410 MYCOBACTERIUM 



reduction system whose E'o is among the lowest reported for systems of biological 

 origin (Ball 1934). The polysaccharides in the ether extract were apparently 

 different from those in the phosphatide or wax. 



A nitrogen-free and phosphorus-free polysaccharide was prepared by Hooper, 

 Renfrew and Johnson (1934) by alkaline hydrolysis of the acetyl product of the 

 crude carbohydrate from a protein-free ultrafiltrate. The molecular weight of 

 the pure polysaccharide has been estimated by Seibert, Pedersen and Tiselius 

 (1938) at 9,000. 



Besides the lipoid material and the polysaccharides, acid-fast bacilli contain 

 proteins that are soluble in water. From cultures of tubercle bacilli on synthetic 

 media Long and Seibert and their colleagues (1926, 1928) isolated various proteins, 

 of which one appears to be the active principle of tuberculin. In this country 

 Gough (1933) obtained evidence of the presence in cultures of human tubercle 

 bacilli of two proteins having different chemical and immunological characters. 

 The later work of Menzel and Heidelberger (1938), who fractionated the residue 

 of frozen and dried bacilli after extraction in the cold with acetone and ether, 

 revealed the great complexity of the proteins in the acid-fast bacilli. Differences 

 were noted between the proteins of saprophytic acid-fast bacilli, avian bacilli, 

 and mammalian bacilli. At least three antigenic components were found among 

 the proteins separated from bacilli of the human type. The protein fraction, 

 PPD-b3, which appears to be responsible for the tuberculin reaction, was found 

 by Seibert, Pedersen and Tiselius (1938) to have a molecular weight of 16,000 

 and to contain 4-4 per cent, of polysaccharide and 3 per cent, of nucleic acid. 

 Further purification by Seibert and Glenn (1941) resulted in the reduction of the 

 nucleic acid to 1-2 per cent, and of the molecular weight to 10,500. 



The cellular reactions to the various fractions extracted from tubercle bacilli 

 have been studied for many years by Sabin and her colleagues (for references, 

 see Sabin 1938 and Sabin and Joyner 1938). They may be summarized briefly 

 as follows. 



Four types of reaction have been noted : (a) exudation of neutrophiles from the blood 

 vessels to the tissues ; (6) stimulation of the phagocytic mononuclear cells of the tissues ; 

 (c) multiplication of fibroblasts ; and {d) local increase in lymphocytes. The polysac- 

 charide, when introduced into the tissues of suitable animals, called forth neutrophiles 

 from the blood vessels, but had no further action. The lipins, in addition to evoking 

 this reaction, stimulated the phagocytic mononuclear cells of the tissues. After injection 

 of the phosphatide fraction tubercles developed consisting of epitheUoid cells and of their 

 multinucleated derivative, the Langhans giant ceU ; caseation sometimes followed. The 

 waxes, higher alcohols, and hydroxy-acids led to a multipUcation of monocytes and their 

 fusion into giant ceUs of the foreign body type. The reaction to the proteins was more 

 complex. They gave rise not to one type of the phagocytic mononuclear cell, but to 

 every type. They induced the formation of monocytes, of " stimulated " monocytes, of 

 macrophages, of epithelioid ceUs singly and in tubercles, and of giant cells, both of the 

 Langhans and the foreign body type. The degree of complexity of the reaction was 

 least with the soluble proteins and greatest with the insoluble. A consistent increase 

 in fibroblasts was found only after the injection of a wax-like fraction isolated from a 

 saprophytic acid-fast organism. The new formation of lymphocytes was observed irregu- 

 larly after injection of the phosphatide and protein fractions. Reactions in general were 

 greater in tuberculous than in normal animals. 



The polysaccharide is non-toxic on intravenous injection into rabbits (Sabin et nl. 

 1931), but like the protein it may play a part in the phenomenon of allergy. It reacts 



