THE STABILITY OF BACTERIAL SUSPENSIONS : V. THE REMOVAL OF ANTIBODY FROM SENSITIZED ORGANISMS.

1. The removal of antibody from Bacillus typhosus is no more complete at pH 3 than at pH 7. 2. Approximately twelve agglutinating doses are firmly combined with the organisms. Immune body in excess of this amount is easily removable by distilled water. 3. A method of testing for the presence of immune body on the organism is described which depends on the difference in the acid agglutination of sensitized and unsensitized organisms. 4. Repeated washing in distilled water will serve to remove all the immune body from sensitized bacteria.     

THE AGGLUTINATION OF RED BLOOD CELLS

1. Unsensitized sheep cells suspended in sugar solutions are agglutinated by electrolytes whenever the potential is depressed to 6 millivolts or less, except in the case of MgCl₂ or CaCl₂. 2. With these salts no agglutination occurs although there is practically no potential. The presence of these salts prevents acid agglutination. This is presumably due to a decrease in the "cohesion" between the cells. 3. Cells which have been sensitized with specific antibody, ricin, colloidal stannic hydroxide, or paraffin oil, are agglutinated whenever the potential is decreased below about 12 millivolts. 4. The agglutination by electrolytes is therefore primarily due to a decrease in the potential whereas agglutination by immune serum, ricin, etc., is due primarily to an increase in the critical potential.     

THE STABILITY OF BACTERIAL SUSPENSIONS : IV. THE COMBINATION OF ANTIGEN AND ANTIBODY AT DIFFERENT HYDROGEN ION CONCENTRATIONS.

1. The amount of immune body required to agglutinate a suspension of Bacillus typhosus increases in direct proportion to the concentration of the suspension. 2. The amount of immune body combined with the organisms is constant from pH 9 to pH 3.7. Below the latter value the amount in combination is decreased. 3. The addition of immune serum to a suspension of Bacillus typhosus at a pH of 2,5 increases the positive charge of the organisms. These results are contradictory to the idea that the combination is caused by a difference in the sign of the charge carried by the immune body and the organism. They agree with the assumption that the immune body forms a film on the surface of the organism and that the effect on the charge is the result of this film.     

THE MECHANISM OF GRANULAR GROWTH OF RABBIT SEPTICEMIA BACILLUS TYPE G

The acid agglutination optimum of Microbes D and G is not independent of the nature of the buffer mixture. Glycocoll-HCl buffer mixtures cause complete flocculation at high C_H+ (2.7 to 2.4), at which points little or no flocculation occurs with the Na lactate-lactic acid buffer series. Beef infusion has the property of broadening the acid agglutination optimum of both Microbes D and G, bacilli of rabbit septicemia. This extension is in the direction of a lower C_H+. There is no evidence that the beef infusion has the power, per se, of agglutinating these organisms. It would seem merely to increase their sensitiveness to sedimentation in the presence of H ions. The data presented explain the mechanism of the granular growth character of Microbe G in liquid media as compared to the diffuse growth of Microbe D. Peptone (Fairchild), contrary to beef infusion, shifts the acid agglutination optimum of Microbes D and G in the direction of a higher C_H+. Strong concentrations of peptone exhibit an inhibitory effect on the agglutination of Microbe D in the optimum zone.     

THE ANTIPROTEOLYTIC ACTIVITY OF SERUM : I. THE NATURE AND EXPERIMENTAL VARIATION OF THE ANTIPROTEOLYTIC ACTIVITY OF SERUM

1. An equation is derived for the calculation of a constant which, experimental results indicate, may be a more reliable index of the antiproteolytic activity of serum than those equations hitherto used. 2. (a) Intramuscular administration of trypsin resulted in a slow rise in the antiproteolytic activity of the serum, followed by a lesser decline. (b) Intravenous administration resulted in no appreciable variation. (c) Oral administration resulted in a rapid rise, which was sustained during the period of administration. (d) Intramuscular, intravenous, or oral administration of denatured trypsin resulted in no appreciable variation. (e) The extent of the local necrosis following subcutaneous injection of trypsin varied inversely with the antiproteolytic activity of the serum. 3. The experimental results indicate that the products of protein hydrolysis in the intestine and parenterally are an important factor in the antiproteolytic activity of the serum. They also indicate that antibodies to trypsin are not an important factor in the antiproteolytic activity of the serum.     

THE ISOELECTRIC POINT OF RED BLOOD CELLS AND ITS RELATION TO AGGLUTINATION

1. The movement of normal and sensitized red blood cells in the electric field is a function of the hydrogen ion concentration. The isoelectric point, at which no movement occurs, corresponds with pH 4.6. 2. On the alkaline side of the isoelectric point the charge carried is negative and increases with the alkalinity. On the acid side the charge is positive and increases with the acidity. 3. On the alkaline side at least the charge carried by sensitized cells is smaller and increases less rapidly with the alkalinity than the charge of normal cells. 4. Both normal and sensitized cells combine chemically with inorganic ions, and the isoelectric point is a turning point for this chemical behavior. On the acid side the cells combine with the hydrogen and chlorine ions, and in much larger amount than on the alkaline side; on the alkaline side the cells combine with a cation (Ba), and in larger amount than on the acid side. This behavior corresponds with that found by Loeb for gelatin. 5. The optimum for agglutination of normal cells is at pH 4.75, so that at this point the cells exist most nearly pure, or least combined with anion and cation. 6. The optimum for agglutination of sensitized cells is at pH 5.3. This point is probably connected with the optimum for flocculation of the immune serum body.     

THE AGGLUTINATION OF RED BLOOD CELLS IN THE PRESENCE OF BLOOD SERA

1. The addition of blood serum displaces the optimum for agglutination of red blood cells in a salt-free medium to the reaction characteristic of flocculation of the serum euglobulin. 2. This effect is not due merely to a mechanical entanglement of the cells by the precipitating euglobulin, since at reactions at which the latter is soluble it protects the cells from the agglutination which occurs in its absence. 3. A combination of some sort appears therefore to take place between sheep cells and sheep, rabbit, and guinea pig serum euglobulin, and involves a condensation of the serum protein upon the surface of the red cell. 4. At the optimal point for agglutination of persensitized cells both mid- and end-piece of complement combine with the cells. 5. Agglutination is closely related to an optimal H ion concentration in the suspending fluid, and probably of the cell membrane, and not to a definite reaction in the interior of the cell.     

THE STABILITY OF BACTERIAL SUSPENSIONS : II. THE AGGLUTINATION OF THE BACILLUS OF RABBIT SEPTICEMIA AND OF BACILLUS TYPHOSUS BY ELECTROLYTES.

1. Measurements have been made of the potential and of the cohesive force at the surface of Bacillus typhosus and the bacillus of rabbit septicemia in solutions of various salts and acids. 2. Electrolytes in low concentration (0.01 N) affect primarily the potential, and in high concentration decrease the cohesive force. 3. As long as the cohesive force is not affected, agglutination occurs whenever the potential is reduced below about 15 millivolts. 4. When the cohesive force is decreased the critical potential is also decreased, and in concentrated salt solution no agglutination occurs even though there is no measurable potential.     

THE COLLOIDAL BEHAVIOR OF SERUM GLOBULIN

1. The globulin prepared from ox serum by dilution and precipitation with carbon dioxide has been found, by electrometric titration experiments, to behave like an amphoteric electrolyte, reacting stoichiometrically with acids and bases. 2. The potential difference developed between a solution of globulin chloride, phosphate, or acetate and a solution of the corresponding acid, free from protein, separated from the globulin by a collodion membrane, was found to be influenced by hydrogen ion concentration and salt concentration in the way predicted by Donnan''s theory of membrane equilibrium. In experiments with sodium globulinate and sodium hydroxide it was found that the potential difference could be similarly explained. 3. The osmotic pressure of such solutions could be qualitatively accounted for by the Donnan theory, but exhibited a discrepancy which is explicable by analogy with certain experiments of Loeb on gelatin. 4. The application of Loeb''s theory of colloidal behavior, which had previously been found to hold in the case of gelatin, casein, egg albumin, and edestin, has thus been extended to another protein, serum globulin.     

THE EFFECT OF PURE PROTEIN SOLUTIONS AND OF BLOOD SERUM ON THE DIFFUSIBILITY OF CALCIUM

1. A comparative study has been made of the diffusibility of calcium in solutions of crystalline egg albumin, serum globulin, and human blood serum. 2. In all three of these solutions, at pH 7.4, molal Ca concentrations within the membrane are greater than the calcium concentrations in the outside solutions, quite in accordance with the Donnan theory. 3. At pH 7.4, the ratio of See PDF for Structure varies directly with the protein concentration whether the solution be one of egg albumin, serum globulin, or blood serum. This is also in accordance with the Donnan theory. 4. On the acid side of the isoelectric point of the proteins, the concentration of Ca outside becomes greater than the concentration in the solution of blood serum or pure protein, as is demanded by the Donnan theory. 5. The magnitude of the Ca ratios on the alkaline and acid sides of the isoelectric points is probably the resultant of the Donnan equilibrium and the formation of complex Ca-protein ions. Northrop and Kunitz have shown the probability of the existence of such ions in the case of Zn⁺⁺, K⁺, and Li⁺, where satisfactory electrodes have been developed for E.M.F. measurements.     

CHANGES IN THE STABILITY AND POTENTIAL OF CELL SUSPENSIONS : II. THE POTENTIAL OF ERYTHROCYTES.

1. Human and sheep erythrocytes, when placed in 0.01 N buffer solutions at reactions more acid than pH 5.2, undergo a progressive change in potential, becoming less electronegative or more electropositive. This change usually occurs within 2 hours at ordinary room temperatures. It did not occur when rabbit erythrocytes were used. 2. This change is due primarily to the liberation of hemoglobin from some of the cells. 3. Hemoglobin, even in very low concentrations, markedly alters the potential of erythrocytes in the more acid reactions. This is due to a combination between the electropositive hemoglobin and the erythrocytes. The effect of the hemoglobin is most marked in the more acid solutions; it occurs only on the acid side of the isoelectric point of the hemoglobin. 4. The isoelectric point of erythrocytes in the absence of salt, or in the presence of salts having both ions monovalent, occurs at pH 4.7. This confirms the observations of Coulter (1920–21). Divalent anions shift the isoelectric point to the acid side. 5. The effect of salts on the potential of erythrocytes is due to the ions of the salts, and is analogous in every way to the effect of salts on albumin-coated collodion particles, as discussed by Loeb (1922–23).     

THE EFFECT OF CERTAIN RESPIRATORY INHIBITORS ON THE RESPIRATION OF CHLORELLA

Chlorella, when made heterotrophic by means of certain sugars, respires like other heterotrophic cells when subjected to the respiratory inhibitors, hydrocyanic acid, hydrogen sulfide, and carbon monoxide. Whether the case of Chlorella is typical for green cells in general remains to be seen. Experiments with various other green organisms are being carried out, in hope of settling this point.     

Immune serum-mediated cytotoxicity against Trypanosoma rhodesiense.

The metabolic integrity of Trypanosoma rhodesiense can be assessed in vitro by the extent of incorporation of radiolabeled leucine into trichloroacetic acid precipitable material or into material retained after filtration on a glass fiber filter. Incorporation is an approximately linear function of time, and the rate of incorporation is linearly dependent on cell concentration in the presence of normal rat serum. Incorporation is completely prevented if the organisms are reacted wiith fresh serum from animals immunized with gamma-irradiated parasites; the degree of inhibition is a function of the dose of immune serum used. This serum-mediated cytotoxic activity is abrogated by heating the serum, but can be fully restored by addition of fresh rat or guinea pig serum to the heated immune serum. The serum activity arises promptly after one to four immunizing doses of irradiated parasites, falls to lower levels by 1 month, but persists for at least 2(1/2) months, and is unaffected by challenge with viable trypanosomes.     

THE IMMUNOLOGICAL SPECIFICITY OF THE EUGLOBULIN AND PSEUDOGLOBULIN FRACTIONS OF HORSE AND H^MAN SERUM

That portion of horse and human serum globulin precipitated by 33 per cent saturation with ammonium sulfate and precipitated on subsequent dialysis was taken as euglobulin; and the fraction precipitated between 33 and 50 per cent saturation and remaining in solution on subsequent dialysis was taken as pseudoglobulin. The sera of rabbits injected with either of these antigens gave precipitation with both. However, two distinct and fraction-specific antibodies could be demonstrated by absorbing the sera with the one antigen, and testing the supernatant fluid with the other. The experimental results are adequately explained on the basis that there are at least two antigenically distinct globulins in serum which we may term globulin I and globulin II and which are largely associated with so called euglobulin and pseudoglobulin respectively. The ordinary methods of salting out and dialysis do not effect complete separation and each globulin preparation contains a trace of the other antigen. The antisera to these euglobulin and pseudoglobulin preparations therefore contain antibodies to both antigens. Each protein solution precipitates all the antibody specific for the one antigen and in addition, by virtue of the trace of contaminating protein, precipitates a portion, and only a portion of the antibody specific for the other antigen. The fact that antisera to whole serum contain these same fraction-specific antibodies suggests that this immunological specificity is an inherent property of two globulins present as such in serum and is not an artifact induced by their precipitation and purification. Lipoids extracted from the globulins by ether, petroleum ether, and alcohol give no demonstrable reaction with antisera to these globulins; antisera absorbed with a large excess of lipoid are not affected as regards their reactivity with the original protein; and globulins extracted with ether and petroleum ether at room temperature are not affected as regards their reactivity with antisera. It is concluded that the immunological specificity of the globulin fractions as evidenced by the precipitation reaction is not determined by lipoids associated with the protein.     

STUDIES OF GAS AND ELECTROLYTE EQUILIBRIA IN THE BLOOD : IX. THE DISTRIBUTION OF ELECTROLYTES BETWEEN TRANSUDATES AND SERUM.

1. Analyses have been made of the electrolytes and proteins of serum and transudates from human subjects. 2. The distribution ratios of HCO₃, Cl, Na, and H⁺ deviated from unity as predicted by the Gibbs-Donnan law for similar heterogeneous systems. 3. Analyses of serum, and of artificial salt solutions approximating edema fluid in composition, after equilibration across collodion membranes showed distributions similar to those between serum and edema fluid in vivo.     

THE ANTIPROTEOLYTIC ACTIVITY OF SERUM : II. PHYSIOLOGICAL SIGNIFICANCE. THE INFLUENCE OF PURIFIED TRYPSIN INHIBITOR ON THE COAGULATION OF THE BLOOD

1. Serum antitrypsin and pancreatic trypsin inhibitor inhibited the coagulation of plasma in vitro. 2. This could be largely prevented by trypsin. 3. The anticoagulant action of the trypsin inhibitor was apparently due to its antiprothrombic action. It had no appreciable antithrombic action. 4. Examination of the blood of two hemophiliacs indicated that the prolonged coagulation time of their blood is not due to an excess of trypsin inhibitor. 5. Examination of the blood of heparinized dogs indicated that heparin does not appreciably contribute to the antiproteolytic activity of the serum.     

AMPHOTERIC COLLOIDS : I. CHEMICAL INFLUENCE OF THE HYDROGEN ION CONCENTRATION.

1. It has been shown in this paper that while non-ionized gelatin may exist in gelatin solutions on both sides of the isoelectric point (which lies for gelatin at a hydrogen ion concentration of C_H = 2.10^–5 or pH = 4.7), gelatin, when it ionizes, can only exist as an anion on the less acid side of its isoelectric point (pH > 4.7), as a cation only on the more acid side of its isoelectric point (pH < 4.7). At the isoelectric point gelatin can dissociate practically neither as anion nor as cation. 2. When gelatin has been transformed into sodium gelatinate by treating it for some time with M/32 NaOH, and when it is subsequently treated with HCl, the gelatin shows on the more acid side of the isoelectric point effects of the acid treatment only; while the effects of the alkali treatment disappear completely, showing that the negative gelatin ions formed by the previous treatment with alkali can no longer exist in a solution with a pH < 4.7. When gelatin is first treated with acid and afterwards with alkali on the alkaline side of the isoelectric point only the effects of the alkali treatment are noticeable. 3. On the acid side of the isoelectric point amphoteric electrolytes can only combine with the anions of neutral salts, on the less acid side of their isoelectric point only with cations; and at the isoelectric point neither with the anion nor cation of a neutral salt. This harmonizes with the statement made in the first paragraph, and the experimental results on the effect of neutral salts on gelatin published in the writer''s previous papers. 4. The reason for this influence of the hydrogen ion concentration on the stability of the two forms of ionization possible for an amphoteric electrolyte is at present unknown. We might think of the possibility of changes in the configuration or constitution of the gelatin molecule whereby ionized gelatin can exist only as an anion on the alkaline side and as a cation on the acid side of its isoelectric point. 5. The literature of colloid chemistry contains numerous statements which if true would mean that the anions of neutral salts act on gelatin on the alkaline side of the isoelectric point, e.g. the alleged effect of the Hofmeister series of anions on the swelling and osmotic pressure of common gelatin in neutral solutions, and the statement that both ions of a neutral salt influence a protein simultaneously. The writer has shown in previous publications that these statements are contrary to fact and based on erroneous methods of work. Our present paper shows that these claims of colloid chemists are also theoretically impossible. 6. In addition to other physical properties the conductivity of gelatin previously treated with acids has been investigated and plotted, and it was found that this conductivity is a minimum in the region of the isoelectric point, thus confirming the conclusion that gelatin can apparently not exist in ionized condition at that point. The conductivity rises on either side of the isoelectric point, but not symmetrically for reasons given in the paper. It is shown that the curves for osmotic pressure, viscosity, swelling, and alcohol number run parallel to the curve of the conductivity of gelatin when the gelatin has been treated with acid, supporting the view that these physical properties are in this case mainly or exclusively a function of the degree of ionization of the gelatin or gelatin salt formed. It is pointed out, however, that certain constitutional factors, e.g. the valency of the ion in combination with the gelatin, may alter the physical properties of the gelatin (osmotic pressure, etc.) without apparently altering its conductivity. This point is still under investigation and will be further discussed in a following publication. 7. It is shown that the isoelectric point of an amphoteric electrolyte is not only a point where the physical properties of an ampholyte experience a sharp drop and become a minimum, but that it is also a turning point for the mode of chemical reactions of the ampholyte. It may turn out that this chemical influence of the isoelectric point upon life phenomena overshadows its physical influence. 8. These experiments suggest that the theory of amphoteric colloids is in its general features identical with the theory of inorganic hydroxides (e.g. aluminum hydroxide), whose behavior is adequately understood on the basis of the laws of general chemistry.     

CYTOPATHIC EFFECT OF THE ATYPICAL PNEUMONIA ORGANISM IN CULTURES OF HUMAN TISSUE.

Eaton, Monroe D. (Harvard Medical School, Boston, Mass.), Ann E. Farnham, Jeana D. Levinthal, and Anthony R. Scala. Cytopathic effect of the atypical pneumonia organism in cultures of human tissue. J. Bacteriol. 84:1330-1337. 1962.-Three strains of the atypical pneumonia agent were adapted to grow in continuous cell cultures of human amnion or human embryonic lung, with production of initial increased acidity followed by destruction of the cells. Evidence is presented that cytopathic effects of the organism were associated with intracellular growth and formation of microcolonies. Clumps of organisms stained specifically with fluorescein-labeled antibody, and showed distinctive tinctorial reactions with the May Grünwald-Giemsa stain. The cytopathic effect was prevented by fresh serum from a rabbit immunized with an egg-passage strain of the atypical pneumonia agent. Heating the immune serum to 56 C for 30 min abolished the neutralizing effect. The significance of heat-labile serum constituents in killing or inhibition of mycoplasma is discussed.     

Trypanosoma gambiense: immunity with thymic cell transfer in mice.

This report deals with the enhanced agglutinin production and protection in thymectomized, lethally irradiated mice (TI-mice) with transferred thymic cells from mice immune to T. gambiense. Such mice, when sensitized with trypanosome antigen showed protection against experimental infection and also produced agglutinins. Thymic cells from cortisone-treated immune mice were able to induce the production of agglutinins in TI-mice subsequently injected with antigen. However, these agglutinin titers were very low. In bovine serum albumin gradient centrifugation experiments, agglutinin production could be efficiently induced by inoculation of TI-mice with a rather high density thymic cell subpopulation taken from immune mice. Fractionated by Sephadex G-200, the agglutinins displayed a division into two parts, a first and second peak. The main agglutination reaction was seen in the first or macroglobulin peak. In the fractionation of serum by DEAE-cellulose column chromatography, agglutinins were eluted in two parts, the 0.0175 M and 0.4 M effluents. The agglutination by the 0.4 M effluent was much stronger than that of the 0.0175 M effluent, in agreement with the gel filtration results. The sera containing agglutinins were able to enhance the phagocytosis of trypanosomes by cultured macrophages from the peritoneal cavity of normal and irradiated mice. Delay of parasitemia was evident in some of the TI-mice having detectable agglutinins. The delayed parasitemia resulted from antigenically altered trypanosomes which were able to withstand the lethal factors of TI-mice. Transplantation of thymic cells was considered to be responsible for agglutinins induced by the antigenic stimulation in TI-mice and for protection against experimental infection.     

THE PROTEIN ASSOCIATED WITH HEMOLYSIN IN RABBIT SERUM AND PLASMA

1. The water-insoluble globulin with which hemolysin is associated, may be separated from immune serum or plasma by dilution and simple dialysis at optimal pH. 2. This optimum in plasma is influenced by the presence of the fibrinogen. 3. Fibrinogen carries no immune body, or only an insignificant amount; when present in immune body solutions in other form than fibrin gel, it depresses the hemolytic activity. The conditions for the formation of fibrin gel are similar to those for the formation of a gel by banana protein. 4. The hemolytic activity is a more labile property of the immune protein than the agglutinating activity; hemolysin is destroyed, hemagglutinin shows an apparent increase, as a result of alcohol extraction.