Oswald T. Avery

Attempts to Produce Vitamin Deficiency Diseases by Feeding Compounds Related Structurally to Vitamins

Summary In contrast to the findings with bacteria, pantothenic acid deficiency of mice and of hamsters was not produced by the feeding of the sulfur analog of pantothenic acid (N-(β,β-dimethyl-α,γ-dihydroxybutyryl)-taurine). Similarly, the feeding of pyridine-3-sulfonic acid did not produce symptoms of nicotinic acid deficiency.

The Rôle of Specific Carbohydrates in Pneumococcus Infection and Immunity

Excerpt Mr. President; Fellows of the American College of Physicians: I count it a great privilege to be able on this occasion to express personally my deep sense of appreciation of the distinguish...

Soluble substance of pneumococcus origin in the blood and urine during lobar pneumonia

A specifically reacting substance of pneumococcus origin has been demonstrated in the nitrates of young cultures of pneumococcus, and also in the blood serum and urine of patients during the course of lobar pneumonia. In bacteria-free filtrates of broth cultures of pneumococcus there is present a soluble material which gives a specific precipitin reaction with antipneumococcus serum. The filtrates from the different types of pneumococcus show the same specificity of reaction with immune serum as do the original cultures from which they are derived. The soluble substance present in the filtrates is undoubtedly of bacterial origin. That it is a product of the life processes of the pneumococcus and not due to its disintegration is shown by the fact that it is present in considerable amounts during the early stage of development of the culture when the organisms are growing at their maximum rate and undergoing little or no cell destruction as indicated by their growth curve. The demonstration of the formation of this soluble substance in cultures of pneumococcus growing actively in vitro suggested the probability of its production in experimentally infected animals and in human beings suffering from lobar pneumonia. If rabbits are infected intraperitoneally with pneumococcus a substance specifically precipitable with antipneumococcus serum can be demonstrated in their blood serum, freed from bacteria by filtration, from within two to six hours following the time of infection. This soluble specific substance is also present in the blood serum during the course of lobar pneumonia in man and gives a precipitin reaction with antipneumococcus serum corresponding in type to the organism with which the individual is infected. This soluble precipitable substance is less frequently present in demonstrable quantities in human serum than in the serum of experimentally infected animals.

Reversion of Avirulent “Rough” Forms of Pneumococcus to Virulent “Smooth” Types.:

The fact that virulent, type-specific, capsulated, “S” forms of pneumococcus can be degraded to avirulent, non-type-specific, unencapsulated, “R” forms is well established. (Friel, 1 Stryker, 2 Griffith, 3 Amoss, 4 and Reimann. 5 ) The reverse process, however, the conversion of avirulent, “R” forms into virulent, “S” forms, has been a matter of some controversy. Felton and Dougherty, 6 by means of an automatic transferring device in a milk-containing medium, were able to restore virulence to a strain of pneumococcus which had become completely avirulent. They employed pure line strains derived from single cells, and report that some cultures showed a maximal increase in virulence while others remained avirulent. Their investigation preceded the recognition of “R” and “S” forms of pneumococcus. Griffith 3 states that “R” strains may revert in all respects to the “S” type or remain unchanged after many generations in subculture or plain blood broth. Single cell cultures, however, were not employed. Reimann 5 reports that he was unable to enhance the virulence of an “R” strain derived from Type I even after 105 mouse passages. Levinthal 7 was able to effect the transformation by growing “R” forms in serum broth at 25° C. and subsequently passing the cultures through mice. For bacteria other than pneumococcus the same differences of opinion appear to exist. Recently, however, Jordan 11 has demonstrated the interconvertibility of “R” and “S” forms of B. paratyphosus B, and Soule 12 has shown the reversibility of the dissociated form of B. subtilis.

Intracutaneous Vaccination of Rabbits with Pneumococcus. III. Hypersensitiveness.

In the preceding communications, the intracutaneous vaccination of rabbits with Pneumococcus (S forms) has been shown to give rise chiefly to the formation of the antiprotein rather than the type specific antibodies, and to the development of an increased resistance to infection with organisms of homologous and heterologous types. The present paper describes briefly the development of an altered tissue reaction to Pneumococcus and its protein derivatives in rabbits which have been inoculated repeatedly into the skin with heat killed suspensions of R and S pneumococci. Mackenzie and Woo 1 have shown that guinea pigs, injected intracutaneously with an alkaline extract of Pneumococcus, develop an allergic reaction in the skin to the bacterial protein; Zinsser and Grinnell 2 have produced allergic sensitization to pneumococcus autolysates in guinea pigs previously injected intradermally or intraperitoneally with the same material. Bull and McKee 3 have recently shown that rabbits, after recovery from infection induced by intranasal inoculation of pneumococci, are highly skin-sensitive to pneumococcus autolysate. The present observations were made in the course of a study of the antibody response and the immunity developed as a result of intracutaneous injection of rabbits. The intracutaneous injection in normal rabbits of 0.2 cc. of a heated vaccine, representing the bacteria from 2 cc. of broth culture, is followed by the appearance locally of a circumscribed slightly raised and indurated nodule, reddish in color, and measuring about 1 cm. in diameter. Upon repeated injection at weekly intervals the reaction changes in character; the size increases, often reaching a maximum of 4 to 6 cm. in diameter, accompanied by a spreading edema and purplish discoloration. The maximum reaction is generally reached after 6 to 8 injections and thereafter each successive lesion tends to, become less intense but to persist longer, often breaking down with the discharge of sterile necrotic material.

The soluble specific substance of a strain of Friedlander bacillus

By a method similar to those previously used in the case of the pneumococcus 1 there has been isolated from a strain of Friedländer bacillus designated as the “E” strain a polysaccharide with specific properties. It differs from the Friedländer carbohydrate obtained by Toenniessen 2 in giving no color with iodine, and from the material reported by Mueller, Smith, and Litarezek 3 in being nitrogen-free. [α]d, is + 100°, the substance, if it be a single substance, is a moderately strong acid, and on hydrolysis it yields 72 per cent of reducing sugars, among which glucose was identified as the osazone and by conversion into potassium acid saccharate. In its present state of purity this specific substance resembles qualitatively that of the Type II pneumococcus; and although there are distinct differences, it actually precipitates Type II anti-pneumococcus serum at as high a dilution as 1:2,000,000, in addition to reacting similarly with its homologous serum.

Intracutaneous Vaccination of Rabbits with Pneumococcus. I. Antibody Response.

Suspensions of heat killed pneumococci were injected into the skin of rabbits at intervals of 7 days during a period of 10 to 14 weeks. The total amount of bacterial substance injected was equivalent to and often greater than that ordinarily employed in the routine immunization of rabbits by the intravenous method. Pneumococci of Types I and III and a degraded “R” strain derived from Type II were used. The sera of the treated rabbits were tested for the presence of agglutinins, precipitins, and protective antibodies. The serum obtained from 85% of the animals immunized intracutaneously with Type I pneumococcus failed to show the presence of any demonstrable type specific antibodies. Virulent cultures of Type I were not agglutinated, nor were solutions of the specific soluble substance from organisms of the homologous type precipitated by these sera even when used in high concentrations. Only rarely did the serum confer any passive protection upon mice infected with a virulent strain of Type I, and in these instances the protective titre was low. In only 15% of animals studied was there any serological evidence of type specific response to repeated intracutaneous inoculation of Type I organisms; in these instances the presence in the serum of specific agglutinins was demonstrable only in low dilutions varying from 1:1 to 1:20. In terms of its capacity to stimulate the formation of type specific antibodies, Type III pneumococcus is at best a poor antigen. It was to be expected, therefore, that organisms of this type, when injected intracutaneously into rabbits, would fail to elicit the type specific response. Such proved to be the case. None of the rabbits treated by the skin method developed demonstrable serum antibodies against Type III.

Intracutaneous Vaccination of Rabbits with Pneumococcus. II. Resistance to Infection.

In the preceding communication it was shown that the antibody response elicited in rabbits vaccinated intracutaneously with specific type strains of Pneumococcus is characterized by the presence in the serum of antiprotein antibodies in excess, together with a corresponding decrease or complete absence of type specific antibodies. That this change in the order of specific antibodies is related to the mode of inoculation was shown by the fact that the same strain (Type I Pneumococcus), when injected intravenously, invariably stimulates the formation of the dominant type-specific antibodies. The present paper summarizes the results of a study of the occurrence and nature of the active resistance to infection which develops in rabbits vaccinated intracutaneously with R and S forms of Pneumococcus. Briefly, it may be stated that, following repeated skin inoculation of heat-killed suspensions of type-specific pneumococci or of the R variants, rabbits acquire a considerable degree of active immunity against infection with virulent strains of homologous and heterologous types. For example, rabbits, immunized intracutaneously with Type I may survive the intravenous injection of a dose as large as 0.2 cc. of homologous culture, the virulence of which is such that 0.0000001 cc. kills normal control animals. Similarly, rabbits immunized in the same manner to an avirulent strain of Type III may survive an infecting dose of from 0.5 to 1 cc. of a culture of the homologous type which by animal passage has been rendered so virulent that 0.001 cc. to 0.0001 cc. is sufficient to kill normal rabbits. Furthermore, animals inoculated intracutaneously with Type III become as resistant to subsequent infection with Type I as animals similarly immunized with homologous organisms. Even when an R strain of Pneumococcus derived from Type II is used for injection, rabbits acquire a high degree of resistance against infection with virulent Type I organisms.

Immunological behavior of the "E" strain of Friedlander bacillus and its soluble specific substance

In the preceding communication the chemical similarity of the specific soluble substance of this strain to that of the Type II pneumococcus was pointed out. These two micro-organisms, widely different in biological characters, were compared immunologically and found to correspond closely. The “E” strain and other serologically related strains of Friedländer bacillus are agglutinated by Type II anti-pneumococcus serum, and not by Type I and Type III antisera; while the Type II pneumococcus is agglutinated by “E” Friedländer antiserum, but not by the antiserum of a strain of Friedländer bacillus serologically distinct from the “E” strain. Absorption of “E” Friedländer antiserum and Type II anti-pneumococcus serum with the homologous organism removes the agglutinins for both, while absorption with the heterologous organism removes only the agglutinins for it, leaving the homologous agglutinins scarcely diminished in titer. The specific soluble substances of the two organisms under comparison react at practically as high dilutions in the opposite antisera as in the antisera to the organisms from which they were derived. Precipitin absorption parallels the agglutinin absorption. Crossprotection was equally striking, Type I1 anti-pneumococcus serum protecting against many thousand lethal doses of highly virulent “E” Friedlander bacillus, and the “E” antiserum protecting against Type I1 pneumococcus at least as well as the Type II antiserum itself. Typical protection protocols follow.

Influence of an artificial peroxidase upon the growth of anaerobic bacilli.

Previous study of the influence of plant tissue upon bacterial growth showed that sterile unheated plant tissue can replace blood in the cultivation of the so called hemophilic organism B. influenzæ. It was also found that plant tissue exerts a marked accelerating action upon the growth of pneumococcus. More recently it has been shown that the addition of unheated vegetable (potato) to plain bouillon makes possible the continued, ærobic growth of certain anærobic bacilli such as B. histolyticus, B. ærofetidis, B. oedematiens, and B. chauvei. This growth promoting action upon three distinct groups of bacteria so widely different in their cultural requirements, indicates that plant tissue meets certain physiological needs of the bacterial cell not wholly provided for by the ordinary culture media. These studies suggest that this action may be dependent not only on the presence of some growth accessory substances in plant tissue but also upon the functioning of certain systems which are concerned in the cellular processes of oxidation and reduction. McLeod and his associates have found that certain ærobic bacteria which are devoid of catalase form hydrogen peroxide when grown in the presence of air. In the case of pneumococcus, which possesses no catalase, hydrogen peroxide is known to accumulate in the fluid of ærobic cultures in concentrations which are bacteriostatic and even bacteriocidal. As far as is known anzrobic bacilli are also devoid of catalase, and hence these cells cannot destroy peroxides. From these relations it seems not unlikely that anærobic bacteria fail to grow in the presence of air, not because atmospheric oxygen as such is a direct poison to the cell, but because of the toxic action of peroxides which may be formed as the result of the union of molecular oxygen with some autoxidizable substance in the bacterial cell.