Immunology of Dermatophytes and Dermatophytosis - Microbiology

BACrERIOLOGICAL REVIEWS, June 1974, p. 222-250Copyright 0 1974 American Society for Microbiology

Vol. 38, No. 2Printed in U.S.A.

Immunology of Dermatophytes and DermatophytosisS. F. GRAPPEL, C. T. BISHOP, AND F. BLANK

Skin and Cancer Hospital, Temple University Health Sciences Center, Philadelphia, Pennsylvania 19140, andDivision of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada KJA OR6

INTRODUCTION.222IMMUNITY IN DERMATOPHYTOSIS.223Acquired Resistance ....... .................................. 223Animals ......................................... 223Humans .......................................... 224

Hypersensitivity ("Trichophytin" Reaction) ..................................... 225Dermatophytid Eruptions ......... ................................ 227Antibodies...................................................................... 228

Animals ......................................... 229Humans ......................................... 230

Nonspecific Resistance ........ ................................. 231IMMUNOCHEMISTRY OF DERMATOPHYTES.232Chemical Composition.232Antigenic Composition ........ ................................. 233

Differentiation of species.233Hypersensitivity factors ("trichophytin") ..................................... 234

Purified Antigenic Components ......................................... 234Isolation of antigens ....... .................................. 234Glycopeptides ......................................... 234Polysaccharides ......................................... 235

Keratinases ......................................... 235Chemistry of antigens ......................................... 235

Glycopeptides ......................................... 235Polysaccharides ....... .................................. 236Keratinases ......................................... 237

Immunological reactivity ............ .............................. 237Relation to structure ........ ................................. 237Taxonomic importance ........... .............................. 239

IMMUNOPROPHYLAXIS AND THERAPY.240Animals ......................................... 240Humans ......................................... 240

SUMMARY.241

INTRODUCTIONThe dermatophytes include species of the

genera Epidermophyton, Microsporum, andTrichophyton. These fungi cause suporficialinfections known as dermatophytosis (ring-worm) in humans and animals. Dermatophytesusually invade and parasitize only the kerati-nized layers of skin, nail, and hair. This highlydeveloped host-parasite relationship is respon-sible for a multitude of clinical manifestationsof dermatophytosis. However, the disease proc-ess is greatly influenced by the host response tothe dermatophyte infection.Sabouraud's (198) fundamental studies on

the structure and taxonomy of the dermato-phytes and their relationships to clinical man-ifestations of dermatophytosis laid the basisfor subsequent studies on changes induced in

the infected host. These investigations beganwith the work of Plato and Neisser in 1902 (173)on "trichophytin" sensitivity, which was fol-lowed by the work of J. Jadassohn and B. Blochon the manifold aspects of the immune responsein man and experimental animals. The earlywork on the immune response of the host wassummarized by B. Bloch in 1928 in German(29). Since then, Gbtz (75) has reviewed theliterature in detail in German, and Lepper (144)has published a concise review in English.Other reviews which have covered immuno-

logical aspects of dermatophytosis are those bySulzberger (217), Kligman and DeLamater(132), Huppert (93), and Salvin (202).This review includes selected references from

both the earlier and more recent literature inorder to illustrate the variety of immune phe-

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VOL. 38, 1974 DERMATOPHYTE AND DERMATOPHYTOSIS IMMUNOLOGY

nomena which occur in dermatophytosis, theantigenic composition of the dermatophytegroup, and the status of immunoprophylaxisand therapy for dermatophytosis.No attempt has been made to change the

nomenclature of the dermatophytes as used bythe authors.Synonyms are listed below with perfect states

in parentheses (Table 1).

IMMUNITY IN DERMATOPHYTOSIS

Acquired ResistanceA state of relative acquired resistance to

reinfection with the same or another species ofdermatophyte may result from cutaneous infec-tion. This resistance may vary both in degreeand duration, depending upon several factors,including the species or strain of dermatophyte(zoophilic or anthropophilic), the host (animalor human), and the site of infection (smoothskin, hair, or nails).

In general, the zoophilic species cause moreinflammatory infections which may heal spon-taneously and result in relative resistance toreinfection. The anthropophilic species usuallycause more chronic, less circumscribed infec-tions which result in less resistance to reinfec-tion.Animals. The characteristics of the primary

infection in experimental infections of severalanimal species by a variety of dermatophytespecies have been reviewed by DeLamater andBenham (52).Bloch (28) was the first to show that cutane-

Name

ous inoculation of guinea pigs with the zoophilicdermatophytes Achorion quinckeanum, Tricho-phyton gypseum, or Microsporum lanosum pro-duced infections which healed spontaneouslyand resulted in a relative resistance to subse-quent reinfection. The phenomenon was latercalled "le phenomene de la reaction acceleree"or "le phenomene de Bruno Bloch" (193). Theresistance resulting from the first infection wasmanifested by an accelerated inflammatoryresponse and course of disease in which fungiwere not always demonstrable. Bloch claimedthat this immunity, although not usually com-plete, was generalized and not confined to thesite of the primary infection. The immunity wasapparent 7 to 9 days postinoculation and per-sisted for 18 months. It was not species-specificInoculation of the host by subcutaneous orintraperitoneal injection of the fungus also al-tered the susceptibility of the skin to cutaneousinfection, but to a lesser degree.This initial report was later confirmed, modi-

fied, and expanded by Bloch and Massini (31),Thardshimanjanz (225), and several other in-vestigators. Hanawa (88) claimed that an accel-eration of the disease process in reinfectedanimals was detectable as soon as 2 to 3 daysafter the first infection and was reflected in thehistopathological changes seen in biopsies of thesite. A longer time interval between the primaryinoculation and the reinfection resulted in agreater modification of the second infection.

Sutter (222), Greenbaum (87), and Kogoj(134) claimed that a local immunity was pro-duced in guinea pigs after a cutaneous infection

Achorion gypseum ....................Achorion quinckeanum ...............Achorion schoenleinii.Epidermophyton Kaufmann-Wolf.

Microsporum lanosum ................Microsporum praecox .................Trichophyton asteroides ..............

Trichophyton faviforme ...............Trichophyton ferrugineum ............Trichophyton granulosum .............

Trichophytongypseum.

Trichophyton interdigitale.

Trichophyton purpureum .............Trichophyton sabouraudii ............Trichophyton sulfureum ..............

Microsporum gypseum (Nannizzia incurvata and N. gypsea)Trichophyton or Microsporum quinckeanumTrichophyton schoenleinii or Trichophyton schonleiniiTrichophyton mentagrophvtes var. interdigitale (Arthrcbenhamiae?)

Microsporum canisSabouraudites praecoxTrichophyton mentagrophytes var. granulosum (Arthrcbenhamiae)

Trichophyton verrucosumMicrosporum ferrugineumTrichophyton mentagrophytes var. granulosum (Arthr(benhamiae)

Trichophyton mentagrophytes var. granulosum (Arthr4benhamiae)

Trichophyton mentagrophytes var. interdigitale (Arthr4benhamiae?)Trichophyton rubrumTrichophyton tonsurans var. sabouraudiiTrichophyton tonsurans var. sulfureum

TABLE 1. Nomenclature of the dermatophytes

Synonym

oderma

oderma

oderma

oderma

oderma

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GRAPPEL, BISHOP, AND BLANK

with A. quinckeanum, T. gypseum, or M. lano-sum which was complete only at the site of thespontaneously healed infection, but not in otherareas, where a second infection resembled thefirst one.

Rivalier (193) avoided trauma to the skinprior to cutaneous inoculation of guinea pigs.He used Trichophyton granulosum and showedthat an autoclaved spore suspension applied 3weeks after the first infection could induce thesame cutaneous reactions, although less pro-nounced, as a second infection with live spores.Thus, the altered host response, rather than theproliferation of the fungus, appeared to beresponsible for the accelerated cutaneous reac-tions seen in a second infection.DeLamater and Benham (53) studied the

dermatophytic infection in guinea pigs exten-sively and characterized the acquired immunityto several strains of dermatophytes. These in-vestigators concluded that in the guinea pig: (i)a cutaneous infection by a virulent strain,whether infection occurred via the skin or theblood stream, altered the animal's subsequentresponse to infection; (ii) the lesion caused by asecond infection resembled the first one, butdeveloped and healed more rapidly and rarelycontained demonstrable fungi; and (iii) theresistance was greatest at the previously in-fected site.These observations in guinea pigs were con-

firmed by Bonk et al. (33) who also showed thatvirulent strains of both zoophilic and anthropo-philic forms of Trichophyton mentagrophytesinduced resistance which was greatest at thepreviously infected site.DeLamater and Benham (53) showed that the

resistance reached a peak 2 to 3 weeks afterhealing of a first infection and corresponded tothe development of delayed cutaneous hyper-sensitivity which also peaked at that time. Byrepeated inoculations of guinea pigs, Catanei(39) showed that the maximal resistance toreinfection was obtained after a second infec-tion.

Several investigators (53, 155, 182) infectedpregnant guinea pigs with dermatophytes andfound that the acquired resistance to infectionwas not passed on to the offspring.

In rabbits and cats, the course of the secondinfection resembled that of the first one. Noevidence was obtained for immunity or cutane-ous sensitization after one infection. Resistanceand cutaneous sensitization were apparent aftermore than one infection in rabbits (53). Reissand Leonard (186) reported that reinfection ofdogs, cats, and rabbits resulted in a shorter

course of infection as compared with the firstinfection. No uniform cutaneous sensitizationwas associated. Kligman (131) found no evidenceof acquired resistance or sensitization to derma-tophytes in mice and rats.

Kielstein (123) and Lepper (145) studied thedevelopment of resistance in cattle by experi-mental infections with Trichophytonverrucosum. The primary infection healedspontaneously, and cattle became resistant tocutaneous reinfection both on previously in-fected sites and noninfected areas. The resist-ance persisted for up to 1 year or more afterrecovery. Lepper (145) observed that althoughthe infection rate was the same, the inflamma-tory response and the resolution of lesions weremore rapid in older animals. Elimination of theinfection was associated with a marked, delayedhypersensitivity reaction.Humas. Bloch (28) and Bloch and Massini

(31) were the first to show that a dermatophyticinfection in humans results in a relative resist-ance to subsequent infection. They infectedsmooth skin with a highly virulent strain of A.quinckeanum, a zoophilic species, which causedinflammation, kerion-like infiltration, and scu-tula formation. The regional lymph nodes be-came enlarged, and the infection healed spon-taneously in 3 months. The infection, even withthis zoophilic species, was less circumscribedand more chronic than in guinea pigs. Reinfec-tion resulted in a highly accelerated, less severecourse of disease. No fungi were isolated, andthere was no scutula formation. These investiga-tors claimed that the acquired resistance wasnot confined to the site of previous infection,but was generalized. The resistance was notspecies-specific.

Subsequent investigations have shown that,in humans, increased resistance usually followsthe severe inflammatory forms of infection suchas kerion formation, usually caused by zoophilicspecies, but does not always follow the morechronic infections caused by anthropophilicspecies (12, 194). Barlow and Chattaway (12)pointed out that fungi which do not invade thehair follicle do not seem to give rise to anequivalent immunity when growing in the hornylayer of the smooth skin.Kligman (130) infected the scalp of children

by using hairs infected with Microsporumaudouinii. Upon reinfection, only two of sevenchildren developed lesions. These lesions wereof a trivial nature and of shorter duration.

Resistance to reinfection was also demon-strated in natural infections. Although childrenwith tinea capitis returned to heavily infected

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environments after cure, they rarely becamereinfected (66, 232).

In contrast, Desai et al. (54) could not demon-strate such acquired immunity in experimentalTrichophyton rubrum infection of smooth skin.The second infection resembled the first oneboth in extent and duration.Although Bloch's early work indicated that

acquired resistance is generalized, other inves-tigators disagreed and demonstrated a stronglocal immunity only at the previously infectedsite and not in skin areas distant from it (87,222). They concluded that experimental infec-tion of the smooth skin by the zoophilic species,A. quinckeanum, M. lanosum, and T. gypseum,produced only a local tissue immunity in hu-mans. The immunity was accompanied by cu-taneous sensitization. Epstein and GrUnmandel(60) also demonstrated a localized immunity inexperimental superficial infections. The im-munity extended to a distance of up to 20 mmsurrounding the focus and was manifested byfailure of the reinfection to take. Resistance wasdemonstrated in almost healed or cured focionly and lasted 3 months.

Hypersensitivity ("Trichophytin" Reaction)The "trichophytin" reaction is the term used

for cutaneous hypersensitivity to dermatophyteantigens injected intradermally in humans orexperimental animals. Both immediate- anddelayed-type reactions occur, but the latter ismost often associated with infection.

Studies on hypersensitivity in dermatophyto-sis started in 1902 when Neisser published thework of his late assistant Plato on the prepara-tion and usefulness of "trichophytins" (173).Plato grew Trichophyton species isolated frompatients with deep-seated trichophytosis in aliquid medium consisting of beef extract, pep-tone, and maltose. After 2 to 3 months at roomtemperature, the growth was ground and fil-tered. Plato called the filtrate "trichophytin."

Plato was unable to infect rabbits with der-matophytes. Injection of "trichophytin" intononinfected rabbits did not cause any irritationat the site of injection, nor did he observe a risein temperature. However, subcutaneous injec-tion of mycelium into the rabbit's ear, followed4 days later by "trichophytin" injection, re-sulted in an elevated temperature.

In patients with deep-seated trichophytosis,Plato (173) found that parenteral injection of"trichophytin" caused signs and symptomsanalogous to those induced in tuberculous pa-tients by injection of tuberculin: general toxicreactions including elevated temperature, per-

spiration, loss of appetite, headache, and painin the joints. There was inflammation, forma-tion of pustules, and burning at the injectionsite. "Trichophytin" did not give a tuberculin-like reaction in tuberculous patients, and pa-tients with superficial dermatophytosis did notreact to tuberculin. Plato, therefore, regardedthe "trichophytin" reaction as specific.Bloch and Massini (31) showed that experi-

mental trichophytosis induced hypersensitivityto "trichophytin" in both humans and in guineapigs. In humans, the cutaneous reactivity to"trichophytin" became apparent 7 to 10 daysafter infection. After healing, the reaction re-mained positive for up to 3 years. The inducedhypersensitivity was specific for dermato-phytes, but was not species-specific. An infec-tion due to T. gypseum caused hypersensitivitynot only to "trichophytin" prepared from thisdermatophyte, but also to favin, prepared fromA. schoenleinii, and to rhicrosporin, preparedfrom M. lanosum.

Sutter (222) showed that the level of cutane-ous sensitivity which developed in guinea pigswas much lower than in humans. Subcutaneousinjection of infected patients with "tricho-phytin" regularly caused a general reactionincluding fever and leukocytosis. However,large doses injected into sensitized guinea pigsdid not cause a rise in temperature or othertoxic reactions. The reaction was as specific asin humans and characterized by the absence ofgeneral toxicity. The guinea pigs reacted only if"trichophytin" was injected intradermally(225). The "trichophytin" reaction was charac-terized by a lentil-shaped, large, inflamed pap-ule with an erythematous halo at 24 h, afterwhich it decreased. Bloch (29) pointed out that.the intensity of the reaction varied considera-bly. He considered the erythematous haloaround the papule very specific. Thardshiman-janz (225) and Sutter (222) found that the"trichophytin" reaction became positive 7 to 9days after infection, corresponding to the heightof infection. It reached its maximum when theinfection subsided.

In humans, repeated inoculation with thesame dermatophyte led to a state of hyposensi-tization or anergy. Both dermatophyte infectionsand "trichophytin" injections produced de-creased inflammatory reactions at previouslyinfected sites (222).The development of delayed cutaneous hy-

persensitivity during the course of cutaneousinfection with T. gypseum was later studied inguinea pigs by DeLamater (50, 51). The "tricho-phytin" reaction peaked at 15 days after in-

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oculation and then started to regress. It in-creased again 2 to 3 weeks after clearing of theinfection, then decreased sharply and remainedat a low level for several months. The site ofprevious infection was relatively anergic asmanifested by a reduced inflammatory reactionto "trichophytin." The ability to react to "tri-chophytin" increased with age, being greatest inadult and least in newborn guinea pigs. Therewas considerable variation in degree of sensitiv-ity in the adult group. The younger animalswere more susceptible to progressive infection.Cruickshank et al. (47) showed that the

delayed hypersensitivity reaction to "tricho-phytin" could be transferred to noninfectedguinea pigs with peritoneal exudate cells ofsensitized donors.Immediate forms of hypersensitivity have

also been demonstrated in experimental ani-mals. W. Jadassohn and associates (108, 109)reported anaplylactic and smooth muscle sen-sitization to "trichophytin" in infected guineapigs. Cruickshank et al. (47) detected immedi-ate cutaneous reactions to "trichophytin" inguinea pigs infected with T. mentagrophytes byusing Evan's Blue dye. Ito and Nishitani (99)showed that repeated intravenous injection ofrabbits with T. mentagrophytes var. asteroidescaused a systemic vascular reaction comparableto allergic vasculitis in humans.According to Jaksch (111) and Kielstein

(121), naturally infected horses and cattle alsodevelop cutaneous hypersensitivity. Lepper(145) studied the development of cutaneousdelayed hypersensitivity in cattle experimen-tally infected with T. verrucosum. The abilityto eliminate infection was associated with thedevelopment of a marked delayed hypersensi-tivity response in four of six cattle 14 days afterinoculation. A mild delayed-type reactiondeveloped on all reinoculated skin sites within48 h. The intravenous injection of 104 viableunits of T. verrucosum resulted in an immedi-ate skin reaction at the original site of infection.The animals also had elevated temperatures.These reactions disappeared 24 h after injec-tion.Neves (175) found mainly delayed-type cu-

taneous hypersensitivity to "trichophytin" inhuman volunteers infected with different spe-cies of dermatophytes. The degree of sensitiza-tion was greater in individuals in whom lesionsbecame more inflammatory. He did not find ahigh incidence of immediate-type reactivity,even in cases of chronic T. rubrum infection.The development of cutaneous hypersensitiv-

ity in an experimental T. rubrum infection in

humans was reported by Ito (96). The delayedcutaneous reaction developed by 14 days afterinoculation. It disappeared after 35 days, atwhich time an immediate-type urticarial reac-tion developed.

Patients with chronic T. rubrum infectionsfrequently have a negative delayed-type or aretarded delayed-type "trichophytin" reaction.Immediate-type "trichophytin" reactions andcirculating antibodies are found more often inthese patients than in those infected with otherdermatophytes (55, 149, 217, 244). The retardeddelayed-type reaction was often found as-sociated with dermatophytid eruptions (46,244).Immediate-type reactions to "trichophytin"

have been found to be exceptionally prevalentin patients infected with T. rubrum. Desai et al.(55) found these reactions in as many as 85% ofinfected adults. However, this immediate-typeurticarial reaction occurred both in patientsinfected by other dermatophytes and in nonin-fected individuals with a similar lower fre-quency (244). It has also been observed in atopicindividuals (154), in individuals with recurrenterysipelas-like eruptions (219), in individualswith recurrent lymphangitis (115), and in pa-tients under treatment with "trichophytin"(220).

Sulzberger and Wise (220) and later Jillsonand Huppert (115) showed that the delayed-type, but not the urticarial reaction, was sup-pressed by repeated intradermal injections of"trichophytin." If the urticarial reaction wasabsent prior to treatment with "trichophytin,"the immediate reaction appeared at the timethe delayed reaction disappeared. A constantsupply of antigen over a long period of time wasconsidered to be important for the induction ofthe immediate-type reaction to "trichophytin."

Sulzberger and Kerr (218) showed that theimmediate-type reaction to "trichophytin" inhumans could be transferred by serum, indicat-ing a circulating antibody reaction. It mightalso be mentioned that Sulzberger (216) ob-served that injection of "trichophytin" intosensitive subjects often provoked allergic reac-tions such as asthma and rhinitis, also as-sociated with circulating reaginic antibodies.Lewis and Hopper (147) compared "tricho-

phytin" reactions in patients with T. rubrumand T. mentagrophytes infections. Of patientswith T. rubrum infections, 53% had immediate-type reaction only, 10% delayed-type reactiononly, 32% both reactions, and 5% no reactions;of patients with T. mentagrophytes infections,1.5% had immediate-type reaction, 72.9% de-

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layed-type reaction, 1.5% both reactions, and10.6% no reactions. T. rubrum infections, inwhich the immediate-type reaction predomi-nates, are more recalcitrant and resistant totreatment than T. mentagrophytes infections inwhich the delayed-type reaction predominates.By using a "trichophytin" purified from T.

mentagrophytes, Jones et al. (116) found nospecific association between immediate-typereactions and T. rubrum infection. However,they did find a strong correlation betweenimmediate-type reactivity and chronic infec-tion. Although 7.0% of individuals with onlydelayed-type hypersensitivity had recurrent in-fections, 75% of those with both immediate- anddelayed-type hypersensitivity or only immedi-ate-type hypersensitivity had recurrent infec-tions.The appearance of the delayed reaction hy-

persensitivity is closely associated with theaccelerated response to reinfection in both ani-mals and humans (183).Balogh et al. (6) reported a correlation be-

tween the results of "in vitro" lymphocytetransformation tests and the spread of thedisease and sensitization of patients with der-matophytosis.The lack of either standardized or pure,

homogeneous "trichophytins" for skin testinghas severely limited the usefulness of the "tri-chophytin" reaction for diagnostic purposes.Wilson (242) also pointed out that the "tricho-phytin" reaction cannot establish or exclude thepresence of dermatophytosis because it maynever develop or may persist long after theinfection is cured. Commercial "trichophytins"are composed mainly of pooled, heat-treated,concentrated culture filtrates of from 1 to 15species. There are more than 10 "trichophytins"available which vary in their manufacturingprocess (208). These crude preparations usuallycontain antigens common to other microorgan-isms as well as dermatophytes.Although the delayed reaction may be most

reliably associated with dermatophytosis, someinvestigators have reported reactions in individ-uals with other sensitivities, including penicillinand tuberculin (181, 206). In addition, W.Jadassohn (105) reported cross-reactions in ashigh as 90% of noninfected individuals.

Dermatophytid EruptionsA dermatophytic infection in humans may

result in the production of secondary skin erup-tions. They occur in a large variety of clinicalforms and are known as dermatophytids, epi-dermophytids, favids, microsporids, trichophy-

tids, or "ids." The lesions occur at a distancefrom the focus of infection. Fungal elements arenot usually demonstrable. The "trichophytin"test is always positive. The secondary eruptionsdisappear after successful treatment of theprimary infection.

J. Jadassohn (102) was the first to recognizethese lesions as allergic reactions of the skinsensitized by the process of dermatophytosis.He chose this term in analogy to Darier'stuberculid after he had originally called thisallergic manifestation "lichen trichophyticus."Williams (241) reviewed the early work of

many investigators who demonstrated the oc-currence of fungi in the blood of individualswith dermatophytid eruptions. Mycelia werevery rarely found in dermatophytid eruptions,but could be demonstrated in fresh lesions lessthan 1 or 2 days old.The "id" lesions are believed to occur as a

result of the hematogenous spread of fungi ortheir allergenic products from the primary focusof infection. The reaction between circulatingantigens and skin-sensitizing antibodies is be-lieved to be responsible for the skin manifesta-tions.According to Bloch (29), the dermatophytid

reaction appears at the height of infection orshortly thereafter due to a massive liberation ofantigens. The eruptions may last from 5 to 20days. Dermatophytids often occur after X-raytreatment, "trichophytin" skin tests, or localirritations.Bloch did not believe that lymphatic spread

of the antigens was of great importance. Trans-port of the antigens by the blood stream wouldexplain (i) the sudden appearance of eruptionsat different parts of the body or the wholeintegument including mucosa, (ii) the symmet-rical distribution of the dermatophytids, (iii)the episodic eruption, (iv) fever, (v) changes inthe blood picture, (vi) the occurrence of subcu-taneous dermatophytids, and (vii) the analogywith tuberculids and syphilids.The fungistatic components of blood and the

body temperature, not optimal for the growth ofdermatophytes, may explain why fungal ele-ments are only very rarely found in dermato-phytid lesions (75). However, the time at whichthe presence of fungal elements is investigatedis of utmost importance. Experiments withguinea pigs showed that spores penetrate theblood stream from 1 to 48 h after cutaneousinfection (103). Sulzberger (215) observed asecond phase of penetration of fungal elementsinto the blood at the height of infection 9 to 13days after cutaneous inoculation of guinea pigs.

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Dermatophytid reactions are most often seen inhumans at the height of infection.The histopathological changes seen in biop-

sies of dermatophytids resemble those observedin tuberculids and have been described in detailby Gans and Steigleder (71) and by Montgom-ery (170). Sulzberger (217) grouped these poly-morphous skin manifestations into 17 differentforms, and Gotz (75) classified them into epi-dermal, cutaneous, subcutaneous, and vascularforms.

J. Jadassohn (102) observed dermatophytidsmainly in children with kerion Celsi and lessfrequently in adults with sycosis barbae. Thedermatophytids were located mainly on thetrunk and more rarely on the extremities andface. They consisted of small, pale to intenselyred, follicular nodules which were disseminatedor arranged in groups. Very fine, spiny projec-tions or pustules sometimes appeared on thenodules. In other cases, plaques resemblingseborrheic dermatitis were observed as well asreddening of the oral mucosa. Scarlatinoideruptions were seen at the onset of dermatophy-tids. More rarely, nodules resembling erythemanodosum were found on the lower extremities.The general health of the patients was alsoaffected. High fever, changes in the blood pic-ture (leukocytosis and lymphocytosis), lym-phadenopathy, splenomegaly, and involvementof the joints accompanied the acute onset of thedermatophytids. Even cases of anaphylaxishave been reported (75).Although Jadassohn's cases were mainly due

to zoophilic dermatophytes, subsequent reportsalso implicated anthropophilic species. Bloch(29) believed that these anthropophilic specieswere abnormally virulent. The majority of der-matophytids had occurred in children whosebodies, according to Bloch, became sensitizedmore easily. However, dermatophytosis, at thattime, occurred more frequently in children,tinea pedis (athlete's foot) not being as wide-spread as it is now. Dostrovsky et al. (57) found"id" reactions in only 0.2% of 6,390 cases oftinea capitis, a disease of childhood. Theseusually occurred following X-ray epilation.

In a group of patients with dermatophytosisseen at the Skin and Cancer Hospital of Phila-delphia, 4.2% of children and 4.6% of adultpatients had dermatophytids. The ratio ofmale-female was 1: 1. The infections were lo-cated on the scalp in childien and on thesmooth skin, usually the feet, in adults. Thecausative organisms were M. audouinii, Micros-porum canis, Trichophyton sulfureum, T.mentagrophytes var. granulosum, Epidermo-

phyton floccosum, T. rubrum, and T.mentagrophytes var. interdigitale. Most ofthese organisms did not cause deep-seated in-fections (F. Blank et al., unpublished data).Bloch (29) gave a male-female ratio of 3: 1. Hepointed out that boys were more often afflictedwith ringworm than girls.Williams (240) and later Jadassohn and Peck

(106) reported that many dermatophytids onthe hands are the sequel of infections of the feet(athlete's foot). This is probably the mostcommon location of dermatophytids seen pre-sently in patients.

Successful treatment of the primary focus ofinfection results in the disappearance of the"id" lesion. However, corticosteroids will hastenthe involution of the "id" reaction and aid inhyposensitization.Dermatophytids do not usually occur in infec-

tions of animals; however, Kuroda (141, 142)and Inaba (95) observed such lesions on the earsof rabbits after experimental cutaneous reinfec-tion with T. mentagrophytes var. asteroides.Their appearance corresponded to a high levelof complement-fixing antibodies and cutaneoushypersensitivity. Dermatophytids could also beinduced experimentally by intravenous injec-tion of large amounts of fungi after a primaryinfection. Ito and Kuroda (98) studied thehistopathological changes in these trichophy-tids and found them comparable to those seenin some human dermatophytids.Trichophytids have also been produced ex-

perimentally in guinea pigs. Bloch (29) showedthat intracardial reinoculation caused tubercu-lid-like skin papules. Later Henrici (90) foundthat a trichophytid could be produced in theguinea pig by a single, cutaneous infection withT. mentagrophytes followed after several weeksby parenteral injection of a massive dose ofspores, cell extract, or crude polysaccharideantigen. The "ids" were localized in the areas ofthe healed infection or extended beyond andoccurred on hairless areas, usually paws andears. The reaction consisted of a generalizederythema followed by scaling and peeling. Noorganism was cultured from the lesions.

AntibodiesIn spite of the superficial nature of most

infections, circulating antibodies have beendemonstrated in sera from both animals andhumans with either natural or experimentaldermatophytosis.Disagreement has arisen among some inves-

tigators as to their presence or the specificity ofthese antibodies for the infecting organism. The

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differences in experimental results may be ac-counted for by (i) the variety of methods usedfor analyses, including Prausnitz-Kiistner, ag-glutination, precipitation, complement-fixa-tion, immunofluorescence, and "in vitro"growth inhibition tests; (ii) the different testantigens used, including concentrated culturefiltrates, commercial "trichophytins," mycelialextracts and crude polysaccharides, or otherfractions; (iii) the time during the course of in-fection at which blood was drawn from the host;and (iv) the nature of the infection (chronicversus acute).Animals. Citron (41) was the first to report

the presence of circulating antibodies to der-matophytes in sera of experimental animals. Heinjected rabbits with suspensions of live T.gypseum and detected precipitating antibodiesto extracts prepared from this species. Theantibodies also reacted with extracts of Acho-rion schoenleinii and A. quinckeanum.His studies were followed by several unsuc-

cessful attempts to demonstrate antibodies insera of animals with dermatophytosis (44, 143,222).

Verotti (234) was the first to demonstratecirculating antibodies in serum from an animalwith a natural cutaneous infection. He detectedcomplement-fixing antibodies to M. lanosum inthe serum of a dog. These antibodies also re-acted with other species of dermatophytes.Brocq-Rousseu et al. (34) infected guinea pigs

with Trichophyton equinum, T. granulosum,and A. gypseum. They found that commercial"trichophytin" was not a good test antigen forserological reactivity, but by using homologousspore suspensions as test antigens, they de-tected complement-fixing antibodies in sera of85% of the infected guinea pigs. Injections with"trichophytin" increased the circulating anti-body level in the infected guinea pigs, but didnot produce detectable levels in controls. Theyalso detected complement-fixing antibodies inone of nine horses with natural Microsporumequinum infections. Serological reactions werenegative when heterologous dermatophyte spe-cies were used as test antigens.

Martenstein (155, 157) described a factor(antibody) which could be found in serum orextracted from epithelial cells of guinea pigsinfected with A. quinckeanum. The factor wasthermostabile and when mixed with live spores"in vitro" at 37 C, a "toxic substance" wasformed. Intradermal injection of the incubatedmixture into normal guinea pigs resulted in aninflammatory infiltration without producingdermatophytosis.

Several investigators have demonstrated theproduction of agglutinins, precipitins, and com-plement-fixing antibodies by immunization ofanimals, especially rabbits, with killed der-matophyte cells. The earliest reports were thoseof Kolmer and Strickler (136), Fuke (67-69),and Sharp (210).

Later, Wharton et al. (238) detected precipi-tins in sera of rabbits experimentally infectedwith Tnchophyton purpureum or immunizedwith killed suspensions of this species. Theyfound that resistance to reinfection decreasedwith disappearance of precipitins.Kuroda (139) detected agglutinins in both

sera of rabbits cutaneously infected and sera ofrabbits immunized with killed T. mentagro-phytes var. asteroides. He used an antigen ob-tained by mechanical disruption of the my-celium. By using a similar antigen, Tomomatsuand Inaba (229) demonstrated precipitins andagglutinins in sera of cutaneously infected rab-bits.

Ito and Kashima (97) also found agglutininsin sera of rabbits infected with T. purpureumwhich reacted most strongly to an antigenprepared from the homologous species. Thesestudies were extended and reviewed by Ito (96).Cox and Moore (45) infected rabbits with T.

verrucosum. The experimental rabbit infectionran a short course as compared with the morechronic natural infection of cattle. Precipitinswere frequently detected, appearing after 24days following infection and persisting up to 13weeks. Low complement-fixing antibody titersrose after several reinfections. No apparentrelationship between the circulating antibodytiter and susceptibility to reinfection in therabbits was found.

Kielstein (123, 124) regularly detected anti-bodies to T. verrucosum in sera of infectedcattle by passive hemagglutination tests byusing coated, tanned sheep erythrocytes. Sera ofinfected cattle had titers above 1/32, whereasnormal animals had titers below 1/32. Anti-bodies were detected less regularly by comple-ment-fixation or precipitation in agar.The gamma globulins of 4 of 5 sera of infected

cattle significantly inhibited the oxygen uptakeof T. mentagrophytes "in vitro" (122). Again,no apparent relationship between the antibodytiter and the severity of infection was found.The author speculated that strong immunereactions in trichophytosis of cattle are proba-bly due to cell-bound antibodies which areresponsible for recovery and rarity of reinfec-tion.

Evidence for cell-bound antibodies at the site

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of previous infections was obtained in guineapigs infected with T. mentagrophytes var.granulosum (43). By indirect fluorescent anti-body tests, using fluorescent rabbit antibody toa keratinase isolated from this species, antibodyreactions to the keratinase could be detected.The reactions were at the level of the externalsheath of the hair follicle in sections of thepreviously infected skin. These reactions mightbe due to circulating antibodies with highaffinity for tissue.Humans. Kolmer and Strickler were the first

to demonstrate circulating antibodies in humandermatophytosis in 1915 (136). They detectedcomplement-fixing antibodies in 78% of serafrom children with tinea capitis due to M.audouinii. They also claimed that 2 of 3 sera ofpatients with favus due to T. schoenleinii hadcomplement-fixing antibodies. Sutter (222)stated that there are no complement-fixing orprecipitating antibodies detectable in sera frommost cases of human ringworm nor in any casesof animal ringworm. He found antibodies onlyin sera from isolated cases of deep-seated tineaof humans and only at the height of infection.Later, Carol (37) detected complement-fixingantibodies in sera from severe cases of deeptrichophytosis by using "trichophytin" as thetest antigen. Sera from patients with superficialinfections were occasionally positive, and serafrom favus cases were always negative.Nathan (172) found complement-fixing and

precipitating antibodies in sera from cases ofdeep trichophytosis, but considered them non-specific. Blumenthal and von Haupt (32) alsodetected complement-fixing antibodies in serafrom cases of deep-seated tinea, but rarely inthose from superficial infections. They observedthat the circulating antibody titers were relatedto the severity of the disease. Similar resultswere obtained by FoldvAri (65).Greenbaum (87) found that sera from pa-

tients with superficial ringworm did not giveclearly positive complement fixation, eventhough he used antigens prepared from theinfecting species.

Sulzberger and Kerr (218) and Sulzberger(216) demonstrated circulating antibodies inthe sera of patients with positive immediate"trichophytin" skin tests by using the Praus-nitz-Kistner test. These results were later con-firmed by Jadassohn and Suter (110) who alsodemonstrated circulating reagins by thismethod.

Jessner and Hoffmann (113) observed thatserum of patients with deep-seated trichophyto-sis contained substances which decreased thevirulence of the dermatophyte when inoculated

into guinea pigs. The serum also inhibited thegrowth of the fungus on culture media. Theinhibitor was stabile when heated at 56 C.Later, Ayres and Anderson (5) demonstrated"fungicidal" antibodies in sera of patients withdermatophytids. The growth of dermatophytestaken from a primary focus was inhibited onSabouraud medium containing 8.0% of suchpatient serum. Sera from patients without der-matophytids were not inhibitory.

Miller et al. (166) used "undenatured tricho-phytin" extracted from washed mycelium astest antigens. They detected mainly precipitinsin 11 of 15 sera, but rarely complement-fixingantibodies in sera of patients.

Kallenberg (117) detected complement-fixingantibodies in sera from 52 of 84 patients, but nosuch antibodies in sera from 60 noninfectedadults.

Later, Kuroda (140) used a crude polysaccha-ride antigen from T. mentagrophytes var.asteroides and found complement-fixing andprecipitating antibodies in sera of patients in-fected by Trichophyton interdigitale, T. ru-brum, or E. floccosum. All patients with der-matophytid eruptions had circulating anti-bodies. Similar results were obtained by Tomo-matsu (228) with sera from 20 of 26 patientswith T. mentagrophytes, T. rubrum, or Micros-porum ferrugineum infections. These sera cross-reacted with antigens from 11 different species.Pepys et al. (184) demonstrated precipitins in

50.0% of sera of patients with T. rubrum infec-tions, which not only reacted with mycelialextracts and culture filtrates of T. rubrum andT. mentagrophytes, but cross-reacted with anti-gens of Cladosporium herbarum, Penicilliumnotatum, and Aspergillus fumigatus. Later,Longbottom and Pepys (150) showed that theirglycopeptide fractions prepared from E.floccosum and T. mentagrophytes even reactedwith C-reactive protein of human serum.

Studies of experimental T. rubrum infectionin man (96) showed that precipitins are detecta-ble up to 5 weeks after inoculation. Theydecreased at the time when immediate cutane-ous hypersensitivity developed.Reyes and Friedman (188) detected anti-

bodies in sera of patients by passive hemag-glutination tests with sheep erythrocytes coatedwith mycelial extracts. These antibodies alsoreacted with antigens of species of Penicilliumand Hormodendrum and could be completelyabsorbed with mycelia of these saprophytes.The antibodies detected in sera from patientsby the indirect fluorescent antibody test alsoproved to be nonspecific for dermatophytosis(137, 168, 236). Cross-reactions were obtained

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with other fungi and with sera from noninfectedcontrols.Voldanova et al. (235) analyzed sera of pa-

tients with T. rubrum infections whose clinicalmahifestations resembled those described by H.Blank and Smith (26) with inflammatory proc-esses in the deep layers of the skin. Precipitinswere found in 9 of 12 sera by using a commercial"trichophytin" as test antigen. No comple-ment-fixing or agglutinating antibodies weredetected. More recent investigations includingthose of Schetsiruli (205) and Balogh et al. (6)have indicated the presence of circulating anti-bodies to the causative organism in sera ofpatients with allergic manifestations.

Antibodies to dermatophytes were demon-strated in sera of patients with tinea capitis andtinea corporis by charcoal agglutination, im-munodiffusion, and complement-fixation testsby Grappel et al. (82, 83). The test antigensused were saline extracts of autoclaved myceliaand polysaccharides which did not react withC-reactive protein or antiserum to C substance.The lower titers of the control sera whichreacted by charcoal agglutination tests in-dicated that the reactivities of sera from pa-tients were not due merely to cross-reactingantibodies present in serum, but were specifi-cally induced during dermatophytic infection.Complement-fixing and precipitating anti-bodies were absent in all of the adult controlsera. Precipitins were found in sera from pa-tients with tinea capitis due to M. audouinii, T.mentagrophytes var. granulosum, T. schoen-leinii, and Trichophyton tonsurans, and fromcases of tinea corporis due to T. mentagrophytesvar. granulosum and var. interdigitale, and T.rubrum. Patients' sera with precipitating anti-bodies reacted with saline extracts of M. au-douinii, T. mentagrophytes var. granulosumand var. interdigitale, T. rubrum, T. schoen-leinii, and T. tonsurans. These observationswere not surprising because many investigatorshave shown that dermatophyte species havemany antigenic components in common. Intinea capitis (83), the humoral antibodies werenot restricted to patients with deep-seated in-fections as has often been reported, and, intinea corporis, the zoophilic variety of T.mentagrophytes induced antibody formationmore frequently than the anthropophilic one.Chronic T. rubrum infections were often accom-panied by the formation of precipitating orcomplement-fixing antibodies. Antibodies werealso found in most patients who developed atrichophytid. In some cases, the antibodies per-sisted for only a short period of time. Therefore,the time at which the blood was drawn was

crucial for the detection of circulating anti-bodies.The results of tests for hypersensitivity and

circulating antibodies give indirect evidence forthe immunological induction of inflammatorytissue reactions such as kerion Celsi and ofallergic regional and disseminated eruptions.Zaslow and Derbes (251) obtained direct evi-dence for the presence of antibodies in keriontissue from nine patients by using a directfluorescent antibody test with rabbit antihu-man gamma globulin. Unfortunately, no infor-mation was obtained as to the specificity of thecell-bound gamma globulin.The occurrence of autoimmune-type reac-

tions in dermatophytosis has also been re-ported. Brusilovskaia (35) reported an autoim-mune process in patients with onychomycosisdue to T. rubrum and with other mycoses due toT. gypseum and Trichophyton faviforme, whichalso occurred in experimentally infected ani-mals. Autoimmune antibodies were detected bycomplement fixation tests with skin antigens.Recently, Peck et al. (179) reported on theresults of fluorescent antibody tests on sera of apatient with a T. rubrum infection. Theyshowed that complement-fixing antibodies to acommercial "trichophytin" had an affinity forepithelial tissue. Complete absorption of com-plement-fixing antibodies with "trichophytin"removed the antibodies which adhered to theepithelial cells.

Seeliger (206, 207) pointed out that serologi-cal techniques have not been of value fordiagnostic purposes due to the cross-reactivityof some dermatophyte antigens with those ofother microorganisms. However, as purifiedantigens which are more specific for the der-matophyte group become available, rapid, sen-sitive serological tests may prove to be useful.

Nonspecific ResistanceMany nonspecific factors may account for

natural resistance to infection in humans oranimals. This section will be limited mainly tothe "serum factor," a fungistatic substance inserum of normal individuals and animals. Thisfactor is believed to limit the growth of thedermatophytes to the keratinized layers, i.e.,prevent their invasion of living tissues (91).

Several investigators have demonstrated theantidermatophytic activity of normal serum.Sera of newborns, adults, and animals show thisinhibitory activity (13, 73, 152, 180, 195).

Lorincz et al. (152) and Blank et al. (25)characterized the "serum factor" as an unsta-ble, dialyzable, heat-labile component of freshserum and tissue fluid.

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Lorincz et al. (152) and Goodman et al. (73)planted dialysis bags containing live T.mentagrophytes in the peritoneal cavity of miceor dogs. The growth was inhibited in the cavity,but growth resumed once the bags were re-moved and the contents were planted on media.Rippon and Scherr (191) planted dialysis

bags containing suspensions of T. rubrum andM. audouinii in the peritoneal cavity of rabbits.They observed proliferation of the dermato-phytes, accompanied by a change to a yeast-likegrowth, especially with T. rubrum. The changein morphology was accompanied by the abilityof the dermatophyte to invade deep tissue wheninjected into rabbits and mice, thus resemblingthe yeast phase of some dimorphic fungi. Theyspeculated that the protection afforded by theplastic bag allowed the gradual induction of acultural phase whose environmental optimawere more consistent with those of the host.When cultured on Sabouraud agar at 25 C, thedermatophytes reverted to their original myce-lial form.The dermatophytes do not usually grow in

internal organs of the living body. However,they can easily be cultured on these organs oncethey have been removed (104). Blank et al. (25)cultured human skin, both newborn and adult,as an organized tissue and infected the skinwith dermatophytes to simulate natural humaninfection. After incubation with or withoutserum, the infected tissues were sectioned andthe localization of the dermatophytes in thetissues was studied. The authors found that thehyphae readily invaded all layers of the skinwhen no serum was present, but that the growthof the fungus was restricted to the keratinizedlayers of the skin when serum had been added tothe culture medium. This activity was lost afterdialysis or heating of the serum at 56 C. The"serum factors" were not identified, but neithergamma globulin nor other protein fractions,such as albumin and Cohn fractions II, III, orIV, showed any activity.

Wilson et al. (243) described subcutaneousgranulomatous infections of the leg due to T.rubrum. Bizarre forms of the dermatophytewere observed in the lesions. Later, Blank andSmith (26) reported a case with widespreadgranulomatous lesions due to T. rubrum. Theyalso found abnormal forms of the dermatophytein the lesions. Their patient had an abnormallylow titer of "serum factor."Normal sera from human newborns and

adults, as well as from guinea pigs and rabbits,inhibit the proteolytic enzymes isolated from T.mentagrophytes var. granulosum. The inhibitorfrom human serum was isolated and identified

as alpha-2 macroglobulin. Its significance in theprocess of dermatophytosis is not yet known (77,246; S. F. Grappel, unpublished data).Other factors may contribute to resistance to

dermatophytic infection. Rothman et al. (197)explained the spontaneous cure of tinea capitisat puberty as due to an increase in fungistaticfatty acids present in the sebum of adults.However, this hypothesis has not been generallyaccepted (133). Rothman and Lorincz (196) alsopointed out a possible relationship between theabsence of sebaceous glands and selective local-ization of lesions in tinea pedis.Kligman (131) observed the necessity for

active keratinization for infection of animalswith skin cycles. Animal hairs were not suscep-tible while in the resting phase.

Chronic widespread dermatophyte infectionsoften occur in hosts whose general resistancehas been reduced. A low rate of desquamation ofskin (196), the presence of underlying diseasesincluding Cushing's syndrome (36, 174, 185),malignant lymphoma (148), or diabetes (153)and treatment with immunosuppressive agents(74) may all lead to such widespread, intracta-ble infections.

Anergy, as manifested by a negative "tricho-phytin" test (56, 243), serum-dysproteinemia,including hypoalbuminemia and hypergamma-globulinemia (24, 26, 56, 148), and atopy (116)have also been associated with chronic infec-tions.

IMMUNOCHEMISTRY OFDERMATOPHYTESChemical Composition

Blank (22) used X-ray diffraction to study thecell walls of 15 species of dermatophytes; hefound chitin, but not cellulose. Shah andKnight (209) analyzed purified cell walls of fourspecies by paper chromatography. They foundthese to consist mainly of a glucose-containingpolysaccharide, an N-acetyl glucosamine po-lymer (chitin), mannan, protein, and smallamounts of galactosamine and lipid.Merkel (162-164) analyzed whole mycelia

from T. gypseum, A. schoenleinii, and A.quinckeanum which were frozen and thawed 20times or more and then extracted. He foundthat lipids comprised 60 to 74%; polysaccha-rides, 17 to 30%; and proteins, 8 to 10% of hisextracts. Each species contained glucose, man-nose, and glucosamine and had similar amountsof nitrogen and protein in the form of nucleo-protein.

Analyses of polysaccharide fractions by manyother workers also revealed that the sugars most

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frequently present were glucose, mannose, ga-lactose, and glucosamine (100, 101, 125, 161,188).G6tz and Pascher (76) studied the amino acid

compositions of three dermatophyte species, T.mentagrophytes, T. schonleinii, and T. rubrum,and found that they did not differ qualitativelyor quantitatively to any significant extent, ex-cept for a larger amount of phenylalanine in T.mentagrophytes. This was also the only specieswhich produced a-aminobutyric acid.The chemical composition may be altered by

growth conditions and age of cultures. Swansonand Stock (223) studied the metabolic activityof M. quinckeanum and found that dermato-phytes have a slower rate of metabolism thansaprophytic molds. The maximal total nitrogen,protein, and ribonucleic acid contents werefound at the end of the log phase, whereastrichloroacetic acid-soluble nitrogen increasedwith increasing age and could be part of struc-tural polysaccharides. The rate of carbohydratesynthesis was found to be relatively uniformduring the entire growth cycle. By starvationexperiments, they concluded that lipid ratherthan carbohydrate was the primary storagematerial in this dermatophyte.Stuka and Burrell (214) studied the influence

of nitrogen content of the growth medium andage of the culture on the antigenic compositionof T. rubrum. They concluded that youngcultures grown in low concentrations of mul-tipeptone are most antigenic. Fluorescent anti-body studies indicated that the hyphal tips weremost reactive and that extracellular antigenicmaterial was exuded from the tips.

Flaherty and Burrell (63) showed later thatthe carbohydrate source influenced the polysac-charide composition of the mycelium, especiallywith regard to molecular weight of the majorantigenic components. The optimal antigenicactivity was obtained in medium with lowcarbohydrate concentrations of 15.0 g/liter.This is less than the amount in routine Sabou-raud dextrose medium.

Antigenic CompositionInterest in the separation and characteriza-

tion of antigenic components from dermato-phytes began with the work of Bloch et al. (30).These investigators were the first to identify theactive component of "trichophytin" as a nitro-gen-containing polysaccharide.

Since that time there have been numerouspublications dealing with the isolation of puri-fied "trichophytins" and the serology of anti-genic preparations. However, in most instancesthe antigen preparations that were isolated

were heterogeneous or were not tested forhomogeneity.

Differentiation of species. Jadassohn et al.(107-109) initiated the concept of group-specificand species-specific antigens of dermatophytesand stimulated interest in isolation of antigensfor serological differentiation of species as wellas for identification of factors involved in hyper-sensitivity reactions and the cutaneous mani-festations of dermatophytosis.Jadassohn et al. (108) used an extract, pre-

pared according to the method of Bloch et al.(30), termed "Trockentrichophytin" or "desic-cated trichophytin." They showed that infec-tion of guinea pigs with A. quinckeanum in-duced not only a delayed cutaneous reactivity,but also an immediate-type hypersensitivity tothis antigen preparation which was apparent 40days or more after infection. They also foundthat the uterus of guinea pigs could be sensi-tized by subcutaneous injection of the "desic-cated trichophytin." Uterine strips from guineapigs sensitized by this method were used foranalysis of "trichophytins" prepared from fourspecies of dermatophytes (107, 109). It wasfound that the "trichophytins" thus preparedfrom A. quinckeanum, T. mentagrophytes, Epi-dermophyton Kaufmann-Wolf, and T.schoenleinii were mixtures of antigens and thateach species had one or more antigens in com-mon as well as antigens that were characteristicof the species. However, they had no success inpurifying the species-specific antigens. Never-theless, their demonstration of group-specificand species-specific antigens led to many at-tempts at differentiation of dermatophyte spe-cies by immunological methods.Sharp (211) found extensive cross-reactions

between mycelial extracts of dermatophyte spe-cies by precipitin tests with rabbit antisera.Differences in relative titers could not be usedas a basis for species differentiation.Keeney and Erickson (118) isolated protein

and carbohydrate antigens from culture fil-trates of T. mentagrophytes and T. rubrumgrowth in synthetic medium. They claimed thatthe antigens were species-specific by serologicalmethods (precipitin tests), but showed cross-reactions in tests for dermal sensitivity.

Fischer (62) compared "desiccated tricho-phytins" from additional species by theSchultz-Dale technique. He found both species-specific and common antigens in T. rubrum andE. floccosum.Tomomatsu (226) compared crude polysac-

charide antigens from 11 different species ofdermatophytes by precipitin reactions with rab-bit antiserum with each of these species. He

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concluded that the Trichophyton species couldbe separated from the Epidermophyton andMicrosporum species by differences in serologi-cal reactivities.Miura (167) and Miura and Kasai (169)

showed that fluorescent antibody techniquesusing rabbit antisera to different dermatophytespecies could be used to detect dermatophytesand differentiate them from other groups offungi. However, their technique could not beused to distinguish species.Dyson and Landy (58) grouped species on the

basis of gel diffusion tests with antisera tosonically treated mycelium. Their results show-ing the antigenic differentiation of T.mentagrophytes and T. rubrum paralleled theresults of species differentiation by "in vitro"hair invasion tests (1).

Kielstein (120) analyzed crude polysaccha-rides from 12 dermatophyte species by Ouchter-lony gel diffusion tests. He found three antigensthat were common to all species and severalothers that showed partial identity. The resultscould not be used as a basis for serologicalclassification.

Sagara (201) also used gel diffusion to com-pare mycelial extracts and found that T.mentagrophytes var. interdigitale and var.asteroides could be differentiated from T. ru-brum, but not from each other by this method.Biguet et al. (17) used immunoelectrophoresis

of saline extracts to show that M. ferrugineumwas more closely related to species of the genusMicrosporum than to Trichophyton species.These results agreed with its classification inthe genus Microsporum based upon the forma-tion of spindle-shaped, thick-walled macroco-nidia, which were described later for this speciesby Vanbreuseghem et al. -(233).Andrieu et al. (3) also used immunoelectro-

phoresis for analysis of 17 species. More than 10antigens were detected in extracts of eachspecies by using homologous antisera. Many ofthese antigens cross-reacted with antisera toother species. The large number of antigensdetected by this method indicated that theantigenic components in dermatophytes weremore complex than had been supposed previ-ously.

Hypersensitivity factors ("trichophytin").Most of the early efforts aimed at purification ofantigenic components of dermatophytes weredirected toward obtaining a pure "tricho-phytin." Although most investigators isolatedcrude nitrogen-containing polysaccharide frac-tions which elicited "trichophytin" reactions insensitized animals (11, 70, 128, 129, 162, 224,227, 228), some also suggested that protein

fractions elicited cutaneous "trichophytin"reactions (67, 138). Protein "trichophytins"containing 12 amino acids were isolated fromfive species of dermatophytes by Meyer et al.(165).

Shechter et al. (212) separated protein con-stituents of six species of dermatophytes by discelectrophoresis. However, these fractions werenot isolated or characterized. Fractions specificfor E. floccosum were detected.

Interest in components of dermatophytes in-volved in pathogenicity led to detection ofproteolytic enzymes by several investigators(27, 40, 189, 190, 192). Yu et al. (247, 249)isolated antigenic proteolytic enzymes from T.mentagrophytes. These enzymes also had "tri-chophytin" activity in infected guinea pigs (77).Lipid fractions have also been reported to have"trichophytin" activity (203).

Ito (100, 101) showed the complexity of "tri-chophytin" by isolating 22 fractions with "tri-chophytin" activity from mycelium and culturefiltrate of T. mentagrophytes var. asteroides by0.5% phenol-water extraction and chromatogra-phy on diethylaminoethyl (DEAE)-SephadexA-50. In addition to carbohydrate-peptide com-plexes, these active fractions included ribonu-cleic acid, peptides, and polysaccharides.

Purified Antigenic ComponentsBeginning about 1962, two groups started

definitive studies on the structures of antigensfrom dermatophytes and have attempted toestablish relations between structure and im-munological activity. For this reason, the pres-ent section will be concerned primarily with thework of those two groups. Barker, Cruickshank,and associates (7-9, 14, 92) concentrated all oftheir earlier efforts on glycopeptides, but haverecently studied some polysaccharides. Bishop,Blank, and associates originally confined theirstudies to polysaccharides, but later investi-gated other antigenic components includingproteolytic enzymes (18-21, 23, 78-81, 84,247-250). More recently, a third group, Nozawaet al. (176-178) has concentrated its efforts onpurified polysaccharide-peptide complexes.

Isolation of antigens. Glycopeptides, poly-saccharides, and keratinases were isolated asdescribed below.

Glvcopeptides. Immunologically active gly-copeptides have been extracted from submergedcultures of dermatophytes with ethylene glycolby Codner et al. (42). This is a proceduredeveloped originally for extraction of the "O"antigen of Shigella dysenteriae (171). The mate-rial in the extract was purified by Barker et al.(9) by dialysis and subsequent fractional pre-

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cipitation by cetyltrimethyl ammonium bro-mide from a borate buffer solution of increasingpH according to the method of Barker et al.(10). Under these conditions, the fraction pre-cipitated at pH 7 to 9.5 contained most of thebiological activity (cutaneous hypersensitivitytests) and was homogeneous by cellulose ace-tate strip electrophoresis, by ultracentrifuga-tion, and by gel filtration. However, a subse-quent investigation by Barker et al. (7) showedthat this material could be fractionated furtherinto two glycopeptides by gradient elution fromDEAE-Sephadex A-50. In the same investiga-tion, three glycopeptides were isolated fromsurface cultures of that organism (T.mentagrophytes) by the same procedures. Theethylene glycol extraction procedure has alsobeen used for cultures of T. rubrum, M. canis,T. schoenleinii, Keratinomyces ajelloi, and E.floccosum to yield crude antigens; fractionationby the same procedures gave glycopeptides fromT. rubrum and M. canis (14).Recently, How et al. (92) have isolated glu-

cans from four species of dermatophytes, T.mentagrophytes, T. rubrum, M. canis, and T.schoenleinii. The glucans were purified fromethylene glycol extracts by mild fractionation,at neutral pH, on Bio-Gel P-300 and DEAE-Sephadex A-50.Nozawa et al. (177, 178) have concentrated

their efforts on isolation of immunologicallyactive polysaccharide-peptide complexes fromT. mentagrophytes. These investigators usedthe phenol method of Westphal et al. (237) toextract defatted mycelial mats of T.mentagrophytes grown in Sabouraud broth.The crude extracts were separated and purifiedby gel filtration and DEAE-cellulose columnchromatography.

Polysaccharides. The procedure used byBishop, Blank, and associates for isolation ofpolysaccharides was purposely more drasticthan that used to isolate the glycopeptides,because the objective was to obtain the total,soluble polysaccharides, free from protein andlipid, for serological studies. The mycelia fromculture growth were defatted by extraction withsolvent and deproteinized by digestion withtrypsin, and the polysaccharides were extractedfrom the insoluble residue by hot, dilute alkali(18). The individual polysaccharides in themixtures thus obtained were separated by pre-cipitation of one as its insoluble copper complexand subsequent separation of the other two bychromatography on DEAE-cellulose (19). Theseprocedures yielded three polysaccharides fromeach of the following organisms: T. granulosum,T. interdigitale, M. quinckeanum, T. rubrum,

T. schoenleinii, Microsporum praecox, T. fer-rugineum, Trichophyton sabouraudii, and T.tonsurans. Each of the polysaccharides isolatedwas homogeneous by free-boundary electro-phoresis in borate buffer.

In addition to the neutral polysaccharides, apyruvated glucan was isolated from autoclavedmycelium of M. quinckeanum (61). The myce-lium was extracted with ethylene glycol, yield-ing a mixture of peptidoglycans. The pyruvatedglucan was separated from the mixture byethanol precipitation and was purified by chro-matography on DEAE-Sephadex A-25.

Keratinases. One extracellular and two cell-bound proteases with keratinolytic activity onguinea pig hair were isolated from cultures of T.mentagrophytes var. granulosum grown in aliquid keratin medium with horse hair as solesource of nitrogen (247-249). The extracellularenzyme, keratinase I, was isolated from theculture filtrate by chromatography on DEAE-and carboxy methyl-cellulose columns and puri-fied by gel filtration on Sephadex G-100. Themycelial enzymes, keratinase II and keratinaseIII, were extracted with phosphate-buffered1.0 M NaCl, pH 7.8. They were separated bycolumn chromatography on DEAE-celluloseand carboxy methyl-cellulose and were purifiedby gel filtration on Sephadex G-100.Chemistry of antigens. The chemistry of

glycopeptides, polysaccharides, and keratinaseswas as described below.

GlYcopeptides. Definitive structural work onthe glycopeptides of Barker et al. was done on a-preparation (8) which was shown subsequentlyto be a mixture (7). However, the results arevalid in showing the main structural features ofthis group of allergens. The yields and constitu-tions of the glycopeptides varied with differentcultures, the chief variation being in the D-galactose content which accounted for 9% to20% of the glycopeptides. The particular sampleexamined contained D-galactose (9%), D-man-nose (73%), and protein (9%). Evidence for alimited number of peptide chains was obtainedby the finding of three N-terminal amino acids,glycine, alanine, and threonine. Furthermore,extensive (80 to 90%) hydrolysis of the aminoacids by proteolytic enzymes indicated a pre-dominance of peptide rather than carbohy-drate-amino acid linkages. The glycopeptidescontained the following amino acids: asparticacid, threonine, serine, glutamic acid, proline,glycine, alanine, valine, isoleucine, leucine, ty-rosine, phenylalanine, and lysine. In the subse-quent investigation (7), the glycopeptides fromsubmerged cultures and from surface cultures ofT. mentagrophytes were resolved into two and

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three galactomannan peptides, respectively.The ratios of amino acids were similar in each ofthese products, with the exception of highcontents of proline in the three galactomannanpeptides from the surface cultures. It was sug-

gested that this difference between the surfaceand submerged cultures could have been causedby a difference in proteolytic activity of theorganism under these conditions. It was notedthat the glycopeptides of high proline contentwere more resistant to pronase and ficin thanthe glycopeptides obtained from submergedcultures. Because proteolytic activity is a neces-

sary property of any dermatophytic organism, itis possible that the peptide moieties of theglycopeptides represent a resistant core.

The carbohydrate portion of the glycopeptidewas analyzed by partial hydrolysis, periodateoxidation, and methylation studies (8). Mildhydrolysis of the glycopeptides caused release ofall of the galactose, and its presence as theD-enantiomer was confirmed by the specificreaction with D-galactose oxidase. On periodateoxidation, all of the D-galactose was destroyedand there was an immediate production of 0.13moles of formaldehyde. All of this evidenceindicated that the D-galactose was present inthe furanoside form, probably as terminal units.Periodate oxidation also reduced the mannose

content of the glycopeptides from 89% to 36.5%and, after reduction and hydrolysis of the oxi-dized product, glycerol and erythritol were de-tected. During methylation analysis, the D-

galactose units were lost, probably by hydrol-ysis during work-up, and the following 0-

methyl ethers of D-mannose were found:2,3,4, 6-tetra-0-methyl; 3,4,6-tri- 0-methyl;2,3, 6-tri-O-methyl; and unidentified mixturesof di-O and mono-0-methyl derivatives. Theratios of the tetra-tri-di-mono were

23: 27: 25: 14. From these results, it was inferredthat the main linkages in the mannan portion ofthe molecules were 1 - 2 and 1 4.Nozawa et al. (177) obtained a major glyco-

peptide from their initial crude mycelial extractby gel filtration on Sephadex G-100. Theyconfined their studies to this first fraction anddiscarded subsequent minor fractions. Themajor fraction which amounted to 68% of thecrude extract, and contained 1.8% nitrogen, wassubsequently chromatographed on a DEAE-cel-lulose column equilibrated with 0.01 M boratebuffer and yielded seven glycopeptides.The glycopeptides were analyzed for amino

acid content. Most of the glycopeptides con-

tained a large amount of serine, threonine,proline, glycine, alanine, and aspartic acid and

smaller amounts of glutamic acid, valine, iso-leucine, lysine, and leucine.

Successive digestion of two of the separatedglycopeptides with trypsin and papain reducedthe nitrogen content from 0.32 to 0.04% andfrom 0.91 to 0.13%, respectively.Each of these polysaccharide-peptide com-

plexes contained mannose, galactose, glucose,and glucosamine. However, no fine structuralanalyses were done on the sugar moieties.

Polysaccharides. The structures of the threepolysaccharides isolated from each of five orga-nisms have been described in a series of sixpapers by Bishop, Blank, and associates (2, 18,19, 20, 21, 23). The dermatophyte species ex-amined were T. granulosum, T. interdigitale,M. quinckeanum, T. rubrum, and T.schoenleinii. A subsequent publication byGrappel et al. (81) described the structures ofsimilar polysaccharides from an additional fourspecies of dermatophytes: M. praecox, T. fer-rugineum, T. sabouraudii, and T. tonsurans.The isolation and fractionation procedures

described earlier yielded the same pattern ofpolysaccharides from each of the nine organismslisted above, namely two galactomannans (Iand II) and a glucan. Their properties and theresults of methylation studies provided defini-tive evidence of the structures of these polysac-charides. Thus, the occurrence of D-galactose inthe furanose ring form, inferred in the study byBarker et al. (8), was confirmed by the isolationof 2,3,5, 6-tetra- O-methyl-D-galactose inamounts that accounted for all of the D-galac-tose that was present originally. The results alsoshowed conclusively that these organisms con-tained two different galactomannans. Theywere similar in having nonreducing end groupsof D-galactofuranose and D-mannopyranoseunits and in containing 1 - 6-linked D-man-nopyranose units in the chains. However, galac-tomannans II contained a predominance of 1 -2-linked D-mannopyranose units in the linearportions, and no such units were present in thegalactomannans I. This represents a basicstructural difference between the two groups ofpolysaccharides that could not have arisen byinadvertent chemical modification (e.g., re-moval of the acid-labile D-galactofuranosideunits) during extraction or fractionation.The galactomannans I (20) are branched

polysaccharides with the branch points throughthe C-2 and C-6 hydroxyls of D-mannopyranosepredominantly, except for those from T.sabouraudii and T. tonsurans where the majorbranch points are through the C-6 and C-3hydroxyls. The linear portions of those polysac-

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charides are formed almost exclusively by 1 -6-linked D-mannopyranose units with only asmall number of 1 - 3 linkages; the chains areterminated by D-mannopyranose and D-galac-tofuranose groups.

In the galactomannans II (19), branch pointsoccur through C-6, C-3 and C-6, C-2 disub-stituted D-mannopyranose units to a more equalextent. In the galactomannan II from T. fer-rugineum, the branching is exclusively throughthe C-6, C-2 positions and in that from T.tonsurans exclusively through C-6, C-3. Thelinear units are predominantly 1 -a 2-linkedD-mannopyranose, and there are a significantnumber of 1 - 6 linkages in all except those fromM. praecox, T. ferrugineum, T. sabouraudii,and T. tonsurans. As in the galactomannans I,the galactomannans II have nonreducing termi-nal groups of D-galactofuranose and D-man-nopyranose.The glucans (21) were also branched poly-

saccharides, but with a more consistent patternof substitution. All branches occurred throughthe C-6, C-3 positions, and linear chains wereformed by 1 - 6 and 1 -- 3 linkages. Thevariation in the ratio of these linkages is proba-bly not significant because 1 -- 3-linked poly-saccharides are labile to alkali (239), and alkaliwas used to extract these polysaccharides.The results indicated that the polysaccha-

rides within each of the three groups wereremarkably similar with only minor variationsin their structural features. Thus, in the galac-tomannans I, only three of the nine polysaccha-rides show a significant difference from theothers, and one of the differences is common totwo of these three. In the galactomannans II,there are three significant differences, one ofthem (the linear units) common to four of thepolysaccharides, one (the end groups) shared bytwo polysaccharides, and two distinct differ-ences in branch points. The maximal variationsfrom a common structure occur in the polysac-charides from T. tonsurans, followed in orderof decreasing differences by T. sabouraudii, T.ferrugineum, T. rubrum, and M. praecox.There were no significant differences betweenthe glucans except for the positive specificrotation of the one from T. granulosum. Thespecific rotations may indicate varying pro-portions of a- and ,-anomeric linkages, butthis has not been confirmed.The pyruvated glucan (61) isolated from M.

quinckeanum contained 0.6% pyruvic acid.Methylation studies showed that it was presentas a 4,6 ketal (4,6-O-1-carboxyethylidene)attached to nonreducing terminal D-glucose

units of the polysaccharide. The glucan had aspecific rotation of -15° and contained nonitrogen. Most of the glycosidic linkages were inthe fl-configuration. From methylation anal-yses, it was determined that the glucan con-tained 1 -- 3 and 1 - 6 linkages in a ratio of 2: 1.The glucans isolated by How et al. (92)

differed from those reported earlier by Bishop etal. (21) in that all contained a(1 -- 4)-linkedD-glucose residues as determined by their infra-red spectra. Only the nitrogen content of theglucan from T. rubrum was reported. It con-tained less than 0.1% nitrogen.

Keratinases. The extracellular keratinaseisolated from T. mentagrophytes var.granulosum by Yu et al. was a protein contain-ing 18 amino acids (250). These were alanine,aspartic acid, cystine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methio-nine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. The molecularweight of this enzyme was 48,000.The two cell-bound enzymes isolated from T.

mentagrophytes var. granulosum were both gly-coproteins having molecular weights of 440,000(keratinase II) and 20,300 (keratinase III) (249).Each contained the same 18 amino acids as theextracellular keratinase I, but these also con-tained galactose, mannose, and glucose (Yu etal., unpublished data).Immunological reactivity. Immunological

reactivity in relation to structure and taxo-nomic importance is as described below.

Relation to structure. Hypersensitivity reac-tions have been classified as immediate anddelayed, the former based upon the presence ofcirculating antibodies, the latter as a manifesta-tion of cellular immunity (72, 221).

Barker, Cruickshank, and their colleagueswere able to demonstrate both immediate- anddelayed-type hypersensitivity reactions inguinea pigs sensitized to T. mentagrophytes.The immediate and delayed hypersensitivityreactions were related to the carbohydrate andpeptide moieties, respectively, of their glyco-peptides (7, 9, 14). It was found that degrada-tion of the carbohydrate portion of the glyco-peptides from T. mentagrophytes by periodateoxidation greatly diminished the immediatehypersensitivity reaction; degradation of thepeptide portion by proteolytic enzymes dimin-ished the delayed reaction (9). These resultswere confirmed with the purified glycopeptides(7). Similar studies were made with glycopep-tides from T. mentagrophytes, T. rubrum, T.schoenleinii, M. canis, K. ajelloi, and E.floccosum by Basarab et al. (14). On the basis of

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cutaneous hypersensitivity reactions in animalssensitized with homologous and heterologousspecies, it was concluded that the glycopeptideswere not species-specific, but indeed were verysimilar in different genera of' keratinophilicfungi. It was also shown that polysaccharides(such as yeast mannans) that had structuralfeatures in common with the carbohydrate por-tion of the glycopeptides gave strong immediatehypersensitivity reactions in dermatophyte-sen-sitized animals. Although these results clearlydemonstrated the presence of cross-reactingantigens in the dermatophytes examined, theresults could not be quantified, and low-levelreactions could well have been missed. It hasbeen suggested that "the established techniquesof determining delayed hypersensitivity, eitherby skin reaction or cytotoxicity, have a highthreshold and, therefore, low levels of cellularhypersensitivity escape detection. Claims thatimmunity is separable from delayed hypersen-sitivity may be true if the presence or absenceof a skin reaction serves as the measure fordesensitization" (221). For these reasons itwould seem that the immediate hypersensitiv-ity reaction or, even better, the quantitativeinteraction of antigens with antibodies, wouldoffer better opportunities for assessing theantigenic specificity of the compounds.Nozawa et al. (177, 178) demonstrated both

immediate- and delayed-type cutaneous reac-tivities to their polysaccharide-peptide com-plexes from T. mentagrophytes in patients withtrichophytosis. By using their phenol-water ex-tract partially purified on Sephadex G-100, theyshowed that proteolytic digestion decreased thedelayed hypersensitivity by 54%, but hardlyaffected the immediate hypersensitivity reac-tion in sensitized humans. Precipitin reactionswith rabbit antiserum to the phenol-water ex-tract were hardly affected by the proteolyticdigestion, indicating that the circulating anti-bodies produced were specific mainly for thecarbohydrate moieties.Noguchi et al. (176) studied the rabbit anti-

bodies produced to phenol-water extracts of T.mentagrophytes. They found that IgG gaveprecipitin and complement fixation, but notpassive hemagglutination reactions. IgM gavepassive hemagglutination and less precipita-tion, but no complement-fixation reactions.These investigators did not isolate polysac-

charide-peptide fractions from any other speciesfor comparison.The N-free neutral polysaccharides isolated

by Bishop et al. did not elicit cutaneous hyper-sensitivity reactions in guinea pigs sensitized bycutaneous infection (200). However, these poly-

saccharides were studied immunochemicallyand compared according to serological reactivi-ties.The first paper in that series of investigations

by Grappel et al. (78) showed that each of thethree groups of polysaccharides (galactoman-nans I and II and glucan) from five organismsreacted with antiserum produced in rabbits toautoclaved mycelial suspensions of M.quinckeanum. The serological reactions weremonitored by qualitative precipitation in gels,by quantitative complement fixation, and byimmunoelectrophoresis. The immunodiffusionresults indicated that, within each group, thepolysaccharides from each of the heterologousspecies contained antigenic groups with reactiv-ities that were very similar to those of thehomologous organism M. quinckeanum. Quan-titative complement fixation showed that thefive galactomannans I were very similar, butthat differences occurred in both the galacto-mannans II and the glucans. These differencescould not be related to the minor structuralvariations revealed by methylation studies.The role of the D-galactofuranoside end

groups in the immunological specificities ofgalactomannans from five dermatophytes wasalso investigated (79). Because of the acidlability of furanosidic linkages, it was possibleto remove the D-galactose from the galactoman-nans I and II leaving mannans I and II that werestill serologically active and that could not,therefore, have been degraded extensively. Theserological activities, qualitative and quantita-tive, of the mannans I were the same as thosegiven by the parent galactomannans I, showingthat the D-galactofuranoside units were notimportant determinants in that group of poly-saccharides. The reverse situation was found inthe mannans II, which displayed considerabledifferences from their parent galactomannans IIby immunodiffusion and complement fixationanalyses. Probably the amount (0 to 13%) of thegalactofuranoside units in the galactomannans Iwas not large enough to induce specific anti-bodies, but the higher contents of that unit (22to 33%) in the galactomannans II were recog-nized. The same study revealed a strong cross-reaction between mannans I and mannans II byusing antisera absorbed with galactomannansI or II. This cross-reaction could be explainedif the removal of the D-galactofuranoside unitsfrom galactomannans II unmasked some 1 -6-linked D-mannopyranose units. Mannansfrom three Candida species demonstratedcross-reactivity with the antiserum to M.quinckeanum. This was attributed to the pres-ence of 1 - 2-linked a-D-mannopyranose units

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in the Candida mannans (245), thus providinga structural feature in common with the galac-tomannans II.The pyruvated glucan isolated by Fielder et

al. (61) reacted with rabbit antiserum to auto-claved mycelium of M. quinckeanum. It alsoelicited immediate-type cutaneous reactions inguinea pigs sensitized to M. quinckeanum bydorsal cutaneous infection.This glucan did not cross-react with type

XXVII pneumococcal antiserum containing an-tibodies directed toward the pyruvic ketal,probably because of the low pyruvate content ofthe glucan. Other species of dermatophyteshave not yet been examined for the presence ofsimilar glucans.How et al. (92) showed that the glucan which

they isolated from T. rubrum was capable ofsensitizing guinea pigs when injected in com-plete Freund adjuvant. It elicited significantimmediate-type cutaneous reactions in guineapigs sensitized with ethylene glycol extract orthe purified glucan of T. rubrum. The M. canisglucan, but not the T. mentagrophytes glu-can, elicited immediate cutaneous reactions inguinea pigs sensitized to T. rubrum glucan. Theethylene glycol extract, from which the T.rubrum glucan was isolated, elicited both im-mediate- and delayed-type cutaneous reactionsin such guinea pigs.Gel diffusion analyses, by using antisera pre-

pared in rabbits to the active keratinases I andII, showed that these enzymes had some deter-minant groups in common (249, 250). Rabbitantibodies to these enzymes precipitated, fixedcomplement, and inhibited their proteolyticactivity (S. F. Grappel, unpublished data).Grappel et al. (85) showed that such antibodiescould also cause a retardation in the growth andan alteration in the structure of T. mentagro-phytes in culture. The inhibitory effect of rabbitantibodies on growth and structure was alsodemonstrated with antibodies to autoclavedmycelium. However, in the presence of guineapig complement, antibodies could actuallycause a change in the morphology of dermato-phytes (85, 86).

Antibodies to the keratinase II were occasion-ally detected in sera of infected guinea pigs bycomplement fixation tests, but they did notpersist (S. F. Grappel, unpublished data). Byusing fluorescent antibody tests, Collins et al.(43) demonstrated local antibody reactions tokeratinase II at the level of the external sheathof the hair follicle in biopsy sections of previ-ously infected sites of guinea pigs.Austwick (4) speculated that such dermato-

phyte-specific antibodies diffusing into the hair

bulbs could be responsible for the degenerativechanges observed in the intrapilary hyphae inhealing ringworm lesions.Grappel and Blank (77) heat-inactivated the

keratinases I and II before using them as skintest antigens. Both of the heat-inactivated kera-tinases, the extracellular and the cell-bound,elicited delayed cutaneous hypersensitivityreactions in guinea pigs sensitized by dorsalcutaneous infection with T. mentagrophytesvar. granulosum. The reactions obtained weregreater than those to a commercial "tricho-phytin." Eleuterio et al. (59) studied the natureof the cutaneous reactions to the keratinases.They used macrophage migration inhibitiontests with peritoneal exudate cells according tothe method of David et al. (49). The results oftheir investigation confirmed the specific cellu-lar nature of the reactions to the keratinases.Guinea pigs were also sensitized to the active

keratinases by immunization with the enzymesin Freund complete adjuvant (77). These guineapigs had both circulating antibodies and cu-taneous hypersensitivity. The antibodies thatwere produced inhibited the proteolytic activityof the enzymes in addition to fixing complementand precipitating with the keratinases.Taxonomic importance. Thus far, the chem-

ical structures and the cutaneous reactivities ofdermatophyte glycopeptides in sensitizedguinea pigs have not shown any evidence ofspecies specificity. However, most of the prep-arations isolated from more than one specieswere mixtures and were not compared immuno-chemically.

Further studies on the cross-reactivities of thethree groups of neutral polysaccharides byGrappel et al. (80, 81) revealed that there weredifferences in the antisera that were prepared inrabbits to each of the organisms. The cross-reactivities of the 27 polysaccharides with anti-sera to each of the 9 dermatophyte species fromwhich they were isolated were determined bycomplement fixation (84). The results showedthat the galactomannans I were the most reac-tive of the three groups of polysaccharides anddiffered most in their reactivities with antiserato T. interdigitale, T. sabouraudii, and T.schoenleinii. The galactomannans II were lessreactive than the galactomannans I and differedin their cross-reactivities with antisera to eachspecies. The structural differences are probablysufficient to classify T. tonsurans, T. sabourau-dii, T. ferrugineum, T. rubrum, and M. praecoxas distinct species. However, the significance ofthe serological variations, determined by com-plement fixation, is more difficult to assess. Theantisera were produced in rabbits. Antiserum

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for each species was taken from a single animalfor the tests. Minor differences could thereforerepresent individual differences in the animals'responses. There is also the possibility of varia-tion in the antigen composition in relation tothe time at which the growth was harvested.Changes in the composition of extracellularfungal polysaccharides with time during growthhave also been reported (159, 160).The keratinolytic enzymes have only been

isolated from one species thus far. However,these antigenic proteolytic enzymes may offerthe most promising tools for an improved class-ification of the dermatophytes as well as forelucidating the mechanism of resistance and itsrelationship to hypersensitivity in dermato-phytosis.

IMMUNOPROPHYLAXIS ANDTHERAPY

The evidence presented thus far has indicatedthat acquired resistance to dermatophytic in-fection is accompanied by cutaneous sensitiza-tion (delayed-type hypersensitivity).

AnimalsEarly investigations showed that inoculation

of living or killed dermatophyte cells by routesother than the cutaneous could produce hyper-sensitivity and resistance to subsequent cutane-ous infection. However, injection of guinea pigswith whole cells by intracardial, intramuscular,subcutaneous, or intraperitoneal routes inducedonly a poor or partial protection. Subsequentcutaneous infections took an abortive course,but protection was of relatively short duration.No protection was obtained by injection ofculture filtrates (31, 38, 114, 135, 143, 199).

Hypersensitivity and partial resistance couldalso be obtained in guinea pigs by rubbing theirskin with killed mycelium (222) or by repeatedintradermal injections of a "toxic substance"obtained from the supernatant fluid of mixturesof extracts of infected skin and mycelium in-cubated at 37 C for 24 h (155, 156, 158).

Later, Wharton et al. (238) injected rabbitssubcutaneously with a killed T. rubrum suspen-sion in Freund complete adjuvant. The immu-nized rabbits were completely resistant to in-fection for up to 17 months or more. This resist-ance was greater than that obtained in rabbitsafter cutaneous infections. Precipitating anti-bodies were detected in the resistant animals.Injection of culture filtrate or extract of thefungus did not give protection.

Reiss and Leonard (187) showed that intra-dermal injections of a "trichophytin," even if

incorporated in Freund complete adjuvant, pro-duced hypersensitivity in guinea pigs, but noresistance to infection. Without the adjuvant,neither hypersensitivity nor resistance was ob-served.Topical application of killed, disintegrated T.

mentagrophytes mycelium in an ointment basewas found to induce both hypersensitivity andpartial resistance in guinea pigs. The immunitywas not of long duration (16, 119).

Hydrolysates of killed T. verrucosum myce-lium have been used successfully for pro-phylaxis for cattle. Florian (64) inoculated 2-week-old calves subcutaneously with a vaccineprepared by extraction of mycelium of T.verrucosum ground in CO2 snow. The vac-cinated calves had a 50% lower incidence ofdisease than controls. The lesions in vaccinatedcalves which became infected were less exten-sive and less severe than those in the controls.

Kielstein and Richter (126, 127) preparedseveral vaccines with T. verrucosum or T.mentagrophytes cultures by modifications ofthe method of Florian (64). Extraction of theground mycelium of T. verrucosum with 0.12 NHCl provided the most effective vaccine. Intra-cutaneous or subcutaneous immunization gavepartial or relative protection in heifers whenthey were exposed to infected cattle. Thedegree of immunity could not be correlated withcirculating antibody titers.

Sarkisov et al. (204) demonstrated completeresistance of calves to experimental infection 2weeks after immunization with T. verrucosumvaccine. The immunity lasted 3 to 5 years.When 107 immunized calves were mixed withringworm-infected animals, only 16 (14.9%)developed small lesions which healed spontane-ously.

HumansPlato and Neisser (173) made the first at-

tempt to produce a therapeutic vaccine forhumans. However, their "trichophytin" fromfiltrates of ground Trichophyton cultures, aswell as the later Truffi (231) "trichophytin"from culture filtrate, had no significant thera-peutic value.

Strickler (213) prepared a vaccine from killedand powdered mycelium of T. tonsurans. Hecured 14 of 20 cases of tinea capitis by numerousinjections. Later, Jausion and Sohier (112) pre-pared the claso-vaccine by hydrolyzing myce-lium of several species with nitric acid. Re-peated subcutaneous or intramuscular injec-tions cured 36 of 45 cases. Da Fonseca and deArea Leao (48) also used a claso-vaccine, known

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as "dermatomycol." They cured resistant ony-chomycosis and tinea capitis. Lewis and Hopper(146) also used this vaccine as well as other"trichophytin" vaccines for cases of tinea capi-tis. The cure rate was disappointing with allpreparations and toxic side reactions were ob-served.

Sulzberger and Wise (220) injected a "tricho-phytin" intradermally in order to desensitize 18patients with "id" reactions. Partial desensiti-zation was observed in 15 patients, and 10 ofthese patients benefited from the treatment.Traub and Tolmach (230) treated 135 patientswith "id" reactions by repeated intradermalinjections of "trichophytins." They found thatdesensitization had little effect both on the "id"eruptions and on the course of the disease.Moreover, desensitization was only temporary.Miller et al. (166) also had little therapeuticsuccess with injections of their "undenaturedtrichophytin." Later, Bazyka (15) found thatsmall intracutaneous doses of fungal vaccinesmade with T. rubrum or T. mentagrophytescould reduce dermatoallergic reactions, whereaslarge doses increased resistance of patients.Harada (89) reported that vaccine therapy

with polysaccharide-nucleic acid antigens was

successful in 50% of patients with dermato-phytosis. Longhin and Olaru (151) also reportedsuccessful treatment of 680 patients with pro-

found trichophytosis by repeated subcutaneousinjections of trichloroacetic acid extracts ofTrichophyton mentagrophytes mycelia. The ex-

tracts contained polysaccharides, phospholip-ids, and polypeptides and were capable ofdesensitization of some patients with der-matoallergic symptoms. Five hundred andthirty patients (78%) recovered without anyadditional therapy. In association with griseo-fulvin, 33 cases which had recurred after treat-ment with griseofulvin alone were cured.Although few attempts at immunopro-

phylaxis have been made in humans, thosereported have been successful in producing a

local immunity to experimental infection.Sutter (222) produced a local immunity in

humans by rubbing killed cultures of A.quinckeanum into the skin. The resistance toinfection diminished with increasing distancefrom the site of treatment and was accompaniedby the development of cutaneous hypersensitiv-ity.Huppert and Keeney (94) immunized volun-

teers by repeated topical applications of disinte-grated T. mentagrophytes mycelium in salve toone foot. Upon challenge, only 14% of thetreated feet became infected, whereas 57% of

the untreated contralateral feet became in-fected. In the controls, 86% infection occurredin the right foot and 71% in the left. As inSutter's experimental infections (222), resist-ance was greatest in the immunized area.

SUMMARYDermatophytosis may result in acquired re-

sistance to reinfection, both in animals andhumans. Immunity is usually local and partial.Complete immunity is rare, but may occur atthe infection site. The reinfection of the previ-ously infected site is of shorter duration andshows less inflammation. Fungal elements arerarely detected. Immunity is always accom-panied by cutaneous sensitization. Resistanceoften follows the more inflammatory forms ofinfection usually caused by zoophilic species;immunity occurs less frequently after infectionby anthropophilic species.The "trichophytin" reaction is the term for

the cutaneous hypersensitivity which may de-velop during infection.Both immediate- and delayed-type cutaneous

hypersensitivities occur in infected animals andin humans. The delayed-type reaction appearsearly and is usually associated with the resist-ance to reinfection. Immediate hypersensitivityappears later and is found in chronic infections,especially those due to T. rubrum.The commercially available "trichophytin"

preparations are mixtures of crude extractsfrom dermatophytes and vary in their composi-tion. They are of little value for diagnosticpurposes.The "trichophytin" test is always positive in

patients who develop dermatophytids. Theseare skin eruptions which appear at a distancefrom the focus of infection. They are believed tobe due to the reaction between skin-sensitizingantibodies and fungal antigens carried by thebloodstream. Dermatophytids occur spontane-ously in man and have been experimentallyinduced in rabbits and guinea pigs.

Antibodies are detected in both animals andhumans with dermatophytosis. However, due tothe crude nature of most antigens used, theantibodies detected by the more sensitive tech-niques, such as passive agglutination, are notalways specific for the infecting organism.

Precipitins and complement-fixing anti-bodies appear to be more reliably associatedwith infection, but occur less frequently. Theirrelationship to resistance has not been estab-lished.A natural "serum factor" is believed to con-

tribute to resistance to dermatophytic invasion.

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This component of normal serum is believed torestrict the growth of dermatophytes to thestratum corneum.Dermatophytes have many antigenic deter-

minants in common with each other as well aswith unrelated fungi. Group-specific, species-specific, and "trichophytin"-reactive antigenshave been recognized. However, isolation ofthese components has proved difficult. At-tempts to classify dermatophytes according toserological reactivities have had little success.The same groups of polysaccharides have

been isolated from nine species and display onlyfine differences in structure and serologicalreactivities. These polysaccharides do not have"trichophytin" activity. Glycopeptides whichdo have "trichophytin" activity have also beenisolated. They are similar in chemical structure,and those from several species give similar"trichophytin" reactions in sensitized guineapigs. These glycopeptides were not compared byserological methods, nor was their homogeneitydemonstrated. Keratinases have "trichophytin"activity. However, these have only been isolatedfrom one species thus far.Immunization against dermatophytosis has

been most successful with whole mycelium orcrude extracts. These were injected intra- orsubcutaneously or applied topically. The im-munity which resulted was usually greatest inthe immunized area and was accompanied bycutaneous sensitization.

Progress in the understanding of the immuneprocess in dermatophytosis will greatly dependupon further studies with purified, specificantigens.

ACKNOWLEDGMENTThis work was supported by a grant from the

Brown-Hazen Fund of the Research Corporation, NewYork, N.Y.

LITERATURE CITED1. Ajello, L., and L. Georg. 1957. In vitro hair

cultures for differentiating between atypicalisolates of Trichophyton mentagrophytes andTrichophyton rubrum. Mycopathol. Mycol.Appl. 8:3-17.

2. Alfes, H., C. T. Bishop, and F. Blank. 1963. Thewater-soluble polysaccharides of dermato-phytes. II. A glucan from Microsporumquinckeanum. Can. J. Chem. 41:2621-2627.

3. Andrieu, S., J. Biguet, and B. Laloux. 1968.Analyse immunoelectrophoretique comparbedes structures antigbniques de 17 especes dedermatophytes. Mycopathol. Mycol. Appl.34:161-185.

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9. Barker, S. A., C. N. D. Cruickshank, J. H.Morris, and S. R. Wood. 1962. The isolation oftrichophytin glycopeptide and its structure inrelation to the immediate and delayed reac-tions. Immunology 5:627-632.

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15. Bazyka, A. P. 1966. (Changes in the reactivity ofthe organism and in the morphological struc-tures of the affected skin in patients withmycoses of the feet under the effect of fungusimmune preparations.) Vestn. Dermatol.Venerol. 40:3-9.

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Immunology of Dermatophytes and Dermatophytosis - Microbiology

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