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Twenty-five pairs of male rabbits of the same variety and strain were joined together in aural parabiosis for 13 to 15 days and then separated from one another. Thirteen pairs of the separated parabiotic twins were surgically united for a second period of parabiosis, less successful than the first due to deficient healing and spontaneous separation of tissue in 5 to 15 days at the site of anastomosis. Within 6 to 90 days after spontaneous or surgical separation of all parabionts, the postparabiotic twins which had been united to one another were cross-grafted with musculofascial transplants. 2 weeks later, the animals were sacrificed and microscopic studies made of the organs and sites of transplantation. These studies disclosed that either one or two periods of parabiosis resulted in the same persistent modification of classical host-homograft interactions. This modification was characterized by the replacement of the customary basic or sensitized homologous incompatibility reactions by a reaction more easily interpreted as one of host-graft tolerance or indifference. The intense inflammatory aspects of the usual host-homograft interactions were absent. Penetration of the musculofascial graft by vascularized mesenchyme derived from the host was retarded. When it occurred, the vascularizing reaction was abnormal and unaccompanied by the customary proliferative, resorptive, and repairative activities of admixed cells of the host and the graft. These observations provide another means which may be useful in seeking conditions for increasing the probability of successful homografting between members of a genetically impure species.

Comparative studies were made of the microorganisms present in D. variabilis ticks, some of which served as a control series while the remainder were exposed to infection with D. rickettsi and thereafter maintained under various conditions. All female ticks contained in their ovaries a coccoid intracellular microorganism. About 50 per cent of all ticks after refeeding contained rickettsia-like microorganisms in variable numbers in nearly all organs. The groups of ticks exposed to infection with the virus of spotted fever, in addition to the above mentioned microorganisms, usually harbored large numbers of D. rickettsi, distinguishable with certainty from the non-pathogenic organisms only by their localization in nuclei of tick cells. No influence upon the size, number, or distribution of either the non-pathogenic rickettsiae or D. rickettsi in ticks was attributable to refeeding, variations of the temperature of incubation, or variations of the length of the period of incubation. We conclude from the results of these studies that the non-pathogenic rickettsiae which occur in D. variabilis ticks have no well defined relationship to D. rickettsi since they differ from the latter organism not only in the absence of virulence and immunizing properties, but also in their distribution in tick tissues and inability to multiply in the nuclei of cells.

In tissue cultures grown at 32 degrees C., typhus Rickettsiae increase rapidly within the cytoplasm of infected cells up to about the 14th day. At this time practically every cell is infected and the majority of cells are distended with organisms. This condition remains constant as long as successful cultures of the cells can be maintained (up to 52 days). Loss in virulence does not take place during this period in vitro. The number of Rickettsia-filled cells found in sections and the incubation period of the infection resulting from inoculation of cultures from each age group are definitely correlated. The behavior of typhus Rickettsiae in dividing cells is described and methods of spread of the infection other than by mitosis of cells are discussed. Normal tissues do not become infected in vitroto any considerable extent in spite of prolonged proximity to heavily infected cultures of scrotal sac exudate. Complete anaerobiosis and alterations in pH do not alter the intracellular location of the organism in tissue cultures. The organisms are not seen within nuclei of infected cells. They remain intact and infective for several weeks in cells which are kept alive but not multiplying. They disappear in less than 1 week, however, when the cells undergo degeneration.

Spotted fever infection has been studied in tissue cultures grown at 32 degrees C. The behavior of spotted fever Rickettsiaeis compared and contrasted with that of typhus Rickettsiae under similar conditions. The spotted fever organisms multiply extensively in the nuclei of cells where they form spherical clusters of various sizes. These structures are compared and contrasted with intranuclear inclusion bodies.

A new method for the isolation of large numbers of individual myofibrils from fresh mammalian skeletal and cardiac muscle has been described. Purification of isolated myofibrils was accomplished by differential centrifugation of fresh frozen sections of muscle which had been mechanically agitated after exposure for 30 to 45 minutes at 0 degrees C. to the action of a dilute solution of trypsin in a phosphate buffer solution with a pH of 7.0 and an ionic strength of 0.25. Isolated skeletal myofibrils of the rabbit and man have similar constant solubility properties. They dissolve in an aqueous mixture of 0.5 N potassium chloride and 0.03 N sodium bicarbonate, giving viscous solutions which exhibit conspicuous birefringence of flow. They are soluble in buffer solutions (ionic strength 0.15) on the acid side of pH 4 and alkaline side of pH 10. If the ionic strength of potassium phosphate buffer solutions is increased to 0.5 or if the ionic strength of phosphate-borate buffer solutions is increased to a similar value by addition of potassium chloride, the isolated myofibrils become soluble at neutrality. Hence, it is possible, first to isolate the myofibrils and then dissolve them without deviating appreciably from physiologic ranges of pH. The extent to which myofibrils are modified by the conditions imposed by the method of isolation is unknown. There is no significant change in microscopic structure or optical birefringence. Furthermore, there is retention of a form of physiological reactivity, for when the isolated skeletal myofibrils are immersed in solutions of adenosinetriphosphate, they promptly and irreversibly change from elongated fibrils with distinct structural detail into dense spherical masses without recognizable microscopic structure.

Simultaneous autologous and homologous musculofascial transplants were made in New Zealand white rabbits. The basic pattern of degeneration and granulation tissue organization of both types of transplant was essentially identical. The superposition of two reactions in the framework of organizing granulation tissue served to distinguish homologous from autologous transplants. One reaction to homologous transplants was predominantly characterized by lymphocytic infiltration and the other by angeitis. The principal locus of these reactions was in the musculofascial zone of the transplant, and from this zone the reactions spread to a variable degree through the fascia into the overlying pannus of granulation tissue. When single homologous transplants were made, the lymphocytic and mild angeitic forms of reaction predominated, becoming conspicuous at the end of 2 weeks. When multiple successive homologous transplants were made from the same donor to the same recipient, acute angeitis with thrombosis supervened and the lymphocytic reaction failed to develop or persist. Multiple successive autologous transplants, on the other hand, did not influence the type or degree of reaction to autologous transplants in the same animal. There was no evidence that autologous transplants had any influence upon the sequence of reactions to homologous transplants or that the presence of homologous transplants influenced the nature of the reaction to autologous transplants in the same animal. Until better methods are developed, methods of bio-assay of the type described, though lacking in quantitative precision, offer the best means for further analysis of factors which govern the incompatibility of tissues of one animal for those of another animal of the same species.

Under ordinary conditions musculofascial cross-grafts made between pairs of rabbits of the same strain and species elicited classical host-homograft tissue interactions. When the cross-grafting was done 7 to 10 days after exchange transfusions leading to introduction of about 40 per cent of foreign blood, the classical host-homograft reaction failed to develop. In its stead there was an harmonious interaction characterized by abundant vascularization of each graft, with minimal stromal replacement and without a trace of inflammation. This reaction resembled a common type previously described in cross-grafts made between postparabiotic twins but lacked some conspicuous features of the reaction of an animal to grafts of its own tissues.

Typhus Rickettsiae are found in large numbers in sections of tissue cultures of scrotal sac exudate. Extensive multiplication of the organisms occurs, and new cells become infected. Organisms are seen in cells undergoing mitotic division. The organisms usually become less numerous after the 6th day in vitro, but in one instance organisms were extremely numerous on the 16th and 21st days. Rickettsiae in tissue cultures retain their intracellular location, even when infection is very heavy. They multiply exclusively in non-phagocytic cells which are believed to be of mesothelial origin. Pleomorphism is much more pronounced in tissue cultures than in guinea pig tissues and is entirely comparable to that seen in the louse.

Several methods for the experimental study of the rickettsial diseases have been applied to fièvre boutonneuse. An analysis of the results has indicated that fièvre boutonneuse is a variety of spotted fever and that the etiologic agent is a rickettsia which belongs to the genus Dermacentroxenus and to the species rickettsi (Wolbach (6)). The distinctive morphology of the organism and its characteristic intranuclear clustering in ticks and in tissue cultures are the important criteria upon which this conclusion is based. Immunological, histological, and cytological observations of a confirmatory nature are also reported.

Skeletal myofibrils isolated either by tryptic digestion at 0 degrees C. or by a colloid mill and suspended in buffer solution (pH 7.0, micro; 0.154) containing 20 per cent glycerin and 0.0025 M adenosinetriphosphate at -5 degrees C. contracted slowly and progressively when the temperature was raised above 0 degrees C. Formalin fixation halted this contraction. With the aid of these procedures myofibrils in progressive stages of contraction were then studied with the electron microscope. Electron micrographs showed that uncontracted fibrils isolated by the colloid mill were structurally similar to those described by other workers. Treatment of fibrils with trypsin removed the Z bands and disorganized the I bands. This enzymatic modification of structure did not impair the contractile response. The principal structural changes during contraction consisted of a migration of dense material from the A band into the A-I junction or the Z band, a gradual increase in width of the fibril, a gradual decrease in length of sarcomeres, an apparent increase in the mean diameter of filaments, and a disorientation of these latter from their parallel arrangement.

The temperature at which tissue cultures infected with typhus Rickettsiae are incubated has been shown to exert a marked influence on the intracellular multiplication of Rickettsia prowazeki. At 41 degrees C. the organisms were not found in the cultures histologically on and after the 2nd day in vitro, and the cultures were non-virulent on and after the 3rd day in vitro, in spite of good preservation and growth of the cells. At 37.5 degrees C. organisms were absent from the cultures histologically and the cultures were non-virulent on and after the 11th day in vitro, in spite of good preservation and growth of the cells. At 32 degrees C. good but slow growth of cells occurred and organisms were found in increasing numbers histologically up to about the 21st day in vitro. At this time, 50 to 99 per cent (approximately) of the cytoplasmic volume of the cultures was occupied by Rickettsiae. From the 21st day to the 51st day (the limit to which cultures have been carried successfully) this condition of unrestricted multiplication remained practically unchanged. Inoculation of guinea pigs with single cultures after varying lengths of time in vitro, (up to the 51st day) always resulted in reproduction of typhus in a characteristic manner. At 27 degrees the cell growth was negligible, but many cells remained alive for 10 days or more. Organisms were absent from the cultures histologically and the cultures were non-virulent on and after the 18th day in vitro. The only histological preparations showing unrestricted multiplication of the organisms (infection of the majority of the cells present) were of cultures incubated at 32 degrees C. It is believed that the detrimental effect of the higher temperatures (37.5 degrees and 41 degrees C.) on the multiplication of the organism is exerted indirectly, by stimulation of the defence mechanism of the cells.