STUDIES ON EXPERIMENTAL HYPERTENSION : XIX. THE PRODUCTION OF PERSISTENT HYPERTENSION IN SHEEP AND GOATS

Persistent hypertension has been produced in the goat and sheep by constriction of the main renal arteries. The presence or absence of accompanying uremia depends upon the degree of constriction of the renal arteries. In both sheep and goat, constriction of one main renal artery also caused elevation of the blood pressure which tended to persist longer than in the dog. Excision of the one kidney with main renal artery constricted resulted in a prompt (24 hours) return of the blood pressure to normal. In the animals with hypertension of long duration but without renal excretory insufficiency, (the "benign" phase) no significant arterio- or arteriolosclerosis developed as a result of the hypertension alone. In the animals that had both hypertension and renal excretory insufficiency, (the "malignant" phase) the typical terminal arteriolar lesions developed in many organs. These lesions consisted of necrosis and fibrinoid degeneration of arterioles and necrotizing arteriolitis which should not be confused with arteriolosclerosis. The same humoral mechanism which is responsible for experimental renal hypertension in the dog and other animals also obtains in the pathogenesis of experimental renal hypertension in the sheep and goat.     

A GROWTH INHIBITORY SUBSTANCE FOR THE INFLUENZA GROUP OF ORGANISMS IN THE BLOOD OF VARIOUS ANIMAL SPECIES : THE USE OF THE BLOOD OF VARIOUS ANIMALS AS A SELECTIVE MEDIUM FOR THE DETECTION OF HEMOLYTIC STREPTOCOCCI IN THROAT CULTURES

1. 5 per cent sheep blood agar is a selective medium for beta hemolytic streptococci in throat cultures since sheep blood inhibits the growth of bacillus X (H. hemolyticus) and B. parainfluenzae hemolyticus. The growth of H. influenzae is also inhibited by sheep blood. 2. The inhibitory action of sheep blood resides in the erythrocytes and is thermolabile. Disruption of the cell by laking has no effect upon the inhibitor. 3. The bloods of animals closely related to the sheep, such as the goat and the cow, have a similar inhibitory action on the growth of hemolytic and non-hemolytic members of the influenza group, while human blood contains a similar but less powerful inhibitor for these organisms. 4. Members of the influenza group grow well on unheated rodent blood: rabbit, guinea pig, and rat. 5. These organisms also grow fairly well on unheated horse blood.     

THE INFLUENCE OF EPINEPHRIN UPON THE CORONARY CIRCULATION OF THE MONKEY

Decrease in coronary flow was the constant response of freshly isolated monkey hearts to epinephrin. These hearts were perfused with autogenous hirudinized blood diluted with Locke solution. The results were constant at high or low perfusion pressures, in beating or resting hearts, and with all adequate doses. Increased coronary flow was obtained constantly in rabbit hearts under identical conditions. In the light of previous work upon isolated human coronary arteries, the general conclusion is drawn that, while actively dilating the coronary vessels in the dog, cat, rabbit, ox, sheep, and pig, epinephrin constricts the coronary vessels in man and the monkey. The coronary arteries of the last two species are presumably supplied with constrictor nerves of true sympathetic (thoracicolumbar) origin.     

SPECIES SPECIFICITY OF LEUKOCYTIC PYROGENS

Polymorphonuclear neutrophilic leukocytes of the dog, cat, and goat release leukocytic pyrogen under the same conditions as the heterophile polymorphonuclear leukocytes of the rabbit. The characteristics of the febrile response to an intravenous injection of homologous leukocytic pyrogen in all four species are very similar: a brisk monophasic fever reaching a peak between 30 and 50 min with smooth defervescence to the baseline by 3 hr. Shivering, which is not obvious in the rabbit, is noted in the dog, cat, and goat during the first 30 min. Quantitative differences in response reveal the cat to be the most sensitive of of these species to homologous leukocytic pyrogen, followed by the rabbit, dog, and goat. The response to heterologous pyrogen is in most cases markedly diminished compared to that after equal doses of homologous protein, suggesting the operation of species specificity, although canine and feline pyrogen behaved very similarly in all tests. Species specificity of leukocytic pyrogen is probably related to amino acid substitutions in different species of a common mammalian protein effector molecule.     

EXPERIMENTS ON THE PRODUCTION OF SPECIFIC ANTISERA FOR INFECTIONS OF UNKNOWN CAUSE : I. TYPE EXPERIMENTS WITH KNOWN ANTIGENS—A BACTERIAL HEMOTOXIN (MEGATHERIOLYSIN), THE PNEUMOCOCCUS,AND POLIOMYELITIC VIRUS.

Attempts to produce antisera in animals to combat specific infections are usually deferred until the cause of the infection has been isolated and grown in pure culture to furnish antigen. It has seemed to us that the fulfillment of these conditions might in some cases be rendered unnecessary through the use of infected tissue itself as an antigen, combined with selective absorption of the antiserum to rid it of elements injurious to the species furnishing the tissue. In order to test this possibility type experiments have been carried out with immune sera effective against known antigens of three different sorts: 1. Sera resulting from the injection of rabbits and a goat with normal guinea pig tissues and a bacterial hemotoxin, the megatheriolysin described by Todd, which hemolyzes guinea pig cells. The sera possessed strong antitoxins for the megatheriolysin but were fatal to guinea pigs. By the method of selective absorption they were rendered innocuous to these animals and were successfully used to protect them from lethal doses of the megatheriolysin. 2. Anti-rabbit dog sera containing antibodies protective against pneumococcus infection. Such sera, subjected to repeated absorption with rabbit red cells, proved capable of protecting mice from pneumococcus infection in exactly the same degree as the unexhausted serum; that is to say, they protected against 100 times the dose of pneurnococci that was fatal with normal dog serum. 3. The serum of a monkey recovered from poliomyelitis and repeatedly injected with human red cells and extract of placental tissue. This serum, after selective absorption with human red cells, protected a monkey against an intracerebral dose of poliomyelitic virus fatal to eight other monkeys given it with normal monkey or human serum. The results in these instances, purposely chosen for their simplicity, would seem to indicate for the absorption method some usefulness in the study of immunity to infections of unknown cause. In Part II of our paper the method is applied to one such infection; namely, a sarcoma of the fowl engendered by a filterable agent. A general discussion will be found in connection with this portion of the work.     

SELECTION WITH THE MAGNET AND CULTIVATION OF RETICULO-ENDOTHELIAL CELLS (KUPFFER CELLS)

Methods and apparatus are described where with living Kupffer cells can be procured from the liver of the rabbit and the dog for study and cultivation in vitro. Almost none of these cells can be dislodged from the normal liver by forcible perfusion; but after they have taken up finely particulate matter (India ink, iron oxide), they come away in great numbers. When they have phagocyted ferromagnetic iron oxide they can be selected with a magnet from amongst the blood elements present in suspension with them; and they are obtainable in quantity by this means. They do poorly when plated in a thin plasma clot, failing to multiply or to assume their characteristic shape; but they flourish when allowed to attach themselves to strands of lens paper bathed in serum that is frequently changed. Bacterial infection of serum cultures of Kupffer cells from normal rabbits and dogs occurs only as the result of secondary contamination of the materials, whereas it regularly develops in cultures from animals with fever induced by the injection of nucleic acid or of killed B. prodigiosus. Kupffer cells obtained under such conditions are abnormally active, and some can be washed out of the liver of sick animals in the absence of any preliminary phagocytosis of particulate matter. The facts have a bearing both on the conditions conducing to blood invasion and on the response of the Kupffer cells in the emergency. The characters of the isolated Kupffer cells and the results of tests of their presumptive functions will be described in later papers.     

THE PRESERVATION OF LIVING RED BLOOD CELLS IN VITRO : I. METHODS OF PRESERVATION.

The erythrocytes of some species are much damaged when handled in salt solutions, as in washing with the centrifuge after the ordinary method. The injury is mechanical in character. It may express itself in hemolysis only after the cells have been kept for some days. It is greatest in the case of dog corpuscles, and well marked with sheep and rabbit cells. The fragility of the red cells, as indicated by washing or shaking them in salt solution is different, not only for different species, but for different individuals. It varies independently of the resistance to hypotonic solutions. The protection of fragile erythrocytes during washing is essential if they are to be preserved in vitro for any considerable time. The addition of a little gelatin (⅛ per cent) to the wash fluid suffices for this purpose, and by its use the period of survival in salt solutions of washed rabbit, sheep, and dog cells is greatly prolonged. Plasma, like gelatin, has marked protective properties. Though gelatin acts as a protective for red cells it is not preservative of them in the real sense. Cells do not last longer when it is added to the fluids in which they are kept. Locke''s solution, though better probably than Ringer''s solution, or a sodium chloride solution, as a medium in which to keep red cells, is ultimately harmful. The addition of innocuous colloids does not improve it. But the sugars, especially dextrose and saccharose, have a remarkable power to prevent its injurious action, and they possess, in addition, preservative qualities. Cells washed in gelatin-Locke''s and placed in a mixture of Locke''s solution with an isotonic, watery solution of a sugar remain intact for a long time,—nearly 2 months in the case of sheep cells. The kept cells go easily into suspension free of clumps, they pass readily through paper filters, take up and give off oxygen, and when used for the Wassermann reaction behave exactly as do fresh cells of the same individual. The best preservative solutions are approximately isotonic with the blood serum. If the cells are to be much handled gelatin should be present, for the sugars do not protect against mechanical injury. Different preservative mixtures are required for the cells of different species. Dog cells last longest in fluids containing dextrin as well as a sugar. The mixture best for red cells is not necessarily best for leukocytes. A simple and practical method of keeping rabbit and human erythrocytes is in citrated whole blood to which sugar solution is added. In citrated blood, as such, human red cells tend to break down rather rapidly, no matter what the proportion of citrate. Hemolysis is well marked after little more than a week. But in a mixture of 3 parts of human blood, 2 parts of isotonic citrate solution (3.8 per cent sodium citrate in water), and 5 parts of isotonic dextrose solution (5.4 per cent dextrose in water), the cells remain intact for about 4 weeks. Rabbit red cells can be kept for more than 3 weeks in citrated blood; and the addition of sugar lengthens the preservation only a little. The results differ strikingly with the amount of citrate employed. Hemolysis occurs relatively early when the smallest quantity is used that will prevent clotting. The optimum mixture has 3 parts of rabbit blood to 2 of isotonic citrate solution. In the second part of this paper experiments are detailed which prove that cells preserved by the methods here recorded function excellently when reintroduced into the body.     

AN EXPERIMENTAL STUDY OF THE INFLUENCE OF KIDNEY EXTRACTS AND OF THE SERUM OF ANIMALS WITH RENAL LESIONS UPON THE BLOOD PRESSURE

1. Extracts of the rabbit''s kidney injected into the rabbit cause a slight, increase in blood pressure which is barely more than that due to the mechanical effect of the injection. 2. Extracts of the dog''s kidney injected into the dog cause a decided fall in pressure; an equal fall may be caused by the dog''s urine. A series of control experiments indicates that the fall caused by the kidney extract may be due to the urinary salts which it contains. 3. Extracts of cat''s kidney cause a rise in pressure. As the cat''s urine causes a fall, this rise in pressure indicates the possibility of a kidney extract containing a pressor substance which cannot be influenced by the depressor substance of the urine. 4. Rabbit''s kidney, which in the rabbit produces a slight rise, when injected into the dog causes a drop comparable to that caused by the dog''s kidney itself. Similarly, the dog''s kidney, which injected into the dog causes a drop, produces in the rabbit a rise analogous to that produced by rabbit''s kidney. It is evident therefore that these pressor and depressor substances of the kidneys in question do not have a constant effect on all animals as do the extracts of the adrenal gland. 5. Extracts of kidneys which are the seat of various forms of nephritis cause the same effect as extracts of normal kidneys. 6. The serum of dogs with considerable reduction of kidney substance causes a slight fall in pressure; the serum of dogs with spontaneous nephritis gives divergent results, as does also the serum of rabbits with various forms of acute nephritis. The serum of dogs with chromate nephritis causes a slight rise, while that of dogs with uranium nephritis produces a sharp and decided fall in pressure. Although there is no uniformity in these results, their general character, and especially the experience with uranium and chromate sera of the dog, suggests that pressure-disturbing substances are present in the serum as the result of the kidney lesion. The very slight evidence of the constant presence of a pressor substance, however, offers little support to the theory that such a substance is furnished by the diseased kidney or is due to disturbances of metabolism caused by disease of the kidney.     

STUDIES ON PNEUMOCOCCUS GROWTH INHIBITION : VII. THE RELATION OF OPSONINS TO NATURAL RESISTANCE AGAINST PNEUMOCOCCUS INFECTION.

A study was made of the pneumococcidal action of serum-leucocyte mixtures of pneumococcus-resistant animals with a view to determining whether this property of the blood is to be accounted for by the presence of certain serum constituents or by cellular characteristics which are lacking in the blood of susceptible animals. By means of a method specially developed for this purpose, it was found that, after adequate contact with the serum of pneumococcus-resistant animals, virulent pneumococci were phagocyted actively not only by the homologous leucocytes but also by the leucocytes of other resistant and susceptible animals. On the other hand, pneumococci exposed to the action of the serum of pneumococcus-susceptible animals were not taken up by the leucocytes of either the resistant or susceptible species. All the resistant animals tested, dog, cat, sheep, pig and horse, showed marked opsonic properties in their blood serum which were not found in the serum of susceptible ones, rabbit, guinea pig and human. There appeared, however, to be no essential difference in the phagocytic activity of the leucocytes from the various animals. It was then shown that the pneumococcus-destroying power of serum-leucocyte mixtures was entirely abolished when heated serum was substituted for fresh serum and that such heated serum had lost much of its opsonic potency. Neither the living leucocytes alone nor extracts of the leucocytes were observed to exert any killing action on pneumococci. Further evidence of the controlling influence of opsonic action in the antipneumococcus defence mechanism of the blood, and its importance in natural resistance, was afforded by a study of the opsonin content and leucocytic functions of the blood of full grown and young rabbits as related to their widely varying degrees of pneumococcus susceptibility.     

STUDIES ON EXPERIMENTAL HYPERTENSION : IX. THE EFFECT ON BLOOD PRESSURE OF CONSTRICTION OF THE ABDOMINAL AORTA ABOVE AND BELOW THE SITE OF ORIGIN OF BOTH MAIN RENAL ARTERIES

Constriction of the aorta just above the origin of both main renal arteries invariably resulted in elevation of the carotid systolic and carotid mean pressure. The hypertension was not immediate, but developed in about the same time as after constriction of the main renal arteries (3). Constriction of the aorta just below the origin of both main renal arteries had no significant effect on the carotid systolic or carotid mean pressure. Since these results were first reported (1), Rytand (88, 89) has shown by an indirect method, namely, the demonstration of the development of cardiac hypertrophy, that hypertension in the upper part of the body can be produced in the rat by constriction of the aorta just above the origin of both main renal arteries. The immediate effect of constriction of the aorta either below or above the main renal arteries is a fall of blood pressure (femoral mean pressure) below the site of the clamp, the extent of the fall being directly dependent upon the degree of constriction of the aorta. Of particular interest is the eventual elevation of the femoral mean pressure above the normal in some animals with the aorta constricted or even occluded above the origin of the main renal arteries. This was most pronounced and persistent in those animals in which, in addition, the aorta below the origin of the renal arteries, and, in some animals, the main renal arteries, also were constricted. The most important factors which determined this elevation of blood pressure in the lower part of the body were probably increased flow of blood into the vascular bed below the clamp and peripheral vasoconstriction of renal and humoral origin, as in the case of the hypertension produced by constriction of the main renal arteries alone (2–86). Although elevation of the carotid systolic or carotid mean pressure occurred invariably within 24 to 48 hours after the constriction of the aorta above the site of origin of both main renal arteries, yet there was a tendency, after a variable period, for the elevated blood pressure to become lower or even to drop to the original level. Increased constriction, and finally occlusion of the aorta, above the origin of the main renal arteries, and even constriction or occlusion of the aorta below the renal arteries, in addition, failed to induce hypertension that persisted for a long time at a high level. In order to produce this effect, it was necessary to constrict the main renal arteries as well. The possible explanation of the failure of the hypertension to persist for a long time after constriction of the aorta alone, is that the initial ischemia of the kidneys disappeared due to the improvement of the blood flow through the kidneys as a result of (a) the increase of the natural accessory circulation to the kidneys; (b) the increased blood pressure above the site of the clamp and consequent increased flow of blood into the part of the aorta below the clamp; (c) increased pressure below the site of the clamp due, in great part, to peripheral vasoconstriction, and in part to the increased inflow of blood into the lower part of the body through the aorta and collateral channels. For the dog, this method is not necessary for the production of persistent hypertension. Constriction of the main renal arteries is easily performed and is effective for the production of generalized hypertension (2–11). However, constriction of the aorta in addition to constriction of the renal arteries results in greatly elevated persistent hypertension. Constriction of the aorta alone above the origin of the main renal arteries would be useful in the dog only for the production of relatively short periods of hypertension in the upper part of the body. For small animals it may be a more effective and useful method. In the dog, the only technical difficulty encountered was the erosion of the wall of the aorta by the clamp. This may not occur in small animals. In previous studies (2–11) that have dealt with the constriction of the main renal arteries, this accident rarely occurred. When the constriction of the aorta above the origin of the main renal arteries was of moderate degree, or was gradually made very great, the resultant hypertension was not accompanied by impairment of renal excretory function, as determined by urea clearance or by the quantity of urea, creatinine or non-protein nitrogen in the blood, the benign phase of hypertension (3). When the constriction of the aorta was suddenly made very great, impairment of the renal excretory function usually followed, and the animal developed fatal convulsive uremia and characteristic vascular lesions, the malignant phase of hypertension (9). These facts, are all indicative of the renal origin of the hypertension which results from the constriction of the aorta just above the origin of both main renal arteries. Hypertension did not persist for a sufficiently long time to permit any conclusive comparison between the effect of the high and low pressures on the structure of the vascular system, above and below the site of the clamp, respectively. During the period of survival of these animals, no significant differences were observed between the appearance of the vascular system of the upper part of the body and that of the lower part of the body, and significant cardiac hypertrophy did not develop. In the aorta and large arteries, intimal arteriosclerosis was not observed. In the aorta of one old animal several small plaques of calcification were found in the media, but these were present in the portion of the aorta below, as well as above the clamp, and they were no larger or more abundant than were observed in some old dogs with normal blood pressure. Dogs 3–50 and 3–83, that are still alive, with very high blood pressure above the site of the aortic clamps, and relatively low pressure (though greater than normal) below the site of the aortic clamps, will be valuable for the determination of possible differences between the effects of the two levels of blood pressure in the large and small blood vessels. In these dogs also, it will be possible to determine the effect of the persistently high blood pressure on the myocardium. The possible application of the results of this study to the problem of the pathogenesis of human eclampsia is mentioned here for consideration. Since this condition occurs in pregnancy only at a time when the uterus is greatly enlarged, it is at least possible that the mass may press on the aorta or both main renal arteries sufficiently to produce renal ischemia. The suddenness with which the uremic convulsive phase of eclampsia develops is in keeping with this idea. In the dog, an aggravating effect of pregnancy on an already established hypertension has not been noted. As a matter of fact, most of the hypertensive dogs that have become pregnant, have shown a slight or moderate fall, rather than an increased rise of pressure. Since the dog stands with the body in a horizontal position, and does not lie on its back, pressure of the pregnant uterus on the aorta and blood vessels is less than in human beings who stand erect and frequently lie on their backs. The soundness of this suggestion could be tested by placing pregnant women, in the early stage of eclampsia, in a position which could relieve possible pressure on the aorta and main renal arteries. A possible explanation of the fall of pressure in the pregnant hypertensive dogs is the compensatory effect of the normal kidneys of the pups, as in the case of an animal with one main renal artery constricted and the other kidney normal. As has been shown (3, 31, 72), the presence of one normal kidney in an animal hypertensive due to constriction of the other main renal artery, results, after a variable period, in a return of the blood pressure to normal. How the normal kidney acts to produce this effect is not known.     

THE LYMPHATIC PATHWAY FROM THE NOSE AND PHARYNX : THE ABSORPTION OF DYES

1. In the monkey, dog, cat, and rabbit the cervical lymph duct was cannulated, and then a solution of T-1824, or trypan blue, or a fine graphite suspension, all in physiological saline, was dropped into the nose. T-1824 was used in all four animals, trypan blue in the cat and dog, the graphite suspension (Hydrokollag) in the cat alone. 2. No Hydrokollag was ever found in the cervical lymph. 3. Trypan blue and T-1824 appear in the cervical lymph 15 to 30 minutes after being placed in the nose of the cat and monkey, 51 to 53 minutes in the dog, and 14 minutes in the rabbit. 4. T-1824 and trypan blue were also absorbed from the nose directly into the blood. 5. Neither the dyes nor the Hydrokollag, though left in the nose for as long as 6 hours, were found to pass through the cribriform plate and reach the interior of the cranium. 6. In the monkey cervical lymph passes through a chain of five or more lymph nodes, in the rabbit frequently through two nodes, in the cat through one node except in rare instances, and in the dog through one node.     

EXPERIMENTAL IMMUNITY WITH REFERENCE TO THE BACILLUS OF LEPROSY : PART I. A STUDY OF THE FACTORS DETERMINING INFECTION IN ANIMALS.

Repeated experiments have proven that few, if any, of the ordinary laboratory and domestic animals are immune against infection by Bacillus lepræ. As previously reported, the goat, horse, guinea pig, and many cold-blooded animals (Couret) have been found susceptible to invasion by this organism. Two factors are of great importance in effecting infection. In the first place, a sufficiently large number of organisms must be employed, and, what is still more important, second and subsequent inoculations are more liable to produce leprous lesions than are primary injections. Moderate doses used in the first inoculation of animals are comparatively harmless as regards their ability to induce lesions. Such preliminary doses, whether they consist of living or dead organisms, produce a condition of hypersensitiveness or allergy, which renders it possible by a second injection of viable bacilli to induce the development of a reactionary lesion. Lesions arising as the result of a second inoculation develop more rapidly, increase in size more quickly, and persist for a longer period than those taking place as the result of a single inoculation, even though very large doses are used. Moreover, the bacilli in these lesions are more liable to lead to metastasis and to a generalized infection. We regard the results of these experiments as having considerable bearing upon the development of the disease in human cases, since we find that it is chiefly among those living in prolonged intimate contact with leprous patients that leprosy develops. The proper interpretation of these findings is difficult and becomes, apparently, more complex the longer they are studied. We are accustomed to similar phenomena of anaphylaxis or allergy with protein materials and with certain bacteria, especially the tubercle bacillus. It is not surprising that a specific allergy or altered reaction should take place in animals previously injected with leprosy bacilli, either alive or dead; why, however, lesions should develop in which the bacilli continue to grow in animals which had recovered from previous injections, or the serum of which showed bactericidal properties and contained other specific antibodies, is not so easily understood. It is unnecessary in this paper to discuss the relative value of the different theories brought forward to explain the phenomenon of anaphylaxis. It must, nevertheless, be assumed that either as the result of the splitting of certain essentially non-toxic substances in the bacillus by specific ferment-like substances in the blood, which increase after a sensitizing dose, or by the joining of some body in the serum with certain substances in the bacilli, a toxic body is produced. Following the setting free or development of these injurious bodies, there results a cellular reaction, an area of local necrosis followed usually by the appearance of cells of the lymphoid and epithelioid type, and, especially if dead bacilli are used, by polymorphonuclear leucocytes. Such a reaction is usual in all allergic or anaphylactic conditions; but why the reaction should pre-dispose to the more or less permanent development of parasitic powers on the part of the bacilli is not so plain. The idea has been expressed by us, as the result of observations based chiefly upon the study of the changes in the tissues of human leprosy, that the presence of bacilli within the large multinucleated cells that are characteristic of the lesion is the result of active multiplication of the bacilli in these cells rather than of phagocytosis by the cells, although it is possible that their original entrance may be the result of phagocytic action. If this be the case, it is possible that, through the cellular reaction plus the necrosis of certain of the fixed tissue cells, a pabulum of split protein products results upon which the bacilli feed, and in which they find protection from the antibodies present in the blood serum. In our attempts to induce infection in various mammalian species with cultures of human leprosy, positive results have been obtained in almost every instance after the second injection of large numbers of the organism. In this manner, we have been successful in the production of lesions in the monkey, goat, horse, guinea pig, and mouse. The results of these experiments lead us to believe that the mechanism through which invasion and multiplication follows in these lower animals is similar to if not identical with that in man. A careful study of the progress of the disease in man together with the behavior of the organism in the monkey and the goat would suggest, at least, that the function of toxin production is, after all, of little use to the leprosy bacillus because it has seemingly acquired a highly parasitic existence; instead of which it is not only possible, but highly probable, that its defensive protection, whatever it may be, is enormously developed. After the inoculation of animals with either dead or living bacilli, the production of specific antibodies is induced; these do not develop in any considerable amount and apparently show no marked tendency to increase upon repeated injections. The tests of the sera in vitro indicate that the antisubstances are produced chiefly by the first few injections. Either the existence of some specific bacterial protective body, or what seems more probable, the protection afforded by the host cells in which the bacilli become ensconsed accounts, we believe, for the difficulty of producing in animals an antiserum of high potency. The idea, too, that the bacilli do not multiply to any great extent until they have entered certain cells of the host has, in a large measure, been verified by the artificial cultivation experiments. We know from our studies upon the biology of the organism that it will live for years in the most unfavorable conditions. This would suggest that during their sojourn in the tissue cells death as the result of autolysis rarely occurs, and as long as the cells can withstand the multiplication of the bacilli within and remain alive, so long are the bacilli protected from outside injurious influences, in consequence of which no disintegration of bacilli occurs to induce the production of immune bodies. On the other hand, death of the cells carrying the bacilli must occur from time to time, and this exposes large numbers of the organisms to the action of the body fluids, and, in consequence, to the condition of formation of specific antibodies.     

THE NORMAL FATE OF ERYTHROCYTES : I. THE FINDINGS IN HEALTHY ANIMALS.

The phagocytosis of red corpuscles, while frequent in the normal dog, rat, and guinea pig, is slight in man, the rhesus monkey, and many rabbits. In cats it is always negligible in amount and frequently absent. Phagocytosis will not suffice as a general explanation of normal blood destruction. When the liver, spleen, and bone marrow of the cat, dog, rabbit, or monkey are slowly perfused with defibrinated blood or Locke"s solution, bodies are given off into the fluid which have the appearance of red corpuscles that have lost their hemoglobin but retained the rest of their cell substance. These bodies possess many of the properties supposedly distinctive of red corpuscles. They are the product of disordered parenchymal cells. By a special method, it has proved possible to search the body, organ by organ, and the circulating blood also, for disintegrating red corpuscles. Shadows of red cells are not present anywhere, nor are hemolyzing red cells found. A hemolytic process, in the ordinary sense of the term, can scarcely play an. important part in normal blood destruction. Instead, it is certain that some red corpuscles, at least, are destroyed in another way; namely, by fragmentation. Normal blood regularly contains small numbers of fragmentation forms—microcytes and poikilocytes—and accumulations of them are regularly present in the spleen, but are found only inconstantly in the other organs. The fragments are in evident process of further subdivision. They occur not only in species in which phagocytosis as a means of cell destruction is negligible (cats), but also in animals in which it is an important process (dogs, some rabbits). The method of study that we have employed is well suited to disclose how the blood is destroyed. The importance of cell fragmentation in this connection is indicated by our failure to find any other means of destruction, save only the phagocytosis already known. Further facts indicating the importance of fragmentation are presented in our second paper, where a general discussion will also be found.     

STUDIES ON THE METABOLISM OF CELLS IN VITRO : I. THE TOXICITY OFα-AMINO-ACIDS FOR EMBRYONIC CHICKEN CELLS.

Summing up these results, we found that all the ten α-amino-acids used inhibited the growth of the cells and finally killed the cultures. This inhibition is preceded by a short period of activity. The typical effect on the cells is shown in Figs. 1 and 2. The first (Fig. 1) is a control culture showing the usual growth of cells and their typical spindle shape form. The second (Fig. 2) is a culture in plasma plus asparagine showing the cells rounded off and beginning to undergo dissolution. We do not wish to draw too extensive conclusions from these experiments, but we believe that toxicity of α-amino-acids towards growing cells has been shown beyond a reasonable doubt; while we have found that compounds of higher molecular weight, namely, the peptones of egg yolk and proteins, are non-toxic. This toxicity depends upon the concentration and the time that the cells are exposed to their action. As these factors are reduced, the toxicity is decreased. In this respect, these substances are similar to all cell poisons. Applying these results to the work done on the intravenous injection of digestion mixtures, we believe that we have found a reason for the death of the experimental animals when the hydrolyzed proteins were injected too rapidly. Buglia found that large amounts of α-amino-acids could be injected into the circulation without causing deep-seated changes in the renal and intestinal functions, provided they were injected slowly enough; in fact, that enough of these mixtures could be injected in this way to cover the nitrogen consumption of the body. This injection, however, was always accompanied by an α-amino excretion through the urine and an increase of the peristalsis, of the intestine, with resultant liquid stools. As is well known, a sudden great concentration of these substances in the blood of an animal causes death. These results agree with our findings Folin and Denis demonstrated the fact that α-amino-acids probably pass into the circulation through the intestines. Van Slyke and Meyer, by means of Van Slyke''s nitrogen method, have practically proven this, and Abel, Rowntree, and Turner, and Abderhalden have lately succeeded in obtaining α-amino-acids in crystalline form from the blood. Van Slyke and Meyer have shown that the tissues take up α-amino-acids to a certain point, but that after this the limit of saturation is reached. This is not so in the liver, which continually desaturates itself by metabolizing the α-amino-acids that it has absorbed, and consequently maintains indefinitely its power of removing them from the circulation, as long as they enter it no faster than the liver can metabolize them. Marshall and Rowntree have shown that there is an increase of the α-amino-acid concentration in the blood after injuries to the liver, which have caused deep-seated anatomical changes. Our experiments prove that tissue cells in general are unable to live in the presence of any great concentration of these acids. At the present time we do not feel able to give an explanation of the significance of this evident toxicity. However, the fact in itself seems to indicate that we should expect stimulation from a certain increase of the α-amino-acid concentration in the body, or the concentration of any one of the acids, while a greater increase would lead to marked disturbances of the metabolism.     

OBSERVATIONS ON EXPERIMENTAL TYPHOID INFECTION OF THE GALL BLADDER IN THE RABBIT

1. Besredka''s living sensitized vaccine, given intravenously, does not produce a typhoid lesion of the gall bladder in the rabbit. 2. The first transplant of this vaccine is capable of producing this lesion. Hence this vaccine is not entirely safe to handle. 3. Regular infections of the gall bladder have not been produced by carrying a known pathogenic strain on rabbit blood agar, by successive passage through animals, or by the use of freshly isolated strains. 4. No evidence could be demonstrated in the rabbit of the immunity produced in man by vaccination with a whole killed vaccine. 5. Vaccine treatment did not cure the gall bladder lesion. 6. With the present methods of producing infections in the chimpanzee and the rabbit, neither of these animals is suitable for deciding the problems of the immunization of man by vaccines. These problems must be settled, as some of them already have been settled, by actual experience with large numbers of men kept under close observation.     

IMMUNOGLOBULIN SPOTS ON THE SURFACE OF RABBIT LYMPHOCYTES

Small and medium lymphocytes from the peripheral blood and lymphoid tissues of the rabbit react in suspension with antibodies directed against different immunoglobulin determinants. Through immunofluorescence, it was possible to show that numerous discrete spots on the surface of the positive lymphocytes carry immunoglobulin molecules. The positive lymphocytes are about one-half of all lymphocytes in the different preparations; thymus lymphocytes are all negative. With antisera specific for rabbit IgM as well as with antisera directed against allotypic determinants specific for IgM or IgG, it was possible to show that about nine-tenths of the immunoglobulin-positive lymphocytes carry IgM molecules on their surface. With antisera directed against a- and b-locus determinants, it was also possible to demonstrate that both heavy and light chains were present in the surface immunoglobulins. Furthermore, in animals which were heterozygous at the a or the b locus, it was found that each lymphocyte had immunoglobulins synthesized under the influence of only one of two alleles. A very small proportion of lymphocytes could be shown to have a specific surface reaction with one antigen (horse ferritin); the proportion of these cells increased very much after immunization.     

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.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : VI. THE IMMUNE RESPONSE TO RED BLOOD CELLS IN IRRADIATED RABBITS AFTER ADMINISTRATION OF NORMAL, PRIMED, OR IMMUNE ALLOGENEIC RABBIT BONE MARROW CELLS

Irradiated rabbits given allogeneic bone marrow cells from normal adult donors responded to an injection of sheep red blood cells by forming circulating antibodies. Their spleen cells were also capable of forming many plaques using the hemolysis in gel technique, and were also capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine upon exposure to the specific antigen in vitro. However, irradiated rabbits injected with allogeneic bone marrow obtained from rabbits injected with sheep red blood cells 24 hr prior to sacrifice (primed donors) were incapable of mounting an immune response after stimulation with sheep red cells. This loss of reactivity by the bone marrow from primed donors is specific for the antigen injected, since the immune response of the irradiated recipients to a non-cross-reacting antigen, the horse red blood cell, is unimpaired. Treatment of the bone marrow donors with high-titered specific antiserum to sheep red cells for 24 hr prior to sacrifice did not result in any diminished ability of their bone marrow cells to transfer antibody-forming capacity to sheep red blood cells. The significance of these results, with respect to the origin of the antigen-reactive and antibody-forming cells in the rabbit, is discussed.     

ACTION OF SHAKEN SERUM ON HOMOLOGOUS FIBROBLASTS

The preceding experiments showed that partial or complete inactivation of serum by shaking brought about a marked decrease in the activity of homologous fibroblasts. Generally, serum was not completely inactivated by shaking. In only one instance was the hemo-lytic effect of chicken serum on sheep corpuscles entirely lost after the serum had been shaken for 8 hours. In all other cases, the normal hemolytic power of chicken and dog sera for sheep erythrocytes was merely decreased. The effect of shaking varied according to certain conditions of the serum. It may be compared to the inffuence of heat, which differs widely according to individual sera, even when they are obtained from animals which are apparently in identical condition. Shaken serum always inhibited the activity of homologous fibroblasts more than normal serum. When chicken serum was completely inactivated by shaking, its restraining action on chicken fibroblasts became also more marked. On the contrary, dog serum partly inactivated by shaking was much less toxic for chicken fibroblasts than normal serum. Thus, shaking brought about a change in the condition of serum, against which homologous and heterologous fibroblasts reacted in an opposite manner. At the same time, the normal lytic action of serum on foreign erythrocytes decreased. This last phenomenon is caused, as is well known, by the partial or complete destruction of alexin. The decrease of the restraining effect of shaken serum on foreign fibroblasts may be attributed to the same cause. The increase of the inhibiting action of shaken serum on homologous fibroblasts is due possibly to the disappearance of a substance favoring the activity of homologous cells. A similar hypothesis was advanced for explaining the increase of the growth-inhibiting action of serum under the influence of senescence and of heat. The restraining power of adult serum on cell multiplication may be due to the antagonistic action of growth-promoting and growth-inhibiting substances, the growth-promoting substance being as unstable as alexin and certain tissue juices which have the property of increasing the rate of cell proliferation. Alexin and growth-activating substances contained in embryonic and gland tissue juices have in common the property of being destroyed by heat and by shaking. Leucocytes added to serum under certain conditions increase its hemolytic power on foreign erythrocytes and decrease its inhibiting action on homologous fibroblasts. Variations in the alex-inic activity of serum under other influences are followed also by a change in its action on homologous cells. Long ago, Gengou found that serum from plasma is less bactericidal than serum from blood. We observed that the lytic effect on sheep erythrocytes of dog and chicken serum from blood was sometimes more marked than that of serum from plasma, but there was often no difference in the action of both sera. At the same time, the growth of homologous fibroblasts was always more extensive in the serum from blood. The opposite effect was observed when heterologous fibroblasts were used. The growth-inhibiting power of serum from blood was still less marked on leucocytes than on fibroblasts. But embryonic juice, which greatly enhances the rate of multiplication of homologous cells, did not increase the lytic action of serum on foreign erythrocytes. It may be concluded that, under the conditions of the experiments: 1. Chicken serum partly or completely inactivated by shaking becomes more inhibiting for chicken fibroblasts. 2. Dog serum partly inactivated by shaking becomes less inhibiting for chicken fibroblasts.     

THE EFFECT OF CARROT FEEDING ON THE SERUM PROTEIN CONCENTRATION OF THE RAT

1. The blood cytology of 41 pairs of virgin rabbits was studied, each pair consisting of a buck and a litter mate sister. A comparison of the values for each blood factor of the two groups was made by obtaining the mean difference and the standard error of the mean difference. In addition, correlation coefficients, both zero order and first order eliminating age, and regression equations were calculated for each of the blood cell elements of the bucks and does. 2. It was found that the male values for both the red cell count and the hemoglobin content were higher than the corresponding female values, and these differences were of statistical significance. No differences were observed between the two groups with respect to other cells. Significant positive correlations were obtained between the two sexes for the following blood constituents: Red blood cell count, white blood cell count, hemoglobin content, lymphocytes in both absolute and relative numbers, and neutrophils, basophils, and monocytes in relative numbers. 3. Since all known variables had been held constant, it was concluded that the higher red cell count and hemoglobin value for the male group were the results of constitutional differences between the sexes. Moreover, the significant correlations between the blood cells of male and female litter mates were ascribed to hereditary influences, and thus the female as well as the male rabbit is available for studies on the mechanism involved in the inheritance of the blood formula. In addition, since high correlations were obtained for cells which have been found to be the most reliable indices of the natural immunity of the male host to subsequent inoculation with tumor or syphilis, it was concluded that these cells should give dependable information concerning the natural resistance of the female host to these two diseases.