Lloyd Jones

Vascular Pathology in Rabbits Following Administration of Foreign Protein.

Summary Mild vascular and perivascular alterations of the smaller branches of the coronary arteries were observed in a majority of rabbits following the parenteral injection of horse serum. No rabbit which elicited a necrotic-type of skin reaction failed to show vascular lesions.

Detection of Minute Amounts of Serum Antibody by Agglutination of Antigen-Coated Bacterial Cells

The authors recently described a local serum reaction 1 occurring upon the ear of a rabbit (at the site of a prior injection of serum antigen) when a very small quantity of homologous antiserum was injected intravenously elsewhere in the body. The amount of antiserum injected was so small that it was not possible to detect antibody in the animals serum by the use of the conventional method of testing, i. e., the precipitation test employing the antigen dilution method. In order to determine such minute amounts of circulating antibody in the blood of injected animals it became necessary to devise a technic which would be both accurate and extremely sensitive. This was accomplished by developing a method which involves coating the cells of a suspension of Serratia marcesens with serum antigen and observing the agglutination of such cells upon addition of the animal serum being tested. That bacterial cells can be coated with serum antigen and subsequently agglutinated with an immune serum (homologous to the serum antigen) was reported by Jones. 2 However, to our knowledge, no practical application of this type of antigen-antibody reaction has been made heretofore. It became evident in preliminary experiments, that agar grown cells (washed twice with saline solution, suspended in distilled water and killed by flowing steam) could be satisfactorily coated by suspending them in horse or beef serum (antigen) and incubating the mixture at 37°C for not less than 9 hours. With such sera in order to achieve satisfactory coating it was necessary that the quantitative ratio of serum to cells be of the order of 5 to 1, as based upon the nitrogen contents of these materials. Electrolyte content of serum does not interfere with satisfactory adsorption of antigen upon the cells.

Serologic Agglutination of Antigen-Coated Dye Particles

Summary By adsorption of equine serum (antigen) to suitable particles of carmine a suspension can be made which is specifically agglutinable in rabbit antihorse serum. With suitably prepared suspensions, the open slide method may be conveniently employed in making agglutination tests. Employing this technic it is possible to detect antibody in the fresh sera of immunized rabbits 2 to 6 days earlier than it is by the conventional precipitation test employing the antigen dilution method.

Virulence and Electrophoresis of Bacillus Diphtheriae.

Thirty-two cultures of B. diphtheriae isolated from as many diagnostic throat cultures and 2 laboratory stock cultures (No. 300 and Park No. 8) all of which exhibited characteristic morphology and fermentative reactions with carbohydrates, were examined for virulence and for velocity of electrophoretic migration. All of the cultures fermented dextrose and galactose. Some of the cultures utilized dextrin when first isolated while other strains acquired the ability to utilize this substance after continued laboratory cultivation. None of the strains fermented sucrose. Accordingly, upon the basis of carbohydrate fermentation all cultures were considered typical of B. diphtheriae. Virulence was determined by intracutaneous inoculation of guinea pigs. Electrophoretic mobilities were determined by the use of a Falk micro-electrophoresis apparatus and velocities of migration calculated as micra per second per volt. The data indicate that virulence or lack of virulence of these organisms for the guinea pig is not correlated with the observed velocity of electrophoretic migration, since 27 virulent cultures exhibited widely divergent mobility rates. P. D. varying from 0.260 to 0.750 and similarly in 7 avirulent cultures P. D. varied from 0.340 to 0.737 μ/sec./volt. From several virulent cultures, aberrant avirulent types were developed through forced dissociation and in these latter lack of virulence was not correlated with any significant change in the rate of electrophoretic migration. Agglutinability of cultures with high and with low P. D. in the presence of salts, such as calcium chloride and copper acetate, was determined. In general, cultures having low P. D. as indicated by low velocity of electrophoretic migration, were agglutinated by a lesser concentration of electrolyte than were the cultures having higher P. D. values. However, as noted in the electrophoresis observations agglutinability in salt solution did not bear significant relationship to the virulence of the culture

Use of Synthetic Medium in Study of Antibacterial Effect of Sulfathiazole.

In the present study we have employed the synthetic medium previously reported as being valuable for the study of the antibacterial effect of the thiazole compounds against Salmonella enteritidis and, as originally employed in demonstrating the inhibiting effect of p-aminobenzoic acid upon these drugs. 1 This liquid medium contains the same nutritive materials as are present in Simmons citrate agar and is quite suitable for the cultivation of this organism even though very small inocula are employed. This synthetic medium possesses definite advantages over ordinary broth containing meat extractives and peptone, since it is invariably uniform in composition, devoid of growth factors, essential metabolites, and of such substances as are known to interfere with the activity of the sulfonamide drugs. That this synthetic medium is superior to ordinary broth is further revealed by our experimental observations wherein 0.3 mg % of sulfathiazole prevents growth (as judged by visible turbidity) of S. enteritidis in the former whereas 20 mg % does not prevent growth in the latter medium. Recognizing this superiority, it was deemed of interest to ascertain the relative effects of sulfathiazole upon S. enteritidis in each of these media as would be elicited by a comparison of viable population curves. In Fig. 1 are recorded the growth curves of the organism in meat extract-peptone broth, without drug, and with 5 and 10 mg % sulfathiazole. One of the effects of sulfathiazole is seen as a delay in the inception of the logarithmic phase of multiplication. During this phase, the drug (5 mg %) serves to increase the generation time by about five-fold as is reflected in the slope of the curve. The stationary phase is reached much earlier in control cultures. However, the ultimate number of cells developing in the drug-containing media is about one-fourth the number found in the control.

Treatment of Serum Serving to Remove Its Serum-Sickness-Causing Activity

We have reported previously concerning a reaction appearing on the ears of rabbits 1 subsequent to an injection of either whole horse serum or fractions thereof, which may be considered as analogous to serum-sickness-causing activity of the various serum proteins led to the suggestion that the more important substance concerned in causing serum-sickness may be some agent other than the proteins themselves although this agent appears to be associated with the pseudoglobulin. 3 It has been found that by the addition of adequate quantities of alkali or acid to serum (normal or immune), exposure of the mixture to heat and subsequent neutralization, the serum-sickness-causing activity may be destroyed so that rabbits injected with the customary amounts of serum will not develop manifestations of the disease. In order to minimize or prevent damage to antibody incident to this type of treatment of serum, it is necessary to adjust the various factors concerned in the treatment of the serum. Six factors must be considered: 1, The amount of alkali used (between 0.2 gm. and 0.1 gm. for 100 cc. of serum when using NaOH); 2, the dilution of the mixture (from 1:2 to 1:10); 3, the temperature to which the mixture is exposed (from 45°C. to 55°C.); 4, the time of exposure (from 30 min. to 18 hr.); 5, the type of antibody in the serum; 6, the particular sample of serum being treated. The first 4 factors must be balanced against each other in relation to the last 2 factors. By this method the serum-sickness-causing factor may be destroyed so that rabbits receiving treated serum (10 cc. per kg.) do not develop serum-sickness while rabbits injected with the same untreated serum develop manifestations of serum-sickness in 6 of 10 animals. In sera which are no longer active in causing serum-sickness, from 50% to 100% of the original protective antibody is retained.

Skin Sensitivity and Antibody Production upon Administration of Normal and Immune Serum in Rabbits

In studies upon the antibody response in human subjects following administration of serum, Tuft and Ramsdell 1 , 2 have noted that normal serum is weakly antigenic and immune serum highly antigenic, as indicated in tests made for precipitins, skin sensitivity, anaphylactic antibodies and transferable human and guinea pig skin sensitizing antibodies. Administration of normal horse serum was followed by serum sickness however, identical in its clinical characteristics and about as frequently as was produced by the injection of immune serum. Since it is possible to produce in the rabbit, upon injection of a single large dose of normal or immune serum, a condition closely resembling or identical with serum sickness in man, 3 an attempt was made to compare the antigenicity (as measured by precipitin response and appearance of skin sensitivity) of normal horse serum with that of immune serum, as produced by a single subscapular injection in this species. Three animals were injected with pooled normal horse serum, 5 with the pseudoglobulin fraction of this normal horse serum, 3 with immune serum (collected from the same horses after immunization with diphtheria toxin and containing 1700 units of diphtheria antitoxin per cc.) and 5 with the pseudoglobulin fraction derived from this immune serum. Serum sickness occurred only in some of the animals receiving 75 mg. or more of serum nitrogen per kilo of body weight, and with about the same incidence and severity in those receiving normal serum as in those receiving immune serum. Skin sensitivity and precipitin production resulting from these injections are given in Table I. Skin tests were made by intradermal injection of 0.1 cc. amounts of various dilutions of normal and immune sera or the pseudoglobulin fractions of these sera in the cases of those animals receiving a single large dose of pseudoglobulin fraction. Tests were made for precipitin content of rabbit serum against normal and immune horse sera, or their pseudoglobulin fractions in those cases where the animals received a single large dose of pseudoglobulin. The results indicate that under these conditions skin sensitivity and precipitin production occur in the rabbit and to practically the same extent whether normal or immune serum is used as the antigen.

Production of Rh Antiserum by Inoculation of Guinea Pigs with Human Erythrocytes

Antisera used for detecting the presence of the Rh antigen in human erythrocytes have been of two types: the serum of guinea pigs immunized against the red blood cells of Rhesus monkeys, 1 , 2 and the serum of certain Rh—human beings who have developed antibodies against the Rh antigen. 3 , 4 These latter sera are not readily procurable since human beings possessing the Rh antibody are infrequently encountered and accordingly the incidence of their employment is greatly restricted. Guinea pig anti-rhesus sera also have limitations to their usefulness. Certain differences have been noted in the Rh antigen as found in monkey and human erythrocytes, 5 , 6 and the implications of these differences as yet have not been clarified. Further, the production of such sera in certain laboratories may be hampered by the present war-time scarcity of Rhesus monkeys. For these practical reasons, as well as for certain theoretical considerations, we have undertaken the production of Rh antisera by inoculating experimental animals with Rh + human erythrocytes. Guinea pigs were inoculated with varying doses of red blood cells over a period of 3 months. The cells used were from an Rh+, group O, type MN individual. The presence of the Rh antigen in these cells was assured by the fact that they agglutinated in several absorbed guinea pig anti-rhesus sera and in 6 human anti-Rh sera. Immune serum obtained in this manner when absorbed, according to our previously reported technic 2 with group O, type MN, Rh cells, distinguished sharply between known Rh + and Rh + human erythrocytes of all blood groups. In view of these findings it would appear that the possibilities for producing Rh anti-sera, useful in testing the red blood cells of human beings belonging to any blood group, have been greatly extended.

Protective Effect of Sulfamethylthiazol on Experimental Salmonella enteritidis Infection in Mice

Conclusions Sulfamethylthiazol apparently exerts a definite protective effect in mice which have been experimentally infected with a virulent strain of Salmonella enteritidis. This protective effect is manifested only by an increase in average survival time of medicated animals. Eventually all of the medicated animals succumb to the Salmonella enteritidis infection.

Antigenic Relationships of Diphtheria Bacilli and Derived Variants

The antigenic relationships between diphtheria bacilli and certain derived variants have been investigated by means of the complement fixation reaction. Two strains of virulent diphtheria bacilli, No. 19 and Park No. 18 and avirulent variant strains derived from these strains were used in the experiments, and in addition, a strain of C. hofmanni. Two of the variant strains, Park No. 8 phage a and Park No. 8 phage b, were derived from young cultures of Park No. 8 to which specific bacteriophage had been added. Four of the variant strains were derived in various ways from the No. 19 culture and are designated as follows: 19 ‘antiserum’, derived from culture in broth containing homologous antiserum, 19 ‘broth 45°C.’ from broth culture incubated at 45°C; 19 ‘LiCl’, from culture in broth containing 0.5% lithium chloride; 19 ‘phenol’, from culture in broth containing 0.1% phenol. These variant organisms were found to be stable in type, grew very slowly in broth, and were completely lacking in fermentative ability. The variant colony was much smaller than the typical colony of diphtheria colony. Microscopically, the variant organisms were hardly distinguishable from the parent organisms. Usually they stained more solidly, but many barred and granular forms were observed. Sera taken from some of the rabbits before immunization failed to fix complement except when large amounts of serum were used. The course of immunization consists of 8 to 10 injections of heat-killed organisms. The sera obtained were inactivated by heating them at 60-62 °C. for 30 minutes. The sera were used in 4 amounts: 0.025, 0.012, 0.006, and 0.003 cc. All antigens were used in such an amount that 4 times that amount was not anticomplementary. The smallest amount of serum giving a strong reaction with the homologous antigen was used as a base and the reactions in this amount with other antigens is reported in the tabulation.