https://www.jacionline.org/article/S0021-8707(68)90117-2/pdf

The beta adrenergic theory of the atopic abnormality in bronchial asthma

Francisella tularensis has been recognized as a human pathogen for almost 100 years and is the etiological agent of the zoonotic disease tularemia. Soon after its discovery, it became recognized as an important pathogen in several parts of the world, for example, in the United States and Soviet Union. The number of tularemia cases in the two countries peaked in the 1940s and has thereafter steadily declined. Despite this decline, there was still much interest in the pathogen in the 1950s and 1960s since it is highly infectious and transmissible by aerosol, rendering it a potent biothreat agent. In fact, it was one of the agents that was given the highest priority in the offensive programs of the United States and Soviet Union. After termination of the offensive programs in the 1960s, the interest in F. tularensis diminished significantly and little research was carried out for several decades. Outbreaks of tularemia during the last decade in Europe, for example, in Kosovo, Spain, and Scandinavia, led to a renewed public interest in the disease. This, together with a massive increase in the research funding, in particular in the United States since 2001, has resulted in a significant increase in the number of active Francisella researchers. This article summarizes, predominantly with a historical perspective, the epidemiology and clinical manifestations of tularemia and the physiology of F. tularensis.

Tularemia: History, Epidemiology, Pathogen Physiology, and Clinical Manifestations

Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages. We have examined the interaction of a recent virulent clinical isolate of F. tularensis subsp. tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of the F. tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D. Two to four hours after infection, vacuoles containing live F. tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with live F. tularensis organisms declined dramatically. In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h). We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents. Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F. tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of the F. tularensis compartment revealed that during the first 4 h after uptake, the majority of F. tularensis bacteria reside within phagosomes with identifiable membranes. The cytoplasmic side of the membranes of ∼50% of these phagosomes was coated with densely staining fibrils of ∼30 nm in length. In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment. By 8 h after infection, the majority of live F. tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate that F. tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm. The capacity of F. tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development.

Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

Tularemia is caused by the facultative intracellular bacterium Francisella tularensis. Attenuated live vaccines, such as F. tularensis LVS (live vaccine strain), afford good--although not complete--protection; how to judge the degree of this protection has long been a problem. Both natural infection and vaccination result in immunospecific and long-lasting humoral and cell-mediated immunity. The latter is the crucial protective mechanism, whereas the humoral response protects only against strains of reduced virulence, such as those used in the vaccines. Immune serum has been used to screen for structures of F. tularensis with the ability to induce a protective immune response. This immune serum is, however, primarily directed toward antigens different from those involved in cell-mediated immunity. Serum antibodies from primed individuals recognize carbohydrate capsule antigens of F. tularensis, whereas T lymphocytes recognize membrane polypeptides of the organism. The preparation of membrane polypeptides from F. tularensis is now facilitated by the availability of a capsule-deficient mutant of F. tularensis LVS. In vitro, several membrane polypeptides of the mutant stimulate T lymphocytes from vaccinees and from naturally infected individuals. Further studies of the mechanisms of induction of protective immunity should focus on these membrane polypeptides.

Nature of Protective Immunity to Francisella tularensis

Francisella tularensis is a pathogenic bacterium whose virulence is linked to its ability to replicate within the host cell cytosol. Entry into the macrophage cytosol activates a host-protective multimolecular complex called the inflammasome to release the proinflammatory cytokines interleukin (IL)-1β and -18 and trigger caspase-1–dependent cell death. In this study, we show that cytosolic F. tularensis subspecies novicida (F. novicida) induces a type I interferon (IFN) response that is essential for caspase-1 activation, inflammasome-mediated cell death, and release of IL-1β and -18. Extensive type I IFN–dependent cell death resulting in macrophage depletion occurs in vivo during F. novicida infection. Type I IFN is also necessary for inflammasome activation in response to cytosolic Listeria monocytogenes but not vacuole-localized Salmonella enterica serovar Typhimurium or extracellular adenosine triphosphate. These results show the specific connection between type I IFN signaling and inflammasome activation, which are two sequential events triggered by the recognition of cytosolic bacteria. To our knowledge, this is the first example of the positive regulation of inflammasome activation. This connection underscores the importance of the cytosolic recognition of pathogens and highlights how multiple innate immunity pathways interact before commitment to critical host responses.

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Type I interferon signaling is required for activation of the inflammasome during Francisella infection

/pdf/a-new-organism-resembling-p-tularensis-isolated-from-water-1qhpnaah7k.pdf

A new organism resembling P. tularensis isolated from water.

The primary inflammatory response of rabbits to intradermal injection of bacterial products has been used as the basis of a sensitive assay for endotoxins. Injection of materials into the skin of the lateral surfaces, rather than the ventral surface of the abdomen, was essential. The method was evaluated by comparison with the mouse toxicity test. Statistical examination revealed an excellent correlation between the mean threshold dose for producing skin lesions in rabbits (SLD50) and the median lethal dose for mice (LD50). For potent endotoxins (LD50 = 0.2–0.3 mg.) the skin test dose was about one-thousandth of the lethal dose for mice. Thereafter the LD50 increased in the proportion of about 0.3 mg. per 1 µg. increase in SLD50. The method of testing was devised for endotoxins prepared in various ways from Salmonella enteritidis and S. typhosa but was directly applicable to endotoxins from several other species of enteric Gram-negative bacteria.

/pdf/a-method-for-titrating-endotoxic-activity-in-the-skin-of-41a2b9ybl1.pdf

A method for titrating endotoxic activity in the skin of rabbits

Summary Preparations of cell wall and protoplasm were made from young Bordetella pertussis cells and tested for: a) their ability to immunize mice against fatal B. pertussis infection, b) their ability to sensitize mice to histamine, and c) their toxic properties. It was found that the heat labile toxin was mainly present in the protoplasm whereas the protecting antigen and the histamine sensitizing factor were located principally in the cell wall.

Antigens of Bordetella pertussis. I. Activities of cell walls and protoplasm.

Francis and Felton4 have proposed a measure of virulence, based on the susceptibility of mice, guniea pigs, and rabbits to infection with this organism, which appears to be the most direct and valid of any that have been applied. Approximately 1 to 10 organisms of maximum virulence are sufficient to cause fatal infection in mice, guinea pigs, and rabbits. Diminished virulence first shows itself in the failure of the organisms to kill rabbits. This method of as-

Virulence of Bacterium tularense. I. A study of the virulence of Bacterium tularense in mice, guinea pigs, and rabbits.

Resistance to infections with Mycobacterium tuberculosis has been studied in a number of experimental animals, but recently the suitability of mice for immunity tests has been emphasized. Stamatin and Stamatin (1) concluded that mice were less susceptible than guinea pigs. The use of mice for studies of resistance to tuberculosis was given great impetus by Dubos and associates (2a-2i) , who showed that, by controlling the size of the challenge dose of virulent bacilli injected intravenously, differences in reactions of nonvaccinated mice could be detected. Lethal infections were avoided because differences between the two groups of animals were difficult to detect. In tests to evaluate the immunogenicity of various strains of BCG, the number of organisms in selected tissues was determined because proliferation of the immunizing agent was necessary to produce protection. The degree of resistance was measured by: the comparative numbers of virulent organisms in lungs and spleens; the number of tuberculous lesions in the lungs; and the number of acid-fast organisms in each nodule of control and immunized mice. The application of these methods has been described by various writers (3-7). Crowle (8) has shown that the relative density of the lungs of vaccinated and control mice challenged with virulent tubercle bacilli may be used as an index of immunity. The survival time of control versus vaccinated mice after intravenous challenge with large numbers of virulent bacilli has also been used as a measure of resistance (9-11). Glover (12) showed that mice were susceptible to infection by aerosols of virulent bovine tubercle bacilli and that approximately 100 organisms were an infectious dose and 120,000 a lethal

Carl L. Larson

Papers

A new organism resembling P. tularensis isolated from water.

A method for titrating endotoxic activity in the skin of rabbits

Antigens of Bordetella pertussis. I. Activities of cell walls and protoplasm.

Virulence of Bacterium tularense. I. A study of the virulence of Bacterium tularense in mice, guinea pigs, and rabbits.

Studies of resistance to experimental tuberculosis in mice vaccinated with living attenuated tubercle bacilli and challenged with virulent organisms.