DEMONSTRATION OF TUMOR-SPECIFIC ANTIGENS IN HUMAN COLONIC CARCINOMATA BY IMMUNOLOGICAL TOLERANCE AND ABSORPTION TECHNIQUES

Two methods were used to demonstrate the presence of tumor-specific antigens in adenocarcinomata of the human colon: (a) rabbits were immunized with extracts of pooled colonic carcinomata, and the antitumor antisera thus produced were absorbed with a pooled extract of normal human colon and with human blood components; (b) newborn rabbits were made immunologically tolerant to normal colonic tissue at birth, and were then immunized with pooled tumor material in adult life. Normal and tumor tissues were obtained from the same human donors in order to avoid misinterpretation of results due to individual-specific antigenic differences. The antisera prepared by both methods were tested against normal and tumor antigens by the techniques of agar gel diffusion, immunoelectrophoresis, hemagglutination, PCA, and immunofluorescence. Distinct antibody activity directed against at least two qualitatively tumor-specific antigens, or antigenic determinants, was detected in the antisera prepared by both methods and at least two additional tumor antigens were detected exclusively in antisera prepared by the tolerance technique. Whether these additional antigens were qualitatively different from normal tissue antigens, or merely present in tumor tissue in higher concentrations than in normal tissue has not as yet been determined. Furthermore, it was shown that the tumor-specific antibodies were not directed against bacterial contaminants or against the unusually high concentrations of fibrin found in many neoplastic tissues. It was concluded from these results that the pooled tumor extracts contained tumor-specific antigens not present in normal colonic tissue. Identical tumor-specific antigens were also demonstrated in a number of individual colonic carcinomata obtained from different human donors.     

两种不同抗原在幽门螺杆菌临床诊断中的比较

采用Sephadex－G200凝胶层析和酸性甘氨酸提取法，制备了两组不同的HP抗原，并分别以此为包被抗原，用SPA－ELISA方法检测了经临床生物学特性鉴定，确定为HP感染的阳性标本26例，阴性标本18例。结果以Sephadex－G200凝胶层析和酸性甘氨酸提取的抗原，对HP感染的阳性检出率分别为92％和62％。提示以Sephadex－G200凝胶层析提纯的抗原，在幽门螺杆菌临床诊断中的特异性明显优于酸性甘氨酸提取的抗原。     

A STUDY OF THE ADDITION OF CHOLESTERIN TO THE ALCOHOLIC EXTRACTS OF TISSUES USED FOR ANTIGENS IN THE WASSERMANN REACTION

1. The addition of cholesterin to an alcoholic extract of heart or fetal liver increases the antigenic value of the extracts in the Wassermann reaction. 2. The optimum amount of cholesterin to be added to heart extract or fetal liver extract was found to be 0.4 per cent. 3. Cholesterin-heart extracts are superior to cholesterin-liver extracts and to alcoholic extracts of syphilitic livers, as well as to ether extracts of dried hearts. 4. Cholesterin-heart extracts prepared from different human hearts are practically equal in anticomplementary and antigenic value. Similar extracts prepared from guinea pig hearts have the same antigenic value as those prepared from human hearts. Both the human heart and the guinea pig heart extracts are superior to beef heart extract when the same amount of cholesterin is added to each of the extracts. 5. In testing blood serum for diagnostic purposes, it is not safe to use more than one fourth of the anticomplementary dose of the 0.4 per cent. cholesterin heart extract. In the work here presented, this consisted of a 1 in 10 emulsion. 6. In testing cerebrospinal fluids, 1 in 10 emulsions give slightly better reactions with smaller quantities of the fluid than do 1 in 6 emulsions. 7. Because of the simple preparation, the superior antigenic property, and the constant antigen value of cholesterin-heart extracts prepared from human hearts, we agree with McIntosh and Fildes that this form of extract fulfills the requirements of a standard antigen.     

A CORRELATIVE STUDY BY ELECTRON AND LIGHT MICROSCOPY OF THE DEVELOPMENT OF TYPE 5 ADENOVIRUS : II. LIGHT MICROSCOPY

The evolution of the intranuclear lesion produced by type 5 adenovirus in HEp-2 and HeLa cells is described as seen in the light microscope and the bodies formed in the course of the infection characterized histochemically. Some 12 hours after infection acidophilic protein bodies, without appreciable nucleic acid, first appear in the nucleus and coalesce into a network. Within or in association with this material, DNA-containing masses (viral aggregates) are formed which rapidly increase in amount and then coalesce. At the same time, a protein is produced, histochemically different from that of the acidophilic or basophilic structures mentioned, within the infected nucleus, which constitutes a matrix within which regular cytstals of a protein, (presumably non-viral) materialize. These structural and histochemical features are correlated with details which have been observed in parallel studies with the electron microscope.     

FACTORS INVOLVED IN THE INDUCTION OF NON-SPECIFIC RESISTANCE TO STREPTOCOCCAL INFECTION IN MICE BY ENDOTOXIN

Endotoxins derived from several species of Gram-negative bacteria, while inducing non-specific resistance to typhoid bacilli in mice, failed to increase the resistance of these animals to infection with virulent strains of Group A streptococci. However, if administration of endotoxin was followed by injection of minute amounts of type-specific antiserum, a substantial degree of protection against the streptococcal pathogen was obtained. The same amount of type-specific antiserum given to the animals by itself did not have any effect on the outcome of the streptococcal infection. Fresh rabbit blood, obtained from animals pretreated with endotoxin, together with anti-M protein immune serum, was found strongly bactericidal for streptococci. These observations suggest that, at least with regard to streptococcal infection, both humoral and cellular factors are required for induction of non-specific resistance.     

A CORRELATIVE STUDY BY ELECTRON AND LIGHT MICROSCOPY OF THE DEVELOPMENT OF TYPE 5 ADENOVIRUS : I. ELECTRON MICROSCOPY

Stages in the nuclear changes consequent to infection with type 5 adenovirus are shown and described. Viral development seems to be confined to the nucleus where characteristic particles are found. The shape of the intracellular virus depends upon the method of preservation employed, appearing spherical after osmium tetroxide or freezing-substitution, occasionally exhibiting angulated faces after formalin and often assuming an hexagonal profile after potassium permanganate. The non-viral crystals are encountered in zones of low density, and it is suggested that crystallization results from the accumulation of protein in these areas. An hypothesis is presented to explain why these crystals, in contrast to the insect polyhedra, contain few viral particles.     

IMMUNOFLUORESCENT STUDIES OF ADENOVIRUS 12 TUMORS AND OF CELLS TRANSFORMED OR INFECTED BY ADENOVIRUSES

Complement-fixing (CF) antibody-positive sera from hamsters bearing adenovirus type 12 (Ad. 12)-induced tumors revealed specific immunofluorescent stainable antigens in essentially all Ad. 12 hamster tumor cells. The antigens were primarily in the form of cytoplasmic flecks; less frequent staining was seen as nuclear flecks or homogeneous staining of nucleus and cytoplasm of a small proportion of cells. Tumor cells did not stain with rabbit antisera to crude Ad. 12 virus or A and C antigens. The hamster serum also stained cytoplasmic flecks in an Ad. 12-induced BALB/c mouse tumor and Ad. 12-"transformed" hamster embryo tissue culture cells. The hamster serum also stained fleck-shaped antigens in hamster and human cell cultures inoculated with homologous and heterologous adenovirus types, although the hamster cells did not react with the rabbit Ad. 12 antiserum. Attempts to identify the fluorescent-stainable fleck-shaped antigens indicated that they are not previously recognized viral antigens and that the cytoplasmic antigens formed in hamster cell cultures inoculated with Ad. 12 are different from those in tumors and in acutely infected human cell cultures.     

IMMUNOFLUORESCENT STUDIES OF THE POTENTIATION OF AN ADENOVIRUS-ASSOCIATED VIRUS BY ADENOVIRUS 7

A quantitative immunofluorescent procedure for detection of viral antigen was used to study the potentiation of AAV-1 by Ad.7. AAV viral antigen formed only when the cells were also infected with adenovirus, and only in cell culture systems in which the adenovirus infection proceeded to completion. Ad. 7 infection of AGMK. cell cultures did not potentiate AAV unless the Ad. 7 infection was itself potentiated by SV40. Dose-response studies indicated that a single AAV particle and a single infectious Ad. 7 particle sufficed to initiate AAV antigen synthesis. Sequential inoculation studies showed that AAV antigen formed simultaneously with Ad. 7 viral antigen when the AAV was inoculated any time between 15 hr before to 10 hr after the Ad. 7, both antigens appearing about 15 hr after inoculation of Ad. 7. The AAV-1 antigen formation had a minimum latent period of 5 hr, as seen with Ad. 7 preinfection of 10 hr or more. When UV-irradiated Ad. 7 was used as helper, the AAV antigen still appeared simultaneously with the Ad. 7 viral antigen, even though the latter was delayed by 23 hr compared to nonirradiated virus. When the early replicative events of both viruses were allowed to proceed in FUDR-inhibited cells, and then the FUDR inhibition was reversed, AAV antigen formed within 2 hr, which was 3 hr before the Ad. 7 viral antigen appeared. It was inferred that the event in the adenovirus cycle that renders a cell competent to synthesize AAV occurs after the 10th hr and may be temporally associated with replication of the adenovirus DNA.     

INTESTINAL OBSTRUCTION : II. A STUDY OF THE FACTORS INVOLVED IN THE PRODUCTION AND ABSORPTION OF TOXIC MATERIAIS FROM THE INTESTINE.

1. It is impossible to sterilize the intestine by the use of chemical antiseptics even when these are applied directly to the mucosa of isolated segments. 2. The mucosa of the alimentary tract does not elaborate an internal secretion which is necessary to life, or a secretion which could be disturbed by the conditions of acute obstruction so as to account for the symptom complex of that condition. 3. The substances responsible for the toxemia in acute obstruction are produced by the action of intestinal bacteria on proteins or their split products. 4. An injury to the intestinal mucosa, particularly that resulting from disturbances of the blood supply to the intestine, greatly facilitates the absorption of these poisons. The work of Hartwell and his associates and that of Murphy and Brooks on this point are confirmed.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : IV. THE RESPONSE OF NORMAL AND IMMUNE RABBIT BONE MARROW AND LYMPHOID TISSUE LYMPHOCYTES TO ANTIGENS IN VITRO

Cell suspensions of immune rabbit lymph nodes and spleen were capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine when maintained in culture with the specific antigen in vitro. They did not respond to other, non-cross-reacting antigens. The blastogenic response obtained with immune lymph node cells could be correlated with the antibody synthesizing capacity of fragment cultures prepared from the same lymph nodes. Cell suspensions of immune bone marrow responded to non-cross-reacting antigens only whereas cell suspensions of immune thymus, sacculus rotundus, and appendix did not respond when exposed to any of the antigens tested. On the other hand, neither fragments nor cell suspensions prepared from lymph nodes, spleen, and thymus of normal, unimmunized rabbits responded with antibody formation and blastogenesis when exposed to any of the antigens. However, normal bone marrow cells responded with marked blastogenesis and tritiated thymidine uptake. The specificity of this in vitro bone marrow response was demonstrated by the fact that the injection of a protein antigen in vivo resulted in the loss of reactivity by the marrow cell to that particular antigen but not to the other, non-cross-reacting antigens. Furthermore, bone marrow cells of tolerant rabbits failed to respond to the specific antigen in vitro. It was also demonstrated that normal bone marrow cells incubated with antigen are capable of forming antibody which could be detected by the fluorescent antibody technique. This response of the bone marrow cells has been localized to the lymphocyte-rich fraction of the bone marrow. It is concluded that the bone marrow lymphocyte, by virtue of its capacity to react with blastogenesis and mitosis and with antibody formation upon initial exposure to the antigen, a capacity not possessed by lymphocytes of the other lymphoid organs, has a preeminent role in the sequence of cellular events culminating in antibody formation.     

ON THE NATURE OF TRANSPLANTATION IMMUNITY IN THE ADENOVIRUS TUMOR SYSTEM

The existence of a virus-induced, virus-specific transplantation, antigen in adenovirus 12-induced CBA mouse tumors was demonstrated. The antigen is virus-specific, but not related to structural virion or T antigens. It is a weak antigen, and required immunization with whole, infectious adenovirus 12 to produce considerable immunity. Comparable immunity could not be achieved with homologous cellular or subcellular materials, but some indication of enhancement was produced with low tumor dose. Immunization required at least 2 wk and was mediated by immune lymphoid cells. Serum of immunized animals showed no demonstrable cytotoxicity or enhancement. Animals immunized with virus and Freund's adjuvant showed diminished transplantation immunity, although these animals were actively immunized against adenovirus type 12 structural virion antigens.     

THE RETENTION OF FOREIGN PROTEIN BY THE KIDNEY : A STUDY IN ANAPHYLAXIS.

Extracts of the kidneys of normal rabbits prepared one, two, three, and four days after the intravenous injection of egg-albumin and horse serum have the power to sensitize guinea pigs to a second injection of these proteins. The sensitization by first and second day extracts was constant and intense, that by the third day extracts was less marked and sometimes was not evident, and that by the fourth day extracts was only occasional, and when present was always weak. Comparative studies of the power of the blood, liver, and kidney to sensitize, indicate that this sensitization depends on the content of foreign protein in the circulating blood and not upon its accumulation or fixation in the tissues of an organ. This opinion is supported by other experiments in which the sensitizing power of the blood and of the extracts of unwashed kidneys was compared with the sensitizing power of extracts of washed kidney. The weak sensitizing power of washed kidney extract is taken as evidence that foreign proteins of the kinds used are not held in the tissues of the kidney, and if these results may be applied to nephrotoxic proteins, it follows that nephritis is not due to selective and persisting fixation of a protein by the renal cells, but is due to the action of such protein merely during the process of its elimination. In experimental acute nephritis of the type due to uranium nitrate, the power of sensitization to egg-albumin is prolonged for twenty-four hours, and in the chrornate type for forty-eight hours, thus indicating that in nephritis, of the acute type at least, the elimination of a foreign protein is delayed. Attempts to study by the same methods the elimination of vegetable and bacterial proteins have failed.     

CHARACTERISTICS OF THE DOPAMINE RECEPTORS INVOLVED IN THE ANORECTIC EFFECTS OF APOMORPHINE IN MICE

In food-deprived mice apomorphine injected SC induced a brief (15-30 min) dose-dependent (30-150 micrograms/kg) reduction in food intake. This effect occurred in naive mice as well as in mice habituated to a food deprivation procedure. The anorectic effect of apomorphine (150 micrograms/kg SC) was antagonized by sulpiride (ID50 = 8.6 mg/kg) and by haloperidol (ID50 = 66 micrograms/kg) but domperidone was ineffective (250 micrograms/kg). Mice submitted to a semi-chronic (6 d) blockade of dopamine receptors by haloperidol or injected intracerebroventricularly with 125 micrograms 6-hydroxydopamine 21 d before testing failed to develop a hypersensitivity to the anorectic effect of apomorphine (60 micrograms/kg). Although a single apomorphine injection (5 mg/kg) induced tolerance to the hypothermic effect of a second apomorphine injection of 150 micrograms/kg, it did not modify the anorectic effect. Repeated apomorphine injection (5 x 5 mg/kg) resulted in a slight but significant reduction in apomorphine-induced anorexia. A similarly significant reduction was not observed in mice submitted to repeated injections of dexamphetamine (5 x 5 mg/kg).     

SEROLOGIC EVIDENCE FOR ANTIGENS CONTROLLED BY THE Ir REGION IN MICE

Antibodies produced in B10.D2 mice against soluble lymphocyte membrane antigens of B10.A (H-2^a) mice reacted only with lymphocytes of the strains carrying the Ir^k region, i.e., B10.A(2R), B10.K, B10.BR, B10.HTT, AQR, A.TE, C3H, and CBA; they did not react with cells of strains carrying different Ir regions, i.e., B10.A(4R), B10, B10.M, A.SW, DBA/1. It is therefore concluded that the antigen detected with these antibodies is apparently controlled by the Ir region of the H-2 complex. The antigen is present on some T lymphocytes and absent on B lymphocytes. Its presence or absence seems to correlate with MLC and GVH reactivity.     

RESTRICTION OF THE CAPACITY TO RESPOND TO TWO ANTIGENS BY SINGLE PRECURSORS OF ANTIBODY-PRODUCING CELLS IN CULTURE

Experiments were designed to determine whether or not precursors of antibody-producing cells are restricted in the number of antigens to which they can respond. An in vitro culture system was used, in which the successful production of hemolysin PFC was dependent on the presence of a large number of heavily irradiated spleen cells which did not themselves give rise to PFC, but which supported the production of PFC by a small number of normal spleen cells. All spleen cells were obtained from unimmunized CBA mice. The cells were mixed with either sheep or chicken erythrocytes, or both, cultured for 4 days and analyzed for hemolysin PFC. By reducing the number of unirradiated spleen cells to limiting dilution it was shown that normal spleen cell suspensions contain approximately three times as many precursors capable of responding to chicken erythrocytes as to sheep erythrocytes. In cultures containing both antigens, the number of precursors responding to one antigen was not affected by the presence of the other antigen. In addition, some cultures were positive for PFC-producing hemolysin against chicken erythrocytes, but not against sheep erythrocytes, and vice versa. This pattern of response was independent of the concentration of antigen in the cultures. Thus, the antigen-sensitive precursors for these non-cross-reacting antigens responded independently of each other, indicating that each precursor was restricted in its capacity to respond to more than one antigen prior to stimulation.     

THE REMOVAL OF CALCIUM FROM THE BLOOD BY DIALYSIS IN THE STUDY OF TETANY

We may devise a fluid containing practically all the inorganic diffusible constituents of the blood except calcium, and use it to dialyze normal blood in such a way as to remove from it a large part of its calcium. The dialyzed blood when perfused through an isolated extremity produces an extreme hyperexcitability of the nerves quite like that observed in tetany. Since perfusion with blood dialyzed in precisely the same way against a fluid of the same composition, but containing calcium in the proportion found in the normal blood, causes no hyperexcitability of the nerves, it is evident that the hyperexcitability is due to the lack of calcium. This effect can be attained in only a slight degree by replacing the blood of a whole animal with the dialyzed blood, since under the conditions of the experiment the tissues cannot be sufficiently depleted of their calcium. It seems probable that the parathyroid secretion is not removed by dialysis, but is returned to the body with the dialyzed blood. To bring this result into relation with the condition in tetany following parathyroidectomy, animals in tetany were bled and the blood was replaced in one case with normal blood, in the other with dialyzed blood poor in calcium. The normal blood immediately relieves the tetany and lowers the excitability, while the dialyzed blood does not. We therefore believe that this is a further proof that in the tetany of parathyroidectomy also the twitching and hyperexcitability of the nerves is due to lack of calcium in the blood and tissues.     

THE BEHAVIOR IN VIVO OF CERTAIN RELATIVELY PURE ANTIGENS

The experiments are of interest in several respects. It is clear that crystallized egg albumen is rapidly eliminated from the circulation and in the experiments cited it could no longer be detected after 18 or 19 hours. A considerable portion of it rapidly passes through the kidney in an apparently unaltered state. Evidently this passage begins almost at once and may continue for a day or two. In an experiment not reported in this paper, egg albumen appeared in naturally voided urine 2 hours following its injection into the peritoneal cavity. In the experiments reported no urine was voided until 5½ and 6½ hours following intravenous administration, but in each instance egg albumen was present in considerable amounts. However, sufficient egg albumen must have been utilized to produce antibody. It is hardly to be expected that such a protein, whose elimination is so rapid, could persist unaltered within the body and reappear within the circulation coincident with its antibody. The behavior of the protein cannot be ascribed to alterations which may have taken place during the process of crystallization since Ascoli showed that the proteins of egg white readily pass from the circulation into the urine. Certain observations of the writer confirm this point. The experience of Alexander, Becke, and Holmes who exposed sensitized guinea pigs to sprays of dilute egg white with the result that 80 per cent of the animals developed symptoms of anaphylaxis, further strengthens the contention that certain of the membranes are readily permeable for the proteins of egg. The conditions following the injection of casein are different. There is no appreciable passage through the kidney. Casein is present within the circulation for a considerable period; it could be detected in the blood serum 12 and 13 days after its introduction into the peritoneal cavity. Antibody appeared on the 7th and 8th days, respectively, so that both antigen and antibody were present in the serum for a period of 3 or 4 days. The phenomenon of antigen and antibody occurring together might be explained on the ground that certain proteins are utilized slowly and that the antibody found in the blood, usually after the 7th day, results from the portion of antigen first utilized. During the next few days a continual supply of antibody enters the circulation and during the period there is a steady utilization of the antigenic substance; it is possible that during this time there is constant union of antigen and antibody within the blood, with the slow utilization of the antigen and a slight utilization of the antibody which is made up by a slow increase from the body cells. Thus there would be a period in which considerable antigen would be present with weak antibody, succeeded by a second period when the amount of antigen would be small with well defined antibody, and finally only antibody. Certain observations tend to support such a view. Bayne-Jones injected rabbits whose serum contained precipitin from egg albumen with this substance and noted the occurrence of both antigen and antibody for a period of 48 hours. Some of his experiments in vitro are equally suggestive. In one instance a rabbit well immunized with egg albumen was injected intravenously with this substance. An hour later it was bled and the stored serum refrigerated for a period. During this time there was a slow spontaneous precipitation with a decline in both precipitin and antigen titer, but even after 6 days both were present. After a longer period only antigen remained. P. A. Lewis and D. Loomis have shown that an injection of sheep red blood cells in guinea pigs results in a well defined hemolysin titer about the 9th day, followed by a definite decline, with a secondary rise in hemolysin until the peak is reached on the 20th day. It becomes evident, then, that the reaction of the rabbit to a single injection of a relatively pure protein will depend on the character of the protein injected. When crystallized egg albumen is administered it is rapidly eliminated from the circulation. The rapid disappearance of the egg albumen from the blood stream is partly accounted for by its prompt elimination through the urine. Antibody appears in the serum from the 7th to the 10th day. Casein behaves differently. It persists in the blood for a considerable period; after the 7th or 8th day both antigen and antibody may be demonstrated in the blood. Casein cannot be detected in the urine following its injection into the body. The behavior of casein within the body affords an analogy with the conditions frequently noted after the administration of foreign serum, in both cases both antigen and antibody may be present in the circulation together.     

THE CATABOLISM OF PROTEIN ANTIGENS IN THE NEWBORN AND MATURING RABBIT

The rate of degradation, organ deposition, and blood clearance of SBSA, SRSA, and IRSA has been measured in the newborn, 6-, and 30-day-old rabbits. When the animals were injected with a weight-graded dose of the 3 proteins, differences in their catabolism in the newborns were demonstrable as compared to the 6-, and 30-day-old animals. The capacity to degrade the azo compounds was shown to be incompletely developed at birth. At 6 days of age, however, the rabbits catabolized these proteins much at the same rate as the 30-day-old animals. Addition of the benzenesulfonate moiety determined the rate of degradation organ deposition and excretion rather than the carrier protein when the azo compounds were injected. The biosynthetically labeled S²⁵ rabbit serum albumin (IRSA) was catabolized at a slower rate than the azoproteins in all age groups. Very little difference in the metabolism and organ deposition of the IRSA was shown to exist between the newborn and maturing animals. A dosage schedule, therefore, designed to test the immunological capacity of developing animals may not be valid when calculated upon body weight. The low level of activity of enzyme systems present at birth which degrade anti-genic material may serve as an explanation as to why this period of development is so vulnerable to the induction of tolerance rather than immunity when compared to the adult.