FURTHER OBSERVATIONS ON THE RELATION OF THE EYE TO IMMUNITY IN EXPERIMENTAL SYPHILIS : II. THE DEVELOPMENT OF IMMUNITY AFTER PRIMARY INTRACORNEAL INOCULATION

Two experiments are reported in which an attempt was made to determine the extent to which a primary syphilitic infection of the cornea in rabbits is followed by the development of a local corneal immunity, by the generalization of the virus, and by the development of a general immunity to the infection. Female rabbits were inoculated intracorneally with a virulent strain of T. pallidum and the disease was allowed to run its course until the lesions which had developed at the site of inoculation had healed spontaneously. Popliteal lymph nodes were transferred from about one-half of these animals (in the second experiment only) to normal male rabbits and in almost every instance the nodes were proved by this method to contain virulent treponemes, showing that generalization of the syphilitic infection is the rule after intracorneal inoculation. All animals were treated with arsphenamine after the local lesion had subsided (160 and 275 days after the original inoculation). The rabbits were then reinoculated with the homologous strain of treponemes, injections being made into the cornea originally inoculated and also into the skin of the back. In one experiment both corneas were reinoculated. The incidence of lesions developing in either cornea after reinoculation was higher than the incidence of lesions developing in the skin. The lesions developing in the corneas of the "immune" animals had a longer incubation period and were of shorter duration on the average than the lesions in the control group. As far as intensity of reaction was concerned no difference was observed. Inoculation of the cornea of rabbits with syphilitic virus is often followed by the development of immunity to the homologous strain of organisms. This immunity is imparted to the skin to a greater extent than to either the cornea inoculated originally or to the opposite uninoculated cornea. It persists after treatment with arsphenamine. It appears to be more marked the longer treatment is postponed. It is apparent from Table IV that in one-half of all the test animals both cornea and skin were immune to a second inoculation of homologous syphilitic virus. In addition to these there were 5 animals in which the cornea was immune and the skin nearly so, for the lesions which developed in the skin of these animals were minimal in extent and duration and were in no way comparable to those which were observed in the controls. Thus in two-thirds of our animals there developed in both the skin and the cornea, after a primary intracorneal inoculation, a high degree of resistance toward a second inoculation with homologous syphilitic virus, but syphilitic disease of the cornea does not always impart to the cornea itself an absolute immunity to reinoculated homologous virus.     

THE IN VITRO PRODUCTION OF ANTIBODY BY DELAYED PHASE CORNEAL LIMBAL LYMPHOID FOCI

The intracorneal inoculation of a sufficient quantity of a soluble protein antigen into the eye of a rabbit produces a biphasic allergic reaction in that cornea. The earlier stage, characterized by a diffuse corneal clouding, is a manifestation of delayed hypersensitivity and is accompanied by a limbal infiltrate composed predominantly of lymphocytic-mononuclear elements. The later response, known as the Wessely Phenomenon, is a ring of opacification in the cornea which is visible in the gross. This reaction is dependent upon the presence of specific circulating antibodies and is therefore classified among the immediate types of hypersensitivity. It is accompanied by a dense limbal infiltration of plasma cells. Intervening between the two reactions is a period of several days during which the eye appears relatively normal. Explants containing large numbers of infiltrating lymphocytic-mononuclear elements were removed from the corneal-scleral junction of experimental eyes during the height of the delayed type hypersensitivity reaction and maintained in vitro in tissue culture. At a later date the overlay fluid in which the explants were maintained was shown to contain specific humoral antibodies, demonstrating the capability of cells present at a delayed reaction for the later production of antibodies. The possible linkage of the two modes of immune phenomena is discussed.     

THE COURSE OF EXPERIMENTAL AUTOALLERGIC THYROIDITIS IN INBRED GUINEA PIGS : THE PATHOLOGIC CHANGES AND THEIR RELATIONSHIP TO THE IMMUNE RESPONSE OVER A 2 YEAR PERIOD

Experimental allergic thyroiditis produced in strain 13 histocompatible guinea pigs after a single immunization with thyroid extract and Freund's adjuvant was followed for more than 2 years. The disease appeared as early as 5 days and persisted for the entire period studied, although it regressed in the later stages. Circulating antithyroid antibody was detected at low levels as early as 7 days after immunization, and increased to a peak at the time of most severe disease. Thereafter, antibody decreased, but was still detectable in most animals as late as 2 years. There was no correlation between antibody levels and extent of disease except at the 7 week stage. Delayed sensitivity to thyroid antigen was found as early as 5 days after immunization, and appeared to precede the development of thyroiditis in many animals. It correlated closely with thyroiditis at 5 days and 7 weeks. At 6 months, the delayed skin reaction was decreased, and a modified type of reaction appeared which persisted as long as 26 months. The time relationship of delayed sensitivity, thyroiditis, and circulating antibody continue to confirm the role of delayed sensitivity in the pathogenesis of this disease. The accumulated data demonstrating production of thyroiditis without antibody, and the converse, tend to strengthen this view.     

STUDIES ON NORMAL AND IMMUNE LYMPHOCYTE TRANSFER REACTIONS IN GUINEA PIGS, WITH SPECIAL REFERENCE TO THE CELLULAR CONTRIBUTION OF THE HOST

Using guinea pigs of strains 2 and 13 and their F₁ hybrids as experimental subjects, various lines of evidence have been obtained that in this species, as in all others tested, the only significant cellular antigens with which donor lymphocytes engage when normal and immune lymphocyte reactions are incited are radiosensitive leukocytes. Constitutive cells of the skin are unimportant. (a) The intensities of these reactions in irradiated subjects are dependent upon the peripheral leukocyte concentration. When this falls below a certain threshold no reactions are incitable. (b) Highly leukopenic animals are capable of developing immune lymphocyte transfer (ILT) reactions if normal lymphoid cells of their own genetic constitution are mixed with the putative attacking donor cells, as "supplementing antigen," before inoculation. (c) Radiation-chimeric strain 13 animals having F₁ hybrid leukocytes in their bloodstream give typical ILT reactions when challenged intradermally with strain 13 anti-2 node cells. Exposure of strain 2 animals to 600 R does not prevent their becoming actively immunized if, 24 hr later, they are injected intradermally with strain 13 lymphocytes. However, this sensitization, revealed by the host's capacity to give delayed hypersensitivity reactions, wanes as leukopenia progresses. On the basis of this and other findings it is argued that the flare-up stage of the NLT reaction in preirradiated hosts is mainly an expression of host sensitivity against the transferred alien cells. Two unexpected observations have been made in the course of this study: (a) F₁ hybrid animals developed what appeared to be a strong delayed hypersensitivity after intradermal inoculation with parental strain lymphoid cells or antigenic extracts prepared from them. (b) If strain 13 guinea pigs which had been sensitized against strain 2 tissue antigens by intradermal injection of lymphocytes 7 days beforehand were inoculated intravenously with strain 2 antigenic extract a significant proportion of the animals developed severe delayed necrotizing reactions, recall flares, at some or all of the healed skin inoculation sites.     

THE INFLUENCE OF THE FACTORS OF SEX,AGE, AND METHOD OF INOCULATION UPON THE COURSE OF EXPERIMENTAL SYPHILIS IN THE RABBIT

For convenience the results are summarized in tabular form. Table I shows a comparison of the primary reaction in the groups of female and male rabbits inoculated intradermally. In Table II the incidence of metastatic lesions is shown in the various groups of animals. In Table III are shown the character and date of appearance of the generalized lesions (exclusive of orchitis) in the various groups of animals. The results presented in Tables I, II, and III, together with a study of the individual protocols, permit the following general statements to be made relative to the effect of the factors studied upon the course of experimental syphilis in the rabbit. Sex.—In the group of females inoculated intradermally the lesions at the site of inoculation were in general less marked than in the group of males similarly inoculated. Moreover, they attained their maximum size earlier and began to recede earlier than was the case in the males. In none of the females was there clinical evidence of the production of generalized lesions, while in the corresponding group of males metastatic lesions of skin or bones detectible by clinical examination occurred in one instance, or 14.3 per cent. In both groups there was wide variation in the character of the initial reaction in individual rabbits and in the time required for it to reach its maximum size. See PDF for Structure Age.—In the five young males inoculated intratesticularly the disease was not greatly different from that observed in the older males similarly inoculated. The initial reactions were slightly slower in making their appearance, but the magnitude of the reaction, as judged by enlargement of the testicles, was somewhat greater. The percentage incidence of metastatic orchitis was almost the same in the two groups. The younger animals showed a slightly greater incidence of generalized lesions involving structures other than the testicles, although the difference was insignificant, but the lesions were smaller, fewer in number, and confined entirely to the skin, and there was no instance of severe generalized lesions. Moreover, the occurrence of metastatic lesions (skin) in the younger animals was definitely delayed. Method of Injection.—In the animals inoculated by the intratesticular route the development of the primary reaction and the incidence of generalized lesions involving skin and bone were much greater than in See PDF for Structure the animals inoculated intradermally. Thus, of the latter group only one, or 14.3 per cent, showed a secondary metastatic lesion (skin), while in the group inoculated by the testicular route the incidence of metastatic skin and bone lesions was as high as 71.4 per cent. The difference in the character of the disease exhibited by these two groups of animals inoculated in a different manner is quite striking. Intratesticular inoculation produced a much more violent local reaction and a much greater incidence of generalized lesions than did intradermal inoculation. Effect of Castration.—In the group of animals with unilateral orchitis in which the infection was allowed to run its course without any attempt at suppression by removal of the initial focus, metastatic lesions appeared more promptly and in slightly greater incidence than See PDF for Structure in the group of animals in which the initial local reaction was suppressed by removal of the focus itself through castration. The percentage differences are not very great, but they are constantly in favor of the first group, as is the time interval of appearance. This experiment is not in strict accord with similar experiments observed by Brown and Pearce (3) in the past, and while the reasons for this difference are not clear the fact may be noted that the incidence of generalized lesions in uncastrated animals is somewhat higher than that reported by these authors, also that there was a high percentage of recurrence of primary lesions.     

FURTHER STUDIES ON ADOPTIVE TRANSFER OF SENSITIVITY TO SKIN HOMOGRAFTS

Mice or rats that have been rendered tolerant of skin homografts from an alien donor strain furnish the basis of a very sensitive and objective test system for investigating the competence of cellular inocula from specifically immunized isologous donors to transfer sensitivity adoptively. By means of this test system it has been shown that immunologically "activated" cells, capable of transferring homograft sensitivity, are present in the blood, peritoneal exudates, and regional nodes of animals that have rejected skin homografts. Leucocytes were as effective as regional node cells. Activated cells were first demonstrable in the regional nodes and blood of skin homograft recipients at the same time,—on the 6th postoperative day,—suggesting that these cells must enter the circulation very soon after their formation in the nodes. Moreover, when sensitization was effected by skin homografts, but not by means of splenic cell suspensions inoculated intraperitoneally, activated cells are highly persistent, still being demonstrable in both the blood and the nodes more than a year after sensitization. The finding that thoracic duct cells, which are almost exclusively lymphocytes, were just as effective as leucocytes or regional nodes in transferring sensitivity in rats formally identifies the cell type responsible for transferring sensitivity in the various tissues tested. Attempts to transfer sensitivity to homografts in normal mice or tolerant mice by means of larger dosages of activated lymphoid cells sequestered in Millipore chambers inserted intraperitoneally were unsuccessful. All this, and other evidence presented, lends strength to the thesis that skin homograft immunity is a cell-mediated reaction.     

THE EFFECT OF ANTECEDENT INFECTION AND IMMUNIZATION WITH STREPTOCOCCI UPON THE REACTIVITY OF RABBITS TO HORSE SERUM

1. The cutaneous responses of rabbits to small doses of horse serum intracutaneously is described. After an original injection of 0.1 cc. a secondary reaction often occurs about the 9th day, and tests at 3 day intervals with 0.001 cc. quantities indicate that general skin hypersensitivity is established at this time. Circulating precipitins for horse serum appear later. As the degree of sensitivity increases, lesions resulting from test doses reach a maximum development more quickly. 2. The reactivity of rabbits to horse serum is greatly increased by antecedent sensitization of the animals with repeated small intracutaneous inoculation of indifferent streptococci or immunization with large intravenous injections of either indifferent or hemolytic streptococci. Doses of indifferent streptococci precisely comparable to those producing greatly enhanced reactivity when given intracutaneously, increase reactivity to horse serum irregularly and to slight degree when injected intravenously. 3. Increases in reactivity (allergic irritability) are made evident by the occurrence of larger skin lesions at the site of the primary horse serum injections, the earlier development of more distinct secondary reactions in a higher percentage of animals, and by the more rapid appearance of skin sensitivity to the small test doses which may be first evidenced by delayed reactions at these injection sites. Accompanying these evidences of increased reactivity, there is an early appearance of blood serum precipitins in high titer. 4. There was no evidence of residual inflammation in any of the animals at the time reactivity to horse serum was tested. No attempt was made to determine the duration of this altered reactive capacity. It has been encountered in tests begun from 8 to 14 days after the last preliminary inoculation. 5. It is suggested that the increased reaction described is brought about by a persisting alteration in the functional activity of the reticulo-endothelial system.     

HISTOPATHOLOGICAL AND HISTOCHEMICAL ALTERATIONS IN THE EARLY STAGES OF CORNEAL GRAFT REJECTION

Rejection of corneal grafts was produced in rabbits after skin from the corneal donor was grafted subcutaneously. Clinical observations showed that the graft sickness started at the periphery of the graft after blood vessels from the host cornea reached the scar. Histologic studies demonstrated that the scar tissue was first invaded by vessels and infiltrated by lymphocytes, monocytes, and plasma cells. This infiltrate reached the endothelium through the gap in Descemet's membrane. The early histological picture of the graft sickness was characterized by endothelial destruction and infiltration at its junction with the scar. Keratocytes changed in shape and apparently became active fibroblasts. Decreased amounts of ground substance in the early stages of rejection, as indicated by appearance of haziness in the graft, were indicated in sections by a decrease in metachromatic staining. Diminished uptake of radioactive sulfate also occurred in the early stages of the graft sickness; which was followed by an elevated uptake of sulfate as the graft became opaque and filled with active fibroblasts.     

ETIOLOGY OF OROYA FEVER : VII. THE RESPONSE OF THE SKIN OF MACACUS RHESUS AND ANTHROPOID APES TO INOCULATION WITH BARTONELLA BACILLIFORMIS.

Bartonella bacilliformis failed to induce lesions when merely rubbed on the surface of the intact skin of a chimpanzee, an ourang-utan, and numerous Macacus rhesus monkeys, although when applied to the scarified skin of the same animals it gave rise to extensive lesions. Application of infectious material to the scarified skin did not always induce verruga lesions, but intradermal inoculation almost invariably gave rise to nodule formation. The localization of Bartonella bacilliformis in the skin is not, in experimental animals, determined by mechanical factors, since scarification of the skin or intradermal injection of foreign substances in monkeys infected with Bartonella bacilliformis does not give rise to verruga formation. The degree of susceptibility of the skin tissues appears to be considerably diminished during the course of experimental infection with Bartonella bacilliformis. Inoculation of the scarified skin of infected animals gave uniformly negative results, and intradermal inoculation induced only a mild local reaction. In a few exceptional instances, however, of animals previously infected with the strain of Bartonella bacilliformis derived from a human verruga nodule, reinoculation with the same strain gave rise to unusually marked reactions. The evolution of the skin lesion induced in experimental animals by Bartonella bacilliformis may be divided into four stages, the period of incubation, the initial stage, the mature and vascular stage, and the regression. In the initial stage the lesion is a pure angioendothelioma, but in the stage of full development the histological picture is complicated by connective tissue proliferation and occasionally also by penetration of epidermis into the lesion. The demonstration of Bartonella bacilliformis in the endothelial cells distinguishes the lesion from others which simulate it. The cutaneous lesions known as verruga nodular, verruga mular, and verruga miliar have been reproduced in monkeys.     

FURTHER OBSERVATIONS ON THE RELATION OF THE EYE TO IMMUNITY IN EXPERIMENTAL SYPHILIS : III. THE INFLUENCE OF A NON-SPECIFIC INFLAMMATORY REACTION IN THE CORNEA ON THE DEVELOPMENT OF IMMUNITY IN THAT TISSUE AFTER INTRATESTICULAR INOCULATION

Two experiments are reported in which rabbits originally inoculated with syphilis and treated late in the course of the disease (174th to 210th day) were reinoculated subsequently in both corneas with the homologous strain of syphilitic virus. In each animal one cornea was inoculated with dead tubercle bacilli prior to reinoculation with the syphilitic virus. This procedure was carried out in order to bring about a non-specific inflammatory reaction with resultant vascularization, the intention being to find out if such vascularization would render the cornea more resistant to inoculation with the homologous strain of syphilitic virus. The results of both experiments were similar and while they were not conclusive, they indicated that there was a tendency for corneas which had been injected with dead tubercle bacilli to be more refractory to a subsequent inoculation with homologous syphilitic virus than the corneas of the same animals that had not been injected with dead tubercle bacilli. This tendency may be interpreted as suggestive evidence for the view that in the syphilitic rabbit there develop circulating antibodies toward the homologous strain of T. pallidum.     

TRANSMISSION OF RESPIRATORY ANAPHYLAXIS (ASTHMA) FROM MOTHER TO OFFSPRING

Rabbits immunized intravenously with living culture or nucleoproteins of non-hemolytic streptococci react to subsequent intracutaneous inoculations with homologous streptococci with smaller and harder lesions than are shown by normal animals similarly inoculated; and they do not develop the general manifestations of hypersensitiveness such as are shown by animals previously inoculated into the tissues with the same cultures. A rabbit may react to intracutaneous inoculation with non-hemolytic streptococci in one of four ways, depending on whether it is normal, hypersensitive, immune or cachectic. Most normal animals show a secondary reaction about 10 days after inoculation with suitable strains of non-hemolytic streptococci; hypersensitive, allergic, or hyperergic animals show much larger lesions than do normals with the corresponding doses of the same streptococci, and practically never show secondary reactions; immune animals show smaller and harder early lesions and usually do not have secondary reactions if they are fairly well immunized. Cachectic animals show very soft and rapidly fading primary reactions and no secondary reactions.     

STUDIES ON VARICELLA : THE SUSCEPTIBILITY OF RABBITS TO THE VIRUS OF VARICELLA.

1. The localization of the virus of varicella in the human skin is influenced by irritation. This is indirect evidence that the virus is in the blood. 2. Rabbits are susceptible to a virus recovered from the blood of varicella patients. 3. Testicular emulsions containing the virus are free from ordinary aerobic and anaerobic bacteria, and produce lesions in the cornea, skin, and testicles of rabbits. 4. The virus can be transmitted indefinitely from rabbit to rabbit by means of testicular inoculation, and can be preserved 29 days in 50 per cent glycerol at a low temperature.     

REACTIONS OF RABBITS TO NON-HEMOLYTIC STREPTOCOCCI : II. SKIN REACTIONS IN INTRAVENOUSLY IMMUNIZED ANIMALS.

Rabbits immunized intravenously with living culture or nucleoproteins of non-hemolytic streptococci react to subsequent intracutaneous inoculations with homologous streptococci with smaller and harder lesions than are shown by normal animals similarly inoculated; and they do not develop the general manifestations of hypersensitiveness such as are shown by animals previously inoculated into the tissues with the same cultures. A rabbit may react to intracutaneous inoculation with non-hemolytic streptococci in one of four ways, depending on whether it is normal, hypersensitive, immune or cachectic. Most normal animals show a secondary reaction about 10 days after inoculation with suitable strains of non-hemolytic streptococci; hypersensitive, allergic, or hyperergic animals show much larger lesions than do normals with the corresponding doses of the same streptococci, and practically never show secondary reactions; immune animals show smaller and harder early lesions and usually do not have secondary reactions if they are fairly well immunized. Cachectic animals show very soft and rapidly fading primary reactions and no secondary reactions.     

OBSERVATIONS ON THE RELATION OF THE EYE TO IMMUNITY IN EXPERIMENTAL SYPHILIS

Three experiments are reported in which an attempt has been made to determine the extent to which the eye participates in the general resistance which develops in rabbits during the course of syphilitic infection. Rabbits treated with arsphenamine well after the period when they would be expected to be immune to intratesticular or intracutaneous inoculations were reinoculated with the homologous strain of T. pallidum, the organisms being injected into either the cornea itself or the anterior chamber. Altogether in the three experiments 43 presumably immune animals were injected, 25 into the cornea itself and 18 into the anterior chamber. 41 normal animals were used as controls. In the immune animals 27 or 62 per cent showed lesions in the cornea. 14 of the 43 test animals were inoculated simultaneously in the cornea and in the skin of the back. In 9 of these 14, lesions developed in the cornea although no lesions developed in the skin. The lesions developing in the corneas of the "immune" animals had a longer incubation period on the average, were often of longer duration, and in some instances were more severe than the lesions developing in the control animals. In the case of some animals, also, they showed a greater tendency to recur. The immediate reactions in both the normal and the "immune" animals were entirely comparable and there was no evidence of an accelerated reaction in the test animals. It is concluded that the eye of the syphilitic rabbit does not share to the same extent as other tissues in the general resistant state which develops in that animal during the course of syphilitic infection. Possible explanations for this finding are discussed.     

IN VITRO DEMONSTRATION OF CELLULAR SENSITIVITY IN ALLERGIC ENCEPHALOMYELITIS

Peritoneal cells obtained from animals exhibiting delayed hypersensitivity are inhibited from migrating in vitro by specific sensitizing antigen. This test for the detection of delayed hypersensitivity was applied to the problem of cellular sensitivity in allergic encephalomyelitis (AE). The migration of peritoneal cells obtained from guinea pigs with AE was inhibited specifically by nervous tissue antigens. The specificity of this reaction was further studied. Neonatal rat nervous tissue, which was shown to lack the encephalitogenic antigen, i.e. did not produce AE when injected with complete adjuvant into guinea pigs and rats, did not inhibit the migration of cells from animals with AE. Adult rat nervous tissue, which readily produces AE, and thus contains the encephalitogenic antigen did inhibit the migration of such cells. The finding that cells from animals with AE display hypersensitivity which appears to be directed specifically to the encephalitogenic antigen strongly supports the view that such cells could play an important role in the pathogenesis of this disease.     

Heart muscle performance after experimental viral myocarditis.

As part of an inquiry into possible antecedents of idiopathic cardiomyopathy, acute experimental coxsackie virus myocarditis was studied for late structural and functional sequelae. Myocarditis was induced in 12- and 22-day-old hamsters by inoculation with coxsackie virus B3. Early viremia occurred, followed by virus replication in heart muscle. Maximum peak developed tension (Tpd) of isometrically contracting isolated heart muscle was depressed 17 and 43% in the animals inoculated at 12 days, and studied 18 and 90 days later, respectively, as compared to their uninoculated controls. In both infected groups, less muscle stretch was required to reach the length at which Tpd was produced. Animals studied 180 days after inoculation did not differ from controls. The muscles from animals inoculated at 22 days of age and studied 18 days later showed a 15% depression of Tpd compared to their controls. Glycerinated muscles from this infected group developed 50% less tension than their controls. The muscles of hamsters inoculated with virus at 22 days and studied 90 and 180 days later showed no change in Tpd. The data suggest that contractility and compliance of heart muscle are decreased 18 days after inoculation, but recover by 90 days if the animals are inoculated at age 22 days. However, if the animals are inoculated at a younger age (12 days), depression of myocardial performance persists for at least an additional 90 days. It is concluded that the inflammatory stage of experimental acute coxsackie virus B3 myocarditis in the Syrian golden hamster may be followed by residual alterations in contractile proteins and myocardial function.     

STUDIES ON THE ETIOLOGY OF RABBIT POX : II. CLINICAL CHARACTERISTICS OF THE EXPERIMENTALLY INDUCED DISEASE

The clinical manifestations and course of disease observed in experimental rabbit pox have been described and analyzed. The condition differed from the acute fulminating and rapidly fatal type of experimental infection (1) in that the period of survival was longer, a variety of clinical manifestations developed and a considerable proportion of the cases recovered. The most conspicuous symptom was a generalized papular eruption on the skin and mucocutaneous borders. The production of the disease was associated with routes of inoculation other than the intratesticular or with a small dosage. The majority of cases were inoculated with Berkefeld V filtrates of tissue-virus emulsions and not with the more potent unfiltered emulsions. The local reactions resulting from various routes of inoculation were described. Of special interest were the pronounced cutaneous reactions induced by intradermal injection, the high instance of marked clinical manifestations after intravenous inoculation, the failure of lesions to localize in the lines of scarification of skin and cornea even in cases with a profuse cutaneous eruption, and the development of cytoplasmic inclusion bodies in the epithelial cells of the cornea following scarification and conjunctival instillation of virus. In the character of its clinical manifestations and course of disease, experimental rabbit pox was indistinguishable from cases of the spontaneous pox.     

STUDIES ON HERPETIC INFECTION IN MICE : IV. THE EFFECT OF SPECIFIC ANTIBODIES ON THE PROGRESSION OF THE VIRUS WITHIN THE NERVOUS SYSTEM OF YOUNG MICE

Two-week-old mice inoculated with herpes virus on the pad of a hind foot regularly developed paralysis of the infected limb followed by paraplegia and encephalitis terminating fatally 5 or 6 days after inoculation. Hyperimmune rabbit serum given intraperitoneally at the time virus was inoculated on the foot pad prevented the formation of an herpetic lesion of the foot pad. When the antiserum was given 12 hours after inoculation of the virus, a typical infection of the epithelium of the foot pad developed, but the virus was prevented from causing obvious signs of infection of the nervous system in many of the animals. Amputation of the foot 2 hours after the inoculation of the virus prevented the paralysis of the hind leg. Some of the mice died of a delayed encephalitis. Amputation of the foot at 24 hours neither prevented nor delayed the sequence of paralysis of the hind leg, encephalitis, and death. In order to study immune serum therapy of an infection of the nervous system uncomplicated by a peripheral focus of infection or by traumatic disturbance of the central nervous system, 2-week-old mice were inoculated on the foot pad, the infected feet were amputated 24 hours later, and the immune serum was administered at varying intervals thereafter. Using litter mate controls and statistically significant numbers of mice, it was shown that hyperimmune rabbit serum, administered during the first one-third of the incubation period, retards and, in some cases, arrests the progress of herpetic infection within the nervous system.     

THE PROBLEM OF THE ETIOLOGY OF HERPES ZOSTER

Attempts were made to produce lesions in. animals by the injection of material obtained from the vesicles and involved skin of nine cases of herpes zoster. All the cases, with the exception of one (Case II), were characteristic cases of idiopathic herpes zoster and the question of their being cases of so called zosteriform herpes or symptomatic herpes zoster can hardly be raised. As regards Case II, if this case occurred alone, there might be some doubt as to its nature on account of the mildness of the symptoms and the small area of skin involvement. Taken in connection with Cases III and IV, however, which occurred in the same ward and in patients who were quite closely in contact with Patient II, it seems fairly reasonable to assume that they were all of the same character. Cases of herpes zoster have been extremely rare in this hospital and the occurrence of three cases in the same ward within a very short period of time suggests very strongly a transference of infection from one case to the other. That Case II was not one of herpes simplex also seems fairly certain from the negative results obtained by inoculation of rabbits'' eyes with vesicle material. In making the animal experiments we employed various methods which were suggested largely by the technique used by previous observers, especially by those who have reported results which were considered positive. In making inoculations into the corneas the technique recommended by Lipschütz was employed as far as possible. Young rabbits were used and the material was obtained from fresh vesicles early in the disease and inoculated with as little delay as possible. The material injected into rabbits'' eyes was obtained from seven cases and twenty-four rabbits were used. In judging of the results obtained in this kind of experimentation great caution must be observed. Our experience convinces us that slight opacities occurring along the lines of scarification and mild conjunctivitis cannot be held to indicate the effect of a specific virus. As regards the interpretation of the microscopic changes found, we were quite familiar with the appearance of intranuclear inclusion bodies as seen in the lesions of experimental herpes simplex and the filterable virus (Virus III) indigenous to rabbits described by Rivers and Tillett (5). We also had no difficulty in imding intranuclear inclusions in the sections of skin removed from patients. It is not likely, therefore, that these structures were overlooked in our study of the sections. Briefly stated, although the material studied was satisfactory and in spite of the fact that a considerable number of animals were used for each case, we have been unable to confirm the observations of Lipschütz regarding the experimental production of specific lesions in the corneas of rabbits. We realize that this is only negative evidence and therefore not of conclusive importance in view of Lipschütz''s observations. It indicates, however, that the production of specific lesions in rabbits'' eyes with material from herpes zoster vesicles is extremely difficult and that successful results may be a matter of chance, depending, possibly, on peculiar susceptibility on the part of the rabbits. In view of the fact, however, that a careful analysis of the positive results reported by other observers shows that the conclusions were based on insufficient evidence, we believe that further work is necessary before the successful inoculation of the rabbits'' corneas with herpes zoster virus can be accepted as fully demonstrated. To make the evidence convincing specific lesions should be obtained with a fair degree of regularity and the virus should be successfully transmitted through at least two generations. Apparently the latter was not attempted by Lipschütz. Intracerebral inoculations into three rabbits with material from two cases (Nos. I and IV) were made. Two rabbits were also inoculated intraspinally with material from one case (No. IV). None of these animals showed any reaction. In the case of one of the animals inoculated into the brain (Case I) although this rabbit showed no symptoms, we thought it conceivable that the susceptibility of the species for the virus might be so slight that no obvious lesion had been produced. Nevertheless it was thought that the virus might possibly remain alive at the seat of inoculation and by repeated transfers become adapted to the rabbit. This phenomenon has been observed by Noguchi with vaccine virus, and by Rivers and Tillett with the rabbit virus isolated by these workers. This possibility was tested by us by making serial corneal and brain inoculations. Corneal transfers were carried through fourteen animals in series, and brain transfers through ten. No specific lesions developed in any of the animals. The work of Teague and Goodpasture suggested that the skin might be rendered more susceptible to infection by previous treatment with tar. Material from two cases (Nos. I and VIII) was inoculated into the tarred skin of guinea pigs and rabbits. The material was injected intracutaneously and also rubbed into the scarified skin. No reaction was obtained in any of the animals. Finally, the transmission of herpes zoster to monkeys was attempted. Blanc and Caminopetros, and Bastai and Busacca, as discussed in the review of the literature, inoculated monkeys (Macacus) in various ways, without success. It was thought possible that although monkeys of the genus Macacus might be refractory, monkeys of another genus might prove susceptible. Consequently, besides the inoculation of two Macacus monkeys, attempts were made to infect five vervets. Moreover, in view of the fact that the virus of vaccinia and the rabbit virus of Rivers and Tillett could be successfully cultivated in the testicle, intratesticular inoculations were employed. The testicles were removed at varying periods following inoculation. Numerous sections of these testicles were made and examined, but in no instance were any lesions found which could be interpreted as specific. No cells containing intranuclear inclusion bodies were found. These experiments, therefore, have also led to purely negative results. This report of our work is made at the present time because a considerable amount of literature has been published which gives the impression that herpes zoster has been successfully transmitted to animals. Although the observations of Lipschütz are suggestive, it is important that they be confirmed by further investigations. Until herpes zoster can be regularly transmitted to animals and cross-immunity tests be carried out, the relation of the virus of herpes zoster to that of herpes simplex remains a matter of speculation. In view of the fact that herpes simplex can be easily and regularly transmitted to rabbits, whereas in the hands of a large number of investigators similar experiments with herpes zoster are completely negative, it does not seem likely that the etiological agent concerned in these two diseases can be absolutely identical. The question of the identity or non-identity of herpes zoster and varicella is even more difficult to answer, because at present neither of these infections is readily transmissible to animals. The work of Kundratitz is extremely interesting. His observations, aside from indicating a close immunological relationship between herpes zoster and varicella, are important in that they seem to show the presence of a transmissible virus in the vesicles of herpes zoster. The only question that arises is whether the cases of herpes zoster from which Kundratitz was able to make successful transfers were true cases of idiopathic herpes zoster.     

STUDIES ON THE MECHANISM OF IMMUNITY IN TYPHUS FEVER : II. ALLERGIC AND TOXIC REACTIONS PRODUCED WITH RICKETTSIA PROWAZEKI

The intradermal inoculation of Mexican typhus virus into immune guinea pigs produces a local reaction which is similar in its appearance to the lesion observed in the skin of normal animals submitted to the same treatment. The reaction in the immune animal appears earlier and fades sooner than the lesion in the normal guinea pig. The inoculation of heat-killed or formalin-killed Rickettsiae produces no significant reactions at the site of the intradermal injection in typhus immune guinea pigs. The virus, inoculated intradermally, has been recovered from the local lesion 72 hours after the injection into typhus immune guinea pigs. Normal guinea pigs and persons without a history of typhus fever present a congestion and some swelling of the skin at the site of the intradermal injection of formalinized Mexican Rickettsiae. The reaction appears 24 hours after the inoculation and fades within 48 hours. Heating the formalinized Rickettsia suspensions at 70°C. for 30 minutes renders them inactive in normal men and guinea pigs. From the experiments reported in this paper it seems that the reactions observed in typhus immune guinea pigs submitted to a second inoculation of typhus virus, belong to the group of reactions presented by tuberculous animals (Koch''s phenomenon) and the accelerated takes shown by immune persons submitted to revaccination with vaccinia virus. A heat labile substance has been demonstrated in the formalinized Rickettsia bodies, which produces a reaction in the skin of normal men and guinea pigs.