Sam C. Wong

Further Studies on Type-Specific Protein of Corynebacterium diphtheriae.

Conclusion From the observations made on the chemical and immunological studies of the polysaccharide, lipoid, and protein fractions obtained from representative serological and cultural types it is justifiable to conclude that the alkalisoluble protein is the cellular constituent responsible for type-specificity in C. diphtheria.

Immunological Studies on the Cellular Constituents of C. diphtheriae.

Conclusions A method suitable for the isolation of a type-specific protein in C. diphtheria is described. Both the lipoid and carbohydrate fractions are group-specific while the alkali-soluble protein is type-specific. The type-specific substance is heat-labile, being converted into a group-specific protein by heating at 56°C for 30 minutes.

Sensitizing Capacity of Polysaccharide of Monilia tropicalis.

Conclusion Polysaccharide derived from Monilia tropicalis by acetic-acid hydrolysis is capable of inducing active sensitization of guinea pigs and of eliciting the production of antibodies in rabbits. Its capacity in these respects, however, is inferior to that of the intact cell.

Immunological Studies on Proteins of Corynebacterium diphtheria

Conclusion Diphtheric protein is a complex antigen consisting of at least a type-specific antigen and a group-antigen.

Immunological Studies of Polysaccharides of Encapsulated Bacillus. I. Isolation from Smooth B. rhinoscleromatis.

It has been conclusively proved that the specific polysaccharide of pneumococcus possesses antigenic power. It is generally thought that similar substances obtained from other bacteria were devoid of any antigenic property. In the present study it will be shown that the substance prepared from Bacillus rhinoscleromatis is antigenic. The organism was isolated at the Peiping Union Medical College Hospital from the infected submaxillary lymph gland of a clinically established case of rhinoscleroma infection. The cultural characteristics and biochemical reactions of this organism are similar to those described by Morris and Julianelle 1 for 10 strains of Klebsiella rhinoscleromatis. The organisms were grown on meat infusion agar, pH 7.6, for 48 hours at 37°C. after which they were washed with distilled water and precipitated by the addition of 95% alcohol. After standing in the ice chest over night the organisms were dried over a boiling water bath and then resuspended in distilled water in the proportion of one part of dried bacilli to 150 parts of water. The flask containing the organisms was thoroughly shaken and placed in the ice chest overnight. At the end of that time glacial acetic acid was added to the gelatinous suspension to effect a concentration of one percent. The flask was placed in a boiling water bath for 30 minutes. It is interesting to note that there was a clearing of the suspension after 15 minutes of heating as all the cells had settled to the bottom of the flask. At this point the flask was agitated and hydrolysis continued. After being allowed to cool, the clear yellowish solution obtained from centrifugation was precipitated with 4 volumes of 95% alcohol to which was added one percent sodium acetate.

Studies on Stability of Dilute Purified Tuberculins.

Summary A phosphate buffer is described which has been found to maintain the skin-reactivity of dilute purified tuberculin for at least 4 months at 20 C and 6 months at 8°C.

Type-Specific Polysaccharides of C. diphtheriae.

Polysaccharides derived from gravis, intermediate, and mitis types of C. diphtheriœ were found to be group-specific. 1 Recent findings, 2 however, indicate that any of these different cultural types may be present in a single serological type. Because of this an attempt was made to extend our previous study to include polysaccharides of different serological types of C. diphtheriœ with the object of determining the existence of type-specific polysaccharides. One organism from each of 5 distinct serological types kindly supplied to us by Sia and Huang 2 was employed for the present study. All organisms, with the exception of Park 8 (Type D41) were isolated locally. The various characterizations of these organisms were as follows: All of the cultures were capable of eliciting precipitin antibodies in rabbits from 4 to 5 weeks when administered intravenously as heat-killed vaccines. The cultivation of organisms, the preparation of polysaccharides, and the immunization of rabbits were the same as those described previously. 1 Absorption test was performed by mixing immune serum diluted 1:5 with well-washed living organisms grown on serum broth at 37°C for 48 hours and the mixture was incubated at 45°C waterbath for 2 hours. It was refrigerated overnight after which the serum was separated by rapid centrifugation. It was found that 2 kinds of polysaccharide could be demonstrated by the precipitin reaction and by absorption tests. These polysaccharides will be designated as A and B. Polysaccharide A was present in the types D25, D30, D41, and X. Identical precipitin titers varying from 1:50,000 to 1:100,000 were obtained when polysaccharides A were mixed in the ring test with the homologous as well as the heterologous immune sera prepared with the above types. Cross absorption tests performed by mixing immune serum with organisms belonging to any of the types D25, D30, D41, and X showed a removal of all precipitins without affecting the type-specific agglutinins.

Serological Studies on Polysaccharides Derived from Diphtheroids

Previous findings 1 suggest that polysaccharides prepared from various cultural types of Corynebacterium diphtheriœ are group-specific. It seemed of interest to determine whether polysaccharides of similar nature could be prepared from common diphtheroids. Stock cultures of C. hofmanni, obtained from the London Type-Culture Collection, and C. xerosis, isolated from the conjunctiva of a patient with dacryocytitis in the Peiping Union Medical College Hospital, were employed for this study. Both organisms are nonpathogenic for either guinea pigs or Chinese hamsters and their staining and biochemical reactions are typical for these organisms with the exception that C. xerosis failed to ferment saccharose. The growing of the organisms and the method of preparing the polysaccharides were the same as those reported previously 1 except that only alkali was employed for hydrolysis. The yield of polysaccharide for both organisms was small, the amount being equal to 1 to 2% of the dried weight of organisms. Incidentally it might be stated that this figure is fairly representative of the yield of all polysaccharides prepared from the corynebacterial organisms thus far studied. The polysaccharides obtained from both organisms were white amorphous powders, readily soluble in saline in a concentration of 0.5%, gave strong Molisch reaction, exhibited none of the usual reactions for protein, and did not reduce Fehlings solution. In serological tests the polysaccharide of C. xerosis was found to react equally with all sera prepared against the various cultural types of C. diphtheriœ; these included strains of intermediate, of gravis and of mitis. Precipitin titers (1:100,000) similar to those obtained with homologous polysaccharides were found. On the other hand, the polysaccharide of C. hofmanni, even in concentration of 1:1000, reacted weakly or not at all with the same sera.

Significance of Acetyl Group in Determining Antigenic Activity of Bacterial Polysaccharides

Conclusion Evidence is presented which suggests that the acetyl group is probably not responsible for antigenic activities of the polysaccharides prepared by acetic-acid hydrolysis of E. coli and the capsulated and the non-capsulated forms of K. rhinoscleromatis.

IV. Negative Effect of Colloidal Carriers on Enhancement of Antigenic and Sensitizing Properties of Polysaccharides

The rôle played by inert colloidal substances on the enhancement of antigenic properties of bacterial polysaccharides is still a debatable one. Although positive evidence of such an increase in immunological activity has been reported by Zozaya 1 and by Hoffstadt and Clark, 2 the general opinion 3 , 4 , 5 is that such an effect occurs with polysaccharides that are weakly antigenic themselves. In view of these discrepancies it appears to be of interest to determine whether the sensitizing and antigenic properties of the polysaccharides of Bacillus rhinoscleromatis can be improved by colloidal carriers. The polysaccharides of this organism were thought to be especially suitable for this study because of 2 serologically very similar preparations, one is weakly antigenic in rabbits and weakly sensitizes guinea pigs, while the antigenic properties of the other are insignificant 6 The polysaccharides used for this experiment include a fraction prepared by hydrolysis with 1% acetic acid and a fraction prepared by hydrolysis with 0.5% potassium hydroxide of the organism remaining after acid-hydrolysis. The details of the method of preparation and the chemical and immunological characteristics of the 2 fractions have been previously reported. 6 The colloidal substances included collodion particles, aluminium hydroxide, charcoal, and hog serum. The collodion particles were prepared according to the methods of Zozaya 1 and the aluminium hydroxide according to the method of Hektoen and Welker. 7 Adsorption was carried out by mixing varying concentrations of polysaccharide with the colloidal substance, incubating at 56°C for 2 hours and leaving in the icebox overnight. In the case of hog serum equal volumes of a 1:250 concentration of polysaccharide and serum were mixed and treated in the same manner. Twenty-eight normal rabbits were divided equally into 2 groups: Group I receiving the acid-prepared polysaccharide mixed with colloidal substances and Group II receiving the alkali-prepared polysaccharide.