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.     

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.     

STUDIES IN EXPERIMENTAL SYPHILIS : III. FURTHER OBSERVATIONS ON THE POSSIBILITY OF CURE OF SYPHILIS IN THE RABBIT WITH ARSPHENAMINE.

Syphilitic rabbits can be treated with arsphenamine in such a manner as to render the lymph nodes incapable of transmitting the infection to normal rabbits. This can be accomplished if treatment is begun either early or comparatively late in the course of the disease. If treatment is begun early, the animals are almost uniformly susceptible to a second infection, whereas, if it is begun late, they are almost uniformly refractory to a second infection. It is suggested that this refractory state in rabbits may be explained by the existence of an acquired immunity which persists after the abolition of the disease, rather than to the persistence of the first infection. It would appear that it is possible under certain conditions to reinoculate rabbits and produce generalized infection without producing any lesion at the portal of entry.     

FURTHER OBSERVATIONS ON CERTAIN FEATURES OF EXPERIMENTAL SYPHILIS AND YAWS IN THE RABBIT

1. Lesions of the testicle and scrotum of the rabbit produced by the treponema of syphilis and of yaws afford material for the study of chemotherapy, of serum reactions, and of immunity. 2. On experimental and clinical grounds, it appears that yaws can be cured with a smaller dose of salvarsan than is necessary to cure syphilis. 3. The spirochætæ of syphilis do not become resistant after repeated treatments with salvarsan. 4. Sodium cacodylate has little effect on the treponema of either disease. 5. Mercury, in a single intravenous dose, has a spirillicidal effect, but the necessary dose usually causes the death of the animal later from nephritis. 6. Potassium iodid, in a single intravenous dose, has no spirillicidal effect. 7. The complement fixation reaction is closely correlated with the course of an infection, as is shown by its onset and its disappearance after castration or treatment. 8. Reinfection during the course of a testicular lesion does not occur, but it occurs after regression, treatment, or extirpation. 9. Specific differences between Treponema pallidum and Trepanema pertenue can be demonstrated in infected animals treated with salvarsan and reinoculated.     

OBSERVATIONS ON THE BLOOD CYTOLOGY IN EXPERIMENTAL SYPHILIS : II. THE PERIOD OF DISEASE LATENCY

The mean blood cell levels of 35 latent syphilitic rabbits in which all lesions had undergone spontaneous regression and complete healing were compared with weighted values for normal rabbits. The only differences noted were in the red cell count and hemoglobin content, both of which were significantly lower in the experimental group than the normal values. A parallelism was observed between the blood cell changes of the experimental disease after spontaneous regression of lesions, and the cell changes in the human disease after treatment. This parallelism lends additional weight to deductions drawn from the experimental disease as applied to human syphilis.     

RECIPROCAL EFFECTS OF CONCOMITANT INFECTIONS : II. THE INFLUENCE OF VACCINAL IMMUNITY ON THE REACTION TO EXPERIMENTAL SYPHILIS.

Experiments are reported in which it was shown, first, that the effectiveness of the reaction to experimental syphilis was increased in rabbits immune to vaccine virus, the ensuing disease being less severe than in control animals. It was further shown that a comparable modification of the syphilitic reaction occurred in rabbits inoculated intratesticularly with a mixture of Treponema pallidum and vaccine virus, and it was suggested that this result was due to the influence of a vaccinal immunity and to the local effect of the vaccinal reaction. It was pointed out that the manner in which two concomitant experimental infections are induced is of significance in determining the character of the host''s reaction.     

LOCAL ORGAN HYPERSENSITIVENESS : III. FURTHER OBSERVATIONS ON ITS EXPERIMENTAL PRODUCTION IN THE RABBIT EYE

1. Rabbit eyes sensitized with guinea pig red blood cells or fresh egg white respond with an inflammatory reaction following the intravenous injection of the homologous antigen, but not the heterologous. 2. Two-tenths of 1.0 cc. of a multiple antigen containing ten separate ingredients, or in other words, 0.02 cc. of each foreign protein, when introduced into the rabbit''s anterior chamber, is sufficient to produce an altered ocular reactivity such that when 1 cc. of one of the ten antigens is introduced intravenously the eye shows hyperemia of the iris and conjunctiva with more or less edema and lacrimation during the next 24 hours. 3. For as long as 8 months after sensitization eyes will respond with an inflammatory reaction following the intravenous injection of fractions of the total antigen. 4. Repeated daily intravenous injections of a single antigen usually produce no reaction in the sensitized eye after the first few days. Injection of different antigens intravenously on succeeding days produces a continued sterile inflammatory process in the sensitized eye. After the total number of single antigens has been injected, repetition of these injections now fails to produce a similar response. Instead, the eye reaction is at a much lower level and the inflammatory response is manifested only to a few of the antigens injected intravenously. 5. Unless massive doses of antigen are used to desensitize, permanent desensitization of the eye has not occurred in animals which have been followed for at least 8 months. Animals may develop maximal eye responses following repeated intravenous injections of the same antigen, if sufficient time has elapsed between injections. Nevertheless, the eye reactions which can be elicited 6 or 7 months after sensitization are less intense than the initial responses. 6. The ability of the eye inflammation to light up following the intravenous injection of homologous antigen is not due to an initial tissue injury as proven by the fact that the reaction is specific and anterior chambers injured with typhoid vaccine, iodine, saline, glycerine, or albolene will not respond subsequently when the various proteins used for sensitization of the other eyes are injected intravenously. 7. It has been impossible to demonstrate sensitivity in the eye by the intravenous shocking route until at least the 5th day following introduction of the antigen into the anterior chamber. 8. The eye reaction can be produced by the subcutaneous as well as the intravenous injection of antigen. 9. Rabbits vary considerably in the intensity of the eye reaction which can be elicited in them, but only rarely was an animal found which failed completely to give a reaction.     

HUMAN IMMUNITY TO THE MENINGOCOCCUS : II. DEVELOPMENT OF NATURAL IMMUNITY

Results of the present study suggest that natural immunity to meningococcal disease is initiated, reinforced, and broadened by intermittent carriage of different strains of meningococci throughout life. In young adults, carriage of meningococci in the nasopharynx is an efficient process of immune sensitization. 92% of carriers of serogroup B, C, or Bo meningococci were found to develop increased titers of serum bactericidal activity to their own meningococcal isolate, and 87% developed bactericidal activity to heterologous strains of pathogenic meningococci. The rise in bactericidal titer occurred within 2 wk of onset of the carrier state, and was accompanied by an increase in titer of specific IgG, IgM, and IgA antibodies to meningococci. In early childhood, when few children have antibodies to pathogenic meningococci, active immunization seems to occur as a result of carriage of atypical, nonpathogenic strains. Immunity to systemic meningococcal infection among infants in the neonatal period is associated with the passive transfer of IgG antibodies from mother to fetus. The antigenic determinants which initiate the immune response to meningococci include the group-specific C polysaccharide, cross-reactive antigens, and type-specific antigens.     

OBSERVATIONS ON THE BLOOD CYTOLOGY IN EXPERIMENTAL SYPHILIS : I. THE PERIOD OF DISEASE ACTIVITY

Weekly observations were made on the blood cytology of seven syphilitic and nine normal control rabbits. Each animal was examined seven times prior to and fifteen times after inoculation of the experimental group. Comparisons were made between the mean blood cell values obtained from all counts on the experimental and control groups in the preinoculation and postinoculation periods. The mean blood cell formula of the syphilitic group for the 3½ month period after inoculation was significantly different from the preinoculation mean values observed in the same group in the following respects: higher total white cell count, platelet count, neutrophil count, and monocyte count, and lower lymphocyte count. The mean blood cell formula of the syphilitic group for the 3½ month period after inoculation was significantly different from the mean blood cell formula of the normal control group in the same time interval in the following respects: higher total white cell count, platelet count, neutrophil and monocyte counts, and lower lymphocyte count. From these results it was concluded that during the period of disease activity, the blood cytology of rabbits infected with Tr. pallidum is characterized by an increase in the total white cell count, the platelet, neutrophil, and monocyte counts, and a decrease in the lymphocyte count from normal values. These changes were statistically significant.     

RECIPROCAL EFFECTS OF CONCOMITANT INFECTIONS : III. THE INFLUENCE OF VACCINIA AND OF VACCINAL IMMUNITY ON THE REACTION TO INFECTION WITH EXPERIMENTAL SYPHILIS (INTRACUTANEOUS INOCULATION).

An experiment is reported in which was studied the effects of a concomitant vaccinal infection and of vaccinal immunity upon the reaction to syphilis in rabbits induced by intracutaneous inoculation. The results obtained showed that the reaction was modified by both conditions. A vaccinal infection initiated at the time of syphilitic inoculation was associated with a defensive reaction of lessened efficiency, the ensuing syphilis being more severe than in control animals. A state of vaccinal immunity present at the time of syphilitic inoculation was associated with a reaction of heightened efficiency, the ensuing syphilis being very mild. These results are in harmony with those obtained in other experiments in which the intratesticular route of syphilitic inoculation was employed.     

EXPERIMENTAL OBSERVATIONS ON THE PROPHYLAXIS AND TREATMENT OF SYPHILIS

1. By inoculating the scarified surface of both sides of the scrotum of rabbits with suspensions of Treponema pallidum, 100 per cent of infections were obtained on one side or the other. Infection through the unbroken skin could not be produced. 2. By gland transfers from animals with positive local inoculations, 87.5 per cent of takes were produced. 3. These two methods were used to test the prophylactic value of 30 per cent calomel ointment, (a) Calomel ointment proved efficacious up to 8 hours after inoculation with syphilis, (b) No marked difference appeared between the action of calomel in a base of lanolin and vaseline and in a base of benzoinated lard and wax. (c) Death from mercurial poisoning was produced in rabbits by a single application of a large amount of calomel ointment. 4. The method of gland transfers was used to test the sterilizing effect of arsphenamine and neoarsphenamine on old infections in the rabbit. The infection was completely abolished in every instance, whether by one, two, or four intravenous doses. 5. Natural spirochetosis of rabbits need not be a serious complicating factor in work on syphilis in rabbits, for the following reasons. (a) In natural spirochetosis, the lesions occur on the penis and not on the scrotum. Gland transfers are negative, (b) A scrotal lesion can be produced by inoculation, but it can be distinguished from that of Treponema pallidum infection by its course, (c) In studies of generalized syphilis supposed to involve the genitalia, and in sexual transmission experiments, Treponema cuniculi may be a serious complicating factor.     

FURTHER STUDIES ON IMMUNITY IN EXPERIMENTAL CRYPTOCOCCOSIS

Following infection with 10³ cells of Cryptococcus neoformans, progressive multiplication occurred in all tissues for 7 to 28 days. Thereafter, most mice gained control of the infection so that 3 to 6 months after inoculation, tissues were usually sterile or contained only small numbers of cryptococci. Survivors challenged 1 to 4 months after infection with 10³ cells lived longer than controls and had an increased survival rate. Pretreatment with bacterial endotoxin also resulted in protection against cryptococcal challenge. Endotoxin-protected animals showed no ability to limit the infection until circulating antibody could be demonstrated. Histologic studies revealed an increased cellular infiltration in the brains of specifically protected and endotoxin-protected mice 1 to 2 weeks after challenge. However, control of cryptococcal multiplication could be correlated with the presence of antibody but could not be correlated with the enhanced cellular response. Although protection against cryptococcal challenge appears to be antibody-dependent, it is unclear whether protective antibody is to be found in plasma or is tissue-bound.     

A STUDY OF THE RELATION OF TREPONEMA PALLIDUM TO LYMPHOID TISSUES IN EXPERIMENTAL SYPHILIS

A widespread dissemination of Treponema pallidum from a local focus of inoculation in the rabbit constantly occurs by way of the lymphatics. Spirochetes were regularly recovered from the satellite lymph nodes by animal inoculation after scrotal inoculation; they were present as early as 2 days, when no specific primary reaction was detected, and at later periods of from 5 to 61 days after inoculation. Other superficial nodes at remote sites such as the popliteals and with no syphilitic lesions in the drainage area have also been shown to harbor active organisms. Although spirochetes were found in relatively few of the lymph node emulsions, the orchitis resulting from their injection was of a rapidly progressive type with an incubation period but slightly longer than that produced by a testicular or skin nodule emulsion rich in spirochetes. It has further been shown that a syphilitic infection is sufficiently established in the rabbit body within 48 hours after scrotal inoculation so that the primary lesion is no longer essential for its maintenance. Active treponemata survive in the popliteal lymph nodes for long periods of time and have been regularly recovered from them in cases of true latency. The lymph nodes, therefore, function as reservoirs of the organisms. The ability to recover the spirochetes from lymphoid tissue through successive generations is seen in the serial passage of lymph node emulsion to testicle during an 18 months period. The persistence of spirochetes in lymphoid tissue irrespective of the presence or absence of syphilitic lesions is a characteristic and fundamental feature of syphilis of the rabbit. The existence of infection, therefore, may be demonstrated at any time by the recovery of spirochetes from the popliteal lymph nodes by animal inoculation. This fact is of great practical importance in the therapy of the infection and may be profitably utilized in determining the ultimate effect of a therapeutic agent. These experiments demonstrate that the disease is not confined to the site of local inoculation but that lymphogenous dissemination of treponemata regularly takes place, and that during the course of this process organisms become localized in the lymph nodes and exist there indefinitely irrespective of the occurrence of manifestations of disease. The intimate relation of Treponema pallidum to lymphoid tissue is an essential concept of syphilis of the rabbit, and from this point of view, the infection is primarily one of lymphoid tissue.     

EXPERIMENTAL OBSERVATIONS ON THE "CURE" OF SYPHILIS IN THE RABBIT WITH ARSPHENAMINE

1. The intravenous administration of six doses of arsphenamine to syphilitic rabbits in amounts of 10 mg. per kilo, 127 days after inoculation is sufficient to render the popliteal nodes of such animals incapable of transmitting the infection to normal animals. 2. Syphilitic rabbits that have been treated in this manner and whose popliteal nodes 32 to 38 days after treatment have been shown to be incapable of transmitting the infection to normal animals are, as far as can be judged by the absence of a local lesion, refractory to a second inoculation of active virus of the same strain, if the inoculation be made intradermally at the base of the ear and carried out 209 days after the first inoculation. 3. Untreated syphilitic rabbits are also refractory toward a second inoculation made in a similar manner and at a similar interval following the first inoculation. 4. Evidence is offered in favor of the view that the most satisfactory method of evaluation of antisyphilitic agents in experimental syphilis of the rabbit is that of lymph node transfer. 5. Evidence is offered in support of the view that the state of refractoriness toward a second inoculation exhibited by syphilitic rabbits may be explainable upon the basis of either an acquired immunity or persistence of a focus of living treponemata in the body.     

STUDIES IN EXPERIMENTAL SYPHILIS : I. THE INFLUENCE OF THE SIZE OF INOCULUM ON THE COURSE OF EXPERIMENTAL SYPHILIS IN THE RABBIT.

1. The size of the dose of the infecting material exerts an appreciable effect upon the duration of the incubation period in experimental syphilis of the rabbit. Other things being equal, the larger the dose the shorter is the incubation period. 2. The size of the inoculum was not found to exercise any marked effect upon the course of the disease as a whole. 3. In individual rabbits inoculated with minimal amounts of virus the disease may run a malignant course.     

RELATION OF VARICELLA TO HERPES ZOSTER : II. CLINICAL AND EXPERIMENTAL OBSERVATIONS.

Experiments and clinical observations dealing with the identity of the viruses of varicella and herpes zoster were presented. The results indicate that the etiological agents concerned with these two diseases are in the majority of instances not identical.     

EXPERIMENTAL SYPHILIS IN THE RABBIT : I. PRIMARY INFECTION IN THE TESTICLE.

A study was made of the infections produced in rabbits inoculated in the testicles with two strains of Treponema pallidum which had been carried in rabbits for several years. Infection resulted in all instances; the incubation period varied as a rule between 2 and 6 weeks and under properly chosen conditions could be reduced to approximately 3 weeks or less. The resulting infection pursued a typically cyclic or relapsing course which affected both the spirochetes and the associated lesions in the testicle. The spirochetes in the local lesions exhibited periodic changes less marked and less regular but identical in character with the changes which occur in the blood in cases of relapsing fever. The lesions in the testicle also showed periods of active development and quiescence or regression which followed closely upon the changes exhibited by the spirochetes. The specific reaction in the testicle showed considerable variation in the speed and sharpness with which successive phenomena occurred as well as in the character and extent of the processes themselves. These reactions were of two fundamental types. In one group of animals, the reaction was characterized by an intense cycle of acute exudation and infiltration with a lesser degree of proliferation, followed by crisis and subsequent recurrence of secondary cycles of proliferative reaction of a minor degree. In the other group of animals, the reaction was more chronic in character and consisted largely of infiltration and proliferation. The progress of the reaction was more gradual, and sharp alterations in its course were absent. The infection progressed by a succession of stages with slight and irregular remissions. In a third group of animals, the reaction was subacute, combining at the same time the processes of exudation, infiltration, and proliferation. The first cycle of reaction was fairly acute and terminated in a definite crisis with moderate regression which in turn was followed by recurrence and more or less pronounced secondary cycles of proliferation. In all cases of outspoken infection, there was diffuse involvement of testicle, tunic, epididymis, and cord, but as the infection progressed, the lesions underwent many transformations, so that a variety of lesions was formed from processes which in the beginning were of a common type. Eventually, the reaction became more irregular and the infection became centered in one or more foci which were commonly situated in the epididymis, tunics, scrotum, or mediastinum testis. These centers served as residual foci of infection. The duration of the testicular process was found to be very variable. In some animals, the entire reaction consisted of but a single sharp cycle, and the local infection was terminated by crisis within 4 to 6 weeks after inoculation. As a rule, the period of active infection was from 2 to 4 months, and quiescent or inactive lesions not infrequently lasted for from 4 to 6 months. In exceptional instances, local infection persisted for more than a year.     

STUDIES IN EXPERIMENTAL SYPHILIS : II. THE INFLUENCE OF A NON-SPECIFIC INFLAMMATORY REACTION UPON THE DEVELOPMENT OF THE CHANCRE.

Rabbits can be successfully infected with syphilis by the inoculation of surface wounds with virulent strains of Treponema pallidum. Old granulating wounds in these animals constitute a particularly favorable terrain for syphilis inoculation, the chancre developing relatively soon and attaining a greater size than the lesion occurring on the basis of a fresh wound. Coal tar dermatitis renders the skin susceptible to infection with Treponema pallidum. The possible reasons for these phenomena are discussed and an hypothesis to account for them is advanced.     

OBSERVATIONS ON SOME CAUSES OF GALL STONE FORMATION : III. THE RELATION OF THE REACTION OF THE BILE TO EXPERIMENTAL CHOLELITHIASIS.

As previous papers from our laboratory have shown, there exists a well defined tendency for calcium carbonate to come out of solution in the normal liver bile of the dog, and for it to be deposited on certain nuclei not infrequent in the secretion under pathological circumstances. Gall stones that had arisen in this fashion were a frequent occurrence in the intubated animals we studied. The present paper is concerned with the reasons for the absence of such stones from dogs with an intact biliary tract. The solubility of calcium carbonate is known to be markedly affected by the reaction of the fluid in which it is contained. The normal liver bile, out of which it tends to precipitate, is alkaline, with an average pH of 8.20 but in the gall bladder where conditions might otherwise seem especially favorable to precipitation, the secretion undergoes a change toward the acid side, becoming on long sojourn there, strongly acid to litmus (pH 5.18 to 6.00). From bile as thus altered, no carbonate precipitation takes place, even when it becomes greatly concentrated as in fasting animals or after obstruction of the common duct. Furthermore, carbonate which has precipitated out of liver bile on standing dissolves again in it when the fluid is rendered slightly acid in vitro, or, in some cases merely neutral to litmus. There are several obvious reasons for the absence of carbonate stones from the normal ducts under ordinary conditions,—notably the motility of these latter, the flushing that they undergo from an intermittently quickened bile stream, and the cleansing and possibly antagonistic action of the secretion elaborated by the duct mucosa. In the fasting animal, one at least of these influences is almost done away with, the rate of bile flow is so greatly cut down; while furthermore the calcium concentration of the secretion undergoes a considerable increase. But pari passu with these changes there occurs one in the bile reaction, a diminution in alkalinity so great that the pH often approximates that of the neutral point for litmus. That this change is not a direct consequence of the increase in calcium, may be inferred from the findings with stasis bile, the calcium content and reaction of which were observed to vary independently, if in general in the same direction. These adjustments within the organism, some of which may be thought to exhibit an element of the purposeful, when considered with the test-tube experiments, strongly suggest that the reaction of the bile plays a critical part in determining the occurrence of carbonate stones, as furthermore that their absence from the normal gall bladder is a consequence of the changes in the bile reaction there occurring. The changes come about through a functional activity of the bladder. This being the case, one might suppose that the failure to act would be followed by a formation of carbonate stones. There is sufficient evidence available in the literature to indicate that this happens, in rabbits at least. It is important to know whether changes in the bile reaction play any part in determining the cholelithiasis of man. To determine the matter will require a large material. But this much we have shown, that carbonate spheroliths not infrequently serve in human beings as centers in a formation of secondary stones of carbonate and cholesterol, as further that cholesterol precipitation out of human bladder bile can be induced or prevented by slightly altering the reaction of the fluid toward the alkaline and acid sides, respectively. The possibility that cholelithiasis may be a consequence of sins of omission on the part of the biliary channels and reservoir deserves to be considered.