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The role of fungal lipids in the hypersensitive response Shkurhan, Eugene E. 1967

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THE ROLE OP FUNGAL LIPIDS IN THE HYPERSENSITIVE RESPONSE by EUGENE E. SHKURHAN B.Sc, University of B r i t i s h Columbia, 1964. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Microbiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1967 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e 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 t h a t t h e 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 a g r e e 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 t h e Head o f my Depar tment o r by h.iis 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 t h a t 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 n o t 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 . D e p a r t m e n t o f Mi rvrob-i o l ogv 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 V a n c o u v e r 8, Canada Date September 26. 1967  ABSTRACT Studies made on the l i p i d components of Microsporum  quinckeanum, have shown that 6 day old fermenter-grown mycelia and that of 21 day old s t a t i c cultures grown in Sabouraud's glucose l i q u i d medium have a l i p i d content of approximately 16% by dry weight. Thin layer chromatography showed that the l i p i d f ractions obtained from both methods of c u l t i v a t i o n were i d e n t i c a l . During the p u r i f i c a t i o n of the l i p i d extracts, a lipoprotein was isolated having 16.45$ protein content, and af t e r hydrolysis and chromatography this material demonstrated the presence of eleven amino acids. Dried mycelia, p u r i f i e d l i p i d and lipoprotein were used separately in complete and incomplete Freund's adjuvant to sens i t i z e guinea pigs and rabbits by various routes. Se n s i t i z a t i o n of guinea pigs was achieved also by Bloch's method. A l l animals were challenged by separate intradermal injections of to t a l l i p i d extracts, lipoprotein and the six l i p i d f r a c t i o n s . Delayed hypersen-s i t i v e responses of varying i n t e n s i t i e s were demonstrated by a l l sensitized animals. Further investigations were made using lymph nodes removed from rabbits separately sensitized with dermatophyte l i p i d extracts and li p o p r o t e i n . Protein extracts of lymph nodes from these animals were examined e l e c t r o p h o r e t i c a l l y . The presence of p r e c i p i t a t i n g antibodies - i i i -in sera and lymph node extracts were demonstrated by immuno-di f f u s i o n and p r e c i p i t i n r i n g t e s t s . Passive transfer tests were carried out successfully to conclusively demonstrate the presence of antibodies in the lymph node extracts. - i v — TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 4 I. Stock Cultures 4 I I . Inoculum 4 I I I . Medium 5 IV. Cultures for L i p i d Extraction 5 1. S t a t i c cultures 5 2. Fermenter cultures 5 V. Harvesting of Mycelia 6 VI. Dry Weight Determination ' 6 VII. Extraction of Lipids 6 VIII. P u r i f i c a t i o n of Extracted Lipids 7 IX. Thin Layer Chromatography 7 1. Preparation of plates 7 2. Application of samples 8 3. Development of chroma.togra.ms 8 4. Detection reagents 9 5. Tests f o r purity a f t e r washing 10 X. ;; Fractionation of L i p i d Extract 11 XI. Separation of Lipoprotein 12 XII. I d e n t i f i c a t i o n of Lipoprotein 12 XIII. Protein Determination in Lipoprotein 14 Page XIV. Detection of Phosphate in Lipoprotein Ik XV. Determination of Phospholipid in L i p i d Extract 14 XVI. Preparation of L i p i d Extract f o r Immunological Studies 1H XVII. Preparation of Li p i d Fractions f o r Immunological Studies ' 15 XVIII. S e n s i t i z a t i o n of Guinea Pigs 15 1. Preparation of spores for Bloch's method 16 2. Preparation of myeelia for se n s i t i z a t i o n 16 3 . Routes of s e n s i t i z a t i o n and materials 17 XIX. Hypersensitivity Testing - Guinea Pig Series 17 1. Preparation of animals 17 XX. Se n s i t i z a t i o n of Rabbits 18 XXI. Hypersensitivity Testing - Rabbit Series 20 1. Preparation of animals 20 XXII. Preparation of Antisera 21 XXIII. Use of Dimethylsulfoxide to S o l u b i l i z e L i p i d Extract 21 XXIV. Preparation of Lymph Node Extracts 22 XXV. Qualitative P r e c i p i t a t i o n in Gel 23 XXVI* Ring or I n t e r f a c i a l P r e c i p i t i n Test 23 - v i ~ Page XXVII. Electrophoresis of Lymph Node Extracts 24 XXVIII. Passive S e n s i t i z a t i o n Transfer Test 25 RESULTS AND DISCUSSION 26 I. L i p i d Extraction and P u r i f i c a t i o n 26 I I . Skin Test Reactions 31 I I I . Skin Test Reactions, Rabbit Series 36 SUMMARY 48 BIBLIOGRAPHY 50 - v i i LIST OF FIGURES Figure Page 1. Challenge scheme for guinea pig s e r i e s . 18 2 . Scheme used for challenging the rabbit s e r i e s . 21 3 ; Scheme for the passive s e n s i t i z a t i o n transfer t e s t . 25 4 . Thin layer chromatograph of M.Q.8 l i p i d . 29 5 . Amino acids of M.Q.8 lipoprotein as demonstrated by paper chromatography. 3° 6 . Representative results using Bloch's method of se n s i t i z a t i o n with M.Q.8. 33 7. Representative results obtained using Bloch's method of s e n s i t i z a t i o n with a virulent s t r a i n of M. gypseum. 34 8. Results obtained from a.nimals sensitized with ground mycelia in adjuvant employing M.Q.8 and M. gypseum. 35 9 . Results obtained when control animals were challenged. 36 10. Representative results observed in rabbit groups 1-4, and groups 6 , 7. 38 11. Results obtained for group 5 observed 28 hours a f t e r challenge. 40 12. Results obtained when a non-sensitized control - v i i i -Pigure Page animal was challenged. 40 13. Results of ring p r e c i p i t i n test when lipoprotein was overlaid onto lymph node extracts. 44 14. Ouchterloney gel d i f f u s i o n employing lymph node extracts and lipoprotein extract. 45 15. Passive transfer t e s t , challenged with l i p o p r o t e i n . 46 16. Passive transfer test, challenged with l i p i d . 47 - i x -LIST OF TABLES Table Page I. Hypersensitivity reactions as demonstrated by guinea pig skin t e s t s . 32 I I . Hypersensitivity reactions as demonstrated by rabbit skin t e s t s . 37 I I I . Protein content as demonstrated by the Lowry method in the lymph node extracts obtained. 43 ACKNOWLEDGEMENT My deepest appreciation to my wife f o r her under-standing and encouragement during the course of t h i s study. I would also l i k e to express my appreciation to Dr. J. J . Stock for his guidance and encouragement; also to Mr. James Kalmakoff for his photography and to other members of the department f o r t h e i r welcome suggestions and c r i t i c i s m s . INTRODUCTION The immunogenic properties of l i p i d s were discovered in the course of two series of investigation between 1910 and 1920. In one group of experiments Porssman observed that rabbits injected with suspensions of organs obtained from guinea pigs produced antibodies which caused l y s i s of sheep erythrocytes. The component which was responsible for t h i s Forssman antigen was soluble in alcohol and when injected by i t s e l f this material was only weakly antigenic. At about the same time that the above findings were reported, other workers observed that alco-holic extracts of Treponema pallidum, as well as alcoholic extracts of normal organs such as heart served well as sources of the Wasserman antigen used i n the complement f i x a t i o n test for the diagnosis of s y p h i l i s . A l l of these observations then led to the finding of a strongly reacting, organ-specific l i p i d hapten in brain tissue ( 3 ) . Comparatively l i t t l e i s known concerning the immunology of microbial l i p i d s . Also, many c o n f l i c t i n g reports have been made concerning t h e i r a b i l i t y to act as antigens ( 7 ) . However, i t i s well known that l i p i d s play a role in the virulence of the tubercle b a c i l l u s (4), and other observations have shown that l i p i d materials are associated with the virulence of - 2 -certain mycotic pathogens such as Blastomyces dermatitidis ( 1 0 ) . Comparatively l i t t l e work has been done on l i p i d s of dermatophytes and that which has been reported deals mostly with the characterization of the l i p i d components of the t o t a l l i p i d extract of dermatophytes. Prince (20) has reported on the types of l i p i d found in a s t r a i n of Trichophyton mentagrophytes. Audette et a l (2) presented a quantitative analysis by gas l i q u i d chromatography of f a t t y acids in Trichophyton mentagro-phytes . Wirth et a l (32) reported the presence of brassicasterol and ergosterol in several strains of Trichophyton rubrum. Wirth et a l (31) have also presented a quantitative analysis of the t o t a l f a t t y acids in Trichophyton rubrum by gas l i q u i d chroma-tography. Other workers have been interested in the quantitative studies of l i p i d s in fungi. Al-Doory and Larsh ( l ) , (9) have determined the percentages of acetone-soluble and insoluble l i p i d s contained in 14 species of human pathogenic fungi, 10 of which were dermatophytes. The l i p i d content was found to range from approximately ten to f o r t y percent, on a dry weight basis. Swanson et a l (27) showed that the l i p i d content of Microsporum  quinckeanum mycelia and spores ranged from approximately 13 to 23 percent. The l i p i d content can range from a very low amount to a high content depending on the environment. For instance, the type of carbon source available can influence l i p i d content greatly. When grown on i n u l i n , l i p i d content was found to be - 3 -approximately 5$ on a dry weight basis, whereas on glucose, the l i p i d content was found to be approximately 16$. Prom the l i t e r a t u r e review to date, no reference has been made to the possible role of l i p i d s in animal immune responses. L i t t l e work has been done in this area of investigation. Tomomatsu (29) prepared somatic dermatophyte antigens by two methods, and then separated c e l l constituents into fractions termed as crude poly-saccharide, crude protein, and crude l i p i d . These three f r a c t i o n s , as well as the watery extract were tested in rabbits previously infected with Trichophyton mentagrophytes var asteroides by Bloch's method. As a r e s u l t of these te s t s , he demonstrated a positive hypersensitive reaction to the crude l i p i d extract. Later (30), he again showed a s l i g h t skin test reaction with l i p i d used as antigen intradermally injected into a previously infected animal. Recent experiments on brain l i p i d s have shown that they can also act as haptens ( 1 8 ) . It i s known that dermatophytes are composed of a com-paratively large l i p i d content, and that most s u p e r f i c i a l dermatomycoses produce cutaneous hypersensitivity. Therefore, the purpose of this investigation was to i s o l a t e , purify and fractionate the l i p i d of Microsporum quinckeanum, and to investigate these constituents for their possible role in the hypersensitive response. -4-MATERIALS AND. METHODS STOCK CULTURES Subcultures of Mlcrosporum quinckeanum, s t r a i n #8 (referred as M.Q8 hereafter) and Microsporum gypseum (from F. Blank, Department of Dermatology and Microbiology, Temple University, Philadelphia, Pa.) were maintained on 4$ Sabouraud's glucose agar in Roux flasks grown at 25° for seven days. Subcultures could then be stored up to a period of three months under r e f r i g e r a t i o n at 4°. Inoculum Microconidia were harvested by shaking the Roux flasks containing 20 ml of s t e r i l e 0 .85$ saline and approximately 10 gm of s t e r i l e glass beads of 5mm diameter. The microconidia were prepared as an inoculum by f i l t r a t i o n through a s t e r i l e , coarse, sintered glass funnel (300 ml), observing aseptic techniques. Following f i l t r a t i o n , the microconidia were washed three times by centrifugation (12 ,000 x g for 15 min.) using s t e r i l e 0.85$ saline and observing aseptic techniques. Following the washing procedure, the microconidia were resuspended in s t e r i l e 0.85$ saline to give an absorbance of .840 at 610 mu (Spectronic 20, 1.2 cm l i g h t path). Medium Sabouraud's glucose medium Glucose 40 gm Neopeptone (Difco) 10 gm Agar (Difco) 16 gm Tap Water 1,000 ml pH 5*8 6.0 using 1 N HC1. S t e r i l i z e d by autoclaving at 121°C f o r 20 minutes.' CULTURES FOR LIPID EXTRACTION 1. S t a t i c cultures: Mycelia f o r the extraction of l i p i d were grown in 4$ Sabouraud's glucose l i q u i d medium in 1000 ml flasks containing 150 ml of medium. One ml of the microconidial suspension per 100 ml of medium was used as inoculum. These cultures were incubated at 25°C for 21 days. 2. Fermenter Cultures: Mycelia for l i p i d extraction were also grown in a V i r t i s laboratory fermenter. A 12 l i t e r fermenter jar containing 10 l i t e r s of Sabouraud's k% glucose l i q u i d medium was employed for each batch. One ml of micro-conidia, o p t i c a l density of .840 at 610 mp, per 100 mis of medium was used as inoculum. The cultures were grown for 6 days at 25°C with constant aeration and a g i t a t i o n . Agitation was maintained at 250 rpm on the fermenter rheostat. T r i b u t y l c i t r a t e was used as the foam control agent applied automatically as required. -6-Harvesting of Mycelia Mycelia from both s t a t i c cultures and fermenter cultures were separately harvested on-a-Buchner-funnel using Whatman #41 f i l t e r paper. Afte r separation from-the medium, the mycelial mass was washed separately-with large amounts of- d i s t i l l e d water. Following this step-, • the • mycelial-mat was semi-dried by vacuum f i l t r a t i o n . The semi-dried mycelia were weighed and a portion was employed for dry weight determination of the t o t a l mycelial mass. Dry Weight Determination Wet mycelial elements were put into pre-dried (110°C for 12 hrs.), tared aluminum pans. The pans and mycelia were then heated at 110°C for 2-3 hrs., transferred to evacuated desiccators containing CaCl 2* where they were stored for 24 hrs. at room tem-perature. The pans containing the dried mycelia were then re-weighed . Extraction of Lipids The mycelial mass was homogenized using a V i r t i s homogen-izes employing chloroform: methanol (2:1 v/v) as the solvent. About 75 mis of solvent were used per 100 gm of mycelial mass. The solvent was then separated from the homogenate using a Buchner funnel and Whatman #41 f i l t e r paper. The solvent volume was measured and made up to 250 ml. The solvent and mycelial mass were then put into a Soxhlet extractor whereby the l i p i d s were - 7 -extracted using the method of Al-Doory and Larsh ( l ) with one modification. Instead of using 36 hr. extractions at 65°C, 2k hr. extractions were found to y i e l d the same results and were therefore employed. Prior to p u r i f i c a t i o n contaminants such as amino acids, carbohydrates, and glucosamines were detected by thin layer chromatography. P u r i f i c a t i o n of Extracted Lipids The l i p i d extract was pu r i f i e d by the "washing,technique" of Folch, Lees and Stanley ( 1 2 ) . The method involves thorough mixing of the l i p i d extract with 0 .2 (v/v) of i t s volume of water to which d i f f e r e n t mineral s a l t s may be added.- A biphasic system i s obtained. The upper phase, contains a l l the non-lipid sub-stances and the l i p i d i s contained In the lower phase. The washed l i p i d was concentrated by removing the solvent by fl a s h evapor-ation and then resuspending the l i p i d extract in a reduced volume of chloroform: methanol (2:1 v/v). Thin Layer Chromatography 1. Preparation of plates S i l i c a gel G (According to Stahl) with CaSOij was pre-washed with chloroform: methanol (1:1 v/v) employing the method of Thompson and Kies ( 2 8 ) . • 35 gm of the pre-washedvsilica gel were s l u r r i e d with 70 ml of d i s t i l l e d water and -transferred'to a Camag applicator. A s i l i c a gel layer 0 . 3 mm thick was used. The plates (20 x 20 cm) prepared in the usual "manner (21), ' were allowed to -8-dry at room temperature for several hours (or overnight) and then dried in an oven at 110°-115°C for approximately 0 .5 hr. and cooled. They were then stored in a desiccator over CaClg. Before use the plates were activated at 110°-115°C for 0 .5 hr. just prior to the application of samples. They were allowed to cool and samples were then applied. The pre-washing was necessary to remove organic material present in the s i l i c a gel which otherwise formed a wide, dark band at the solvent front a f t e r the detecting reagent was applied. 2. Application of Samples The samples in chloroform: methanol (2:1 v/v) were applied with micropipettes 2.5 cm from the bottom edge of the plates. The amounts of application ranged from approximately 100-150 pg of the t o t a l l i p i d extracted. Spots were, dried with a stream of warm a i r from a d r i e r . 3. Development of Chromatograms The chromatographic chambers (21.5 x 20.9 x 6 cm) were prepared just p r i o r to insertion of plates by placing small amounts of solvent into the chamber. The chambers were lined on three sides with Whatman #4 f i l t e r paper wetted with the devel-oping, solvent. The plates, which had about, 0 .5 cm of the adsorbent layer removed off the bottom edge, were placed in the chamber and allowed to equilibrate f o r 10-15 mins.. Then the chamber was opened at one corner and 100 ml of solvent were - 9 -slowly poured i n . The system employed was a modified solvent of Dittmer and Lester ( l l ) consisting of chloroform, methanol, pyridine, water (38:12 v/v, 2 $ , 2%) and allowed to move approxi-mately 18-18.5 Cm from the bottom of the plate. The plates were allowed to dry in a ventilated chromatography drying oven at 50°C. 4. Detection Reagents Lipids were detected by spraying the plates with a 10$ solution of phosphomolybdic acid in ethanol. The plates were then heated in a drying oven at 120°C. As a comparison, a 50$ H2SO4 solution was also employed ( 2 2 ) . Amino acids were detected by spraying the plates with a modified ninhydrin reagent containing: (A) 50 ml of a 0.2% anhydrous ethanolic nonhydrin solution, 10 ml of g l a c i a l acetic acid, 2.0 ml of 2 , 4 , 6 - c o l l i d i n e and (B) 1.0$ solution of Cu(N0g) 2. 3H 2 0 in ethanol ( 2 3 ) . 50 parts of A were mixed with 3 parts of B just p r i o r to use. Carbohydrates were detected by spraying the plates with an anisaldehyde s u l f u r i c acid reagent. The reagent was 10 ml of a fr e s h l y prepared mixture consisting of 9 .0 ml of 95$ ethanol, 0 . 5 ml cone, s u l f u r i c acid and 0 .5 ml anisaldehyde. 5 .0 drops of g l a c i a l acetic acid were also added (24). The plates were then heated at 9©°-100°c for 10 mins. As an alternative method, a n i s i -dine phthalate was also employed ( 2 4 ) . Hexosamines and N-acetylhexosamines were detected - .10 , by spraying the thin layer chromatogram with the following -reagents (14), ( l 8 ) . (a) Hexosamines . Reagent (a): 0 . 5 ml acetylacetone in 5 0 ml butanol. Reagent (b): 5 . © ml 5 0 $ NaOH in 2 0 ml ethanol. Combined 0 . 5 ml of reagent (b) and 1 0 ml of reagent (a). After spraying, the chromatogram was heated at 105°C for 5 . 0 mins., cooled, then sprayed with the following reagent. Reagent ( c ) ; 1 . 0 $ dimethylaminobenzaldebyde in 9 * 0 volumes acetone and 1 . 0 volume cone. HC1.. (b) N-acetylhexosamines The chromatogram was sprayed with reagent (b) above, heated at 105°C for 5 mins., cooled then sprayed with reagent ( c ) . 5 . Tests for purity a f t e r "washing" -After washing by the method of Polch et a l ( 1 2 ) the l i p i d extract was checked for the contaminants as outlined in the previous section except that standards were now employed. The following standards and amounts were spotted on the chromatogram in conjunction with the l i p i d extract. A. Amino acids - 0 . 0 5 M, 2 . 0 pi applied. Tryptophane Alanine Phenylalanine B. Lipids - applied - approximately 2 0 jug. -11-Oleic . Stearic Lecithin C. Carbohydrates - a p p l i e d 0 .5 pg. Lactose-Glucose • -Sucrose • D. Hexosamine - applied 50 >Ag. -E. N-acetylhexosamine - applied 50 pg. Fractionation of L i p i d Extract The l i p i d extract was fractionated by thin layer chroma-tography. 20 x 20 cm plates were employed and these were • prepared as previously outlined. Application of the material was modified from the previous procedure in that the extract was applied as a narrow s t r i p approximately 2 .0 cm from the bottom of the plate. The plates were developed in the chambers using the solvent system already outlined. Following drying, the plates were covered with aluminum f o i l except f o r a one-half inch s t r i p on either edge of the plate. The unprotected s t r i p s cf the plates were- then sprayed with phosphomolybdic a c i d in ethanol as previously outlined. Horizontal lines were drawn on the plate separating the six f r a c t i o n s . The sprayed areas were scraped off and discarded while the protected areas were scraped off separately and each f r a c t i o n placed Into a separate container. The l i p i d -12-was eluted from the s i l i c a gel with chloroform: methanol (2 : l ) , by mixing the sl u r r y with gentle shaking of a p a r t i a l l y stoppered f l a s k . The s l u r r y was centrifuged at 10,000 x g for 5 mins. at 4 ° C The supernate was poured off into s t e r i l e pre-weighed v i a l s . . The s i l i c a gel was washed twice more, a l l supernates of each f r a c t i o n pooled, and the six separate fractions were ob-tained. The solvent was driven off using a hot stream of a i r from a chromatography dryer and the v i a l s then placed over CaClg in an evacuated.desiccator overnight. The v i a l s were then re-weighed with t h e i r l i p i d contents. Marcol GX mineral o i l was now added to each f r a c t i o n so that the concentration of l i p i d -in each v i a l was brought to 10 mg/ml. The l i p i d and mineral o i l were now made into a homogeneous suspension using a Vortex mixer. Separation of Lipoprotein The l i p i d extract of mycelia obtained from chloroform: methanol resulted in a biphasic system. During washing, by the method of Polch et a l ( 1 2 ) , the top layer.was washed separately and then a l l the washed fractions were pooled. Upon reducing the volume by flash evaporating to about l / 2 0 t h of o r i g i n a l volume, i t was observed that a residue formed which was now insoluble in chloroform: methanol ( 2 : 1 ) , but was soluble in d i s t i l l e d water. I d e n t i f i c a t i o n of Lipoprotein Hydrolysis of the water-soluble complex was carried out -13-using hydrolysis v i a l s containing 1.0 ml of the complex and 2.5 ml of 6ttPiHCl. The v i a l s were sealed a f t e r evacuation of a i r by vacuum pump and hydrolysis carried out at 100°C for.. 4-6 hours. - " , Following hydrolysis, the contents of the v i a l were removed to small test tubes and the acid removed by a stream of a i r while the tubes were immersed in a hot (90°-95°C) water bath. The residue was washed 3-5 times, or as necessary to remove the HC1, and each time volume was reduced by a stream of a i r over the hot water bath. Afte r the f i n a l washing, the residue was redissolved in 0 . 5 ml of dis t i l l e d • w a t e r and ce n t r i fuged to remove any particulate matter. Subsequent to hydrolysis, paper chromatography was used in the i d e n t i f i c a t i o n of the amino acids. Twenty amino acid standards were employed. Chromatography was carried out- on Whatman #4 ( l 8 x 22 inch) paper using single and two dimen-sional chromatography according to the methods•outlined by Ivor Smith ( 2 6 ) . . For single dimensional chromatography 2 mg/ml standards were employed. 10 ul of the standard amino acids were applied while 50 ; j l of the hydrolysate was applied. For one-way chromatography the following solvents were employed: nVbutanol-acetic acid-water (60:15:25 v/v) and n-butanol-pyridine-water (60:60:60 v/v). The solvent systems for two dimensional chromatography ;were: -n-butanol-a-cetic acid-water -14-(60:15:25 v/v) in the f i r s t d i r e c t i o n , followed by phenol— ammonia (200:1)(26). Detection of the amino acids was carried out by the ninhydrin, c o l l i d i n e reagent previously outlined (see TLC, Detection reagents). Protein Determination in Lipoprotein Protein content in the lipoprotein was determined employing the method of Lowry et a l (17) and using c r y s t a l l i n e bovine albu-min as a standard protein. Detection of Phosphate in Lipoprotein The presence of phosphate in the l i p i d extracts and l i p o -protein complex was detected by spraying a thin layer chromatogram with the following mixture: 1 gm ammonium molybdate dissolved in 8 ml H 2 0, 3 ml 60$ perchloric a c i d , 3 ml cone. HC1. The above was made up to 100 ml with acetone. The mixture was sprayed over the chromatogram, dried and the chromatogram placed under UV. The presence of blue fluorescent spots i n d i -cated the presence of phosphate (26). Determination of Phospholipid in L i p i d Extract Phospholipid content in the l i p i d extract was determined by the method of Al-Doory ( l ) which involved treatment of l i p i d residue with cold acetone. Since phospholipids are insoluble in acetone, the amount can be calculated by weighing the acetone insoluble .precipitate. Preparation of L i p i d Extract f o r Immunological Studies -15-The l i p i d extract was prepared by adding 6.0 mis of Marcol-GX mineral o i l to 6.892 gms of l i p i d extract. The pur-pose was to reduce i t s v i s c o s i t y for subsequent handling and preparation as antigenic material. Preparation of L i p i d Fractions for Immunological Studies The s i x l i p i d f r a c t i o n s were suspended in Marcol-GX mineral o i l to give a concentration of approximately 10 mg/ml. Sen s i t i z a t i o n of Guinea Pigs The albino guinea pigs used in these series of experiments were 250-300 gm in weight. Sensitization of the guinea pigs was carried out by two methods. ( i ) Bloch's method - s e n s i t i z a t i o n by infecti o n (25). The method involves s c a r i f y i n g the flank of an animal with coarse sandpaper and then applying a suspension of spores and mycelia in honey. Several days are required f o r i n i t i a t i o n of infection with the peak being at about the eleventh day. After t h i s the reaction becomes less v i v i d indicating convalescence. A t o t a l of 4-5 weeks therefore i s necessary to insure that the animals have f u l l y developed the hypersensitive state. ( i i ) Subcutaneous i n j e c t i o n of 1.0 ml of mycelial-adjuvant suspension in the scapular region. Because of the l i p i d nature of the material, d i f f u s i o n i s slow, and therefore 5 weeks were allowed before challenge in order to make sure that the hypersensitive state was obtained. -16-Preparation of Spores f o r Bloch fs Method Cultures were grown in Roux flasks on Sabouraud's glucose agar as previously outlined and the spores were harvested as previously reported. The suspension of spores, hyphal elements and saline was centrifuged at 5000 x g, the supernate removed and the p e l l e t added to a small amount of honey. This suspension was then warmed to reduce v i s c o s i t y and mixed thoroughly employing a glass rod. The same pro-cedure was followed for M. quinckeanum #8 and M. gypseum. Preparation of Mycelia for Se n s i t i z a t i o n Cultures were grown in the usual manner in flasks of k% Sabouraud's glucose l i q u i d medium (previously outlined). The 21 day old mycelia were harvested a s e p t i c a l l y , washed repeatedly with d i s t i l l e d s t e r i l e water, pre-dried by vacuum f i l t r a t i o n and then l e f t to dry in an evacuated desiccator over anhydrous CaClg. The dried mycelia then were transferred to a s t e r i l e mortar, and observing aseptic techniques, the mycelia were ground to a fine powder. The ground product was then weighed and a known amount was used for suspension with adjuvant. 50 mg of powdered mycelia (approximately 1.92 mg N/ml) were suspended per 1.0 ml of Freund's incomplete adjuvant (Difeo). This procedure was repeated f o r mycelia of M. gypseum. -17-Guinea Pig Series - Routes of Sensitization and Materials The guinea pig series Involved 7 groups' of 6 animals each, and were sensitized according to the following schedule: Group #1: Spores of M.Q.8 - method of Bruno Bloch. 1 1 #2: Spores of M. gypseum - method of Bruno Bloch. " #3: As group #1 with M.Q.8 l i p i d added. •' " #4: As group #2 with M.Q.8 l i p i d added. " #5: Powdered mycelia. of M.Q.8 in Freund's incomplete adjuvant, subcutaneous injection- route. " #6: Powdered mycelia of M. gypseum in Freund's incom-plete adjuvant, subcutaneous inje c t i o n route. • " #7: Controls. Groups #5 and #6 received 1.0 ml of the suspension in the scapular region once weekly f o r a. t o t a l of three weeks. Hypersensitivity Testing - Guinea Pig Series  Preparation of Animals Five weeks following the l a s t s e n s i t i z i n g i n j e c t i o n the animals were prepared f o r hypersensitivity testing by c l i p p i n g the hair on the backs of the animals, and complete removal of hair was completed by the use of a d e p i l i t a t i n g paste (Nair) the day prior to challenge. Then the animals were challenged by intracutaneous i n j e c t i o n of 0.1 ml of the following: l i p i d extract from M.Q.8, the l i p i d f ractions and Marcol-GX mineral o i l as a contr o l . -18-Th e d e p i l i t a t e d area of the animals back was divided into 10 equal regions employing a f e l t - t i p p e d marking pen. The scheme of challenge i s shown in Pig. 1. out at |, 1, 2, 4, 6, 18, 24, 36, 48 and 72 hours a f t e r challenge i n j e c t i o n s . S e n s i t i z a t i o n of Rabbits Zealand White s t r a i n with the animals weighing from 2-3 kg. The series included 8 separate groups of 4 animals each. S e n s i t i z a -tion was carried out by i n j e c t i n g 1.0 ml of suspension in 0.2 ml quantities subcutaneously at f i v e locations. Inoculations were repeated three times at one week i n t e r v a l s . The s e n s i t i z i n g material employed for each group was the following: Pig. <L: Challenge scheme f o r guinea pig s e r i e s : A-P: Six lip-id f r a c t i o n s . X: Marcol-GX mineral o i l used for sus-pending l i p i d and l i p i d f r a c t i o n s . Observations and recording of observations were carried Rabbits used for this series of experiments were the New -19-Group 1: 3.0 ml Freund's incomplete adjuvant (Difco), 2.0 ml l i p i d suspension and 1.0 ml of s t e r i l e s a l i n e . Group 11: 4.0 ml Preund's incomplete adjuvant, 2.0 ml l i p i d and 4.0 ml l i p o p r o t e i n . Group 111: 10.0 ml Preund's incomplete adjuvant, 500 mg fine ground mycelia and 2.0 ml l i p i d suspension. Group IV: 10.0 ml Preund's incomplete adjuvant and 10.0 ml of l i p o p r o t e i n . Group V: 60 mg c r y s t a l l i n e bovine serum albumin, 60 mg (approx.) of l i p i d extract, 6.0 ml of s t e r i l e physiological saline and 6.0 ml Preund's incomplete adjuvant. Material allowed to equilibrate f o r three days pr i o r to use in s e n s i t i z a t i o n of animals. In a l l cases where adjuvant was included in the s e n s i t i z i n g material thorough homogenization of the suspensions was carried out to insure an even suspension of the antigenic material. Group VI: This group was sensitized by lipoprotein which had been s t e r i l i z e d by raillipore f i l t r a t i o n using 0.3 u porosity membrane. Sensitization was carried out by s t a r t i n g with intravenous injections of the marginal vein and l a t e r continuing s e n s i t i z a t i o n by intraperitoneal injections according to the following schedule. -20-Day 1: 0.2 ml, ear vein n 4. 0.4 ml, ear vein » 7. 0.4 ml, ear vein " 10: 0.5 ml, ear vein " 14: 1.-0 ml, ear vein " 21: 1.5 ml, intraperitoneal " 28: 1.5 ml, n Group VII: 3.0 ml Preund's complete adjuvant, 2.0 ml l i p i d suspension and 1.0 ml s t e r i l e physiological s a l i n e . S e n s i t i z a t i o n same as groups I to V. Group V I I I : These animals were control animals and therefore were not s e n s i t i z e d . Hypersensitivity Testing - Rabbit Series  Preparation of Animals Five weeks following the completion of s e n s i t i z a t i o n the animals were prepared f o r hypersensitivity testing in the same manner that was used for guinea pig d e p i l i t a t i o n . The animals were challenged by 0.1 ml intracutaneous injections of the following materials: t o t a l l i p i d extract, six separate l i p i d components, lip o p r o t e i n , Marcol-GX mineral o i l , and in Groups IV to VIII a lipid-bovine albumin-Marcol-GX emulsion was also added. The challenge scheme for the rabbit series i s shown in F i g . 2. -21 A B C D E F l i p i d l i p o l i p i d i p r o t. and • a l b . F i g . 2: Scheme used f o r c h a l l e n g i n g the r a b b i t s e r i e s : A-F: denotes l i p i d f r a c t i o n s . X: denotes Marcol-GX mineral o i l . Observations and reco r d i n g of r e s u l t s were c a r r i e d out at |, 1, 2, 4, 6, 8, 18, 20, 24, 36, 48 and 72 hours a f t e r the ch a l l e n g i n g i n j e c t i o n s were completed. Preparation of A n t i s e r a The r a b b i t s were bled 4-| weeks a f t e r the l a s t s e n s i t i z a -t i o n . The blood was allowed t o c l o t i n the col d and the serum was removed, c e n t r i f u g e d to remove any remaining c e l l s and frozen u n t i l used. Groups of animals chosen f o r t h i s purpose were from Groups I I , IV, V, V I , V I I , and V I I I as p r e v i o u s l y o u t l i n e d . Use of DMSO (d i m e t h y l s u l f o x i d e ) to S o l u b i l i z e L i p i d E x t r a c t The l i p i d e x t r a c t , p u r i f i e d and suspended i n mineral o i l as p r e v i o u s l y o u t l i n e d was added to a minimal volume of a 1/10 d i l u t i o n of DMSO. This suspension was then used as a n t i g e n i c -22-material in the gel d i f f u s i o n technique for the detection of p r e c i p i t a t i n g antibodies. Preparation of Lymph Node Extracts Sensitized and non-sensitized rabbits were s a c r i f i c e d and p o p l i t e a l and inguinal lymph nodes were excised using aseptic techniques. The excised nodes were removed to s t e r i l e p e t r i dishes which were then placed on ice and l e f t for approximately one hour. A f t e r cooling, the nodes were firm, while f a t t y and connective tissue remained s o f t . Working at ice-bath tempera-tures, the nodes were removed from the connective t i s s u e . The nodes were then homogenized with minimal d i s t i l l e d water for two minutes. The homogenate was then centrifuged at k ° C at 18,000 x g for 30 minutes. The supernate was removed and the p e l l e t was again homogenized in the same manner. The supernates were pooled and centrifuged at 4°C, 18,000 x g for one hour in c e l l u -lose n i t r a t e tubes. During this step, the l i p i d present in the homogenate concentrated as a s o l i d mass at the surface. A s t e r i l e guage 18 syringe needle with cutdown bevel was inserted in the bottom of the centrifuge tube and the contents were drained into another s t e r i l e tube, leaving the l i p i d material in the o r i g i n a l centrifuge tube. These aqueous extracts were reduced in volume by placing them in d i a l y s i s tubing at k ° C and allowing streams of a i r to blow over t h e i r surface from a fan. When only a. few mis remained, -23-the contents were emptied into s t e r i l e tubes and the volume made up to 10.0 ml using s t e r i l e .85$ s a l i n e . The procedure was repeated f o r a t o t a l of 6 animals, 1 from each group chosen. The protein content of each extract was now determined using the method of Lowry et a l (17). The animals s a c r i f i c e d here were the ones which had previously been immunized and bled for the preparation of antisera. Qualitative P r e c i p i t a t i o n in Gel Rabbit antisera and lymph node extracts were screened separately for p r e c i p i t a t i n g antibodies against l i p i d and l i p o -protein by the immunodiffusion method of Ouchterlony (5) using 0.85$ Ionagar No. 2 (Colab Laboratories, Inc., Chicago Heights, 111.) dissolved in s t e r i l e borate-saline solution with merthiolate added (1:10,000). The plates were prepared according to a pattern comprising 6 equidistant circumferential wells and 1 central well in the agar, using penicylinders. When the plates had set 0.2 ml of lipoprotein (or l i p i d ) was placed in the centre well, whereas the circumferential wells were prepared by introducing 0.2 mis of lymph node extract (or antisera) from the six d i f f e r e n t groups of rabbits used. Diffusion was allowed to take place at 30°C for 4-10 days in a humidifier. Ring or I n t e r f a c i a l P r e c i p i t i n Test Rabbit antisera and lymph node extracts were examined for pr e c i p i t a t i n g antibodies against l i p i d and lipoprotein by the use - 2 4 -of the r i n g or i n t e r f a c i a l test according to the method of Campbell et a l (6). Test tubes used for the test were 6 x 50 mm. The lymph node extracts for t h i s test were diluted 1:1 with borate saline solution (95 parts .85$ saline and 5 parts borate buffer, pH 8.4). Lipoprotein extract was used undiluted. The lipoprotein (.3 ml) was overlaid onto the lymph node extracts (0.5 ml). Observations were made at regular 10 minute inter v a l s f o r 90 minutes a f t e r which time periods were increased to 20 minutes for another two hours. A l l negative tubes were observed for periods of 18 hours before discarding. Mixtures showing negative results were tested again using d i f f e r e n t d i l u t i o n s of lipoprotein (or l i p i d ) and lymph node extracts (or a n t i s e r a ) . Electrophoresis of Lymph Node Extracts Electrophoresis was carried out on lymph node extracts employing the Gelman Electrophoresis apparatus (Rapid e l e c t r o -phoresis chamber #51101) and Sepraphore 111, a c e l l u l o s e poly-acetate support membrane (Gelman Instrument Co., Ann Arbor, Michigan). The method employed was that outlined in Manual #70176-A (Rapid Electrophoresis, Gelman Instrument Co.) "Pro-cedure for iSerum Protein Electrophoresis". The amount of each sample spotted on the c e l l u l o s e polyacetate s t r i p s was 10 yx\. The buffer used was Gelman HR buffer. Later a buffer composed of 0.1 M sodium diethyl barbiturate and 0.02 M diethyl barbituric - 2 5 -acid at pH 8 .6 was employed ( 1 6 ) . Passive S e n s i t i z a t i o n Transfer Test Eight guinea pigs were employed for this t e s t . The backs of the animals were d e p i l i t a t e d by the method previously described and then divided into 12 zones using a f e l t - t i p p e d marker. In the six areas on the right side of the animal 0.1 ml of antisera was injected intradermally. On the l e f t side of the animal, 0.1 ml of lymph node extract (the sera and the lymph node extract were from the same animal) was injected i n t r a -dermally. See F i g . 3-F i g . 3 : Scheme for the Pa,s-sive S e n s i t i z a t i o n Transfer Test. A-F denotes antisera-from 6 d i f f e r e n t rabbits. A'-F' de-notes lymph node extracts corresponding to the antisera. Five hours l a t e r , the animals were Injected i n t r a -dermally with 0.1 ml of l i p i d or lipoprotein near the previous i n j e c t i o n s i t e s where antisera and lymph node extracts were -26-introduced. Pour of the eight animals were challenged with l i p i d extract and the other four were challenged with l i p o -protein. Observations were carried out at 15 min. intervals for the f i r s t 2 hours and then at 30 min. intervals f o r another 6 hours. RESULTS AND DISCUSSION Lip i d Extraction and P u r i f i c a t i o n Extraction of l i p i d from the mycelia of Microsporum  quinckeanum, s t r a i n #8, grown under s t a t i c and submerged fermentation conditions revealed, a f t e r p u r i f i c a t i o n , that the t o t a l l i p i d content of the mycelia was 16$ on a dry weight basis regardless of growth techniques when cultivated in the same medium. This value i s well within the range of values reported, by various workers, f o r dermatophytes and other fungi. Values from 1.0 to kO.0% have been reported ( l ) , (9). Some variation in l i p i d content was expected, since this could ar i s e as a re s u l t of di f f e r e n t methods of extraction. It was assumed that the procedure used would produce maximum extraction, as there i s no assurance that any one method w i l l extract l i p i d completely from fungal mycelia. Tests carried out on the gross l i p i d extract f o r amino acids, carbohydrates and hexosamines revealed that these were present in r e l a t i v e l y large amounts. However, when the l i p i d extract was p u r i f i e d , no detectable amounts of these "common11 -27-contaminants were observed. Thin layer chromatography was employed as a test for purity, since this was the method of choice, due to the s o l u b i l i t y problem of l i p i d s . The use of standards revealed that this method was v a l i d as a means of testing for purity of the l i p i d extract. Chemical assays for the detection of protein or carbohydrate could not be used owing to the lack of s o l u b i l i t y of l i p i d s in aqueous reagents, and in assays where acids were involved, such as the Anthrone reaction, charring of the l i p i d occurred. Thin layer chromatography of the pur i f i e d l i p i d extracts from both s t a t i c and submerged fermentation cultures revealed that s i x f r a c t i o n were present, and that these were i d e n t i c a l under both conditions of mycelial growth when using the same medium (Fi g . 4). The presence of phosphate was detected in Fraction A, and i t was shown to be a phospholipid since the spot fluoresced under u l t r a v i o l e t l i g h t . When known l i p i d s were used on TLC, the Rf of Fraction A and l e c i t h i n were almost i d e n t i c a l . It was therefore assumed that Fraction A could be a l e c i t h i n or a l e c i t h i n - l i k e material. When the six l i p i d fractions were separated by the use of TLC and subsequently eluted, tests for the presence of con-taminants showed no detectable amino acids, carbohydrates or hexosamines to be present. It was therefore concluded that - 2 8 -the "washing" procedure employed was e f f e c t i v e , and that the separation on TLC of the pu r i f i e d l i p i d provided an additional step in i t s p u r i f i c a t i o n . Subsequently, TLC and elution were employed as a means of obtaining the six l i p i d fractions in separate and pu r i f i e d amounts large enough f o r further experi-mentation. The "washing" procedure used yielded a compound which was i d e n t i f i e d as a lipoprotein complex by demonstrating the presence of proteinaceous material, l i p i d and phosphate. According to Hanahan (13) > lipoprotein possesses these con-stituents in various concentrations, the phosphate most l i k e l y a phospholipid. Subsequent protein determinations demonstrated a protein content of 16.45$ on a. dry weight basis. The use of the phosphate detecting spray revealed the presence of a small amount of phosphate which was most l i k e l y a phospholipid. TLC and a detecting spray for l i p i d also revealed the presence of l i p i d material. Acid hydrolysis of the lipoprotein followed by paper chromatography ( F i g . 5 , single and two dimensional using standard amino acids) and several d i f f e r e n t solvent systems showed the presence of the following eleven amino acids: cysteine, cystine, aspartic, glutamic, serine, glycine, threo-nine, h i s t i d i n e , l y s i n e , arginine and proline. Cystine appeared in very low concentrations while proline appeared in - 2 9 -solvent front 4 E A 4 0 * 4 : * A A i F i g . 4 : Thin layer chromatograph of-M.Q.#8 L i p i d . L e f t : s t a t i c culture; r i g h t : submerged, fermentation culture. Solvent: chloroform-: methanol (38:12 v/v) 2% pyridine, 2$ d i s -t i l l e d water. ( x | of o r i g i n a l ) . -30-E < O-i OJ l l proline f arginine £ lysine | h i s t i d i n e 1 « sei glycine threonine serine cystine cysteine glutamine aspartic #1 BuA Pig. 5: Amino acids of M.Q.#8 lipoprotein as demonstrated by paper chromatography, - 3 1 -comparatively large concentrations. I d e n t i f i c a t i o n of the amino acids was aided by the use of standards in conjunction with the test material and a detecting spray which results in each amino acid having its. own ch a r a c t e r i s t i c color. Skin Test Reactions A n t i g e n i c i t y of the six l i p i d f r a c t i o n s , the t o t a l l i p i d , and the lipoprotein was demonstrated by means of skin test observations carried out on sensitized guinea pigs and rabbits. Guinea pigs were sensitized with both Microsporum  quinckeanum s t r a i n #8 (M.Q.8) and Microsporum gypseum. In two series of animals, extra M.Q.8 l i p i d was added to the spore-honey suspension for Bloch's method (25) of s e n s i t i z a t i o n to determine If i t s presence would provide an aggravated inflam-matory response in the infectious process. Sensitization;pro-cedures and the results f o r the guinea pig series are outlined in Table 1. Results revealed that a hypersensitive reaction occurred in a l l cases where animals were sensitized by some method, while the controls showed no reaction. The challenge material in a l l cases was the s i x individual l i p i d f r a c t i o n s , the t o t a l l i p i d extract and the lipoprotein f r a c t i o n (Table 1 ) . Regardless of the s e n s i t i z i n g agent, whether i t was an active i n f e c t i o n or whether i t was ground mycelia in adjuvant, upon skin test challenge the reactions obtained were of a delayed hypersensitive response, and a l l sensitized animals reacted in Table 1 Hypersensitivity Reactions as Demonstrated by Guinea Pig Skin Tests GROUP SENSITIZING MATERIAL AND NO. .. . AND USED ROUTE OP SENSITIZATION CHALLENGING MATERIAL RESULTS 1 (6) Spores of M.Q.8, method of Bruno Bloch 2 (6) Spores of M. gypseum, method of Bruno Bloch 3 (6) As group 1 with M.Q.8 l i p i d added 4 (6) As group 2 with M.Q.8 l i p i d added 5 (6) Powdered mycelia of M.Q.8 in Freund's, Subcutaneous 6 (6) Powdered mycelia of M. gypseum in Freund's, SUbcutaneous M.Q.8 l i p i d components (6) Total l i p i d , lipoprotein As above As above As above As above As above Pos i t i v e , necrosis to l i p i d and lipoprotein As above As above i IV) I As above Positive f o r a l l com-ponents As for group 5 7 (6) CONTROLS, no s e n s i t i z a t i o n As above Negative - 3 3 -a s i m i l a r manner and to a similar degree of r e a c t i v i t y . It becomes obvious from the results obtained that the l i p i d and lipoprotein participate in the induction of a hypersensitive state. The addition of t o t a l , p u r i f i e d l i p i d to the spore suspension did not appear to Increase the degree of r e a c t i v i t y as had been anticipated i f the l i p i d had been toxic or possessed the a b i l i t y to enhance degree of immunogenic r e a c t i v i t y . Necrosis was observed in areas where the animals were challenged with the t o t a l l i p i d extract and the li p o p r o t e i n . This may sug-gest that either the concentration of the l i p i d fractions was too low to produce a severe enough reaction or that the necrosis was not due to the l i p i d but rather to a l i p i d - p r o t e i n complex and therefore necrosis was due to the protein moiety. Figure 6 shows representative results obtained using Bloch's method of se n s i t i z a t i o n with M.Q.8, and therefore d i s -plays a homologous skin test reaction. F i g . 6 : Shows representative results obtained using Bloch's method of s e n s i t i z a t i o n with M.Q.8. - 3 4 -Figure 7 shows r e p r e s e n t a t i v e r e s u l t s again using Bloch's method of s e n s i t i z a t i o n , but s e n s i t i z a t i o n was made with a v i r u l e n t s t r a i n of M. gypseum. Therefore a lack of immuno-specificity has been demonstrated by the l i p i d and l i p o p r o t e i n e x t r a c t s obtained from two d i f f e r e n t Microsporum speci e s . Since r e p o r t s have been made that d i f f e r e n t f u n g i possess the same l i p i d s ( 2 ) (31) t h i s observation i s not unusual that c r o s s - r e a c t i v i t y can occur. This i s a l s o i n accordance with r e p o r t s which s t a t e that once the a l l e r g i c s t a t e ( h y p e r s e n s i t i v e s t a t e ) has been created i t can be demonstrated by various methods, such as the a p p l i c a t i o n of an emulsion of k i l l e d f u n g i , or s t i l l more simply by the intracutaneous i n j e c t i o n of a crude a l l e r g e n i c e x t r a c t such as T r i c h o p h y t i n or M i c r o s p o r i n . These observations correspond F i g . 7: Shows re p r e s e n t a t i v e r e s u l t s obtained using Bloch's method of s e n s i t i z a t i o n with a v i r u l e n t s t r a i n of M. gypseum. For challenge scheme f o r guinea pig s e r i e s r e f e r to methods, F i g . 1. - 3 5 -c l o s e l y to those obtained in the present series of te s t s . One obtains therefore, inflammatory reactions with the sensitized animal. Similar results were again shown by animals sensitized with ground mycelia in adjuvant employing M.Q.8 and M. gypseum (Fig . 8). The control animals showed no reaction upon challenge as demonstrated in F i g . 9» It i s to be noted that in a l l the res u l t s obtained in thi s series, when skin test challenge of sensitized animals was made with t o t a l l i p i d component and with lipoprotein f r a c t i o n , a necrotic reaction was obtained. No such reaction was obtained for the six in d i v i d u a l , p u r i f i e d l i p i d f r a c t i o n s . Although no necrosis was present the results were nevertheless positive and indicated either a weaker response or a di f f e r e n t type of immu-nologic reaction than to the t o t a l l i p i d . F i g . 8: Results obtained from animals sensitized with ground mycelia in adjuvant employing M.Q.8 and M. gypseum. - 3 6 -F i g . 9: Results obtained when control animals were challenged. No v i s i b l e reactions were obtained to either of the six l i p i d f r a c t i o n s , the t o t a l l i p i d extract or the l i p o p r o t e i n . Skin Test Reaction, Rabbit Series Preliminary observations with the use of rabbits showed that the skin test reactions seemed to provide as sensitive a means of hypersensitivity reaction detection as did guinea pigs. As outlined in Table 11, rabbits were sensitized in groups of four with the complete l i p i d extract by various routes, and other groups of four by two di f f e r e n t routes with the l i p o -protein extract. It may be noted that another group of animals was sensitized with a suspension of lipid-bovine albumin which was emulsified and allowed to remain at room temperature for three days prior to use in s e n s i t i z a t i o n . The purpose of this type of s e n s i t i z a t i o n was to determine whether l i p i d acted as a Table II Hypersensitivity Reactions as Demonstrated by Rabbit Skin Tests GROUP SENSITIZING MATERIAL AND NO. AND USED ROUTE OF SENSITIZATION 1 (4) L i p i d from M.Q.8, incom-plete Freund's adjuvant; subcutaneous 2 (4) Li p i d from M.Q.8, l i p o -protein, incomplete Freund's adjuvant; sub-cutaneous 3 (4) L i p i d from M.Q.8, dried mycelia from M.Q.8, incom-plete Freund !s adjuvant; subcutaneous 4 (4) Lipoprotein and Freund's adjuvant; subcutaneous 5 (4) L i p i d , bovine albumin and-incomplete Freund's adju-vant; subcutaneous 6 (4) Lipoprotein, intravenously and i n t r a p e r i t o n e a l l y 7 (4) L i p i d and Freund's adju-vant; subcutaneous 8 (4) CONTROLS, no s e n s i t i z a t i o n CHALLENGING MATERIAL Li p i d components ( 6 ) , t o t a l l i p i d , l i p o p r o t e i n , Marcol-GX As above As above As above plus a- lipid-bovine albumin-Marcol-GX emulsion As in Group 4 As in Group 4 As in Group 4 RESULTS Po s i t i v e , necrosis to l i p i d and l i p i d protein As above As in Group 4 As above Pos i t i v e , necrosis to lipoprotein P o s i t i v e , necrosis to lipid-albumin emulsion Pos i t i v e , l i g h t necrosis to lipoprotein Positive, necrosis to l i p i d , l i p o p r o t e i n , l i p i d albumin emulsion No reactions i oo I - 3 8 -b e t t e r antigen i n presence of a c a r r i e r p r o t e i n . Niedieck et a l ( 1 8 ) , while working with myelin l i p i d hapten, reported that i n the presence of c a r r i e r p r o t e i n (bovine serum albumin) myelin cerebroside produced an t i b o d i e s i n r a b b i t s which would p r e c i p i t a t e with c e r e b r o s i d e - c h o l e s t e r o l - l e c i t h i n emulsions. Animals which did not receive i n j e c t i o n s with c a r r i e r p r o t e i n but c h o l e s t e r o l alone did not produce an t i b o d i e s which would p r e c i p i t a t e with t h i s a n t i g e n i c complex. Their r e s u l t s i n d i -cate that cerebroside i s a hapten according to the d e f i n i t i o n given by Landsteiner ( 8 ) . P i g . 10: Representative r e s u l t s observed i n r a b b i t groups 1-4, and groups 6, 7. For c h a l -lenge scheme i n the r a b b i t s e r i e s r e f e r to Fig.2 . S e n s i t i z a t i o n procedures and the r e s u l t s f o r the r a b b i t s e r i e s are o u t l i n e d i n Table I I . Representative r e s u l t s of groups 1 to 4 and of groups 6 and 7, e n t a i l i n g 24 animals can be observed i n F i g . 10. A l l animals i n these groups showed a -39-delayed type of hypersensitive reaction to the six individual l i p i d components, the l i p i d and the l i p o p r o t e i n . However, at about 24 hours a f t e r challenge, necrosis was observed at the t o t a l l i p i d and the lipoprotein challenge s i t e s and t h i s pro-gressively increased in i n t e n s i t y u n t i l 36 hours, and then slowly diminished. In group 6, however, there was no necrosis to the t o t a l l i p i d as well as the individual l i p i d f r a c t i o n s . This could be expected, since the animals in this group were sensitized with lipoprotein alone. Since a positive reaction without necrosis was nevertheless obtained i t may be assumed that either the l i p i d component i s somewhat common to the t o t a l l i p i d extract or that l i p i d induced reactions are not s p e c i f i c and that variable c r o s s - r e a c t i v i t y occurs. The necrotizing a c t i v i t y in t h i s case may also be due to an immunologic s p e c i f i c protein moiety. Pig . 11 shows t y p i c a l results obtained for group 5 observed at 23 hours a f t e r challenge exemplary of a l l four animals in this group and depicts an increase in necrosis when l i p i d was added to bovine albumin prior to s e n s i t i z a t i o n . These results seem to indicate that in the presence of albumin, the l i p i d may complex with the protein and therefore act as better antigenic material than when used alone. These res u l t s support the observations obtained by Niedieck et a l (18). F i g . 12 shows t y p i c a l r e s u l t s of skin test challenge using a non-40-sensitized or control rabbit demonstrating absence of immu nologic r e a c t i v i t y . F i g . 11: Results obtained for group 5 observed 28 hours a f t e r challenge. Note the increase in necrosis to the lipid-bovine alrbumin emulsion. Sensitization induced by lipid-bovine albumin emulsion. F i g . 12: Results obtained when a non-sensitized control animal was challenged in the usual manner. -41-Prom the results presented u n t i l now, delayed hyper-s e n s i t i v i t y reactions were obtained with a l l skin test challenge antigenic materials in sensitized animals. Since the control animals showed no reactions, i t was concluded that sensitized animals when challenged displayed a delayed hypersensitive reaction due to the presence of antibodies produced against an antigenic determinant s i t e conferred by the l i p i d i t s e l f , the lipoprotein and/or lipid-serum protein complexes. An immediate hypersensitive response was not demonstrated. Necrosis, how-ever, was obtained at the lipoprotein challenge s i t e . Since a l l the immunologic reactions obtained so f a r appeared to be of the delayed hypersensitive type, a.ntisera was obtained from representative animals of the various groups in an attempt to demonstrate the presence or absence of c i r c u -l a t i n g antibodies. Antisera were obtained from groups 2, 4, 5, 6, 7 and 8 as outlined in Table I I . As l i p i d s are insoluble in aqueous solutions, a problem arose when an attempt was made to test antisera for antibodies against the l i p i d and l i p i d f r a c t i o n s . The lack of s o l u b i l i t y was overcome by the use of dimethyl sulfoxide (DMSO). In an attempt to test for Inter-ference from DMSO in gel d i f f u s i o n reactions, known antigen-antibody systems were treated with DMSO and tested by gel di f f u s i o n techniques outlined in methods. The known immuno-logic systems evaluated were: -42-Beef serum: Rabbit antibeef serum Horse serum: Rabbit antihorse serum Bean plant v i r u s : Rabbit bean plant virus antiserum The presence of DMSO in these systems did not interfere with the reactions and very clear p r e c i p i t i n lines were obtained. Subsequently rabbit antisera. obtained from aforementioned se n s i -tized groups were tested against the t o t a l l i p i d , , the l i p i d f r a c t i o n s , a.nd lipoprotein by the gel d i f f u s i o n technique. No p r e c i p i t i n l i n e s were obtained by this method even a f t e r 10 days in a humidifying chamber. It was therefore concluded at th i s stage that the system was either not a precipitable system, or the l i p i d could not act as a p r e c i p i t a t i n g antigen without serum protein, or that there were no c i r c u l a t i n g antibodies in the antisera. The surprising observation was that there -did not appear to be any p r e c i p i t a t i n g antibodies against the l i p o -protein, since antisera is sometimes used to test for l i p o -protein classes (14). However, since the reaction to l i p o -protein was also of the delayed type i t was thought that' there were no c i r c u l a t i n g antibodies present. Since delayed hypersensitive responses were obtained, and since in such responses i t has been shown that i n j e c t i o n of lymphocytes and other nucleated c e l l s from the blood and lymphoid tissue of a sensitive individual w i l l confer, - 4 3 -passively, the same delayed-type hypersensitivity on the r e c i p i e n t , i t was thought that any antibodies that were present were " t i s s u e - f i x e d " in the lymphoid ti s s u e . In an attempt to demonstrate either the presence or absence of such " t i s s u e - f i x e d " antibodies the following groups of rabbits were chosen. Groups 2, k, 5> 6, 7* and S as out-lined in Table I I . The lymph node extracts were examined fo r protein content by the Lowry method (17). The results are shown in Table I I I . Table III Protein content as demonstrated by the Lowry method in the lymph node extracts obtained. One animal per group was used to obtain the extract. Group No. Protein(mg/ml) 2 6.00 4 3.20 5 2.25 6 3.50 7 5.00 8 6.60 P o p l i t e a l and inguinal lymph node extracts were examined by electrophoretic techniques on c e l l u l o s e acetate s t r i p s and only very s l i g h t differences in band formation were observed when compared to the controls. Consequently the p r e c i p i t i n r i n g test was chosen in an attempt to elucidate whether antibodies were present in these extracts. Lipoprotein e x t r a c t s were o v e r l a i d onto s o l u t i o n s of lymph node e x t r a c t s from the s i x animals o u t l i n e d and the r e s u l t s obtained can be seen i n F i g . 13 showing strong p r e c i p i t i n r e a c t i o n s , except where the lymph node e x t r a c t obtained from a n o n - s e n s i t i z e d animal was employed. F i g . 13: Results of r i n g p r e c i p i t i n t e s t when l i p o p r o t e i n was o v e r l a i d onto lymph node e x t r a c t s . The numbers of the tubes correspond to lymph node e x t r a c t s of animals s e n s i -t i z e d i n the f o l l o w i n g : 1, i n t r a p e r i t o n e a l l y with l i p o -p r o t e i n ; 2, subcutaneously with l i p o p r o t e i n i n complete Freund's adjuvant; 3> subcutaneously with Freund's incom-ple t e adjuvant with l i p i d and bovine serum albumin; 4, subcutaneously with Freund's incomplete adjuvant with l i p i d and l i p o p r o t e i n ; 5, c o n t r o l : non s e n s i t i z e d ; 6, not shown, s e n s i t i z e d to l i p i d contained i n complete Freund's adjuvant, subcutaneously. From the r e s u l t s obtained, i t appears that p r e c i p i t a t i n g a n t i bodies were present i n the lymphoid t i s s u e against the l i p o -p r o t e i n . A s i m i l a r t e s t was attempted using l i p i d and the s i x l i p i d f r a c t i o n s , but due to the i n s o l u b i l i t y of the l i p i d m a t e r i a l no r e s u l t s were obtained. When, i n the presence of DMSO, -45-s o l u b i l i t y was achieved but no r e s u l t s were observed. I t was thus concluded that e i t h e r t h i s was not a p r e c i p i t a t i n g system (no c a r r i e r animal p r o t e i n present) or there was i n t e r f e r e n c e from the DMSO. A lack of lymphoid-bound an t i b o d i e s was not considered since a l l s k i n t e s t challenges produced very strong r e a c t i o n s i n a l l s e n s i t i z e d animals as p r e v i o u s l y shown. I t was concluded that t h i s system was not a p p l i c a b l e to the m a t e r i a l s i n v o l v e d . To f u r t h e r demonstrate the presence of lymphoid con-tained a n t i b o d i e s against the l i p o p r o t e i n , the Ouchterlony g e l d i f f u s i o n technique i n agar was employed. The center w e l l contained the l i p o p r o t e i n e x t r a c t and the c i r c u m f e r e n t i a l w e l l s contained various sources of lymph node e x t r a c t s as p r e v i o u s l y o u t l i n e d . The r e s u l t s of t h i s t e s t are shown i n F i g . 14. F i g . 14: Ouchterlony g e l d i f f u s i o n employing lymph node e x t r a c t s i n c i r c u m f e r e n t i a l wells and l i p o p r o t e i n e x t r a c t i n the center w e l l . Numbers 1-6 r e f e r to the sources of e x t r a c t s as o u t l i n e d f o r F i g . 13. -46-I t appears, t h e r e f o r e , that cell-bound a n t i b o d i e s are present against the l i p o p r o t e i n i n lymph node e x t r a c t s as demonstrated by the previous t e s t s . Since a h y p e r s e n s i t i v e response of the delayed type was observed i n a l l animals s e n s i t i z e d , f u r t h e r experiments were attempted to e l u c i d a t e the presence of cell-bound a n t i -bodies. The passive t r a n s f e r technique was attempted i n which a n t i s e r a and corresponding lymph node e x t r a c t s were se p a r a t e l y i n j e c t e d i n t r a d e r m a l l y . These s i t e s then were challenged with l i p i d and l i p o p r o t e i n (4 animals i n each group). The r e s u l t s to these t e s t s can be observed i n P i g s . 15 and 16. P i g . 15: Passive t r a n s f e r t e s t . Rt. side of animal contains intradermal i n j e c t i o n of sera corresponding to lymph node e x t r a c t s as p r e v i o u s l y o u t l i n e d . L t . side contains lymph node e x t r a c t s as p r e v i o u s l y o u t l i n e d . Challenged with l i p o p r o t e i n . - 4 7 -P i g . 16: Passive t r a n s f e r t e s t . Rt. side of animal contains intradermal i n j e c t i o n of sera corresponding to lymph node e x t r a c t s as p r e v i o u s l y o u t l i n e d . L t . side contains lymph node e x t r a c t s i n j e c t e d i n t r a d e r m a l l y as p r e v i o u s l y o u t l i n e d . Challenged with l i p i d . These r e s u l t s i n d i c a t e that lymphoid tissue-bound a n t i -bodies were present i n the lymph node e x t r a c t s as demonstrated by the passive t r a n s f e r of a n t i b o d i e s to non-sensitized animals, and that they were present not only to l i p o p r o t e i n as p r e v i o u s l y demonstrated by g e l d i f f u s i o n and p r e c i p i t i n r i n g t e s t s but were a l s o present against the t o t a l l i p i d e x t r a c t . I t i s thought therefore that they were not demonstrable against the l i p i d by other t e s t s , as e i t h e r the system was not a p r e c i p i t a t i n g one, or c a r r i e r p r o t e i n s are necessary. Since the s i t e of the animal where serum was i n j e c t e d i n t r a d e r m a l l y produced no r e a c t i o n , i t i s concluded that the r e a c t i o n s observed are due to passive t r a n s f e r of lymphoid-bound a n t i b o d i e s and not due to any t o x i c e f f e c t s of the l i p i d or the l i p o p r o t e i n . -48-SUMMARY Under the conditions of the experiments carried out the observations made may be summarized as follows: the l i p i d content of Microsporum quinckeanum s t r a i n #8 i s 16$ regardless of growth techniques when the same medium i s used. A l i p o -protein complex was also present regardless of growth techniques. Whether grown as s t a t i c culture or as submerged culture in a fermenter, the l i p i d components were the same as demonstrated by thin layer chromatography. Acid hydrolysis of the l i p o -protein revealed the presence of eleven amino acids, namely, cysteine, cystine, aspartic, glutamic, serine, glycine, threonine, h i s t i d i n e , l y s i n e , arginine and proline. Positive skin test reactions were obtained for the l i p i d extract and for the ind i v i d u a l l i p i d components regardless of the route of s e n s i t i z a t i o n . Lack of immunospecificity for the l i p i d complex was also demonstrated. The addition of bovine albumin to the l i p i d complex resulted in a stronger skin test reaction when challenged with the same material. Since non-sensitized control animals demonstrated no reaction upon challenge with the l i p i d or lipoprotein i t appears that the pur i f i e d l i p i d does act either as an antigen or as a hapten. Serum protein may act as a c a r r i e r of the haptenic moiety. There is a lack of immunospecificity as shown by the results when lipoprotein was used as the antigenic material. Species s p e c i f i c a n t i -- 4 9 -genicity does not appear to be present in this c e l l u l a r f r a c t i o n which has been demonstrated for the f i r s t time with this organism. Cell-bound antibodies were present to the lipoprotein as demon-strated by the ring p r e c i p i t i n t e s t , the gel d i f f u s i o n technique and the passive transfer technique. It is concluded that the same i s true f o r the l i p i d complex as demonstrated by the results obtained by the skin test reactions, however, because of the lack of s o l u b i l i t y of these compounds this could not be demonstrated in any other manner. -50-BIBLIOGRAPHY 1. Al-Doory, Y., and H. W. Larsh. 1962. Quantitative studies of t o t a l l i p i d s of pathogenic fungi. Appl. Microbiol. 10: 192-195. 2. Audette, R. C. S., R. M. Baxter, and G. C. Walker. 1961. A study of the l i p i d content of Trichophyton menta- grophytes. Can. J. Microbiol. 7: 2b2-2«3. 3. Brady, R. 0 . , and E. G. Trams. 1964. The Chemistry of L i p i d s . Ann. Rev. Biochem. 33: 75-82. 4. Burrows, W. 1963. Textbook of Microbiology. W. B. Saunders Co.: 750-751. 5. Campbell, D. H., J . S. Garvey, N. E. Cremer, and D. H. Sussdorf. 1963. Methods in Immunology. W. A. Benjamin, Inc.: 143-149. 6. Campbell, D. H., J. S. Garvey, N. E. Cremer, and D. H. Sussdorf. 1963. Methods in Immunology. W. A. Benjamin, Inc.: 131-135. 7. Carpenter, P. L. 1965. Immunology and Serology. W. B. Saunders Co.: 64-65. 8. Carpenter, P. L. 1965. Immunology and Serology. W. B. Saunders Co.: p. 40. 9. Cochrane, V. W. 1963. Physiology of Fungi. John Wiley and Sons, Inc.: 45-49. 10. DiSalvo, A. F., and J. Fred Denton. 1963. L i p i d Content of Four Strains of Blastomyces dermatiditis of Different Mouse Virulence. J. B a c t e r i d . 35: 927-9"31. 11. Dittmer, J. C , and R. L. Lester. 1964. A simple s p e c i f i c assay for the detection of phospholipids on TLC. J. L i p i d Research 5: 126-127. 12. Folch, J . , M. Lees, and G. H. J . Stanley. 1957. A simple method for the i s o l a t i o n and p u r i f i c a t i o n of t o t a l lipids, from animal ti s s u e s . J . B i o l . Chem. 226: 497. 13. Hanahan, D. J . i960. L i p i d Chemistry. John Wiley and Sons: 266-267. - 5 1 -14. Hanahan, D. J . i 9 6 0 . L i p i d Chemistry. John Wiley and Sons.: 281 . 15. Kabat, E. A., and M. M. Mayer. 1961. Experimental Immune-chemistry. Charles C. Thomas.: 582-583. 16. Kabat, E. A., and M. M. Mayer. 1961. Experimental Immuno-chemistry. Charles C. Thomas.: 629. 17. Lowry, 0 . H., N.'F. Rosebrough, A. L. F a r r , and R. J . R a n d a l l . 1951. P r o t e i n measurement with t h e ' F o l i n phenol reagent. J . B i o l . Chem. 193: 265-275. 18. N i e d i e c k , B., E. Kuwert, 0 . P a l a c i o s and 0 . Drees. 1965. Immunochemical and s e r o l o g i c a l studies on the l i p i d hapten of myelin with r e l a t i o n s h i p to experimental a l l e r g i c encephalomyelitis (EAE). Ann. N.Y. Acad. S c i . 122:266-276. 19. P a r t r i d g e , S. M. 1948. F i l t e r - p a p e r p a r t i t i o n chromatog-raphy of sugars. Biochem. J . 42_: 238-253. 2 0 . P r i n c e , H. M. i 9 6 0 . T o t a l l i p i d s of Trichophyton menta-grophytes. J . B a c t e r i o l . 79.: 154. 21 . Randerath, K. 1963. Thin Layer Chromatography. Academic Press, p. 25 . 2 2 . Randerath, K. 1963. Thin. Layer Chromatography. Academic Press, p. 126-127. 23. Randerath, K. 1963. Thin Layer Chromatography. Academic Press, p. 9 4 . 2 4 . Randerath, K. 1963. Thin Layer Chromatography. Academic Press, p. 200. 2 5 . R i v a l i e r , E. i 9 6 0 . Phenomenes d'immunite dans l e s derma-t o p h y t e s . Path. B i o l . 8 : 307-312. 2 6 . Smith, I . i 9 6 0 . Chromatographic and E l e c t r o p h o r e t i c Techniques. V o l . 1. Chromatography. I n t e r s c i e n c e P u b l i s h e r s , Inc. p. 82-100. 2 7 . Swanson, R., and J . J . Stock. 1966. Biochemical a l t e r -a t i o n s of"dermatophytes during growth. A p p l . M i c r o b i o l . 14: 433-444. -52-28. Thompson, E. B., and M. W. Kies. 1965. Current studies on the l i p i d s and proteins of myelin. Ann. N. Y. Acad. S c i . 122: 129. 29. Tomomatsu, S.. 1959. A s e r o l o g i c a l study of experimental dermatomycosis using an antigen prepared from fungus" mechanically disintegrated. B u l l . Pharmaceutical Res. Inst. No. 23: 16-23. 30. Tomomatsu, S.. 19-61. A ser o l o g i c a l study of experimental dermatomycosis using an antigen prepared from fungus mechanically disintegrated. B u l l . Pharmaceutical Res. Inst. No. 35: 2-11. 31. Wirth, J . C , and S. R. Anand. '1964. The f a t t y acids of Trichophyton rubrum. Can. J. Microbiol. 10: 23-27. 32. Wirth, J . C , T. Beesley, and W. M i l l e r . 1961. The i s o l a t i o n of a unique s t e r o l from'mycelium of a s t r a i n of Trichophyton rubrum. J. Invest. Dermatol. 37: 153-159. ~~" 

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