Leighton E. Cluff, Chairman CARL LAMANNA1. the ciliary beat, reference is not made to secondary. would be among the most, if not the most, potent

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1 PART VI. IMMUNOLOGY AND PUBLIC HEALTH Leighton E. Cluff, Chairman IMMUNOLOGICAL ASPECTS OF AIRBORNE INFECTION: SOME GENERAL CONSIDERATIONS OF RESPONSE TO INHALATION OF TOXINS CARL LAMANNA1 Naval Biological Laboratory, School of Public Health, University of California, Berkeley, California Knowledge of the effects in animals following exposure to aerosols of microbial toxins suffers from a paucity of data. This probably is attributable to the fact that natural exposure to microbial toxins, free of microorganisms, does not occur. Interest in the subject, therefore, stems only from curiosity, idle or scientific, about a theoretical possibility or from concern over exposure to microbial toxins in the laboratory, in industry, or aerosolized intentionally as an act of war. In a sense, one can speak of respiratory toxins produced by microorganisms in vitro or in vivo which are capable of affecting adversely the activity of the respiratory tract. But the questions have never been seriously entertained as to whether or not these materials are more potent when introduced into the host by inhalation as experimental aerosols, by natural infection, or by other means. In addition, there are few observations related to the capacity of respiratory toxins to poison cells other than those resident in the respiratory tract. It is clear, however, that inhaled microbial toxins are harmful, independent of their capacity to poison respiratory tissue cells. In considering the nature of toxic materials affecting the respiratory tract, I have been struck by an inability to find described the existence of material of microbial origin which specifically interferes with the mechanism of the ciliary beat. For example, I found no reference to such a material in a recent extensive bibliography of 1,335 references, prepared by Rivera (12), on cilia and ciliated epithelium. In the evolution of respiratory infection, specific microbial toxins have an ample opportunity to appear and 1 The author's research was supported by the Office of Naval Research under a contract with the Regents of the University of California. Reproduction in whole or in part is permitted for any purpose of the United States government. Present address: Army Research Office, Washington 25, D.C. potentiate the effects of the pathogens. Do such toxins not exist or have they not been sought by appropriate means? It should be clear that in speaking of a microbial poison directly affecting the ciliary beat, reference is not made to secondary effects on ciliary activity resulting from interference with the general tissue environment and attributable to such events as edema, increased mucus, viscosity, or tissue necrosis. If toxins specifically directed against the ciliary beat existed, it might well prove that inhalation would be among the most, if not the most, potent route of exposure of animals. FACTORS INFLUENCING POTENCY OF TOXINS UPON INHALATION What general considerations enter into the picture of the potency of toxins prepared as aerosols? Poisoning of an animal by any mode of exposure to a toxin, including inhalation, will depend upon the outcome of a sequence of four events: first, the capacity of the toxin to escape destruction inherent in the technique of application; second, the capacity of the toxin to get to and to remain at the site of deposition where effective systemic absorption can take place; third, the capacity of the toxin after absorption to be transported to the primary sites of poisoning; and fourth, the capacity of the toxin to resist specific or nonspecific forces of detoxification en route to the sites of action. The generation of aerosols of toxins presents formidable problems, since the toxins tend to be detoxified both in the process of aerosolization and during persistence in the airborne state. The contrivance for dispersal of toxins in aerosols, the nature and quantities of accompanying extraneous matter, and the temperature and humidity of the atmosphere all influence the preservation of toxicity. But these problems need not concern us further since our primary interest here is in what happens upon inhalation of the active toxin remaining in an aerosol. 323

2 324 CARL LAMANNA [VOL. 25 TABLE 1. Effect of particle size on inhalation anaphylaxis in the hypersensitized guinea pig Particle size range Exposure Inhaled dose Rate of Group no. time inhalation NTo. responding/no. exposed Dyspnea Convulsions Death 4 w inin mg mg/lsin 77.4% from 0.0 to /3 3/3 2/3 2 3/3 3/3 2/3 3 3/3 3/3 3/3 4 3/3 3/3 3/3 5 3/3 3/3 3/3 89% from 7.5 to /3 0/3 0/3 2 3/3 1/3 1/3 3 3/3 1/3 0/3 4 3/3 0/3 0/3 5 3/3 0/3 0/3 None of the nonsensitized controls were affected. Egg albumin was employed as the allergen (C. W. Beard and W. R. Kerpsack, personal communication). Upon inhalation, the depth of penetration of aerosols of toxins into the respiratory tree seems to present no problems that are not common for aerosols in general. The belief is that the deeper the penetration of the toxin beyond the nasopharyngeal barrier, the greater is the potency exhibited by the aerosol. In the case of allergens whose action is directly on lung tissue, the importance of small particle size has been neatly demonstrated recently, for example, by C. W. Beard, M. E. Prickett, P. Irwin, and W. R. Kerpsack at Fort Detrick, with guinea pigs sensitized to egg albumin. Data kindly supplied by Beard and Kerpsack are recorded in Table 1. A. H. Corwin of The Johns Hopkins University, working with a material of plant origin, ricin, has also demonstrated a relationship between particle size of aerosolized toxin and severity of reaction. He found that clouds with no particles above a mass median diameter of 2.1.A are 2.75 times as toxic as clouds with a maximal particle size of 4.2 1,u and that the most certain method for increasing potency is to decrease the particle size of the powdered ricin contained in aerosols. Microbial toxins, such as botulinal and tetanal neurotoxins, inhaled into and penetrating the deeper recesses of the respiratory tract, probably have a greater opportunity for systemic absorption. This appears to be logical, because the thickness of the membranes to be penetrated by the toxin is less in the deeper reaches of the respiratory tract, and the toxin is not rapidly swept out of the respiratory tract by the activity of the ciliated epithelium. Once the toxin penetrates the respiratory barriers, the dose required to kill an animal with the symptoms of poisoning may be expected to be equivalent to the dose required to produce these effects when introduced directly into the blood stream. Death may be postponed, however, because of the lag in time necessary for a lethal quantity of the toxin to be absorbed. Legroux, Levaditi, and Jeramec (11) have presented data on the variation in rapidity of death resulting from the injection by different routes of the same dose of type B botulinal toxin, which lend some support to this argument (Table 2). PERMEABILITY OF RESPIRATORY TRACT TO TOXIN The increased potency of aerosols of small particle size should not be taken to imply that toxins are not poisonous unless they pass beyond the nasopharyngeal area. Although absorption of toxin from the upper respiratory tract and the contiguous alimentary tract area may be poorer than in alveoli, it does occur. Thus, intranasal instillation of botulinal and tetanal toxins kills mice, although by this technique relatively heavier contamination of the upper respiratory tract and nasopharyngeal area may be expected than with contamination by inhalation of small particles. With tetanal toxin in mice I have found intranasal instillation to be of the order of not less than 10,000 times more potent than instillation per os. Fifty intraperitoneal LD5o Will

3 19611 IMMUNOLOGICAL RESPONSE TO INHALATION OF TOXINS 325 TABLE 2. Order of rapidity of death of rabbits by various routes of injection of botulinum toxin Route' Time of Death days hr min Intravenous Intra-arterial Intramuscular Intracerebral Intrapulmonary Occipital Subcutaneous Eye anterior chamber 8 15 Intraperitoneal Intrasciatique Intragastric Intrarectal Symptoms of poisoning were said to be similar by the various routes of injection (Legroux, Levaditi, and J6ramec (11)). kill half or more of mice by the intranasal route. With botulinal toxin, a poison we consider to be a natural oral poison, intranasal instillation is more harmful to the victim than ingestion. About 60 intraperitoneal LD5o constitute 1 LD5o intranasal dose for the type A crystalline botulinal toxin. The high potency exhibited by these toxins when instilled intranasally points to a significant potential capacity for toxic protein to be absorbed from the upper respiratory and nasopharyngeal areas. Unfortunately reports of definitive studies of the efficiencies of absorption of microbial toxins from various isolated regions of the respiratory tract have not been found in a search of the literature. One may also raise the question of the relative potencies of toxin where comparisons are made of the effects resulting from inhalation of similarsized droplets of toxin in the dried state and in solutions, but there are no definitive data on this subject. No matter how deliquescent dried material might be, rapidity of complete solution would be a function of particle size. Decrease in particle size is associated with increase in surface energy and hence an increase in the tendency for small particles to dissolve. Before solution and subsequent systemic absorption could take place, more of this dried material might be expelled into the oropharyngeal area and swallowed than would be the case for droplets of toxin already in true solution. This suggests that there may be variations in potency because of differences in the opportunities for absorption in the more premeable respiratory tree than in the alimentary system. The respiratory tract as a whole may be considered to be permeable to soluble protein. As long ago as 1905, in Haliburton's Handbook of Physiology, Segalos and Meyer were quoted as having observed practically instantaneous absorption of water introduced intratracheally in dogs and rabbits. It is probable that absorption of water in such circumstances includes uptake of small amounts of water-soluble compounds although in some inverse proportion to their molecular size. In 1922 D'Aunoy (3) confirmed the possibilities for efficient precipitin production by intratracheal exposure of guinea pigs and rabbits to protein solutions. It is hard to believe that, in opposition to the tendency of the ciliated epithelium to move material up and out of the respiratory tree, the absorption of protein by the intratracheal injection of material will be limited in time to only that protein finding its way into the lower and nonciliated regions and alveoli of the respiratory apparatus. Hogben (6) has neatly stated a philosophy relevant to the problem of the penetration of tissue barriers by large-sized molecules such as microbial toxins: "Passage across cell membranes, must be considered in statistical terms of likelihood and unlikelihood. Given a sufficiently sensitive method, any substance can be shown to cross a boundary." With bacterial toxins, for example the neurotoxins, extremely small rates of passage of protein across tissue barriers can have pathological consequences. This means we cannot treat the passage of toxic proteins in the same vein as the physiologist who, in considering permeability of tissues to proteins, is generally focusing his attention on orders of magnitude of penetration considerably beyond those of concern to the bacteriologist, immunologist, and pathologist. Based on available information, it seems that, except for possibly the unbroken healthy skin, no external body surface and exposed mucosal surface should be inferred to be absolutely impermeable to bacterial toxins until proved otherwise. This is intuitively a safe point of view to adopt and has practical significance in field situations where exposure to aerosols of toxin can result in widespread contamination of the external body surfaces as well as those of the respiratory and alimentary tracts. As long ago as 1924, type

4 326 CARL LANIANNA livol. 25 A botulinal toxin in naturally contaminated food was shown by Geiger (4) to be absorbed by injured, abraded, or depilated skin and by normal mucosal tissue such as the vaginal surface. Even a substance we might logically consider to be highly impenetrable, the enamel of teeth, is claimed to be penetrable by toxin; this was reported by Berggren and Hedstr6m (2) for tetanal toxin. They also showed the degree of penetration of the enamel by toxin to be favored by glucose and fructose. The latter observation suggests a more general and, to my knowledge, unexplored question. How do the chemical constituents of the vehicle in which toxin is suspended for aerosolization affect the absorption rates of the inhaled toxin impinged on various tissue surfaces? This question seems an appropriate one for future consideration in studies of aerosols of toxins and seems to involve broader questions of how watersoluble proteins and other high molecular weight compounds including the bacterial toxins penetrate across diverse tissue barriers. QUANTITATIVE STUDIES Qualitatively, it has been clearly established, for those toxins of bacterial and plant origin which have been studied, that inhalation can result in poisoning of a variety of animals. Yet critical or exhaustive knowledge of the quantitative aspects of the subject is not available because few investigations have been published. A reviewer must depend for the most part on personal communication and cannot be certain he has tapped all the sources of quantitative experiences. A need exists for the publication of results and comment by workers who have studied problems involving inhalation of toxins. Such publication would be a welcome stimulus to studies oriented to the needs of public health, civilian defense, and laboratory safety. The quantitative data available to me are few and will be briefly summarized. W. G. Roessler, D. A. Kautter, and B. J. Wilson of Fort Detrick have found a ratio of 1:22.2:3.3 in the doses required to cause emesis in 50 % of exposed animals by the intravenous oral, and inhalation routes of administration of staphylococcal enterotoxin in rhesus monkeys (M11acaca mulatta). Employing a partially purified toxin prepared by methods developed at the University of Chicago (1), the weights of toxin yielding these results were 9, 200, and 30,ug, respectively, for the intravenous, oral, and inhalation routes. By the intravenous and aerosol exposures some monkeys actually were killed with an accompanying lung edema. But whether or not this edema is to be associated with the enterotoxin or impurities is not known. A significant sidelight of these experiments was the development of enterotoxin intoxication in several of the investigators handling animals following the aerosol exposure. More extensive work has been done with botulinal toxins, employing guinea pigs. The toxin preparations employed were of varying degree of purity and it is not possible to relate the results obtained in units of weight that would permit one to compare potency in absolute terms, namely the number of molecules per fatal dose. Tables 3 and 4 record results from guinea pig studies that were made available to us by workers at Fort Detrick (M. A. Cardella and J. V. Jemski). These data show that although the toxicity by the respiratory route is less than by a direct parenteral route, it is generally greater than by the natural oral route of poisoning, this difference being especially prominent for the type E toxin. TABLE 3. Toxicity of botulinum toxins for the guinea pig by various routes Type Mouse intraperitoneal (ip) units ip Oral Respiratory A B C D E , (M. A. Cardella and J. V. Jemski, personal communication.) TABLE 4. Toxicity of botulinum toxins for the guinea pig by various routes Type Guinea pig intraperitoneal units Oral Respiratory A B C D E 6, (M. A. Cardella and J. V. Jemski, personal conimnunication.)

5 1961 i IMMUNOLOGICAL RESPONSE TO INHALATION OF TOXINS 327 It is also evident that differences in sensitivity to the five toxin types are not as great by the respiratory as by the oral route. It should be noted that the data in Table 3 are expressed in standardized terms of LD5o for the intraperitoneal dose for mice, whereas those of Table 4 are in terms of the guinea pig intraperitoneal LD5o. The relative ranking of potency of the toxin types by the oral and respiratory routes is not the same in these tables. Thus, the relative sensitivity of a species to the different types of botulism toxins seems to vary with the standard of reference or basis on which comparisons are made. In addition, the relative ranking by parenteral exposure is shown not to be a reliable clue to the ranking for the inhalatory and oral doses. Assuming similarity in the state of dispersal of the toxins, the latter result could be interpreted to mean that there are differences in the biological efficiency either of absorption of the different botulinal toxin types from the oral and respiratory tracts or in transport of toxin to the sites of action in vivo. If the efficiencies of these mechanisms were the same, then the parenteral route of exposure should predict the relative ranking of sensitivity by both the oral and respiratory routes. Another possibility is that the differences observed are due to differential rates of destruction of the toxin types deposited in the alimentary and respiratory tracts. These considerations are raised as academic questions, to answer which no data are available. Toxic POTENCY AND BODY WEIGHT (AGE) OF EXPERIMENTAL ANIMALS A persistent and difficult question faced by students of bacterial toxins is the proper mode of expression of potencies for making interspecies and intraspecies comparisons, a problem we have recently reviewed (9, 10). By the parenteral route of injection the lethality of a bacterial toxin can be independent of the weight (age) of a susceptible animal. We have shown this to be true for intraperitoneal injection of botulinal and tetanal toxins in mice. Yet it would be improper to generalize this experience for other routes of injection. For example, both these neurotoxins, when administered to mice per os, in contrast to what common sense might suggest, tend to be required in larger amounts to kill the youthful mouse than the older heavier mouse (10). This tendency seems to be greater for the type A botulinal toxin than for tetanal toxin. Stimulated by these experiences we have determined the LD5o dosage of given solutions of toxin with mice of different weight employing intranasal instillation. The results are recorded in Table 5. Here in contradistinction to prior experience with the intraperitoneal and oral routes the potency by the respiratory route is affected in rough proportion to the weight of the victim so that the larger mouse requires a larger dose of toxin for lethality than the smaller mouse. Without further and extensive study one cannot read too much into these data by way of any attempted explanation of why a respiratory route should show a direct proportional relation between age or weight of the mouse which is absent for the intraperitoneal and oral routes. The explanation may be one of anatomy, physiology, technique, or any possible combination of these. The upper mucosal tissues of young mice might be more permeable to systemic absorption of protein than those of older mice. Simply a subtle aspect of technique may be involved. Possibly, in spite of our unwillingness to admit it, the larger mouse is a sly creature who is quite successful in expelling a portion of intranasally instilled fluid without obvious notice to the investigator, or in franker colloquial terms, in pulling the wool over the observer's eyes. The anatomy and the mechanics of breathing may favor deeper penetration and retention in the respiratory tree of the small than large mouse when the toxic fluid is intranasally instilled. It also cannot be ignored that intranasal instillation is done under light anaesthesia, and it is not known if these results would be duplicated if unanaesthetized mice were directly exposed to aerosols. What is important is the fundamental unanswered question these data illustrate, namely, what is the most physiologically meaningful way for stating comparative respiratory doses of toxin in animals of different age (weight) and species? The information at hand makes one cognizant of the possible danger in any attempt to extrapolate directly from data on potency based on a parenteral route of exposure to potency by inhalation. IMMUNIZATION Having recognized the capacity of toxins to cause harm by inhalation, it becomes important to learn how specific antitoxin can act prophylactically and therapeutically against this route

6 328 CARL LATMANNA [VOL. 25 TABLE 5. Mortality of female mice of varying weight upon intranasal instillation of toxins Weights of mice Response to toxin Toxin Large Small Ratio: large/ small No. deaths/no. injected Large Small Ratio of toxin potency for small to large mice* Tetanal Range Avg... SD... Range... Avg... SD.. Type A botulinal Range... Avg... SD... Range... Avg... SD... Range... Avg... SD g g /89 19/54 45/60 13/60 20/60 52/89 35/60 56/60 22/60 39/60 Not calculable * Based on LD50 values obtained by titration of given toxin solutions in small and large mice. Values larger than one indicate greater potency for small (younger) mice. SD = standard deviation. of poisoning. Again there are only a few publications on the subject and these provide scanty quantitative information. Passive and active immunization can protect experimental animals. As expected, the efficiency of protection is best if antitoxin is present before any symptoms of poisoning develop. Jakolev (7) could not protect with antitoxin given later than 10 to 12 hr after the inhalation of types A and B botulinal toxin by mice and guinea pigs. He found the protection by the antitoxin given after inhalation of toxin to be related to the length of time it takes one lethal respiratory dose to kill a particular species. Thus, the mouse, which dies in 3 to 4 days after exposure, could receive a protective parenteral dose of antitoxin after inhalation of toxin somewhat later than the guinea pig, which dies in 1 to 2 days. Of greater interest and in need of further confirmation is Jakolev's (7, 8) finding that higher levels of antitoxin are required for prophylaxis against inhalation of botulinal toxin than against poisoning by other routes of exposure. One antitoxin unit protected the mouse against only one lethal respiratory dose, whereas it protected against 20 lethal subcutaneous doses and 50 lethal oral doses. This result could imply a quantitative difference in the opportunity for the toxin reaching the effector sites to contact antitoxin. We should recall that by all these routes, death results from poisoning of the breathing apparatus. Might it be that absorption of botulinal toxin from the respiratory tract and transport to the neural sites of poisoning of the musculature of the breathing apparatus excludes the hematogenous route for the most part? After all, the level of antitoxin is higher in the blood than in other body fluids, and by the subcutaneous and oral routes it is probable that the absorbed toxin is, during the course of normal events, picked up in the blood before final deposition is made in the tissue fluid bathing the neural sites of fatal toxic action. Understanding would be assisted if detailed knowledge were available on the routes of transport carrying absorbed aerosolized toxin away from the mucosal surface of the respiratory tree. It is unfortunate, too, that we do not know if the experience with botulinal toxin is duplicated by inhalation of aerosols of other kinds of toxins. Other possible explanations must be con-

7 19611J IMMUNOLOGICAL RESPONSE TO INHALATION OF TOXINS 329 sidered before any final conclusions can be and treatment against exposure to aerosols of reached. It should be recalled that it takes more toxin. tetanal toxin to kill by intranasal instillation in To conclude I would like to express a sense of mice than by the intraperitoneal route. In one dissatisfaction with the accomplishment of my trial, a fixed amount of passively introduced task. A mole hill rather than a mountain of data antitoxin resulted in a 25-fold decrease in the has been mined. It may justify some satisfaction intraperitoneal LD5o dose with only a 5-fold to have had the opportunity to cast suspicion decrease in the intranasal LD5o dose. Consequently it does seem that a given amount of situation or ecology of the toxemia of bacterial upon any tendency of knowledge of the natural antitoxin might be more protective against a disease to shut our eyes to the potentialities for parenteral than a respiratory route of injection. poisoning by an unnatural route such as exposure But, might this merely be a reflection of the fact to aerosols. Some solace can rest on the hope that that a greater weight of toxin constitutes the along the way in revealing this potentiality, intranasal dose? This question could be considered intelligently if it were known how much been been raised. Two of these questions would possibly questions of broad scientific interest have of the difference between a parenteral and be: what are the mechanisms of permeability of respiratory lethal dose actually represents the respiratory tract tissues to high molecular weight difference in the amounts of systemic absorption substances and what are the routes of transport of toxin and in turn how much of this absorbed of large molecules away from the areas of penetration? toxin is effective. By effective toxin we mean those molecules actually in a position to make their way to effector sites for mortal action. LITERATURE CITED In oral toxicity it seems certain that only a 1. BERGDOLL, M. S., B. LEVIN, M. SURGALLA, small portion of the ingested toxin escapes the AND G. M. DACK Chromatography in intestinal barrier (5) and therefore it is not the preparation of staphylococcal enterotoxin. Science 116: surprising that antibody in vivo is more protective against ingestion than against other modes of 2. BERGGREN, H., AND J. HEDSTROM Experimental studies in vivo on the permea- poisoning, including respiratory routes. For oral toxicity another reason for the greater bility of enamel with particular reference to protective value of antibody may be the association of a slow rate of absorption with the presence the effect of sugar solutions. J. Dental Research 30: D'AUNOY, R Antibody production by of antitoxin in the saliva, which is being constantly swallowed. Thus, a portion of ingested fectious Diseases 30: intratracheal injection of antigen. J. In- toxin can be specifically neutralized before 4. GEIGER, J. C The possible danger of systemic absorption takes place. Sugg and Neill absorption of toxin of B. botulinus through (13) long ago proved the presence of diphtheria fresh wounds and from mucous surfaces. antitoxin in the saliva of naturally immune Am. J. Public Health 14: human subjects and estimated the daily output 5. HECKLY, R. J., G. J. HILDEBRAND, AND C. of salivary antitoxin to be 0.07% of the total LAMANNA On the size of the toxic amount of antitoxinin the blood. Most interesting particle passing the intestinal barrier in was the finding of a constant ratio of antitoxin in botulism. J. Exptl. Med. 111: saliva to antitoxin in serum of about 1.4 X 10-s. 6. HOGBEN, C. A. M The alimentary tract. I know of no comparable studies on the presence Ann. Rev. Physiol. 22: of antitoxin in the mucous fluid bathing the 7. IAKOLEV, A. M The importance of antitoxic immunity for the defense of the body external surfaces of the epithelium of the respiratory tree and how this might vary with age and in respiratory penetration of bacterial toxins. I. The role of passive immunity in the other circumstances, such as location in the different areas of the tract. ease caused by Clostridium botulinum toxins. defense of the body against respiratory dis- Poorly explored are the possibilities for utilization of inhalation of antitoxin under definable lish translation) 29: J. Microbiol. Epidemiol. Immunobiol. (Eng- conditions which might prove more effective than 8. IAKOLEV, A. M The importance of antitoxic, immunity for the defense of the parenteral injection of antitoxin for protection body

8 330 CARL LAMANNA [VOL. 25 in respiratory penetration by bacterial toxins. II. Tests of antitoxic immunity in white mice and guinea pigs upon intranasal administration of Clostridium botulinum toxins. J. Microbiol. Epidemiol. Immunobiol. (English translation) 29: LAMANNA, C The most poisonous poison. Science 130: LAMANNA, C Oral poisoning by bacterial exotoxins exemplified on botulism. Ann. N. Y. Acad. Sci. 88: LEGROUX, R., J. C. LEVADITI, AND C. JeRA- MEC Influence de voies d'introduction de la toxine sur le botulisme expdrimentale du lapin. Ann. inst. Pasteur 71: RIVERA, J. A Cilia, ciliated epithelium and ciliary activity. (A bibliographic review.) Pergamon Press, New York (in press). 13. SUGG, J. Y., AND J. M. NEILL Loss of immune substances from the blood. II. Diphtheria antitoxin in human saliva. J. Immunol. 20: