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Activation of Complement by Pityrosporum orbiculare
Feb. 1983
ACTIVATION OF COMPLEMENT BY P. ORBICULARE
immunization markedly enhance the degree of co ntact sensitivity in sensitized animals a nd reverse tolerance in unl'esponsive animals [20-22]. In t h e present study, we observed that t h e unl'esponsiveness obtained by topical a pplication of DNCB on PUV A-treated skin was also reversed by intraperitoneal a dministration of cycloph osph amide. Consequently it can be assumed th at the tolerance in t hese a nimals is also a specific immunologic unresponsiveness media ted by suppressor cells. Recently we confirm ed in mice tha t the unresponsiven ess can be transferred to syngeneic recipients using spleen cells and that t h e suppression by spleen cells was a brogated by antibody to T cell-specific antigen (Thy 1.2) and complement treatm ent (unpublished data) . REFERENCES 1. Morison WL, Parrish JA , Epstein, JH: Photoimmunology. Arch D ermatol 115:350-355, 1979 2. Morison WL: Photoimmunology. J Invest D ermato l 77:71-76, 198 1 3. Greene MI, Sy MS, Kripke ML, Benacerraf B: Impairme nt of a ntigen-presenting cell function by ultraviolet radiat ion. Proc N a t! Acad Sci USA 76:6591-6595, 1979 4. Toews GB, Bergstresser PA, Streilein JW: Epidermal Langerha ns ce ll density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 124:445-453, 1980 5. Morison WL, P a rrish JA, McAuliffe DJ , Bloch KJ : Sensitivity of mononuclear cells to PUVA: effect on subsequent stimulation with mitogens a nd on exclusion of tJ:ypan blue dye. Clin Exp Derma tol 6:273-277, 1981 6. Morison WL, Parrish JA, McAuliffe DJ , Bloch KJ : Sensitiv ity of mononucl ear ce lls to UV radiation: effect of subseq uent stimu lation with phytohemagglu tinin. Phochem P hotobiol 32:99-101, 1980 . 7. Morison WL, Parris h JA , B loc h KJ, Krugler JI: In vivo eft'ects of PUV A on lymphocyte fun ction. Br J D ermatol 104:405-413, 1981 8. Morison WL, Parrish JA, Bloch KJ , Krugler JI: In vivo effect of UVB on lymphocyte fun ction. Br J Dermatol 101:513-519, 1979 9. Kripke ML, Moriso n WL, Parris h JA: Differe nces in the immuno-
93
logic r eactivity of mice treated with UVB or methoxsal en plus UV A radiatio n. J Invest Dermatol 76:445-448, 1981 10. Robe rts LK, Schmi tt M , Daynes RA: Tumor susceptibili ty generated in mi ce t reated with s ubcarcinogenic doses of 8- methoxypso ralen a nd long-wave ul traviol et light. J Invest D ermato l 72:306-309, 1979 11. Okamoto H, Horio T: The effect of 8-methoxypsorale n a nd longwave ultraviolet light on Langerhans cell. J Invest Dermatol 77:345-346, 1981 12. H orio T , Okamoto H : The mec ha nisms of inhibitory effect of 8methoxypsoralen a nd long-wa ve ultraviolet light on experimental contact sensitization. J Invest Dermatol 78:402-405, 1982 13. Lynch DH, GUl'ish MF, Daynes RA: Relationship betwee n epidermal Langerha ns cell density ATPa e activity a nd the indu ction of contact hypersensitivity. J Immunol 126:1892-1897, 1981 14. M eyer J C, Grundmann HI', Weiss H: Inhibi tory effect of 8-methoxypsoralen plus UV A (PUV A) on the systemic indu cti on of contact sensit ivity to dinitrochlorobenzene (DNCB) in guin ea pigs. Arc h Dermatol R es 268:189-195,1980 15. Briffa DV, Parker D, Tosca N, Turk JL, Greaves MW: The effect of photochemo t herapy (PUVA) on ce ll med iated immunity in . t he guinea pig. J Invest Dermatol 77:377-380, 1981 16. M orison WL, Parrish JA, Woeh ler ME, B loch KJ: The influence of ul traviolet radiation on alle rgic contact dermatitis in the guinea 't~~1 II. Psoralen/ UVA radiation. Br J Dermatol 104:165-168, 17. Moriso n WL, Parrish JA, Woehler ME, Krugler JI, B loch KJ: Influ ence of PUVA a nd UVB radiation on delayed hypersensit ivity in t he guinea pig. J Invest Dermatol 76:484-488, 1981 18. Frey JR, D e Weck AL, Geleick H : Immunological unrespo nsiveness in allergic contact dermatit is to dini trochlorobe nzene in guinea pigs. J Invest D ermatol 42:41-47, 1964 19. Frey JR, D e Weck AL, Ge leick H: Inhibi tion of t he contact reaction to dinitrochlorobenzene by intravenou s injection of dini trochlorobenzene s ulfonate in guinea pigs sensitized to dini trochl orobe nzene. J Invest D ermatol 42:189-196, 1964 20. Polak L, TUl'k JL: Reversal of immunological tolerance by cyclophospha mide through the inhibit ion of suppresso r ce ll activity. N ature 249:654- 656, 1974 . 21. Po la k L, Geleick H: Differi ng mecha nisms of toleran ce a nd dese nsit ization to DNCB in guinea pigs. Eur J Immunol 5:94-99, 1975 22. Pola k L: Suppressor ce lls in different types of unresponsiveness to DNCB contact sensitivity in guinea pigs. Clin Exp Immunol 19:543- 549, 1975
0022-202X/ 83/ 8002-0093$02.00/0 THE JOURNAL OJ' I NVESTIGATI VE OEI1MA'I'OLOGY,
80:93-97. 1983
Vol. 80. No . 2
:::opyrighL© 1983 by Th e Willia ms & Wilkins Co.
Prill/ ed ill U. S .A.
Activation of Complement by Pityrosporum orbiculare PETER
G.
SOHNLE,
M.D.
AND CATHLEEN COLLINS-LECH,
B.A.
Section of In.fectious Diseases, Department of M edicine, The M edical Co llege of Wiscons,:n, Milwaukee, and the Medical and R esearch Services, Veterans Administration M edical Centel ~ Wood (Milwauhee), Wisconsin, U.S.A.
The ability to activate complement in human serum was evaluated for the two yeast-like urganisms Pityrosporum orbiculare, the presumed etiologic agent of tinea versicolor, and Candida albicans. Complement activation was measured by: (a) using inhibition of rabbit red blood cell lysis by human serum after incubation with the organisms, and (b) quantitation of the amount of C3 deposited on the surface of the yeast by an enzymelinked immunoabsorbent assay. It was found that both organisms had approximately equal ability to activate complement in normal serum or serum having only the Ma nuscrip t received Decembe r 14, 1981; accepted for publication June 11, 1982. This work was supported by the Vetera ns Administra tion a nd by Gra nt AI-16559 from t he N ational Instit utes of H ealt h. Reprin t requ ests to: Dr. Peter G. Sohnl e, R esear ch Service/ l 51.
alternative pathway intact, even though extracts of C. albicans contained significantly greater amounts of both carbohydrate and antigenic material capable of combining with the antibody present in normal human serum. The marked difference in inflammation in the cutaneous lesions produced by these two organisms does not app ear to be related to their complement-activating ability and is more likely due to some other factor such as differences in invasiveness or in ability to elicit other immunologic reactions. Veterans Adm inistration Med ical Center, Woorl, Wisconsin 53193. Abbrev iations: EGTA: ethylene glyco l-bis-( beta-a mino ethyl ether)-N ,N '- t.etraacetic acid ELISA: enzy me- linked immul1oabsorbent assay PBS: phosphate- buffe red saline RB C: red-blood cells
94
Vo l. 80, No. 2
SORNLE AND COLLIN S-LECR
T inea versicolor is a s uperfic ial fun gal infectio n t hat e vokes very lit tle in fl a m matory r esp o n se by t h e host. In con trast, s u perfi cial c u ta n eous infections wit h Candida albican s a r e highly infla m matory, p ro du cin g n e u t r o phil-co n taining e pidermal m icroabscesses in acu te infectio n s [1] a nd marke d d erm al mon o n uclear cell in fll tratio n in ch ronic infectio n s [2]. Co mple me n t activatio n via t h e alternative p athway h as b een implicated in t h e produ ction of t h e former t y p e o f infla mmat ory r eactio n to cu tan eou s Cand ida infe ctions [3]. Howev er , t h e inflamm atio n produ ced in an oth er type o f s upe rficia l mycosis, i.e., exp erime n tal cu ta n eo u s Trichophyton m en tagrophytes infectio n , appears to be d u e mainly to cell-m ediated immuni ty [4]. In a n att e mpt to elucidate t h e d iffer e n ces in cap acity to produ ce inflammatio n of Candida a nd P ityrosporum orbicu lare (th e apparen t etiologic age nt o f tinea v ersicolor) , w e h a v e previously com pared t h e a n t igenicity of t h e t w o o rga nis m s in lymp h ocyte tran sform ati o n tests. These experim e nts d e m o n strated t h at Candida was significa ntly more a n tige ni c t h a n was Pityrosporum in t his syste m [5]. Compleme n t activatio n m ay b e involve d in micro a bscess form atio n , an d Can dida infectio n s are associa t e d with t his t ype of ac u te infil trate w h er eas infectio n s ca u sed by P ityrosp orum are not. T h er efor e, t h e presen t study w as d esign e d to compare compleme n t activatio n by t h e two or ganism s. Pityrosp orum is a yeast-like orga nism t h at h as a n absolu te growth r e quire m en t for added lip ids i n vitro. I n p r evio u s studi es, w e h ave fo und t h at most of t h e yeast grown in a 2- phase sy s t e m will b eco m e ph ysically associated w it h t h e oil [5]. The r efore , this or gani s m 's sUl'face m igh t be con sid era bly differ e n t fro m t h at o f Can d id a a n d t hus less able to activate co mple m e n t a nd elicit ac u te infl ammatio n .
serum was then incubated at a 1:5 dilutio n wit h r ab bit R BC that had been obtained in he parin and then washed and suspend ed to approximate ly 1.5 X lOB ceLls/ ml of ge latin-verona l-saline at pH 7.6 co nta ining 5 >< 10- " M EG TA and 5 X 10- " M MgCb. T he incubation was a llowed to proceed for 2 hr at 37 °C, after which t ime 2.5 ml of 10- 2 M EGTA in saline was added. The t ubes were t hen cen trifuged and t he r esulting supern atants read at 413 nm in a s pectr ophotometer . The results wer e compared to t hose from sa mples in which 100% RBC lysis was o btained with distilled water. Resul ts wer e ex pressed as percent inhibi t ion of hemolys is as a function of either t he dry weight or pac ked volume of orga nism s employed. The a moun t r eq uired to produce 20% or 50% inh ib ition of hemolysis was obtained usin g a se ri es of dilu t ions of t he yeast sus pension.
MATERIALS AND METHODS
Determination of A ntigen Content in Yeast. Extracts by Inhibition of Antibody Activit.y
Organisms Five isolates of P. orbiculare were obtained fro m ski n scra pings of tinea versicolor lesions by cu lt UJ"e on mycosel agar (BBL, D iv. Becton, Dickinso n, and Co., Cockeysville, Mru·ylan d) overla id wit h ste ril e olive oil. S ubc ul t ures were made f!"Om emerging colonies and identili ed as P . orbiculare by morp hologic appear ance a nd a stri ct requirement for added lipid. All 5 isolates grew prefere nt ia lly at t he oil- water in te rface of broth cult ures, although 2 a lso grew in t he wate r phase a nd could be centri fuged free of oil if 0.1% Tween 80 were added to t he med ium. Five isolates of C. albicans were obtained fro m clinical isolates a nd ma intained by repeated passage on Sabouraud's aga r. Both organisms were grown in bul k in AOAC synthetic broth (D ifco La borato ri es, Detroit, Michigan) containi ng 1% dex trose, chlora mph enicol (20 f1.g/ ml) , cyclohexim ide (250 f1.g/ ml ), 0.1 % T wee n 80, and overlaid wi t h sterile oli ve oil. At t he end of t he cul ture period (14 days for P . orbiculare and 5 days fo r C. albicans), t he oil and wate r phases were separated by centrifugation and t he organisms in the water phase washed 3 times in 0.9% NaC I. T he q uantity of orga nisms prese nt was expressed as eit her m l of packed cells or as dry we ight dete rmined a fter lyophilization of t he yeast suspensions. These two methods were used in case one or t he other a lone might have given a n inacc urate estimate of t he quantity of organisms p resent.
P reparation of Ext.racts Extracts of t he ·two organisms were prepar ed as described previously [5] using eit her t he ether extraction method or sonication. P rotei n concentrations of the extracts were dete rmined by the Lowry techniqu e a nd car bo hydrates by t he ac id-phenol method [6]. Measurement of Complement Activation Using Hemolysis of R a bbit Red Blood Cells (RBC) H uman sera we re incubated with known· qu antities of yeast or yeast extracts and then evalu ated fo r t heir a bility to hemolyze ra bbit RBC by the alte rnative pathway using a modification of the method of Platts-M ills and Ishizaka [7). In brief, t he yeast or yeast extracts were incubated for 30 min at 37°C wit h normal hum an serum , hum an se rum with 5 X 10- " Methylene glyco l-bis-(beta-amino et hyl ether)-N,N'tetraacetic acid (EGTA) conta ining 5 X 10- " M of MgC I2, or C2-deficient human serum (kin dly provided by Dr. S usa n Koethe, Med ical College of Wisconsin). T he yeasts were removed by centrifugation, a nd t he
Measuremen t of Complem ent. Acti vation by Qua.ntitating C3 Dep osition on the Y east S urface An enzy me- linked immunoa bsorbent assay (ELISA) was developed to measure t he amoun t of C3 deposited on kn own qu ant it ies of yeast during incubation wi th t he 3 human serum preparations used above. Various dilu tions of yeast suspensions wer e first heated to 100°C for 60 min to remove endogenous per oxidase activity and then incubated in 0.1 ml volumes wi t h 0.1 mI of se rum for 30 min at 37°C. The yeast was next washed 3 t imes in 0.05 M phosphate- bu ffer ed saline (PBS ) conta ining 0.05% Twee n 20. They were then incubated wi t h 0.5 ml of a 1:1000 dilu t ion of peroxidase-conju gated goa t a nti huma n C3 (Cappe U La boratories, Coc hranville, Pennsylva nia ) for 30 min at 37°C and again washed 3 t imes wi th PBS-Tween 20. After removal to a fresh test tube, 1.0 ml of substrate was added (1 mg/ ml O- phenylenedia min e, Eastm a n K oda k Co., Hocheste r, N ew York , in 0.05 M citra te buffe r, pH 4.5, a nd 0.05% H, 0 2). After 40 min of incubat ion at room temper atw·e, th e supernatants were se parated by cent rifugation and read in a spectr ophoto mete r at 420 nm. Data wer e expr essed as opt ical density t imes 1000 per f1.g of yeast.
An E LIS A-type antibody assay was set up for determining ant ibody activi ty aga inst extracts of C. a lbicans a nd P . orb iculare in norma l hum an serum . Polysty rene t ubes we re coated wit h 1.0 ml of a ppropriate dilu t ions of eit her extract in 0.05 M P BS at pH 7.6 fo r 1 hr at roo m temperature. The tu bes were washed 3 t imes wi t h PBS-Tween 20, treated with 1% bovine se rum albumin, a nd washed again. Following t his step, 0.5 m l of a 1:50 dilu t ion of a pool of se rum 1'r om 10 norm al subj ects was added and incuba ted at 37 °C for 30 min. The tubes we re washed 3 t imes wi th PBS-Tween 20, and 0.5 ml of a 1:200 dilu t ion of peroxidase- la beled, a ffini ty- purified goat ant ihuman IgG (Li tton B ionetics, Kensin gto n, Maryland) was added. After incubation for 30 min at 37°C, t he t ubes wer e aga in washed 3 t imes wi t h PBS-Tween 20, and 1.0 ml of the substrate described a bove was add ed . The t ubes were incubated fo r 40 m in and read at 420 nm. Bla nks wer e set up wit hout t he hum an se rulTI and t hese va lu es sub t racted fro m t he experimenta l valu es. The ex perim ents were cruTied out by incubating the pooled hum an serum with various amounts of 4 extracts eac h of C. a lbicans and P . orbiculare and t hen testing the serum for residua l antibody activity aga inst t he corresponding orga nism. The resul t ing percent inhibi tion of t he op t ical density obtained was plotted aga inst t he qu antity of extract employed (based on protein plus carbohydrate co ntent) to lind the amoun t necessary for 50% inhibi t ion. Controls for t he spec ificity of the peroxidase- la beled antihuman heavy-chain reage nts were ca rried out in our syste m as fo llows: purified immunoglobulins of th e 3 classes (CappeLl La boratories, Cochranville, P ennsylva nia ) were coated onto plasti c tubes at a concentration of 10 f1.g/ ml, washed, a nd incubated wit h dilu t ions of t he 3 peroxidase- labeled reagents. Good agreement was ob tained betwee n the stated s pecificity and actua l antibody activity aga inst t he a ppropr iate immunoglobulin class in this test.
Human Sera Three sera fr om norma l persons and one fTom an oth erwise normal, but C2-deficient perso n were used for t he studies of co mpl ement activation. These sera were stored in a liquots at -70°C. The pool of norma l sera was prepru·ed using equ a l volumes of serum fr om 10 norm a l subjects wit hout a history of superfi cial fun ga l infec tions. These sera were kept at -20°C. We have prev iously demonstra ted t hat normal human serum has a nt ibody aga inst Pityrosp orum as well as Cand ida [8).
Feb. 1983
ACTI VATION OF COMPLEMENT BY P. ORBICULARE
Statistics
TABLE
In the complement assays, 4 different yeast prepa ratio ns representing 2 isolates each of the two organis ms were used. For t he studies comparing protein a nd carbohydrate contents of extracts, 6 ether extracts a nd 4 sonicates were tested, representing 5 isolates of each organism in all The extracts that were used in t he studies of a ntigen conte nt a nd complement-activating a bili ty consisted of 2 ether extr acts and 2 sonicates representing 2 isolates of each organism. Co mparisons were m ade between t he various groups usin g the unpaired, two-ta iled t-test.
Sl S2 E1 E2 Sl S2 E1 E2
Complement Activation by C. albicans and P . orbiculare In Table I, the ability of the two organisms to activate complem en t is expressed as th e percent inhibition of rabbit RBC h emolysis after incubation of 5.0% and 0.5% yeast suspensions with the serum. No significant differences were found between the two organisms at eit h er concentration using normal serum or serum lacking the classical pathway by virtue of calcium chelation or C2 deficiency. Similar results were obtained when the yeasts were quantified after lyophilization of aliquots a nd weighing (results not shown). It should be no ted that h e molysis by C2-deficient serum was affected less by incubation with th e two yeasts or with inulin than was normal serum with or without M g-EGTA. This finding may be related to the mildly deficient function of C2-deficient serum in t his system as has been r epor ted previously .[9]. As shown in Table II, h eatin g at 56 °C for I/~ hr significantly suppressed t he a bility of either normal or C2-deficient serum to cause he molysis of rabbit RBC. The a bility of extracts of t he two organisms to activate complem e nt was also evaluated using the rabbit RBC system. TABLE
I. Percent inhibition of rabbit REC h emolysis by hu.man
serum, complement u.sing constant volumes of yeast Serum
Candida
P':ly rosporu m.
(N = 4)
(N = 4)
5,0% yeast by vo lume (mean ± SE of % inhibition) N ormal 91.0 ± Normal + Mg-EGTA 83,5 ± C2-defi cient 33.8 ± 0.5% yeast by vo lume (mean ± SE of % inh ibition) Normal 30.5 ± Normal + Mg-EGTA 32,8 ± C2-deficie nt 1.0 ±
2,7 6,1 10.4
85.3 ± 2,7 86.8 ± 2,0 49.8 ± 9,0
NS NS NS
2. 1 11 ,3 0,6
26,8 ± 3.2 34.3 ± 12.1 6,0 ± 1.5
NS NS p < ,02
Rabbit REC H emo lysis
Norma l C2-defi cient
Percent of
Un heated (% lysis )
(% lysis)
activ ity remaining
74.1% 58.8%
1.7% 2.4%
2.3% 4,1%
C3-0eposition o n Yeast Surface as M easured by the ELISA Assay Serum
Candida Normal C2-deficient Pityrosporwn Normal C2-deficient
Normal hum.an serum 22.2" 27.7 11.1
25,0 7.0 0,0 0.0
12.4 No rm.al human serum + Mg-EGTA 26,7 20.4 38.0 7.8 2,0 5.4 2.0 22.3
" Extracts represent 2 sonicates (Sl and S2) a nd 2 ether extracts (E 1 a nd E2) of each organism, " Dat.a represe nt % inhibition of he molysis aft.er in cubation wit h 500 /lg/ ml of t he indicated extract.. 400 ,---------------------------------____~ f(fJ
w
>-
•
CA NDID A
iii
PITYROSPORUM
300
01
~
a a a
x
>-
f-
200
(fJ
Z
w
o
-'
100
f0.
N OR MAL SERUM
H eated
PityroHpOrU11l
o
II. Comparison of unheated serum vs. serum heated at 56°C for 30 /II,in. in. the assays of complem eniactivation
Serum
Candida
Statistica l s ignifica nce
N ote: Inulin at 10 mg/ ml produced 94% s uppression of hemolysis using normal seru m, 95% using normal se rum + Mg-EGTA, a nd 28% using C2-defi cient serum , TA BLE
III, Percent inlu:bition of rabbit REC hemolysis by human . serum. using 500 /lg/m.l of yeast extracts
Extract"
RESULTS
95
Un heat ed (00 X 1000)
H eated (00 X 1000)
Percen t of activ ity remaining
770 470
60 63
7,8% 13.4 %
947 782
138 163
14,6% 20.8%
These data ar e from 1 representative experimen t of 3 that were performed.
NORMAL SE RUM PLU S Mg - EGTA
C 2-0 EFICIENT SE RUM
FIG 1. D eposition of C3 on t he surface of C. albicans and P. o/'b iculare (4 isolates each) using normal huma n serum, normal human seru m plus Mg-EGTA, or C2-deficient huma n serum, D eposition of C3 was determined using a n ELISA system as out lined in Mate rials and Methods a nd t he results expressed as optical density x 1000/ /lg of yeast. The differ ences in C3 deposition were not significan tly different for t he two types of yeast with a ny of t he 3 t.ypes of serum, For Candida, but not Pityrosporwn, t here was a significa nt differ ence between C3 deposition using normal serum vs. either Mg-EGTAtreated serum or C2-deficient serum (p < 0,02 in both cases).
Two eth er extracts a nd 2 sonicates of each organism were tested in this system at a concentration of 500 flg / ml (combined protein a nd carbohydrate contents), t h e approxin1ate amount at which whole yeast produced 20% inhibition of h emolysis after incubation with t he test serum. As shown in Table III, th e results were quite variable, alt hough some complement-activating a bility was evident for some of the extracts. Complement activation was a lso measured by determining the amount of C3 deposited on the yeast cell surface. This technique was developed because of t he possibility t hat inhibition of rabbit RBC lysis by t h e yeast could have been due to a n actual complement inhibitor of some kind rather th an to th e consumption of complem ent components by t heir activation. As shown in Fig 1, th ere ar e no significant differences in the amo unt of C3 deposited on the sw-face of the two organisms after incubation with huma n serum . It would appeal' that more C3 is deposited on th e yeast swface if serum with an intact classical pathway is used. The differ ence between t h e r esults obtained with normal serum vs. t h at treated with Mg-EGTA or C2-deficient serum was significant for Candida (p < 0.02 in
96
Vol. 80, No.2
SOHNLE AND COLLINS-LECH
TABLE IV. Protein and carbohydrate contents o(Candida and Pityrosporum extracts Protein/ Carbohydrate Organism Protein carbohydrate Sonicates" Candida 13.58 ± 3.75 0.93 ± 0.34 9.30± 0.82 Pityrosporum 1.44 ± 0.68 3.54 ± 0.43 4.71 ± 1.76 p < 0.02 p < 0.01 Statistical sig0.1 > p > 0.05 nificance b Ether extracts" 5.67 ± 1.30 0.80 ± 0.37 Candida 3.57 ± 1.21 3.94 ± 0.77 Pityrosporum 2.57 ± 0.61 0.69 ± 0.13 Statistical sigp < 0.01 p < 0.01 NS nificance" " Comparisons were made between protein and carbohydrate contents (expressed as mg/ml of organisms ± S.E.) for 4 sonicates and 6 ether extracts of each organism. " Statistical significance by the unpaired, two-tailed I-test was taken atp < 0.05. TABLE V. Antigenic activity o(Candida and Pityrosporum extracts in an ELISA assay
Organism Candida Pityrosporum
M of Extract for 50% inhibiiion fl
Log 1o values
70.0 ± 28.1 822.6 ± 260.4 p < 0.05
1.73 ± 0.2 2.83 ± 0.2 p < 0.01
Statistical significance" " Combined protein and carbohydrate contents for 2 ether extracts and 2 sonicates of each organism required for 50% inhibition of antibody activity (± S.E.). . " Statistical significance by the unpaired, two-tailed t-test. both cases) but not for Pityrosporum, perhaps because of greater variability in the results obtained with the latter. As shown in Table II, heating serum to 56°C for '/2 11.1' significantly reduced the amount of C3 deposited on the surface of both yeasts using either normal or C2-deficient serum.
Protein, Carbohydrate, and Antigenic Contents of Candida and Pityrosporum Extracts As shown in Table IV, extracts of both organisms contained comparable protein contents, whereas those of Candida contained significantly more carbohydrate. This factor resulted in lower protein to carbohydrate ratios for extracts of Candida than for those of Pityrosporum. Antigenic contents of the extracts were measured using inhibition of antibody activity of pooled human serum against the organisms in an ELISA system as described under Materials and Methods. The results demonstrated that Candida appears to have a significantly higher content of antigen than does Pityrosporum (Table V), a finding compatible with our previous studies using the lymphocytetransformation system [5]. DISCUSSION These experiments indicate that the two yeasts, C. albicans and P. orbiculare, appear to have approximately equal abilities . to activate complement using either the rabbit RBC hemolytic system to measure alternative pathway activity left in human serum after incubation with the organisms or measurement of C3 deposition on the yeast .s urface using an ELISA system. Complement-activating ability appeared to be comparable for the two organisms in both systems using either normal human serum or serum with a nonfunction'a l classical pathway because of C2 deficiency or the addition of Mg-EGT A. In addition, these studies have demonstrated that extracts of Candida contain more carbohydrate and antigenic activity than do those of Pityrosporum. Many microorganisms that are pathogenic for humans initiate complement activation via the alternative pathway. These include: (a) other Candida species in addition to C. albicans
[10]; (b) Cryptococcus neoformans [11]; (c) Paracoccidioides brasiliensis [12]; (d) Streptococcus pneumonia [13]; and (e) the sindbis virus [14]. In addition, complement activation has been suggested as a possible cause of inflammation in acne [15]. Finally, complement activation, particularly that occurring via the alternative pathway, has been demonstrated to be important in the defense against experimental intravascular infections with C. albicans [16] and pneumococci [17]. Whether or not this process is really important in the host defense against these pathogens causing human disease is unclear. This situation is comparable to the status of alternative pathway activation by small molecules as a cause of some cases of asthma-an attractive hypothesis but without solid evidence backing it [18]. The results of the present investigation indicate that the lack of inflammation in the lesions of tinea versicolor is not due to the inability of the causative organism, P. orbiculare, to activate complement. This organism is approximately as capable in initiating complement activation by both pathways as is C. albicans, a producer of highly inflammatory cutaneous lesions. This finding is interesting inasmuch as Pityrosporum appears to contain significantly less extractable carbohydrate and antigenic material than does Candida. The ability to activate complement is obviously not a property singularly sufficient for an infecting organism to produce inflammatory skin lesions. Although in biopsies of Candida- and Pityrosporum-infected skin in humans the organisms are generally found at the same locations, i.e., confined to the stratum corneum [2,19], it is possible that a greater capacity of Candida for deeper invasion through the stratum corneum may be the reason for the differences in inflammation produced by the two organisms. Evidence to support such an explanation has been generated in studies of experimental cutaneous infections of mice produced by C. albicans and other Candida species [3]. In addition, it is possible that Pityrosporum does not release soluble complement-activating factors that can diffuse across the stratum corneum to encounter a source of activatable complement. However, an alternative explanation is that some factor instead of or in addition to complement activation is important for the production of inflammation in superficial fungal infections. Differences in the quantity of antigen present and the ability to elicit immunologic reactions could account for the differences in inflammatory potential of these two organisms, as we have suggested previously [5]. The authors thank Dr. Susan Koethe (Medical College of Wisconsin, Milwaukee, Wisconsin) for the gift of the C2-deficient serum and Victoria Gollish for typing the manuscript. REFERENCES 1. Rebora A, Marples RR, Kligman AM: Experimental infection with Candida albican.s. Arch Dermatol 108:69-73, 1973 2. Kirkpatrick CH, Rich RR, Bennett JE: Chronic mucocutaneous candidiasis: model building in cellular immunity. Ann Intern Med 74:955-978, 1971 3. Ray TL, Wuepper KD: Experimental cutaneous candidiasis in rodents. II. Role of the stmtum corneum barrier p.nd serum complement as a mediator of a protective inflammatory response. Arch Dermatol 114:539-543, 1978 4. Kerbs S, Gl'eenberg J, J esrani K: Temporal correlation of lymphocyte blastogenesis, skin test responses, and erythema during dermatophyte infections. Clin Exp Immunol 27:526-530, 1977 5. Sohnle PG, Collins-Lech C: Relative antigenicity of P. orbiclllare and C. albican.s. J Invest Dermatol 75:279-283, 1980 6. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith E: Colorimetric method for determination of sugars and related substances. Anal Chern 28:350-356, 1956 7. Platts-Mills TAE, Ishizaka K: Activation of the alternate pathway of human complement by rabbit cells. J Immunol 113:348-358, 1974 8. DaMert GJ, Kirkpatrick CH, Sohnle PG: Comparison of antibody responses in chronic mucocutaneous candidiasis and tinea versicolor. Int Arch Allergy Appl Immunol 63:97-104, 1980 9. Polhill HB, Pruitt KM, Johnston RB, Jr: Kinetic assessment of alternative complement pathway activity in a hemolytic system. I. Experimental and mathematical analyses. J Immunol 121:363-368, 1978
Feb. 1983
RAT SKIN ODC ANTIZYME
10. Ray TL, Wu ep per KD: Activation of the a lte rnative (properd in) pathway of compl e men t by Candida albicans a nd re lated s pecies. J Invest Dermatol 67:700-703, 1976 11. Dia mond RD, May JE, Kane MA, Frank MM, Be nn ett JE: The role of t he class ical a nd a ltern ate complement pathways in host d efenses aga inst C,yptococcus n eofonnans infection. J Immunol 1l2:~ ::SO-2270 , 1974 12. Calich VLG, Kipnis TL, M a riano M, Neto CF, Dias Da Silva W: Th e activation of t he co mplement system by Paracoccidioides brasiliensis in vitro: its opsonic effect a nd possible significa nce for an in vivo model of infection. Clin Immunol Immunopathol 12:20-30, 1979 13. Winke lstein JA, Tomasz A: Activation of t he alternate pathway by pneumococcal ce ll wa lls. J ImmunoI1l8:45J-454, 1977 14. Hirs h RL, Winke lste in JA, Griffin DE: The rol e of comp le men t in viral infections. III. Activation of t he classica l and alternative
15. 16. 17. 18. 19.
97
complement pathways by s indbis virus. J Immunol 124 :2507-2510, 1980 Webste r GF, Leyden JJ, Nilsson UR: Complemen t activation in ac ne vulgaris: co nsump tion of co mple ment by comedones. Infect Immun 26:183-186, 1979 Ge lfand JA, Hurley DL, Fauci AS, Frank MM: Role of co mplement in host de fense aga inst experimen ta l disse minated ca ndidiasis. J Infect Dis 138:9-16, 1978 Hosea SW, Brown EJ, Frank MM: The critica l role of complement in ex perimental pneumococca l sepsis. J Infect Dis 142:903-909, 1980 Wilson MR, Salvaggio JE: Activation of the a lternative pathway of compl ement: a mec hanism in search of diseases. J Allergy Clin Immunol 65:319-321, 1980 Mon tes LF: Systemic a bnorma lities a nd t he intrace llul ar s ite of infections of the stratum corneum. JAMA 213: 1469-1472, 1970
0022-202X/ 83/8002-0097$02.00/ 0 TBE JOURNAL OF I NVESTl (;A TI VE [)t;IlMATOLO(:Y.
Vo l. 80, No.2
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Printed in U.S.A.
Copyright © 1983 by Th e Williams & Wilkins Co.
Antizyme Release Is an Early Event in Ornithine Decarboxylase Induction by Hair Plucking* JEANNE LESIEWI CZ, PH.D. AND LOWELL
A. GOLDSMITH, M.D .
Division olDennatology, Departrnent of Jltfedicin e, Dulle University Medical Center, Durham, North Carolina, U.S.A.
Plucking of hair from the dorsal skin of rats resulted in a rapid decrease in ornithine decarboxylase (ODe) activity. A significant loss of activity did not occur in other skin enzymes under the same conditions and in vivo incorporation of [3H]-leucine in skin was not significantly decreased 60 min immediately following hair plucking. Treatment of ODe enzyme preparations with 10% (NILhSo', resulted in recovery of approximately 75 % greater ODe activity than in untreated samples, suggesting the presence of an inhibitor (antizyme). ODe inhibitor was detected in plucked skin; inhibitor levels increased after treatment of plucked skin extracts with 10% (NH4 hSo',.
In the rat, hair growth occurs in a cyclic manner. P lucking of hair during the telogen phase induces high levels of ornithine decarboxylase (L-ornithine carboxylase, Ee 4 .1.1.1 7) (ODe) [1] within 4 hr [2]. However, during the 60 'min immediately following hail' plucking ODe activity is observed to decr ease [1,3]. The cause of this initial loss of ODe enzyme activity is the' subject of this report.
M a nuscript received January 21, 1982; acce pted for publication June 14 , 1982. Supported in part by NIH grant AM-17253. • Pub lication No. 104 of the Dermatological Research Laboratories of Duke University M edi ca l Center. R eprin t requ ests to: Dr. L. A. Goldsmith , Department of Med icine, Dermatology Unit, Un iversity of Rochester School of Med icin e and Dentistry, 601 E lmwood Avenu e, Rochester, New York 14642. Abbreviations: DDT: dithiothre ito l EDTA: (ethyle ned initrilol-tetraacetic acid, disod ium salt ODC: ornit hine decarboxy lase PLP: pyridoxal-5' -phos ph ate TCA: trichloroacetic acid Tris: trih ydroxyam inomethane
MATERIALS AND METHODS Ch.emicals Trihydroxyaminomethane (Tris) , pyridoxal-5'-phosphate (PLP), and cyclohex imide, were purchased from Sigma Chemica l Co., St. Louis, Missouri. DL-[l-"'C]-ornithine (51.2 mC/ mm ol), L-[4,5-"H]-leucine (12 Ci/mmol) , [1,4-'4C]-putrecine (89.9 mCi/mmo l), phenethylamine, and Aqu aso l II were from New England Nuclear, Boston, Massachusetts. Enzyme-grade sucrose a nd ultra pure ammonium s ulfate were fTo m Schwartz-Mann, S prin g Valley, New York. Dithiothreitol (OTT) was from Bachem Feinchemikalien, L iesta l, Switzerland. Biogel-PlOO was obtained fTom Bio-Rad Laboratories, Richmond, California . Anim.als Juvenile ma le CD strain rats weighing 80-100 g (24-28 days old) were obtained from Charles R iver Breeding Laboratories, Wilmington, Massachusetts. For ODC enzyme studi es the animals were used in t he early a nagen ph ase of ha ir growth, which was defined as dorsa l skin fo r which t he most ventral edge of a patch was within 1 cm of the discernib le border of new hair el·up tio n. For inhibitor studies, a nima ls in the telogen phase were used. This phase was defined as s kin for which no border of new ha ir growth was detected on t he an ima l's dorsum.
Time Co u.rse of Plucking R esponse Animals were anesthesized with ether and the ha ir plu cked from their dorsal skin in patches 60, 45, 30, 15, 5, a nd 0 (immediately) min before death by decapitation. H a ir plucking was accomp lished using hemostat forceps which had t hin-walled rubber tubing forced over the ends. The hair co uld t hen be grasped t ightly a nd jerked free in clumps. A 2.5 x 2.5 cm skin patch can be plucked bare in 15-20 sec by t his method. Th e skin patches were excised, cleaned of underly ing musclllature, weighed, a nd homogenized 1:9 (weight:volume) in ODe buffer (10 mM Tris pH 7.0, 0.5 mM EDTA pH 7.0, 0.01 mM PLP, and 5 mM DTT) as previously described [2]. ODC assays a nd turbidim etric protein determinations were performed as previously described [3]. One uni t of ODC activity is ca pable of releasing 1 Ilmol "'COz/hr.
Response of Other Shin Enzym.es to H air Pluching Anima ls were anesthesized with ether and t he ha ir plucked from one half of their dorsal skin 60 a nd 0 min before death by decapitation. Th e
ACTIVATION OF COMPLEMENT BY P. ORBICULARE
immunization markedly enhance the degree of co ntact sensitivity in sensitized animals a nd reverse tolerance in unl'esponsive animals [20-22]. In t h e present study, we observed that t h e unl'esponsiveness obtained by topical a pplication of DNCB on PUV A-treated skin was also reversed by intraperitoneal a dministration of cycloph osph amide. Consequently it can be assumed th at the tolerance in t hese a nimals is also a specific immunologic unresponsiveness media ted by suppressor cells. Recently we confirm ed in mice tha t the unresponsiven ess can be transferred to syngeneic recipients using spleen cells and that t h e suppression by spleen cells was a brogated by antibody to T cell-specific antigen (Thy 1.2) and complement treatm ent (unpublished data) . REFERENCES 1. Morison WL, Parrish JA , Epstein, JH: Photoimmunology. Arch D ermatol 115:350-355, 1979 2. Morison WL: Photoimmunology. J Invest D ermato l 77:71-76, 198 1 3. Greene MI, Sy MS, Kripke ML, Benacerraf B: Impairme nt of a ntigen-presenting cell function by ultraviolet radiat ion. Proc N a t! Acad Sci USA 76:6591-6595, 1979 4. Toews GB, Bergstresser PA, Streilein JW: Epidermal Langerha ns ce ll density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 124:445-453, 1980 5. Morison WL, P a rrish JA, McAuliffe DJ , Bloch KJ : Sensitivity of mononuclear cells to PUVA: effect on subsequent stimulation with mitogens a nd on exclusion of tJ:ypan blue dye. Clin Exp Derma tol 6:273-277, 1981 6. Morison WL, Parrish JA, McAuliffe DJ , Bloch KJ : Sensitiv ity of mononucl ear ce lls to UV radiation: effect of subseq uent stimu lation with phytohemagglu tinin. Phochem P hotobiol 32:99-101, 1980 . 7. Morison WL, Parris h JA , B loc h KJ, Krugler JI: In vivo eft'ects of PUV A on lymphocyte fun ction. Br J D ermatol 104:405-413, 1981 8. Morison WL, Parrish JA, Bloch KJ , Krugler JI: In vivo effect of UVB on lymphocyte fun ction. Br J Dermatol 101:513-519, 1979 9. Kripke ML, Moriso n WL, Parris h JA: Differe nces in the immuno-
93
logic r eactivity of mice treated with UVB or methoxsal en plus UV A radiatio n. J Invest Dermatol 76:445-448, 1981 10. Robe rts LK, Schmi tt M , Daynes RA: Tumor susceptibili ty generated in mi ce t reated with s ubcarcinogenic doses of 8- methoxypso ralen a nd long-wave ul traviol et light. J Invest D ermato l 72:306-309, 1979 11. Okamoto H, Horio T: The effect of 8-methoxypsorale n a nd longwave ultraviolet light on Langerhans cell. J Invest Dermatol 77:345-346, 1981 12. H orio T , Okamoto H : The mec ha nisms of inhibitory effect of 8methoxypsoralen a nd long-wa ve ultraviolet light on experimental contact sensitization. J Invest Dermatol 78:402-405, 1982 13. Lynch DH, GUl'ish MF, Daynes RA: Relationship betwee n epidermal Langerha ns cell density ATPa e activity a nd the indu ction of contact hypersensitivity. J Immunol 126:1892-1897, 1981 14. M eyer J C, Grundmann HI', Weiss H: Inhibi tory effect of 8-methoxypsoralen plus UV A (PUV A) on the systemic indu cti on of contact sensit ivity to dinitrochlorobenzene (DNCB) in guin ea pigs. Arc h Dermatol R es 268:189-195,1980 15. Briffa DV, Parker D, Tosca N, Turk JL, Greaves MW: The effect of photochemo t herapy (PUVA) on ce ll med iated immunity in . t he guinea pig. J Invest Dermatol 77:377-380, 1981 16. M orison WL, Parrish JA, Woeh ler ME, B loch KJ: The influence of ul traviolet radiation on alle rgic contact dermatitis in the guinea 't~~1 II. Psoralen/ UVA radiation. Br J Dermatol 104:165-168, 17. Moriso n WL, Parrish JA, Woehler ME, Krugler JI, B loch KJ: Influ ence of PUVA a nd UVB radiation on delayed hypersensit ivity in t he guinea pig. J Invest Dermatol 76:484-488, 1981 18. Frey JR, D e Weck AL, Geleick H : Immunological unrespo nsiveness in allergic contact dermatit is to dini trochlorobe nzene in guinea pigs. J Invest D ermatol 42:41-47, 1964 19. Frey JR, D e Weck AL, Ge leick H: Inhibi tion of t he contact reaction to dinitrochlorobenzene by intravenou s injection of dini trochlorobenzene s ulfonate in guinea pigs sensitized to dini trochl orobe nzene. J Invest D ermatol 42:189-196, 1964 20. Polak L, TUl'k JL: Reversal of immunological tolerance by cyclophospha mide through the inhibit ion of suppresso r ce ll activity. N ature 249:654- 656, 1974 . 21. Po la k L, Geleick H: Differi ng mecha nisms of toleran ce a nd dese nsit ization to DNCB in guinea pigs. Eur J Immunol 5:94-99, 1975 22. Pola k L: Suppressor ce lls in different types of unresponsiveness to DNCB contact sensitivity in guinea pigs. Clin Exp Immunol 19:543- 549, 1975
0022-202X/ 83/ 8002-0093$02.00/0 THE JOURNAL OJ' I NVESTIGATI VE OEI1MA'I'OLOGY,
80:93-97. 1983
Vol. 80. No . 2
:::opyrighL© 1983 by Th e Willia ms & Wilkins Co.
Prill/ ed ill U. S .A.
Activation of Complement by Pityrosporum orbiculare PETER
G.
SOHNLE,
M.D.
AND CATHLEEN COLLINS-LECH,
B.A.
Section of In.fectious Diseases, Department of M edicine, The M edical Co llege of Wiscons,:n, Milwaukee, and the Medical and R esearch Services, Veterans Administration M edical Centel ~ Wood (Milwauhee), Wisconsin, U.S.A.
The ability to activate complement in human serum was evaluated for the two yeast-like urganisms Pityrosporum orbiculare, the presumed etiologic agent of tinea versicolor, and Candida albicans. Complement activation was measured by: (a) using inhibition of rabbit red blood cell lysis by human serum after incubation with the organisms, and (b) quantitation of the amount of C3 deposited on the surface of the yeast by an enzymelinked immunoabsorbent assay. It was found that both organisms had approximately equal ability to activate complement in normal serum or serum having only the Ma nuscrip t received Decembe r 14, 1981; accepted for publication June 11, 1982. This work was supported by the Vetera ns Administra tion a nd by Gra nt AI-16559 from t he N ational Instit utes of H ealt h. Reprin t requ ests to: Dr. Peter G. Sohnl e, R esear ch Service/ l 51.
alternative pathway intact, even though extracts of C. albicans contained significantly greater amounts of both carbohydrate and antigenic material capable of combining with the antibody present in normal human serum. The marked difference in inflammation in the cutaneous lesions produced by these two organisms does not app ear to be related to their complement-activating ability and is more likely due to some other factor such as differences in invasiveness or in ability to elicit other immunologic reactions. Veterans Adm inistration Med ical Center, Woorl, Wisconsin 53193. Abbrev iations: EGTA: ethylene glyco l-bis-( beta-a mino ethyl ether)-N ,N '- t.etraacetic acid ELISA: enzy me- linked immul1oabsorbent assay PBS: phosphate- buffe red saline RB C: red-blood cells
94
Vo l. 80, No. 2
SORNLE AND COLLIN S-LECR
T inea versicolor is a s uperfic ial fun gal infectio n t hat e vokes very lit tle in fl a m matory r esp o n se by t h e host. In con trast, s u perfi cial c u ta n eous infections wit h Candida albican s a r e highly infla m matory, p ro du cin g n e u t r o phil-co n taining e pidermal m icroabscesses in acu te infectio n s [1] a nd marke d d erm al mon o n uclear cell in fll tratio n in ch ronic infectio n s [2]. Co mple me n t activatio n via t h e alternative p athway h as b een implicated in t h e produ ction of t h e former t y p e o f infla mmat ory r eactio n to cu tan eou s Cand ida infe ctions [3]. Howev er , t h e inflamm atio n produ ced in an oth er type o f s upe rficia l mycosis, i.e., exp erime n tal cu ta n eo u s Trichophyton m en tagrophytes infectio n , appears to be d u e mainly to cell-m ediated immuni ty [4]. In a n att e mpt to elucidate t h e d iffer e n ces in cap acity to produ ce inflammatio n of Candida a nd P ityrosporum orbicu lare (th e apparen t etiologic age nt o f tinea v ersicolor) , w e h a v e previously com pared t h e a n t igenicity of t h e t w o o rga nis m s in lymp h ocyte tran sform ati o n tests. These experim e nts d e m o n strated t h at Candida was significa ntly more a n tige ni c t h a n was Pityrosporum in t his syste m [5]. Compleme n t activatio n m ay b e involve d in micro a bscess form atio n , an d Can dida infectio n s are associa t e d with t his t ype of ac u te infil trate w h er eas infectio n s ca u sed by P ityrosp orum are not. T h er efor e, t h e presen t study w as d esign e d to compare compleme n t activatio n by t h e two or ganism s. Pityrosp orum is a yeast-like orga nism t h at h as a n absolu te growth r e quire m en t for added lip ids i n vitro. I n p r evio u s studi es, w e h ave fo und t h at most of t h e yeast grown in a 2- phase sy s t e m will b eco m e ph ysically associated w it h t h e oil [5]. The r efore , this or gani s m 's sUl'face m igh t be con sid era bly differ e n t fro m t h at o f Can d id a a n d t hus less able to activate co mple m e n t a nd elicit ac u te infl ammatio n .
serum was then incubated at a 1:5 dilutio n wit h r ab bit R BC that had been obtained in he parin and then washed and suspend ed to approximate ly 1.5 X lOB ceLls/ ml of ge latin-verona l-saline at pH 7.6 co nta ining 5 >< 10- " M EG TA and 5 X 10- " M MgCb. T he incubation was a llowed to proceed for 2 hr at 37 °C, after which t ime 2.5 ml of 10- 2 M EGTA in saline was added. The t ubes were t hen cen trifuged and t he r esulting supern atants read at 413 nm in a s pectr ophotometer . The results wer e compared to t hose from sa mples in which 100% RBC lysis was o btained with distilled water. Resul ts wer e ex pressed as percent inhibi t ion of hemolys is as a function of either t he dry weight or pac ked volume of orga nism s employed. The a moun t r eq uired to produce 20% or 50% inh ib ition of hemolysis was obtained usin g a se ri es of dilu t ions of t he yeast sus pension.
MATERIALS AND METHODS
Determination of A ntigen Content in Yeast. Extracts by Inhibition of Antibody Activit.y
Organisms Five isolates of P. orbiculare were obtained fro m ski n scra pings of tinea versicolor lesions by cu lt UJ"e on mycosel agar (BBL, D iv. Becton, Dickinso n, and Co., Cockeysville, Mru·ylan d) overla id wit h ste ril e olive oil. S ubc ul t ures were made f!"Om emerging colonies and identili ed as P . orbiculare by morp hologic appear ance a nd a stri ct requirement for added lipid. All 5 isolates grew prefere nt ia lly at t he oil- water in te rface of broth cult ures, although 2 a lso grew in t he wate r phase a nd could be centri fuged free of oil if 0.1% Tween 80 were added to t he med ium. Five isolates of C. albicans were obtained fro m clinical isolates a nd ma intained by repeated passage on Sabouraud's aga r. Both organisms were grown in bul k in AOAC synthetic broth (D ifco La borato ri es, Detroit, Michigan) containi ng 1% dex trose, chlora mph enicol (20 f1.g/ ml) , cyclohexim ide (250 f1.g/ ml ), 0.1 % T wee n 80, and overlaid wi t h sterile oli ve oil. At t he end of t he cul ture period (14 days for P . orbiculare and 5 days fo r C. albicans), t he oil and wate r phases were separated by centrifugation and t he organisms in the water phase washed 3 times in 0.9% NaC I. T he q uantity of orga nisms prese nt was expressed as eit her m l of packed cells or as dry we ight dete rmined a fter lyophilization of t he yeast suspensions. These two methods were used in case one or t he other a lone might have given a n inacc urate estimate of t he quantity of organisms p resent.
P reparation of Ext.racts Extracts of t he ·two organisms were prepar ed as described previously [5] using eit her t he ether extraction method or sonication. P rotei n concentrations of the extracts were dete rmined by the Lowry techniqu e a nd car bo hydrates by t he ac id-phenol method [6]. Measurement of Complement Activation Using Hemolysis of R a bbit Red Blood Cells (RBC) H uman sera we re incubated with known· qu antities of yeast or yeast extracts and then evalu ated fo r t heir a bility to hemolyze ra bbit RBC by the alte rnative pathway using a modification of the method of Platts-M ills and Ishizaka [7). In brief, t he yeast or yeast extracts were incubated for 30 min at 37°C wit h normal hum an serum , hum an se rum with 5 X 10- " Methylene glyco l-bis-(beta-amino et hyl ether)-N,N'tetraacetic acid (EGTA) conta ining 5 X 10- " M of MgC I2, or C2-deficient human serum (kin dly provided by Dr. S usa n Koethe, Med ical College of Wisconsin). T he yeasts were removed by centrifugation, a nd t he
Measuremen t of Complem ent. Acti vation by Qua.ntitating C3 Dep osition on the Y east S urface An enzy me- linked immunoa bsorbent assay (ELISA) was developed to measure t he amoun t of C3 deposited on kn own qu ant it ies of yeast during incubation wi th t he 3 human serum preparations used above. Various dilu tions of yeast suspensions wer e first heated to 100°C for 60 min to remove endogenous per oxidase activity and then incubated in 0.1 ml volumes wi t h 0.1 mI of se rum for 30 min at 37°C. The yeast was next washed 3 t imes in 0.05 M phosphate- bu ffer ed saline (PBS ) conta ining 0.05% Twee n 20. They were then incubated wi t h 0.5 ml of a 1:1000 dilu t ion of peroxidase-conju gated goa t a nti huma n C3 (Cappe U La boratories, Coc hranville, Pennsylva nia ) for 30 min at 37°C and again washed 3 t imes wi th PBS-Tween 20. After removal to a fresh test tube, 1.0 ml of substrate was added (1 mg/ ml O- phenylenedia min e, Eastm a n K oda k Co., Hocheste r, N ew York , in 0.05 M citra te buffe r, pH 4.5, a nd 0.05% H, 0 2). After 40 min of incubat ion at room temper atw·e, th e supernatants were se parated by cent rifugation and read in a spectr ophoto mete r at 420 nm. Data wer e expr essed as opt ical density t imes 1000 per f1.g of yeast.
An E LIS A-type antibody assay was set up for determining ant ibody activi ty aga inst extracts of C. a lbicans a nd P . orb iculare in norma l hum an serum . Polysty rene t ubes we re coated wit h 1.0 ml of a ppropriate dilu t ions of eit her extract in 0.05 M P BS at pH 7.6 fo r 1 hr at roo m temperature. The tu bes were washed 3 t imes wi t h PBS-Tween 20, treated with 1% bovine se rum albumin, a nd washed again. Following t his step, 0.5 m l of a 1:50 dilu t ion of a pool of se rum 1'r om 10 norm al subj ects was added and incuba ted at 37 °C for 30 min. The tubes we re washed 3 t imes wi th PBS-Tween 20, and 0.5 ml of a 1:200 dilu t ion of peroxidase- la beled, a ffini ty- purified goat ant ihuman IgG (Li tton B ionetics, Kensin gto n, Maryland) was added. After incubation for 30 min at 37°C, t he t ubes wer e aga in washed 3 t imes wi t h PBS-Tween 20, and 1.0 ml of the substrate described a bove was add ed . The t ubes were incubated fo r 40 m in and read at 420 nm. Bla nks wer e set up wit hout t he hum an se rulTI and t hese va lu es sub t racted fro m t he experimenta l valu es. The ex perim ents were cruTied out by incubating the pooled hum an serum with various amounts of 4 extracts eac h of C. a lbicans and P . orbiculare and t hen testing the serum for residua l antibody activity aga inst t he corresponding orga nism. The resul t ing percent inhibi tion of t he op t ical density obtained was plotted aga inst t he qu antity of extract employed (based on protein plus carbohydrate co ntent) to lind the amoun t necessary for 50% inhibi t ion. Controls for t he spec ificity of the peroxidase- la beled antihuman heavy-chain reage nts were ca rried out in our syste m as fo llows: purified immunoglobulins of th e 3 classes (CappeLl La boratories, Cochranville, P ennsylva nia ) were coated onto plasti c tubes at a concentration of 10 f1.g/ ml, washed, a nd incubated wit h dilu t ions of t he 3 peroxidase- labeled reagents. Good agreement was ob tained betwee n the stated s pecificity and actua l antibody activity aga inst t he a ppropr iate immunoglobulin class in this test.
Human Sera Three sera fr om norma l persons and one fTom an oth erwise normal, but C2-deficient perso n were used for t he studies of co mpl ement activation. These sera were stored in a liquots at -70°C. The pool of norma l sera was prepru·ed using equ a l volumes of serum fr om 10 norm a l subjects wit hout a history of superfi cial fun ga l infec tions. These sera were kept at -20°C. We have prev iously demonstra ted t hat normal human serum has a nt ibody aga inst Pityrosp orum as well as Cand ida [8).
Feb. 1983
ACTI VATION OF COMPLEMENT BY P. ORBICULARE
Statistics
TABLE
In the complement assays, 4 different yeast prepa ratio ns representing 2 isolates each of the two organis ms were used. For t he studies comparing protein a nd carbohydrate contents of extracts, 6 ether extracts a nd 4 sonicates were tested, representing 5 isolates of each organism in all The extracts that were used in t he studies of a ntigen conte nt a nd complement-activating a bili ty consisted of 2 ether extr acts and 2 sonicates representing 2 isolates of each organism. Co mparisons were m ade between t he various groups usin g the unpaired, two-ta iled t-test.
Sl S2 E1 E2 Sl S2 E1 E2
Complement Activation by C. albicans and P . orbiculare In Table I, the ability of the two organisms to activate complem en t is expressed as th e percent inhibition of rabbit RBC h emolysis after incubation of 5.0% and 0.5% yeast suspensions with the serum. No significant differences were found between the two organisms at eit h er concentration using normal serum or serum lacking the classical pathway by virtue of calcium chelation or C2 deficiency. Similar results were obtained when the yeasts were quantified after lyophilization of aliquots a nd weighing (results not shown). It should be no ted that h e molysis by C2-deficient serum was affected less by incubation with th e two yeasts or with inulin than was normal serum with or without M g-EGTA. This finding may be related to the mildly deficient function of C2-deficient serum in t his system as has been r epor ted previously .[9]. As shown in Table II, h eatin g at 56 °C for I/~ hr significantly suppressed t he a bility of either normal or C2-deficient serum to cause he molysis of rabbit RBC. The a bility of extracts of t he two organisms to activate complem e nt was also evaluated using the rabbit RBC system. TABLE
I. Percent inhibition of rabbit REC h emolysis by hu.man
serum, complement u.sing constant volumes of yeast Serum
Candida
P':ly rosporu m.
(N = 4)
(N = 4)
5,0% yeast by vo lume (mean ± SE of % inhibition) N ormal 91.0 ± Normal + Mg-EGTA 83,5 ± C2-defi cient 33.8 ± 0.5% yeast by vo lume (mean ± SE of % inh ibition) Normal 30.5 ± Normal + Mg-EGTA 32,8 ± C2-deficie nt 1.0 ±
2,7 6,1 10.4
85.3 ± 2,7 86.8 ± 2,0 49.8 ± 9,0
NS NS NS
2. 1 11 ,3 0,6
26,8 ± 3.2 34.3 ± 12.1 6,0 ± 1.5
NS NS p < ,02
Rabbit REC H emo lysis
Norma l C2-defi cient
Percent of
Un heated (% lysis )
(% lysis)
activ ity remaining
74.1% 58.8%
1.7% 2.4%
2.3% 4,1%
C3-0eposition o n Yeast Surface as M easured by the ELISA Assay Serum
Candida Normal C2-deficient Pityrosporwn Normal C2-deficient
Normal hum.an serum 22.2" 27.7 11.1
25,0 7.0 0,0 0.0
12.4 No rm.al human serum + Mg-EGTA 26,7 20.4 38.0 7.8 2,0 5.4 2.0 22.3
" Extracts represent 2 sonicates (Sl and S2) a nd 2 ether extracts (E 1 a nd E2) of each organism, " Dat.a represe nt % inhibition of he molysis aft.er in cubation wit h 500 /lg/ ml of t he indicated extract.. 400 ,---------------------------------____~ f(fJ
w
>-
•
CA NDID A
iii
PITYROSPORUM
300
01
~
a a a
x
>-
f-
200
(fJ
Z
w
o
-'
100
f0.
N OR MAL SERUM
H eated
PityroHpOrU11l
o
II. Comparison of unheated serum vs. serum heated at 56°C for 30 /II,in. in. the assays of complem eniactivation
Serum
Candida
Statistica l s ignifica nce
N ote: Inulin at 10 mg/ ml produced 94% s uppression of hemolysis using normal seru m, 95% using normal se rum + Mg-EGTA, a nd 28% using C2-defi cient serum , TA BLE
III, Percent inlu:bition of rabbit REC hemolysis by human . serum. using 500 /lg/m.l of yeast extracts
Extract"
RESULTS
95
Un heat ed (00 X 1000)
H eated (00 X 1000)
Percen t of activ ity remaining
770 470
60 63
7,8% 13.4 %
947 782
138 163
14,6% 20.8%
These data ar e from 1 representative experimen t of 3 that were performed.
NORMAL SE RUM PLU S Mg - EGTA
C 2-0 EFICIENT SE RUM
FIG 1. D eposition of C3 on t he surface of C. albicans and P. o/'b iculare (4 isolates each) using normal huma n serum, normal human seru m plus Mg-EGTA, or C2-deficient huma n serum, D eposition of C3 was determined using a n ELISA system as out lined in Mate rials and Methods a nd t he results expressed as optical density x 1000/ /lg of yeast. The differ ences in C3 deposition were not significan tly different for t he two types of yeast with a ny of t he 3 t.ypes of serum, For Candida, but not Pityrosporwn, t here was a significa nt differ ence between C3 deposition using normal serum vs. either Mg-EGTAtreated serum or C2-deficient serum (p < 0,02 in both cases).
Two eth er extracts a nd 2 sonicates of each organism were tested in this system at a concentration of 500 flg / ml (combined protein a nd carbohydrate contents), t h e approxin1ate amount at which whole yeast produced 20% inhibition of h emolysis after incubation with t he test serum. As shown in Table III, th e results were quite variable, alt hough some complement-activating a bility was evident for some of the extracts. Complement activation was a lso measured by determining the amount of C3 deposited on the yeast cell surface. This technique was developed because of t he possibility t hat inhibition of rabbit RBC lysis by t h e yeast could have been due to a n actual complement inhibitor of some kind rather th an to th e consumption of complem ent components by t heir activation. As shown in Fig 1, th ere ar e no significant differences in the amo unt of C3 deposited on the sw-face of the two organisms after incubation with huma n serum . It would appeal' that more C3 is deposited on th e yeast swface if serum with an intact classical pathway is used. The differ ence between t h e r esults obtained with normal serum vs. t h at treated with Mg-EGTA or C2-deficient serum was significant for Candida (p < 0.02 in
96
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SOHNLE AND COLLINS-LECH
TABLE IV. Protein and carbohydrate contents o(Candida and Pityrosporum extracts Protein/ Carbohydrate Organism Protein carbohydrate Sonicates" Candida 13.58 ± 3.75 0.93 ± 0.34 9.30± 0.82 Pityrosporum 1.44 ± 0.68 3.54 ± 0.43 4.71 ± 1.76 p < 0.02 p < 0.01 Statistical sig0.1 > p > 0.05 nificance b Ether extracts" 5.67 ± 1.30 0.80 ± 0.37 Candida 3.57 ± 1.21 3.94 ± 0.77 Pityrosporum 2.57 ± 0.61 0.69 ± 0.13 Statistical sigp < 0.01 p < 0.01 NS nificance" " Comparisons were made between protein and carbohydrate contents (expressed as mg/ml of organisms ± S.E.) for 4 sonicates and 6 ether extracts of each organism. " Statistical significance by the unpaired, two-tailed I-test was taken atp < 0.05. TABLE V. Antigenic activity o(Candida and Pityrosporum extracts in an ELISA assay
Organism Candida Pityrosporum
M of Extract for 50% inhibiiion fl
Log 1o values
70.0 ± 28.1 822.6 ± 260.4 p < 0.05
1.73 ± 0.2 2.83 ± 0.2 p < 0.01
Statistical significance" " Combined protein and carbohydrate contents for 2 ether extracts and 2 sonicates of each organism required for 50% inhibition of antibody activity (± S.E.). . " Statistical significance by the unpaired, two-tailed t-test. both cases) but not for Pityrosporum, perhaps because of greater variability in the results obtained with the latter. As shown in Table II, heating serum to 56°C for '/2 11.1' significantly reduced the amount of C3 deposited on the surface of both yeasts using either normal or C2-deficient serum.
Protein, Carbohydrate, and Antigenic Contents of Candida and Pityrosporum Extracts As shown in Table IV, extracts of both organisms contained comparable protein contents, whereas those of Candida contained significantly more carbohydrate. This factor resulted in lower protein to carbohydrate ratios for extracts of Candida than for those of Pityrosporum. Antigenic contents of the extracts were measured using inhibition of antibody activity of pooled human serum against the organisms in an ELISA system as described under Materials and Methods. The results demonstrated that Candida appears to have a significantly higher content of antigen than does Pityrosporum (Table V), a finding compatible with our previous studies using the lymphocytetransformation system [5]. DISCUSSION These experiments indicate that the two yeasts, C. albicans and P. orbiculare, appear to have approximately equal abilities . to activate complement using either the rabbit RBC hemolytic system to measure alternative pathway activity left in human serum after incubation with the organisms or measurement of C3 deposition on the yeast .s urface using an ELISA system. Complement-activating ability appeared to be comparable for the two organisms in both systems using either normal human serum or serum with a nonfunction'a l classical pathway because of C2 deficiency or the addition of Mg-EGT A. In addition, these studies have demonstrated that extracts of Candida contain more carbohydrate and antigenic activity than do those of Pityrosporum. Many microorganisms that are pathogenic for humans initiate complement activation via the alternative pathway. These include: (a) other Candida species in addition to C. albicans
[10]; (b) Cryptococcus neoformans [11]; (c) Paracoccidioides brasiliensis [12]; (d) Streptococcus pneumonia [13]; and (e) the sindbis virus [14]. In addition, complement activation has been suggested as a possible cause of inflammation in acne [15]. Finally, complement activation, particularly that occurring via the alternative pathway, has been demonstrated to be important in the defense against experimental intravascular infections with C. albicans [16] and pneumococci [17]. Whether or not this process is really important in the host defense against these pathogens causing human disease is unclear. This situation is comparable to the status of alternative pathway activation by small molecules as a cause of some cases of asthma-an attractive hypothesis but without solid evidence backing it [18]. The results of the present investigation indicate that the lack of inflammation in the lesions of tinea versicolor is not due to the inability of the causative organism, P. orbiculare, to activate complement. This organism is approximately as capable in initiating complement activation by both pathways as is C. albicans, a producer of highly inflammatory cutaneous lesions. This finding is interesting inasmuch as Pityrosporum appears to contain significantly less extractable carbohydrate and antigenic material than does Candida. The ability to activate complement is obviously not a property singularly sufficient for an infecting organism to produce inflammatory skin lesions. Although in biopsies of Candida- and Pityrosporum-infected skin in humans the organisms are generally found at the same locations, i.e., confined to the stratum corneum [2,19], it is possible that a greater capacity of Candida for deeper invasion through the stratum corneum may be the reason for the differences in inflammation produced by the two organisms. Evidence to support such an explanation has been generated in studies of experimental cutaneous infections of mice produced by C. albicans and other Candida species [3]. In addition, it is possible that Pityrosporum does not release soluble complement-activating factors that can diffuse across the stratum corneum to encounter a source of activatable complement. However, an alternative explanation is that some factor instead of or in addition to complement activation is important for the production of inflammation in superficial fungal infections. Differences in the quantity of antigen present and the ability to elicit immunologic reactions could account for the differences in inflammatory potential of these two organisms, as we have suggested previously [5]. The authors thank Dr. Susan Koethe (Medical College of Wisconsin, Milwaukee, Wisconsin) for the gift of the C2-deficient serum and Victoria Gollish for typing the manuscript. REFERENCES 1. Rebora A, Marples RR, Kligman AM: Experimental infection with Candida albican.s. Arch Dermatol 108:69-73, 1973 2. Kirkpatrick CH, Rich RR, Bennett JE: Chronic mucocutaneous candidiasis: model building in cellular immunity. Ann Intern Med 74:955-978, 1971 3. Ray TL, Wuepper KD: Experimental cutaneous candidiasis in rodents. II. Role of the stmtum corneum barrier p.nd serum complement as a mediator of a protective inflammatory response. Arch Dermatol 114:539-543, 1978 4. Kerbs S, Gl'eenberg J, J esrani K: Temporal correlation of lymphocyte blastogenesis, skin test responses, and erythema during dermatophyte infections. Clin Exp Immunol 27:526-530, 1977 5. Sohnle PG, Collins-Lech C: Relative antigenicity of P. orbiclllare and C. albican.s. J Invest Dermatol 75:279-283, 1980 6. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith E: Colorimetric method for determination of sugars and related substances. Anal Chern 28:350-356, 1956 7. Platts-Mills TAE, Ishizaka K: Activation of the alternate pathway of human complement by rabbit cells. J Immunol 113:348-358, 1974 8. DaMert GJ, Kirkpatrick CH, Sohnle PG: Comparison of antibody responses in chronic mucocutaneous candidiasis and tinea versicolor. Int Arch Allergy Appl Immunol 63:97-104, 1980 9. Polhill HB, Pruitt KM, Johnston RB, Jr: Kinetic assessment of alternative complement pathway activity in a hemolytic system. I. Experimental and mathematical analyses. J Immunol 121:363-368, 1978
Feb. 1983
RAT SKIN ODC ANTIZYME
10. Ray TL, Wu ep per KD: Activation of the a lte rnative (properd in) pathway of compl e men t by Candida albicans a nd re lated s pecies. J Invest Dermatol 67:700-703, 1976 11. Dia mond RD, May JE, Kane MA, Frank MM, Be nn ett JE: The role of t he class ical a nd a ltern ate complement pathways in host d efenses aga inst C,yptococcus n eofonnans infection. J Immunol 1l2:~ ::SO-2270 , 1974 12. Calich VLG, Kipnis TL, M a riano M, Neto CF, Dias Da Silva W: Th e activation of t he co mplement system by Paracoccidioides brasiliensis in vitro: its opsonic effect a nd possible significa nce for an in vivo model of infection. Clin Immunol Immunopathol 12:20-30, 1979 13. Winke lstein JA, Tomasz A: Activation of t he alternate pathway by pneumococcal ce ll wa lls. J ImmunoI1l8:45J-454, 1977 14. Hirs h RL, Winke lste in JA, Griffin DE: The rol e of comp le men t in viral infections. III. Activation of t he classica l and alternative
15. 16. 17. 18. 19.
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complement pathways by s indbis virus. J Immunol 124 :2507-2510, 1980 Webste r GF, Leyden JJ, Nilsson UR: Complemen t activation in ac ne vulgaris: co nsump tion of co mple ment by comedones. Infect Immun 26:183-186, 1979 Ge lfand JA, Hurley DL, Fauci AS, Frank MM: Role of co mplement in host de fense aga inst experimen ta l disse minated ca ndidiasis. J Infect Dis 138:9-16, 1978 Hosea SW, Brown EJ, Frank MM: The critica l role of complement in ex perimental pneumococca l sepsis. J Infect Dis 142:903-909, 1980 Wilson MR, Salvaggio JE: Activation of the a lternative pathway of compl ement: a mec hanism in search of diseases. J Allergy Clin Immunol 65:319-321, 1980 Mon tes LF: Systemic a bnorma lities a nd t he intrace llul ar s ite of infections of the stratum corneum. JAMA 213: 1469-1472, 1970
0022-202X/ 83/8002-0097$02.00/ 0 TBE JOURNAL OF I NVESTl (;A TI VE [)t;IlMATOLO(:Y.
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Antizyme Release Is an Early Event in Ornithine Decarboxylase Induction by Hair Plucking* JEANNE LESIEWI CZ, PH.D. AND LOWELL
A. GOLDSMITH, M.D .
Division olDennatology, Departrnent of Jltfedicin e, Dulle University Medical Center, Durham, North Carolina, U.S.A.
Plucking of hair from the dorsal skin of rats resulted in a rapid decrease in ornithine decarboxylase (ODe) activity. A significant loss of activity did not occur in other skin enzymes under the same conditions and in vivo incorporation of [3H]-leucine in skin was not significantly decreased 60 min immediately following hair plucking. Treatment of ODe enzyme preparations with 10% (NILhSo', resulted in recovery of approximately 75 % greater ODe activity than in untreated samples, suggesting the presence of an inhibitor (antizyme). ODe inhibitor was detected in plucked skin; inhibitor levels increased after treatment of plucked skin extracts with 10% (NH4 hSo',.
In the rat, hair growth occurs in a cyclic manner. P lucking of hair during the telogen phase induces high levels of ornithine decarboxylase (L-ornithine carboxylase, Ee 4 .1.1.1 7) (ODe) [1] within 4 hr [2]. However, during the 60 'min immediately following hail' plucking ODe activity is observed to decr ease [1,3]. The cause of this initial loss of ODe enzyme activity is the' subject of this report.
M a nuscript received January 21, 1982; acce pted for publication June 14 , 1982. Supported in part by NIH grant AM-17253. • Pub lication No. 104 of the Dermatological Research Laboratories of Duke University M edi ca l Center. R eprin t requ ests to: Dr. L. A. Goldsmith , Department of Med icine, Dermatology Unit, Un iversity of Rochester School of Med icin e and Dentistry, 601 E lmwood Avenu e, Rochester, New York 14642. Abbreviations: DDT: dithiothre ito l EDTA: (ethyle ned initrilol-tetraacetic acid, disod ium salt ODC: ornit hine decarboxy lase PLP: pyridoxal-5' -phos ph ate TCA: trichloroacetic acid Tris: trih ydroxyam inomethane
MATERIALS AND METHODS Ch.emicals Trihydroxyaminomethane (Tris) , pyridoxal-5'-phosphate (PLP), and cyclohex imide, were purchased from Sigma Chemica l Co., St. Louis, Missouri. DL-[l-"'C]-ornithine (51.2 mC/ mm ol), L-[4,5-"H]-leucine (12 Ci/mmol) , [1,4-'4C]-putrecine (89.9 mCi/mmo l), phenethylamine, and Aqu aso l II were from New England Nuclear, Boston, Massachusetts. Enzyme-grade sucrose a nd ultra pure ammonium s ulfate were fTo m Schwartz-Mann, S prin g Valley, New York. Dithiothreitol (OTT) was from Bachem Feinchemikalien, L iesta l, Switzerland. Biogel-PlOO was obtained fTom Bio-Rad Laboratories, Richmond, California . Anim.als Juvenile ma le CD strain rats weighing 80-100 g (24-28 days old) were obtained from Charles R iver Breeding Laboratories, Wilmington, Massachusetts. For ODC enzyme studi es the animals were used in t he early a nagen ph ase of ha ir growth, which was defined as dorsa l skin fo r which t he most ventral edge of a patch was within 1 cm of the discernib le border of new hair el·up tio n. For inhibitor studies, a nima ls in the telogen phase were used. This phase was defined as s kin for which no border of new ha ir growth was detected on t he an ima l's dorsum.
Time Co u.rse of Plucking R esponse Animals were anesthesized with ether and the ha ir plu cked from their dorsal skin in patches 60, 45, 30, 15, 5, a nd 0 (immediately) min before death by decapitation. H a ir plucking was accomp lished using hemostat forceps which had t hin-walled rubber tubing forced over the ends. The hair co uld t hen be grasped t ightly a nd jerked free in clumps. A 2.5 x 2.5 cm skin patch can be plucked bare in 15-20 sec by t his method. Th e skin patches were excised, cleaned of underly ing musclllature, weighed, a nd homogenized 1:9 (weight:volume) in ODe buffer (10 mM Tris pH 7.0, 0.5 mM EDTA pH 7.0, 0.01 mM PLP, and 5 mM DTT) as previously described [2]. ODC assays a nd turbidim etric protein determinations were performed as previously described [3]. One uni t of ODC activity is ca pable of releasing 1 Ilmol "'COz/hr.
Response of Other Shin Enzym.es to H air Pluching Anima ls were anesthesized with ether and t he ha ir plucked from one half of their dorsal skin 60 a nd 0 min before death by decapitation. Th e