the increased diuresis caused by the injection of this substance. 5. The injection of adrenalin causes a temporary increase in the osmotic pressure of the blood, which gradually returns to normal. Under certain conditions, after the injection of adrenalin, there is a tendency toward maintaining the higher osmotic pressure of the blood serum, even up to the end of the experiment. We have reason to believe that this increase in the osmotic pressure of the blood is the main factor in increasing the absorption of fluid from the peritoneal cavity. 6. In experiments in which 0.85 per cent. sodium chloride solution has been injected intraperitoneally, either with or without adrenalin, there exists a tendency of the peritoneal fluid to attain a greater osmotic pressure than the blood serum, in spite of the fact that the injected fluid is slightly hypotonic as compared with the blood serum. We note a similar condition in cases of general edema in man, in which the osmotic pressure of the ascitic fluid is greater than that of the other edematous fluids, or even that of the blood serum. There exists, therefore, a mechanism that causes the passage of osmotically active substances from the blood or from the tissues into the peritoneal cavity, and that causes the osmotic pressure of the peritoneal fluid to become higher than that of the blood. It follows from our experiments that this mechanism, which causes the ascites in edematous persons to have such a high osmotic pressure, is not dependent upon certain pathological changes in the lining membranes or upon other pathological conditions, but exists already in normal animals. 7. The addition of 1.22 per cent. calcium chloride solution to 0.83 per cent. sodium chloride solution, in such proportions as we used in our infusion experiments, in which we determined the transudation into the peritoneal cavity, delays the absorption of fluid from the peritoneal cavity but very slightly. Therefore, calcium chloride increases directly the transudation into the peritoneal cavity and does not cause an increase in the amount of fluid in the peritoneal cavity merely by inhibiting the absorption. 8. It follows that adrenalin does not increase the amount of peritoneal transudate found after the intravenous infusion of large quantities of sodium chloride solution, to which adrenalin has been added, by delaying the absorption from the peritoneal cavity. The increased amounts of peritoneal fluid must be due to increased transudation into the peritoneal cavity; and the adrenalin, in view of its marked effect on absorption from the peritoneal cavity, must increase the movement of fluid into the peritoneal cavity much more strongly than could be assumed from the figures obtained in the infusion experiments.     

CELLULAR MECHANISMS OF PROTEIN METABOLISM IN THE NEPHRON : IV. THE PARTITION OF SUCCINOXIDASE AND CYTOCHROME OXIDASE ACTIVITIES IN THE CELLS OF THE PROXIMAL CONVOLUTION OF THE RAT AFTER INTRAPERITONEAL INJECTION OF EGG WHITE

1. Shifts of enzymatic activity have been followed during the formation and evolution of the droplets that form in the cells of the proximal convolution of the nephron of the rat after the injection of a 50 per cent solution of egg white in isotonic saline. 2. Twelve hours after injection there is a 35 to 40 per cent decrease in succinoxidase and cytochrome oxidase activities in the fraction containing the larger particles; i.e. mitochondria and droplets in equal concentration. Although after 30 hours the quantitative proportion of droplets and mitochondria is the same as previously, the activities of the fraction have returned to the normal observed originally in the uninjected rat in a corresponding fraction consisting of mitochondria only. 3. The microsome fraction shows an average increase of 35 per cent in oxidative enzyme activities during the early period following injection, and decreases to the original figure in the later period of droplet formation. 4. It is concluded from the shifting pattern of localization of oxidative enzyme activity within the cell particulates that the absorption droplets arise by the incorporation of the mitochondrial elements, which originally contain the highest enzyme activity, with absorbed protein through the intermediate stage of smaller (microsomal) particles.     

A STUDY OF THE THERAPEUTIC MECHANISM OF ANTIPNEUMOCOCCIC SERUM ON THE EXPERIMENTAL DERMAL PNEUMOCOCCUS INFECTION IN RABBITS : II. A COMPARISON OF THE THERAPEUTIC EFFECT OF UNREFINED,ANTIPNEUMOCOCCIC SERUM WITH THAT OF ITS VARIOUS PROTEIN FRACTIONS: THE R?LE OF THE NON-ANTIBACTERIAL FACTOR

The experiments of the preceding communication showed that the therapeutic action of antipneumococcic serum depends to a considerable extent upon a certain non-antibacterial factor. The experiments reported in the present communication had two main objects; the first was to determine the distribution of the non-antibacterial factor among the various protein fractions of the serum, and incidentally to correlate this property of the factor with that of certain known antibodies, as well as to learn whether or not the protein fractions commonly excluded from refined preparations of antipneumococcic serum, have any therapeutic value; the second was to determine the rôle of the non-antibacterial factor in the therapy of pneumococcus infection, as exemplified by the experimental, dermal, pneumococcus infection in rabbits. To determine the distribution of the non-antibacterial factor, Type I antipneumococcic serum was fractionated with (NH₄)₂SO₄, and the antibacterial bodies were absorbed by concentrated suspensions of heat-killed pneumococci. The activity of the non-antibacterial factor in the different protein fractions was then tested for by adding varying amounts of the absorbed supernatant liquids to a certain constant, subeffective dose of serum. Even though the method of titration has certain inherent faults it was possible to ascertain that the non-antibacterial factor was apparently associated with all the globulin fractions to a similar degree, and to a certain extent with the albumen as well. In this respect the non-antibacterial factor resembles neither the antibacterial bodies of antipneumococcic serum, nor, for example, the known diphtheria or tetanus antitoxins. The determination of the relative importance of the antibacterial and non-antibacterial factors in the therapy of pneumococcus infection in rabbits, was rendered possible by the fact that the various globulin fractions differ in their relative content of the two factors. Thus whereas the water-insoluble fraction of the globulin, precipitated when the saturation of (NH₄)₂SO₄ is raised from 30 per cent to 50 per cent, contains about 90 per cent of the antibacterial (mouse protective) bodies, it apparently contains no more of the non-antibacterial factor than the water-soluble fraction. The minimal effective doses as well as the mouse protective unit content of the different fractions were determined. Before the tests were performed, it seemed that if the therapeutic effect of a given dose were to depend chiefly upon its content of mouse protective antibody, the amounts required for 1 M.E.D. would be determined by the number of mouse protective units it contained; on the other hand, if it depended primarily on the non-antibacterial factor, one would expect the amounts required for 1 M.E.D. to be of approximately the same volume. Actually the therapeutic effect was found not to depend entirely upon either factor, alone; the M.E.D.''s varied both in volume and m.p.u. content, the absolute concentration of either factor being inconstant and almost proportional to the relative concentration of the other. Thus a relative abundance of non-antibacterial factor made up for a scarcity of mouse protective antibody (as in the water-soluble globulin), and vice versa (as in the water-insoluble globulin). It appears, therefore, that when a relatively larger amount of antibacterial bodies are acting, less of the non-antibacterial factor is necessary; conversely, when more of the non-antibacterial factor is available, less of the antibacterial bodies are necessary. The last experiment dealt with the practical question as to whether any protein fraction or combination of fractions is as good a therapeutic agent as the whole, unrefined serum from which it was derived. Two considerations were in mind in making the comparison; one had to do with the quantitative recovery of potency, the other with the qualitative effect of the various fractions as regards the lesion, course, and duration of the experimental disease. The results may be summarized as follows: (a) the total globulin was not only as good, but in this instance, definitely better than the original serum; (b) the total water-insoluble globulin, although almost as potent as the original serum, was qualitatively and quantitatively less effective than the total globulin; (c) the removal of the 30 per cent (NH₄)₂SO₄ fraction not only diminished the potency of the remaining globulin to a considerable extent (at least 25 per cent), but also seems to have disturbed a certain equilibrium, which resulted in an undesirable zone phenomenon. As a result of this zone phenomenon, which, it is important to note, was not observed in the agglutination or mouse protection tests on the same fractions, the range of effective dosage is very narrowly limited. Although the results of agglutination tests correlated well with those of the mouse protection tests, both of these showed no parallelism with the therapeutic effects of the various fractions on the experimental, dermal, pneumococcus infection in rabbits.     

ULTRACENTRIFUGATION STUDIES OF YELLOW FEVER VIRUS

1. It was possible to study in the ultracentrifuge by optical methods the behavior of yellow fever virus particles directly in the unaltered serum from infected monkeys. 2. The virus showed an extremely high light absorption in the spectral range of 320 to 440 mµ, which seemed to be its intrinsic property. In a 1 cm. thickness of fluid, the small amount of virus present in unaltered infective serum absorbed about as much light (approximately 25 per cent) in the middle of this range as did all the normal serum proteins present in a combined concentration some 1000 times as great. 3. The concentration of virus in the unaltered serum was found to be of the order of 0.00005 gm. per cc. 1 cc. of a 10^–9 dilution, which, as has been shown, may constitute a minimal infective dose for monkeys, would contain approximately 10,000 virus particles. The probability that most of the virus particles were in the inactive form is discussed. 4. In infective serum having a viscosity of 14 millipoises, the particles sediment with a blurred boundary at rates lying between approximately 18 and 30 x 10^–13 cm./sec./dyne. Evidence indicates that this spread is the result of an aggregation or association phenomenon. 5. Computations of size are in approximate agreement with those made from ultrafiltration studies. On the assumption that the density of the virus particle is near that of protein, its volume is computed to be at least that of a spherical particle having a diameter of 12 mµ. An assumed density of 1.15 gm. per cc. yields a diameter of 19 mµ, considering the shape as spherical.     

A METHOD FOR THE DETERMINATION OF THE SPECIFIC GRAVITY OF RED BLOOD CELLS

1. A simple method for determining the specific gravity of red blood cells is described. 2. It is more rapid than and as accurate as pycnometer determinations. 3. The marked chemical and physical interactions between corpuscles and suspension fluids in other methods are avoided.