STUDIES ON THE ANTIBACTERIAL ACTION OF THE SULFONAMIDE DRUGS : I. THE RELATION OFp-AMINOBENZOIC ACID TO THE MECHANISM OF BACTERIOSTASIS

The following observations have been made which substantiate the theory that the sulfonamide drugs used in the treatment of bacterial infections exert their bacteriostatic effect by competing with the essential metabolite, p-amino-benzoic acid, for an important enzyme site on the bacterial cell. 1. p-Aminobenzoic acid was shown to nullify the bacteriostatic effect of all of the six sulfonamide compounds studied even though the drugs exhibited marked differences in chemical structure. 2. The bacteriostatic potency of each sulfonamide drug was found to be directly proportional to its ability to counteract the antibacteriostatic action of p-aminobenzoic acid. 3. In the case of each drug tested over a wide range of concentrations the minimum amount of p-aminobenzoic acid needed to prevent bacteriostasis was such that the ratio of p-aminobenzoic acid to drug was constant. 4. The linear relationship between p-aminobenzoic acid and drug was interpreted as indicating the competitive inhibition of an essential enzyme reaction by a substance chemically related to the substrate. This interpretation was supported by the fact that the equation derived on purely theoretical grounds relating drug and acid expressed the same linear relationship as that observed experimentally

STUDIES ON THE MECHANISM OF RECOVERY IN PNEUMOCOCCAL PNEUMONIA : I. THE ACTION OF TYPE SPECIFIC ANTIBODY UPON THE PULMONARY LESION OF EXPERIMENTAL PNEUMONIA

A uniformly fatal lobar pneumonia was produced in white rats by inoculation of the left main bronchus with virulent Type I pneumococci suspended in mucin. All of the animals succumbed in less than 5 days, half of them dying within 48 hours. In only 5 of 40 rats was the lesion confined to the left lung, and all but one developed pleurisy, pericarditis, or both. All had bacteriemia at the time of death. The pathogenesis of the pulmonary lesion was studied by examining the lungs of 35 rats killed at various intervals following inoculation. The pneumonic process spread rapidly until most of the left lung was involved in 36 hours. Frequent blood cultures showed invasion of the blood stream in a few rats at 6 hours and in over 90 per cent at the end of the first day. The first signs of pleurisy usually appeared at 18 hours. Microscopic examination of the actively spreading lesion revealed three characteristic zones: (1) an outer "edema zone" in which the alveoli contained many pneumococci floating freely in edema fluid, (2) a middle zone where both leucocytes and organisms were present, many of the latter being phagocytized, and (3) an inner zone of advanced consolidation in which the alveoli contained many leucocytes but no organisms and where there were already local areas of early resolution. Study of numerous lesions, at intervals of from 12 to 36 hours after inoculation, indicated that the pneumococci spread into normal alveoli principally by way of the infected edema fluid in the outer zone. Pneumococcus-laden edema fluid in large bronchi and in alveoli beneath the pleura suggested the mode of spread of the infection to other lobes and possibly to the pleural cavity. No adequate explanation could be found for the presence of active phagocytosis in the lungs of animals with bacteriemia and presumably without circulating antibodies, but this conspicuous phagocytic reaction was obviously responsible for the clearing of the central part of the spreading lesion. The action of type specific antibody upon the pulmonary lesion of experimental lobar pneumonia was studied in rats similarly infected but treated with antipneumococcal serum. When injected intravenously in a single dose within 18 hours after inoculation the antiserum was found to protect all of the rats against the otherwise fatal pneumonia. It stopped the spread of the pneumonic lesion, cleared the blood stream of organisms, and prevented the extension of early pleurisy. The antibody caused agglutination and capsular swelling of the pneumococci in the lung, particularly in the edema zone at the margin of the lesion where they were most numerous. Apparently immobilized by agglutination the organisms were overtaken by leucocytes and destroyed by phagocytosis. The phagocytic reaction was greatly accelerated by the specific opsonins of the antiserum, and the pneumococci were destroyed by polymorphonuclear leucocytes before many macrophages appeared in the alveolar exudate. Within a week after treatment resolution of the pulmonary lesion was well in progress. Both horse and rabbit antibody were shown to penetrate the lung, and immune bodies were demonstrated in the alveoli within 10 minutes after the start of treatment. The relation of the observed phenomena to the curative action of anti-pneumococcal serum has been briefly discussed, and it is pointed out that the principal effect of antiserum is to cause immobilization of the pneumococci in the advancing edema zone. Experiments to be reported in a later publication have shown that sulfapyridine exerts a similar effect through a different mechanism

STUDIES ON THE PATHOGENESIS OF FEVER : I. THE PRESENCE OF TRANSFERABLE PYROGEN IN THE BLOOD STREAM FOLLOWING THE INJECTION OF TYPHOID VACCINE

The rate of clearance of intravenously injected typhoid vaccine was studied in unsensitized, sensitized, and pyrogen-tolerant rabbits by means of a passive transfer technique. The blood of unsensitized rabbits which had not been previously exposed to bacterial pyrogen remained pyrogenic for normal recipients throughout a period of 2 hours following the injection. In contrast, rabbits sensitized by having received either one or two injections of the vaccine at least 3 weeks prior to the experiment cleared their blood of the test vaccine within 30 minutes despite the fact that they exhibit the same febrile response as unsensitized rabbits. After 1 hour, however, a transferable pyrogenic substance was again demonstrable in the sera of this group. Reasons are discussed for believing that this newly appearing substance may be of endogenous origin and may be the factor which directly affects the thermoregulatory centers of the brain. Rabbits which are made tolerant by repeated daily injections of vaccine have a characteristically depressed febrile response. Not only were the blood streams of such animals cleared of the injected vaccine within less than 5 minutes, but samples of their sera obtained 1 and 2 hours after the injection also failed to contain demonstrable quantities of the secondary pyrogen observed in sensitized animals. The latter observation is in keeping with the suggestion that the secondary pyrogen may play a critical role in the production of fever

STUDIES ON THE PATHOGENESIS OF FEVER : VI. THE INTERACTION OF LEUCOCYTES AND ENDOTOXIN IN VITRO

Study in vitro of the interaction of bacterial endotoxin with rabbit polymorphonuclear leucocytes has resulted in the following findings: 1. Incubation of endotoxin and leucocytes in saline results in: (a) the release of leucocytic pyrogen, and (b) the inactivation of endotoxin. 2. Cell-free extracts of leucocytes also inactivate endotoxin. 3. Incubation of leucocytes in "physiological" saline causes rapid discharge of leucocytic pyrogen. In contrast, relatively little pyrogen is released by leucocytes incubated in fresh serum. 4. The release of leucocytic pyrogen in serum is markedly stimulated by the presence of endotoxin. 5. Leucocytes obtained from tolerant rabbits interact with endotoxin in essentially the same manner as leucocytes from normal rabbits. The pertinence of these findings to the pathogenesis of fever and to related information concerning human leucocytes has been discussed

CELLULAR MECHANISMS OF ANTIBACTERIAL DEFENSE IN LYMPH NODES : I. PATHOGENESIS OF ACUTE BACTERIAL LYMPHADENITIS

Acute pneumococcic lymphadenitis produced in rats by intradermal inoculation of the foot-pad is characterized by rapid infiltration of polymorphonuclear leucocytes into the intermediary sinuses of the node, and prompt phagocytosis of pneumococci by both the macrophages of the sinuses and the recently arrived leucocytes. After 5 to 7 hours the polymorphonuclear leucocytes are found densely congregated about the hilar region, and 9 hours after inoculation most of the phagocyted organisms have been digested. At the end of the 24 hour period the popliteal node presents the picture of a subsiding inflammation with a marked macrophage reaction and regenerating lymph follicles. Phagocytosis of encapsulated pneumococci in the foot-pad and popliteal node occurs in less than 30 minutes after inoculation. It is assumed that this prompt phagocytosis is effected by the non-antibody mechanism of "surface phagocytosis." The majority of polymorphonuclear leucocytes that enter the sinuses of the inflamed node appear to come from capillaries within the node itself rather than from the primary site of inflammation in the foot-pad. The prompt inflammatory response of the nodal tissues serves as an active defense against lymph-borne infection. Macrophages invade nodal sinuses only after most of the pneumococci have been destroyed by polymorphonuclear leucocytes. It is suggested that the macrophage reaction follows removal of the primary inflammatory stimulus by the granulocytes, and thus constitutes only a late phase of recovery. Fibrin formation in the sinuses of the lymph node is rare during acute lymphadenitis. This finding may be related to the observation that within 5 minutes after entrance of bacteria into the node, heparin-containing granules from mast cells are strewn throughout the sinuses

STUDIES ON THE PATHOGENESIS OF FEVER : II. IDENTIFICATION OF AN ENDOGENOUS PYROGEN IN THE BLOOD STREAM FOLLOWING THE INJECTION OF TYPHOID VACCINE

Further studies have been made of a pyrogenic substance which appears in the circulation of rabbits during the course of experimental fever induced by injection of typhoid vaccine. With the use of a passive transfer method and pyrogen-tolerant recipients, the biological properties of this substance have been differentiated from those of the uncleared vaccine in the circulation. The newly identified factor resembles leucocytic pyrogen in the rapidity with which it produces fever and in its failure to exhibit cross-tolerance with bacterial pyrogen. This striking similarity of properties suggests that the circulating factor is of endogenous origin and may arise from cell injury. A close correlation between its presence in the circulation and the existence of fever has been demonstrated. The possible relationship of these findings to the pathogenesis of fever is evident

STUDIES ON THE ANTIBACTERIAL ACTION OF THE SULFONAMIDE DRUGS : II. THE POSSIBLE RELATION OF DRUG ACTIVITY TO SUBSTANCES OTHER THANp-AMINOBENZOIC ACID

1. In cultures of Staphylococus aureus in a synthetic medium nicotinamide and cozymase were shown to block the bacteriostatic action of chemically unrelated sulfonamide drugs as well as the chemically related compound sulfapyridine. The antibacterial properties of organic dyes totally unrelated to the sulfonamide compounds (methylene blue and thionine) were also nullified by the addition of cozymase to the culture medium. 2. The antagonistic action of the pyridine-containing coenzyme, cozymase, was found, by quantitative study, to be no greater against sulfapyridine than against other structurally dissimilar sulfonamide compounds. 3. The antidrug effects of nicotinamide and cozymase in staphylococcus cultures were observed to be directly proportional to their ability to stimulate the growth of the organism in the synthetic medium. When tested in cultures of B. coli in which they failed to accelerate bacterial growth, these same substances failed to influence the bacteriostatic action of the sulfonamide drugs. 4. The in vitro action of the coenzyme, cocarboxylase, as measured in the Warburg respirometer, was shown to be unaffected by the chemically related drug, sulfathiazole, even when the latter was present in great excess. The above observations fail to support the theory that sulfapyridine, sulfathiazole, and sulfadiazine prevent bacterial growth by interfering with the functioning of the chemically related coenzymes, cozymase, and cocarboxylase. The mode of action of sulfanilamide and its more common derivatives is discussed in the light of these observations, and a tentative theory is offered to explain the differences in bacteriostatic potency exhibited by the various sulfonamide compounds

STUDIES ON THE MECHANISM OF RECOVERY IN PNEUMONIA DUE TO FRIEDLÄNDER'S BACILLUS : I. THE PATHOGENESIS OF EXPERIMENTAL FRIEDLÄNDER'S BACILLUS PNEUMONIA

Experimental pneumonia due to Friedländer's bacillus was produced in white rats by the intrabronchial inoculation of the bacilli suspended in mucin. The pneumonia was lobar in type, was almost uniformly fatal, and simulated the acute form of the natural disease in human beings. The pathogenesis of the pneumonic lesion was studied by examination of microscopic sections of the lungs of animals killed at frequent intervals during the course of the infection. The histologic characteristics of the various stages of the pneumonia were essentially the same as those previously described in experimental pneumococcal (Type I) pneumonia except for the following differences: (1) In isolated areas of the lung in Friedländer's pneumonia many more bacteria were encountered in the alveoli than were ever noted in experimental pneumococcal pneumonia. (2) Abscess formation was common in the late stages of Friedländer's infection, whereas it was not noted in the pneumococcal lesion. (3) Organization of the alveolar exudate, rarely observed in experimental pneumococcal pneumonia, was a prominent feature of the pneumonia due to Friedländer's bacillus. The mechanism of spread of Friedländer's lesion appeared to be the same as that of pneumococcal pneumonia. Likewise there was noted the same phagocytosis of organisms in the lungs of even bacteremic animals dying of the infection

STUDIES ON THE PATHOGENESIS OF FEVER : IX. THE PRODUCTION OF ENDOGENOUS PYROGEN BY POLYMORPHONUCLEAR LEUCOCYTES

Determination of the dose-response curve for rabbit leucocytic pyrogen reveals a hyperthermic "ceiling" at which there is a marked insensitivity to dosage. This finding has important implications in relation to the quantitative assay of leucocytic pyrogen. Polymorphonuclear leucocytes separated from normal rabbit blood possess the capacity to produce less than 5 per cent of the pyrogen generated by the same number of rabbit granulocytes collected from acute peritoneal exudates. Blood granulocytes, separated in the cold from the buffy coat, contain no detectable preformed pyrogen. The amount of preformed pyrogen within exudate granulocytes represents but a small fraction of the pyrogen which the cells are capable of generating when incubated in normal saline at 37°C. It is suggested that the active pyrogen is formed from an inactive precursor within the cells. Under the conditions tested, cell fragments of rabbit granulocytes fail to produce endogenous pyrogen. The fact that the production of pyrogen is blocked at 4°C is in keeping with the hypothesis that it involves metabolic reactions within the cell