SPOTTED FEVER : III. THE IDENTIFICATION OF DERMACENTROXENUS RICKETTSI AND ITS DIFFERENTIATION FROM NON-PATHOGENIC RICKETTSIAE IN TICKS

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.     

ON THE PRESERVATION OF TYPHUS FEVER RICKETTSIAE IN CULTURES

1. Cultures of typhus fever rickettsiae were as a rule found to remain viable and virulent after being stored at 37° and –20°C. for several months, whereas they failed to survive when stored at the intermediate temperatures of 20° and –4°C. for 4 weeks and 10 days respectively. In one instance cultures were stored at 37°C. for 8 months without having been transferred, and were subsequently found to be viable and infectious. 2. The conditions influencing such long survival of an organism which seems to require living tissue for multiplication (as filterable viruses do in general) are discussed. 3. Typhus-infected tissues (minced guinea pig tunica) suspended in a serum-Tyrode mixture in sealed flasks remained infectious at 37°C. for at least 10 weeks. 4. Additional evidence for the etiological significance of the rickettsiae in typhus fever is obtained from the experiments described.     

TYPHUS FEVER : V. THE EFFECT OF TEMPERATURE ON THE MULTIPLICATION OF RICKETTSIA PROWAZEKI IN TISSUE CULTURE

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°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°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°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° 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°C. It is believed that the detrimental effect of the higher temperatures (37.5° and 41°C.) on the multiplication of the organism is exerted indirectly, by stimulation of the defence mechanism of the cells.     

IMMUNITY STUDIES OF ROCKY MOUNTAIN SPOTTED FEVER : I. USEFULNESS OF IMMUNE SERUM IN SUPPRESSING AN IMPENDING INFECTION.

From the results of the experiments presented it is evident that in guinea pigs an early administration of immune rabbit serum will suppress the infection; that is, if it is given within the period of incubation, the effect being proportionately greater the earlier the serum is administered. Almost no beneficial effect is observed when the serum is given after the onset of the disease. In the animals inoculated with 10 to 100 M.L.D. the incubation period is shorter than when 1 M.L.D. is injected; nevertheless 1 cc. of the immune serum saved the animals as late as 96 hours from the time of the introduction of the virus into the system. When administered within 24 hours in the case of 100 M.L.D. and within 48 hours in the case of 10 M.L.D., the serum completely neutralized the virus, and the animals escaped infection altogether. On the other hand, the same quantity of the serum only modified the infection into a non-fatal one when given a day or two later. In the animals which were inoculated with 1 M.L.D. the incubation period was a day or two longer, and the neutralizing effect of the serum was much more powerful. Here animals were saved as late as 5, 6, and 7 days and with a much smaller quantity of the serum (0.1 cc.). As to the usefulness of such an immune serum in human cases, the relative susceptibility of man and the guinea pig must first be considered. In a large number of experimental infections carried out with guinea pigs in the past 6 years almost never has a naturally refractory animal been encountered. The mortality is nearly 80 per cent with most strains, although as low as 50 per cent with some. The strain used in the present study caused death in nearly 80 per cent of the animals. Hence the susceptibility of guinea pigs is at least as great as that of man, in whom the mortality in the Bitter Root Valley is estimated to be about 70 per cent. The relative length of the incubation period in guinea pig and in man is another point which requires analysis. In guinea pigs it varies somewhat according to the number of passages, being as short as 3 days when 100 M.L.D. or more of an adapted virus are inoculated. On the other hand, when the infection is the result of 1 M.L.D. or the bite of an infected tick, the incubation period is much longer, being 5, 6, or 7 days in the former and 7 to 8½ days in the latter instance, as with the present strain. In man the infection is brought on by the bite of an infected tick, and the period of incubation varies from 3 to 10 days but is usually 7 days; i.e., it is about the same as in guinea pigs infected with 1 M.L.D. Hence we may regard the susceptibility of man and the guinea pig as nearly equal. The final point to be considered is the quantity of the immune serum that may be recommended for use in human cases. To prevent the infection in a guinea pig weighing 500 gm., 0.1 cc. of the serum was sufficient. This quantity protected the animal against 1 M.L.D. even as late as 5, 6, or 7 days. Calculated on this basis, 16 cc. of the serum would be required for a man weighing 80 kilos (about 160 pounds); that is, 16 cc. of an immune rabbit serum, administered before onset of the disease, should theoretically be sufficient to save a man of average weight against an infection brought about by the bite of an infected tick or by a laboratory accident. It would probably be best to administer the serum intravenously. The titer of the immune serum should be previously determined in guinea pigs, and 1 cc. should neutralize 100 M.L.D. completely and 0.1 and 0.01 cc. render the infection non-fatal. Such a serum is easily produced in rabbits (a rabbit weighing 2,500 gm. will yield 50 to 60 cc. of the serum) and probably will remain active a year or longer when kept at refrigerator temperature.     

ANTIBODY PRODUCTION BY CELLS IN TISSUE CULTURE : I. MORPHOLOGICAL EVOLUTION OF LYMPH NODE AND SPLEEN CELLS IN CULTURE

An organ culture technique was used to investigate the migration and the morphological evolution of lymphocytes from lymphopoietic tissues. This evolution was compared with the behavior of cells extracted from the tissue and kept in nutritive medium in vitro. It was found that cells were continuously migrating from the fragments of lymph nodes or spleen, and were attaching to the glass. They spread on glass, their protoplasm enlarged and their nucleus became clearer. The evolution towards blastoid cells was identical with that described under artificial stimulation by PHA for example. Cytological identification of the cells actively engaged in antibody synthesis (as detected by local hemolysis in gum) at the time of staining, showed that several distinct cellular types were active, including plasma cells and macrophagelike cells. It is assumed that the stimulated lymphocytes, after spontaneous migration from the tissue are able to evolve into an "immunoblast" stage and then, eventually after fixation upon a physical support, to initiate antibody synthesis.     

IMMUNITY STUDIES OF ROCKY MOUNTAIN SPOTTED FEVER : II. PROPHYLACTIC INOCULATION IN ANIMALS.

Freshly prepared mixtures of spotted fever virus and immune rabbit serum in neutral or superneutral proportions confer complete immunity on guinea pigs. The mixtures undergo a considerable loss in immunizing power when heated to 60°C. for 20 minutes, but are still capable, if used in sufficient quantity, of conferring a degree of immunity on the vaccinated animal such that a subsequent experimental infection is rendered less severe and non-fatal. Unheated mixtures which had been preserved in the refrigerator at 4°C. for a period of 32 days still retained a certain degree of immunizing property. The virus alone, or mixed with normal rabbit serum, when allowed to die out by prolonged preservation at refrigerator temperature, or when killed either by heating at 60°C. for 20 minutes or by chemicals (chloroform, ether, xylene) does not induce immunity in guinea pigs.     

GRAFT-VS.-HOST REACTION IN TISSUE CULTURE

The primary purpose of this study has been to validate the in vitro graft-vs.-host reaction as an experimental system. Time-dose studies have been presented for cells obtained from spleen, thymus, cortisone-treated thymus, inguinal lymph node, mesenteric lymph node, thoracic duct, and bone marrow cells. Both the degree of splenomegaly and the onset of spleen enlargement were found to be dependent on the number and source of cells tested. The effect of several immunosuppressive agents was examined. Amantadine was found to suppress completely the graft-vs.-host reaction in vitro when present at a concentration of 75 µg/ml. Pretreatment of effector cells with mitomycin C prevented their subsequent ability to cause a graft-vs.-host reaction. The effect of X irradiation on immunocompetence of spleen cells in vitro paralleled the known effect of irradiation on in vivo immunocompetence. Preimmunization did not increase the number or effectiveness of immunocompetent cells when measured under standard in vitro conditions. Preimmunization did, however, permit persistence of immunocompetence after immunosuppressive doses of X irradiation. Studies using congenic lines, moreover, indicated that the preimmunization effect could be demonstrated in strain combinations differing only in factors determined by the H-2 complex of genes. A weak graft-vs.-host reaction could be detected in strain combinations not involving differences at the H-2 locus. The potential of the in vitro graft-vs.-host reaction as a highly reproducible, quantifiable, internally controlled, and experimentally accessible system for study of such critical problems as cell differentiation and cell interactions is discussed.     

FURTHER EXPERIMENTS IN TYPHUS FEVER : IV. INFECTION WITH WASHED MEXICAN RICKETTSIAE AND IMMUNITY TO EUROPEAN TYPHUS

Precise interpretation of our experiments seems to impose the following conclusions: Guinea pigs inoculated with washed Rickettsiae from Mexican typhus fever develop a disease identical with that resulting from inoculations with whole tunica scrapings, blood or other virulent material, and become thereby immunized to European typhus fever. The etiological agent of Mexican typhus fever is the Rickettsia body of the type described by Mooser (5) in the tunica vaginalis of infected guinea pigs; and it is likely that the etiological agent of European typhus fever is an organism similar to this, but not identical with it in some of its minor biological characteristics.     

INTERFERENCE BETWEEN VIRUSES IN TISSUE CULTURE

The influence of one virus on the growth of another in tissue culture was investigated. The 17DD High strain of yellow fever virus was found capable of completely suppressing the growth of both the Asibi strain of the same virus and of the heterologous West Nile virus, even when these were added to the cultures in large amounts. The 17DD High strain of yellow fever virus and the West Nile virus produced either partial or complete suppression of growth of the Venezuelan equine encephalomyelitis virus, depending upon the quantity of the latter inoculated into the cultures. Owing to lack of methods for the detection of interference except in a single direction, reciprocal interference with these viruses could not be investigated. The 17DD High strain of yellow fever virus and the West Nile virus were able to suppress completely, or almost completely, the growth of influenza A virus added to the infected cultures in maximal amounts. Interference in the reverse direction, even with the use of small amounts of the neurotropic viruses, was not demonstrable. Cultures infected with the 17DD High strain of yellow fever virus were examined for the presence of neutralizing antibodies and non-specific antiviral substances; neither was found present.     

TISSUE CULTURE STUDIES ON BACTERIAL HYPERSENSITIVITY : I. TUBERCULIN SENSITIVE TISSUES

1. A high degree of cellular sensitivity to tuberculin toxicity was demonstrated when explants from tuberculous animals were grown in media containing that substance. 2. Similar degrees of sensitivity were noted in cells derived from animals infected with either virulent or relatively lowly virulent strains of tubercle bacilli. 3. The specificity of the tuberculin cytotoxicity was proven by testing with other bacterial cytotoxic materials. 4. Tuberculin sensitive cells grown in vitro in normal media showed, when tested with tuberculin, persistence of this cellular sensitivity through several transplantations during which time many new generations of cells developed. 5. There was a depression of the initial growth energy of explants from animals during the toxic phase of the disease. During the healing stage the initial growth energy returned to normal although marked sensitivity to tuberculin persisted. 6. The degree of cellular sensitivity to tuberculin in vitro did no parallel the acuity of the infectious process but represented a more or less permanent acquired characteristic impressed on the cell as a result of the infection.     

BIOCHEMICAL STUDY OF CELLULAR ANTIGEN-ANTIBODY REACTION IN TISSUE CULTURE : I. ACTIVATION AND RELEASE OF A PROTEASE

The correlation between morphological and biochemical changes produced by the antigen-antibody reaction was studied in cultures of tissue monocytes taken from sensitized animals. The cells were grown under conditions which allowed collection of samples from the culture fluid as well as microscopic observation. Introduction of the antigen into the culture medium causes rapid release of a protease characterized by its susceptibility to sulfhydryl block and its optimum pH in the neutral range. Protease activation occurs simultaneously with morphological changes in the cytoplasm of the cultured cells. Delayed changes affecting the mitochondria and Golgi bodies appear after the peak of the proteolytic reaction and may be secondary to it. The gradual inactivation of the protease observed in the course of the antigen-antibody reaction will be discussed in a separate paper.     

CHARACTERISTICS OF FROG CARCINOMA IN TISSUE CULTURE

The adenocarcinoma of leopard frogs may be cultivated with ease in plasma media. In such cultures two types of growth occur with regularity. The first is in the form of tubules which promptly grow out in the solid medium and retain their tubular form as long as they remain completely enveloped by plasma. When, however, they make contact with the surface of the glass, they adhere to it, the part in contact becomes flat, and its cells now grow no longer as tubules but as membranes. The manner of growth in vitro resembles the growth of transplants of the same tumor in the anterior chamber of the living eye, thus suggesting that in each case the habit of growth is determined by the same morphogenetic factors, i.e. those inherent in the cells themselves, and those depending on interfacial forces. The malignant cells of the frog carcinoma have the attributes which in general distinguish malignant cells from normal cells of corresponding type. In comparison with adult kidney cells, their normal homologues, the conspicuous properties of frog carcinoma cells are: larger and more variable size and shape of cell body, of nucleus, and nucleolus; coarser and denser structure of cytoplasm, of nucleoplasm, and of nuclear membrane; increase in number of mitochondria, and more frequent occurrence of mitosis. These cytological characteristics remain unaltered in cultures maintained for as long as six months. Frog carcinoma is a transmissible disease due to an agent which induces inclusion bodies, and which has other attributes indicating that it is a virus. The general correspondence in character between its cells and the malignant cells of mammalian tumors of diverse origin suggests that neoplastic phenomena are essentially alike, no matter in what group of animals they occur or what their causal factors may be.     

PROPAGATION OF RABIES VIRUS IN TISSUE CULTURE

Rabies virus has been propagated in serum-Tyrode solution containing either embryo mouse brain or embryo chick brain. The culture virus reached a titre of 3 x 10^–5 cc. after 4 days'' incubation at 37°C., and survived at least 2 months at 5°C. in the liquid or dry state.     

A CYTOLOGICAL STUDY OF THE NATURE OF RICKETTSIA IN ROCKY MOUNTAIN SPOTTED FEVER

The Rickettsia of Rocky Mountain spotted fever were easily differentiated from mitochondria, phagocytosed blood pigment, nuclear debris, and all other known cellular constituents. Although they were lodged within the cytoplasm of endothelial cells, they were not observed to establish any definite relations with the nucleus or with other cellular components. Their number varied in contiguous cells which sustained the same degree of injury as evidenced by nuclear changes, and alterations in their mitochondria content. The mitochondria, on the other hand, showed similar modifications, characterized by a decrease in number and a rounding up into spherules, in all the endothelial cells seen in a section of an affected blood vessel. Diplobacillary forms were most abundant in the early stages of the reaction and single bacillary bodies towards its termination. Other slight differences in morphology from Wolbach''s account were noted in the organisms as seen in the tissues of both ticks and guinea pigs. His study of the distribution of specific lesions with accompanying organisms in the tissues of guinea pigs was confirmed and extended.     

TYPHUS FEVER : IV. FURTHER OBSERVATIONS ON THE BEHAVIOR OF RICKETTSIA PROWAZEKI IN TISSUE CULTURES

In tissue cultures grown at 32°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.     

ETIOLOGY OF YELLOW FEVER : I. SYMPTOMATOLOGY AND PATHOLOGICAL FINDINGS OF THE YELLOW FEVER PREVALENT IN GUAYAQUIL.

The clinical and pathological features of the yellow fever prevalent in Guayaquil conform with those described by other investigators of this disease as it has occurred elsewhere, both epidemically and endemically.     

昆明地区发热患者血培养与抗生素药敏试验观察

目的了解昆明地区临床发热患者血液培养与抗生素药敏试验结果,为临床诊治传染性疾病提供参考依据。方法用全自动血培养系统和细菌鉴定及纸片扩散法,对临床3 126例血液标本进行了培养分析及其细菌鉴定和药敏试验。结果从3 126例血液标本中培养分离出2 188株菌,35个种属,阳性率为69.9%。在2 188株细菌中,革兰阳性球菌436株,占19.9%;革兰阴性杆菌1 447株,占66.1%;真菌193株,占8.8%;厌氧菌37株,占1.69%。共检出218株产β内酰氨酶菌株,产酶菌株对氨苄西林等10种临床常用抗生素耐药率均为100%。结论昆明地区发热病人血液标本细菌培养阳性率较高,分离菌株以革兰阴性菌为主,细菌耐药性比较严重。     

STUDIES ON INFECTION AND IMMUNITY IN EXPERIMENTAL TYPHOID FEVER : I. TYPHOID FEVER IN CHIMPANZEES ORALLY INFECTED WITH SALMONELLA TYPHOSA

A disease resembling human typhoid fever has been induced by feeding live cultures of Salmonella typhosa to young chimpanzees, thus confirming the classical reports of Grünbaum and of Metchnikoff and Besredka. Detailed clinical observations, results of stool and blood cultures, and serological studies have confirmed the impression that the disease produced in chimpanzees closely resembles the mild form of human typhoid fever frequently seen in childhood. Gross and histologic examination of intestines, mesenteric lymph nodes, liver, spleen, and other organs of orally infected chimpanzees has demonstrated that the pathological findings are essentially indistinguishable from those seen in mild typhoid fever in man. The clinical spectrum of disease seen in chimpanzees ranged from moderately severe illness, through transitory illness, to afebrile infection with or without bacteriemia (but invariably with an antibody response), occasionally leading to the development of persisting biliary infection and the carrier state. Thus the range of illness observed in chimpanzees resembled that seen in man, except that the severe and complicated forms of typhoid fever were not observed in the chimpanzee. A reason for this difference is proposed and discussed. In contrast to the limitations imposed upon the interpretation of human epidemiologic observations, it has been possible to demonstrate in the chimpanzee that clinical variation in disease pattern from animal to animal may occur despite the administration of the same dose of the same bacterial strain simultaneously to an entire group of animals under study; in other words, variation in clinical pattern is dependent on inherent, non-specific host factors as well as on dose, strain or preceding state of immunity. Variation in dose and in challenge strain of S. typhosa employed also appeared to have an effect upon the likelihood of producing febrile as against afebrile infection in chimpanzees. The dose required to produce clinical disease, even with the more virulent strain, was excessively large compared to what is believed to be the dose required to produce illness in man; the limitations of this assumption, and suggested explanations for the findings, are discussed. The production of the spectrum of typhoid fever in the chimpanzee has made possible the study of basic problems in this disease which are not amenable to definitive study through the use of prevailing laboratory techniques.     

CARTILAGE MATRIX DEPLETION BY RHEUMATOID SYNOVIAL CELLS IN TISSUE CULTURE

Articular cartilage fragments were added to monolayer cultures of synovial membrane cells. After 3 wk of incubation, the cartilage fragments were examined histologically for metachromasia and basophilia, and for fluorescent staining using a rabbit antiserum to cartilage protein-polysaccharide. Cartilage incubated with cells derived from rheumatoid synovial membranes showed striking loss of metachromasia and basophilia as well as diminished to absent fluorescent staining. Cartilage fragments incubated with cells from normal synovia, or with cells from the synovial membrane of a patient with Reiter's syndrome, did not show these changes and resembled control cartilage incubated in tissue culture medium alone. It appears, therefore, that rheumatoid synovial cells in tissue culture are able to deplete the matrix of articular cartilage.     

CONNECTIVE TISSUE SYNTHESIS BY SCLERODERMA SKIN FIBROBLASTS IN CELL CULTURE

Skin fibroblasts from subjects with scleroderma and control subjects were grown in tissue culture to compare the characteristics of connective tissue metabolism. A striking increase in soluble collagen (media hydroxyproline) was observed in eight of nine scleroderma cultures when they were compared with identically handled control cultures matched for the age and sex of the donor and the anatomic site of the donor skin. Glycoprotein content as estimated by hexosamine and sialic acid was also significantly increased in the scleroderma cultures. Estimations of protein-polysaccharide content by uronic acid determinations were low in all cultures and not significantly increased in scleroderma cultures. This report demonstrates the feasibility of using fibroblast cell cultures to study chronic rheumatic and connective tissue disorders. The initial results suggest a net increase in collagen and glycoprotein synthesis in scleroderma fibroblast cultures. The implications of an abnormality of connective tissue metabolism by skin fibroblasts propagated in vitro in the acquired disorder scleroderma are discussed.