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A Study of the Host-Parasite Relationship of Trichophyton Mentagrophytes and Trichophyton Rubrum When Introduced Into the Granuloma Pouch of Rats1
A STUDY OF THE HOST-PARASITE RELATIONSHIP OF TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RUBRUM WHEN INTRODUCED INTO THE GRANULOMA POUCH OF RATS* VICTOR D. NEWCOMER, M.D., EDWIN T. WRIGHT, M.D. AND THOMAS H. STERNBERG, M.D.
One of two theories is generally offered to explain why the superficial dermato-
phytes usually limit their growth to the keratin of the epidermis and its accessory structures. One proposes that the fungus lives entirely in the dead stratum corneum because of nutritional requirements; the other that the host actively and successfully resists any invasive efforts of the dermatophytes, and the stratum corneum and the structures of the skin composed of keratin represent sites of growth by necessity. Sulzberger (1) in 1929 reviewed the early work and presented his own data in regards to this problem. Employing Achorion quinckeanum, he was able to demonstrate living fungal elements from the blood and in internal organs of guinea pigs following both intracardial injections of spores and after the production of cutaneous infections. Fungi could be cultured from internal organs for a period of at least 12 days following intracardial injection. It was not possible to produce local damage to internal organs of such a nature that active growth of the superficial dermatophyte occurred. He agreed with Werner Jadassohn that superficial dermatophytes grow in the keratin not because of any positive factors attributed to keratin as such but because living tissue offers unfavorable growth conditions. Recently Sternberg, et al. (2), have reviewed the evidence regarding the fact that certain superficial fungi are capable of active invasion of the deeper tissues. They inoculated mice intraperitoneally with Trichophyton rubrum and were able to reisolate the organism from the mice 25 days later. The organisms were readily identified with the use of the Hotchkiss-McManus stain in granulomatous lesions in the omentum, liver, spleen, and muscles and they questioned the present classification of this organism as an obligatory superficial dermatophyte. Lewis, et al. (3) have recently emphasized the fact that the host's resistance is an active factor in the control of superficial dermatophytes. The literature regarding the clinical exceptions to the rule that superficial dermatophytes do not invade deeper than the epidermal structures has been reviewed by Wilson, et al. (4) and they presented fourteen cases of nodular granulomatous perifolliculitis of the legs caused by T. rubrum in which large *
From the Medical Service, Veterans Administration Center, General Medical and Surgical Hospital, Los Angeles 25, California, and the Division of Dermatology, Department of Medicine, University of California Medical Center, Los Angeles 24, California.
This research was supported in part by a grant from the Squibb Institute for Medical Research, New Brunswick, N. J. Presented at the fifteenth annual meeting of The Society for Investigative Dermatology, Inc., San Francisco, California, June 20, 1954. 3.59
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masses of healthy fungi were found in the corium far removed from any demon-
strable keratin material. They pointed out that in their study fungi were not seen in healthy corium and that necrotic material was present in all involved areas. Consequently the view that they cannot thrive by digesting living tissue may still be valid. This paper presents our preliminary results employing the granuloma pouch and pneumoderma techuics as described by Selye (5—10) in a study directed toward a better understanding of the relationship between the so-called superficial dermatophytes and those factors in the animal which tend to limit their existence to the epidermis. MATERIALS AND METHODS
Wistar and Long-Evans strains of rats were employed; males and females being used interchangeably. The weight of the Wistar rats varied between 200—250 grams and those of the Long-Evans strain between 150—200 grams. Purina Laboratory Chow® and water was allowed ad libitum. Pouches were prepared by three different means. Granuloma pouches were produced in the first series of animals in the following manner: The animals were etherized and 1 cc of a 1 per cent solution of croton oil* in Mazola oil® was injected into the subcutaneous tissue of the dorsal region between the shoulder blades. The syringe containing tbe oils was disconnected from the 22-gauge needle which was kept in situ. A 30 cc syringe was then connected and 25 cc of air injected. After a period of 7—10 days the wall of the granuloma pouches was well developed and the pouch contained a sero-
sanginous fluid, the latter having almost completely replaced the air. At this time a prepared suspension of either T. mentagrophytes or '1'. rubruni was injected into the granuloma pouch. The animals were again anesthetized and a 22-gauge 1j-inch needle attached to an empty syringe was inserted into the normal subcutaneous tissue posterior to the pouch and extending anteriorly until it penetrated the wall of the pouch. Two cc of fluid were removed and the syringe was disconnected leaving the needle in place. Another syringe containing 2 cc of the fungus suspension was connected to the in situ needle and the suspension was
injected. Pouches were prepared differently in a second series of rats. Two cc of a suspension of fungi were injected subcutaneously between the shoulder blades of anesthetized rats and this was immediately followed by 25 cc of air. In a third series of animals, pouches were prepared by first injecting a suspension of the
fungus, then 1 cc of a solution of hydrocortisone acetatet containing 25 mg., followed by 25 cc of air. Suspensions of fungi were prepared by growing stock strains of T. mentagrophytez and T. rubrum on slants of Difco Sabourauds Dextrose Agar® at room temperature. Freshly mature colonies of these organisms were mechanically removed from the surface of the slants with as little adhering agar as possible and placed in sterile Ten Brock tissue grinder. Ten cc of saline was then added and the material ground to a fine particle size. Crystalline penicillin
G Potassium, U.S.P.t and Streptomycin, were added to the suspensions so that the final concentration was 100 units of each per cc. The material was then inoculated into the pouches utilizing the methods described above. Two strains of T. mentagrophytez and three strains of T. rubrum were used in this study. Rats in which the granuloma pouch was prepared with the use of croton oil were sacrificed * National
Croton Oil Formulary, Magnus, Mabee, and Reynard, Inc., (Manufacturers) t Hydrocortone, Merck and Co., Inc., Rahway, N. J. t Bristol Laboratories, Inc., Syracuse, N. Y. § Merck & Co., Inc., Rahway, N. J.
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at intervals from 1—3 weeks post-inoculation. The rats in which pouches were prepared with the organism and air were sacrificed between 2—4 weeks, and those rats in which pouches
were prepared with the organism, hydroeortisone acetate, and air were sacrificed two weeks following inoculation. All animals were sacrificed by etherization, then immersed in 70 per cent alcohol for 15 minutes following which they were placed on a dissecting board saturated with Phenolor®. The abdominal cavity was first opened and a portion of the spleen, liver, and kidney was removed and placed on Difco Sabouraud's Dextrose Agar for culture. The chest was then examined and a portion of the lung removed and placed on Sabouraud's media. The granuloma pouch was dissected and its contents observed grossly. Wet mount preparations were examined for organisms and material was taken for culture. Various portions of its contents
and wall were placed in 10 per cent formaldehyde solution for histological preparation. The histological sections were stained with hematoxylin and eosin and the Kligman (11) and Gridley (12) modifications of the periodic acid-Schiff stain. RESULTS
The post-inoculation course was variable in some of those animals whose pouches had been prepared with croton oil 7—10 days previously. Necrosis involved the dorsal surface of the pouch in some of these animals in approximately 6—10 days and caused the experiment to be terminated. Early evidence of impending necrosis consisted of induration and discolorization of the superior portion of the wall of the pouch. If the process was allowed to continue, a large segment of the wall sloughed with subsequent collapse of the pouch. All animals appeared in a state of good health at the termination of these experiments and there was no clinical evidence to suggest that systemic dissemination had occurred. No evidence of disease at the time of autopsy could be found
in the liver, spleen, kidneys, or lungs, and the walls of all the pouches were intact. The pouch and its contents varied considerably depending upon the method of preparation. Those pouches prepared with croton oil were well developed, firm, and contained a moderate amount of straw colored fluid. The wall of the pouch was fairly thick and it could be easily dissected from the surrounding tissues. Usually there was a variable amount of "cheesy" material loosely adherent to the inner surface of the wall. Pouches prepared by the injection of fungi and air had a tendency to decrease in size over the approximately two-week period of observation. They were not as large nor as firm as those prepared with the croton oil. Their walls were thinner, little or no fluid was present, and in some, air was still present in the pouch at the time of autopsy. There was less cheesy material present. Pouches which were prepared with fungi, air, and hydrocortisone acetate were,
at the time of autopsy, much smaller in size than the preceding two types of pouches. In some animals there was little gross evidence of a pouch at the end of the two-week period. Fluid and air were either absent or scanty. The amount of
cheesy material was considerably less and usually located on the floor of the pouch. The wall was very thin and difficult to dissect out. The cultural results and the factors involved in the inoculation of the pouches of the rats with T. mcntagrophytes and T. rubrum are presented in Table I. Pouches of 14 rats which were prepared with croton oil were inoculated with
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TABLE I Factors and cultural results front pouches of rats inoculated with different strains of T. mentagrophytes and T. rubruni No.ot
Fungus Type &
Type of Pouch
No. of days in pouch
3 3 1 1 1
2 1
2 2 2 2 2 I
3 1 1 1 1 1
1
2 2 2 2
T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. meritag-1 T. mentag-2 T. mentag-1 T. mentag-2 T. rubrum-1 T. rubrum-1 T. rubrum-1 T. rubrum-1 T. rubruni-2 T. rubrum-2 T. rubrum-3 T. rubrum-3 T. rubrum-2 T. rubrum-3 T. rubrum-2 T. rubrum-3
Croton oil Croton oil Croton oil Croton oil Croton oil Croton oil Croton oil Croton oil
air air Hydrocortisone Hydrocortisone eroton oil croton oil croton oil croton oil croton oil croton oil croton oil croton oil air
air 1{ydrocortisone
Hydrocortisone
6 9 12 18 21 22 26 40 14 14 14 14
5 6 9 13
6 7 14
20 14 14 14 14
No. of Positive Cultural results in: __________ __________________ Pouch Kidney Liver Spleen Lung
3 3 1 1 1 2 1 2
2 2 2 2 0 3 1 1 0
0 0 0 0 0 2 0
0 0
0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0
0 0 0 0 0
2
2
0 0 0
0 0
0 0 0
0
0
0
0
0 0 0 0
0 0 0 0
0 0
0 0 0 0 0 0 0 0 0 0 0
0
0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0
one strain of T. mentagrophytes. The animals were sacrificed 6—40 days later. Fungi were cultured from all of these pouches. Organisms from the majority of these pouches could be identified in wet mount preparations at the time of au-
topsy. In addition fungi were cultured 14 days after inoculation from four pouches which were prepared with two strains of T. mentagrophytes and air, and from four pouches prepared with two strains of T. mentagrophytes, hydrocortisone acetate and air. Three strains of T. rubrum were inoculated into ten pouches prepared with croton oil and the animals were sacrificed at intervals from 5 to 20 days later. Positive cultures were obtained from five of these animals. All the positive cultures were from one strain and these animals were sacrificed on the 6th, 9th, and 13th day post-inoculation. All cultures taken from the four pouches which were prepared with two different strains of T. rubrum and air were negative. Fungi were cultured from two of the pouches prepared by injecting one of two strains of T. rubrum, air, and hydrocortisone acetate. Fungi were cultured from the liver and spleen of three rats. The pouches of
TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RUBRUM
363
these rats had been prepared with the use of croton oil and inoculated with T. mentagrophytes. They were sacrificed on the 6th day post-inoculation. In no other instance were fungi cultured from the spleen, liver, kidneys or lungs. HISTOLOGICAL EXAMINATION
The outer and main portion of the wall of those pouches prepared with the use of croton oil was composed of a dense zone of proliferating fibroblasts arranged with their long axis parallel to the wall of the pouch. External to and throughout this area was an extensive vascular bed which was most prominent at the base of the pouch. Extending inward toward the center and forming an ill-defined middle zone of the wall of the pouch was a relatively avascular area composed of large histiocytes throughout which were numerous large vacuolated spaces (Plate I, Fig. 1). There were focal and diffuse areas of lymphocytic infiltration, occasional giant cells and a few polymorphonuclear leukocytes. On the inner aspect of this zone was one composed of eosinophilic staining amorphous material in which hematoxylin staining granular debris was distributed diffusely and in strands. Polymorphonuclear leucocytes were heavily distributed in this zone and were in all stages of degeneration (Plate II, Fig. 1). The viable fungal elements were readily identified with the use of the two modifications of the Hotchkiss-McManus stain. No distinction could be made between the histological appearance of T. mentagrophytes and T. rubrnm. In all instances the majority of the fungi were located in the inner zone of necrotic debris and polymorphonuclear leukocytes. To a lesser extent individual and aggregates of hyphal elements were found deeper in the histiocytic zone where they formed a nidus for polymorphonuclear leukocytes. The predominant form of the organism was that of hyphal elements, branching and intertwining so that an extensive mesh was formed upon the inner surface of the pouch (Plates I and III). Dispersed among these normal hyphal elements were hyphae in which the individual cells had swollen to become chains of rounded intercalary spores (Plate III, Fig. 2). Such chains showed evidence of fragmentation to individual spores. Some of these were in various stages of germination (Plate III, Fig. 3). In some areas these rounded spores assumed a rather large size of approximately 20 u in diameter (Plate III, Fig. 4). These fungal elements stained clearly with both Kligman's and Gridley's modifications of the Hotchkiss-McManus stain. The individual cells composing the hyphal elements appeared to be in a good state of turgor and the cell walls and septae stained clearly. This in addition to positive cultural evidence formed the basis on which we interpreted them as being viable organisms. There was also histological evidence indicating that a large percentage of the fungi died. Such evidence was present as early as the 5th post-inoculation day. Death was manifested by a decrease in their ability to stain clearly. There was a collapse of the individual cells and a narrowing of the diameter so that the hyphae appeared stringy. There was no evidence of branching or individual cell enlargement which was characteristic of viable organisms. Finally there was
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fragmentation of varying degrees to an end-product of Hotchkiss-McManus staining dust (Plate II, Fig. 2). The majority of dead organisms were found in the innermost avascular zone in which they formed centers of tremendous accumulations of polymorphonuclear leukocytes in all stages of degeneration (Plate II, Fig. 1 and 2). Many of the polymorphonuclear leukocytes had ingested the Hotchkiss-McManus positive dust. This material was also observed to a lesser degree in the middle zone where it was located primarily in histiocytes. Many of these cells had ingested this material to the point that the cytoplasm of the cell stained a purple-pink color which caused it to be sharply differentiated from the surrounding tissue (Plate II, Fig. 3). No evidence of fungal material was found in the outermost zone. There were, however, a variable number of cells which stained positively with the Hotchkiss-McManus stain. These were interpreted as being mast cells.
The fungi appeared essentially the same in all the three types of pouches. Although there were definite gross differences in the three types of prepared pouches, these differences were not as evident in those sections which we examined microscopically. However, in general, there was considerably less fibroblastic proliferation in the pouches prepared with hydrocortisone acetate. DISCUSSION
This study presents cultural and histological data which lends support to the
concept that in the animal keratin is not an essential growth substance for T. mentagrophytes or T. rubrum and that under certain conditions they may actively grow below the epidermis. T. mentagrophytes was re-isolated from granuloma pouches which had been inoculated 40 days previously. The histological studies suggest that this survivability was not due to the survival of highly resistant spores formed on the Sabouraud's media since active proliferating hyphal elements were observed in all histological sections made from culturally positive
pouches. Spore formation was observed in all histological sections in which hyphae were present and this was characterized by the formation of intercalary or terminal spores. This type of spore formation is suggestive of the type which is frequently seen in positive skin scrapings of T. mentagrophytes and T. rubrum and may account for the "rejuvenation" of the colonies which was noted. No macroconidia or characteristic microconidia were observed histologically. Apparently these types of spores are more suited to survival in conditions other than those found in or on the animal. The early death of many of our inoculums, particularly those of T. rubrum, indicated to us that mere survival of vegetative spores played a minor role and that the host's defense mechanisms were actively and in some instances successfully engaged in destroying the organism.
It was our impression that in all instances a large percentage of fungi died shortly after inoculation into the pouch. We are unable as yet to account for this initial high mortality. Probably several factors were responsible. The organisms were grown on Difco's Dextrose Sabouraud's Agar at room temperature and prior to inoculation they were subjected to grinding to a fine particle size. The change from Sabouraud's media to the animal per se and the change
TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RUBRIJM
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from room temperature to the rat's temperature were undoubtedly important factors in this early death. However we do not feel that these factors accounted for the death in some instances of all the organisms. Death of the majority of the inoculated organisms occurred either in the fluid portion or in the innermost zone of the wall of the pouch which was composed predominately of polymorphonuclear leucocytes. The hyphal elements in all stages of degeneration were always the center of large aggregates of polymorphonuclear leucocytes and their debris. This zone of polymorphonuclear leukocytes and dead organisms was prominent in all three types of pouches. At present we are unable to assign an
exact role to the fluid and the polymorphonuclear leucocytes in the death of the fungi. The dead organisms rapidly disintegrated and fragmented so that Hotchkiss-
McManus positive fragmented and granular material was observed to be diffusely scattered throughout this polymorphonuclear leukocyte zone. On occasion these cells were observed to be actively ingesting this material. Identical material was also seen in the cytoplasm of the histiocytes directly below the above
area. None of the mechanisms of allergy has been cited to explain any of our observations, but it is interesting to speculate that, in view of the tremendous amount of fungal material ingested by these fixed tissue cells, they might react violently to future administration of the same or similar material. No HotchkissMcManus positive fungal material was found in the outer portion of the pouch wall, and we do not know the ultimate disposition of this material; however, in speculation we feel that it is slowly digested by the local histiocytes and that breakdown products are ultimately released. Active proliferating fungal elements were observed in most sections in spite of the high mortality. The majority were located in the inner zone but many ex-
tended into the middle histiocytic zone. In all instances proliferating hyphae were observed which suggested to us that the subsequent positive cultures obtained from pouches were not the result of surviving vegetative spores. Selye (8, 13) has shown that the wall of the granuloma pouch which is produced by the use of croton oil reaches a high degree of resistance to subsequent injections of the same irritant. This protection is specific but in such a pouch the ability to resist other types of irritants may be less. He employs this explanation to account for the early necrosis which occurred in such granuloma pouches subsequently inoculated with a different type of irritant of such a dosage that it normally would not produce necrosis. We employed predominately the granuloma pouch prepared with croton oil in the hope that this lowered resistance to
other irritants might greatly alter the host-parasite relationship to the superficial dermatophytes. Although we did encounter early necrosis of the wall of the pouch after inoculation with T. mentagrophytes and T. rubrum, we do not feel that the host-parasite relationship was radically altered by employing such pouches.
We are unable to evaluate the exact histological response of the host to the fungus. In those rats in which the pouch was formed with the use of croton oil, the histological response was well developed by the seventh to the tenth day at
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the time the pouches were inoculated with the fungus. When pouches were made using suspensions of fungi and air, we could not be absolutely certain that there had not been some contamination with agar. However, when comparison was
made between the two types of pouches the histological response was quite similar. There was cultural evidence of dissemination to the liver and spleen from the granuloma pouch in three animals. The pouches had been prepared with the use of croton oil and their pouches inoculated with T. mentagrophfjtes. The animals were sacrificed on the sixth day. Although we have not investigated this finding
specifically, apparently dissemination from the pouch can occur and in this study did so shortly after inoculation. Apparently the local host's response is successful in preventing late continued dissemination. CONCLUSIONS
1. T. mentagrophytes and T. rubrum. can survive and grow beneath the epidermis in the granuloma pouch. In this study T. mentagrophyte.s was re-cultured from the granuloma pouches of two rats 40 days post-inoculation.
2. Growth of these fungi occurred primarily by the proliferation of hyphal elements. These hyphal elements were observed histologically in pouches which by culture revealed the presence of viable organisms.
3. Spore formation occurred and was of the intercalary and terminal type. The formation of macroconidia and microconidia was not observed. 4. A large percentage of the fungi died shortly after inoculation into the pouch. 5. The polymorphonuclear leukocyte was the predominant cell found in association with the dying fungal elements. 6. The fixed tissue histiocytes appeared to play an active role in the ultimate disposition of fungal debris. 7. Dissemination occurred in the liver and spleen in three animals inoculated with T. mentagrophytes. REFERENCES 1. SULZBERGER, M. B.: Experimentelle untersuchungen uber die derinatotropie der trichophytonplize. Arch. f. Dermat. u. Syph., 157: 345—357, 1929. 2. STERNBERG, T. H., TARBET, J. E., NEWCOMER, V. D. AND WIRER, L. H.: Deep infection
of mice with trichophyton rubruin (purpureum). J. Invest. Dermat., 19:373—384, 1952. 3. Lxwis, G. M., HOPPER, M. E. AND SCOTT, M. J.: Generalized trichophyton rubrum infec-
tion associated with systemic lymphoblastoma. Report of three cases. Arch. Dermat. & Syph., 67: 247—259, 1953.
4. WILSON, J. W., PLUNKETT, 0. A. AND GREGERSEN, A.: Nodular granulomatous perifolli-
culitis of the legs caused by trichophyton rubrum. Arch. Dermat. & Syph., 69: 258— 277, 1954.
5. SELYR, H. AND HORAVA, A.: Second Annual Report on Stress., Montreal, Acta Inc., Med. Pub., 268, 1952.
6. SELYE, H.: Use of granuloma pouch technic in the study of antiphlogistic corticoid. Proc. Soc. Exper. Biol. & Med., 82: 328—333, 1953.
7. SELYE, H.: Fourth Conference on the Adrenal Cortex. Nov. 13—15, 29—97, 1952, New York, Josiah Macy Foundation.
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SELYN, H.: Induction of topical resistance to acute tissue injury. An experimental study with the granuloma pouch technique. Surg. Clinc. No. America, 33: 1417—1146, 1953. 9. SELYE, H.: On the acquisition of tissue resistance to digestion by gastric juice. Gastro8.
enterology, 26: 221—229, 1954.
10. SELTE, H.: The effect of topically administered hydrocortisone upon dystrophic skin, as revealed by the pneumoderma technic. J. Invest. Dermat., 21: 91—98, 1953. 11. KLIGMAN, A. M. AND MESCON, H.: The periodic acid-schiff stain for the demonstration of fungi in animal tissue. J. Bact. 60: 415—421, 1950. 12. GRIDLEY, M. F.: A stain for fungi in tissue sections. Am. J. Clin. Path., 23: 303, 1953.
13. SELYE, H.: The part of inflammation in the local adaptation syndrome. The mechanism of inflammation, pp 53—74. G. Jasmin and A. Robert, Acta Inc., Medical Publishers, Montreal, Canada.
DISCUSSION Du. STEPHEN ROTHMAN, Chicago, ill.: Were those fungi in the pouches viable; and if so, how long? If the experimental period was Dot long enough it still re-
mains to prove that they are permanently viable outside the horny material. Dn. J. WALTER WILsoN, Los Angeles, Calif.: We, too, saw these large, spheri-
cal, Hotchkiss-McManus staining bodies in our perifollicular granuloma areas. Dr. Wright, do you have any impression as to whether these are fungi in some process of dying, or are they viable? DR. EDWIN T. WRIGHT (in closing): I would like to thank the discussors for their comments. Dr. Rothman's question can best be answered by giving the summary which, due to the limited time, I failed to present. 1. T. mentagrophytes and T. rubrum csn survive and grow beneath the epidermis in the granuloma pouch. 2. Growth occurred primarily by the proliferation of hyphal elements. 3. The polymorphonuclear leukocyte was the predominant cell found in association with the dying fungal elements. 4. The fixed tissue histiocytes appeared to play an active role in the ultimate disposition of fungal debris. Dr. Wilson asked the question, "Are the large cells viable?" We think that they probably are, primarily because they take on the same characteristics as do the other fungal elements which we consider to be viable. In closing I would like to reiterate the differences between viable and dead organisms as noted in this study. Viable organisms were characterized by 1) Good staining (the cell walls, septae and spores stained clearly and were well outlined). 2) There was evidence of branching and individual cell enlargement. 3) Positive cultures were obtained as late as 40 days post-inoculation. Dead organisms were characterized by: 1) Poor staining quality. 2) Collapse of individual cells and narrowing of the diameter so that the hyphae appeared stringy. 3) No evidence of branching or individual cell enlargement.
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PLATE I
FIG. 1. T. mentagrophytes 21 days in pouch prepared with croton oil. Hotchkiss-McManus
stain. The large empty spaces are probably due to the injected oil. 25X FIG. 2. T. mentagrophytes 14 days in pouch prepared with 25mg. of hydrocortisone acetate
and air. Hotchkiss-McManus stain. Upper dark layer and the lighter staining vacuolated area represents a zone of polymorphonuclear leukocytes, hyphal elements and debris from both. Fibroblastic proliferation is greatly inhibited. 30X FIG. 3. T. rubrum 9 days in granuloma pouch produced with croton oil. HotchkissMcManus stain. Active proliferating hyphae are seen. 125X
TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RLTBRTJM
PLATE
I
369
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
PLATE II
Fro. 1. T. rubrum 14 days in pouch prepared with hydrocortisone and air. Hematoxylin and eosin stain. Demonstrates zone composed of polymorphonuclear leukocytes and necrotic debris in the wall. 35X FIG. 2. Alternate section of Fig. 1. Hotehkiss-McManus stain. Demonstrates necrotic fungal debris scattered throughout and in strands in the same zone. 35X FIG. 3. High power of Fig. 2, demonstrating fungal debris concentrated in histiocytes of middle zone. 650X
371
TRICHOPHYTON MENTAGROPHYTES AND TEICHOPHYTON RUBRUM
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PLATE III
Fiu. 1. T. mentaqrophytes 21 days in pouch prepared with croton oil. Hotchkiss-McManus stain. Demonstrates usual type of hyphal proliferation. 650X Fin. 2. Same slide as Fig. 1 but from a different area showing intercalary spore formation. 650X
Fin. 3. T. mentegrophytes 14 days in pouch prepared with hydrocortisone and air. Demonstrates spore germination. 650X FIG. 4. T. mentagrophytes 18 days in pouch prepared with croton oil. llotchkiss-McManus train showing unusually large spores. 650X
373
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TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RUBRTJM
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
One of two theories is generally offered to explain why the superficial dermato-
phytes usually limit their growth to the keratin of the epidermis and its accessory structures. One proposes that the fungus lives entirely in the dead stratum corneum because of nutritional requirements; the other that the host actively and successfully resists any invasive efforts of the dermatophytes, and the stratum corneum and the structures of the skin composed of keratin represent sites of growth by necessity. Sulzberger (1) in 1929 reviewed the early work and presented his own data in regards to this problem. Employing Achorion quinckeanum, he was able to demonstrate living fungal elements from the blood and in internal organs of guinea pigs following both intracardial injections of spores and after the production of cutaneous infections. Fungi could be cultured from internal organs for a period of at least 12 days following intracardial injection. It was not possible to produce local damage to internal organs of such a nature that active growth of the superficial dermatophyte occurred. He agreed with Werner Jadassohn that superficial dermatophytes grow in the keratin not because of any positive factors attributed to keratin as such but because living tissue offers unfavorable growth conditions. Recently Sternberg, et al. (2), have reviewed the evidence regarding the fact that certain superficial fungi are capable of active invasion of the deeper tissues. They inoculated mice intraperitoneally with Trichophyton rubrum and were able to reisolate the organism from the mice 25 days later. The organisms were readily identified with the use of the Hotchkiss-McManus stain in granulomatous lesions in the omentum, liver, spleen, and muscles and they questioned the present classification of this organism as an obligatory superficial dermatophyte. Lewis, et al. (3) have recently emphasized the fact that the host's resistance is an active factor in the control of superficial dermatophytes. The literature regarding the clinical exceptions to the rule that superficial dermatophytes do not invade deeper than the epidermal structures has been reviewed by Wilson, et al. (4) and they presented fourteen cases of nodular granulomatous perifolliculitis of the legs caused by T. rubrum in which large *
From the Medical Service, Veterans Administration Center, General Medical and Surgical Hospital, Los Angeles 25, California, and the Division of Dermatology, Department of Medicine, University of California Medical Center, Los Angeles 24, California.
This research was supported in part by a grant from the Squibb Institute for Medical Research, New Brunswick, N. J. Presented at the fifteenth annual meeting of The Society for Investigative Dermatology, Inc., San Francisco, California, June 20, 1954. 3.59
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masses of healthy fungi were found in the corium far removed from any demon-
strable keratin material. They pointed out that in their study fungi were not seen in healthy corium and that necrotic material was present in all involved areas. Consequently the view that they cannot thrive by digesting living tissue may still be valid. This paper presents our preliminary results employing the granuloma pouch and pneumoderma techuics as described by Selye (5—10) in a study directed toward a better understanding of the relationship between the so-called superficial dermatophytes and those factors in the animal which tend to limit their existence to the epidermis. MATERIALS AND METHODS
Wistar and Long-Evans strains of rats were employed; males and females being used interchangeably. The weight of the Wistar rats varied between 200—250 grams and those of the Long-Evans strain between 150—200 grams. Purina Laboratory Chow® and water was allowed ad libitum. Pouches were prepared by three different means. Granuloma pouches were produced in the first series of animals in the following manner: The animals were etherized and 1 cc of a 1 per cent solution of croton oil* in Mazola oil® was injected into the subcutaneous tissue of the dorsal region between the shoulder blades. The syringe containing tbe oils was disconnected from the 22-gauge needle which was kept in situ. A 30 cc syringe was then connected and 25 cc of air injected. After a period of 7—10 days the wall of the granuloma pouches was well developed and the pouch contained a sero-
sanginous fluid, the latter having almost completely replaced the air. At this time a prepared suspension of either T. mentagrophytes or '1'. rubruni was injected into the granuloma pouch. The animals were again anesthetized and a 22-gauge 1j-inch needle attached to an empty syringe was inserted into the normal subcutaneous tissue posterior to the pouch and extending anteriorly until it penetrated the wall of the pouch. Two cc of fluid were removed and the syringe was disconnected leaving the needle in place. Another syringe containing 2 cc of the fungus suspension was connected to the in situ needle and the suspension was
injected. Pouches were prepared differently in a second series of rats. Two cc of a suspension of fungi were injected subcutaneously between the shoulder blades of anesthetized rats and this was immediately followed by 25 cc of air. In a third series of animals, pouches were prepared by first injecting a suspension of the
fungus, then 1 cc of a solution of hydrocortisone acetatet containing 25 mg., followed by 25 cc of air. Suspensions of fungi were prepared by growing stock strains of T. mentagrophytez and T. rubrum on slants of Difco Sabourauds Dextrose Agar® at room temperature. Freshly mature colonies of these organisms were mechanically removed from the surface of the slants with as little adhering agar as possible and placed in sterile Ten Brock tissue grinder. Ten cc of saline was then added and the material ground to a fine particle size. Crystalline penicillin
G Potassium, U.S.P.t and Streptomycin, were added to the suspensions so that the final concentration was 100 units of each per cc. The material was then inoculated into the pouches utilizing the methods described above. Two strains of T. mentagrophytez and three strains of T. rubrum were used in this study. Rats in which the granuloma pouch was prepared with the use of croton oil were sacrificed * National
Croton Oil Formulary, Magnus, Mabee, and Reynard, Inc., (Manufacturers) t Hydrocortone, Merck and Co., Inc., Rahway, N. J. t Bristol Laboratories, Inc., Syracuse, N. Y. § Merck & Co., Inc., Rahway, N. J.
TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RUBRUM
361
at intervals from 1—3 weeks post-inoculation. The rats in which pouches were prepared with the organism and air were sacrificed between 2—4 weeks, and those rats in which pouches
were prepared with the organism, hydroeortisone acetate, and air were sacrificed two weeks following inoculation. All animals were sacrificed by etherization, then immersed in 70 per cent alcohol for 15 minutes following which they were placed on a dissecting board saturated with Phenolor®. The abdominal cavity was first opened and a portion of the spleen, liver, and kidney was removed and placed on Difco Sabouraud's Dextrose Agar for culture. The chest was then examined and a portion of the lung removed and placed on Sabouraud's media. The granuloma pouch was dissected and its contents observed grossly. Wet mount preparations were examined for organisms and material was taken for culture. Various portions of its contents
and wall were placed in 10 per cent formaldehyde solution for histological preparation. The histological sections were stained with hematoxylin and eosin and the Kligman (11) and Gridley (12) modifications of the periodic acid-Schiff stain. RESULTS
The post-inoculation course was variable in some of those animals whose pouches had been prepared with croton oil 7—10 days previously. Necrosis involved the dorsal surface of the pouch in some of these animals in approximately 6—10 days and caused the experiment to be terminated. Early evidence of impending necrosis consisted of induration and discolorization of the superior portion of the wall of the pouch. If the process was allowed to continue, a large segment of the wall sloughed with subsequent collapse of the pouch. All animals appeared in a state of good health at the termination of these experiments and there was no clinical evidence to suggest that systemic dissemination had occurred. No evidence of disease at the time of autopsy could be found
in the liver, spleen, kidneys, or lungs, and the walls of all the pouches were intact. The pouch and its contents varied considerably depending upon the method of preparation. Those pouches prepared with croton oil were well developed, firm, and contained a moderate amount of straw colored fluid. The wall of the pouch was fairly thick and it could be easily dissected from the surrounding tissues. Usually there was a variable amount of "cheesy" material loosely adherent to the inner surface of the wall. Pouches prepared by the injection of fungi and air had a tendency to decrease in size over the approximately two-week period of observation. They were not as large nor as firm as those prepared with the croton oil. Their walls were thinner, little or no fluid was present, and in some, air was still present in the pouch at the time of autopsy. There was less cheesy material present. Pouches which were prepared with fungi, air, and hydrocortisone acetate were,
at the time of autopsy, much smaller in size than the preceding two types of pouches. In some animals there was little gross evidence of a pouch at the end of the two-week period. Fluid and air were either absent or scanty. The amount of
cheesy material was considerably less and usually located on the floor of the pouch. The wall was very thin and difficult to dissect out. The cultural results and the factors involved in the inoculation of the pouches of the rats with T. mcntagrophytes and T. rubrum are presented in Table I. Pouches of 14 rats which were prepared with croton oil were inoculated with
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TABLE I Factors and cultural results front pouches of rats inoculated with different strains of T. mentagrophytes and T. rubruni No.ot
Fungus Type &
Type of Pouch
No. of days in pouch
3 3 1 1 1
2 1
2 2 2 2 2 I
3 1 1 1 1 1
1
2 2 2 2
T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. mentag-1 T. meritag-1 T. mentag-2 T. mentag-1 T. mentag-2 T. rubrum-1 T. rubrum-1 T. rubrum-1 T. rubrum-1 T. rubruni-2 T. rubrum-2 T. rubrum-3 T. rubrum-3 T. rubrum-2 T. rubrum-3 T. rubrum-2 T. rubrum-3
Croton oil Croton oil Croton oil Croton oil Croton oil Croton oil Croton oil Croton oil
air air Hydrocortisone Hydrocortisone eroton oil croton oil croton oil croton oil croton oil croton oil croton oil croton oil air
air 1{ydrocortisone
Hydrocortisone
6 9 12 18 21 22 26 40 14 14 14 14
5 6 9 13
6 7 14
20 14 14 14 14
No. of Positive Cultural results in: __________ __________________ Pouch Kidney Liver Spleen Lung
3 3 1 1 1 2 1 2
2 2 2 2 0 3 1 1 0
0 0 0 0 0 2 0
0 0
0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0
0 0 0 0 0
2
2
0 0 0
0 0
0 0 0
0
0
0
0
0 0 0 0
0 0 0 0
0 0
0 0 0 0 0 0 0 0 0 0 0
0
0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0
one strain of T. mentagrophytes. The animals were sacrificed 6—40 days later. Fungi were cultured from all of these pouches. Organisms from the majority of these pouches could be identified in wet mount preparations at the time of au-
topsy. In addition fungi were cultured 14 days after inoculation from four pouches which were prepared with two strains of T. mentagrophytes and air, and from four pouches prepared with two strains of T. mentagrophytes, hydrocortisone acetate and air. Three strains of T. rubrum were inoculated into ten pouches prepared with croton oil and the animals were sacrificed at intervals from 5 to 20 days later. Positive cultures were obtained from five of these animals. All the positive cultures were from one strain and these animals were sacrificed on the 6th, 9th, and 13th day post-inoculation. All cultures taken from the four pouches which were prepared with two different strains of T. rubrum and air were negative. Fungi were cultured from two of the pouches prepared by injecting one of two strains of T. rubrum, air, and hydrocortisone acetate. Fungi were cultured from the liver and spleen of three rats. The pouches of
TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RUBRUM
363
these rats had been prepared with the use of croton oil and inoculated with T. mentagrophytes. They were sacrificed on the 6th day post-inoculation. In no other instance were fungi cultured from the spleen, liver, kidneys or lungs. HISTOLOGICAL EXAMINATION
The outer and main portion of the wall of those pouches prepared with the use of croton oil was composed of a dense zone of proliferating fibroblasts arranged with their long axis parallel to the wall of the pouch. External to and throughout this area was an extensive vascular bed which was most prominent at the base of the pouch. Extending inward toward the center and forming an ill-defined middle zone of the wall of the pouch was a relatively avascular area composed of large histiocytes throughout which were numerous large vacuolated spaces (Plate I, Fig. 1). There were focal and diffuse areas of lymphocytic infiltration, occasional giant cells and a few polymorphonuclear leukocytes. On the inner aspect of this zone was one composed of eosinophilic staining amorphous material in which hematoxylin staining granular debris was distributed diffusely and in strands. Polymorphonuclear leucocytes were heavily distributed in this zone and were in all stages of degeneration (Plate II, Fig. 1). The viable fungal elements were readily identified with the use of the two modifications of the Hotchkiss-McManus stain. No distinction could be made between the histological appearance of T. mentagrophytes and T. rubrnm. In all instances the majority of the fungi were located in the inner zone of necrotic debris and polymorphonuclear leukocytes. To a lesser extent individual and aggregates of hyphal elements were found deeper in the histiocytic zone where they formed a nidus for polymorphonuclear leukocytes. The predominant form of the organism was that of hyphal elements, branching and intertwining so that an extensive mesh was formed upon the inner surface of the pouch (Plates I and III). Dispersed among these normal hyphal elements were hyphae in which the individual cells had swollen to become chains of rounded intercalary spores (Plate III, Fig. 2). Such chains showed evidence of fragmentation to individual spores. Some of these were in various stages of germination (Plate III, Fig. 3). In some areas these rounded spores assumed a rather large size of approximately 20 u in diameter (Plate III, Fig. 4). These fungal elements stained clearly with both Kligman's and Gridley's modifications of the Hotchkiss-McManus stain. The individual cells composing the hyphal elements appeared to be in a good state of turgor and the cell walls and septae stained clearly. This in addition to positive cultural evidence formed the basis on which we interpreted them as being viable organisms. There was also histological evidence indicating that a large percentage of the fungi died. Such evidence was present as early as the 5th post-inoculation day. Death was manifested by a decrease in their ability to stain clearly. There was a collapse of the individual cells and a narrowing of the diameter so that the hyphae appeared stringy. There was no evidence of branching or individual cell enlargement which was characteristic of viable organisms. Finally there was
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fragmentation of varying degrees to an end-product of Hotchkiss-McManus staining dust (Plate II, Fig. 2). The majority of dead organisms were found in the innermost avascular zone in which they formed centers of tremendous accumulations of polymorphonuclear leukocytes in all stages of degeneration (Plate II, Fig. 1 and 2). Many of the polymorphonuclear leukocytes had ingested the Hotchkiss-McManus positive dust. This material was also observed to a lesser degree in the middle zone where it was located primarily in histiocytes. Many of these cells had ingested this material to the point that the cytoplasm of the cell stained a purple-pink color which caused it to be sharply differentiated from the surrounding tissue (Plate II, Fig. 3). No evidence of fungal material was found in the outermost zone. There were, however, a variable number of cells which stained positively with the Hotchkiss-McManus stain. These were interpreted as being mast cells.
The fungi appeared essentially the same in all the three types of pouches. Although there were definite gross differences in the three types of prepared pouches, these differences were not as evident in those sections which we examined microscopically. However, in general, there was considerably less fibroblastic proliferation in the pouches prepared with hydrocortisone acetate. DISCUSSION
This study presents cultural and histological data which lends support to the
concept that in the animal keratin is not an essential growth substance for T. mentagrophytes or T. rubrum and that under certain conditions they may actively grow below the epidermis. T. mentagrophytes was re-isolated from granuloma pouches which had been inoculated 40 days previously. The histological studies suggest that this survivability was not due to the survival of highly resistant spores formed on the Sabouraud's media since active proliferating hyphal elements were observed in all histological sections made from culturally positive
pouches. Spore formation was observed in all histological sections in which hyphae were present and this was characterized by the formation of intercalary or terminal spores. This type of spore formation is suggestive of the type which is frequently seen in positive skin scrapings of T. mentagrophytes and T. rubrum and may account for the "rejuvenation" of the colonies which was noted. No macroconidia or characteristic microconidia were observed histologically. Apparently these types of spores are more suited to survival in conditions other than those found in or on the animal. The early death of many of our inoculums, particularly those of T. rubrum, indicated to us that mere survival of vegetative spores played a minor role and that the host's defense mechanisms were actively and in some instances successfully engaged in destroying the organism.
It was our impression that in all instances a large percentage of fungi died shortly after inoculation into the pouch. We are unable as yet to account for this initial high mortality. Probably several factors were responsible. The organisms were grown on Difco's Dextrose Sabouraud's Agar at room temperature and prior to inoculation they were subjected to grinding to a fine particle size. The change from Sabouraud's media to the animal per se and the change
TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RUBRIJM
365
from room temperature to the rat's temperature were undoubtedly important factors in this early death. However we do not feel that these factors accounted for the death in some instances of all the organisms. Death of the majority of the inoculated organisms occurred either in the fluid portion or in the innermost zone of the wall of the pouch which was composed predominately of polymorphonuclear leucocytes. The hyphal elements in all stages of degeneration were always the center of large aggregates of polymorphonuclear leucocytes and their debris. This zone of polymorphonuclear leukocytes and dead organisms was prominent in all three types of pouches. At present we are unable to assign an
exact role to the fluid and the polymorphonuclear leucocytes in the death of the fungi. The dead organisms rapidly disintegrated and fragmented so that Hotchkiss-
McManus positive fragmented and granular material was observed to be diffusely scattered throughout this polymorphonuclear leukocyte zone. On occasion these cells were observed to be actively ingesting this material. Identical material was also seen in the cytoplasm of the histiocytes directly below the above
area. None of the mechanisms of allergy has been cited to explain any of our observations, but it is interesting to speculate that, in view of the tremendous amount of fungal material ingested by these fixed tissue cells, they might react violently to future administration of the same or similar material. No HotchkissMcManus positive fungal material was found in the outer portion of the pouch wall, and we do not know the ultimate disposition of this material; however, in speculation we feel that it is slowly digested by the local histiocytes and that breakdown products are ultimately released. Active proliferating fungal elements were observed in most sections in spite of the high mortality. The majority were located in the inner zone but many ex-
tended into the middle histiocytic zone. In all instances proliferating hyphae were observed which suggested to us that the subsequent positive cultures obtained from pouches were not the result of surviving vegetative spores. Selye (8, 13) has shown that the wall of the granuloma pouch which is produced by the use of croton oil reaches a high degree of resistance to subsequent injections of the same irritant. This protection is specific but in such a pouch the ability to resist other types of irritants may be less. He employs this explanation to account for the early necrosis which occurred in such granuloma pouches subsequently inoculated with a different type of irritant of such a dosage that it normally would not produce necrosis. We employed predominately the granuloma pouch prepared with croton oil in the hope that this lowered resistance to
other irritants might greatly alter the host-parasite relationship to the superficial dermatophytes. Although we did encounter early necrosis of the wall of the pouch after inoculation with T. mentagrophytes and T. rubrum, we do not feel that the host-parasite relationship was radically altered by employing such pouches.
We are unable to evaluate the exact histological response of the host to the fungus. In those rats in which the pouch was formed with the use of croton oil, the histological response was well developed by the seventh to the tenth day at
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the time the pouches were inoculated with the fungus. When pouches were made using suspensions of fungi and air, we could not be absolutely certain that there had not been some contamination with agar. However, when comparison was
made between the two types of pouches the histological response was quite similar. There was cultural evidence of dissemination to the liver and spleen from the granuloma pouch in three animals. The pouches had been prepared with the use of croton oil and their pouches inoculated with T. mentagrophfjtes. The animals were sacrificed on the sixth day. Although we have not investigated this finding
specifically, apparently dissemination from the pouch can occur and in this study did so shortly after inoculation. Apparently the local host's response is successful in preventing late continued dissemination. CONCLUSIONS
1. T. mentagrophytes and T. rubrum. can survive and grow beneath the epidermis in the granuloma pouch. In this study T. mentagrophyte.s was re-cultured from the granuloma pouches of two rats 40 days post-inoculation.
2. Growth of these fungi occurred primarily by the proliferation of hyphal elements. These hyphal elements were observed histologically in pouches which by culture revealed the presence of viable organisms.
3. Spore formation occurred and was of the intercalary and terminal type. The formation of macroconidia and microconidia was not observed. 4. A large percentage of the fungi died shortly after inoculation into the pouch. 5. The polymorphonuclear leukocyte was the predominant cell found in association with the dying fungal elements. 6. The fixed tissue histiocytes appeared to play an active role in the ultimate disposition of fungal debris. 7. Dissemination occurred in the liver and spleen in three animals inoculated with T. mentagrophytes. REFERENCES 1. SULZBERGER, M. B.: Experimentelle untersuchungen uber die derinatotropie der trichophytonplize. Arch. f. Dermat. u. Syph., 157: 345—357, 1929. 2. STERNBERG, T. H., TARBET, J. E., NEWCOMER, V. D. AND WIRER, L. H.: Deep infection
of mice with trichophyton rubruin (purpureum). J. Invest. Dermat., 19:373—384, 1952. 3. Lxwis, G. M., HOPPER, M. E. AND SCOTT, M. J.: Generalized trichophyton rubrum infec-
tion associated with systemic lymphoblastoma. Report of three cases. Arch. Dermat. & Syph., 67: 247—259, 1953.
4. WILSON, J. W., PLUNKETT, 0. A. AND GREGERSEN, A.: Nodular granulomatous perifolli-
culitis of the legs caused by trichophyton rubrum. Arch. Dermat. & Syph., 69: 258— 277, 1954.
5. SELYR, H. AND HORAVA, A.: Second Annual Report on Stress., Montreal, Acta Inc., Med. Pub., 268, 1952.
6. SELYE, H.: Use of granuloma pouch technic in the study of antiphlogistic corticoid. Proc. Soc. Exper. Biol. & Med., 82: 328—333, 1953.
7. SELYE, H.: Fourth Conference on the Adrenal Cortex. Nov. 13—15, 29—97, 1952, New York, Josiah Macy Foundation.
TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RTJBIUJM
367
SELYN, H.: Induction of topical resistance to acute tissue injury. An experimental study with the granuloma pouch technique. Surg. Clinc. No. America, 33: 1417—1146, 1953. 9. SELYE, H.: On the acquisition of tissue resistance to digestion by gastric juice. Gastro8.
enterology, 26: 221—229, 1954.
10. SELTE, H.: The effect of topically administered hydrocortisone upon dystrophic skin, as revealed by the pneumoderma technic. J. Invest. Dermat., 21: 91—98, 1953. 11. KLIGMAN, A. M. AND MESCON, H.: The periodic acid-schiff stain for the demonstration of fungi in animal tissue. J. Bact. 60: 415—421, 1950. 12. GRIDLEY, M. F.: A stain for fungi in tissue sections. Am. J. Clin. Path., 23: 303, 1953.
13. SELYE, H.: The part of inflammation in the local adaptation syndrome. The mechanism of inflammation, pp 53—74. G. Jasmin and A. Robert, Acta Inc., Medical Publishers, Montreal, Canada.
DISCUSSION Du. STEPHEN ROTHMAN, Chicago, ill.: Were those fungi in the pouches viable; and if so, how long? If the experimental period was Dot long enough it still re-
mains to prove that they are permanently viable outside the horny material. Dn. J. WALTER WILsoN, Los Angeles, Calif.: We, too, saw these large, spheri-
cal, Hotchkiss-McManus staining bodies in our perifollicular granuloma areas. Dr. Wright, do you have any impression as to whether these are fungi in some process of dying, or are they viable? DR. EDWIN T. WRIGHT (in closing): I would like to thank the discussors for their comments. Dr. Rothman's question can best be answered by giving the summary which, due to the limited time, I failed to present. 1. T. mentagrophytes and T. rubrum csn survive and grow beneath the epidermis in the granuloma pouch. 2. Growth occurred primarily by the proliferation of hyphal elements. 3. The polymorphonuclear leukocyte was the predominant cell found in association with the dying fungal elements. 4. The fixed tissue histiocytes appeared to play an active role in the ultimate disposition of fungal debris. Dr. Wilson asked the question, "Are the large cells viable?" We think that they probably are, primarily because they take on the same characteristics as do the other fungal elements which we consider to be viable. In closing I would like to reiterate the differences between viable and dead organisms as noted in this study. Viable organisms were characterized by 1) Good staining (the cell walls, septae and spores stained clearly and were well outlined). 2) There was evidence of branching and individual cell enlargement. 3) Positive cultures were obtained as late as 40 days post-inoculation. Dead organisms were characterized by: 1) Poor staining quality. 2) Collapse of individual cells and narrowing of the diameter so that the hyphae appeared stringy. 3) No evidence of branching or individual cell enlargement.
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PLATE I
FIG. 1. T. mentagrophytes 21 days in pouch prepared with croton oil. Hotchkiss-McManus
stain. The large empty spaces are probably due to the injected oil. 25X FIG. 2. T. mentagrophytes 14 days in pouch prepared with 25mg. of hydrocortisone acetate
and air. Hotchkiss-McManus stain. Upper dark layer and the lighter staining vacuolated area represents a zone of polymorphonuclear leukocytes, hyphal elements and debris from both. Fibroblastic proliferation is greatly inhibited. 30X FIG. 3. T. rubrum 9 days in granuloma pouch produced with croton oil. HotchkissMcManus stain. Active proliferating hyphae are seen. 125X
TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RLTBRTJM
PLATE
I
369
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
PLATE II
Fro. 1. T. rubrum 14 days in pouch prepared with hydrocortisone and air. Hematoxylin and eosin stain. Demonstrates zone composed of polymorphonuclear leukocytes and necrotic debris in the wall. 35X FIG. 2. Alternate section of Fig. 1. Hotehkiss-McManus stain. Demonstrates necrotic fungal debris scattered throughout and in strands in the same zone. 35X FIG. 3. High power of Fig. 2, demonstrating fungal debris concentrated in histiocytes of middle zone. 650X
371
TRICHOPHYTON MENTAGROPHYTES AND TEICHOPHYTON RUBRUM
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372
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
PLATE III
Fiu. 1. T. mentaqrophytes 21 days in pouch prepared with croton oil. Hotchkiss-McManus stain. Demonstrates usual type of hyphal proliferation. 650X Fin. 2. Same slide as Fig. 1 but from a different area showing intercalary spore formation. 650X
Fin. 3. T. mentegrophytes 14 days in pouch prepared with hydrocortisone and air. Demonstrates spore germination. 650X FIG. 4. T. mentagrophytes 18 days in pouch prepared with croton oil. llotchkiss-McManus train showing unusually large spores. 650X
373
PLATE
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TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON RUBRTJM
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
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