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Immunochemical studies on the antigenic properties of the cell wall of Trichophyton mentagrophytes Al-Rammahy, Abdul Khaliq Abdullah 1978

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IMMUNOCHEMICAL STUDIES ON THE ANTIGENIC PROPERTIES OF THE CELL WALL OF TRICHOPHYTON MENTAGROPHYTES by ABDUL KHALIQ ABDULLAH^AL-RAMMAHY B.V.M. & S. U n i v e r s i t y of Baghdad, 1969 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1978 Abdul K h a l i q Abdullah Al-Rammahy In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f MICROBIOLOGY The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 A p r i l , 7. 1978 i ABSTRACT C e l l w a l l preparations of Trichophyton mentagrophytes were digested w i t h c h i t i n a s e f o l l o w i n g which various f r a c t i o n s were i s o l a t e d by u l t r a f i l t r a t i o n and Sephadex g e l f i l t r a t i o n . A l l f r a c t i o n s i s o l a t e d contained both polysaccharide and peptide m a t e r i a l . A c o r r e l a t i o n was seen between those f r a c t i o n s capable of e l i c i t i n g immediate and delayed s k i n r e a c t i o n s i n s e n s i t i z e d guinea pigs and those capable of s t i m u l a t i n g the ±n v i t r o p r o l i f e r a t i o n of lymphocytes taken from s e n s i t i z e d guinea p i g s . These immunologically a c t i v e f r a c t i o n s a l s o developed p r e c i p i t i n l i n e s w i t h antiserum taken from s e n s i t i z e d animals. Amino a c i d a n a l y s i s of an immunologically a c t i v e f r a c t i o n of low molecular weight i n d i c a t e d that the peptide content comprised a l i m i t e d array of amino a c i d s . This f r a c t i o n , found to be completely r e a c t i v e immunologically ( m ^ C a ) ) , appeared to have a molecular weight i n the range of 2,000-4,000 as assessed by u l t r a f i l t r a t i o n and g e l f i l t r a t i o n s t u d i e s . This f r a c t i o n , ( U T i ^ C a ) ) was f u r t h e r degraded by treatment w i t h e i t h e r a combination of pronase and carboxypeptidase A or w i t h t r y p s i n . Peptides were separated from the carbohydrate-rich f r a c t i o n by u l t r a f i l t r a t i o n . The carbohydrate-r i c h f r a c t i o n r e t a i n e d the a b i l i t y to induce both immediate and delayed s k i n r e a c t i o n s i n s e n s i t i z e d guinea pigs and to s t i m u l a t e the p r o l i f e r a t -i o n of s e n s i t i z e d lymphocytes i n v i t r o . The peptide moieties r e t a i n e d r e a c t i v i t y i n that they caused delayed r e a c t i o n s and lymphocyte p r o l i f e r a t i o n but were unable to induce immediate or Arthus r e a c t i o n s i n s e n s i t i z e d animals. T r y p t i c peptides from UM 9(a) were p u r i f i e d by ion exchange chromatography. A high p r o p o r t i o n of these peptides demonstrated immunological a c t i v i t y at both the c e l l u l a r and humoral l e v e l s i n c e they were capable of inducing delayed r e a c t i o n s and/or lymphocyte t r a n s f o r m a t i o n , as w e l l as being capable of b l o c k i n g the complement f i x a t i o n r e a c t i o n between UM„(a) and s p e c i f i c antiserum. i i i TABLE OF CONTENTS Page SECTION I : Genral Introduction. 1 Dermatophytes 1 Acquired r e s i s t a n c e 1 Composition & Chemistry of dermatophytic 3 Antigens. H y p e r s e n s i t i v i t y r e a c t i o n s 6 Immunological r e a c t i v i t y . 8 SECTION I I : I s o l a t i o n and c h a r a c t e r i z a t i o n of immunolo-g i c a l l y r e a c t i v e f r a c t i o n s from c h i t i n a s e 12 Digested c e l l w a l l of T_. mentagrophytes. I n t r o d u c t i o n 12 M a t e r i a l and Methods. 14 Organism and Growth 14 Pre p a r a t i o n of m y c e l i a l c e l l w a l l 14 Di g e s t i o n and f r a c t i o n a t i o n of c e l l w a l l 15 Q u a n t i t a t i v e a n a l y s i s 18 Immunization of animals 18 Skin t e s t s 23 In v i t r o lymphocyte s t i m u l a t i o n 23 Immunodiffusion 24 Results-N . . 25 D i s c u s s i o n 38 iv Page SECTION I I I : C h a r a c t e r i z a t i o n of immunologically a c t i v e 41 peptides from the c e l l w a l l of T. mentagrophytes I n t r o d u c t i o n 41 M a t e r i a l and Emthods 43 Antigen 43 Enzyme d i g e s t i o n 43 Pronase-Carboxypeptidase 43 Try p s i n 44 Peptide p u r i f i c a t i o n 44 Immunization 45 Skin t e s t s and lymphocyte s t i m u l a t i o n 45 Polyacrylamide g e l e l e c t r o p h o r e s i s 46 Thin l a y e r chromatography 46 Complement f i x a t i o n 51 Hapten i n h i b i t i o n 51 Results 52 Di s c u s s i o n 68 SECTION IV : General Conclusions. 72 C h i t i n a s e D i g e s t i o n 72 Immunological a c t i v i t y of various peptides 72 Studies on UM 2(a) 73 Immunological behaviour of T r y p t i c 74 peptides from UM2(a) References s 76 L i s t of Tables Table Page 1. Q u a n t i t a t i v e a n a l y s i s of the anthronepositive and Lowry p o s i t i v e m a t e r i a l of the hydroly s a t e and of 26 the f r a c t i o n obtained a f t e r u l t r a f i l t r a t i o n and g e l f i l t r a t i o n . T r i t i a t e d thymidine i n c o r p o r a t i o n by lymph node c e l l s i n response to T\_ mentagrophytes c e l l w a l l f r a c t i o n 34 a f t e r 24 h and 1 day d i g e s t i o n w i t h c h i t i n a s e 3. Amino a c i d composition of the f r a c t i o n s U M ^ C b ) and ^7 UM^a) 4. Chemical a n a l y s i s of U W ^ C a ) f r a c t i o n a f t e r anzyines ^ d i g e s t i o n . Lymph node c e l l 96 h i n c o r p o r a t i o n of t r i t i a t e d thymidine i n response to UM„(a) s u b - f r a c t i o n s a f t e r enzymes treatment and f r a c t i o n a t i o n 56 6. S e n s i t i z e d lymph nqde c e l l 96 h i n c o r p o r t i o n of t r i t i a t e d thymidin^ i n response to t r y p t i c peptides ^ fromUM^a) 66 7. The amino a c i d compositions of some of the t r y p t i c peptides from (a) 8. I n h i b i t i o n of complement f i x a t i o n by t r y p t i c peptides. 67 v i L i s t of Fi g u r e s . Page Figure (1) Pr e p a r a t i o n of a n t i g e n i c m a t e r i a l s from 17 T. mentagrophytes c e l l w a l l Figure (2) E l u t i o n p r o f i l e of high molecular weight 20 m a t e r i a l (UM^Q r e t e n t a t e ) on Sephadex G-150 Figure (3) E l u t i o n p r o f i l e of low molecular weight 22 m a t e r i a l (UM2 ret e n t a t e ) on Sephadex G—25 Figure (4)a Cutaneous a l l e r g i c r e a c t i o n s i n immunized 29 guinea pigs (whole column) compared to that of unimmunized animals (shaded) to T\_ mentagrophytes c e l l w a l l f r a c t i o n s a f t e r 24 h and 7 days d i g e s t i o n .. w i t h c h i t i n a s e 30 minutes a f t e r i n t r a d e r m a l i n j e c t i o n (immediate r e a c t i o n ) . (b) Cutaneous a l l e r g i c r e a c t i o n s 5 h a f t e r 30 int r a d e r m a l i n j e c t i o n (Arthus r e a c t i o n ) . (c) Cutaneous a l l e r g i c r e a c t i o n s 24 h a f t e r i n t radermal 31 i n j e c t i o n (delayed h y p e r s e n s i t i v i t y r e a c t i o n ) . Figure (5) uTl^Ca) antigen t i t r a t i o n by the i n v i t r o lymphocyte 33 transformation. Figure (6) Immunodiffusion of a l l f r a c t i o n s a f t e r u l t r a f i l t r a t i o n and g e l f i l t r a t i o n of the c h i t i n a s e d i g e s t . 36 Antiserum used was that of the immunized guinea p i g s . ' Figure (7) Disc g e l e l e c t r o p h o r e s i s p a t t e r n of 24 h (UNF),> UM2(a) f r a c t i o n s . TD i s the p o s i t o n of the t r a c k i n g dye. 48 Figure (8) Complement f i x a t i o n t i t r a t i o n . c u r v e of UM2(a) 50 f r a c t i o n Figure (9) Cutaneous a l l e r g i c r e a c t i o n s of the enzyme di g e s t s 55 of of UM2(a) f r a c t i o n i n immunized guinea pigs (whole column) compared to that of the unimmunized animals Figure (10) E l u t i o n p r o f i l e of t r y p t i c peptides on DOWEX 50 W 58 x8 column. Figure (11) Thin l a y e r chromatography of the t r y p t i c peptides. 61 Figure (12) Cutaneous a l l e r g i c r e a c t i o n s of the t r y p t i c from 63 UM2(a) i n immunized guinea pigs compared to that of unimmunized animals at 30 min (A),5 h (B) and 24 h ( c ) . v i i Ackn owledgemen t s I would l i k e to express my s i n c e r e thanks to Dr. J u l i a Levy f o r her d i r e c t i o n and encouragements during the course of the experimental work and the w r i t i n g of t h i s t h e s i s . I would a l s o l i k e to express my a p p r e c i a t i o n to Ms. Barbra K e l l y f o r her help and c o n s t r u c t i v e suggestions. G r a t i t u d e are a l s o expressed to Ms. K h a d i j a A b d o l a l l f o r her patience i n the t y p i n g of t h i s t h e s i s . S p e c i a l thanks to my w i f e Maysoon. 1 S e c t i o n 1 General I n t r o d u c t i o n Dermatophytes The dermatophytes are species of the genera Trichophyton , Microsporia! > and Epidermophyton. The dermatophytic fu n g i are pathogenic only to the s u p e r f i c i a l s k i n , n a i l s and h a i r i n human beings and animals. They do not invade the deeper t i s s u e or i n t e r n a l organs. These i n f e c t i o n s are known as dermatophytesis (ringworm). They produce r e l a t i v e l y non-inflammatory i n f e c t i o n s i n the case of A n t h r o p o p h i l l i c f u n g i and inflammatory i n f e c t i o n s i n the z o o p h i l i c f u n g i . However, the disease process i s g r e a t l y i n f l u e n c e d by the host response to the dermatophytic i n f e c t i o n . Acquired Resistance: Cutaneous i n f e c t i o n s w i t h dermatophytes may induce r e s i s t a n c e to r e - i n f e c t i o n w i t h the same or another species. This r e s i s t a n c e depend on the degree and du r a t i o n of i n f e c t i o n , upon the species and s t r a i n of dermatophytes (Zoophylic or A n t h r o p o p h i l i c ) , the host (animal or human), and the s i t e of i n f e c t i o n . In an attempt to understand the l a c k of p a t h o g e n i c i t y of derma-tophytes to the deeper t i s s u e , L o r i n c z e t a l , (45) showed that derma-tophytes were i n h i b i t e d when implanted i n the abdomen of h e a l t h y mice, but grew v i g o r o u s l y when t r a n s f e r r e d to c u l l u r e medium i n d i c a t i n g the i n h i b i t o r y p r o p e r t i e s of the deep t i s s u e environment. Blank et a l . (13) demonstrated that dermatophytes grew p r o f u s e l y i n a l l l a y e r s of v i a b l e f u l l t h i c k n e s s s k i n explants maintained i n short term t i s s u e c u l t u r e . However, growth was prevented by bathing the explants i n f r e s h 2 species on the b a s i s of g e l d i f f u s i o n t e s t s w i t h a n t i s e r a to s o n i c a l l y t r e a t e d mycelia. However, l i t t l e d e f i n i t i v e i n f o r m a t i o n i n t h i s area has been forthcoming . I n t e r e s t i n the p u r i f i c a t i o n of the a n t i g e n i c components of dermatophytes s t a r t e d i n 1962 w i t h the work of Barker ejt al (6) who p u r i f i e d the glycopeptides from the c e l l w a l l by e x t r a c t i o n w i t h ethylene g l y c o l as shown by Codner at a l (20). The e x t r a c t s were p r e c i p i t a t e d by c e t y l t r i m e t h y l ammonium bromide from a borate b u f f e r s o l u t i o n by i n c r e a s i n g the pH. The glycopeptides contained the f o l l o w i n g amino a c i d s ; a s p a r t i c a c i d , threonine, 'serine, glutamic a c i d , p r o l i n e , g l y c i n e , a l a n i n e , v a l i n e , i s o l e u c i n e , l e u c i n e , t y r o s i n e , phenylalanine and l y s i n e . The carbohydrate p a r t of the glycopeptide contained e i t h e r D-galactose or D-mannose 9.0-20 and 73% r e s p e c t i v e l y . The p r o t e i n content was 9% . How e_t al (37) have i s o l a t e d glucans from four species of dermatophytes, among them was T. mentagrophytes, these glucans were p u r i f i e d from ethylene g l y c o l e x t r a c t s by m i l d f r a c t i o n a t i o n at n e u t r a l pH, on Bio-Gel P-300 and DEAE Sephadex A-50 . Polysaccharide-peptide complexes from T_ mentagrophytes were i s o l a t e d by Nozawa e_t a l . (54) by using the phenol e x t r a c t i o n method of Westphal (71). The crude e x t r a c t s were separated and p u r i f i e d by g e l f i l t r a t i o n and DEAE c e l l u l o s e chromatography. In most f r a c t i o n s they found a la r g e amount of s e r i n e , threonine, p r o l i n e , g l y c i n e , a l a n i n e , and a s p a r t i c a c i d ; and s m a l l amounts of glutamic a c i d , v a l i n e , i s o l e u c i n e , l y s i n e and l e u c i n e . Each of these polysaccharide-peptide complexes contained mannose, galac t o s e , glucose and glucosamine. 3 Composition & Chemistry of Dermatpphytic Antigens Over f i f t y years ago Bloch et a l (16) characterized the antigenic components of trichophytine (crude extracts of various dermatophyte preparation) as being a nitrogen containing polysaccharide. Since that time, there have been numerous publications dealing with the isolation and purification of these components and the serology of the antigenic preparations. The early work was directed mainly toward differentiating the different species of dermatophytes according to the chemical and the serological characteristics of their antigenic components^ Sharp (61) found extensive cross reactivity between mycelial extracts from different species by precipitin tests with rabbit antisera. Carbohydrates and protein antigens were isolated by keeney ejt a l (43) from culture f i l t r a t e s of T^ mentagrophytes and T_^_ rub rum. They showed cross reactions in tests for dermal sensitivity. Dermatophytes could be differentiated from other fungi by a fluorescent antibody technique using rabbit antisera to different dermatophyte species (47,48) but could not be used to differentiate between dermatophyte species because of the extensive cross reactivity. Andrieu et_ al_ (2) used immunoelecr trophoresis for the analysis of 17 species. More than 10 antigens were detected in extracts of each species by using homologous antisera. Many of these antigens cross-reacted with antisera to other species. Various methods have been used for the isolation and characterization of dermotophyte antigens. Shecter et a l (62) separated protein constituents of six species of dermatophytes by disc electrophoresis. However, these fractions were not isolated or characterized. Dyson et_ a l (26) grouped 4 to be responsible for the accelerated reaction appearing after secondary infection. Acquired resistance to dermatophytic infection was shown by De Lamater (22) not to be passed on to the offspring of infected pregnant guinea pigs, indicating that the altered host response following infection is probably c e l l mediated rather than antibody-mediated. In humans, increased resistance usually folows the severe inflammatory forms of infection by zoophylic species but does not always follow the more chronic infections caused by anthropophillic species (9). The same author pointed out that fungi which do not invade the hair f o l l i c l e s do not seem to give rise to an equivalent immunity when growing in the horny layer of the smooth skin. Resistance in natural infections was also shown by Friedman et al (27) who noticed that from children cured tinea capitis never became reinfected when they returned Lto a heavily infected environment. King e_t aL (44) characterized a factor present in normal human serum (serum inhibitory factor). This inhibitory factor was non dialyzable, heat stable et 56 C for 4 hours and i s fungistatic. It would appear that resistance following either experimental or natural infections with dermatophytes i s mediated by cell-mediated immunity (since i t i s not passed to the foetus), as well as soluble factors which may include specific antibody as well as non-specific factors. 5 human serum. Jessner and Hoffman (40), as well as Per and Braude (57) have also demonstrated the inhibitory effect of serum on the grouith of dermatophytes. They attributed this inhibition to the presence of fungicidal antibodies with in the serum possibly arising from previous subclinical exposure to dermatophytic infection. Bloch (15) , showed that cutaneous inoculation of giunea pigs with the zoophylic dermatophytes, Achorion quinkeanum, Trichophyton gypsium or Mlcrosporum Lanosum produced infections which healed spontaneously and resulted in a relative resistance to subsequent infection. He claimed that the immunity was generalized and not confined to the site of the primary infection. It was also noted that prior subcutaneous or intraperitoneal injection of fungus decreased the susceptibility of the skin to cutaneous infection, thus supporting his contention of generalized resistance resulting from prior infection. Greenbaun (33), on the other hand, claimed that a local immunity was produced in guinea pigs after cutaneous infection with dermatophytes. This resistance to infection was only complete at the site of the spontaneously healed areas. Reinjection at f i r s t site demonstrated complete resistance whereas injection of the animal at another site gave rise to typical infectious lesions. Revalier (58), showed that an autoclaved spore suspension of t. granulosum applied three weeks after the f i r s t infection could induce a similar but less pronounced cutaneous reaction when compared to a second infection with live spores. Thus, the altered host response, probably cell-mediated immunity, rather than proliferation of the fungus, appeared 6 Bishop e t a l (12) i s o l a t e d three polysaccharides from many species of dermatophytes. They d e p r o t e i n i z e d the def a t t e d m y c e l i a by d i g e s t i o n n w i t h t r y p s i n , and the polysaccharides were e x t r a c t e d from the i'soluble residue by hot a l k a l i . Anderson e t a l (1) f r a c t i o n a t e d the crude t r i c h o p h y t i n and obtained the f o l l o w i n g f r a c t i o n s ; po l y s a c c h a r i d e -peptide complex, t o t a l l i p i d without f r e e f a t t y a c i d s , and f a t t y a c i d s . They showed t h a t , i n a d d i t i o n to the polysaccharide-peptide complex, free f a t t y acids were res p o n s i b l e f o r p o s i t i v e s k i n r e a c t i o n s but not the t o t a l l i p i d without the free f a t t y a c i d s . From these s t u d i e s i t would appear that many species of dermat-ophytes cont a i n s i m i l a r , i f not i d e n t i c a l , antigens i n t h e i r c e l l w a l l , s i n c e c r o s s - r e a c t i v i t y between species i s a common observation. Attempts a t f r a c t i o n a t i o n o f dermatophyte antigens have demonstrated the complexity of these organisms both i n t h e i r p r o t e i n antigens and i n t h e i r glycopepetides which presumably c o n s t i t u t e c e l l w a l l components. H y p e r s e n s i t i v i t y Reactions: Bloch and h i s a s s o c i a t e s (17) recognized the importance of r h y p e r s e n s i t i v i t y r e a c t i o n s i n dermatophyte i n f e c t i o n and concluded that the a c t i v e p r i n c i p l e , e x t r a c t a b l e from the mycelia of the causative f u n g i , was a carbohydrate-protein complex. Studies on h y p e r s e n s i t i v i t y i n dermatophytesis s t a r t e d i n 1902 when N e i s s e r and P l a t o (51) published t h e i r work on the pr e p a r a t i o n and usefulness of t r i c h o p h y t i n s . T r i c h o p h y t i n i s the a n t i g e n i c p r e p a r a t i o n of the fung a l mycelia and/or concentrated c u l t u r e f i l t r a t e s which i s used f o r cutaneous hypersensit-i v i t y t e s t i n g by in t r a d e r m a l i n j e c t i o n . Both immediate and delayed 7 r e a c t i o n s occur, but the l a t t e r i s most oft e n a s s o c i a t e d w i t h i n f e c t f - -.: io n s . Commercial t r i c h o p h y t i n s are composed mainly of pooled concentrated c u l t u r e , f i l t r a t e s of 1-15 species. There are more than ten t r i c h o p h y t i n s a v a i l a b l e which vary i n t h e i r manufacturing process (63). S e n s i t i z a t i o n r e s u l t i n g from dermatophytic i n f e c t i o n i s s p e c i f i c f o r dermatophytes but i s not species s p e c i f i c . An i n f e c t i o n due to T.  gypseum w i l l cause h y p e r s e n s i t i v i t y to t r i c h o p h y t i n prepared from t h i s dermatophyte but a l s o to f a v i n prepared from A^ s c h o e n l e i n i i and microsporin prepared from M. lanosum (17). De Lamater (23,24) s t u d i e d the development of delayed type h y p e r s e n s i t i v i t y during the course of cutaneous i n f e c t i o n w i t h M.  gypseum i n guinea p i g s . The t r i c h o p h y t i n r e a c t i o n peaked at 15 days a f t e r i n o c u l a t i o n and then s t a r t e d to regress. The s i t e of previous i n f e c t i o n was r e l a t i v e l y anergic as shown by reduced inflammatory r e a c t i o n to t r i c h o p h y t i n . The same author showed that the a b i l i t y to react to t r i c h o p h y t i n increased w i t h age, being greatest i n adult and l e a s t i n newborn guinea p i g s . I t o (38) detected delayed cutaneous r e a c t i o n i n an experimental _T. rub rum i n f e c t i o n i n humans by 14 days a f t e r i n o c u l a t i o n which disappeared a f t e r 35 days at which time an antibody-mediated immediate type u r t i c a r i a l r e a c t i o n developed. Immediate forms of h y p e r s e n s i t i v i t y have al s o been demonstrated by others. Cruickshank et_ a l (21) detected immediate r e a c t i o n to t r i c h o p h y t i n i n guinea pigs i n f e c t e d w i t h T.  mentagrophytes by u s i n g Evans blue dye. H a n i f i n et_ a l (35) compared 8 the type of cutaneous r e a c t i o n i n p a t i e n t s w i t h T. mentagrophytes and T. rubrum. They found- most of the p a t i e n t s w i t h T. mentagrophytes e x h i b i t e d delayed r e a c t i o n to t r i c h o p h y t i n w h i l e most of the p a t i e n t s w i t h 1\_ rub rum lacked delayed r e a c t i o n s but manifested an immediate r e a l r e a c t i o n t o t r i c h o p h y t i n . P a t i e n t s w i t h c h r o n i c dermatophytosis were found to have a r e l a t i v e l y s p e c i f i c defect i n delayed h y p e r s e n s i t i v i t y to t r i c h o p h y t i n and t h e i r c e l l mediated responses to other antigens were somewhat decreased (64). Cruickshank e_t a l (21) showed th a t the delayed h y p e r s e n s i t i v i t y r e a c t i o n to t r i c h o p h y t i n could be t r a n s f e r r e d t o non i n f e c t e d guinea p i g s w i t h p e r i t o n e a l exudate c e l l s of s e n s i t i z e d donors, i n d i c a t i n g that a c l a s s i c a l c ell-mediated response was i n v o l v e d . Immediate-type r e a c t i o n to t r i c h o p h y t i n i n humans could be t r a n s f e r r e d by serum i n d i c a t i n g a c i r c u l a t i n g antibody r e a c t i o n ;(66). Thus, both ce l l - m e d i a t e d and humoral immunity appear to be i n v o l v e d i n the immune response to these f u n g i . d The l a c k of e i t h e r stander'ized or pure homogenous t r i c h o p h y t i n s f o r s k i n t e s t i n g has s e v e r e l y l i m i t e d the usefulness of the t r i c h o p h y t i n r e a c t i o n f o r d i a g n o s t i c purposes (32). Immunological R e a c t i v i t y : Barker, Cruickshank and t h e i r colleagues demonstrated that the immediate r e a c t i o n was r e l a t e d to the carbohydrate p o r t i o n of dermat-ophyte antigens w h i l e delayed r e a c t i o n was r e l a t e d to the peptide moiety of the glycopeptide (6,8,11). I t was a l s o shown that yeast or other polysaccharides that had s i m i l a r s t r u c t u r a l features i n common w i t h the carbohydrate p o r t i o n gave strong Immediate r e a c t i o n i n s e n s i t i z e d 9 I n d i v i d u a l s . Nozawa et a l (55) showed that p r o t e o l y t i c d i g e s t i o n of t h e i r phenol e x t r a c t e d , p a r t i a l l y p u r i f i e d p olysaccharide-peptide complexes from T_. mentagrophytes decreased the delayed h y p e r s e n s i t i v i t y r e a c t i o n but h a r d l y a f f e c t e d the immediate r e a c t i o n . P r e c i p i t i n r e a c t i o n was the same before and a f t e r p r o t e o l y t i c enzyme treatment. These authors concluded that t h i s i n d i c a t e d that the a n t i b o d i e s were s p e c i f i c mainly f o r the carbohydrate m o i e t i e s . The n i t r o g e n free polysaccharides i s o l a t e d by Bishop et_ a l (12) d i d not e l i c i t cutaneous h y p e r s e n s i t i v i t y r e a c t i o n s i n guinea pigs s e n s i t i z e d by cutaneous i n f e c t i o n . They showed that each of the three groups of polysaccharides (galactomannan 1 and galactomannan 2 and glucan) reacted w i t h antiserum produced i n r a b b i t from autoclaved mycelia. They monitored the s e r o l o g i c a l r e a c t i o n s by q u a l i t a t i v e p r e c i p i t a t i o n i n g e l , complement f i x a t i o n and Immunoelectrophoresis (29,59) How e t a l (37) showed that the ethylene g l y c o l e x t r a c t of T. rubrum mycelia, the p u r i f i e d glucan, and the mixture of glycopepetides, i s o l a t e d from the e x t r a c t , were each capable of s e n s i t i z i n g guinea p i g s . A l l three groups of animals thus s e n s i t i z e d gave good immediate and delayed responses when challenged w i t h the ethylene g l y c o l e x t r a c t of the mycelium or the glycopeptide components. The glucan, however, e l i c i t e d s i g n i f i c a n t immediate responses i n animals s e n s i t i z e d w i t h the glucan or the ethylene g l y c o l e x t r a c t , but i n s i g n i f i c a n t delayed responses i n a l l three groups of animals. Keratinase I & 11 have been shown to have common determinant groups 10 by gel diffusion analysis using antisera prepared in rabbits to active Keratinase I & 11. (73,74). Grappel et a l (30,31) showed that these rabbit antibodies could cause retardation in the growth and alteration in the morphology of dermatophytes. Austwick (3) speculated that these dermatophyte specific antibodies diffusing into the hair bulbs could be responsible for the degenerative changes observed in the intrapapillary hyphae in healing ringworm lesions. Balogh e_t al (4) showed a correlation between the degree of positivity of the lymphocyte transformation test, the spread of the mycotic process and the sensitization of the organism. Hanifin et al (35) showed the correlation of positive lymphocyte responses and the presence of delayed but not immediate cutaneous reactions in naturally infected human beings. Svejgaard et a l (67) studied the lymphocyte response of patients to different dermatophyte antigens as well as to some bacterial and fungal mitogens. The latter were used to show that the lymphocytes respond normally to these mitogens and patients suffered no functional lympholyte deficiency. They showed that lymphocytes from patients responded more strongly to the dermatophyte antigens than did these from non-immune controls. They observed that in most patients suffering from T. mentagrophytes infection, responses to T_. mentagrbphytes antigens were significantly stronger than that in other petients. On this basis, these workers felt that the lymphocyte transformation assay could demonstrate specificity to antigens not detected by other assay procedures, which usually showed extensive cross reactivity. 11 Attempts to c h a r a c t e r i z e antigens of dermatophytes i n terms of h the r e a c t i o n s they evoke have i n d i c a t e d that the carboydrate moieties of these antigens may be resp o n s i b l e to a large extent, f o r humoral immunity, whereas the peptide f r a c t i o n s are thought to be re s p o n s i b l e f o r s t i m u l a t i n g c e l l mediated immunity. Since few i n v e s t i g a t o r s have worked w i t h h i g h l y p u r i f i e d antigens, such observations cannot be regarded as co n c l u s i v e . In t h i s work an attempt was made to i s o l a t e the a n t i g e n i c components of T\_ mentagrophytes by enzymatic d i g e s t i o n of the m y c e l i a l c e l l w a l l w i t h c h i t i n a s e , since c h i t i n has been reported to be one of the major c e l l w a l l components of dermatophytes, (53,55,60) and to compare s k i n t e s t i n g w i t h lymphocyte transformation and immune p r e c i p i t a t i o n as measures of the immune responses i n s e n s i t i z e d guinea p i g s . The immunological r o l e of the polysaccharide peptide moieties of the a n t i g e n i c components of the c e l l w a l l of T\_ mentagrophytes was als o i n v e s t i g a t e d . 12 Section II Isolation and Partial characterization of Immunologically Reactive Fractions from Chitinase Digested c e l l wall of Trichophyton mentagrophytes. Introduction Different approaches have been followed by many workers in the isolation of the antigenic components of dermatophytes. Most of the methods used were chemical and include; ethylene glycol (7,8,20,37), hot dilute a l k a l i (12) and phenol (52,54). Using these methods, many investigators isolated crude nitrogen containing polysaccharide fractions which were used for detecting skin reactivity in sensitized animals (7,8) or naturally infected human beings (35,42,64). The immunological roles of the polysaccharide and peptide moieties of this material have been investigated by several workers. According to Barker and co-workers (7,8) immediate and delayed type hypersensi-t i v i t y were attributable to the carbohydrate and peptide moieties of the Isolated galactomaman-peptide respectively. Nozawa and co-workers (51), showed that proteolytic digestion of phenol water extracted material decreased the delayed hypersensitivity. Glucans isolated by How et a l . , (37) from Trichophton rub rum and Microsporum quiekeanum sensitized guinea pigs and eli c i t e d immediate skin reactions. The l i p i d fraction of the crude trichophytin of the c e l l wall of T_. mentagrophytes was shown by Anderson et^ a l . , (1) to cause inflammatory delayed allergic skin reactions in sensitized guinea pigs. In addition to serological techniques, immediate and delayed type 13 h y p e r s e n s i t i v i t y as measured by s k i n r e a c t i v i t y has been, u n t i l r e c e n t l y , the main immunological t e s t used by other workers to study the a n t i g e n i c components of the dermatophytes. Se v e r a l i n v e s t i g a t o r s (4,35,67), showed that lymphocyte t r a n s -formation was a s e n s i t i v e method f o r the demonstration of mycotic s e n s i t i z a t i o n and e l u c i d a t i o n of the c e l l mediated immunological responses to dermatophyte antigens. In t h i s work an attempt was made to i s o l a t e the a n t i g e n i c components of _T. mentagrophytes by enzymatic d i g e s t i o n of the m y c e l i a l c e l l w a l l w i t h c h i t i n a s e , s i n c e c h i t i n has been reported to be one of the major c e l l w a l l components of dermatophytes, (53,55,60), and to compare s k i n t e s t i n g w i t h lymphocyte transformation and immune p r e c i p i t a t i o n as measures of the immune responses i n s e n s i t i z e d guinea p i g s . 14 MATERIALS AND METHODS Organism and Growth Conditions-Trichophyton mentagrophytes Var asteroides i s one of the stock c u l t u r e c o l l e c t i o n s of the Department of M i c r o b i o l o g y , U n i v e r s i t y of B r i t i s h Columbia. The fungus was grown i n a 30 l i t r e fermenter i n 4% glucose, 1% neopeptone and incubated at 30°C f o r 90 h w i t h continuous s t i r r i n g and a e r a t i o n . The inoculum was prepared by seeding e i g h t two l i t r e f l a s k s , each c o n t a i n i n g 800 ml of growth medium which were incubated i n a shaking waterbath at 30°C f o r 6 days. A l l c u l t u r e s were checked under the microscope to exclude the p o s s i b i l i t y of contamination. The c u l t u r e was t r e a t e d w i t h m e r t h i o l a t e at a f i n a l c oncentration of 1:7000 f o r 2 h p r i o r to f u r t h e r manipulation. This treatment has been shown to e f f e c t i v e l y k i l l a l l fungal c e l l s . The c u l t u r e was then f i l t e r e d through two l a y e r s of cheese c l o t h and the fungal mat was washed e x t e n s i v e l y w i t h d i s t i l l e d water u n t i l the absorbance at 280 nm of the f i l t r a t e was <0.03 . P r e p a r a t i o n of M y c e l i a l C e l l W a l l The fungal mat was d e f a t t e d according to the method of B a r t i n i c k i -G a r c i a & Nickerson (10). Fat free mycelia were freeze d r i e d and the dry weight was 26.25 g. The d r i e d m y c e l i a were then s o n i c a t e d i n c i t r a t e phosphate b u f f e r at a s e t t i n g of 80 (Biosonic) 5 times f o r 30 seconds each w i t h a 20 second i n t e r v a l between each treatment. M y c e l i a l sonicates were c e n t r i f u g e d at 600 x g i n a S o r v a l l c e n t r i f u g e . The p e l l e t was 15 exposed to freezing and thawing with liquid nitrogen using glass beads (Operlin o . l - o . l l mm) in a mortar u n t i l 95% breakage of mycelia was demonstrated by microscopic examination. This method has been found to be efficient for breakage by others (14). The mycelial c e l l wall suspension was centrifuged at 10,000 x g and the pellet was washed three times to remove a l l cytoplasmic debris, and then freeze dried. This dry material was used in the preparation of the antigenic fractions. Digestion and Fractionation of Cell Wall The antigenic fractions of the c e l l wall were prepared by chitinase digestion as illustrated in Fig. 1. The dried c e l l wall material was Streptomyces digested for 24 h with chitinase/(Calbiochem) at a concentration of 0.5 mg per cent of the substrate mixture at 37 C in a shaking waterbath following which the remaining pellet was washed several times with 0.05M citrate buffer pH 6.0 and the same amount of fresh enzyme was added and incubated for another 6 days with a few drops of toluene to prevent con-tamination. Microscopic examination of the digests showed that no bact-e r i a l contamination had occurred. The 24 h and 7 day digestions did not show obvious differences in terms of the molecular size of the components in the chitinase hydrolysate as shown by u l t r a f i l t r a t i o n followed by gel f i l t r a t i o n . Digests were centrifuged at 30,000 x g for 60 min. prior to fraction-ation. High molecular weight materials were concentrated by a UMin Amicon Figure 1 Preparation of antigenic materials from Trichophyton  mentagrophytes cell walls. C e l l w a l l p e l l e t was homogenized i n 0.5 M c i t r a t e phosphate b u f f e r pH 6.0, incubated w i t h c h i t i n a s e at 0.5 mg/ml at 37°C i n a shaking water bath f o r 24 hrs or 7 days. Hydrolysates were c l e a r e d by c e n t r i f u g a t i o n at 10,000 g . Hydrolysates were concentrated by u l t r a f i l t r a t i o n on Amicon UM^Q f i l t e r s Retentate (UM^Q concentrated) Sephadex G 150 ( F i g . 2) Peaks a and b rated by UM-^ Q were concent-f i l t r a t i o n UM 1 0 (a) re t e n t a t e UM 1 Q (b) r e t e n t a t e (UT^ F i l t r a t e passed f r a c t i o n was concentrated on UM^ f i l t e r ) Sephadex G 25 ( F i g . 3) UM2 (a) UM2 (b) UM2 (c) 'UM2.(d) UM2 (e) Peaks were concentrated by UM2 f i l t e r UM passed f r a c t i o n s were freeze d r i e d and des a l t e d on Sephadex G 25, and again freeze d r i e d -> PUM 2(c) PUM 2(a) PUM 2(b): 18 f i l t e r (MW > 10,000) and the retentate further fractionated on a Sephadex G 150 column (100 x 2.5 cm) which had been equilibrated and eluted with 0.85% NaCl in 5 ml fractions (Fig. 2). Material in the G-150 peaks (U^Q ( a) a n (* (b) were again concentrated by UM-^ Q f i l t r a t i o n and stored at -20°C . Digested material of molecular weight < 10,000 which passed through UM-^ Q f i l t e r s was concentrated on a UM^  f i l t e r (MW > 1,000) and fractionated on Sephadex G 25 column (60 x 2.0 cm) in a 4 ml fractions (Fig. 3). The peaks, (a) through (e) were again concentrated by UTi, f i l t r a t i o n , and the rentates were stored at -2Cr°C . The f i l t r a t e s , which passed through the UM^  f i l t e r s (ie., PU^ fractions) were concentrated by lyophilization. Chitinase activity was recovered only in the UM^Q (a) fraction. Chemical analyses were performed on the digested and sized fractions from the c e l l wall material to determine their sugar and protein content. Total sugar content was determined by Anthrone reagent (50) using glucose as a standard. Protein was measured by the method of Lowry et^ al., (46) Amino acid analyses were performed on the isolated fractions. Samples were hydrolysed with 6 N HCl in sealed evacuated ampules at 108°C for 20 h . The hydrolysates were repeatedly washed with d i s t i l l e d water and evaporated using a flash evaporator to remove the HCl, and the amino acid composition was determined on a Beckman-Spinco model 120 automatic amino acid analyzer. Immunization of Animals Albino guinea pigs weighing 400-500 g were immunized with the whole dried isolated c e l l wall material. Each animal received three 19 Figure 2. E l u t i o n p r o f i l e of high molecular weight m a t e r i a l (UM. retentate) on Sephadex G-150 . 20 FRACTION NUMBER 21 F igure 3. E l u t i o n p r o f i l e of low molecular weight m a t e r i a l (UM, r e t e n t a t e ) on Sephadex G—25 . 22 1 5 3 0 4 5 6 0 FRACTION NUMBER 23 injections of 0.2 ml of 2.0 mg/ml c e l l wall antigen in 50% complete Freund's adjuvant (CFA, Difco). Two injections were given intramuscu-larl y and one intraperitonealy The animals were boosted with the same preparation and amount four weeks after the f i r s t injection. Skin Tests The isolated antigenic fractions used for skin testing were dissolved in 0.85% NaCl solution. Intradermal injections of 25 yg of Lowry positive test antigens in 0.2 ml were administered into the skin of sensitized and control animals whose flanks had been shaved and subsequently treated with Nair (Carter-Wallace N.S., Inc., Toronto, Canada). This volume was used in order to minimize inaccuracies due to faulty delivery and leakage at the site of inoculation. Cutaneous reactions were measured after 30 min for an immediate reaction, after 5 h for an Arthus reaction and after 24 h for delayed hypersensitivity reaction. Measurements were made with calipers under strong light. Animals were coded to prevent prejudicial measurement. Reactions were considered positive when the difference in the diameter of the wheal or induration between sensitized and control animals was > 8 mm. Skin measurements after 48 h were not significantly different from those measured at 24 h . For this reason data are routinely presented at 24 h. In Vitro Lymphocyte Stimulation In these studies lymphocyte transformation tests were done in vitro to measure the antigen specific lymphocyte stimulation and to correlate the results with those skin reactivity. Animals used in these studies had been primed with antigen 10 - 20 days prior to sacrifice. The 24 procedures used have been described i n d e t a i l elsewhere (34). B r i e f l y , 5 5 x 10 lymph node c e l l s per m i c r o t i t e r w e l l (Linbro) were c u l t u r e d i n 0.25 ml 1640 medium w i t h d i f f e r e n t concentrations of antigens, and i n -0 cubated f o r 24 or 96 h at 37 C i n a humidifed incubator s u p p l i e d w i t h 3 5% • Eighteen hours p r i o r to c u l t u r e h a r v e s t , .H-thymidine (New England Nuclear) (2 UCi per w e l l i n 0.05 ml 1640) was added. Harvesting was done by microharvester, (Otto H i l l e r , Inc., Madison, Wise.) and thymidine i n c o r p o r a t i o n was measured by l i q u i d s c i n t i l l a t i o n counter. A l l t e s t s were run i n t r i p l i c a t e . Comparisons were made between c u l t u r e s c o n t a i n i n g antigen, and those c o n t a i n i n g no antigen, using lymphocytes from immunized and unimmunized animals. S t a t i s t i c a l analyses of data were c a r r i e d out using Student's t - t e s t . Immunodiffusion Double d i f f u s i o n s l i d e s using 1% agarose i n barbitone b u f f e r were used. A l l f r a c t i o n s were t e s t e d f o r the presence of p r e c i p i t a b l e antigens w i t h the antiserum from the immunized guinea p i g s . Serum was taken from those animals s a c r i f i c e d and used f o r lymphocyte transformation s t u d i e s . S l i d e s were incubated i n a humidifer at 4 C f o r 48 h, washed w i t h s a l i n e f o r two days w i t h frequent changes, and f i n a l l y washed w i t h d i s t i l l e d water, d r i e d and s t a i n e d w i t h Coomassie blue 0.25% i n ethanol. 0 25 Results The effect of chitinase on the isolated mycelial wall of T_. mentagrophytes was monitored by measuring the carbohydrate and protein content of the supernatants after each step of digestion. Table 1 shows the quantitative analysis of the original hydrolysate and of the different fractions obtained after u l t r a f i l t r a t i o n and gel f i l t r a t i o n . Very l i t t l e difference was found between the ratio of cmthrone positive to protein positive fractions in the 24 h and 7 days digestions. The immunological activity of the different fractions as assessed by intradermal skin testing of sensitized guinea pigs is shown in Fig. 4a, b, c, which demonstrate the immediate, Arthus and delayed reactions of the 24 h and 7 day digests and the various fractions derived from them. It can be seen that both immediate and delayed reactions were evoked from essentially a l l UTL^ Q fractions as well as the UH^(2) fraction. Table 2 shows the t r i t i a t e d thymidine incorporation by lymph node cells in the presence of the various fractions. Stimulation indices after 24 h and 96 h incubations were calculated by comparing the ratio of CPM of sensitized cells with and without antigen, and theiratio of CPM of unsensitized cells with and without antigen. Tests were run.at both 5.0 and 25.0 ug/ml of Lowry protein. Because the higher dose was optimal for stimulation only these figures are shown. None of the fractions tested caused non-specific proliferation in unsensitized animals and increased thymidine uptake in unsensitized cells (data not shown), so i t was concluded that increased uptakes were indications of specific responsiveness. Stimulation indices of > 2.0 were shown to be significant (p= < 0.05), since SEM for t r i p l i c a t e counts was never greater than 10% Lowry p o s i t i v e m a t e r i a l of the hydrolysate and of the f r a c t i o n s obtained a f t e r u l t r a f i l t r a t i o n and g e l f i l t r a t i o n . 24 Hour D i g e s t i o n F r a c t i o n No. Lowry p o s i t i v e mg recovered % Anthrone p o s i t i v e recovered 7 Days D i g e s t i o n F r a c t i o n Lowry % No. p o s i t i v e mg recovered Anthrone p o s i t i v e mg recovered UNF EG(a)' EG(b)' ^ 0 ^ 0 UM2 IfrL, m? UM, PUM, PUM, PUM, (a) 3 (b) (a) (b) (c) (d) :(e) (a) (b) (c) 218 1.4 4 891 5.6* 26 1.3 4 37 1.8* 14 0.7* 4 0.2 4 112 51.3 40 4.5 16 7.3 196 22.0 29 13.3 99 11.1 4 1.8 80 8.9 5 2.3 118 13.2 22 10.1 72 8.0 16 7.3 112 12.6 27 12.4 105 11.8 4 1.8 0 0 2 0.9 0 0 1 c h i t i n a s e d i g e s t . two peaks recovered from Sephade x G-150 UNF 116 0.77 540 3.6^ UM UM 1 C UM„ UM, UM, t UM, (a) 1 0 ( b ) (a) !(c) :(c) !(d) PUM2 PUM„ PUM, (b) :(c) 66 56.9 39 7.2 30 25.9 120 22.2 10 8.6 39 7.2 2 1.7 '19 3.5 7 6 193 35.7 4 3.4 13 2.4 17 14.6 .:24 4.4 4 3.4 6 1.1 10 8.6 0 0 g l y c o l (date not shown). 3 See Figure (1) f o r d e s c r i p t i o n of the i n d i v i d u a l f r a c t i o n s tested. +These %s r e l a t e to the amount of m y c e l i a l dry weight before c h i t i n a s e d i g e s t i o n and ethylene g l y c o l e x t r a c t i o n . A l l other %s r e l a t e to the amounts recovered from UNF a f t e r d i g e s t i o n 27 I t can be seen that there i s reasonably good c o r r e l a t i o n between those f r a c t i o n s capable of i n d u c i n g s k i n r e a c t i v i t y i n s e n s i t i z e d animals and those capable of inducing lymphocyte p r o l i f e r a t i o n . No major d i f f e r -ences i n immunological a c t i v i t y between the high molecular weight f r a c t i o n s and UM 2(a) low mol wt) are e v i d e n t , although s l i g h t l y lower s t i m u l a t i o n i n d i c e s were observed w i t h t h i s f r a c t i o n i n comparison t o the higher molecular weight m a t e r i a l s . F i g . 5 shows the dose response t i t r a t i o n curve of the uT^a) f r a c t i o n . The maximum lymphocyte response occurred a f t e r 96 h i n c u b a t i o n w i t h an optimum antigen concentration of 25 yg/ml. F i g . 6 shows the immunodiffusion a n a l y s i s w i t h anti-whole m y c e l i a l c e l l w a l l guinea p i g serum and the various f r a c t i o n s . I t i s of i n t e r e s t to note that the UK, (a) f r a c t i o n , although i t i s of low molecular weight produces stronger p r e c i p i t i n l i n e s than the equivalent \M^(a) and UM 1 Q(b) f r a c t i o n s . The r e s u l t s of amino a c i d a n a l y s i s of UML^ Ca) and UM^(b) are shown i n Table 3. UM^g(a) was considered to c o n t a i n most of the enzyme c h i t i n a s e and was t h e r e f o r e not analysed. I t can be seen that the amino a c i d r a t i o s of these two f r a c t i o n s are q u i t e d i s t i n c t from each other. In the UlLjCa) f r a c t i o n , there i s a l i m i t e d array of amino a c i d s (no Phe or Tyr) i n d i c a t i n g that the peptide moiety of t h i s m a t e r i a l may be of l i m i t e d s i z e . This i s not s u r p r i s i n g since the molecular weight of U i l ^ a ) may be roughly estimated at somewhere between 2000 and 4000. Moreover, the r a t h e r high r a t i o of p r o l i n e i n t h i s f r a c t i o n i s of i n t e r e s t , s i n c e high p r o l i n e r a t i o s i n the c e l l w a l l s of dermatophytes have been observed by other i n v e s t i g a t o r s (60) . 28 Figure 4 (a) Cutaneous a l l e r g i c r e a c t i o n s i n immunized guinea pigs (whole column) compared to tha t of unimmunized animals (shaded) to T_. mentagrophytes c e l l w a l l f r a c t i o n s a f t e r 24 h r and 7 days d i g e s t i o n w i t h c h i t i n a s e 30 minutes a f t e r intradermal i n j e c t i o n (immediate r e a c t i o n ) . EG. ( a ) , (b) are the two peaks recovered from Sephadex G-150 a f t e r e x t r a c t i o n w i t h ethylene g l y c o l . F r a c t i o n s 5-13 are from 1 day c i t i n a s e d i g e s t wherease f r a c t i o n s 14-22 are from the 7 days c h i t i n a s e d i g e s t , (b) Cutaneous a l l e r g i c r e a c t i o n s i n immunized guinea pigs {whole column) compared to that of unimmunized animals (shaded) to T_. mentagrophytes c e l l w a l l f r a c t i o n s a f t e r 24 h r and 7 days d i g e s t i o n w i t h c h i t i n a s e 5 h r a f t e r i n t r a d e r m a l i n j e c t i o n (Arthus r e a c t i o n ) . (c) Cutaneous a l l e r g i c r e a c t i o n s i n immunized guinea pigs (whole column) compared to that of unimmunized animals (shaded) to T. mentagrophytes c e l l w a l l f r a c t i o n s a f t e r 24 h r and 7 days d i g e s t i o n w i t h c h i t i n a s e 24 h r a f t e r i n t r a d e r m a l i n j e c t i o n s (delayed h y p e r s e n s i t i v i t y r e a c t i o n ) . These r e s u l t s represent averages of 12 immunized guinea pigs and 12 unimmunized c o n t r o l s . Standard d e v i a t i o n s f o r i n d i v i a d -u a l antigens t e s t e d d i d not exceed 20% when a l l data were averaged and 10% f o r unimmunized c o n t r o l s . Each animal recei v e d a maximum of 12 i n j e c t i o n s , 11 t e s t samples and one s a l i n e c o n t r o l . Therefore, each antigen was t e s t e d 5 times and data represent an average of these measurements. O 00 o LESION DIAMETER (mm) o — ^ — ro o o> o ro ]UNF 24 hrs ro ]UNF 7 days GO EG(a) EG(b) 0 1 UM 10(a) UMlO ( b ) oo ]uM2 ( a ) UM2(b) oo tn UM2(c) UM2(d) UM2(e) PUM2(a) PUM2(b) r UM10(a) lUMlOto Z Z Z Z Z Z H UM 2(a) UM2(b) » ^ S 8 U M 2 ( c ) 5 ^ IUM2(d1 PUM2(a) ]pUM2(b) ]PUM 2(c) SALINE o 3 " 3 0) ro a 3 -<E' a in o 3 O 3 " 3 0) (A a Q-CD cn O 3 ro oo o ~~T~ ro o oo o 3 3 LESION DIAMETER (mm) 32 Figure 5 UM2(a) antigen titration by the in vitro lymphocyte trans f o r m a t i o n . (© ® ) ( E 3 m ) = t r i t i a t e d thymidine i n c o r p o r a t i o n by the s e n s i t i z e d c e l l s a f t e r 24 h r and 96 hr i n c u b a t i o n r e s p e c t i v e l y . (o O) (• •) = i n c o r p o r a t i o n by the n o n - s e n s i t i z e d c e l l s a f t e r 24 hr and 96 hr i n c u b a t i o n r e s p e c t i v e l y . 33 Table (2). T r i t i a t e d thymidine i n c o r p o r a t i o n by lymph node c e l l s i n response to Trichophyton mentagrophytes c e l l w a l l f r a c t i o n s a f t e r 24 hr and 7 days d i g e s t i o n w i t h c h i t i n a s e F r a c t i o n No Response at 24 h to 25 Ug/.ml Lowry p r o t e i n Response at 96 h to 25 Ug/ml Lowry p r o t e i n CPM S.I.* CPM S.I. 24 h dige s t UM 1 0(a) 39,133 4.99 124,561 9.14 UMio.(b) 25,200 3.22 108,940 8.00 UM 2(a) 17,126 2.18 64,028 4.70 UM 2(b) 12,093 NS** 18,558 NS UM 2(c) 7,730 NS 13,793 NS UM (d) 2 9,540 NS 15,231 NS UM (e) 2 10,050 NS 15,401 NS PUM (a) 2 1,910 NS 13,078 NS PUM„(b) 2 6,680 NS 14,815 NS PUM 2(c) 6,400 NS 15,305 NS 7 days d i g e s t UM i ( )(a) 29,200 3.73 93,950 6.9 UM 1 Q(b) 41,330 5.27 11,003 NS UM 2(a) 18,756 2.39 32,310 2.37 UM 2(b) 12,093 NS 7,611 NS UM 2(c) 6,466 NS 11,283 NS UM 2(d) 9,363 NS 1,451 NS PUM 2(a) 18,426 2.35 7,701 NS PUM 2(b) 11,566 NS 8,091 NS PUM 2(c) 16,696 2.13 17,996 NS No Antigen 7838 13,623 * S t i m u l a t i o n Index ** Not s i g n i f i c a n t when compared to c o n t r o l counts -FIGURE 6 Immunodiffusion of the f r a c t i o n s a f t e r u l t r a f i l t r a t i o n and g e l f i l t r a t i o n of the c h i t i n a s e d i g e s t . Antiserum used was that of the immunized guinea p i g s . A l l antigens were tested at two d i f f e r e n t concentrations (20, 40 Ug). a & b 24 h di g e s t c & d 7 day d i g e s t e & f 7 day UM 1 Q(a) g & h 24 h UM 1 0(a) i & j 24 h UM 1 0(b) k & 1 24 h UM 2(a) Table 3. Amino Acid compositions of the fractions UM^g(b) and UM2(a). Amino Acid ^ 0 (b) . UM2(a) Umoles Molar Ratio Umoles Molar R Aspartic acid .068 2.7 .088 3.9 Threonine .139 5.4 .063 2.8 Serine .170 6.7 .061 2.7 Glutamic acid .084 3.3 .083 3.7 Proline .026 1.0 .151 6.7 Cysteine 0 - 0 -Glycine .115 4.5 .098 4.3 Alanine .248 9.7 .041 1.8 Valine .101 3.9 .040 1.8 Methionine 0 - 0 -Isoleucine .027 1.0 .023 1.0 Leucine .169 6.6 .024 1.1 Tyrosine .063 2.5 0 -Phenylalanine .016 .61 0 -Histidine .052 2.03 .050 2.2 Lysine .022 .85 .049 2.2 Arginine .264 10.30 .018 0.8 * Calculated on the basis of 61 amino acid residues being present : which Pro, l i e , Lys and Phe have a molar ratio of 1.0. Calculated on the basis of 25 amino acid residues being present in which He, Leu, and Arg have a molar ratio of 1.0 38 D i s c u s s i o n There has been a considerable amount of work done on the immunological p r o p e r t i e s of chemically e x t r a c t e d dermatophytic my c e l i a . By and l a r g e , those r e s u l t s obtained w i t h e x t r a c t e d m a t e r i a l s have been i n agreemnt when assessed by t h e i r a b i l i t y to cause immediate or delayed s k i n r e a c t i o n s i n s e n s i t i z e d guinea p i g s . In the present study c e l l w a l l m a t e r i a l of T. mentagrophytes was freed from cytophlasmic contamination and the l i p i d content was removed, f o l l o w i n g which, i t was t r e a t e d w i t h c h i t i n -ase. I t was assumed that enzymatic degradation of c e l l w a l l components would not modify the a n t i g e n i c determinants wherease chemical e x t r a c t i o n might do so. In previous s t u d i e s , the time during which the enzyme was permitted to d i g e s t fungal c e l l w a l l s ranged from 12 h to 7 days (25,49, 53,55,56). Our choice of enzymatic treatment of 24 h and 7 days was based on the assumption that a 24 d i g e s t i o n might give f r a c t i o n s which would not represent the a n t i g e n i c components of the c e l l w a l l due to the r e s i s t a n c e of the c e l l w a l l to enzymatic a t t a c k , and that prolonged d i g e s t i o n might y i e l d more r e p r e s e n t a t i v e fragments. E s s e n t i a l l y a l l UM^Q (high molecular weight) and UM^a) f r a c t i o n s from the 24 h and 7 days d i g e s t i o n s were a c t i v e i n both s k i n t e s t s as w e l l as i n lymphocyte tran s f o r m a t i o n s t u d i e s . Our data support the f i n d i n g of other i n v e s t i g a t o r s (7,8,54) who found that peptide or p r o t e i n r i c h f r a c t i o n s gave strong delayed s k i n r e a c t i o n s . I t was n o t i c e a b l e t h a t some f r a c t i o n s w i t h high p r o t e i n contents were s l i g h t l y more a c t i v e than f r a c t i o n s c o n t a i n i n g low amounts of p r o t e i n . However, t h i s was not a c o n s i s t e n t o b s e r v a t i o n , and no 39 conclusions can be drawn from t h i s . Assuming that lymphocyte transformation i s a measure of the T c e l l a c t i v i t y (34) , our lymphocyte transformation s t u d i e s are i n accordance w i t h the s k i n t e s t r e s u l t s , f o r those f r a c t i o n s capable of inducing delayed s k i n r e a c t i o n s a l s o causedlymphocyte p r o l i f e r a t i o n w i t h s e n s i t i z e d lymphocytes. These r e s u l t s also confirm the observation of H a n i f i n et a l . , (35) and Svejaard et a l . , (67) who have s t a t e d that dermatophytic c e l l w a l l e x t r a c t s can induce p r o l i f e r a t i o n of s e n s i t i z e d lymphocytes. The amino a c i d analyses of UM^(b) and Ubl^a) were q u i t e d i s t i n c t . The UML^b) f r a c t i o n contained e s s e n t i a l l y a l l the amino acids commonly found i n p r o t e i n s . This f r a c t i o n i s probably composed of a v a r i e t y of c e l l w a l l fragments which may be qu i t e heterogeneous. The IM^ia) f r a c t i o n on the other hand had a q u i t e d i s t i n c t i v e amino a c i d composition i n t h a t i t contained extremely h i g h l e v e l s of p r o l i n e and a l i m i t e d array of amino acids (phenylalanine and t y r o s i n e were not pr e s e n t ) . This f r a c t i o n i s of qu i t e low molecular weight, p o s s i b l y between 2,000 to 4,000 as assessed by i t s f i l t r a t i o n p r o p e r t i e s . The amino a c i d composition of t h i s m a t e r i a l i s suggestive of the p o s s i b i l i t y of a r e l a t i v e l y s m a l l peptide being associated w i t h the polysaccharide of the c e l l w a l l . I t i s of i n t e r e s t to note t h a t the a n a l y s i s of UH^ia) i s i n agreement w i t h those reported by others f o r i s o l a t e d glycopeptides from the c e l l w a l l of T_. mentagrophytes. 40 In summary, d i g e s t i o n of the c e l l w a l l of T_. mentagrophytes w i t h c h i t i n a s e releases fragments which contain l a r g e amounts of Anthrone p o s i t i v e and Lowry p o s i t i v e m a t e r i a l . These fragments were shown to e x h i b i t immunological a c t i v i t y by e l i c i t i n g immediate and delayed type h y p e r s e n s i t i v i t y , by inducing lymphocyte p r o l i f e r a t i o n and by y i e l d i n g immune p r e c i p i t a t e s w i t h a n t i s e r a taken from s e n s i t i z e d guinea pi g s . J 41 S e c t i o n I I I C h a r a c t e r i z a t i o n of Immunologically A c t i v e Peptide from the C e l l W a l l of Trichophyton mentagrophytes I n t r o d u c t i o n I ..The peptide moiety of the n i t r o g e n - c o n t a i n i n g polysaccharides of dermatophytes has been shown by many i n v e s t i g a t o r s to be r e s p o n s i b l e f o r the delayed-type h y p e r s e n s i t i v i t y r e a c t i o n (7,8,37,54). Nozawa et a l . , (54) showed that the peptide contents of the polysaccharide-peptide complex e x t r a c t e d from the c e l l w a l l of T_. mentagrophytes a f f e c t e d the delayed h y p e r s e n s i t i v i t y r e a c t i o n . They showed that f r a c t i o n s c o n t a i n i n g 11.4% - 18.7% peptide were much more a c t i v e than those c o n t a i n i n g 0.25% -0.81% peptide. These i n v e s t i g a t o r s were not c e r t a i n that the peptide moiety had an e f f e c t on the immediate r e a c t i o n , s i n c e the a n t i g e n i c substances t e s t e d contained both polysaccharide and peptide m a t e r i a l . Because animals are capable of responding immunologically at both the humoral and c e l l u l a r l e v e l to very s m a l l amounts of a n t i g e n i c m a t e r i a l , the data presented by these workers, although i n d i c a t i v e of a separate immunologi-c a l r o l e f o r the peptide and carbohydrate moieties of dermatophyte c e l l w a l l m a t e r i a l , are not c o n c l u s i v e . A n a l y s i s of the a n t i g e n i c i t y of d i f f e r e n t p r o t e i n species i s o l a t e d from T. mentagrophytes have been done by I t o (39), who i s o l a t e d 22 p r o t e i n f r a c t i o n s by phenol-water e x t r a c t i o n and chromatography. C h r i s t i a n s e n and Svejgaard (67) analyzed the a n t i g e n i c contents of four dermatophyte species by cross-immunoelectrophoresis using submerged c u l t u r e s homogenized by mechanical methods. They used the whole m y c e l i a l e x t r a c t as the antigen f o r r a i s i n g antiserum and f o r immune e l e c t r o p h o r e s i s . 42 These workers showed 26 antigens from t h e i r p r e p a r a t i o n of T_. mentagro- phytes. D i s c e l e c t r o p h o r e s i s was used by Shechter ejt _ a l . , (62) to compare the p r o t e i n species i n c u l t u r e f i l t r a t e s and m y c e l i a l c e l l w a l l e x t r a c t s of many dermatophytes. They found no d e f i n i t e r e l a t i o n s h i p between the p r o t e i n f r a c t i o n s from the same organism c u l t u r e d i n two d i f f -erent media and suggested that the c u l t u r e medium i n f l u e n c e s the genetic expression of the species. I have shown p r e v i o u s l y that a r e l a t i v e l y low molecular weight f r a c t i o n taken from a c e l l w a l l hydrolysate of T_. mentagrophytes (UTi^a), i s a peptide c o n t a i n i n g polysaccharide which can stiumulate i n v i t r o s e n s i t i z e d lymphocytes and can evoke immediate and delayed s k i n h y p e r s e n s i t i v i t y as w e l l as form a p r e c i p i t i n l i n e w i t h antibody r a i s e d i n guinea pigs to a p r e p a r a t i o n of whole m y c e l i a l c e l l w a l l of T_. mentagrophytes. In t h i s s e c t i o n f u r t h e r s t u d i e s were made on that f r a c t i o n and the immunological r o l e s o f the p o l y s a c c h a r i d e and peptide m o i e t i e s of t h i s f r a c t i o n of the c e l l w a l l h y d r o l y s a t e . 43 M a t e r i a l s and Methods Antigen: UM^ (a) i s one of the f r a c t i o n s which was i s o l a t e d from the c e l l w a l l of Trichophyton mentagrophytes a f t e r c h i t i n a s e d i g e s t i o n , u l t r a - f i l t r a t i o n and g e l f i l t r a t i o n . The procedures f o r d i g e s t i o n and f r a c t i o n a t i o n have been described p r e v i o u s l y . UTLjCa) m a t e r i a l was i s o l a t e d from 24 hr d i g e s t s . This m a t e r i a l i s the major low molecular weight f r a c t i o n which was shown to be immunologically comparable to the higher molecular weight f r a c t i o n s , i n that i t s t i m u l a t e d high l e v e l immediate and delayed s k i n r e a c t i o n s , formed a p r e c i p i t i n l i n e w i t h s p e c i f i c antiserum, and s t i m u l a t e d the p r o l i f e r a t i o n of s e n s i t i z e d lymphocytes i n v i t r o . Therefore, the U^Ca) f r a c t i o n was chosen and used f o r f u r t h e r c h a r a c t e r i z a t i o n , i n an attempt to understand the immunological r o l e s of the carbohydrate and peptide m o i e t i e s of t h i s c e l l w a l l complex. Enzyme Digestions, Pronase - Carboxypeptidase: This procedure was c a r r i e d out i n an attempt to remove most of the peptide moiety. The UK, (a) f r a c t i o n was digested f i r s t w i t h pronase (Calbiochem) at an enzyme concentration of 0.5 - 1.0% of the weight of the s u b s t r a t e ( c a l c u l a t e d as per cent Lowry p r o t e i n ) to be d i g e s t e d , and increased during the course of d i g e s t i o n to 2% . Incubation was continued f o r 6 days at 37°C at pH 7.8 i n the presence of 0.0015 M C a C l 2 . The enzyme dig e s t was then put through a UM-^ Q amicon (Amicon Corporation, Lexington, Mass.) f i l t e r to remove the enzyme. Subsequent t e s t i n g of f i l t r a t e s showed them to be f r e e of enzyme a c t i v i t y . The f i l t r a t e was put through a UM„ f i l t e r and washed three times w i t h d i s t i l l e d water. 44 ate The filter'was freeze-dried while the retained fraction was subjected for further enzyme treatment with carboxypeptidase A (Worthington Biochem-i c a l Investigations) in phosphate buffer 0.02 M, pH 8.0 at a concentration of 1% and incubated for 24 h at 37°C . The enzyme was removed as for pronase. The UTL, retained fraction was considered to consist mainly of the uTL^ Ca) carbohydrate moiety. Peptide and carbohydrate contents of a l l fractions were derermined by quantitative ninhydrin and Anthrone tests respectively. Trypsin: To test the antigenic role of the peptide moiety in uTl^C3)* tryptic degradation of the complex was performed. This enzyme (Sigma) was chosen because of the specificity of the enzyme, so that sizable peptides could be recovered from the digest for immunological testing. Previous amino acid analysis of bit,(a) showed lysine but not arginine to be present at significant levels, so that i t was possible that substantial digestion could be achieved. The enzyme content of the incubation mixture was approximately 2% the amount of the substrate. Digestion was carried out in 0.04M phosphate buffer at pH 7.0 for 24 h at 37°C . Separation of the enzyme from the peptides was performed as described above by u l t r a f i l t r a t i o n . Peptide Purification The tryptic peptides were purified chromatographically according to the method of Canfield (18) for peptide separation using a Dowex 50W X8 resin in a 0.7 x 150.0 cm water-jacketed column equilibrated with starting buffer. Each sample was adjusted to pH 2.8 with formic acid, applied to the column and washed in with three 1.0 ml volumes of starting buffer. Elution was performed using an eight-chambered gradient system with each 45 buffer at a volume of 200 ml. Gradient elution with an increasing pH and salt concentration was used with pyridine-acetate buffers ranging from 0.1 N acetic acid, pH 3.8, up to 2.0 N acetic acid, pH 5.18. Fractions of 4 ml were collected and 0.3 ml from every other tube were taken and analyzed by the quantitative ninhydrin reaction after alkaline hydrolysis according to the method of Hirs et^ a l . (36). Eluted material accounted for 100 per cent of starting material. Each ninhydrin positive peak was pooled and dried by flash evaporation at 45°C, washed with d i s t i l l e d water at least three times, freeze-dried and dissolved in normal saline. These fractions were used for skin tests, lymphocyte stimulation, complement fixation tests and amino acid analysis. Immunization: Twelve guinea pigs to be used for immunological testing were immunized with c e l l wall material from T_. mentagrophytes as described previously. Serum was collected from immunized guinea pigs at the time of sacrifice inactivated at 56°C for 30 min. and stored at -20°C. Skin tests and lymphocyte stimulation: These were carried out as described previously. A l l peptide fractions collected were tested for their immunological reactivity by intradermal skin test and for their a b i l i t y to stimulate lymphocytes taken from immunized guinea pigs. Unimmunized animals were used as controls. A l l tests were run at 10 and 25 ug peptide material. A total of 12 immunized animals were used in this study. Amino acid analyses Peptides that were shown to have been antigenic by skin test and lymphocyte transformation and which were shown to be a single peptide by 46 Polyacrylamide g e l e l e c t r o p h o r e s i s : The method; of Weber and Osborn (70) was followed. The UTi^a) f r a c t i o n was found to conta i n two s u b f r a c t i o n s , F i g (7) which d i f f e r i n t h e i r s t a i n i n g i n t e n s i t y . Each sub f r a c t i o n was shown to contain peptide and carbohydrate moieties by coomassie b r i l l i a n t blue and periodate a c i d s c h i f f s t a i n s r e s p e c t i v e l y . Thin l a y e r chromatography: The peptide f r a c t i o n s of pronase and t r y p s i n d i g e s t s were ana-l y z e d by t h i n l a y e r chromatography. Precoated p l a t e s w i t h s i l i c a g e l G 250 microns were used. The solvent was bu t a n o l , a c e t i c a c i d , water 3:1:1 r e s p e c t i v e l y according to Wunch et a l (72) D i s c g e l e l e c t r o p h o r e s i s p a t t e r n of 24 hr (UNF), UM2(a) f r a c t i o n s . TD i s the p o s i t i o n of the t r a c k i n g dye. 24 hr(UNF) UM2 ( a) Figure 8 Complement fixation t i t ration curve of U M 2 ( a ) fraction. 50 51 t h i n - l a y e r chromatography were analyzed f o r t h e i r amino a c i d composition i n the Beckman Spinco automatic amino a c i d analyzer. Complement F i x a t i o n : Q u a n t i t a t i v e complement f i x a t i o n t e s t s were performed by a m o d i f i c a t i o n of the method described by Kabat and Mayer (1961). P r e c i s e methods have been published elsewhere (28). L y o p h i l i z e d guinea p i g complement (Flow L a b o r a t o r i e s ) was used at a d i l u t i o n of 1:100 . Veronal b u f f e r at pH 7.5 was used as a d i l u e n t . The antigen d i l u t i o n s used were 0.1, 0.2 - 3.2 ug/ml and antiserum d i l u t i o n s from 1:100 to 1:1600 . A 2% suspension of washed sheep e r y t h r o c y t e s was s e n s i t i z e d by mixing them w i t h an equal volume of 1:100 d i l u t i o n of hemolysin (Difco) f o r at -l e a s t 15 min . The d i l u t i o n of complement producing 50% hemolysis was obtained by the method of p r o b i t s as described p r e v i o u s l y (68). T i t r a t i o n of UTy^a) w i t h guinea p i g antiserum i s shown In f i g . 8 . Hapten I n h i b i t i o n : Hapten i n h i b i t i o n s t u d i e s were performed as described by Gerwing and Thompson (28) . B r i e f l y , the t e s t i n v o l v e d i n c u b a t i o n f o r 24 h at 4°C of a n t i whole m y c e l i a l c e l l w a l l guinea p i g serum (at 1:1000) w i t h the various t e s t peptides (at 50 Ug per ml). Subsequently, whole antigen (UK^a) at a p r e v i o u s l y determined optimal concentration (0.25 ug/ml) f o r complement f i x a t i o n and guinea p i g complement at a d i l u t i o n of 1:75 was added to each t e s t . Incubation at 4°C was continued f o r a f u r t h e r 24 h a f t e r which time each mixture was assayed f o r f r e e complement. Appropriate c o n t r o l s of the pep t i d e s , antigen and antibody were c a r r i e d out i n each i n s t a n c e . 52 Results We have shown i n s e c t i o n I I that a low molecular weight f r a c t i o n (UM-jCa)) from T. mentagrophytes c e l l w a l l c o n t a i n i n g both polysaccharide u and peptide gave i n ginea pigs strong delayed s k i n r e a c t i v i t y as w e l l as a s t i m u l a t i o n to s e n s i t i z e d lymph node c e l l s a f t e r 96 h i n c u b a t i o n . I t was thought that the e l i m i n a t i o n of the peptide part would present an oppo r t u n i t y to study the immunological r o l e of the carbohydrate moiety. To t h i s end enzyme d i g e s t i o n s were performed on the WL C^a) f r a c t i o n . Table 4 shows q u a n t i t a t i v e l y the amount of Anthrone p o s i t i v e and n i n h y d r i n p o s i t i v e m a t e r i a l remaining a f t e r pronase-carboxypeptidase and t r y p s i n d i g e s t i o n s . The r e s u l t s i n d i c a t e that most of the peptide m a t e r i a l was removed by pronase-carboxypeptidase treatment, wherease t h i s was not so w i t h t r y p s i n treatment. Figure 9 shows the r e s u l t s of those f r a c t i o n s which evoked hyper-s e n s i t i v i t y r e a c t i o n s i n immunized guinea p i g s . I t can be seen t h a t the undigested u l ^ r e t e n t a t e m a t e r i a l c o n t a i n i n g mainly carbohydrate induced both immediate and delayed s k i n r e a c t i o n s , wherease the peptide f r a c t i o n s could not induce immediate but d i d give both Arthus and delayed r e a c t i o n s , i n d i c a t i n g that both the pronase and t r y p s i n digested peptides r e t a i n e d some immunological s p e c i f i c i t y . Table 5 shows the i n v i t r o response of s e n s i t i z e d lymphocytes to the f r a c t i o n s obtained a f t e r enzymatic treatment. The CPM of the s e n s i t i z e d c e l l s i n the presence of peptide fragments was compared to c e l l s incubated without peptide. C e l l s from unimmunized animals were a l s o incubated w i t h Table 4 . Chemical a n a l y s i s of UM 2(a) f r a c t i o n a f t e r enzyme d i g e s t i o n . Figures represent t o t a l values f o r each f r a c t i o n . Enzyme Try p s i n Pronase Pronase + Carboxypeptidase Mg CH 20 before Mg P r o t e i n before Mg CH20 a f t e r Mg P r o t e i n a f t e r Mg P r o t e i n passed d i g e s t i o n d i g e s t i o n d i g e s t i o n d i g e s t i o n and u l t r a f i l t r a t i o n (UM 2(a)) 62 62 54 UM2 Amicon f i l t e r (UM 2(a)) 22 22 10.46 (PCUM2R) 55.5 54.0 46.5 • (PCUM2R) 12.24 10.46 2.42 (PCUM2P) 9.75 (1) Anthone t e s t (2) Q u a n t i t a t i v e n i n h y d r i n t e s t (3) no carbohydrate was det e c t a b l e i n t h i s f r a c t i o n 54 Figure 9 Cutaneous a l l e r g i c reactions of the enzyme digests of UM2(a) f r a c t i o n i n immunized guinea pigs (whole column) compared to that of the unimmunized animals (shaded). A = UM2(a) f r a c t i o n B = Pronase-carboxypeptidase UK, retained f r a c t i o n (PCUM2R) C = Pronase-carboxypeptidase UK, passed f r a c t i o n (PCuK,^) D = Trypsin UM2 retained (TUM2R) E = Trypsin UM2 passed (TUM2P) F = Saline 56 Table 5. Lymph node c e l l 96 hour i n c o r p o r a t i o n of t r i t i a t e d thymidine i n response to UM^Ca) s u b - f r a c t i o n s a f t e r enzyme treatment and f r a c t i o n a t i o n . Lymphocyte response at various p r o t e i n l e v e l s Antigen 5 yg/ml S.I? 25 yg/ml S.I.* 125 ug/lml UM 2(a) 16,063 2.55 16,280 2.59 17,183 2.73 PCUM2R 20,036 3.036 21,916 3.49 19,260 3.06 PCUM2P 16,343 2.60 16,373 2.60 12,066 1.92 TUM2R 20,466 3.26 22,520 3.59 18,670 2.97 TUM2P 6,766 NS 16,793 2.67 17,890 2.85 No Antigen 6,286 * S t i m u l a t i o n Index E l u t i o n p r o f i l e of t r y p t i c peptides on Dowex 50W X8 column. (© © ) = absorbance at 570 urn (x x) = absorbance at 440 um . 59 and without the t e s t f r a c t i o n s . A l l f r a c t i o n s were capable of inducing lymphocyte p r o l i f e r a t i o n of c e l l s from immunized animals at 96 h showing that immunological s p e c i f i c i t y was r e t a i n e d and r e s i d e d i n both the carbohydrate-rich and peptide f r a c t i o n s . However, there was v i r t u a l l y no measurable response at 24 h i n c u b a t i o n . Cultures of c e l l s from unimmunized animals showed no p r o l i f e r a t i v e response (data not shown). The e u l t i o n p r o f i l e of the p u r i f i e d t r y p t i c peptides a f t e r i on exchange chromatography i s shown i n Figure 10. The s k i n r e a c t i o n s to the pooled peptide peaks are shown i n Figure 12a,b,c . Out of 17 peptides t e s t e d , i t can be seen t h a t 5 gave f a i r l y good delayed s k i n r e a c t i o n s w h i l e none of them induced r e a c t i o n s at 30 minutes. Table 6 shows the r e s u l t s of the lymphocyte response to each peptide. About h a l f of the i s o l a t e d peptides induced lymphocyte p r o l i f e r a t i o n i n c u l t u r e w i t h immunized c e l l s . Most peptide f r a c t i o n s which showed p o s i t i v e s k i n r e a c t i o n s were capable of s t i m u l a t i n g p r o l i f e r a t i o n , although there was not an absolute c o r r e l a t i o n . Except f o r T^, T^, T ^ and T^, a l l other peptide peaks showed one spot on t h i n l a y e r chromatography ( F i g 11). The f r a c t i o n s w i t h more than one spot were not analyzed f o r t h e i r amino a c i d content. The amino a c i d analyses of some of the peptides shown to have been a n t i g e n i c by s k i n t e s t and/or lymphocyte s t i m u l a t i o n are presented i n Table 7 . Some of the i s o l a t e d peptides were at too low a y i e l d to permit analyses. Immunodiffusion and complement f i x a t i o n s t u d i e s w i t h anti-whole m y c e l i a l c e l l w a l l guinea p i g serum showed that a number of peptides were 60 Figure 11 . Thin l a y e r chromatography of the t r y p t i c peptides. g-/ 1 — 1 \ l O O f \ / Cutaneous a l l e r g i c r e a c t i o n s of the t r y p t i c peptides from UlljCa) i n immunized guinea pigs (whole column) compared to that of unimmunized animals (shaded) at 30 min (A), 5 h (B) and 24 h (C) . 6 3 32.0 -P8.0 424.0 20.0 16.0 12.0 Tl 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 S PEPTIDE NUMBER UM2(OJ 64 apparently large enough to form Immune precipitates or to f i x complement (data not shown). These peptides were numbers 13, 14, 15 and 16 and were not included in the hapten inhibition test. The results of the inhibition of complement fixation by tryptic peptides is shown in Table 8 . It can be seen that most of the peptides tested ( i . e . t h o s e which did not f i x complement in their own right) were capable of inhibiting the reaction of UlL^a) with specific antiserum indicating that these peptides contain at least one intact antigenic determinant recognized by antibodies formed against whole c e l l wall material. 65 Table 6. Sensitized lymph node c e l l 96 hour incorporation of t r i t i a t e d thymidine in response to tryptic peptides from UM2(a) . 25 yg/ml 125 yg/ml CPM S.I. CPM S . I . UM2(a) 63,076 3.37 52,876 2.69 T l 50,563 2.70 18,183 NS T2 49,713 2.65 40,506 2.06 T3 49,816 2.67 30,350 NS T4 39,253 2.09 6,933 NS T5 40,970 2.19 14,763 NS T6 32,820 NS 14,873 NS T 7 39,763 2.12 53,769 2.73 T 8 32,466 NS 22,953 NS T 9 21,630 NS 21,443 NS T 10 22,710 NS 11486 NS T 11 16,210 NS 2,163 NS T 12 24,073 NS 18,336 NS T 13 50,666 2.7 30,980 NS T 14 31,776 NS 23,090 NS T 15 29,632 NS 34,060 NS T 16 44,173 2.36 54,040 2.75 T 17 58,860 3.14 48,618 2.47 N. Ag. 18,740 19,680 *S.I. - Stimulation index Table 7. The amino a c i d compositions of some of the t r y p t i c peptides from Ift^Ca) T2 T3 T13 T16 T17 UM 2(a) Umoles molar Umoles molar Umoles molar Umoles molar Umoles molar Umoles molar r a t i o r a t i o r a t i o r a t i o r a t i o r a t i o A s p a r t i c Acid .053 1.04 .071 2.96 .04 1.02 .088 3.9 Threonine .052 2.16 .063 2.8 Serine .058 1.15 .069 2.88 .061 2.7 Glutamic a c i d .047 1.96 .051 2.21 .020 1.4 .077 1.97 .083 3.7 P r o l i n e 143 2.81 1028 1.21 .151 6.7 Glycine .040 2.7 .098 4.3 Alanine .065 2.83 .041 1.8 Valine .040 1.74 .058 4.0 .040 1.8 Isoleucine .053 2.20 .027 1.8 .023 1.0 Leucine .030 2.0 .024 1.1 H i s t i d i n e .050 2.2 Lysine .010 0.2 .010 0.43 .011 0.8 .049 2.2 Arginine .018 0.8 Table 8 . I n h i b i t i o n of complement f i x a t i o n by t r y p t i c peptides Peptide No. % of I n h i b i t i o n T l 54 T2 57 T 3 85 T4 71 T 5 53 T 6 71 T 7 80 T 8 69 T 9 100 T 79 10 T l l 68 T 1 2 75 68 Discus s i o n The p r o t e o l y t i c enzyme d i g e s t i o n o f the U l ^ a ) f r a c t i o n , f o l l o w e d by u l t r a f i l t r a t i o n , was shown to be e f f e c t i v e i n the removal of a s u b s t a n t i a l amount of peptide from the polysaccharide peptide complex. pronase on Wherease e i t h e r t r y p s i n , o r t h e i r own removed approximately h a l f the peptide content, the combination of pronase plus carboxypeptidase removed about 90% (Table 4). When the carbohydrate-rich f r a c t i o n l e f t a f t e r p r o t e o l y t i c d i g e s t i o n was t e s t e d immunologically i t appeared that i t was capable of e l i c i t i n g immediate and Arthus s k i n r e a c t i o n s i n " s e n s i t i z e d guinea p i g s , both of which are i n d i c a t i o n s of humoral immunity. However, t h i s c arbohydrate-rich f r a c t i o n was a l s o capable of e l i c i t i n g delayed s k i n r e a c t i o n s and lymphocyte transformation of s e n s i t i z e d c e l l s to l e v e l s comparable w i t h the i n t a c t ITC^a) f r a c t i o n . These data imply that c e l l u l a r (T c e l l ) immunity i n s e n s i t i z e d animals i s d i r e c t e d toward the carbohydrate moiety of the c e l l w a l l of T_. mentagrophytes. These f i n d i n g s are i n disagreement w i t h those of Barker and Cruickshank (7,8,), whose work i n d i c a t e d t h a t the humoral response r a t h e r than the c e l l - m e d i a t e d response was s t i m u l a t e d by the carbohydrate of dermatophyte c e l l w a l l s . Our f i n d i n g are a l s o i n disagreement w i t h those of Nozawa et_ al ., (54), who found that p r o t e o l y t i c d i g e s t i o n of c e l l w a l l m a t e r i a l decreased the delayed h y p e r s e n s i t i v i t y r e a c t i o n by 54% . While these f i n d i n g s are not i n agreement w i t h those of other i n v e s t i g a t o r s , i t should be emphasized that no conclusions can be drawn on the immunological r o l e of the carbohydrates of t h i s f r a c t i o n s i n c e even a f t e r p r o t e o l y t i c d i g e s t i o n , a measurable amount of peptide m a t e r i a l remained (approximately 5%) and i t i s conceivable but 69 u n l i k e l y that the c e l l - m e d i a t e d r e a c t i o n s demonstrated were a t t r i b u t a b l e e x c l u s i v e l y to the peptide moiety remaining . The peptide m a t e r i a l s which were separated from the carbohydrate polymer by f i l t r a t i o n through a U^ L, amicon f i l t e r appeared to be a c t i v e immunologically. These f r a c t i o n s were not able to induce immediate s k i n r e a c t i o n s i n s e n s i t i z e d animals, but d i d cause the development of delayed r e a c t i o n s . This observation i s i n agreement w i t h the i m p l i c a t i o n s of other i n v e s t i g a t o r s (y,8,54). I t was somewhat s u r p r i s i n g that the peptide m a t e r i a l i s o l a t e d from the pronase-carboxypeptidase d i g e s t was capable of causing s k i n r e a c t i v i t y and of s t i m u l a t i n g lymphocyte t r a n s f o r m a t i o n , s i n c e : I t was f e l t that t h i s k i n d of degradation would probably r e s u l t i n the formation of peptides too s m a l l to r e t a i n any immunological i n t e g r i t y . The t r y p t i c peptide f r a c t i o n was f u r t h e r c h a r a c t e r i z e d by i o n exchange chromatography. The i n d i v i d u a l peptides thus i s o l a t e d were t e s t e d f o r t h e i r Immunological p r o p e r t i e s . The data showed c l e a r l y that those peptides capable of inducing delayed s k i n r e a c t i o n s were a l s o those which u s u a l l y induced lymphocyte t r a n s f o r m a t i o n , thus i n d i c a t i n g a c o r r e l a t i o n between the i n v i v o and i n v i t r o assay f o r c e l l - m e d i a t e d immunity. I t should be mentioned that t h i s c o r r e l a t i o n was not absolute s i n c e some peptides reacted p o s i t i v e l y i n only one of the t e s t systems. The reasons f o r t h i s are not c l e a r . This observation i s i n general agreement w i t h those of H a n i f i n et a l . , (35) who showed the consistency of lymphocyte transformation and delayed s k i n r e a c t i v i t y . They found that p a t i e n t s w i t h e i t h e r T_. mentagrophytes or T_. rub rum i n f e c t i o n who gave delayed s k i n r e a c t i o n s a l s o had p e r i p h e r a l blood lymphocytes which underwent transformation i n 70 > the presence of antigen a f t e r 5 days i n v i t r o . The f i n d i n g that these i s o l a t e d peptides d i d not induce immediate s k i n r e a c t i o n s was not s u r p r i s i n g , s i n c e most of them were ob v i o u s l y too s m a l l to be able to e f f e c t a c l a s s i c a l histamine mediated immediate r e a c t i o n , s i n c e i t i s assumed t h a t mast c e l l d e granulation w i l l only take place i n the presence of aggregated antigen. However, i t has been shown p r e v i o u s l y , (65,69) that s m a l l haptenic peptides c o n t a i n i n g s i n g l e a n t i -genic determinants are capable of inducing c e l l - m e d i a t e d r e a c t i o n s i n s e n s i t i z e d animals. The p o s s i b i l i t y that these peptides were f u n c t i o n a l i n humoral immunity was i n v e s t i g a t e d by t e s t i n g the t r y p t i c peptides f o r t h e i r a b i l i t y to i n h i b i t complement f i x a t i o n by U l ^ a ) w i t h s p e c i f i c antiserum and f o r t h e i r a b i l i t y to form p r e c i p i t a t e s w i t h the antiserum. I t was somewhat s u r p r i s i n g to note t h a t v i r t u a l l y a l l of the i s o l a t e d peptides were a c t i v e e i t h e r i n i n h i b i t i n g the r e a c t i o n between U l l ^ a ) and the antiserum, which i m p l i e s a s i n g l e a n t i g e n i c determinant; or by forming a p r e c i p i t i n l i n e w i t h the antiserum which i m p l i e s a peptide of two or more determinants. Thus, w h i l e i t may be accurate to s t a t e that the peptide f r a c t i o n does not cause immediate s k i n r e a c t i o n s i n s e n s i t i z e d animals, i t i s apparent that c i r c u l a t i n g antibody i n s e n s i t i z e d animals does re a c t s t r o n g l y w i t h the peptides. Thus, i t i s c l e a r that the peptide p o r t i o n of the c e l l w a l l i s a c t i v e at the l e v e l of both humoral and cell-mediated immunity. The amino a c i d analyses of some of the i s o l a t e d peptides are shown i n t a b l e 7 as w e l l as the a n a l y s i s of UM 9(a). These data do not c o n s t i t u t e .71 a complete picture of the tryptic peptides associated with this fraction but they do provide information regarding the amino acid composition of the major immunologically active peptides. Peptides and were active in the lymphocyte stimulation assay as well as in the complement fixation inhibition test, whereas only was capable of inducing a positive delayed skin reaction. It is clear that both and are unique peptides. Peptides T 1 0, T.,. and T. _ caused lymphocyte transformation 13 lo 1/ and delayed skin reactions and were active in the complement fixation test. The analysis of these peptides showed clearly that these also have quite distinct amino acid sequences. From this somewhat limited study of the amino acid compositions of these peptides, i t would appear that the peptide portions of the UT^Ca) fraction contain a diverse array of amino acid sequences which constitute antigenic determinants recognized at both the humoral and cellular level of immune responsiveness. 72 S e c t i o n IV General Conslusions C h i t i n a s e D i g e s t i o n The d i g e s t i o n of clean c e l l w a l l p r e p a r a t i o n of T\_ mentagrophytes w i t h c h i t i n a s e y i e l d e d a v a r i e t y of heterogeneous m a t e r i a l s which could be p a r t i a l y f r a c t i o n a t e d according to t h e i r molecular s i z e by u l t r a f i l -t r a t i o n and g e l f i l t r a t i o n . A l l the f r a c t i o n s i s o l a t e d i n t h i s manner contained both peptide and polysaccharide m a t e r i a l . When comparisons were made between m a t e r i a l which had been digested f o r e i t h e r 24 hours or 7 days w i t h c h i t i n a s e , i n terms of the f r a c t i o n s obtained and t h e i r immunological a c t i v i t i e s no d i f f e r e n c e s were observed. This i n d i c a t e s that extensive d i g e s t i o n occurs during the f i r s t 24 hours, and prolonged treatment w i t h the enzyme has very l i t t l e obvious e f f e c t . Immunological a c t i v i t y of various f r a c t i o n s A l l the f r a c t i o n s from the 24 hour c h i t i n a s e d i g e s t i o n obtained a f t e r u l t r a f i l t r a t i o n and sephadex f i l t r a t i o n were tested f o r t h e i r a b i l i t y to evoke immediate, Arthus-type, or delayed s k i n r e a c t i o n s i n guinea pigs s e n s i t i z e d to whole c e l l w a l l fragments of mentagrophytes. They were a l s o t e s t e d f o r t h e i r a b i l i t y to s t i m u l a t e lymphocyte p r o l i f e r a t i o n i n c e l l s from s e n s i t i z e d guinea p i g s . F r a c t i o n s which had molecular weight of greater than 10,000 (as estimated by t h e i r a b i l i t y to pass through a UM10 f i l t e r gave s t r o n g s k i n r e a c t i v i t y and good lymphocyte s t i m u l a t i o n . Only one f r a c t i o n of lower molecular weight (IM^ia)) showed s t r o n g immunological r e a c t i v i t y by both s k i n t e s t i n g and i n v i t r o lymphocyte s t i m u l a t i o n . This f r a c t i o n had a molecular weight of between 2 and 10,000 as estimated by i t s a b i l i t y to pass through a UM10 f i l t e r but not a Wi£a)filter, and by i t s e l u t i o n w i t h the v o i d volume on Sephadex G—25 . 73 Other UK, f r a c t i o n s which were in c l u d e d i n Sephadex G-25 columns d i d not show complete immunological r e a c t i v i t y . As a r e s u l t of these observations, f u r t h e r s t u d i e s were c a r r i e d out on the Ull^Ca) f r a c t i o n s i n c e i t was the m a t e r i a l of minimum molecular s i z e to e x h i b i t complete r e a c t i v i t y . Studies on UM 2(a) An attempt was made to determine whether the carbohydrate and peptide moieties of t h i s f r a c t i o n played d i f f e r e n t r o l e s i n immune responsiveness. UM^a m a t e r i a l was digested w i t h e i t h e r t r y p s i n or a combination of pronase and carboxypeptidase A. A f t e r d i g e s t i o n the m a t e r i a l was washed on a UK, f i l t e r . The r e t a i n e d m a t e r i a l contained e s s e n t i a l l y a l l the polysaccharide part of the f r a c t i o n wherease peptides were found i n the f i l t r a t e . The polysaccharide f r a c t i o n which had been digested w i t h carboxypeptidase A and pronase r e t a i n e d only about 5% of the o r i g i n a l peptide m a t e r i a l . Tests were c a r r i e d out to determine the immunological r e a c t i v i t y of each f r a c t i o n . I t was found that the polysaccharide r i c h m a t e r i a l s r e t a i n e d t h e i r a b i l i t y to induce both immediate and delayed s k i n r e a c t i o n s i n s e n s i t i z e d guinea pigs as w e l l as to s t i m u l a t e lymphocyte p r o l i f e r a t i o n . These observations i n d i c a t e that no separate r o l e can be a t t r i b u t e d to the polysaccharide moiety i n s t i m u l a t i n g e i t h e r humoral or c e l l mediated immunity. However, because a small amount of peptide remained i n these polysaccharide r i c h f r a c t i o n s , these observations are not c o n c l u s i v e . The peptide m a t e r i a l r e s u l t i n g from the d i g e s t i o n s were of r e l a t i v e l y low molecular weight s i n c e they had passed through a 74 f i l t e r . These f r a c t i o n s , however, were found to be capable of s t i m u l a t i n g delayed s k i n r e a c t i o n s and lymphocyte p r o l i f e r a t i o n but not immediate and Arthus r e a c t i o n s . While these observations, s u p e r f i c i a l y , might be taken to i n d i c a t e that peptide m a t e r i a l s were only i n v o l v e d i n c e l l mediated immunity, t h i s i s not n e c e s s a r i l y the case. These m a t e r i a l s are of low molecular weight. I t i s thought that mast c e l l d e g ranulation, which i n i t i a t e s the events l e a d i n g to an immediate s k i n r e a c t i o n , i n v o l v e s the complexing of antigen and antibody on the mast c e l l s u r f a ce. I t has been shown that haptens and monovalent antigens can block t h i s r e a c t i o n , s i n c e i t i s thought that m u l t i v a l e n c y of antigen i s required to acheive s u f f i c i e n t p e r t u r b a t i o n of the mast c e l l membrane to i n i t i a t e degranulation. Therefore, the observation that these peptides could not cause immediate s k i n r e a c t i o n s should not be regarded as proof f o r t h e i r l a c k of p a r t i c i p a t i o n i n humoral immunity. Immunological Behaviour of T r y p t i c Peptides from uTt^Ca) The t r y p t i c d i g e s t of UT^a was chromatographed on an i o n exchange column i n an attempt to p u r i f y i n d i v i d u a l peptides so that t h e i r r e a c t i v i t y could be test e d . I s o l a t e d f r a c t i o n s were t e s t e d f o r homogeneity by t h i n l a y e r chromatography. Only those f r a c t i o n s which s a t i s f i e d t h i s c r i t e r i o n were analysed f o r t h e i r a b i l i t y to induce s k i n r e a c t i v i t y i n s e n s i t i z e d guinea p i g s , to s t i m u l a t e lymphocyte p r o l i f e r a t i o n , and to bl o c k complement f i x a t i o n between I f l ^ a and s p e c i f i c antiserum. E s s e n t i a l l y no peptide induced immediate or Arthus r e a c t i o n s . However, a very high p r o p o r t i o n of them were capable of 75 inducing delayed r e a c t i o n s , s t i m u l a t i n g lymphocyte p r o l i f e r a t i o n or b l o c k i n g complement f i x a t i o n . These f i n d i n g s showed that a number of these peptides were r e a c t i v e at both the humoral and c e l l mediated l e v e l of immune responsiveness, showing c l e a r l y that these two elements cannot be separated i n terms of the r o l e s of d i f f e r e n t f r a c t i o n s of T_. mentagrophytes c e l l w a l l components. The amino a c i d analyses which were c a r r i e d out on some of the t r y p t i c peptides i s o l a t e d showed c l e a r l y that the peptide moiety of UT^a d i d not c o n s t i t u t e a s m a l l repeating subunit but r a t h e r a complex peptide s t r u c t u r e . Previous reports i n t h i s area have i n d i c a t e d t h a t the polysaccharide p o r t i o n s of dermatophyte c e l l w a l l s are r e s p o n s i b l e f o r inducing antibody-mediated (immediate) r e a c t i o n s , whereas the peptide p o r t i o n s are r e s p o n s i b l e f o r inducing the c e l l - m e d i a t e d r e a c t i o n s . The f i n d i n g s reported here do not support t h i s view, and show th a t immune responsive-ness to dermatophyte antigens can not be c l a s s i f i e d i n t h i s way. Rather, both polysaccharide and peptide moieties of the c e l l w a l l appear to s t i m u l a t e both humoral and cell-mediated immunity. 76 References Anderson, B.A., H e l l g r e n , L., and Vincent, J . (1976) A l l e r g i c delayed s k i n r e a c t i o n s from l i p i d f r a c t i o n s of t r i c h o p h y t i n . Sabouraudia 14 : 237-244 Anderieu, S., B i g u r t , J . , and L a l u x , B., (1968) Analyse immunoelectrophoretique compare'e des s t r u c t u r e s antigeniques des 17 especes de dermatophytes mycopathol. Mycol. appl. 34: 161-185 A u s t u r i c k , P.K.C. (1972) The p a t h o g e n i c i t y of fungi pgs.251-278 In H. Smith and J.H. Pearce (ed). M i c r o b i a l p a t h o g e n i c i t y i n man and animals. The u n i v e r s i t y p r e s s , Cambridge. Balogh, E., Meszaros, Cs. and Halmy, K. Die Anwendung des lymphocyten-tra n s f o r m a t i o n - t e s t e s b e i des Untersechung der mykotischen s e n s i -b i l i s a t i o n Mykosen 14: 207-211 Barker, S.A., Stacey, M. and I w e i f e l (1957) a The s e p a r a t i o n of n e u t r a l p o l y s a c c h r i d e s Chem. Ind. (London) Page 330. Barker, S.A., Cruickshank, C.M.D, M o r r i s , J.H, and Wood, S.R. (1962) The i s o l a t i o n of t r i c h o p h y t i n glycopeptide and i t s s t r u c t u r e i n r e l a t i o n to the immediate and delayed r e a c t i o n Immunology 5: 627-632 . 76 References Anderson, B.A., H e l l g r e n , L., and Vincent, J . (1976) A l l e r g i c delayed s k i n r e a c t i o n s from l i p i d f r a c t i o n s of t r i c h o p h y t i n . Sabouraudia 14 : 237-244 Anderieu, S., B i g u r t , J . , and L a l u x , B., (1968) Analyse immunoelectrophoretique compare'e des s t r u c t u r e s antigeniques des 17 especes de dermatophytes mycopathol. Mycol. appl. 34: 161-185 A u s t u r i c k , P.K.C. (1972) The p a t h o g e n i c i t y of fungi pgs.251-278 In H. Smith and J.H. Pearce (ed). M i c r o b i a l p a t h o g e n i c i t y i n man and animals. The u n i v e r s i t y p r e s s , Cambridge. Balogh, E., Meszaros, Cs. and Halmy, K. Die Anwendung des lymphocyt tra n s f o r m a t i o n - t e s t e s b e i des Untersechung der mykotischen s e n s i -b i l i s a t i o n Mykosen 14: 207-211 Barker, S.A., Stacey, M. and I w e i f e l (1957) a The s e p a r a t i o n of n e u t r a l polysaccharides Chem.- Ind. (London) Page 330. Barker, S.A., Cruickshank, C.M.D, M o r r i s , J.H, and Wood, S.R. (1962) The i s o l a t i o n of t r i c h o p h y t i n glycopeptide and i t s s t r u c t u r e i n r e l a t i o n to the immediate and delayed r e a c t i o n Immunology 5: 627-632 • 77 (7) Barker, S.A., Cruickshank, C.N.D. and Holden, J.H (1963) Structure of galactomannan-peptlde allergen from I. mentagrophytes. 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